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Archive for the ‘Cardiomyopathy’ Category


Christopher J. Lynch, MD, PhD, the New Office of Nutrition Research, Director

Curator: Larry H. Bernstein, MD, FCAP

 

Christopher J. Lynch to direct Office of Nutrition Research

National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

http://www.nih.gov/news-events/news-releases/christopher-j-lynch-direct-office-nutrition-research

 

Christopher J. Lynch, Ph.D., has been named the new director of the Office of Nutrition Research (ONR) and chief of the Nutrition Research Branch within the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Lynch officially assumed his new roles on Feb. 21, 2016. NIDDK is part of the National Institutes of Health.

Lynch will facilitate nutrition research within NIDDK and — through ONR — across NIH, in part by forming and leading a trans-NIH strategic working group. He will also continue and extend ongoing efforts at NIDDK to collaborate widely to advance nutrition research.

“Dr. Lynch is a leader in the nutrition community and his expertise will be vital to guiding the NIH strategic plan for nutrition research,” said NIH Director Francis S. Collins, M.D., Ph.D.  “As NIH works to expand nutrition knowledge, Dr. Lynch’s understanding of the field will help identify information gaps and create a framework to support future discoveries to ultimately improve human health.”

NIH supports a broad range of nutrition research, including studies on the effects of nutrient and dietary intake on human growth and disease, genetic influences on human nutrition and metabolism and other scientific areas. ONR was established in August 2015 to help NIH develop a strategic plan to expand mission-specific nutrition research.

NARRATIVE:
Our laboratory is dedicated to developing cures for metabolic diseases like Obesity, Diabetes and MSUD. We have several projects:
Project 1: How Antipsychotic Drugs Exert Obesity and Metabolic Disease Side effects
Project 2: Impact of Branched Chain Amino Acid (BCAA) signaling and metabolism in obesity and diabetes.
Project 3: Adipose tissue transplant as a treatment for Maple Syrup Urine Disease.
Project 4: How Gastric Bypass Surgery Provides A Rapid Cure For Diabetes And Other Obesity Co-Morbidities Like Hypertension
Project 5: Novel Mechanism Of Action Of Cannabinoid Receptor 1 Blockers For Improvement Of Diabetes

Timeline

  1. Klingerman CM, Stipanovic ME, Hajnal A, Lynch CJ. Acute Metabolic Effects of Olanzapine Depend on Dose and Injection Site. Dose Response. 2015 Oct-Dec; 13(4):1559325815618915.

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  1. Lynch CJ, Kimball SR, Xu Y, Salzberg AC, Kawasawa YI. Global deletion of BCATm increases expression of skeletal muscle genes associated with protein turnover. Physiol Genomics. 2015 Nov; 47(11):569-80.

View in: PubMed

  1. Lynch CJ, Xu Y, Hajnal A, Salzberg AC, Kawasawa YI. RNA sequencing reveals a slow to fast muscle fiber type transition after olanzapine infusion in rats. PLoS One. 2015; 10(4):e0123966.

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  1. Shin AC, Fasshauer M, Filatova N, Grundell LA, Zielinski E, Zhou JY, Scherer T, Lindtner C, White PJ, Lapworth AL, Ilkayeva O, Knippschild U, Wolf AM, Scheja L, Grove KL, Smith RD, Qian WJ, Lynch CJ, Newgard CB, Buettner C. Brain Insulin Lowers Circulating BCAA Levels by Inducing Hepatic BCAA Catabolism. Cell Metab. 2014 Nov 4; 20(5):898-909.

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  1. Lynch CJ, Adams SH. Branched-chain amino acids in metabolic signalling and insulin resistance. Nat Rev Endocrinol. 2014 Dec; 10(12):723-36.

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  1. Olson KC, Chen G, Xu Y, Hajnal A, Lynch CJ. Alloisoleucine differentiates the branched-chain aminoacidemia of Zucker and dietary obese rats. Obesity (Silver Spring). 2014 May; 22(5):1212-5.

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  1. Zimmerman HA, Olson KC, Chen G, Lynch CJ. Adipose transplant for inborn errors of branched chain amino acid metabolism in mice. Mol Genet Metab. 2013 Aug; 109(4):345-53.

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  1. Olson KC, Chen G, Lynch CJ. Quantification of branched-chain keto acids in tissue by ultra fast liquid chromatography-mass spectrometry. Anal Biochem. 2013 Aug 15; 439(2):116-22.

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  1. She P, Olson KC, Kadota Y, Inukai A, Shimomura Y, Hoppel CL, Adams SH, Kawamata Y, Matsumoto H, Sakai R, Lang CH, Lynch CJ. Leucine and protein metabolism in obese Zucker rats. PLoS One. 2013; 8(3):e59443.

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  1. Lackey DE, Lynch CJ, Olson KC, Mostaedi R, Ali M, Smith WH, Karpe F, Humphreys S, Bedinger DH, Dunn TN, Thomas AP, Oort PJ, Kieffer DA, Amin R, Bettaieb A, Haj FG, Permana P, Anthony TG, Adams SH. Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity. Am J Physiol Endocrinol Metab. 2013 Jun 1; 304(11):E1175-87.

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  1. Klingerman CM, Stipanovic ME, Bader M, Lynch CJ. Second-generation antipsychotics cause a rapid switch to fat oxidation that is required for survival in C57BL/6J mice. Schizophr Bull. 2014 Mar; 40(2):327-40.

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  1. Carr TD, DiGiovanni J, Lynch CJ, Shantz LM. Inhibition of mTOR suppresses UVB-induced keratinocyte proliferation and survival. Cancer Prev Res (Phila). 2012 Dec; 5(12):1394-404.

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  1. Lynch CJ, Zhou Q, Shyng SL, Heal DJ, Cheetham SC, Dickinson K, Gregory P, Firnges M, Nordheim U, Goshorn S, Reiche D, Turski L, Antel J. Some cannabinoid receptor ligands and their distomers are direct-acting openers of SUR1 K(ATP) channels. Am J Physiol Endocrinol Metab. 2012 Mar 1; 302(5):E540-51.

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  1. Albaugh VL, Singareddy R, Mauger D, Lynch CJ. A double blind, placebo-controlled, randomized crossover study of the acute metabolic effects of olanzapine in healthy volunteers. PLoS One. 2011; 6(8):e22662.

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  1. She P, Zhang Z, Marchionini D, Diaz WC, Jetton TJ, Kimball SR, Vary TC, Lang CH, Lynch CJ. Molecular characterization of skeletal muscle atrophy in the R6/2 mouse model of Huntington’s disease. Am J Physiol Endocrinol Metab. 2011 Jul; 301(1):E49-61.

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  1. Fogle RL, Hollenbeak CS, Stanley BA, Vary TC, Kimball SR, Lynch CJ. Functional proteomic analysis reveals sex-dependent differences in structural and energy-producing myocardial proteins in rat model of alcoholic cardiomyopathy. Physiol Genomics. 2011 Apr 12; 43(7):346-56.

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  1. Zhou Y, Jetton TL, Goshorn S, Lynch CJ, She P. Transamination is required for {alpha}-ketoisocaproate but not leucine to stimulate insulin secretion. J Biol Chem. 2010 Oct 29; 285(44):33718-26.

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  1. Agostino NM, Chinchilli VM, Lynch CJ, Koszyk-Szewczyk A, Gingrich R, Sivik J, Drabick JJ. Effect of the tyrosine kinase inhibitors (sunitinib, sorafenib, dasatinib, and imatinib) on blood glucose levels in diabetic and nondiabetic patients in general clinical practice. J Oncol Pharm Pract. 2011 Sep; 17(3):197-202.

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  1. Li J, Romestaing C, Han X, Li Y, Hao X, Wu Y, Sun C, Liu X, Jefferson LS, Xiong J, Lanoue KF, Chang Z, Lynch CJ, Wang H, Shi Y. Cardiolipin remodeling by ALCAT1 links oxidative stress and mitochondrial dysfunction to obesity. Cell Metab. 2010 Aug 4; 12(2):154-65.

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  1. Culnan DM, Albaugh V, Sun M, Lynch CJ, Lang CH, Cooney RN. Ileal interposition improves glucose tolerance and insulin sensitivity in the obese Zucker rat. Am J Physiol Gastrointest Liver Physiol. 2010 Sep; 299(3):G751-60.

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  1. Hajnal A, Kovacs P, Ahmed T, Meirelles K, Lynch CJ, Cooney RN. Gastric bypass surgery alters behavioral and neural taste functions for sweet taste in obese rats. Am J Physiol Gastrointest Liver Physiol. 2010 Oct; 299(4):G967-79.

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  1. Lang CH, Lynch CJ, Vary TC. BCATm deficiency ameliorates endotoxin-induced decrease in muscle protein synthesis and improves survival in septic mice. Am J Physiol Regul Integr Comp Physiol. 2010 Sep; 299(3):R935-44.

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  1. Albaugh VL, Vary TC, Ilkayeva O, Wenner BR, Maresca KP, Joyal JL, Breazeale S, Elich TD, Lang CH, Lynch CJ. Atypical antipsychotics rapidly and inappropriately switch peripheral fuel utilization to lipids, impairing metabolic flexibility in rodents. Schizophr Bull. 2012 Jan; 38(1):153-66.

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  1. Fogle RL, Lynch CJ, Palopoli M, Deiter G, Stanley BA, Vary TC. Impact of chronic alcohol ingestion on cardiac muscle protein expression. Alcohol Clin Exp Res. 2010 Jul; 34(7):1226-34.

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  1. Lang CH, Frost RA, Bronson SK, Lynch CJ, Vary TC. Skeletal muscle protein balance in mTOR heterozygous mice in response to inflammation and leucine. Am J Physiol Endocrinol Metab. 2010 Jun; 298(6):E1283-94.

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  1. Albaugh VL, Judson JG, She P, Lang CH, Maresca KP, Joyal JL, Lynch CJ. Olanzapine promotes fat accumulation in male rats by decreasing physical activity, repartitioning energy and increasing adipose tissue lipogenesis while impairing lipolysis. Mol Psychiatry. 2011 May; 16(5):569-81.

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  1. Lang CH, Lynch CJ, Vary TC. Alcohol-induced IGF-I resistance is ameliorated in mice deficient for mitochondrial branched-chain aminotransferase. J Nutr. 2010 May; 140(5):932-8.

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  1. She P, Zhou Y, Zhang Z, Griffin K, Gowda K, Lynch CJ. Disruption of BCAA metabolism in mice impairs exercise metabolism and endurance. J Appl Physiol (1985). 2010 Apr; 108(4):941-9.

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  1. Herman MA, She P, Peroni OD, Lynch CJ, Kahn BB. Adipose tissue branched chain amino acid (BCAA) metabolism modulates circulating BCAA levels. J Biol Chem. 2010 Apr 9; 285(15):11348-56.

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  1. Li P, Knabe DA, Kim SW, Lynch CJ, Hutson SM, Wu G. Lactating porcine mammary tissue catabolizes branched-chain amino acids for glutamine and aspartate synthesis. J Nutr. 2009 Aug; 139(8):1502-9.

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  1. Lu G, Sun H, She P, Youn JY, Warburton S, Ping P, Vondriska TM, Cai H, Lynch CJ, Wang Y. Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells. J Clin Invest. 2009 Jun; 119(6):1678-87.

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  1. Nairizi A, She P, Vary TC, Lynch CJ. Leucine supplementation of drinking water does not alter susceptibility to diet-induced obesity in mice. J Nutr. 2009 Apr; 139(4):715-9.

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  1. Meirelles K, Ahmed T, Culnan DM, Lynch CJ, Lang CH, Cooney RN. Mechanisms of glucose homeostasis after Roux-en-Y gastric bypass surgery in the obese, insulin-resistant Zucker rat. Ann Surg. 2009 Feb; 249(2):277-85.

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  1. Culnan DM, Cooney RN, Stanley B, Lynch CJ. Apolipoprotein A-IV, a putative satiety/antiatherogenic factor, rises after gastric bypass. Obesity (Silver Spring). 2009 Jan; 17(1):46-52.

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  1. She P, Van Horn C, Reid T, Hutson SM, Cooney RN, Lynch CJ. Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism. Am J Physiol Endocrinol Metab. 2007 Dec; 293(6):E1552-63.

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  1. She P, Reid TM, Bronson SK, Vary TC, Hajnal A, Lynch CJ, Hutson SM. Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle. Cell Metab. 2007 Sep; 6(3):181-94.

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  1. Vary TC, Lynch CJ. Nutrient signaling components controlling protein synthesis in striated muscle. J Nutr. 2007 Aug; 137(8):1835-43.

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  1. Vary TC, Deiter G, Lynch CJ. Rapamycin limits formation of active eukaryotic initiation factor 4F complex following meal feeding in rat hearts. J Nutr. 2007 Aug; 137(8):1857-62.

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  1. Vary TC, Anthony JC, Jefferson LS, Kimball SR, Lynch CJ. Rapamycin blunts nutrient stimulation of eIF4G, but not PKCepsilon phosphorylation, in skeletal muscle. Am J Physiol Endocrinol Metab. 2007 Jul; 293(1):E188-96.

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  1. Vary TC, Lynch CJ. Meal feeding stimulates phosphorylation of multiple effector proteins regulating protein synthetic processes in rat hearts. J Nutr. 2006 Sep; 136(9):2284-90.

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  1. Lynch CJ, Gern B, Lloyd C, Hutson SM, Eicher R, Vary TC. Leucine in food mediates some of the postprandial rise in plasma leptin concentrations. Am J Physiol Endocrinol Metab. 2006 Sep; 291(3):E621-30.

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  1. Albaugh VL, Henry CR, Bello NT, Hajnal A, Lynch SL, Halle B, Lynch CJ. Hormonal and metabolic effects of olanzapine and clozapine related to body weight in rodents. Obesity (Silver Spring). 2006 Jan; 14(1):36-51.

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  1. Vary TC, Lynch CJ. Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation. Am J Physiol Endocrinol Metab. 2006 Apr; 290(4):E631-42.

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  1. Vary TC, Goodman S, Kilpatrick LE, Lynch CJ. Nutrient regulation of PKCepsilon is mediated by leucine, not insulin, in skeletal muscle. Am J Physiol Endocrinol Metab. 2005 Oct; 289(4):E684-94.

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  1. Vary TC, Lynch CJ. Biochemical approaches for nutritional support of skeletal muscle protein metabolism during sepsis. Nutr Res Rev. 2004 Jun; 17(1):77-88.

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  1. Lynch CJ, Halle B, Fujii H, Vary TC, Wallin R, Damuni Z, Hutson SM. Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR. Am J Physiol Endocrinol Metab. 2003 Oct; 285(4):E854-63.

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  1. Lynch CJ, Hutson SM, Patson BJ, Vaval A, Vary TC. Tissue-specific effects of chronic dietary leucine and norleucine supplementation on protein synthesis in rats. Am J Physiol Endocrinol Metab. 2002 Oct; 283(4):E824-35.

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  1. Lynch CJ, Patson BJ, Anthony J, Vaval A, Jefferson LS, Vary TC. Leucine is a direct-acting nutrient signal that regulates protein synthesis in adipose tissue. Am J Physiol Endocrinol Metab. 2002 Sep; 283(3):E503-13.

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  1. Vary TC, Lynch CJ, Lang CH. Effects of chronic alcohol consumption on regulation of myocardial protein synthesis. Am J Physiol Heart Circ Physiol. 2001 Sep; 281(3):H1242-51.

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  1. Lynch CJ, Patson BJ, Goodman SA, Trapolsi D, Kimball SR. Zinc stimulates the activity of the insulin- and nutrient-regulated protein kinase mTOR. Am J Physiol Endocrinol Metab. 2001 Jul; 281(1):E25-34.

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Global deletion of BCATm increases expression of skeletal muscle genes associated with protein turnover.

Lynch CJ1Kimball SR2Xu Y2Salzberg AC3Kawasawa YI4.   Author information
Physiol Genomics. 2015 Nov;47(11):569-80.  http://dx.doi.org:/10.1152/physiolgenomics.00055.2015

Consumption of a protein-containing meal by a fasted animal promotes protein accretion in skeletal muscle, in part through leucine stimulation of protein synthesis and indirectly through repression of protein degradation mediated by its metabolite, α-ketoisocaproate. Mice lacking the mitochondrial branched-chain aminotransferase (BCATm/Bcat2), which interconverts leucine and α-ketoisocaproate, exhibit elevated protein turnover. Here, the transcriptomes of gastrocnemius muscle from BCATm knockout (KO) and wild-type mice were compared by next-generation RNA sequencing (RNA-Seq) to identify potential adaptations associated with their persistently altered nutrient signaling. Statistically significant changes in the abundance of 1,486/∼39,010 genes were identified. Bioinformatics analysis of the RNA-Seq data indicated that pathways involved in protein synthesis [eukaryotic initiation factor (eIF)-2, mammalian target of rapamycin, eIF4, and p70S6K pathways including 40S and 60S ribosomal proteins], protein breakdown (e.g., ubiquitin mediated), and muscle degeneration (apoptosis, atrophy, myopathy, and cell death) were upregulated. Also in agreement with our previous observations, the abundance of mRNAs associated with reduced body size, glycemia, plasma insulin, and lipid signaling pathways was altered in BCATm KO mice. Consistently, genes encoding anaerobic and/or oxidative metabolism of carbohydrate, fatty acids, and branched chain amino acids were modestly but systematically reduced. Although there was no indication that muscle fiber type was different between KO and wild-type mice, a difference in the abundance of mRNAs associated with a muscular dystrophy phenotype was observed, consistent with the published exercise intolerance of these mice. The results suggest transcriptional adaptations occur in BCATm KO mice that along with altered nutrient signaling may contribute to their previously reported protein turnover, metabolic and exercise phenotypes.

 

RNA sequencing reveals a slow to fast muscle fiber type transition after olanzapine infusion in rats.

Lynch CJ1Xu Y1Hajnal A2Salzberg AC3Kawasawa YI4. Author information
PLoS One. 2015 Apr 20;10(4):e0123966. http://dx.doi.org:/10.1371/journal.pone.0123966. eCollection 2015.

Second generation antipsychotics (SGAs), like olanzapine, exhibit acute metabolic side effects leading to metabolic inflexibility, hyperglycemia, adiposity and diabetes. Understanding how SGAs affect the skeletal muscle transcriptome could elucidate approaches for mitigating these side effects. Male Sprague-Dawley rats were infused intravenously with vehicle or olanzapine for 24h using a dose leading to a mild hyperglycemia. RNA-Seq was performed on gastrocnemius muscle, followed by alignment of the data with the Rat Genome Assembly 5.0. Olanzapine altered expression of 1347 out of 26407 genes. Genes encoding skeletal muscle fiber-type specific sarcomeric, ion channel, glycolytic, O2- and Ca2+-handling, TCA cycle, vascularization and lipid oxidation proteins and pathways, along with NADH shuttles and LDH isoforms were affected. Bioinformatics analyses indicate that olanzapine decreased the expression of slower and more oxidative fiber type genes (e.g., type 1), while up regulating those for the most glycolytic and least metabolically flexible, fast twitch fiber type, IIb. Protein turnover genes, necessary to bring about transition, were also up regulated. Potential upstream regulators were also identified. Olanzapine appears to be rapidly affecting the muscle transcriptome to bring about a change to a fast-glycolytic fiber type. Such fiber types are more susceptible than slow muscle to atrophy, and such transitions are observed in chronic metabolic diseases. Thus these effects could contribute to the altered body composition and metabolic disease olanzapine causes. A potential interventional strategy is implicated because aerobic exercise, in contrast to resistance exercise, can oppose such slow to fast fiber transitions.

 

Brain insulin lowers circulating BCAA levels by inducing hepatic BCAA catabolism.

Shin AC1Fasshauer M1Filatova N1Grundell LA1Zielinski E1Zhou JY2Scherer T1Lindtner C1White PJ3Lapworth AL3,Ilkayeva O3Knippschild U4Wolf AM4Scheja L5Grove KL6Smith RD2Qian WJ2Lynch CJ7Newgard CB3Buettner C8. Author information
Cell Metab. 2014 Nov 4;20(5):898-909. http://dx.doi.org:/10.1016/j.cmet.2014.09.003   Epub 2014 Oct 9

Circulating branched-chain amino acid (BCAA) levels are elevated in obesity/diabetes and are a sensitive predictor for type 2 diabetes. Here we show in rats that insulin dose-dependently lowers plasma BCAA levels through induction of hepatic protein expression and activity of branched-chain α-keto acid dehydrogenase (BCKDH), the rate-limiting enzyme in the BCAA degradation pathway. Selective induction of hypothalamic insulin signaling in rats and genetic modulation of brain insulin receptors in mice demonstrate that brain insulin signaling is a major regulator of BCAA metabolism by inducing hepatic BCKDH. Short-term overfeeding impairs the ability of brain insulin to lower BCAAs in rats. High-fat feeding in nonhuman primates and obesity and/or diabetes in humans is associated with reduced BCKDH protein in liver. These findings support the concept that decreased hepatic BCKDH is a major cause of increased plasma BCAAs and that hypothalamic insulin resistance may account for impaired BCAA metabolism in obesity and diabetes.

 

Branched-chain amino acids in metabolic signalling and insulin resistance.

Lynch CJ1Adams SH2Author information
Nat Rev Endocrinol. 2014 Dec; 10(12):723-36. http://dx.doi.org:/10.1038/nrendo.2014.171

Branched-chain amino acids (BCAAs) are important nutrient signals that have direct and indirect effects. Frequently, BCAAs have been reported to mediate antiobesity effects, especially in rodent models. However, circulating levels of BCAAs tend to be increased in individuals with obesity and are associated with worse metabolic health and future insulin resistance or type 2 diabetes mellitus (T2DM). A hypothesized mechanism linking increased levels of BCAAs and T2DM involves leucine-mediated activation of the mammalian target of rapamycin complex 1 (mTORC1), which results in uncoupling of insulin signalling at an early stage. A BCAA dysmetabolism model proposes that the accumulation of mitotoxic metabolites (and not BCAAs per se) promotes β-cell mitochondrial dysfunction, stress signalling and apoptosis associated with T2DM. Alternatively, insulin resistance might promote aminoacidaemia by increasing the protein degradation that insulin normally suppresses, and/or by eliciting an impairment of efficient BCAA oxidative metabolism in some tissues. Whether and how impaired BCAA metabolism might occur in obesity is discussed in this Review. Research on the role of individual and model-dependent differences in BCAA metabolism is needed, as several genes (BCKDHA, PPM1K, IVD and KLF15) have been designated as candidate genes for obesity and/or T2DM in humans, and distinct phenotypes of tissue-specific branched chain ketoacid dehydrogenase complex activity have been detected in animal models of obesity and T2DM.

 

Leucine and protein metabolism in obese Zucker rats.

She P1Olson KCKadota YInukai AShimomura YHoppel CLAdams SHKawamata YMatsumoto HSakai RLang CHLynch CJAuthor information
PLoS One. 2013;8(3):e59443. http://dx.doi.org:/10.1371/journal.pone.0059443   Epub 2013 Mar 20.

Branched-chain amino acids (BCAAs) are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1-(14)C]-leucine metabolism, tissue-specific protein synthesis and branched-chain keto-acid (BCKA) dehydrogenase complex (BCKDC) activities. Male obese Zucker rats (11-weeks old) had increased body weight (BW, 53%), liver (107%) and fat (∼300%), but lower plantaris and gastrocnemius masses (-21-24%). Plasma BCAAs and BCKAs were elevated 45-69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%), leucine (Leu) turnover and proteolysis [35% per g fat free mass (FFM), urinary markers of proteolysis: 3-methylhistidine (183%) and 4-hydroxyproline (766%)] and decreased BCKDC per g kidney, heart, gastrocnemius and liver (-47-66%). A process disposing of circulating BCAAs, protein synthesis, was increased 23-29% by obesity in whole-body (FFM corrected), gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193-418%) than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and protein turnover along with impaired BCKDC activity. Elevated BCAAs/BCKAs may contribute to observed elevations in protein synthesis and BCAA oxidation.

 

Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity.

Lackey DE1Lynch CJOlson KCMostaedi RAli MSmith WHKarpe FHumphreys SBedinger DHDunn TNThomas APOort PJKieffer DAAmin RBettaieb AHaj FGPermana PAnthony TGAdams SH.
Am J Physiol Endocrinol Metab. 2013 Jun 1; 304(11):E1175-87. http://dx.doi.org:/10.1152/ajpendo.00630.2012

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

 

Some cannabinoid receptor ligands and their distomers are direct-acting openers of SUR1 K(ATP) channels.

Lynch CJ1Zhou QShyng SLHeal DJCheetham SCDickinson KGregory PFirnges MNordheim UGoshorn SReiche D,Turski LAntel J.   Author information
Am J Physiol Endocrinol Metab. 2012 Mar 1;302(5):E540-51.
http://dx.doi.org:/10.1152/ajpendo.00258.2011

Here, we examined the chronic effects of two cannabinoid receptor-1 (CB1) inverse agonists, rimonabant and ibipinabant, in hyperinsulinemic Zucker rats to determine their chronic effects on insulinemia. Rimonabant and ibipinabant (10 mg·kg⁻¹·day⁻¹) elicited body weight-independent improvements in insulinemia and glycemia during 10 wk of chronic treatment. To elucidate the mechanism of insulin lowering, acute in vivo and in vitro studies were then performed. Surprisingly, chronic treatment was not required for insulin lowering. In acute in vivo and in vitro studies, the CB1 inverse agonists exhibited acute K channel opener (KCO; e.g., diazoxide and NN414)-like effects on glucose tolerance and glucose-stimulated insulin secretion (GSIS) with approximately fivefold better potency than diazoxide. Followup studies implied that these effects were inconsistent with a CB1-mediated mechanism. Thus effects of several CB1 agonists, inverse agonists, and distomers during GTTs or GSIS studies using perifused rat islets were unpredictable from their known CB1 activities. In vivo rimonabant and ibipinabant caused glucose intolerance in CB1 but not SUR1-KO mice. Electrophysiological studies indicated that, compared with diazoxide, 3 μM rimonabant and ibipinabant are partial agonists for K channel opening. Partial agonism was consistent with data from radioligand binding assays designed to detect SUR1 K(ATP) KCOs where rimonabant and ibipinabant allosterically regulated ³H-glibenclamide-specific binding in the presence of MgATP, as did diazoxide and NN414. Our findings indicate that some CB1 ligands may directly bind and allosterically regulate Kir6.2/SUR1 K(ATP) channels like other KCOs. This mechanism appears to be compatible with and may contribute to their acute and chronic effects on GSIS and insulinemia.

 

Transamination is required for {alpha}-ketoisocaproate but not leucine to stimulate insulin secretion.

Zhou Y1Jetton TLGoshorn SLynch CJShe PAuthor information
J Biol Chem. 2010 Oct 29;285(44):33718-26. http://dx.doi.org:/10.1074/jbc.M110.136846

It remains unclear how α-ketoisocaproate (KIC) and leucine are metabolized to stimulate insulin secretion. Mitochondrial BCATm (branched-chain aminotransferase) catalyzes reversible transamination of leucine and α-ketoglutarate to KIC and glutamate, the first step of leucine catabolism. We investigated the biochemical mechanisms of KIC and leucine-stimulated insulin secretion (KICSIS and LSIS, respectively) using BCATm(-/-) mice. In static incubation, BCATm disruption abolished insulin secretion by KIC, D,L-α-keto-β-methylvalerate, and α-ketocaproate without altering stimulation by glucose, leucine, or α-ketoglutarate. Similarly, during pancreas perfusions in BCATm(-/-) mice, glucose and arginine stimulated insulin release, whereas KICSIS was largely abolished. During islet perifusions, KIC and 2 mM glutamine caused robust dose-dependent insulin secretion in BCATm(+/+) not BCATm(-/-) islets, whereas LSIS was unaffected. Consistently, in contrast to BCATm(+/+) islets, the increases of the ATP concentration and NADPH/NADP(+) ratio in response to KIC were largely blunted in BCATm(-/-) islets. Compared with nontreated islets, the combination of KIC/glutamine (10/2 mM) did not influence α-ketoglutarate concentrations but caused 120 and 33% increases in malate in BCATm(+/+) and BCATm(-/-) islets, respectively. Although leucine oxidation and KIC transamination were blocked in BCATm(-/-) islets, KIC oxidation was unaltered. These data indicate that KICSIS requires transamination of KIC and glutamate to leucine and α-ketoglutarate, respectively. LSIS does not require leucine catabolism and may be through leucine activation of glutamate dehydrogenase. Thus, KICSIS and LSIS occur by enhancing the metabolism of glutamine/glutamate to α-ketoglutarate, which, in turn, is metabolized to produce the intracellular signals such as ATP and NADPH for insulin secretion.

 

Effect of the tyrosine kinase inhibitors (sunitinib, sorafenib, dasatinib, and imatinib) on blood glucose levels in diabetic and nondiabetic patients in general clinical practice.

Agostino NM1Chinchilli VMLynch CJKoszyk-Szewczyk AGingrich RSivik JDrabick JJ.
J Oncol Pharm Pract. 2011 Sep; 17(3):197-202. http://dx.doi.org:/10.1177/1078155210378913

Tyrosine kinase is a key enzyme activity utilized in many intracellular messaging pathways. Understanding the role of particular tyrosine kinases in malignancies has allowed for the design of tyrosine kinase inhibitors (TKIs), which can target these enzymes and interfere with downstream signaling. TKIs have proven to be successful in the treatment of chronic myeloid leukemia, renal cell carcinoma and gastrointestinal stromal tumor, and other malignancies. Scattered reports have suggested that these agents appear to affect blood glucose (BG). We retrospectively studied the BG concentrations in diabetic (17) and nondiabetic (61) patients treated with dasatinib (8), imatinib (39), sorafenib (23), and sunitinib (30) in our clinical practice. Mean declines of BG were dasatinib (53 mg/dL), imatinib (9 mg/dL), sorafenib (12 mg/dL), and sunitinib (14 mg/dL). All these declines in BG were statistically significant. Of note, 47% (8/17) of the patients with diabetes were able to discontinue their medications, including insulin in some patients. Only one diabetic patient developed symptomatic hypoglycemia while on sunitinib. The mechanism for the hypoglycemic effect of these drugs is unclear, but of the four agents tested, c-kit and PDGFRβ are the common target kinases. Clinicians should keep the potential hypoglycemic effects of these agents in mind; modification of hypoglycemic agents may be required in diabetic patients. These results also suggest that inhibition of a tyrosine kinase, be it c-kit, PDGFRβ or some other undefined target, may improve diabetes mellitus BG control and it deserves further study as a potential novel therapeutic option.

 

Cardiolipin remodeling by ALCAT1 links oxidative stress and mitochondrial dysfunction to obesity.

Li J1Romestaing CHan XLi YHao XWu YSun CLiu XJefferson LSXiong JLanoue KFChang ZLynch CJWang HShi Y.    Author information
Cell Metab. 2010 Aug 4;12(2):154-65. http://dx.doi.org:/10.1016/j.cmet.2010.07.003

Oxidative stress causes mitochondrial dysfunction and metabolic complications through unknown mechanisms. Cardiolipin (CL) is a key mitochondrial phospholipid required for oxidative phosphorylation. Oxidative damage to CL from pathological remodeling is implicated in the etiology of mitochondrial dysfunction commonly associated with diabetes, obesity, and other metabolic diseases. Here, we show that ALCAT1, a lyso-CL acyltransferase upregulated by oxidative stress and diet-induced obesity (DIO), catalyzes the synthesis of CL species that are highly sensitive to oxidative damage, leading to mitochondrial dysfunction, ROS production, and insulin resistance. These metabolic disorders were reminiscent of those observed in type 2 diabetes and were reversed by rosiglitazone treatment. Consequently, ALCAT1 deficiency prevented the onset of DIO and significantly improved mitochondrial complex I activity, lipid oxidation, and insulin signaling in ALCAT1(-/-) mice. Collectively, these findings identify a key role of ALCAT1 in regulating CL remodeling, mitochondrial dysfunction, and susceptibility to DIO.

 

BCATm deficiency ameliorates endotoxin-induced decrease in muscle protein synthesis and improves survival in septic mice.

Lang CH1Lynch CJVary TC.   Author information
Am J Physiol Regul Integr Comp Physiol. 2010 Sep; 299(3):R935-44.
http://dx.doi.org:/10.1152/ajpregu.00297.2010

Endotoxin (LPS) and sepsis decrease mammalian target of rapamycin (mTOR) activity in skeletal muscle, thereby reducing protein synthesis. Our study tests the hypothesis that inhibition of branched-chain amino acid (BCAA) catabolism, which elevates circulating BCAA and stimulates mTOR, will blunt the LPS-induced decrease in muscle protein synthesis. Wild-type (WT) and mitochondrial branched-chain aminotransferase (BCATm) knockout mice were studied 4 h after Escherichia coli LPS or saline. Basal skeletal muscle protein synthesis was increased in knockout mice compared with WT, and this change was associated with increased eukaryotic initiation factor (eIF)-4E binding protein-1 (4E-BP1) phosphorylation, eIF4E.eIF4G binding, 4E-BP1.raptor binding, and eIF3.raptor binding without a change in the mTOR.raptor complex in muscle. LPS decreased muscle protein synthesis in WT mice, a change associated with decreased 4E-BP1 phosphorylation as well as decreased formation of eIF4E.eIF4G, 4E-BP1.raptor, and eIF3.raptor complexes. In BCATm knockout mice given LPS, muscle protein synthesis only decreased to values found in vehicle-treated WT control mice, and this ameliorated LPS effect was associated with a coordinate increase in 4E-BP1.raptor, eIF3.raptor, and 4E-BP1 phosphorylation. Additionally, the LPS-induced increase in muscle cytokines was blunted in BCATm knockout mice, compared with WT animals. In a separate study, 7-day survival and muscle mass were increased in BCATm knockout vs. WT mice after polymicrobial peritonitis. These data suggest that elevating blood BCAA is sufficient to ameliorate the catabolic effect of LPS on skeletal muscle protein synthesis via alterations in protein-protein interactions within mTOR complex-1, and this may provide a survival advantage in response to bacterial infection.

 

Alcohol-induced IGF-I resistance is ameliorated in mice deficient for mitochondrial branched-chain aminotransferase.

Lang CH1Lynch CJVary TCAuthor information
J Nutr. 2010 May;140(5):932-8. http://dx.doi.org:/10.3945/jn.109.120501

Acute alcohol intoxication decreases skeletal muscle protein synthesis by impairing mammalian target of rapamycin (mTOR). In 2 studies, we determined whether inhibition of branched-chain amino acid (BCAA) catabolism ameliorates the inhibitory effect of alcohol on muscle protein synthesis by raising the plasma BCAA concentrations and/or by improving the anabolic response to insulin-like growth factor (IGF)-I. In the first study, 4 groups of mice were used: wild-type (WT) and mitochondrial branched-chain aminotransferase (BCATm) knockout (KO) mice orally administered saline or alcohol (5 g/kg, 1 h). Protein synthesis was greater in KO mice compared with WT controls and was associated with greater phosphorylation of eukaryotic initiation factor (eIF)-4E binding protein-1 (4EBP1), eIF4E-eIF4G binding, and 4EBP1-regulatory associated protein of mTOR (raptor) binding, but not mTOR-raptor binding. Alcohol decreased protein synthesis in WT mice, a change associated with less 4EBP1 phosphorylation, eIF4E-eIF4G binding, and raptor-4EBP1 binding, but greater mTOR-raptor complex formation. Comparable alcohol effects on protein synthesis and signal transduction were detected in BCATm KO mice. The second study used the same 4 groups, but all mice were injected with IGF-I (25 microg/mouse, 30 min). Alcohol impaired the ability of IGF-I to increase muscle protein synthesis, 4EBP1 and 70-kilodalton ribosomal protein S6 kinase-1 phosphorylation, eIF4E-eIF4G binding, and 4EBP1-raptor binding in WT mice. However, in alcohol-treated BCATm KO mice, this IGF-I resistance was not manifested. These data suggest that whereas the sustained elevation in plasma BCAA is not sufficient to ameliorate the catabolic effect of acute alcohol intoxication on muscle protein synthesis, it does improve the anabolic effect of IGF-I.

 

Impact of chronic alcohol ingestion on cardiac muscle protein expression.

Fogle RL1Lynch CJPalopoli MDeiter GStanley BAVary TCAuthor information
Alcohol Clin Exp Res. 2010 Jul;34(7):1226-34. http://dx.doi.org:/10.1111/j.1530-0277.2010.01200.x

BACKGROUND:

Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved.

METHODS:

The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT). Following the reaction with the ICAT reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches.

RESULTS:

Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system.

CONCLUSIONS:

Based on the changes in proteins, we speculate modulation of cardiac muscle protein expression represents a fundamental alteration induced by chronic alcohol consumption, consistent with changes in myocardial wall thickness measured under the same conditions.

 

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Calcium Channel Blocker Potential for Angina

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Pranidipine    

ANTHONY MELVIN CRASTO, PhD

str1

https://newdrugapprovals.files.wordpress.com/2015/12/str116.jpg

 

File:Pranidipine structure.svg

Pranidipine , OPC-13340, FRC 8411

Acalas®

NDA Filing in Japan

A calcium channel blocker potentially for the treatment of angina pectoris and hypertension.

 

CAS No. 99522-79-9

  • Molecular FormulaC25H24N2O6
  • Average mass 448.468

 

see dipine series………..http://organicsynthesisinternational.blogspot.in/p/dipine-series.html

manidipine

 

PAPER

Der Pharmacia Sinica, 2014, 5(1):11-17

https://newdrugapprovals.files.wordpress.com/2015/12/str113.jpg

pelagiaresearchlibrary.com/der-pharmacia-sinica/vol5-iss1/DPS-2014-5-1-11-17.pdf

 

Names
IUPAC name

methyl (2E)-phenylprop-2-en-1-yl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate
Other names

2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid O5-methyl O3-[(E)-3-phenylprop-2-enyl] ester
Identifiers
99522-79-9 Yes
ChEMBL ChEMBL1096842 
ChemSpider 4940726 
Jmol interactive 3D Image
MeSH C048161
PubChem 6436048
UNII 9DES9QVH58 Yes

 

 

 

PATENT SUBMITTED GRANTED
Process for the preparation of 1,4 – dihydropyridines and novel 1,4-dihydropyridines useful as therapeutic agents [US2003230478] 2003-12-18
Advanced Formulations and Therapies for Treating Hard-to-Heal Wounds [US2014357645] 2014-08-19 2014-12-04
METHODS OF TREATING CARDIOVASCULAR AND METABOLIC DISEASES [US2014322199] 2012-08-06 2014-10-30
Protein Carrier-Linked Prodrugs [US2014323402] 2012-08-10 2014-10-30
sGC STIMULATORS [US2014323448] 2014-04-29 2014-10-30
TREATMENT OF ARTERIAL WALL BY COMBINATION OF RAAS INHIBITOR AND HMG-CoA REDUCTASE INHIBITOR [US2014323536] 2012-12-07 2014-10-30
Agonists of Guanylate Cyclase Useful For the Treatment of Gastrointestinal Disorders, Inflammation, Cancer and Other Disorders [US2014329738] 2014-03-28 2014-11-06
METHODS, COMPOSITIONS, AND KITS FOR THE TREATMENT OF CANCER [US2014335050] 2012-05-25 2014-11-13
ROR GAMMA MODULATORS [US2014343023] 2012-09-18 2014-11-20
High-Loading Water-Soluable Carrier-Linked Prodrugs [US2014296257] 2012-08-10 2014-10-02 

 

 

Synthesis, isolation and use of a common key intermediate for calcium antagonist inhibitors

Neelakandan K.a,b, Manikandan H.b , B. Prabhakarana*, Santosha N.a , Ashok Chaudharia *, Mukund Kulkarnic , Gopalakrishnan Mannathusamyb and Shyam Titirmarea
a API Research Centre, Emcure Pharmaceutical Limited, Hinjawadi, Pune, India bDepartment of Chemistry, Annamalai University, Chidhambaram, India cDepartment of Chemistry, Pune University, Pune, India _________________________________________________________________________________

Pelagia Research Library     www.pelagiaresearchlibrary.com      Der Pharmacia Sinica, 2014, 5(1):11-17

 

The compound (3) synthesized from Nitrobenzaldehyde, tertiary butyl acetoacetate and piperidine can be used as a common intermediate for the production of calcium channel blockers like Nicardipine hydrochloride (1) and Pranidipine hydrochloride (2) with high purity.

 

The last twenty years have witnessed discoveries of calcium antagonists associated with multicoated pharmacodynamics potential which include not only antihypertensive and antiarrhythmic effects of the drugs but also action against excessive calcium entry in the cell of cardiovascular system and subsequent cell damage. Among many classes of calcium channel blockers, 1,4-dihydropyrimidine based drug molecules represented by Felodipine, Clevidipine, Benidipine, Nicardipine and Pranidipine are by far the best to reduce systemic vascular resistance and arterial pressure.

The reported synthetic approaches however proceed with complicated work ups, laborious purification procedures, highly expensive chemicals and low overall yields. (Scheme-I).

Synthetic scheme of Nicardipine and Pranidipine In view of the draw backs associated with previous synthetic approaches there is a strong need for environmentfriendly high yielding process applicable to the multi-kilogram production of calcium antagonist inhibitors. Herein, we report a scalable synthesis for Nicardipine hydrochloride (1) and Pranidipine hydrochloride (2) in fairly high overall yield using key intermediate 3-nitro benzylidene acid (3).Compound (3) was synthesized in two steps using 3-nitrobenzaldehyde, tertiary butyl acetoacetate and piperidine as a base to furnish tertiary butyl ester derivative (10). This was followed by hydrolysis of (10) in TFA and DCM to furnish compound (3) which would serve as a precursor for synthesis of versatile calcium antagonist inhibitors (Scheme-II).

Reported routes for synthesis of Benidipine,1,2 Lercanadipine,3-6 Nimodipine,7-11 Barnidipine12-14 and Manidipine15-16 were explored in our laboratory which involve reaction of nitro benzaldehyde with tertiary butyl acetoacetate using piperidine as a base to get tertiary butyl ester derivative (10). This is further treated with respective reagents to get various calcium channel blockers as shown in scheme 4. Since reported procedures involve in-situ generation of intermediate (3) and its reaction with corresponding fragments, it results in the formation of by-products which ultimately decrease the yield and increase the cost of API.

A novel process of manufacturing benzylidine acid derivative (3) was developed. Use of this intermediate was demonstrated by synthesis of Nicardipine and Pranidipine. This protocol may be employed for synthesis of other calcium channel blockers. In conclusion, a highly efficient, reproducible and scalable process for the synthesis of calcium channel blockers has been developed using (3) as the key intermediate.

 

[1] US 63 365 (Kyowa Hakko; appl.15.4.1982; J-prior.17.4.1981). [2] US 4 448 964 (Kyowa Hakko;15.5.1984; J-prior.17.4.1981). [3] Leonardi, A. et al.: Eur. J. Med.Chem. (EJMCA5) 33,399 (1988). [4] EP 153 016 (Recordati Chem. and Pharm.; appl. 21.1.1985; GB-prior. 14.2.1984). [5] US 4 705 797 (Recordati;10.11.1987; GB-prior. 14.2.1984). [6] WO 9 635 668 (Recordati Chem. and Pharm.; appl. 9.5.1996; I-prior. 12.5.1995). [7] DOS 2 117 571 (Bayer; appl. 10.4.1971). [8] DE 2 117 573 (Bayer; prior.10.4.1971) [9] US 3 799 934 (Bayer;26.3.1974;D-prior.10.4.1971). [10] US 3 932 645 (Bayer;13.1.1976;D-prior.10.4.1971). [11] Meyer, H. et al.: Arzneim.-Forsch. (ARZNAD) 31, 407 (1981); 33, 106 (1983). [12] DE 2 904 552 (Yamanouchi Pharm.; appl. 7.2.1979; J-prior.14.2.1978). [13] US 4 220 649 (Yamanouchi;2.9.1980; J-prior.14.2.1978). [14] CN 85 107 590( Faming Zhuanli Sheqing Gonhali S.; appl. 11.10.1985; J-prior.24.1.1985). [15] EP 94 159 (Takeda; appl. 15.4.1983; J-prior. 10.5.1982). [16] US 4 892 875 (Takeda;9.1.1990; J-prior. 10.5.1982, 11.1.1983).

 

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Biochemistry and Dysmetabolism of Aging and Serious Illness

Curator: Larry H. Bernstein, MD, FCAP

 

White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer’s Disease

Lauren P. Klosinski, Jia Yao, Fei Yin, Alfred N. Fonteh, Michael G. Harrington, Trace A. Christensen, Eugenia Trushina, Roberta Diaz Brinton
http://www.ebiomedicine.com/article/S2352-3964(15)30192-4/abstract      DOI: http://dx.doi.org/10.1016/j.ebiom.2015.11.002
Highlights
  • Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production.
  • Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females.
  • Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy.

The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer’s, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.

 

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer’s. Age remains the greatest risk factor for Alzheimer’s and the prevalence of age-related late onset Alzheimer’s is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer’s risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer’s and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

3. Results

  1. 3.1. Pathway of Mitochondrial Deficits, H2O2 Production and cPLA2 Activation in the Aging Female Brain
  2. 3.2. cPLA2-sphingomyelinase Pathway Activation in White Matter Astrocytes During Reproductive Senescence
  3. 3.3. Investigation of White Matter Gene Expression Profile During Reproductive Senescence
  4. 3.4. Ultra Structural Analysis of Myelin Sheath During Reproductive Senescence
  5. 3.5. Analysis of the Lipid Profile of Brain During the Transition to Reproductive Senescence
  6. 3.6. Fatty Acid Metabolism and Ketone Generation Following the Transition to Reproductive Senescence

 

4. Discussion

Age remains the greatest risk factor for developing AD (Hansson et al., 2006, Alzheimer’s, 2015). Thus, investigation of transitions in the aging brain is a reasoned strategy for elucidating mechanisms and pathways of vulnerability for developing AD. Aging, while typically perceived as a linear process, is likely composed of dynamic transition states, which can protect against or exacerbate vulnerability to AD (Brinton et al., 2015). An aging transition unique to the female is the perimenopausal to menopausal conversion (Brinton et al., 2015). The bioenergetic similarities between the menopausal transition in women and the early appearance of hypometabolism in persons at risk for AD make the aging female a rational model to investigate mechanisms underlying risk of late onset AD.

Findings from this study replicate our earlier findings that age of reproductive senescence is associated with decline in mitochondrial respiration, increased H2O2 production and shift to ketogenic metabolism in brain (Yao et al., 2010, Ding et al., 2013, Yin et al., 2015). These well established early age-related changes in mitochondrial function and shift to ketone body utilization in brain, are now linked to a mechanistic pathway that connects early decline in mitochondrial respiration and H2O2 production to activation of the cPLA2-sphingomyelinase pathway to catabolize myelin lipids resulting in WM degeneration (Fig. 12). These lipids are sequestered in lipid droplets for subsequent use as a local source of ketone body generation via astrocyte mediated beta-oxidation of fatty acids. Astrocyte derived ketone bodies can then be transported to neurons where they undergo ketolysis to generate acetyl-CoA for TCA derived ATP generation required for synaptic and cell function (Fig. 12).

Thumbnail image of Fig. 12. Opens large image

http://www.ebiomedicine.com/cms/attachment/2040395791/2053874721/gr12.sml

Fig. 12

Schematic model of mitochondrial H2O2 activation of cPLA2-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA2-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H2O2, in the prodromal/preclinical phase of Alzheimer’s with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA2-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.

Biochemical evidence obtained from isolated whole brain mitochondria confirms that during reproductive senescence and in response to estrogen deprivation brain mitochondria decline in respiratory capacity (Yao et al., 2009, Yao et al., 2010, Brinton, 2008a, Brinton, 2008b, Swerdlow and Khan, 2009). A well-documented consequence of mitochondrial dysfunction is increased production of reactive oxygen species (ROS), specifically H2O2 (Boveris and Chance, 1973, Beal, 2005, Yin et al., 2014, Yap et al., 2009). While most research focuses on the damage generated by free radicals, in this case H2O2 functions as a signaling molecule to activate cPLA2, the initiating enzyme in the cPLA2-sphingomyelinase pathway (Farooqui and Horrocks, 2006, Han et al., 2003, Sun et al., 2004). In AD brain, increased cPLA2 immunoreactivity is detected almost exclusively in astrocytes suggesting that activation of the cPLA2-sphingomyelinase pathway is localized to astrocytes in AD, as opposed to the neuronal or oligodendroglial localization that is observed during apoptosis (Sun et al., 2004, Malaplate-Armand et al., 2006, Di Paolo and Kim, 2011, Stephenson et al., 1996,Stephenson et al., 1999). In our analysis, cPLA2 (Sanchez-Mejia and Mucke, 2010) activation followed the age-dependent rise in H2O2 production and was sustained at an elevated level.

Direct and robust activation of astrocytic cPLA2 by physiologically relevant concentrations of H2O2 was confirmed in vitro. Astrocytic involvement in the cPLA2-sphingomyelinase pathway was also indicated by an increase in cPLA2 positive astrocyte reactivity in WM tracts of reproductively incompetent mice. These data are consistent with findings from brains of persons with AD that demonstrate the same striking localization of cPLA2immunoreactivity within astrocytes, specifically in the hippocampal formation (Farooqui and Horrocks, 2004). While neurons and astrocytes contain endogenous levels of cPLA2, neuronal cPLA2 is activated by an influx of intracellular calcium, whereas astrocytic cPLA2 is directly activated by excessive generation of H2O2 (Sun et al., 2004, Xu et al., 2003, Tournier et al., 1997). Evidence of this cell type specific activation was confirmed by the activation of cPLA2 in astrocytes by H2O2 and the lack of activation in neurons. These data support that astrocytic, not neuronal, cPLA2 is the cellular mediator of the H2O2 dependent cPLA2-sphingomyelinase pathway activation and provide associative evidence supporting a role of astrocytic mitochondrial H2O2 in age-related WM catabolism.

The pattern of gene expression during the shift to reproductive senescence in the female mouse hippocampus recapitulates key observations in human AD brain tissue, specifically elevation in cPLA2, sphingomyelinase and ceramidase (Schaeffer et al., 2010, He et al., 2010, Li et al., 2014). Further, up-regulation of myelin synthesis, lipid metabolism and inflammatory genes in reproductively incompetent female mice is consistent with the gene expression pattern previously reported from aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). In these analyses of gene expression in aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus down regulation of genes related to mitochondrial function, and up-regulation in multiple genes encoding for enzymes involved in ketone body metabolism occurred (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). The comparability across data derived from aging female mouse hippocampus reported herein and those derived from male rodent brain, female nonhuman brain and human AD brain strongly suggest that cPLA2-sphingomyelinase pathway activation, myelin sheath degeneration and fatty acid metabolism leading to ketone body generation is a metabolic adaptation that is generalizable across these naturally aging models and are evident in aged human AD brain. Collectively, these data support the translational relevance of findings reported herein.

Data obtained via immunohistochemistry, electron microscopy and MBP protein analyses demonstrated an age-related loss in myelin sheath integrity. Evidence for a loss of myelin structural integrity emerged in reproductively incompetent mice following activation of the cPLA2-sphingomyelinase pathway. The unraveling myelin phenotype observed following reproductive senescence and aging reported herein is consistent with the degenerative phenotype that emerges following exposure to the chemotherapy drug bortezomib which induces mitochondrial dysfunction and increased ROS generation (Carozzi et al., 2010, Cavaletti et al., 2007,Ling et al., 2003). In parallel to the decline in myelin integrity, lipid droplet density increased. In aged mice, accumulation of lipid droplets declined in parallel to the rise in ketone bodies consistent with the utilization of myelin-derived fatty acids to generate ketone bodies. Due to the sequential relationship between WM degeneration and lipid droplet formation, we posit that lipid droplets serve as a temporary storage site for myelin-derived fatty acids prior to undergoing β-oxidation in astrocytes to generate ketone bodies.

Microstructural alterations in myelin integrity were associated with alterations in the lipid profile of brain, indicative of WM degeneration resulting in release of myelin lipids. Sphingomyelin and galactocerebroside are two main lipids that compose the myelin sheath (Baumann and Pham-Dinh, 2001). Ceramide is common to both galactocerebroside and sphingomyelin and is composed of sphingosine coupled to a fatty acid. Ceramide levels increase in aging, in states of ketosis and in neurodegeneration (Filippov et al., 2012, Blazquez et al., 1999, Costantini et al., 2005). Specifically, ceramide levels are elevated at the earliest clinically recognizable stage of AD, indicating a degree of WM degeneration early in disease progression (Di Paolo and Kim, 2011,Han et al., 2002, Costantini et al., 2005). Sphingosine is statistically significantly elevated in the brains of AD patients compared to healthy controls; a rise that was significantly correlated with acid sphingomyelinase activity, Aβ levels and tau hyperphosphorylation (He et al., 2010). In our analyses, a rise in ceramides was first observed early in the aging process in reproductively incompetent mice. The rise in ceramides was coincident with the emergence of loss of myelin integrity consistent with the release of myelin ceramides from sphingomyelin via sphingomyelinase activation. Following the rise in ceramides, sphingosine and fatty acid levels increased. The temporal sequence of the lipid profile was consistent with gene expression indicating activation of ceramidase for catabolism of ceramide into sphingosine and fatty acid during reproductive senescence. Once released from ceramide, fatty acids can be transported into the mitochondrial matrix of astrocytes via CPT-1, where β-oxidation of fatty acids leads to the generation of acetyl-CoA (Glatz et al., 2010). It is well documented that acetyl-CoA cannot cross the inner mitochondrial membrane, thus posing a barrier to direct transport of acetyl-CoA generated by β-oxidation into neurons. In response, the newly generated acetyl-CoA undergoes ketogenesis to generate ketone bodies to fuel energy demands of neurons (Morris, 2005,Guzman and Blazquez, 2004, Stacpoole, 2012). Because astrocytes serve as the primary location of β-oxidation in brain they are critical to maintaining neuronal metabolic viability during periods of reduced glucose utilization (Panov et al., 2014, Ebert et al., 2003, Guzman and Blazquez, 2004).

Once fatty acids are released from myelin ceramides, they are transported into astrocytic mitochondria by CPT1 to undergo β-oxidation. The mitochondrial trifunctional protein HADHA catalyzes the last three steps of mitochondrial β-oxidation of long chain fatty acids, while mitochondrial ABAD (aka SCHAD—short chain fatty acid dehydrogenase) metabolizes short chain fatty acids. Concurrent with the release of myelin fatty acids in aged female mice, CPT1, HADHA and ABAD protein expression as well as ketone body generation increased significantly. These findings indicate that astrocytes play a pivotal role in the response to bioenergetic crisis in brain to activate an adaptive compensatory system that activates catabolism of myelin lipids and the metabolism of those lipids into fatty acids to generate ketone bodies necessary to fuel neuronal demand for acetyl-CoA and ATP.

Collectively, these findings provide a mechanistic pathway that links mitochondrial dysfunction and H2O2generation in brain early in the aging process to later stage white matter degeneration. Astrocytes play a pivotal role in providing a mechanistic strategy to address the bioenergetic demand of neurons in the aging female brain. While this pathway is coincident with reproductive aging in the female brain, it is likely to have mechanistic translatability to the aging male brain. Further, the mechanistic link between bioenergetic decline and WM degeneration has potential relevance to other neurological diseases involving white matter in which postmenopausal women are at greater risk, such as multiple sclerosis. The mechanistic pathway reported herein spans time and is characterized by a progression of early adaptive changes in the bioenergetic system of the brain leading to WM degeneration and ketone body production. Translationally, effective therapeutics to prevent, delay and treat WM degeneration during aging and Alzheimer’s disease will need to specifically target stages within the mechanistic pathway described herein. The fundamental initiating event is a bioenergetic switch from being a glucose dependent brain to a glucose and ketone body dependent brain. It remains to be determined whether it is possible to prevent conversion to or reversal of a ketone dependent brain. Effective therapeutic strategies to intervene in this process require biomarkers of bioenergetic phenotype of the brain and stage of mechanistic progression. The mechanistic pathway reported herein may have relevance to other age-related neurodegenerative diseases characterized by white matter degeneration such as multiple sclerosis.

Blood. 2015 Oct 15;126(16):1925-9.    http://dx.doi.org:/10.1182/blood-2014-12-617498. Epub 2015 Aug 14.
Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias.
Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.
Oncogene. 2015 Oct 12.   http://dx.doi.org:/10.1038/onc.2015.394. [Epub ahead of print]
Tumour-suppression function of KLF12 through regulation of anoikis.
Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.
Mol Cell. 2015 Oct 14. pii: S1097-2765(15)00764-9. doi: 10.1016/j.molcel.2015.09.025. [Epub ahead of print]
PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.
Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.
Mol Cancer Res. 2015 Oct;13(10):1408-20.   http://dx.doi.org:/10.1158/1541-7786.MCR-15-0048. Epub 2015 Jun 16.
Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.
Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway.  This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy. Mol Cancer Res; 13(10); 1408-20. ©2015 AACR.
SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.
BACKGROUND:  Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.
METHODS:   In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.

RESULTS:

We found that the SDHB(R46Q) mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4±63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.

CONCLUSIONS:

These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

 

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

  1. L. Figarola, J. Singhal, J. D. Tompkins, G. W. Rogers, C. Warden, D. Horne, A. D. Riggs, S. Awasthi and S. S. Singhal.

J Biol Chem. 2015 Nov 3, [epub ahead of print]

 

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

  1. W. Miller, D. R. Soto-Pantoja, A. L. Schwartz, J. M. Sipes, W. G. DeGraff, L. A. Ridnour, D. A. Wink and D. D. Roberts.

J Biol Chem. 2015 Oct 9, 290 (41): 24858-74.

 

Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

  1. Sun, A. Zhu, L. Zhang, J. Zhang, Z. Zhong and F. Wang.

Int J Mol Sci. 2015 Oct 15, 16 (10): 24574-87.

 

EglN2 associates with the NRF1-PGC1alpha complex and controls mitochondrial function in breast cancer

  1. Zhang, C. Wang, X. Chen, M. Takada, C. Fan, X. Zheng, H. Wen, Y. Liu, C. Wang, R. G. Pestell, K. M. Aird, W. G. Kaelin, Jr., X. S. Liu and Q. Zhang.

EMBO J. 2015 Oct 22, [epub ahead of print]

 

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual’s predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency. Cancer Res; 75(20); 4429-36. ©2015 AACR.

 

Cancer Lett. 2015 Oct 29. pii: S0304-3835(15)00656-4.    http://dx.doi.org:/10.1016/j.canlet.2015.10.025. [Epub ahead of print]
Carboxyamidotriazole inhibits oxidative phosphorylation in cancer cells and exerts synergistic anti-cancer effect with glycolysis inhibition.

Targeting cancer cell metabolism is a promising strategy against cancer. Here, we confirmed that the anti-cancer drug carboxyamidotriazole (CAI) inhibited mitochondrial respiration in cancer cells for the first time and found a way to enhance its anti-cancer activity by further disturbing the energy metabolism. CAI promoted glucose uptake and lactate production when incubated with cancer cells. The oxidative phosphorylation (OXPHOS) in cancer cells was inhibited by CAI, and the decrease in the activity of the respiratory chain complex I could be one explanation. The anti-cancer effect of CAI was greatly potentiated when being combined with 2-deoxyglucose (2-DG). The cancer cells treated with the combination of CAI and 2-DG were arrested in G2/M phase. The apoptosis and necrosis rates were also increased. In a mouse xenograft model, this combination was well tolerated and retarded the tumor growth. The impairment of cancer cell survival was associated with significant cellular ATP decrease, suggesting that the combination of CAI and 2-DG could be one of the strategies to cause dual inhibition of energy pathways, which might be an effective therapeutic approach for a broad spectrum of tumors.

 

Cancer Immunol Res. 2015 Nov;3(11):1236-47.    http://dx.doi.org:/10.1158/2326-6066.CIR-15-0036. Epub 2015 May 29.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies.

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236-47. ©2015 AACR.

 

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

  1. L. Judge, K. M. Owens, S. J. Pollock, C. F. Woeller, T. H. Thatcher, J. P. Williams, R. P. Phipps, P. J. Sime and R. M. Kottmann.

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 15, 309 (8): L879-87.

 

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

  1. Yun, E. Mullarky, C. Lu, K. N. Bosch, A. Kavalier, K. Rivera, J. Roper, Chio, II, E. G. Giannopoulou, C. Rago, A. Muley, J. M. Asara, J. Paik, O. Elemento, Z. Chen, D. J. Pappin, L. E. Dow, N. Papadopoulos, S. S. Gross and L. C. Cantley.

Science. 2015 Nov 5, [epub ahead of print]

 

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells

  1. Zhang, J. Wang, H. Xing, Q. Li, Q. Zhao and J. Li.

Mol Cell Biochem. 2015 Nov 6, [epub ahead of print]

 

J Mol Cell Cardiol. 2015 Oct 23. pii: S0022-2828(15)30073-0.     http://dx.doi.org:/10.1016/j.yjmcc.2015.10.002. [Epub ahead of print]
GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

 

Br J Pharmacol. 2015 Oct 27. doi: 10.1111/bph.13377. [Epub ahead of print]
All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the Erk2 signalling pathway.
BACKGROUND AND PURPOSE:

Doxorubicin (Dox) is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. It is known that all-trans retinoic acid (ATRA) plays important roles in many cardiac biological processes, however, the protective effects of ATRA on doxorubicin cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and underlying mechanisms.

EXPERIMENTAL APPROACHES:

Cellular viability assays, western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR (Polymerase Chain Reaction) and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA’s effects on doxorubicin cardiotoxicity.

KEY RESULTS:

ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species (ROS) generation, and restored the expression level of mRNA and proteins in phase II detoxifying enzyme system: Nrf2 (nuclear factor-E2-related factor 2), MnSOD (manganese superoxide dismutase), HO-1 (heme oxygenase1) as well as mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers, mitochondrial respiration capacity, biogenesis and dynamics). Both Erk1/2 (extracellular signal-regulated kinase1/2) inhibitor (U0126) and Erk2 siRNA, but not Erk1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells.

CONCLUSION AND IMPLICATION:

ATRA protected cardiomyocytes against doxorubicin-induced toxicity by activating the Erk2 pathway without compromising the anticancer efficacy of doxorubicin. Therefore, ATRA may be a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.

 

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation

  1. Colak, O. Pougovkina, L. Dai, M. Tan, H. Te Brinke, H. Huang, Z. Cheng, J. Park, X. Wan, X. Liu, W. W. Yue, R. J. Wanders, J. W. Locasale, D. B. Lombard, V. C. de Boer and Y. Zhao.

Mol Cell Proteomics. 2015 Nov 1, 14 (11): 3056-71.

 

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

  1. Pancrazi, G. Di Benedetto, L. Colombaioni, G. Della Sala, G. Testa, F. Olimpico, A. Reyes, M. Zeviani, T. Pozzan and M. Costa.

Proc Natl Acad Sci U S A. 2015 Oct 27, 112(45): 13910-5.

 

Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

  1. E. Hjelm, B. Rollins, F. Mamdani, J. C. Lauterborn, G. Kirov, G. Lynch, C. M. Gall, A. Sequeira and M. P. Vawter.

Mol Neuropsychiatry. 2015 Nov 1, 1 (4): 201-219.

 

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

  1. Bernard, N. J. Logsdon, S. Ravi, N. Xie, B. P. Persons, S. Rangarajan, J. W. Zmijewski, K. Mitra, G. Liu, V. M. Darley-Usmar and V. J. Thannickal.

J Biol Chem. 2015 Oct 16, 290 (42): 25427-38.

 

J Biol Chem. 2015 Oct 23;290(43):25834-46.    http://dx.doi.org:/10.1074/jbc.M115.658815. Epub 2015 Sep 4.
Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

 

FASEB J. 2015 Oct 19.    http://dx.doi.org:/pii: fj.15-276360. [Epub ahead of print]
Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

 

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.
Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

 

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Empagliflozin

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Empagliflozin Benefits in EMPA-REG Explored in Diabetics Initially With or Without Heart Failure

Marlene Busko

http://www.medscape.com/viewarticle/854542

 

ORLANDO, FL — Patients with type 2 diabetes and established CVD who received the antidiabetic sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (Jardiance, Lilly/Boehringer Ingelheim), as opposed to placebo, had a reduced risk of being hospitalized for heart failure or dying from CVD during a median follow-up of 3.1 years. The finding was strongest in patients without heart failure at baseline[1]. The finding is noteworthy in part because associated heart failure has been a concern, justified or not, with some other diabetes medications.

In these high-risk patients, empagliflozin resulted in a “consistent benefit” in these outcomes, Dr Silvio E Inzucchi (Yale University School of Medicine, New Haven, CT) said, presenting these findings from a prespecified secondary analysis of the EMPA-REG OUTCOME trial at theAmerican Heart Association (AHA) 2015 Scientific Sessions.

Unlike the gasps and applause that greeted him when he presented the trial’s primary outcome results at the European Association for the Study of Diabetes (EASD) 2015 Meeting in Stockholm in mid-September, the audience reaction this time was more measured. The trial had also been published at about the time of its EASD presentation [2].

The principal findings showed that compared with patients who took placebo, those who were randomized to empagliflozin had a 38% (P<0.001) reduced risk of CV death and a 35% P=0.002) reduced risk of hospitalization for HF, at a median follow-up of 3.1 years.

In the current secondary analysis, the 90% of patients who were free of heart failure at study entry showed a steep and significant drop in HF hospitalizations during the trial. There was also a drop in HF hospitalizations with active therapy in the minority who had HF at baseline, but it failed to reach significance.

“I think metformin is likely to remain our first-line oral therapy for patients with type 2 diabetes,” Dr Donald M Lloyd-Jones (Northwestern University Feinberg School of Medicine, Chicago, IL), cochair at an AHA press briefing, told heartwire from Medscape. “There is an alphabet soup of diabetes medications,” with multiple agents that effectively lower blood glucose and reduce patients’ risk of retinopathy, nephropathy, and neuropathy.

However, “it was . . . unexpected that [empagliflozin], as reported recently [at the EASD meeting and] in the New England Journal of Medicine [has an] effect on CV death and other CV events.” This is still an early stage of research, he cautioned, and it is not known how the drug exerts its CV effects and whether there is a class effect. “But [this] could be a game changer, because we would love to have [antidiabetic] medications that not only control blood sugar but also reduce death and [other] hard events,” he said.

 

First CV Outcomes Trial in this Drug Class

Until now, none of the antiglycemic medications has also been shown to improve HF outcomes, Inzucchi explained. “We’ve actually been searching decades for a diabetes medicine that will not only lower blood glucose but also reduce cardiovascular complications,” he said in a press briefing. “And I would remind you that based on the 2008 FDA guidance to industry, all new diabetes medications need to be tested for cardiovascular safety before being allowed on the market,” he added.

EMPA-REG OUTCOME is the first published, large CV-outcome trial of an SGLT-2 inhibitor.

As previously described, the trial randomized 7028 adult patients who had type 2 diabetes and established CVD to receive 10 mg/day or 25 mg/day empagliflozin or placebo. The CVD included prior MI (46.6%), CABG (24.8%), stroke (23.3%), and peripheral artery disease (PAD) (20.8%).

The patients were also required to have an HbA1c level between 7% and 10%, body-mass index (BMI) <45, and, because the drug exerts its effects via the kidney, estimated glomerular filtration rate (eGFR) >30 mL/min/1.73 m2.

“Importantly, study medication was given upon a backdrop of standard care—antihyperglycemia therapy, as well as other evidence-based cardiovascular therapies such as statins, ACE inhibitors, and aspirin,” Inzucchi stressed.

 

Spotlight on HF Outcomes

The current analysis dove deeper into the heart-failure outcomes in the trial.

The risk of hospitalization for HF or CV death was consistently significantly lower in patients who received empagliflozin vs placebo, in subgroup analyses related to age, kidney function, and medication use (ACE inhibitors/angiotensin receptor blockers [ARBs], diuretics, beta-blockers, or mineralocorticoid-receptor antagonists).

Overall, the patients who received empagliflozin had a 34% reduced risk of being hospitalized for HF or dying from CV causes and a 39% reduced risk of being hospitalized for or dying from HF.

Risk of Hospitalization or Death, Empagliflozin vs Placebo

Outcome HR (95% CI) P
Hospitalization for HF or CV death 0.66 (0.55–0.79) <0.00001
Hospitalization for or death from HF 0.61 (0.47–0.79) <0.00001

Most patients (90%) did not have HF at baseline.

In the patients without HF at baseline, “as you might expect, [HF] hospitalizations were relatively small in number” (1.8% of patients on the study drug and 3.1% of patients on placebo), said Inzucchi. There was a statistically significant 41% reduced risk of HF hospitalization in patients without HF at baseline on the study drug vs placebo (HR 0.59, 95% CI 0.43–0.82).

In the smaller number of patients who did have HF at baseline, the rate of hospitalizations for HF was much higher (10.4% of patients on the study drug and 12.3% of patients on placebo). But in this case, the difference between patients on the study drug vs placebo was not statistically significant (HR 0.75, 95% CI 0.48–1.19).

The results were similar when the analysis was repeated for the combined outcome of hospitalization for HF or CV death.

“Not surprisingly,” adverse events were more common in sicker patients with baseline HF; genital infections, a well-known adverse event in drugs that increase glucose in the urine, were three times more common in those patients, said Inzucchi.

“I think these are very compelling data, but early days,” said Lloyd-Jones.

Inzucchi receives research grants from Genzyme and honoraria from Boehringer Ingelheim, Merck Sharp & Dome, Sanofi, Amgen, and Genzyme, and he is a consultant on advisory boards for Boehringer Ingelheim, Sanofi, and Amgen. Disclosures for the coauthors are listed in the abstract. Lloyd-Jones has no relevant financial relationships.

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MYBPC3 gene and the heart

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

MYBPC3 myosin binding protein C, cardiac [ Homo sapiens (human) ]

http://www.ncbi.nlm.nih.gov/gene/4607

MYBPC3 provided by HGNC
Official Full Name – myosin binding protein C, cardiac provided by HGNC
Primary source – HGNC:HGNC:7551 ;
See related Ensembl:ENSG00000134571; HPRD:02980; MIM:600958; Vega:OTTHUMG00000166986
Gene type protein coding RefSeq status
REVIEWED Organism Homo sapiens
LineageEukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo
Also known asFHC; CMH4; CMD1MM; LVNC10; MYBP-C

SummaryMYBPC3 encodes the cardiac isoform of myosin-binding protein C. Myosin-binding protein C is a myosin-associated protein found in the cross-bridge-bearing zone (C region) of A bands in striated muscle. MYBPC3, the cardiac isoform, is expressed exclussively in heart muscle. Regulatory phosphorylation of the cardiac isoform in vivo by cAMP-dependent protein kinase (PKA) upon adrenergic stimulation may be linked to modulation of cardiac contraction. Mutations in MYBPC3 are one cause of familial hypertrophic cardiomyopathy. [provided by RefSeq, Jul 2008]

 

 

 

What is the official name of the MYBPC3 gene?

The official name of this gene is “myosin binding protein C, cardiac.”

MYBPC3 is the gene’s official symbol. The MYBPC3 gene is also known by other names, listed below.

Read more about gene names and symbols on the About page.

 

What is the normal function of the MYBPC3 gene?

The MYBPC3 gene provides instructions for making the cardiac myosin binding protein C (cardiac MyBP-C), which is found in heart (cardiac) muscle cells. In these cells, cardiac MyBP-C is associated with a structure called the sarcomere, which is the basic unit of muscle contraction. Sarcomeres are made up of thick and thin filaments. The overlapping thick and thin filaments attach to each other and release, which allows the filaments to move relative to one another so that muscles can contract. Regular contractions of cardiac muscle pump blood to the rest of the body.

In cardiac muscle sarcomeres, cardiac MyBP-C attaches to thick filaments and keeps them from being broken down. Cardiac MyBP-C has chemical groups called phosphate groups attached to it; when the phosphate groups are removed, cardiac MyBP-C is broken down, followed by the breakdown of the proteins of the thick filament. Cardiac MyBP-C also regulates the rate of muscle contraction, although the mechanism is not fully understood.

 

Does the MYBPC3 gene share characteristics with other genes?

The MYBPC3 gene belongs to a family of genes called fibronectin type III domain containing(fibronectin type III domain containing). It also belongs to a family of genes called immunoglobulin superfamily, I-set domain containing (immunoglobulin superfamily, I-set domain containing). It also belongs to a family of genes called MYBP (myosin binding proteins).

A gene family is a group of genes that share important characteristics. Classifying individual genes into families helps researchers describe how genes are related to each other. For more information, see What are gene families? in the Handbook.

http://ghr.nlm.nih.gov/gene/MYBPC3

 

Aliases for MYBPC3 Gene

http://www.genecards.org/cgi-bin/carddisp.pl

  • Myosin Binding Protein C, Cardiac 2 3
  • C-Protein, Cardiac Muscle Isoform 3 4
  • CMD1MM 3 6
  • LVNC10 3 6
  • CMH4 3 6
  • Myosin-Binding Protein C, Cardiac-Type 3
  • Myosin-Binding Protein C, Cardiac 2
  • Cardiac MyBP-C 4
  • MYBP-C 3
  • FHC 3

 

GO – Molecular functioni

http://www.uniprot.org/uniprot/Q14896

GO – Biological processi

Keywords – Molecular functioni

Muscle protein

Keywords – Biological processi

Cell adhesion

Keywords – Ligandi

Actin-binding, Metal-binding, Zinc

Enzyme and pathway databases

 

Organization and Sequence of Human Cardiac Myosin Binding Protein C Gene (MYBPC3) and Identification of Mutations Predicted to Produce Truncated Proteins in Familial Hypertrophic Cardiomyopathy

Lucie CarrierGisele BonneEllen BahrendBing YuPascale RichardFlorence NielBernard Hainque, et al.

Circulation Research.1997; 80: 427-434   http://dx.doi.org:/10.1161/01.res.0000435859.24609.b3

Cardiac myosin binding protein C (MyBP-C) is a sarcomeric protein belonging to the intracellular immunoglobulin superfamily. Its function is uncertain, but for a decade evidence has existed for both structural and regulatory roles. The gene encoding cardiac MyBP-C (MYBPC3) in humans is located on chromosome 11p11.2, and mutations have been identified in this gene in unrelated families with familial hypertrophic cardiomyopathy (FHC). Detailed characterization of the MYBPC3 gene is essential for studies on gene regulation, analysis of the role of MyBP-C in cardiac contraction through the use of recombinant DNA technology, and mutational analyses of FHC. The organization of human MYBPC3 and screening for mutations in a panel of French families with FHC were established using polymerase chain reaction, single-strand conformation polymorphism, and sequencing. The MYBPC3 gene comprises >21 000 base pairs and contains 35 exons. Two exons are unusually small in size, 3 bp each. We found six new mutations associated with FHC in seven unrelated French families. Four of these mutations are predicted to produce truncated cardiac MyBP-C polypeptides. The two others should each produce two aberrant proteins, one truncated and one mutated. The present study provides the first organization and sequence for an MyBP-C gene. The mutations reported here and previously in MYBPC3 result in aberrant transcripts that are predicted to encode significantly truncated cardiac MyBP-C polypeptides. This spectrum of mutations differs from the ones previously observed in other disease genes causing FHC. Our data strengthen the functional importance of MyBP-C in the regulation of cardiac work and provide the basis for further studies.

Cardiac MyBP-C is a member of a family comprising isoforms specific for slow-skeletal, fast-skeletal, and cardiac muscles. The skeletal isoforms were initially described in 1971 [1] and later came to be recognized as proteins with specific myosin- and titin-binding properties located in the A bands of the thick filaments of all vertebrate cross-striated muscle and forming a series of seven to nine transverse stripes, 43 nm apart, in the crossbridgebearing region. [2-5] Subsequent cloning of the three isoforms showed them to belong to the intracellular immunoglobulin superfamily and to share a conserved domain pattern consisting of IgI set domains and fn-3 domains. [6-11]

Comparison of the cardiac and the skeletal MyBP-C isoform sequences reveals three distinct regions that are specific to the cardiac isoform: the N-terminal domain C0 IgI containing 101 residues, the MyBP-C motif (a 105-residue stretch linking the C1 and C2 IgI domains), and a 28-residue loop inserted in the C5 IgI domain. [7,12,13] The MyBP-C motif is not specific to the cardiac isoform, but the alignment of skeletal and cardiac sequences revealed the addition of a nine-residue loop in the cardiac variant, which is the key substrate site for phosphorylation by both protein kinase A and a calmodulin-dependent protein kinase associated with the native protein. [7] As for the 28-residue loop, it is strictly cardiac specific. [14,15] The major myosin-binding site of MyBP-C resides in the C-terminal C10 IgI domain and is mainly restricted to the last 102 amino acids. [16-18] The titin-binding site is also located in the C-terminal region, spanning the C8 to C10 IgI domains of the molecule. [6,13]

The function of MyBP-C is uncertain, but for a decade evidence has existed to indicate both structural and regulatory roles. It should be stressed, however, that most studies were performed on skeletal muscles and that very little functional data exist for cardiac muscle. Several investigators have shown that MyBP-C modulates in vitro the shape and the length of sarcomeric thick filaments [19-21] and that depending on ionic strength and the molar ratio of actin and myosin in solution, the addition of MyBP-C can modulate the actin-activated ATPase activity of skeletal and cardiac myosins. [3,22,23] Partial extraction of the MyBP-C from rat skinned cardiac myocytes and rabbit skeletal muscle fibers alters Ca2+-sensitive tension, supporting the view that contractile function is affected by MyBP-C. [24] This view was very recently strengthened by the elegant studies of Weisberg and Winegrad. [25] These authors showed that phosphorylation of cardiac MyBP-C alters myosin crossbridges in native thick filaments isolated from rat ventricles and suggested that MyBP-C can modify force production in activated cardiac muscles.

The gene encoding the cardiac isoform in humans (MYBPC3) was assigned to the chromosomal location 11p11.2 [7] in a region where we had identified the CMH4 disease locus in FHC. [26] Recently, three mutations in MYBPC3 have been identified in unrelated families with FHC by our group [27] and others. [28] FHC is a genetically and phenotypically heterogeneous disease, transmitted as an autosomal-dominant trait. None of the previous hypotheses of the pathophysiological mechanisms would have predicted that defects in sarcomeric protein genes could be a possible molecular basis for the disease. The results of molecular genetic studies have nevertheless shown that many forms of the disease involve mutations in genes encoding sarcomeric proteins (for reviews, see [29-31]), and the findings that MYBPC3 is one of these disease genes are consistent with the view that cardiac MyBP-C may play a more important role in the regulation of cardiac contraction than was previously thought.

Detailed characterization of the MYBPC3 gene is essential for studies of gene regulation, analysis of the role of cardiac MyBP-C in the sarcomere structure and function through the use of recombinant DNA technology, and, finally, mutational analyses and further studies in FHC. In the present work, we have determined the organization and sequence of the human MYBPC3 gene and shown it to exceed 21 000 bp in size and to contain 35 exons, out of which 34 are coding. We also report that six new mutations in the MYBPC3 gene are associated with FHC in seven unrelated French families. Four of these mutations are predicted to produce truncated cardiac MyBP-C polypeptides in these families. The two others should each produce two aberrant proteins, one truncated and the other mutated or deleted.

 

Screening the Human MYBPC3 Gene for Mutations

The primers were constructed on the basis of flanking intron sequences and were used to amplify each exon (see Table 1). The touchdown PCR was performed (as described above according to the conditions reported in Table 1) on genomic DNA from unrelated FHC patients. For SSCP, PCR products were denatured for 5 minutes at 96 degrees C in a standard denaturing buffer, kept on ice for 5 minutes, loaded onto 6% to 10% polyacrylamide gels, and then run at 6 mA and at 7 degrees C or 20 degrees C in a Hoeffer apparatus. The bands were visualized after silver staining of the gels (Bio-Rad). Sequencing was performed as described above.

Table 1.

Oligonucleotide Primers and PCR Conditions for Detection of Mutations in Human MYBPC3 Gene

RNA Isolation, cDNA Synthesis, and MYBPC3 cDNA Amplifications

Total cellular RNA was isolated from human lymphoblastoid cell lines using RNA Plus (Bioprobe Systems), and the cDNA synthesis was performed as previously described. [27] The cDNA products were amplified in a 50-micro L PCR reaction using two outer primers (see Table 2). A second round of PCR was performed with a final dilution of 1:100 of the first round products, using nested primers (see Table 2). The primers were determined according to the cDNA sequence (EMBL accession number X84075), and cDNA fragments were amplified using a touchdown PCR protocol between 70 degrees C and 60 degrees C. Sizes of normal and mutated cDNA-PCR fragments were assessed, followed by size-fractionation on agarose gels. After extraction and purification of the normal and the putative mutated cDNAs, they were cloned using pGEM-T System II (Promega) and then sequenced as described above.

Table 2.

Oligonucleotide Primers for MYBPC3 cDNA Amplifications

Genomic Organization and Sequence of Human MYBPC3

The size of introns was first estimated by PCR amplification of DNA segments between exons from control genomic DNA, followed by size-fractionation of the PCR products on agarose gels. The exon/intron boundaries and the entire intronic sequences were then determined by sequencing. The sequences have been deposited with EMBL (accession number Y10129). The schematic organization of the human MYBPC3 gene and the alignment of exons with structural domains in the protein are shown in Figure 1. The gene comprises >21 000 bp and contains 35 exons, out of which 34 are coding. A (GT) repeat was found in intron 20 (data not shown). The 101-residue N-terminal extra IgI domain is encoded by exons 1 to 3; the proline-rich domain (51 residues), by exons 3 and 4; the C1 IgI domain (104 residues), by exons 4 to 6; the MyBP-C motif (105 residues), by exons 6 to 12; the C2 IgI domain (91 residues), by exons 12 to 16; the C3 IgI domain (91 residues), by exons 16 to 18; the C4 IgI domain (90 residues), by exons 18 to 20; the linker (11 residues), by exons 20 and 21; the C5 IgI domain (127 residues), by exons 21 to 24; the C6 fn-3 domain (98 residues), by exons 24 to 26; the C7 fn-3 domain (101 residues), by exons 26 to 28; the C8 IgI domain (95 residues), by exons 28 to 30; the C9 fn-3 domain (115 residues), by exons 30 to 32; and the C-terminal C10 IgI domain (94 residues), by exons 32 to 34.

Figure 1.

Schematic organization of the human MYBPC3 gene and alignment of exons with structural domains of the protein. Top, The structural domains of cardiac MyBP-C. The high-affinity myosin heavy chain domain (confined to the C10 IgI repeat), the titin binding site (C8 to C10), and the phosphorylation sites are indicated. Middle, The mRNA with the limits of exons. Bottom, the schematic organization of the gene with locations of exons shown by boxes and introns shown by horizontal lines. The exons are numbered from the 5 prime end of the gene, with exon 1 containing the first codon ATG. The exons coding for structural domains are indicated by interrupted lines.

The sizes of exons and introns are summarized in Table 3. The exon sizes, excluding the 5 prime and 3 prime untranslated regions, vary between 3 and 267 bp. Two of the exons, ie, exons 10 and 14, are unusually small and contain three nucleotides each. The remaining 32 exons vary in size between 18 and 267 bp. Twenty-seven exons finish with a split codon (see Table 3). The intron sizes vary between 85 and [nearly =]2000 bp. The major consensus donor splice site is GTGAG in 53% of the cases, and the major consensus acceptor splice site is CAG in 91% of the cases. Twenty-seven of the 34 introns contain putative branch point sequences located -14 to -51 upstream from each splice acceptor site. Introns 1, 4, 11, 14, 16, 24, and 31 do not contain any known consensus branch point sequence.

Table 3.

Exon-Intron Boundaries in the Human MYBPC3 Gene Identification of Mutations in MYBPC3 Gene Associated With FHC

Because the families were not large enough to assess linkage on the basis of a statistically significant Lod score, we used haplotype analysis to define the disease locus responsible for FHC in each family. Linkage was established on the basis of the transmission of a common haplotype in affected individuals and exclusion on the basis of affected recombinant individuals. Families 717 and 740 presented linkage only to CMH4, and the other five families (families 702, 716, 731, 750, and 754) were less informative but at least potentially linked to CMH4 (data not shown).

All the exon-intron boundaries were analyzed by PCRSSCP according to the conditions described in Table 2. A total of six new mutations were identified in MYBPC3 associated with FHC in seven unrelated French families (Figure 2 andFigure 3, Table 4).

Table 4.

Consequences at mRNA Level of MYBPC3 Mutations

Figure 2.

Pedigrees of families with MYBPC3 gene mutations. Clinical affection status is indicated: darkened, affected; clear, unaffected; and clear with a cross, indeterminate. Genetically affected status is indicated by an asterisk. The mutations (M) are as follows: M1, GTGAG[arrow right]GTGAA splice donor site mutation in intron 7; M2, GAA[arrow right]CAA mutation in exon 17; M3, GT[arrow right]AT splice donor site mutation in intron 23; M4, TGAT[arrow right]TGGT transversion in the branch point consensus sequence of intron 23; M5, [-GCGTC] deletion in exon 25; and M6, duplication [+TTCAAGAATGGC]/deletion [-ACCT] in exon 33.

Figure 3.

Normal and mutated cardiac MyBP-C polypeptides. N indicates the normal structure of human cardiac MyBP-C; M1 to M6 correspond to the predicted products of the aberrant MyBP-C cDNAs resulting from the different mutations.

M1 is a GTGAG[arrow right]GTGAA transition in the 3 prime splice donor site of intron 7 in family 717. The G residue at position +5 in the intron is a highly conserved nucleotide in the splice donor consensus sequence. [33] The G[arrow right]A mutation inactivates this donor site. Amplification of MYBPC3 cDNA from patients’ lymphocytes identified the skipping of the 49-bp exon 7 that produces a frameshift. No alternative splice donor site was found in intron 7. The aberrant cDNA encodes 258 normal cardiac MyBP-C residues, followed by 25 new amino acids, and a premature termination of translation. This should produce a large truncated protein (-80%) lacking the MyBP-C motif containing the phosphorylation sites and the titin and myosin binding sites.

M2 is a G[arrow right]C transversion at position 1656 in exon 17 in families 702 and 750 that produces a mutated polypeptide in the C3 domain at the position 542 (Glu[arrow right]Gln). Otherwise, this mutation affects the last nucleotide of the exon, which is part of the consensus splicing site. [34] A common feature in human exon-intron boundaries is that 80% of exons finish with a guanine (85% in MYBPC3). This mutation also results in an aberrant transcript in lymphocytes (with the skipping of exon 17) that directly introduces a stop codon. The aberrant cDNA encodes 486 normal cardiac MyBP-C residues, leading to a truncated protein (-62%) that lacks the titin and myosin binding sites.

M3 is a GT[arrow right]AT transition in the 3 prime splice donor site of intron 23 in family 716 that inactivates this splicing site. This mutation produces the skipping of the 160-bp exon 23. No alternative splice donor site was found in lymphocytes. The mutated cDNA identified in lymphocytes encodes 717 normal residues and then 51 novel amino acids, followed by premature termination of the translation in the C5 domain, leading to a potential truncated protein (-44%) that loses the titin and myosin binding domains.

M4 is a TGAT[arrow right]TGGT transition in intron 23 in family 740. This A[arrow right]G mutation inactivates a potential branch point consensus sequence (URAY). Although three potential branch points exist upstream from the mutation, they do not seem to be used, since analysis of the transcripts in lymphocytes indicates the existence of two aberrant cDNAs. One corresponds to the skipping of the 105-bp exon 24 without frameshift and encodes a polypeptide depleted of 35 amino acids in the C6 domain (-50% of C6). The other still contains the 724-bp intron 23. This mutant cDNA is associated with a frameshift: it encodes 770 normal cardiac MyBP-C residues and then 100 novel amino acids, followed by a stop codon, and the corresponding truncated protein (-40%) should not interact with either titin or myosin.

M5 is a 5-bp deletion (-GCGTC) in exon 25 in family 731. This deletion also produces a frameshift: the aberrant cDNA identified in the lymphocytes encodes 845 normal MyBP-C residues and then 35 novel amino acids, followed by a premature stop codon in the C6 domain that should produce a truncated protein (-34%), losing the C-terminal region containing both the titin- and myosin-binding sites.

M6 is a 12-bp duplication (+TTCAAGAATGGC)/4-bp deletion (-ACCT) in exon 33 in family 754. This modification introduces a frameshift at position 3691 that leads to 1220 normal MyBP-C residues and then 19 novel amino acids, followed by a premature stop codon in the last third part of the C10 domain. The predicted truncated protein (-4%) should also lose part of its myosin binding site.

All these six mutations were absent in 200 samples from control unrelated subjects without FHC and also in 42 unrelated probands with FHC (out of which 8 have mutations in MYBPC3, 8 have mutations in the beta-myosin heavy chain gene [MYH7], 1 has a mutation in the cardiac troponin T gene, and 25 have presently undefined mutations).

Discussion

The present work describes the first genomic organization for an MyBP-C. The gene is over 21 000 bp and contains 35 exons. An interesting feature of the organization of this gene is that there is a striking correspondence between the limits of the exons and those of structural domains (Figure 1). The IgI and fn-3 domains are encoded by two or three exons. The linker region between the IgI C4 and IgI C5 domains corresponds to exon 20. Twenty-six of the 28 cardiac-specific amino acids of the IgI C5 domain correspond to exon 22. Finally, the MyBP-C motif is encoded by the most complex exon structure: the nine cardiac-specific amino acids correspond to exon 8, and the four phosphorylation sites described by Gautel et al [7] are encoded by six exons and are located at the end or at the junction of two exons (phosphorylation sites: A, junction of exons 7 and 8; B, end of exon 8; C, end of exon 9, exon 10, and beginning of exon 11; and D, end of exon 12). The correlation between exonic organization and protein structure has also recently been described concerning the titin, [35] suggesting a common feature for the intracellular immunoglobulin superfamily.

We suggest that the new mutations described here cause FHC because they segregate with the disease, are not present in controls, and result in aberrant transcripts that are predicted to encode significantly altered cardiac MyBP-C polypeptide structure and/or function. They are all transcribed into mRNAs in lymphocytes. However, because most, if not all, genes in humans are thought to be transcribed at very low levels in lymphocytes (“illegitimate transcription”), [36] these results do not address the hypothesis that these mutations are expressed in the diseased myocardium. Since cardiac MyBP-C is specifically expressed in heart, ventricular tissue is needed to address this issue, and we had no access to any myocardial specimens. One study documented the expression of a missense mutation in the mRNA for the beta-myosin heavy chain in myocardial tissue from an affected patient with FHC. [37] Because the beta-myosin heavy chain is normally expressed in slow-twitch skeletal fibers, skeletal muscle biopsies can also be used to show that the mutated myosin is produced in the muscle and that the mutation alters the function of the beta-myosin and the contractile properties of the muscle fibers. [38,39] One might thus reasonably assume that the MYBPC3 gene mutations are expressed in the myocardium and that they exert their effect by altering the multimeric complex assembly of the cardiac sarcomere via at least one of these mechanisms: (1) They can act as “poison polypeptides” through a dominant-negative effect. The altered proteins would be incorporated in the sarcomere and would alter the assembly of the sarcomeric filaments, since most truncated MyBP-Cs are unable to cross-link the titin and/or myosin molecules. (2) They can act as “null alleles,” potentially leading to haplo insufficiency; the production of insufficient quantities of normal cardiac MyBP-C would produce an imbalance in stoichiometry of the thick-filament components that would be sufficient to alter the sarcomeric structure and function. (3) Since myosin, titin, and MyBP-C might be translated and assembled cotranslationally, one can also assume that the misfolded, mutated MYBPC3 mRNAs may disturb the translation of the other sarcomeric components that would interfere with the proper assembly of sarcomeric structures.

The full spectrum of mutations of the FHC disease genes is far from known, but it is intriguing to note that most mutations found so far in MYH7 are missense ones, whereas most of those in MYBPC3 disrupt the reading frame and produce premature stop codons. Both genes are large ones, composed of [nearly =]40 exons, and there are no reasons for different types of mutations in the two genes. Thus, one might hypothesize that mutations leading to truncated proteins exist also for MYH7 in humans but have no deleterious effect. In support of this are the reports of two deletions in the C-terminal part of the beta-myosin heavy chain molecule with almost no phenotype. One is a 2.4-kbp deletion including part of intron 39 and exon 40 containing the 3 prime untranslated region and the polyadenylation signal, which was reported in a small pedigree. [40] Only the proband had developed clinically diagnosed hypertrophic cardiomyopathy at a very late onset (age, 59 years), and the other genotypically affected family members had not developed the disease at 10, 32, and 33 years. The other one is a large deletion leaving only a short variant of the beta-myosin heavy chain constituting only the first 53 residues of the molecule (out of 1935). This deletion was found by chance in an unaffected individual. [41] For MYBPC3, in contrast, the majority of the mutations described so far produce the C-terminal truncation of the cardiac MyBP-C polypeptides and are associated with an FHC phenotype. However, no definitive conclusion can be drawn at this stage concerning the pathogenic mechanisms of mutations in these two genes. The present work provides the molecular basis for the production of transgenic animals for cardiac MyBP-C that will help to resolve some of these issues.

Footnotes
  • Received December 2, 1996; accepted January 10, 1997.

  • This manuscript was sent to Laurence Kedes, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

  • Selected Abbreviations and Acronyms
    EMBL
    European Molecular Biology Laboratory
    FHC
    familial hypertrophic cardiomyopathy
    fn-3
    fibronectin III
    MyBP-C
    myosin binding protein C
    PCR
    polymerase chain reaction
    SSCP
    single-strand conformation polymorphism analysis
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MYBPC3 – Hypertrophic Cardiomyopathy Testing

http://www.cincinnatichildrens.org/workarea/downloadasset.aspx?id=90111
Hypertrophic Cardiomyopathy (HCM) is relatively common, with a prevalence of 1 in 500 adults (1). HCM is a primary disorder of heart muscle characterized by left ventricular hypertrophy. The most classic finding in HCM is asymmetric septal hypertrophy, with or without left ventricular outflow tract obstruction. The disease demonstrates extensive clinical variability with regard to age of onset, severity and progression of disease. HCM can affect infants and children although it is more typically identified in adolescence or adulthood (2,3).

The MYBPC3 gene codes for cardiac myosin binding protein C. Phosphorylation of this protein modulates contraction and is an important component of the sarcomere (4). The MYBPC3 gene contains 35 exons and is located at chromosome 11p11.2. Up to 40% of individuals with a clinical diagnosis of HCM have MYBPC3 mutations (2). MYBPC3 mutations are inherited in an autosomal dominant manner. The majority of individuals inherit the MYBPC3 from a parent, although de novo mutations do occur. Mutations in MYBPC3 and MYH7 genes are the most common causes of HCM. However, the disease is genetically heterogeneous and sequencing additional genes should be considered if familial HCM is suspected or the underlying etiology remains unknown. Approximately 50-65% of individuals with a known or suspected diagnosis of familial HCM have a mutation in one of a number of genes encoding components of the sarcomere and cytoskeleton (3). Compound heterozygous mutations have been reported in MYBPC3 and other genes associated with HCM (5). Mutations in the MYBPC3 gene have been primarily associated with HCM, but can also be associated with other types of heart muscle disease including dilated cardiomyopathy, restrictive cardiomyopathy and left-ventricular non-compaction (6).
Indication MYBPC3 testing is utilized to confirm a diagnosis of HCM in patients with clinically evident disease. Genetic testing also allows for early identification and diagnosis of individuals at greatest risk prior to the expression of typical clinical manifestations. If a mutation is identified in an asymptomatic individual, regular and routine outpatient follow up is indicated. If clinically unaffected members of a family with an identified mutation for HCM are found not to carry that mutation, they can be definitely diagnosed as unaffected and reassured that neither they nor their children will be at higher risk compared to the general population to develop symptoms related to HCM. A negative test result in an individual with a known familial mutation also eliminates the need for routine follow up.
Methodology:
All 35 exons of the MYBPC3 gene, as well as the exon/intron boundaries and a portion of untranslated regions of the gene are amplified by PCR. Genomic DNA sequences from both forward and reverse directions are obtained by automatic fluorescent detection using an ABI PRISM® 3730 DNA Analyzer. Sequence variants different from National Center for Biotechnology Information GenBank references are further evaluated for genetic significance. If a mutation is identified, a known familial mutation analysis will be available for additional family members.
Sensitivity & Accuracy:
Greater than 98.5% of the mutations in exon 1-35 of MYBPC3 are detectable by sequence based methods. Sequencing does not detect deletions or duplications. Mutations in MYBPC3 account for up to 40% of cases of idiopathic hypertrophic cardiomyopathy.
References:
1. Maron BJ, Gardin JM, Flack JM, Gidding SS, Kurosaki TT, Bild DE. Prevalence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the cardia study. Coronary artery risk development in (young) adults. Circulation. 1995;92:785-789.
2. Kaski JP, Syrris P, Esteban MT, Jenkins S, Pantazis A, Deanfield JE, McKenna WJ, Elliott PM. Prevalence of sarcomere protein gene mutations in preadolescent children with hypertrophic cardiomyopathy. Circulation Cardiovascular Genetics. 2009;2:436441.
3. Morita H, Rehm HL, Menesses A, McDonough B, Roberts AE, Kucherlapati R, Towbin JA, Seidman JG, Seidman CE. Shared genetic causes of cardiac hypertrophy in children and adults. The New England Journal of Medicine. 2008;358:1899-1908.
4. van Dijk SJ, Dooijes D, dos Remedios C, Michels M, Lamers JM, Winegrad S, Schlossarek S, Carrier L, ten Cate FJ, Stienen GJ, van der Velden J. Cardiac myosin-binding protein c mutations and hypertrophic cardiomyopathy: Haploinsufficiency, deranged phosphorylation, and cardiomyocyte dysfunction. Circulation. 2009;119:1473-1483.
5. Van Driest SL, Vasile VC, Ommen SR, Will ML, Tajik AJ, Gersh BJ, Ackerman MJ. Myosin binding protein c mutations and compound heterozygosity in hypertrophic cardiomyopathy. Journal of the American College of Cardiology. 2004;44:1903-1910.
6. Hershberger RE, Norton N, Morales A, Li DX, Siegfried JD, Gonzalez-Quintana J. Coding sequence rare variants identified in MYBPC3, MYH6, TPM1, TNNC1, and TNNI3 from 312 patients with familial or idiopathic dilated cardiomyopathy. CirculationCardiovascular Genetics. 2010;3:155-161.

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Cardiac Resynchronization Therapy (CRT) Improves Symptoms and Reduces Mortality and Readmission among Selected Patients with Heart Failure and Left Ventricular Systolic Dysfunction

Reporter: Aviva Lev-Ari, PhD, RN

QRS Duration, Bundle-Branch Block Morphology, and Outcomes Among Older Patients With Heart Failure Receiving Cardiac Resynchronization Therapy

Pamela N. Peterson, MD, MSPH1,2,3; Melissa A. Greiner, MS4; Laura G. Qualls, MS4; Sana M. Al-Khatib, MD, MHS4,5; Jeptha P. Curtis, MD6; Gregg C. Fonarow, MD7; Stephen C. Hammill, MD8; Paul A. Heidenreich, MD9; Bradley G. Hammill, MS4; Jonathan P. Piccini, MD, MHS4,5; Adrian F. Hernandez, MD, MHS4,5; Lesley H. Curtis, PhD4,5; Frederick A. Masoudi, MD, MSPH2,3

Importance  The benefits of cardiac resynchronization therapy (CRT) in clinical trials were greater among patients with left bundle-branch block (LBBB) or longer QRS duration.

Objective  To measure associations between QRS duration and morphology and outcomes among patients receiving a CRT defibrillator (CRT-D) in clinical practice.

Design, Setting, and Participants  Retrospective cohort study of Medicare beneficiaries in the National Cardiovascular Data Registry’s ICD Registry between 2006 and 2009 who underwent CRT-D implantation. Patients were stratified according to whether they were admitted for CRT-D implantation or for another reason, then categorized as having either LBBB or no LBBB and QRS duration of either 150 ms or greater or 120 to 149 ms.

Main Outcomes and Measures  All-cause mortality; all-cause, cardiovascular, and heart failure readmission; and complications. Patients underwent follow-up for up to 3 years, with follow-up through December 2011.

Results  Among 24 169 patients admitted for CRT-D implantation, 1-year and 3-year mortality rates were 9.2% and 25.9%, respectively. All-cause readmission rates were 10.2% at 30 days and 43.3% at 1 year. Both the unadjusted rate and adjusted risk of 3-year mortality were lowest among patients with LBBB and QRS duration of 150 ms or greater (20.9%), compared with LBBB and QRS duration of 120 to 149 ms (26.5%; adjusted hazard ratio [HR], 1.30 [99% CI, 1.18-1.42]), no LBBB and QRS duration of 150 ms or greater (30.7%; HR, 1.34 [99% CI, 1.20-1.49]), and no LBBB and QRS duration of 120 to 149 ms (32.3%; HR, 1.52 [99% CI, 1.38-1.67]). The unadjusted rate and adjusted risk of 1-year all-cause readmission were also lowest among patients with LBBB and QRS duration of 150 ms or greater (38.6%), compared with LBBB and QRS duration of 120 to 149 ms (44.8%; adjusted HR, 1.18 [99% CI, 1.10-1.26]), no LBBB and QRS duration of 150 ms or greater (45.7%; HR, 1.16 [99% CI, 1.08-1.26]), and no LBBB and QRS duration of 120 to 149 ms (49.6%; HR, 1.31 [99% CI, 1.23-1.40]). There were no observed associations with complications.

Conclusions and Relevance  Among fee-for-service Medicare beneficiaries undergoing CRT-D implantation in clinical practice, LBBB and QRS duration of 150 ms or greater, compared with LBBB and QRS duration less than 150 ms or no LBBB regardless of QRS duration, was associated with lower risk of all-cause mortality and of all-cause, cardiovascular, and heart failure readmissions.

Clinical trials have shown that cardiac resynchronization therapy (CRT) improves symptoms and reduces mortality and readmission among selected patients with heart failure and left ventricular systolic dysfunction. Following broad implementation of CRT, it was recognized that one-third to one-half of patients receiving the therapy for heart failure do not improve.1 Identification of patients likely to benefit from CRT is particularly important, because CRT defibrillator (CRT-D) implantation is expensive, invasive, and associated with important procedural risks.

A primary question regarding optimal patient selection for CRT is whether patients with longer QRS duration or left bundle-branch block (LBBB) morphology derive greater benefit than others. Current guidelines recommend selection of patients primarily on the basis of QRS duration and morphology based predominantly on meta-analyses and subgroup analyses of clinical trials evaluating either QRS duration or morphology. Only 1 study specifically evaluated the combination of QRS duration and morphology but did not assess meaningful patient outcomes.2 Thus, the role of QRS duration and morphology in the selection of patients for CRT in contemporary clinical practice remains unclear.

The objectives of this study were to determine the long-term outcomes of unselected patients undergoing CRT-D implantation in real-world settings and associations between combinations of QRS duration and presence of LBBB and longitudinal outcomes, including mortality, readmission, and complications following CRT-D implantation in a large population of Medicare beneficiaries who received CRT-Ds.

SOURCE

http://jama.jamanetwork.com/article.aspx?articleid=1728715&utm_content=sidebar-related&utm_term=alsolike&utm_source=Silverchair%20Information%20Systems&utm_medium=email&utm_campaign=JAMA:OnlineFirst08/30/2015

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle


Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD

 

Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

http://www.amazon.com/dp/B012BB0ZF0.

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

https://pharmaceuticalintelligence.com/biomed-e-books/

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 

 

Summary 

Epilogue

 

 

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Premature Ventricular Contraction percentage predicts new Systolic Dysfunction and clinically diagnosed CHF and overall Mortality

Reporter: Aviva Lev-Ari, PhD, RN

 

Cardiovascular Health Study (CHS)

This study has been completed. ClinicalTrials.gov processed this record on July 13, 2015
Sponsor:
Information provided by:
National Heart, Lung, and Blood Institute (NHLBI)
ClinicalTrials.gov Identifier:
NCT00005133
First received: May 25, 2000
Last updated: May 1, 2009
Last verified: May 2009
  Purpose

To determine the extent to which known risk factors predict coronary heart disease and stroke in the elderly, to assess the precipitants of coronary heart disease and stroke in the elderly, and to identify the predictors of mortality and functional impairments in clinical coronary disease or stroke.

SOURCE

https://clinicaltrials.gov/ct2/show/NCT00005133?term=Cardiovascular+Health+Study&rank=2

Although links between frequent PVCs and ongoing heart failure have been observed, the current analysis, based on a cohort from the Cardiovascular Health Study (CHS), provides “the first evidence that PVC percentage predicts new systolic dysfunction, as well as clinically diagnosed CHF and overall mortality,” say the authors in their report, published in the July 14, 2015 issue of the Journal of the American College of Cardiology. It also raises the issue of whether PVCs might sometimes be an appropriate target for treatments aimed at preventing heart failure.

The observational study can’t demonstrate causality, note the authors, led by Dr Jonathan W Dukes (University of California, San Francisco). But overall, the findings “suggest that PVCs might be an important cause of occult or ‘idiopathic’ cardiomyopathy and might be an important determinant of incident CHF among those with other established CHF risk factors.”

Ablate PVCs in HF, LVEF Can Improve

“There’s this general notion that PVCs are very benign, which is certainly what I was taught, even in my general cardiology fellowship, before the more recent data that came out of the electrophysiology labs,” senior author Dr Gregory M Marcus (UCSF) said in an interview with heartwire from Medscape.

In recent years, he said, it’s been appreciated that ablation of PVCs in patients with lots of them can improve quality of life by alleviating symptoms such as syncope. And there are series of patients with PVCs and primarily nonischemic cardiomyopathy in the EP literature suggesting that “if you ablate those PVCs, their heart failure improves and often their reduced ejection fraction normalizes,” according to Marcus. “Many of us have seen that and witnessed it firsthand in many of our own patients.”

Although the analysis tried to control for such factors, she said, the question remains “whether PVCs are causing deterioration in EF and HF or if they are simply a marker of underlying disease. If the former is true, then treating PVCs would help. But if the latter is true, then treating PVCs may not make a difference.”

Marcus acknowledges that PVCs may be simply a risk marker in people with sick hearts. “But even if that’s the case, I think it’s potentially a very useful marker.” He said he hopes the report will help “motivate future research in potentially two different directions. One, might ablation be an effective therapy to prevent heart failure in the right patients? Alternatively, could this be used to help predict heart failure and implement other strategies, such as beta-blockers, to prevent heart failure in those patients?”

CASTing a New Light on Treatment of PVCs

The Cardiac Arrhythmia Suppression Trial (CAST), Marcus noted, “taught us a lot of important lessons. More generally, it was a great example of the need to look at hard outcomes rather than secondary or surrogate outcomes.”

As cardiology textbooks have since noted, CAST randomized about 2300 patients who had asymptomatic or only mildly symptomatic PVCs after acute MI to receive one of three antiarrhythmic agents or placebo. The drugs, which included the class Ic agents encainide and flecainide, were mostly effective at suppressing PVCs. But over a mean 10 months of follow-up, patients who had received those drugs showed steep rise in rate of arrhythmic death (the primary end point) as well as nonfatal cardiac arrest, almost certainly due to proarrhythmic effects.

The widely learned lesson: post-MI suppression of PVCs, a surrogate for the pathology behind sudden cardiac death in ischemic heart disease, doesn’t lower its risk; in fact, treatment of surrogate markers can make things a lot worse. (Importantly, CAST was conducted in the early days of arrhythmia ablation and implantable defibrillators, which were not options for its patients.)

As a result, according to Marcus, class Ic agents are generally avoided in patients with structural heart disease. “I think that while the proarrhythmic effects of those drugs were known, they weren’t fully appreciated, and CAST taught us to be wary of them.”

 

The CHS is sponsored by the National Heart, Lung, and Blood Institute. Dukes and Marcus report that they have no relevant financial relationships; disclosures for the other authors are in the report. Santangeli and Marchlinski report that they have no relevant financial relationships. Al-Khatib says she has no relevant financial relationships with industry.

 

SOURCE

http://www.medscape.com/viewarticle/847859?nlid=84244_2562&src=wnl_edit_medp_card&uac=93761AJ&spon=2&impID=760872&faf=1

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Heart-Lung-Kidney: Essential Ties

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

Introduction

The basic functioning of the heart, and the kidney have been covered in depth elsewhere, and pulmonary function less, except in this series.  The relationship between them on the basis of endocrine, signaling, and metabolic balance is the focus in this piece.

Other elated articles can be found in http://pharmaceuticalintelligence.com:

The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide – Part I
https://pharmaceuticalintelligence.com/2012/11/26/the-amazing-structure-and-adaptive-functioning-of-the-kidneys/

Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II
https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-and-inos-have-key-roles-in-kidney-diseases/

Stroke and Bleeding in Atrial Fibrillation with Chronic Kidney Disease
https://pharmaceuticalintelligence.com/2012/08/16/stroke-and-bleeding-in-atrial-fibrillation-with-chronic-kidney-disease/

Risks of Hypoglycemia in Diabetics with Chronic Kidney Disease (CKD)
https://pharmaceuticalintelligence.com/2012/08/01/risks-of-hypoglycemia-in-diabetics-with-ckd/

Acute Lung Injury
https://pharmaceuticalintelligence.com/2015/02/26/acute-lung-injury/

Neonatal Pathophysiology
https://pharmaceuticalintelligence.com/2015/02/22/neonatal-pathophysiology/

Altitude Adaptation
https://pharmaceuticalintelligence.com/2015/02/24/altitude-adaptation/

Action of Hormones on the Circulation
https://pharmaceuticalintelligence.com/2015/02/17/action-of-hormones-on-the-circulation/

Innervation of Heart and Heart Rate
https://pharmaceuticalintelligence.com/2015/02/15/innervation-of-heart-and-heart-rate/

Neural Activity Regulating Endocrine Response
https://pharmaceuticalintelligence.com/2015/02/13/neural-activity-regulating-endocrine-response/

Adrenal Cortex
https://pharmaceuticalintelligence.com/2015/02/07/adrenal-cortex/

Thyroid Function and Disorders
https://pharmaceuticalintelligence.com/2015/02/05/thyroid-function-and-disorders/

Highlights in the History of Physiology
https://pharmaceuticalintelligence.com/2014/12/28/highlights-in-the-history-of-physiology/

The Evolution of Clinical Chemistry in the 20th Century
https://pharmaceuticalintelligence.com/2014/12/13/the-evolution-of-clinical-chemistry-in-the-20th-century/

Complex Models of Signaling: Therapeutic Implications
https://pharmaceuticalintelligence.com/2014/10/31/complex-models-of-signaling-therapeutic-implications/

Cholesterol and Regulation of Liver Synthetic Pathways
https://pharmaceuticalintelligence.com/2014/10/25/cholesterol-and-regulation-of-liver-synthetic-pathways/

A Brief Curation of Proteomics, Metabolomics, and Metabolism
https://pharmaceuticalintelligence.com/2014/10/03/a-brief-curation-of-proteomics-metabolomics-and-metabolism/

Natriuretic Peptides in Evaluating Dyspnea and Congestive Heart Failure
https://pharmaceuticalintelligence.com/2014/09/08/natriuretic-peptides-in-evaluating-dyspnea-and-congestive-heart-failure/

Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease
https://pharmaceuticalintelligence.com/2014/07/06/omega-3-fatty-acids-depleting-the-source-and-protein-insufficiency-in-renal-disease/

Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases
https://pharmaceuticalintelligence.com/2014/05/10/summary-part-2-volume-4-translational-medicine-in-cardiovascular-diseases/

More on the Performance of High Sensitivity Troponin T and with Amino Terminal Pro BNP in Diabetes
https://pharmaceuticalintelligence.com/2014/01/20/more-on-the-performance-of-high-sensitivity-troponin-t-and-with-amino-terminal-pro-bnp-in-diabetes/

Diagnostic Value of Cardiac Biomarkers
https://pharmaceuticalintelligence.com/2014/01/04/diagnostic-value-of-cardiac-biomarkers/

Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker
https://pharmaceuticalintelligence.com/2013/12/10/epo-as-therapeutics-for-anemia-in-chf/

The Young Surgeon and The Retired Pathologist: On Science, Medicine and HealthCare Policy – Best writers Among the WRITERS
https://pharmaceuticalintelligence.com/2013/12/10/the-young-surgeon-and-the-retired-pathologist-on-science-medicine-and-healthcare-policy-best-writers-among-the-writers/

Renal Function Biomarker, β-trace protein (BTP) as a Novel Biomarker for Cardiac Risk Diagnosis in Patients with Atrial Fibrillation
https://pharmaceuticalintelligence.com/2013/11/13/renal-function-biomarker-%CE%B2-trace-protein-btp-as-a-novel-biomarker-for-cardiac-risk-diagnosis-in-patients-with-atrial-fibrilation/

Leptin signaling in mediating the cardiac hypertrophy associated with obesity
https://pharmaceuticalintelligence.com/2013/11/03/leptin-signaling-in-mediating-the-cardiac-hypertrophy-associated-with-obesity/

The Role of Tight Junction Proteins in Water and Electrolyte Transport
https://pharmaceuticalintelligence.com/2013/10/07/the-role-of-tight-junction-proteins-in-water-and-electrolyte-transport/

Selective Ion Conduction
https://pharmaceuticalintelligence.com/2013/10/07/selective-ion-conduction/

Translational Research on the Mechanism of Water and Electrolyte Movements into the Cell
https://pharmaceuticalintelligence.com/2013/10/07/translational-research-on-the-mechanism-of-water-and-electrolyte-movements-into-the-cell/

Landscape of Cardiac Biomarkers for Improved Clinical Utilization
https://pharmaceuticalintelligence.com/2013/09/22/landscape-of-cardiac-biomarkers-for-improved-clinical-utilization/

Calcium-Channel Blocker, Calcium as Neurotransmitter Sensor and Calcium Release-related Contractile Dysfunction (Ryanopathy)
https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/

Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease
https://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-exchange-mechanism-in-health-and-disease/

Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage
https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-sepsis-and-the-cardiovascular-system-at-its-end-stage/

The Cardio-Renal Syndrome (CRS) in Heart Failure (HF)
https://pharmaceuticalintelligence.com/2013/06/30/the-cardiorenal-syndrome-in-heart-failure/

More…

Sodium homeostasis

Icariin attenuates angiotensin IIinduced hypertrophy and apoptosis in H9c2 cardiomyocytes by inhibiting reactive oxygen speciesdependent JNK and p38 pathways

H Zhou, Y Yuan, Y Liu, Wei Deng, Jing Zong, Zhou‑Yan Bian, Jia Dai and Qi‑Zhu Tang
Exper and Therapeutic Med 7: 1116-1122, 2014
http://dx.doi.org:/10.3892/etm.2014.1598

Icariin, the major active component isolated from plants of the Epimedium family, has been reported to have potential protective effects on the cardiovascular system. However, it is not known whether icariin has a direct effect on angiotensin II (Ang II)‑induced cardiomyocyte enlargement and apoptosis. In the present study, embryonic rat heart‑derived H9c2 cells were stimulated by Ang II, with or without icariin administration. Icariin treatment was found to attenuate the Ang II‑induced increase in mRNA expression levels of hypertrophic markers, including atrial natriuretic peptide and B‑type natriuretic peptide, in a concentration‑dependent manner. The cell surface area of Ang II‑treated H9c2 cells also decreased with icariin administration. Furthermore, icariin repressed Ang II‑induced cell apoptosis and protein expression levels of Bax and cleaved‑caspase 3, while the expression of Bcl‑2 was increased by icariin. In addition, 2′,7’‑dichlorofluorescein diacetate incubation revealed that icariin inhibited the production of intracellular reactive oxygen species (ROS), which were stimulated by Ang II. Phosphorylation of c‑Jun N‑terminal kinase (JNK) and p38 in Ang II‑treated H9c2 cells was blocked by icariin. Therefore, the results of the present study indicated that icariin protected H9c2 cardiomyocytes from Ang II‑induced hypertrophy and apoptosis by inhibiting the ROS‑dependent JNK and p38 pathways.

Short-term add-on therapy with angiotensin receptor blocker for end-stage inotrope-dependent heart failure patients: B-type natriuretic peptide reduction in a randomized clinical trial

Marcelo E. Ochiai, ECO Brancalhao, RSN Puig, KRN Vieira, et al.
Clinics. 2014; 69(5):308-313
http://dx.doi.org:/10.6061/clinics/2014(05)02

OBJECTIVE: We aimed to evaluate angiotensin receptor blocker add-on therapy in patients with low cardiac output during decompensated heart failure. METHODS: We selected patients with decompensated heart failure, low cardiac output, dobutamine dependence, and an ejection fraction ,0.45 who were receiving an angiotensin-converting enzyme inhibitor. The patients were randomized to losartan or placebo and underwent invasive hemodynamic and B-type natriuretic peptide measurements at baseline and on the seventh day after intervention. ClinicalTrials.gov: NCT01857999. RESULTS: We studied 10 patients in the losartan group and 11 patients in the placebo group. The patient characteristics were as follows: age 52.7 years, ejection fraction 21.3%, dobutamine infusion 8.5 mcg/kg.min, indexed systemic vascular resistance 1918.0 dynes.sec/cm5.m2, cardiac index 2.8 L/min.m2, and B-type natriuretic peptide 1,403 pg/mL. After 7 days of intervention, there was a 37.4% reduction in the B-type natriuretic peptide levels in the losartan group compared with an 11.9% increase in the placebo group (mean difference, – 49.1%; 95% confidence interval: -88.1 to -9.8%, p = 0.018). No significant difference was observed in the hemodynamic measurements. CONCLUSION: Short-term add-on therapy with losartan reduced B-type natriuretic peptide levels in patients hospitalized for decompensated severe heart failure and low cardiac output with inotrope dependence.

Development of a Novel Heart Failure Risk Tool: The Barcelona Bio-Heart Failure Risk Calculator (BCN Bio-HF Calculator)

Josep Lupon, Marta de Antonio, Joan Vila, Judith Penafiel, et al.
PLoS ONE 9(1): e85466. http://dx.doi.org:/10.1371/journal.pone.0085466

Background: A combination of clinical and routine laboratory data with biomarkers reflecting different pathophysiological pathways may help to refine risk stratification in heart failure (HF). A novel calculator (BCN Bio-HF calculator) incorporating N-terminal pro B-type natriuretic peptide (NT-proBNP, a marker of myocardial stretch), high-sensitivity cardiac troponin T (hs-cTnT, a marker of myocyte injury), and high-sensitivity soluble ST2 (ST2), (reflective of myocardial fibrosis and remodeling) was developed. Methods: Model performance was evaluated using discrimination, calibration, and reclassi-fication tools for 1-, 2-, and 3-year mortality. Ten-fold cross-validation with 1000 bootstrapping was used. Results: The BCN Bio-HF calculator was derived from 864 consecutive outpatients (72% men) with mean age 68.2612 years (73%/27% New York Heart Association (NYHA) class I-II/III-IV, LVEF 36%, ischemic etiology 52.2%) and followed for a median of 3.4 years (305 deaths). After an initial evaluation of 23 variables, eight independent models were developed. The variables included in these models were age, sex, NYHA functional class, left ventricular ejection fraction, serum sodium, estimated glomerular filtration rate, hemoglobin, loop diuretic dose, β-blocker, Angiotensin converting enzyme inhibitor/Angiotensin-2 receptor blocker and statin treatments, and hs-cTnT, ST2, and NT-proBNP levels. The calculator may run with the availability of none, one, two, or the three biomarkers. The calculated risk of death was significantly changed by additive biomarker data. The average C-statistic in cross-validation analysis was 0.79. Conclusions: A new HF risk-calculator that incorporates available biomarkers reflecting different pathophysiological pathways better allowed individual prediction of death at 1, 2, and 3 years.

TNF and angiotensin type 1 receptors interact in the brain control of blood pressure in heart failure

Tymoteusz Zera, Marcin Ufnal, Ewa Szczepanska-Sadowska
Cytokine 71 (2015) 272–277
http://dx.doi.org/10.1016/j.cyto.2014.10.019

Accumulating evidence suggests that the brain renin-angiotensin system and proinflammatory cytokines, such as TNF-α, play a key role in the neuro-hormonal activation in chronic heart failure (HF). In this study we tested the involvement of TNF-α and angiotensin type 1 receptors (AT1Rs) in the central control of the cardiovascular system in HF rats. Methods: we carried out the study on male Sprague–Dawley rats subjected to the left coronary artery ligation (HF rats) or to sham surgery (sham-operated rats). The rats were pretreated for four weeks with intracerebroventricular (ICV) infusion of either saline (0.25 µl/h) or TNF-α inhibitor etanercept (0.25 µg/0.25 µl/h). At the end of the pretreatment period, we measured mean arterial blood pressure (MABP) and heart rate (HR) at baseline and during 60 min of ICV administration of either saline (5 µl/h) or AT1Rs antagonist losartan (10 µg/5 µl/h). After the experiments, we measured the left ventricle end-diastolic pressure (LVEDP) and the size of myocardial scar. Results: MABP and HR of sham-operated and HF rats were not affected by pretreatments with etanercept or saline alone. In sham-operated rats the ICV infusion of losartan did not affect MABP either in saline or in etanercept pretreated rats. In contrast, in HF rats the ICV infusion of losartan significantly decreased MABP in rats pretreated with saline, but not in those pretreated with etanercept. LVEDP was significantly elevated in HF rats but not in sham-operated ones. Surface of the infarct scar exceeded 30% of the left ventricle in HF groups, whereas sham-operated rats did not manifest evidence of cardiac scarring. Conclusions: our study provides evidence that in rats with post-infarction heart failure the regulation of blood pressure by AT1Rs depends on centrally acting endogenous TNF-α.

Statins in heart failure—With preserved and reduced ejection fraction. An update

Dimitris Tousoulis , E Oikonomou, G Siasos, C Stefanadis
Pharmacology & Therapeutics 141 (2014) 79–91
http://dx.doi.org/10.1016/j.pharmthera.2013.09.001

HMG-CoA reductase inhibitors or statins beyond their lipid lowering properties and mevalonate inhibition exert also their actions through a multiplicity of mechanisms. In heart failure (HF) the inhibition of isoprenoid intermediates and small GTPases, which control cellular function such as cell shape, secretion and proliferation, is of clinical significance. Statins share also the peroxisome proliferator-activated receptor pathway and inactivate extracellular-signal-regulated kinase phosphorylation suppressing inflammatory cascade. By down-regulating Rho/Rho kinase signaling pathways, statins increase the stability of eNOS mRNA and induce activation of eNOS through phosphatidylinositol 3-kinase/Akt/eNOS pathway restoring endothelial function. Statins change also myocardial action potential plateau by modulation of Kv1.5 and Kv4.3 channel activity and inhibit sympathetic nerve activity suppressing arrhythmogenesis. Less documented evidence proposes also that statins have antihypertrophic effects – through p21ras/mitogen activated protein kinase pathway – which modulate synthesis of matrix metalloproteinases and procollagen 1 expression affecting interstitial fibrosis and diastolic dysfunction. Clinical studies have partly confirmed the experimental findings and despite current guidelines new evidence supports the notion that statins can be beneficial in some cases of HF. In subjects with diastolic HF, moderately impaired systolic function, low B-type natriuretic peptide levels, exacerbated inflammatory response and mild interstitial fibrosis evidence supports that statins can favorably affect the outcome. Under the lights of this evidence in this review article we discuss the current knowledge on the mechanisms of statins’ actions and we link current experimental and clinical data to further understand the possible impact of statins’ treatment on HF syndrome.

Since 1980 when the first 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor or statin was introduced in clinical practice, statins have been extensively used in the treatment of patients with dyslipidemia as well as of those with coronary artery disease (CAD). Importantly, large scale trials and metanalysis have documented their significant benefits in terms of primary and secondary CAD prevention which out-weigh any potential side effects. Statins’ benefits extend, according to recent studies, even in patients with normal or low cholesterol levels and beyond their lipid lowering effects, indicating their multiple protective mechanisms.

Heart failure (HF) is a complex syndrome with different definitions and its diagnosis is based on a combination of symptoms, clinical signs and imaging or laboratory data. different categorization schemes have been used dividing HF in acute or chronic, in systolic or diastolic, and in ischemic or dilated simply reflecting the complexity of the syndrome and the multiplicity of the pathophysiologic mechanisms implicated in the disease development and progression. In addition to the diverse pathophysiology of HF the syndrome is also characterized by high morbidity and mortality. Recent treatment advantages such as angiotensin converting enzyme inhibitors and beta blockers have not yet proven their clinical benefit in subjects with diastolic HF.

As the most common cause of HF is CAD and statins have proven their benefits in a wide spectrum of diseases directly or indirectly associated with atherosclerotic cardiovascular disease, HMG-CoA reductase inhibitors have been tested in subjects with HF. Interestingly, non-randomized, observational and retrospective early studies in subjects with HF of ischemic and non-ischemic etiology have suggested that statins are associated with improved outcomes. Thereafter, two large scale randomized control trials failed to demonstrate any benefits in mortality of HF patients treated with rosuvastatin and subsequently current HF guidelines do not include recommendations for statin use except from when they are indicated for comorbidities, such as established CAD.

Statins inhibit HMG-CoA reductase. This enzyme catalyzes the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to L-mevalonic acid, which is the rate-limiting step in the cholesterol synthesis pathway. Inhibition of the mevalonate pathway and of cholesterol synthesis triggers an increase in LDL receptor activity by stimulating production of mRNA for LDL receptor in liver. The induction of LDL receptors is responsible for the observed increase in plasma clearance of LDL cholesterol. CAD is the cause of approximately two-thirds of cases of systolic HF. The beneficial effects of statins-induced LDL reduction are well established in patients with atherosclerosis and CAD. Nevertheless, the results from statin treatment, even in ischemic HF cases, are not straightforward and several mechanisms have been proposed for this paradox.

multiplicity of HMG CoA reductase inhibitors mechanisms and their effects

multiplicity of HMG CoA reductase inhibitors mechanisms and their effects

The figure demonstrates the multiplicity of HMG CoA reductase inhibitors mechanisms and their effects. ↓: decrease; ↑ increase; FPP: farnesyl pyrophosphate: GGPP: geranylgeranyl pyrophosphate; Ras, Rac, Rho; small GTPases; eNOS: endothelial nitric oxide synthase; ATP: adenosine triphosphate; PI-3 kinase: phosphatidylinositol 3-kinase; AMPK: AMP activated protein kinase; GTP: Guanosine triphosphate; NADPH: Nicotinamide adenine dinucleotide phosphate; ERK: extracellular-signal-regulated kinase; Shadow box represents adverse mechanism and actions of HGM CoA reductase inhibitors.

The anti-inflammatory effects of HMG CoA reductase inhibitors in atherosclerosis have been early recognized. Statins also have a potent anti-inflammatory effect in HF models. Importantly, there is a link between inflammation and HF pathogenesis and is now widely accepted that pro-inflammatory cytokines cause systolic dysfunction, myocardial hypertrophy, activate a fetal gene program in cardiac myocytes, disturb extracellular matrix structure, cause cardiac cachexia etc. In addition, data from the Vesnarinone trial (VEST) in 384 patients with HF demonstrate a decline in survival with increasing TNFα levels confirming the notion that circulating cytokines are associated with adverse prognosis of HF patients.

The proposed, by the aforementioned mechanisms, anti-inflammatory effects of statins have been confirmed experimentally. Indeed, in a rat HF model with preserved ejection fraction (EF), treatment with rosuvastatin resulted in a significant additional improvement in HF and cardiac remodeling, partly due to decreased myocardial inflammation. In rats after acute myocardial infarction simvastatin treatment for 4 weeks beneficially modified the levels of TNFα, interleukin (IL)-1, 6 and 10 in the infarct regions. Importantly, in 446 patients with systolic HF, followed up for a period of 24 months, statins’ treatment was associated with a decrease in serum levels of C-reactive protein (CRP), IL-6 and tumor necrosis factor-alpha receptor II. Recently, in a randomized study of 22 subjects with ischemic HF short term atorvastatin treatment achieved a significant decrease in serum levels of intracellular adhesion molecule-1.

Taken together we can conclude that HMG CoA reductase inhibitors can modify inflammatory status by modulation of PRAP and ERK pathways by down regulating Toll like receptor 4 mRNA expressions and LDL oxidation and by reducing soluble lipoprotein-associated phospholipase A2 mass and activity. Importantly, the theoretical anti-inflammatory properties were confirmed in experimental and clinical HF models.

Endothelial dysfunction contributes to the pathogenesis of HF and can enhance adverse left ventricle (LV) remodeling and increase afterload in subjects with HF. Interestingly, statins have been constantly associated with improved endothelial function in subjects with a variety of cardiovascular diseases. Endothelium derived nitric oxide (NO) is an important determinant of endothelial function and HMG-CoA reductase inhibitors can up regulate endothelial NO synthase (eNOS) by different mechanisms.

Statins induce down regulation of Rho/Rho kinase signaling pathways, increasing the stability of eNOS mRNA and its expression . In addition, in human endothelial cells the Rho-kinase inhibitor, hydroxyfasudil leads to the activation of the phosphatidylinositol 3-kinase/Akt/eNOS pathway. Statins also induce activation of eNOS through the rapid activation of the serine–threonine protein kinase Akt. The beneficial effects of Akt activation are not limited to eNOS phoshorylation but extend to the promotion of new blood vessels growth. HMG CoA reductase inhibitors can further affect endothelial function through their effect on caveolin-1. Caveolin-1 binds to eNOS inhibiting NO production. Incubation of endothelial cells with atorvastatin promotes NO production by decreasing caveolin-1 expression, regardless of the level of extracellular LDL-cholesterol. These effects were reversed with mevalonate highlighting the therapeutic potential of inhibiting cholesterol synthesis in peripheral cells to correct NO-dependent endothelial dysfunction associated with hypercholesterolemia and possibly other diseases.

Although the experimentally confirmed benefits of HMG CoA reductase inhibitors in diastolic dysfunction and left ventricle stiffness, few data exist concerning the underlying mechanisms. As diastolic dysfunction precedes myocardial hypertrophy the anti-hypertrophic pathways mentioned in the previous section (inhibition of RhoA/Ras/ERK, PRAPγ pathways, inhibition of a large G(h) protein-coupled pathway etc.), may also contribute to the restoration of diastolic function. Moreover, in angiotensin II induced diastolic dysfunction in hypertensive mice, pravastatin not only improved diastolic function but also down-regulated collagen I, transforming growth factor-beta, matrix metalloproteinases (MMPs)-2 and -3, atrial natriuretic factor, IL-6 TNFα, Rho kinase 1 gene expression, and upregulated eNOS gene expression. These findings suggest the potential involvement of Rho kinase 1 in the beneficial effects of pravastatin in diastolic HF. Taken together data suggest that HMG CoA reductase inhibitors might be beneficial in patients with diastolic HF, a hypothesis that remains to be confirmed by clinical studies. Nevertheless, mechanistic studies have not fully explored the pathways affecting diastolic function and most data until now are indirect. Therefore efforts should be focus on the underline mechanisms affecting collagen synthesis, MMPs activity extracellular matrix synthesis and overall diastolic function in HF subjects under statin treatment.

Statins through inhibition of small GTPases can modulate MMPs activity in several cell types such as endothelial cells and human macrophages. In rat and human cardiac fibroblasts, stimulated with either transforming growth factor β1 or angiotensin II, atorvastatin reduced collagen synthesis and α1-procollagen mRNA as well as gene expression of the profibrotic peptide connective tissue growth factor 4. This antifibrotic action may contribute to the anti-remodelling effect of statins. In mouse cardiac fibroblasts treated with angiotensin II, the combination of pravastatin and pioglitazone blocked angiotensin II p38 MAPK and p44/42 MAPK activation and procollagen expression-1.

Several studies have documented the impact of statin treatment on arrhythmia potential. The arrhythmic protective effects of statins can be attributed not only to anti-inflammatory properties but also to changes in myocardial action potential plateau by modulation of Kv1.5 and Kv4.3 channel activity. Atorvastatin and simvastatin block Kv1.5 and Kv4.3 channels shifting the inactivation curve to more negative potentials following a complex mechanism that does not imply the binding of the drug to the channel pore. Moreover, in hypertrophied neonatal rat ventricular myocytes simvastatin alleviated the reduction of Kv4.3 expression, I(to) currents in subepicardial myocardium from the hypertrophied left ventricle. Furthermore, pravastatin in an animal model attenuated reperfusion induced lethal ventricular arrhythmias by inhibition of calcium overload.

Taking together experimental and cellular evidence supporting an effect of statin treatment in myocardial contractility is spare and for the time being we cannot definitively conclude on the clinical impact of HMG CoA reductase inhibitors in myocardial systolic performance.

Half of the cases of HF are attributed to diastolic dysfunction and the prognosis of HF with preserved EF is as ominous as the prognosis of HF with systolic dysfunction. Unfortunately, no treatment has yet been shown, convincingly, to reduce morbidity and mortality in patients with HF and preserved EF, while this group of patients is usually excluded from large prospective randomized trials and accordingly few data exist for the role of statins in this heterogeneous population.

As there is substantially lack of evidence concerning the effects of HMG CoA reductase inhibitors in subjects with HF and preserved EF the first indirect hypothesis was extrapolated from observational prospective studies in subjects with ischemic heart disease and no evidence of congestive HF. Indeed, in a cohort of 430 consecutive patients with ischemic heart disease and a mean EF of 57% Okura et al. observed that subjects under HMG CoA reductase inhibitors treatment had decreased E/E′ ratio—corresponding to a better diastolic function—and a significantly higher survival rate (Okura et al., 2007). According to the authors those beneficially effects can be attributed to improved endothelial function and vasodilatory response to reactive hyperemia, attenuation of myocardial hypertrophy, and interstitial fibrosis.

Despite the positive results from mechanistic and experimental studies clinical studies have failed to confirm a definitive role of HMG CoA reductase inhibitors in HF. Nevertheless, by extrapolating experimental and mechanistic data in clinical settings we further understand how HMG-CoA reductase inhibitors can beneficially affect subgroups of HF subjects such as those with preserved EF, low B-type natriuretic peptide levels, exacerbated inflammatory response and limited interstitial fibrosis. Nevertheless, as a definitive mechanism is lacking, there is uncertainty about the decisive mode of action and further mechanistic studies are needed to reveal how HMG-CoA reductase inhibitors act in HF substrate.

Pro- A-Type Natriuretic Peptide, Proadrenomedullin, and N-Terminal Pro-B-Type Natriuretic Peptide Used in a Multimarker Strategy in Primary Health Care in Risk Assessment of Patients with Symptoms of Heart Failure

Urban Alehagen, Ulf Dahlstr€Om,  Jens F. Rehfeld, And Jens P. Goetze
J Cardiac Fail 2013; 19(1):31-39. http://dx.doi.org/10.1016/j.cardfail.2012.11.002

Use of new biomarkers in the handling of heart failure patients has been advocated in the literature, but most often in hospital-based populations. Therefore, we wanted to evaluate whether plasma measurement of N-terminal pro-B-type natriuretic peptide (NT-proBNP), midregional pro-A-type  atriuretic peptide (MR-proANP), and midregional proadrenomedullin (MR-proADM), individually or combined, gives prognostic information regarding cardiovascular and all-cause mortality that could motivate use in elderly patients presenting with symptoms suggestive of heart failure in primary health care. Methods and Results: The study included 470 elderly patients (mean age 73 years) with symptoms of heart failure in primary health care. All participants underwent clinical examination, 2-dimenstional echocardiography, and plasma measurement of the 3 propeptides and were followed for 13 years. All mortality was registered during the follow-up period. The 4th quartiles of the biomarkers were applied as cutoff values. NT-proBNP exhibited the strongest prognostic information with 4-fold increased risk for cardiovascular mortality within 5 years. For all-cause mortality MR-proADM exhibited almost 2-fold and NTproBNP 3-fold increased risk within 5 years. In the 5e13-year perspective, NT-proBNP and MR-proANP showed significant and independent cardiovascular prognostic information. NT-proBNP and MR-proADM showed significant prognostic information regarding all-cause mortality during the same time. In those with ejection fraction (EF) !40%, MR-proADM exhibited almost 5-fold increased risk of cardiovascular mortality with 5 years, whereas in those with EF O50% NT-proBNP exhibited 3-fold increased risk if analyzed as the only biomarker in the model. If instead the biomarkers were all below the cutoff value, the patients had a highly reduced mortality risk, which also could influence the handling of patients. Conclusions: The 3 biomarkers could be integrated in a multimarker strategy for use in primary health care.

Novel Biomarkers in Heart Failure with Preserved Ejection Fraction

Kevin S. Shah, Alan S. Maisel
Heart Failure Clin 10 (2014) 471–479
http://dx.doi.org/10.1016/j.hfc.2014.04.005

KEY POINTS

  • Heart failure with preserved ejection fraction (HFPEF) is a common subtype of congestive heart failure for which therapies to improve morbidity and mortality have been limited thus far.
  • Numerous biomarkers have emerged over the past decade demonstrating prognostic significance in HFPEF, including natriuretic peptides, galectin-3, soluble ST2, and high-sensitivity troponins.
  • These markers reflect the multiple mechanisms implicated in the pathogenesis of HFPEF, and future research will likely use these markers to not only help determine heart failure phenotypes but also target specific therapies.

Heart failure (HF) is a global epidemic, defined as an abnormality of cardiac function leading to the inability to deliver oxygen at a rate adequate to meet the requirements of tissues. It is truly a clinical syndrome of symptoms and signs resulting from this cardiac abnormality. Over the past decade, further characterization into 2 entities has occurred: HF with preserved ejection fraction (HFPEF) and HF with reduced ejection fraction (HFREF). HFPEF, previously termed diastolic HF, encompasses the syndrome of HF with a preserved ejection fraction. Cutoffs for this ejection fraction typically are from 45% to 50%. The prevalence of HF is upward of 1% to 2% of the adult population, with an increased prevalence found in elderly and female patients. Multiple studies have shown that the prevalence of HFPEF is actually comparable with the number of patients with HFREF. As expected, most deaths from HFPEF are cardiovascular, comprising 51% to 70% of mortality.

The pathophysiology of HFPEF is controversial and remains poorly understood. Originally, HFPEF was thought to be a primary manifestation of diastolic dysfunction of the left ventricle. However, patients with HFREF are known to also commonly have impaired ventricular relaxation. The primary mechanism of left ventricular (LV) dysfunction is based on structural remodeling and endothelial dysfunction, lending itself to LV stiffness, and increased left atrial pressure. This pressure change is what drives pulmonary venous congestion and subsequent symptomatology. The ventricular stiffness commonly seen in HFPEF is attributed to multiple mechanisms, including fibrosis, excessive collagen deposition, cardiomyocyte stiffness, and slow LV relaxation.

The natriuretic peptides (NPs) are the cornerstone biomarker in congestive HF (CHF). Many of the details of the role of NPs are covered in an article – Florea VG, Anand IS. Biomarkers. Heart Fail Clin 2012;8(2):207–24. The Breathing Not Properly trial originally helped establish the role of B-type natriuretic peptide (BNP) in the diagnosis of CHF. BNP and the N-terminal prohormone BNP (NT-proBNP) have been shown in numerous trials to be an excellent tool for ruling out CHF as a cause of acute dyspnea. Aside from a strong negative predictive value, NPs correlate with HF severity, prognostication, outpatient CHF management, and screening. When attempting to use NPs specifically to distinguish between HFPEF and HFREF, results have shown that NPs do not have a particular cutoff, but are typically elevated in HFPEF in comparison with patients without HF. These levels of NPs in HFPEF are typically lower than levels in patients with HFREF.

Although the role of novel renal biomarkers has not been fully explored specifically in HFPEF, they likely have an impactful role in the assessment and management of acute kidney injury (AKI) and the cardiorenal syndrome. Two biomarkers are briefly discussed here: neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C. NGAL is a 25-kDa protein in the lipocalin family of proteins with a role in inflammation and immune modulation.

The future of biomarkers and their utility in HF is very promising, starting with the potential for using biomarkers as end points in trials. Biomarkers serve as surrogates for various pathophysiologic mechanisms, and there are potential benefits in using them as trial end points. Advantages include the ability to obtain quick and early data, as well as possibly better understand the nature of the disease. However, the counterargument against using biomarkers as trial end points includes whether treatment effects on a biomarker reliably predict effects on a clinically meaningful end point.
Reduced cGMP signaling activates NF-κB in hypertrophied hearts of mice lacking natriuretic peptide receptor-A

Elangovan Vellaichamy, Naveen K. Sommana, Kailash N. Pandey
Biochemical and Biophysical Research Communications 327 (2005) 106–111
http://dx.doi.org:/10.1016/j.bbrc.2004.11.153

Mice lacking natriuretic peptide receptor-A (NPRA) develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for cardiac hypertrophic growth in the absence of NPRA signaling are not yet known. We sought to determine the activation of nuclear factor-κB (NF-κB) in Npr1 (coding for NPRA) gene-knockout (Npr1-/-) mice exhibiting cardiac hypertrophy and fibrosis. NF-κB binding activity was 4-fold greater in the nuclear extract of Npr1-/-mutant mice hearts as compared with wild-type (Npr1+/+) mice hearts. In parallel, inhibitory κB kinase-b activity and IκB-α protein phosphorylation were also increased 3- and 4-fold, respectively, in hypertrophied hearts of mutant mice. cGMP levels were significantly reduced 5-fold in plasma and 10-fold in ventricular tissues of mutant mice hearts  relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates NF-κB binding activity associated with hypertrophic growth of mutant mice hearts.

Regulation of guanylyl cyclase/natriuretic peptide receptor-A gene expression

Renu Garg, Kailash N. Pandey
Peptides 26 (2005) 1009–1023
http://dx.doi.org:/10.1016/j.peptides.2004.09.022

Natriuretic peptide receptor-A (NPRA) is the biological receptor of the peptide hormones atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The level and activity of this receptor determines the biological effects of ANP and BNP in different tissues mainly directed towards the maintenance of salt and water homeostasis. The core transcriptional machinery of the TATA-less Npr1 gene, which encodes NPRA, consists of three SP1 binding sites and the inverted CCAAT box. This promoter region of Npr1 gene has been shown to contain several putative binding sites for the known transcription factors, but the functional significance of most of these regulatory sequences is yet to be elucidated. The present review discusses the current knowledge of the functional significance of the promoter region of Npr1 gene and its transcriptional regulation by a number of factors including different hormones, growth factors, changes in extracellular osmolarity, and certain physiological and patho-physiological conditions.

Atrial natriuretic peptide (ANP), a member of natriuretic peptide family is a polypeptide consisting of 28 amino acids and was discovered as a potent vasodilator and diuretic hormone produced in granules of the atrium. The natriuretic peptide family consists of the peptide hormones ANP, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), each of which is derived from a separate gene. ANP and BNP are cardiac derived peptides, which are secreted and up-regulated in myocardium in response to different patho-physiological stimuli, while CNP is an endothelium-derived mediator that plays an important paracrine role in the vasculature. All of these natriuretic peptides elicit a number of vascular, renal, and endocrine effects mainly directed towards the maintenance of blood pressure and extracellular fluid volume by binding to their specific cell surface receptors. ANP exerts its effects at a number of sites including the kidney, where it produces natriuretic and diuretic responses; the adrenal gland, where it inhibits aldosterone synthesis and secretion; vascular smooth muscle cells, where it produces vasorelaxation; the endothelial cells, where it may regulate vascular permeability; gonadal cells, where it affects synthesis of androgen and estradiol. Each of these target sites of ANP activity has been shown to possess specific high affinity receptors for ANP. To date, three different subtypes of natriuretic peptide receptors have been characterized, purified, and cloned, i.e. natriuretic peptide receptors A, B, and C also designated as NPRA, NPRB, and NPRC, respectively. ANP and BNP specifically bind to NPRA, which contains guanylyl cyclase catalytic activity and produces intracellular secondary messenger cGMP in response to hormone binding.

NPRA is considered the biological receptor of ANP and BNP because most of the physiological effects of these hormones are triggered by generation of cGMP or its cell permeable analogs. Recent studies with mice lacking the Npr1 gene, demonstrated that genetic disruption of NPRA increases the blood pressure and causes hypertension in these animals. On the other hand, the effect of ANP was found to be increased linearly in Npr1 gene-duplicated mice
in a manner consistent with gene copy number. All this clearly indicates that the level of NPRA expression determines the extent of the biological effects of ANP and BNP. But the intervention strategies aimed at controlling NPRA expression are limited by the paucity of studies in this area. The cDNA and gene encoding NPRA designated as Npr1 has been cloned and characterized in mouse, rat, bull frog, euryhaline eel, and medaka fish. The primary structure of this gene is essentially same in all the different species and contains 22 exons interrupted by 21 introns.  The Npr1 gene sequence has been found to be interspersed with a number of repetitive elements including (SINES), (MER2), and tandem repeat elements in all the different species.

Although the Npr1 gene transcriptional regulation is only poorly understood, the activity and expression of NPRA assessed primarily through ANP stimulated cGMP accumulation are found to be regulated by a number of factors including auto-regulation by natriuretic peptides themselves, other hormones such as endothelin, glucocorticoids, and angiotensin II (ANG II), growth factors, changes in extracellular ion composition, and certain physiological and patho-physiological conditions.

The core molecular machinery of the TATA-less Npr1 gene consisting of SP1 binding sites and the inverted CCAAT box has been authenticated to be indeed functional in rat promoter element. It has been shown that the molecular machinery that regulates the basal expression of Npr1 gene consists of three SP1 binding sites in conjunction with an inverted CCAAT box present in the proximal promoter region. Mutation in any of these SP1 binding sites which
are located within 350 bp upstream of transcription start site in rat Npr1 promoter inhibited SP1 and SP3 binding and decreased the promoter activity by 50–75%, while simultaneous mutation of all the three led to a >90% reduction in promoter activity. The proximal SP1 binding site was much more effective than the distal sites in inducing the expression implying that the proximity to the core transcriptional machinery contributes to the magnitude of the observed effect. The over-expression of either SP1 or SP3 resulted in the induction of the wild type Npr1 promoter, confirming that these transcription factors serve as positive regulators of the Npr1 gene expression.

A number of natriuretic peptides such as ANP, BNP, CNP, and urodilatin (i.e. ANP95–126) can down-regulate ligand dependent NPRA activity after as little as 2 h prior exposure to the ligand, which remains suppressed until 48 h of exposure in cultured cells. The early reduction of NPRA activity is independent of changes in Npr1 gene expression as the pretreatment of cultured cells with actinomycin D (an inhibitor of transcription) for 1 h failed to block the response to ANP implying that ligand acts, at least early on, through a post transcriptional mechanism in reducing NPRA activity. The sustained reduction of NPRA activity, on the other hand, has been shown in fact due to reduction in NPRA mRNA levels (∼50%) by treatment with 100nM ANP for 48 h. This reduction could also be affected by treatment of cultured cells with 8-Br-cGMP with similar kinetic response and was amplified by phosphodiesterase inhibitors, but was not shared by NPRC-selective ligand cANF, suggesting that the down regulation of Npr1 gene expression is mediated by elevations of intracellular cGMP involving either NPRA or NPRB. .. The cGMP regulatory region was pinpointed to position−1372 to−1354 bp from the transcription start site of Npr1 by gel shift assays and footprinting analysis, which indicated its interaction with transcriptional factor(s). Further cross-competition experiments with mutated oligonucleotides led to the definition of a consensus sequence (−1372 bp AaAtRKaNTTCaAcAKTY −1354 bp) for the novel cGMP-RE, which is conserved in the human (75% identity) and mouse (95% identity) Npr1 promoters. The combination of these transcriptional and post-transcriptional ligand-dependent regulatory mechanisms provides the cells with greater flexibility in both initiating and maintaining the suppression of NPRA activity.

The peptide hormone Ang II is an important component of renin-angiotensin system (RAS) and exerts its biological effects such as blood pressure regulation, vasoconstriction, and cell proliferation in many tissues including the kidney, adrenal glands, brain, and vasculature. The two vasoactive peptide hormones, Ang II (vasoconstrictive) and ANP (vasodilatory), interact and mutually antagonize the biological effects of each other at various levels. ANP has been shown to inhibit Ang II-induced contraction of isolated glomeruli and cultured mesangial cells, as well as Ang II-stimulated activation of protein kinase C and mitogen activated protein kinase in vascular smooth muscle cells in a cGMP-dependent manner. Inversely, Ang II has been shown to down-regulate guanylyl cyclase activity of the biological receptor of ANP, NPRA, by activating protein kinase C and/or by stimulating protein tyrosine phosphatase activity, thereby inhibiting the ANP stimulated cGMP accumulation. Ang II also reduces the ANP dependent cGMP levels by stimulating cGMP hydrolysis, apparently
via a calcium dependent cGMP phosphodiesterase.

Endothelin is a vasoconstrictor peptide that was originally isolated from porcine endothelial cells. It is produced as three isoforms (ET1-3) that bind to two receptor subtypes (ETA and ETB). ET is produced in the kidney and subject to regulation by a number of local and systemic factors including immune cytokines and extracellular tonicity. Since, endothelin is avidly expressed in the nephron segment, where NPRA is up-regulated by osmotic stimulus, it was investigated whether endothelin plays a role in the control of basal or osmotically regulated Npr1 gene expression in these cells. The endogenous endothelin and not the exogeneously administered endothelin inhibit the basal but not osmotically stimulated expression of Npr1. The type A (BQ610) and type B (IRL 1038) endothelin receptor antagonists increased the level of NPRA mRNA by two to three-fold, whereas co-administration of exogenous endothelin resulted in partial reversal of this stimulatory effect of receptor antagonists. The increase in extracellular tonicity reduces the endothelin mRNA accumulation (∼15% of control levels) in inner medullary collecting duct cells but this reduction is not found to be linked to the stimulation of NPRA activity/expression in response to osmotic stress.

Glucocorticoids influence the cardiovascular system and induce a rapid increase in blood pressure. Glucocorticoids are known to regulate
transcription in many systems, possibly by interacting with glucocorticoid responsive elements and associated chromatin proteins. These have been shown to affect the atrial endocrine system by regulating both the synthesis and secretion of ANP in vitro and in vivo. Thus, it seems plausible that glucocorticoid may also interact with the atrial endocrine system by modulating ANP receptor levels. The stimulation of vascular smooth muscle cells from rat mesenteric artery with dexa-methasone (a highly specific synthetic glucocorticoid agonist) caused an increase in NPRA mRNA levels in a time dependent manner which reached a plateau after 48 h of glucocorticoid administration. This mRNA increase was mimicked by cortisol and inhibited by glucocorticoid receptor antagonists RU38486. Also cGMP generated by NPRA in dexamethasone treated cells was higher than in control cells and this production was mimicked by cortisol and blocked by RU 38486. These results suggest that glucocorticoids exert a positive effect on NPRA transcription in rat mesenteric arteries.

Previous studies have shown that guanylyl cyclase activity of NPRA is either activated, or inhibited by an increase in extracellular tonicity. Though none of these studies were definitive in terms of elucidating the mechanisms involved, they suggested that the activation predominates with longer exposure (∼24 h), while the inhibition with short-term exposure (minutes) to the osmotic stimulus. More recently, the mechanism(s) underlying the activation of NPRA expression by osmotic stimulus has been investigated. The NaCl (75 mM) or sucrose (150 mM), but not osmotically inert solute, urea (150 mM) increased NPRA activity, gene expression, and promoter activity after as early as 4 h reaching a maximum at 24 h in inner medullary collecting duct cells. The osmotic stimulus also activated extracellular signal regulated kinase (ERK), c-Jun-NH2-terminal kinase (JNK), and p38 mitogen activated protein kinase- (p38 MAPK-β). The inhibition of p38 MAPK-βwith SB20580 completely  blocked the osmotic stimulation of receptor activity and expression, and caused a dose-dependent reduction in promoter activity, whereas inhibition of ERK with PD98059 had no effect.

The expression of NPRB, the biological receptor of CNP, has been shown to be regulated by a number of factors including natriuretic peptide ligands, intracellular cAMP levels, water deprivation, TGF-1, dexamethasone treatment, as well as renal sodium status, as its mRNA levels were upregulated in the renal cortex of sodium depleted animals. NPRB expression has also been found to be regulated by alternative splicing. Three isoforms of NPRB have been identified of which NPRB1 is the full length form and responds maximally to CNP, NPRB2 isoform contains a 25 amino acid deletion in protein kinase homology domain and NPRB3 contains a partial extracellular ligand binding domain and fails to bind the ligand. The relative expression levels of the three isoforms vary across different tissues. Since, the smaller splice variants of NPRB act as dominant negative isoforms by blocking formation of active NPRB1 homodimers, these isoforms might play important role in the tissue specific regulation of receptor, NPRB.

The NPRC expression has also been found to be down-regulated by its ligands and their secondary messenger, cGMP, hormones, growth factors, dietary salt supplementation, β-adrenergic blocker, and physiological as well as patho-physiological conditions. On the other hand, NPRC expression gets augmented by TGF-β1, 1,25-dihydroxy VitaminD3 and during conditions like chronic heart failure.

Hypertension is the leading cause of human deaths in today’s world. The natriuretic peptide system plays a well defined role in the regulation of blood pressure and fluid volume. The cellular and physiological effects of natriuretic peptides (ANP, BNP, and CNP) are mediated by their specific receptors NPRA, NPRB, and NPRC. The transcriptional regulation of these receptors has been studied since their identification, but still remains poorly understood. Better understanding and the elucidation of different molecular mechanisms responsible for the regulation of NPRA expression would provide us the framework to develop the therapeutic strategies to manipulate the expression levels of this receptor and to control the biological actions of ANP and BNP during different patho-physiological conditions.

Inhibition of Heat Shock Protein 90 (Hsp90) in Proliferating Endothelial Cells Uncouples Endothelial Nitric Oxide Synthase Activity

Jingsong Ou, Zhijun Ou, AW Ackerman, KT Oldham, & KA Pritchard, Jr.
Free Radical Biol Med 2003; 34(2):269–276
PII S0891-5849(02)01299-6

Dual increases in nitric oxide (•NO) and superoxide anion (O2•-) production are one of the hallmarks of endothelial cell proliferation. Increased expression of endothelial nitric oxide synthase (eNOS) has been shown to play an important role in maintaining high levels of •NO generation to offset the increase in O2•- that occurs during proliferation. Although recent reports indicate that heat shock protein 90 (hsp90) associates with eNOS to increase •NO generation, the role of hsp90 association with eNOS during endothelial cell proliferation remains unknown. In this report, we examine the effects of endothelial cell proliferation on eNOS expression, hsp90 association with eNOS, and the mechanisms governing eNOS generation of •NO and O2•-. Western analysis revealed that endothelial cells not only increased eNOS expression during proliferation but also hsp90 interactions with the enzyme. Pretreatment of cultures with radicicol (RAD, 20 µM), a specific inhibitor that does not redox cycle, decreased A23187-stimulated •NO production and increased Lω-nitroargininemethylester (L-NAME)-inhibitable O2•-generation. In contrast, A23187 stimulation of controls in the presence of L-NAME increased O2•- generation, confirming that during proliferation eNOS generates •NO. Our findings demonstrate that hsp90 plays an important role in maintaining •NO generation during proliferation. Inhibition of hsp90 in vascular endothelium provides a convenient mechanism for uncoupling eNOS activity to inhibit •NO production. This study provides new understanding of the mechanisms by which ansamycin antibiotics inhibit endothelial cell proliferation. Such information may be useful in the development and design of new antineoplastic agents in the future.

Natriuretic Peptides, Ejection Fraction, and Prognosis – Parsing the Phenotypes of Heart Failure

James L. Januzzi, JR
J Amer Coll Cardiol 2013; 61(14): 1507-9
http://dx.doi.org/10.1016/j.jacc.2013.01.039

Since the first pivotal studies introduced the natriuretic peptides as biomarkers for the diagnosis of heart failure (HF), use of both B-type natriuretic peptide (BNP) and its N-terminal equivalent (NT-proBNP) has grown not only for this indication, but also for establishing HF prognosis as well. Indeed, a vast array of studies has established the natriuretic peptides as the biomarker gold standard to prognosticate risk for a wide array of relevant complications in HF (ranging from ventricular arrhythmias to pump failure). In these studies, the prognostic information provided by BNP and NT-proBNP in HF was independent of a number of relevant covariates, including left ventricular ejection fraction (LVEF).

It has been known for quite a while that patients with heart failure and preserved ejection fraction (HFpEF) typically have lower natriuretic peptide values than do those with heart failure and reduced ejection fraction (HFrEF). A conundrum is thus present: whereas both BNP and NTproBNP tend to be lower in HFpEF, when these peptides are elevated in this setting, they remain prognostic; this intriguing circumstance has been relatively poorly studied. It is in this setting that van Veldhuisen et al. examined the impact of LVEF on the prognostic merits of BNP in the COACH (Coordinating Study Evaluating Outcomes of Advising and Counseling in Heart Failure) study in the present issue of the Journal. The investigators found—as expected—that BNP levels were lower in HFpEF, but for a given BNP concentration, prognosis of those with HFpEF in COACH was just as poor as those with HFrEF at matched BNP values. Stated differently, a high BNP in a patient with HFpEF imparted similar prognostic information as it would in someone with HFrEF. Actually, whereas LVEF was not obviously prognostically impactful, when considered across the range of ventricular function, an elevated BNP concentration in the most normal range of LVEF seemed to be associated with a higher risk than at the lower ranges of pump function. Although it is previously established that BNP or NT-proBNP are prognostic independently of LVEF, the well-executed analysis by van Veldhuisen et al. (van Veldhuisen DJ, Linssen GCM, Jaarsma T, et al. B-type natriuretic peptide and prognosis in heart failure patients with preserved and reduced ejection fraction. J Am Coll Cardiol 2013;61:1498–506.) allows for a more in-depth examination of this phenomenon and raises some important questions.

Phenotypic Definition of the Patient With Heart Failure

Phenotypic Definition of the Patient With Heart Failure

Phenotypic Definition of the Patient With Heart Failure

Natriuretic Peptides in Heart Failure with Preserved Ejection Fraction

Mark Richards, James L. Januzzi Jr, Richard W. Troughton
Heart Failure Clin 10 (2014) 453–470
http://dx.doi.org/10.1016/j.hfc.2014.04.006

KEY POINTS

  • Threshold values of B-type natriuretic peptide (BNP) and N-terminal prohormone B-type natriuretic peptide (NT-proBNP) validated for diagnosis of undifferentiated acutely decompensated heart failure (ADHF) remain useful in patients with heart failure with preserved ejection fraction (HFPEF), with minor loss of diagnostic performance.
  • BNP and NT-proBNP measured on admission with ADHF are powerfully predictive of in-hospital mortality in both HFPEF and heart failure with reduced EF (HFREF), with similar or greater risk in HFPEF as in HFREF associated with any given level of either peptide.
  • In stable treated heart failure, plasma natriuretic peptide concentrations often fall below cut-point values used for the diagnosis of ADHF in the emergency department; in HFPEF, levels average approximately half those in HFREF.
  • BNP and NT-proBNP are powerful independent prognostic markers in both chronic HFREF and chronic HFPEF, and the risk of important clinical adverse outcomes for a given peptide level is similar regardless of left ventricular ejection fraction.
  • Serial measurement of BNP or NT-proBNP to monitor status and guide treatment in chronic heart failure may be more applicable in HFREF than in HFPEF.

 

The bioactivity of atrial NP (ANP) and B-type NP (BNP) encompasses short-term and longterm hemodynamic, renal, neurohormonal, and trophic effects. The relationship between cardiac hemodynamic load, plasma concentrations of ANP and BNP, and the cardioprotective profile of NP bioactivity have led to investigation of both biomarker and therapeutic potential of

NPs in HF.

PlasmaBNPandNT-proBNP thresholds (100pg/mL and 300 pg/mL, respectively) used in the diagnosis of undifferentiated ADHF retain good diagnosticperformance for acute HFPEF

 

Plasma NPs are related to multiple echo indicators of cardiac structure and function in both HFREF and HFPEF.
Box 1Causes of increased plasma cardiac natriuretic peptides

Cardiac

Heart failure, acute and chronic

Acute coronary syndromes

Atrial fibrillation

Valvular heart disease

Cardiomyopathies

Myocarditis

Cardioversion

Left ventricular hypertrophy

Noncardiac

Age

Female sex

Renal impairment

Pulmonary embolism

Pneumonia (severe)

Obstructive sleep apnea

Critical illness

Bacterial sepsis

Severe burns

Cancer chemotherapy

Toxic and metabolic insults

 

BNP and NT-proBNP fall below ADHF thresholds in stable HFREF in approximately 50% and 20% of cases, respectively. Levels in stable HFPEF are even lower, approximately half those in HFREF.
Whereas BNPs have 90% sensitivity for asymptomatic LVEF of less than 40% in the community (a precursor state for HFREF), they offer no clear guide to the presence of early community based HFPEF.
Guidelines recommend BNP and NT-proBNP as adjuncts to the diagnosis of acute and chronic HF and for risk stratification. Refinements for application to HFPEF are needed.
The prognostic power of NPs is similar in HFREF and HFPEF. Defined levels of BNP and NT-proBNP correlate with similar short-term and long-term risks of important clinical adverse outcomes in both HFREF and HFPEF.
Diagnostic algorithm for suspected heart failure presenting either acutely or nonacutely

Diagnostic algorithm for suspected heart failure presenting either acutely or nonacutely

Diagnostic algorithm for suspected heart failure presenting either acutely or nonacutely. a In the acute setting, mid-regional pro–atrial natriuretic peptide may also be used (cutoff point 120 pmol/L; ie, <120 pmol/L 5 heart failure unlikely). b Other causes of elevated natriuretic peptide levels in the acute setting are an acute coronary syndrome, atrial or ventricular arrhythmias, pulmonary embolism, and severe chronic obstructive pulmonary disease with elevated right heart pressures, renal failure, and sepsis. Other causes of an elevated natriuretic level in the nonacute setting are old age (>75 years), atrial arrhythmias, left ventricular hypertrophy, chronic obstructive pulmonary disease, and chronic kidney disease. c Exclusion cutoff points for natriuretic peptides are chosen to minimize the false-negative rate while reducing unnecessary referrals for echocardiography. d Treatment may reduce natriuretic peptide concentration, and natriuretic peptide concentrations may not be markedly elevated in patients with heart failure with preserved ejection fraction. BNP, B-type natriuretic peptide; ECG, electrocardiogram; NT-proBNP, N-terminal prohormone of B-type natriuretic peptide. (From McMurray JJ, Adamopoulos S, Anker SD, et al. The task force for the diagnosis and treatment of acute and chronic heart failure 2012 of the European Society of Cardiology. ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012. Eur Heart J 2012;33:1787–847; with permission.)

Natriuretic Peptide Receptor-A Negatively Regulates Mitogen-Activated Protein Kinase and Proliferation of Mesangial Cells: Role of cGMP-Dependent Protein Kinase

Kailash N. Pandey, Houng T. Nguyen, Ming Li, and John W. Boyle
Biochem Biophys Res Commun 271, 374–379 (2000)
http://dx.doi.org:/10.1006/bbrc.2000.2627

peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (NPRA) on mitogen-activated protein kinase/extracellular signal-regulated kinase 2 (MAPK/ERK2) activity in rat mesangial cells overexpressing NPRA. Agonist hormones such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), angiotensin II (ANG II), and endothelin-1 (ET-1) stimulated 2.5- to 3.5-fold immunoreactive MAPK/ERK2 activity in these cells. ANP inhibited agonist-stimulated activity of MAPK/ERK2 by 65–75% in cells overexpressing NPRA, whereas in vector transfected cells, its inhibitory effect was only 18–20%. NPRA antagonist A71915 and KT5823, a specific inhibitor of cGMP-dependent protein kinase (PKG) completely reversed the inhibitory effect of ANP on MAPK/ERK2 activity. ANP also inhibited the PDGF stimulated [3H]thymidine uptake by almost 70% in cells overexpressing NPRA, as compared with only 20–25% inhibition in vector-transfected cells. These
results demonstrate that ANP/NPRA system negatively regulates MAPK/ERK2 activity and proliferation of mesangial cells in a PKG-dependent manner.

 

Regulation of lipoprotein lipase by Angptl4

Wieneke Dijk and Sander Kersten
Trends in Endocrin and Metab, Mar2014; 25(3):146-155
http://dx.doi.org/10.1016/j.tem.2013.12.005

Triglyceride (TG)-rich chylomicrons and very low density lipoproteins (VLDL) distribute fatty acids (FA) to various tissues by interacting with the enzyme lipoprotein lipase (LPL). The protein angiopoietin-like 4 (Angptl4) is under sensitive transcriptional control by FA and the FA-activated peroxisome proliferator activated receptors (PPARs), and its tissue expression largely overlaps with that of LPL. Growing evidence indicates that Angptl4 mediates the physiological fluctuations in LPL activity, including the decrease in
adipose tissue LPL activity during fasting. This review focuses on the major ambiguities concerning the mechanism of LPL inhibition by Angptl4, as well as on the physiological role of Angptl4 in lipid metabolism, highlighting its function in a variety of tissues, and uses this information to make suggestions for further research.

Box 1. LPL and TG metabolism

LPL belongs to a family of lipases that also includes hepatic lipase, pancreatic lipase, and endothelial lipase. Because LPL is essential in the lipolytic processing of chylomicrons and VLDL, LPL is primarily expressed in tissues that either require large amounts of FA as fuel or are responsible for TG storage, which include heart, skeletal muscle, and adipose tissue. Upon production by the underlying parenchymal cells, LPL is released into the subendothelial space and is transported to the luminal side of the capillary endothelium by the GPI-anchored protein GPIHBP1, which after transport continues to anchor LPL to the capillary endothelium. The essential role for LPL in the clearance of plasma TG is well-demonstrated by the severe hypertriglyceridemia of patients carrying homozygous mutations in the LPL gene. Generalized deletion of LPL in mice results in severe hypertriglycer-idemia, resulting in the premature death of pups within 24 h after birth. Analogous to the deletion of LPL, the mislocalization of LPL to the subendothelial spaces due the absence or misfolding of GPIHBP1 also results in severe chylomicronemia and hypertriglyceridemia. The LPL enzyme is catalytically active as a non-covalent head-to-tail dimer with a catalytic N-terminal domain and a non-catalytic C terminal domain. Folding of LPL into its dimer conformation occurs in the endoplasmic reticulum, chaperoned by lipase maturation factor 1, calreticulin, and calnexin. In its active 3D conformation, the catalytic site of LPL is postulated to be covered by a lid, which can be opened by the binding of chylomicrons and VLDL to the C terminus. The active LPL dimers rapidly exchange subunits, indicating that a dynamic equilibrium exists between LPL dimers and dimerization-competent monomers. Dimerization-competent monomers have, however, not yet been isolated, and it is unclear whether this monomer is catalytically active. The enzymatic activity of LPL is lost when the LPL dimer is converted into inactive, folded monomers. This conversion to inactive monomers is mainly regulated via post-translational mechanisms and is dependent on nutritional state. Enzymatic activity of inactive monomers can be regained in vitro by the addition of calcium, indicating that inactivation of LPL is a reversible process.

One of the key questions is whether (patho)physiological variations in LPL activity are mediated via regulation of Angptl4 cleavage and/or oligomerization, and which factors are involved in modulating Angptl4 in vivo. Recent biochemical evidence suggests that FA may be able to promote dissociation of oligomers, which, by destabilizing the protein, would impair its ability to inhibit LPL. Destabilization of Angptl4 by FA is, however, seemingly at odds with the marked stimulatory effect of FA on Angptl4 production observed in vitro and in vivo.

The currently accepted molecular model for the inhibition of LPL by Angptl4 is that Angptl4 stimulates the conversion of catalytically active LPL dimers into inactive monomers – following in vitro studies showing that coincubation of LPL and Angptl4 increases the abundance of LPL monomers. Subsequent studies revealed that the proportion of LPL dimers is reduced in post-heparin plasma of mice that overexpress Angptl4 in favor of LPL monomers, providing in vivo support for the dimer-to monomer conversion. The elucidation of the purported biochemical mechanism has strengthened the status of Angptl4 as a LPL inhibitor, but several questions related to the in vivo mechanism remain unanswered. Whereas the original in vitro experiments favored the hypothesis that Angptl4 enzymatically and irreversibly catalyzes the LPL dimer-to-monomer conversion, an in vivo study of Angptl4 transgenic mice suggested that Angptl4 is physically bound to LPL monomers, thereby driving the LPL dimer–monomer equilibrium towards inactive monomers. The latter study also revealed that the relative decrease in post-heparin plasma LPL activity upon Angptl4 overexpression is much more pronounced than the relative decrease in heparin-releasable LPL dimers, pointing to an additional or alternative mechanism. In support, a recently published study suggests that Angptl4, instead of acting as a catalyst, functions as a conventional, non-competitive inhibitor that binds to LPL to prevent the hydrolysis of substrate LPL and Angptl4 are regulated by changes in nutritional state in a tissue-specific manner, reflecting the different functions of these tissues and the corresponding variations in physiological requirements for lipids. Below, we discuss current knowledge on the regulation of Angptl4 and LPL in response to various physiological stimuli and address the importance of Angptl4 in lipid uptake. An overview of the role of Angptl4 in physiological regulation of lipid metabolism is presented in Figure 2.

model for mechanisms of lipoprotein lipase (LPL) inhibition by Angptl4.

model for mechanisms of lipoprotein lipase (LPL) inhibition by Angptl4.

Figure 1. Hypothetical model for mechanisms of lipoprotein lipase (LPL) inhibition by Angptl4. Angiopoietin-like 4 (Angptl4) and LPL are expressed in the parenchymal cells of muscle, heart, and adipose tissue. Following secretion of LPL and Angptl4 into the subendothelial space, transport of LPL to the capillary lumen is mediated by two mechanisms. The principal transport mechanism (1) relies on GPIHBP1 [glycosylphosphatidylinositol (GPI)-anchored high density lipoprotein-binding protein] picking up LPL from the subendothelial space and transporting it to the capillary lumen. This action by GPIHBP1 is opposed by Angptl4, which is bound to extracellular matrix (ECM) proteins and which retains and inhibits LPL. In the presence of GPIHBP1, high expression levels of Angptl4 are needed to overcome the competition with GPIHBP1. Angptl4 secreted into the capillary lumen, primarily as N-terminal truncation fragment generated by cleavage by proprotein convertases (PCs), inhibits LPL activity on the endothelium by promoting the irreversible conversion of LPL dimers into inactive monomers and/or via a reversible mechanism that requires binding of Angptl4 to LPL. The second transport mechanism involves a so far unidentified carrier and can be disrupted by Angptl4. In the absence of GPIHBP1, Angptl4 fully retains LPL in the subendothelial space (a). The additional loss of Angptl4 liberates LPL and allows it to be transported to the endothelial surface via the unidentified carrier (b). This model suggests that Angptl4 and LPL start interacting before arrival in the capillary lumen, either in the parenchymal cells or in the subendothelial space. Abbreviation: HSPG, heparan sulfate proteoglycan.

Regulation and role of angiopoietin-like 4 (Angptl4)

Regulation and role of angiopoietin-like 4 (Angptl4)

Figure 2. Regulation and role of angiopoietin-like 4 (Angptl4) in lipid metabolism. Angptl4 is expressed in parenchymal cells of white adipose tissue (WAT), liver, intestine, heart and muscle, as well as in macrophages, where it is subject to cell- and tissue-specific regulation. Angptl4 is a sensitive target of peroxisome proliferator-activated receptor (PPAR) transcription factors in several tissues. In WAT the expression of Angptl4 is induced during fasting and by the transcription factors PPARg, glucocorticoid receptor (GR), and hypoxia inducible factor 1a (HIF1a). In WAT Angptl4 stimulates lipolysis of stored triglycerides (TG) and inhibits lipoprotein lipase (LPL) activity. Expression of Angptl4 in liver is stimulated by PPARa, PPARd, and GR. Because the liver does not express LPL, Angptl4 is mainly released into the blood, affecting LPL activity in peripheral tissues. Angptl4 may also impact upon hepatic lipase activity in liver. Expression of Angptl4 in heart and skeletal muscle is potently induced by fatty acids (FA) via PPARd activation. Angptl4 inhibits LPL activities in cardiac and likely skeletal muscle. FA also stimulate Angptl4 expression in macrophages via PPARd, leading to local inhibition of LPL activity. We hypothesize that macrophage LPL enables uptake of remnant particles containing lipid antigens, which are subsequently presented to natural killer T cells. In the intestine, FA stimulate Angptl4 expression via one of the PPARs. Angptl4 produced by enterocytes may be released towards the lumen and inhibit pancreatic lipase activity. Angptl4 produced by enteroendocrine cells is released towards the blood and may inhibit LPL in distant tissues.

Box 2. Outstanding questions

  1. What is the importance of Angptl4 cleavage and oligomerization to Angptl4 function in vivo?
  2. What is the precise biochemical mechanism behind the inhibition of LPL activity by Angptl4?
  3. At which cellular location(s) does the inhibition of LPL by Angptl4 occur and, if at multiple locations, what is the relative contribution of both tissue-produced Angptl4 compared to circulating Angptl4 with respect to inhibition of tissue LPL activity.
  4. What is the interplay between GPIHBP1 and Angptl4 in the regulation of LPL activity?
  5. What is the protein structure of Angptl4 and LPL?
  6. Does Angptl4 also regulate LPL activity in brown adipose tissue and skeletal muscle and, if so, how is the expression of Angptl4 regulated in these tissues?
  7. What is the potential of Angptl4 as a biomarker in the context of disorders of lipid metabolism?

In the past decade, angiopoietin-like proteins have been demonstrated to regulate plasma TG levels powerfully in mice and humans. The elucidation of these proteins as inhibitors of LPL activity has led to a paradigm shift in how clearance of circulating TG and thereby tissue uptake of FA are regulated. Most of our understanding of angiopoietin-like proteins has resulted from detailed study of Angptl4.

A major portion of the physiological variation in LPL activity in various tissues can be attributed to regulation of Angptl4 production. We predict that Angptl4 will turn out to be equally important for governing LPL activity in muscle during exercise, in brown adipose tissue during cold, and in several tissues during fasting.

Besides the increasing recognition of the pivotal role of Angptl4 in lipid metabolism as an inhibitor of LPL, major insight has been gained into the molecular mechanism of action of Angptl4. Key questions remain, however, especially related to the interaction between LPL, GPIHBP1, and Angptl4 on the endothelium and in the subendothelial space. Several points of interest have been highlighted throughout the text; these include the elucidation of the molecular structure for LPL and Angptl4 by X-ray crystallography and the clarification of in vivo Angptl4 cleavage and oligomerization.

Native Low-Density Lipoprotein Induces Endothelial Nitric Oxide Synthase Dysfunction: Role of Heat Shock Protein 90 And Caveolin-1

Kirkwood A. Pritchard, Jr., Allan W. Ackerman, Jingsong Ou, et al.
Free Radical Biol & Med 2002; 33(1):52–62 PII S0891-5849(02)00851-1

Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide (•NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O2•-), which inactivates •NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for •NO, O2•-, phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O2•-by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by [1]34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (-85%) and cytosol (-15%). Only 3–6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O2•-, which scavenges •NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O2•-.

In conclusion, n-LDL decreases the association of hsp90 with eNOS, increases phospho-eNOS levels, and increases eNOS-dependent O2•-generation. These findings suggest that activation of eNOS without adequate levels of hsp90 may signal eNOS to switch from •NO to O2•-generation. Such changes in eNOS radical product generation may play an important role in impairing endothelial and vascular function.

New insights into IGF-1 signaling in the heart

Rodrigo Troncoso, C Ibarra, JM Vicencio, E Jaimovich, and S Lavandero
Trends in Endocrin and Metab, Mar 2014; 25(3):128-131
http://dx.doi.org/10.1016/j.tem.2013.12.002

Insulin-like growth factor 1 (IGF-1) signaling regulates contractility, metabolism, hypertrophy, autophagy, senescence, and apoptosis in the heart. IGF-1 deficiency is associated with an increased risk of cardiovascular disease, whereas cardiac activation of IGF-1 receptor (IGF-1R) protects from the detrimental effects of a high-fat diet and myocardial infarction. IGF-1R activates multiple pathways through its intrinsic tyrosine kinase activity and through coupling to heterotrimeric G protein. These pathways involve classic second messengers, phosphorylation cascades, lipid signaling, Ca2+ transients, and gene expression. In addition, IGF-1R triggers signaling in different subcellular locations including the plasma membrane, perinuclear T tubules, and also in internalized vesicles. In this review, we provide a fresh and updated view of the complex IGF-1 scenario in the heart, including a critical focus on therapeutic strategies.

The hormone insulin-like growth factor 1 (IGF-1) is a small peptide of 7.6 kDa, which is composed of 70 amino acids and shares 50% homology with insulin. IGF-1 plays key roles in regulating proliferation, differentiation, metabolism, and cell survival. It is mainly synthesized and secreted by the liver in response to hypothalamic growth hormone (GH); its plasma concentration is finely regulated (Box 1). However, other tissues also produce IGF-1, which acts locally as an autocrine and paracrine hormone. IGF-1 exhibits pleiotropic effects in many organs and is also involved in the development of several pathologies.

Box 1. IGF-1 synthesis and biodisponibilityInsulin-like growth factor 1 (IGF-1) is a 70 amino acid peptide

hormone with endocrine, paracrine, and autocrine effects. It shares

>60% structure homology with IGF-2 and 50% with pro-insulin. IGF-

1 is mainly synthesized in the liver in response to hypothalamic

growth hormone (GH). In the peripheral circulation it exerts negative

feedback on the somatotrophic axis suppressing pituitary GH

release. IGF-1 can also be generated in almost all tissues, but liver

synthesis accounts for nearly 75% of circulating IGF-1 levels. As a

hormone with a wide range of physiological roles, IGF-1 circulating

levels must be strictly controlled. Around 98% of circulating IGF-1 is

bound to insulin-like growth factor binding protein (IGFBP). Six

forms of high affinity IGFBP have been described, with IGFBP3

binding approximately 90% of circulating IGF-1. Also, IGFBP1–6 and

their fragments have significant intrinsic biological activity independent

of IGF-1 interaction.

Canonical and noncanonical IGF-1 signaling pathways Activation of IGF-1R requires the sequential phosphorylation of three conserved tyrosine residues within the activation loop of the catalytic domain. From these phosphorylated motifs, tyrosine 950 contained in an NPXY motif provides a docking site for the recruitment of adaptor proteins, such as insulin receptor substrate-1 (IRS-1) and Shc, as an obligatory step to initiate signaling cascades. Two canonical pathways are activated by IGF-1R in cardiomyocytes – the phosphatidylinositol-3 kinase (PI3K)/Akt pathway and the extracellular signal-regulated kinase (ERK) pathway. Both pathways have been extensively studied, and their involvement in the pro-hypertrophic and pro-survival actions in cardiomyocytes is well established. Interestingly, a noncanonical signaling mechanism for IGF-1R in cardiomyocytes has been described in several recent studies. These studies show that some of the effects of IGF-1 are inhibited by the heterotrimeric Gi protein blocker Pertussis toxin (PTX) in several cell lines, suggesting that IGF-1R is a dual-activity receptor that triggers tyrosine-kinase-dependent responses as well as Gi-protein-dependent pathways. This duality has been reported in cultured neonatal cardiomyocytes; IGF-1R can activate ERK and Akt but also phospholipase C (PLC), which increases inositol 1,4,5 triphosphate (InsP3; IP3) leading to nuclear Ca2+ signals.

The cardiac effects of IGF-1 are mediated by activation of the plasma membrane IGF-1R, which belongs to the receptor tyrosine kinase (RTK) family. IGF-1R comprises a α2β2 heterotetrameric complex of approximately 400 kDa. Structurally, IGF-1R has two extracellular a-subunits that contain the ligand-binding sites. Each α-subunit couples to one of two membrane-spanning β-subunits, which contain an intracellular domain with intrinsic tyrosine kinase activity. Both subunits of IGF-1R are the product of one single gene, which is synthesized as a 180 kDa precursor. The immature IGF-1R full peptide is further glycosylated, dimerized, and proteolytically processed for assembly of the mature receptor isoforms a and b. In neonatal and adult rat cardiomyocytes, the IGF-1R precursor peptide and the processed α and β receptor subunits have been detected. Binding of IGF-1 to its receptor initiates a complex signaling cascade in cardiomyocytes.

Figure 1. not shown. Canonical and noncanonical signaling pathways activated by insulin-like growth factor 1 (IGF-1) in cardiomyocytes. Binding of IGF-1 to plasma membrane IGF-1 receptor (IGF-1R) leads to receptor autophosphorylation in the intracellular β-subunits. Docking of Grβ2 to the phosphorylated IGF-1Rβ subunits leads to extracellular signal-regulated kinase (ERK) phosphorylation through the Ras/Raf/Mitogen-activated protein kinase (MEK) axis. Phosphorylated ERK can translocate to the nucleus to control gene expression. Phosphorylated β-subunits also provide docking sites for insulin receptor substrate-1 (IRS-1), which mediates phosphatidylinositol-3 kinase (PI3K) activation and Akt phosphorylation. Downstream targets of activated Akt are mechanistic target of rapamycin (mTOR), which suppresses autophagy and promotes protein synthesis by activating S6K and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Akt also phosphorylates and inactivates Bad, thus inhibiting apoptosis. IGF-1R activation also promotes its interaction with a Pertussis-toxin-sensitive heterotrimeric Gi protein, which mediates the activation of phospholipase C (PLC) and hydrolysis of plasma membrane phosphatidylinositol 4,5 biphosphate (PIP2) to form inositol 1,4,5 triphosphate (InsP3; IP3) which activates InsP3 receptors located at the endoplasmin reticulum (ER)/nuclear envelope Ca2+ store, producing nucleoplasmic and cytoplasmic Ca2+ increases. The former is involved in the regulation of specific target genes and the latter promotes mitochondrial Ca2+ uptake, which increases mitochondrial respiration and metabolism, further preventing apoptosis and regulating autophagy. Canonical signaling pathways include the ERK and Akt axes, and are shown in red, whereas the noncanonical G protein pathway is shown in blue. Both pathways interact as Ca2+ contributes to ERK activation and additionally both Akt and ERK can compensate each other’s activation. Abbreviations: MEK, Mitogen-activated protein kinase; mTOR, mechanistic target of rapamycin; 4EBP1, eukaryotic translation initiation factor 4E binding protein 1; PIP2, phosphatidylinositol 4,5 biphosphate.

Figure 2. not shown. Classical versus proposed models of nuclear Ca2+ signaling in cardiomyocytes. The insulin-like growth factor 1 receptor (IGF-1R) can specifically regulate nuclear Ca2+ signaling independently of the role of Ca2+ on excitation–contraction coupling. On the classic model, inositol 1,4,5 triphosphate (InsP3; IP3) produced after IGF-1R activation travels from the peripheral plasma membrane to the nucleus, where it activates InsP3 receptors. In this model InsP3 bypasses its receptors present on the sarcoplasmic reticulum, which would lead to cytosolic Ca2+ signals. The novel model that we propose is based on recent findings, where the IGF-1R signaling complex is present in T-tubule invaginations toward the nucleus. In these compartments, IGF-1R activation leads to locally restricted InsP3 production that allows nuclear Ca2+ signals to regulate gene expression of genes associated with the development of cardiomyocyte hypertrophy. Abbreviations: RyR, ryanodine receptor; ECC, excitation–contraction coupling; PLC, phospholipase C; DHPR, dihydropyridine receptor.

The beneficial roles of IGF-1 in the cardiovascular system largely explain the interest in the development of new IGF-1-based treatments for cardiovascular disease. So far the FDA has approved two drugs for the treatment of IGF-1 deficiency: mecasermin (Increlex1), a human recombinant IGF-1 analog; and mecasermin rinfabate (IPLEX1), a binary protein complex of human recombinant IGF-1 and human recombinant IGBP-3. The safety of a chronic systemic IGF-1 therapy is open to question because it could promote severe adverse effects, such as an increased risk of cancer. To avoid these problems, several researchers have selectively overexpressed IGF-1 and IGF-1R in the heart.

Box 2. Outstanding questionsInsulin-like growth factor 1 (IGF-1) is an old friend of the heart. Despite the well-known protective effects of IGF-1 on cardiac function and the antiapoptotic effects of this peptide, novel evidence opens new questions to this longstanding relationship.

·       How do the multiple signaling pathways triggered by IGF-1 receptor (IGF-1R) interact with each other?

·       What lies further than extracellular signal-regulated kinase (ERK)/Akt/Ca2+ activation toward heart function?

·       Do these signaling pathways regulate cardiac fibroblast or endothelial cell function?

·       Which are the specific downstream signaling pathways of the different pools of IGF-1R and their role in regulating cardiomyocyte survival, hypertrophy, metabolism, proliferation?

·       What drives IGF-1R to such specific subcellular compartments?

·       What is the relevance of the hybrid IGF-1R/insulin receptors on cardiovascular disease?

·       Does a crosstalk exist between insulin receptor and IGF-1R in the heart under physiological and pathological conditions?

·       Is one pathway more beneficial than the other?

·       Will stem cell therapy of cardiac progenitors be able to provide concrete treatment opportunities?

·       Is IGF-1 a key regulator of this outcome?

Abundant evidence supports the key physiological roles of IGF-1 in the heart. In cardiomyocytes, IGF-1 activates multiple downstream signaling pathways for controlling cell death, metabolism, autophagy, differentiation, transcription, and protein synthesis (Figure 1). Of great interest are the findings that the entire IGF-1R complex is strategically located in perinuclear sarcolemmal invaginations that locally control nuclear Ca2+ signaling and transcriptional upregulation (Figure 2). This novel evidence changesmthe classical paradigm of IGF-1 signaling and adds a new level of complexity that may be relevant for other signaling receptors in the heart: interorganelle communication between plasma membrane invaginations and the nucleus.
The strategic localization of IGF-1R in these structures and the association with heterotrimeric G proteins may explain the differences in the phenotypic response induced by IGF-1 and others agonists, like endothelin-1 and angiotensin II, that also signal through intracellular Ca2+. By activating a noncanonical, selective mechanism of nuclear Ca2+ release, IGF-1 can regulate the expression of a specific set of cardiac genes via the generation of a particular signal-encoding pattern, leading to adaptive cardiac hypertrophy, antiapoptotic effects, and metabolic adaptation.

Pulmonary Hypertension in Heart Failure with Preserved Ejection Fraction – any Pathophysiological Role of Mitral Regurgitation

Marco Guazzi
http://dx.doi.org:/10.1016/j.jacc.2009.04.088

read with interest the study by Lam et al. (1) as an important contribution to the pathophysiological and clinical impact of pulmonary hypertension (PH) in hypertensive patients with heart failure and preserved left ventricular ejection fraction (HFpEF). Recent guidelines on arterial PH recognize HFpEF as a growing cause of left-sided PH, but a definitive appreciation of its true prevalence and prognostic relevance is lacking. The present study provides some new important information on this subject.

It is noteworthy that HFpEF was associated, in a high rate of cases (83%), with a typical hemodynamic pattern of precapillary PH, and a strong correlation was found between pulmonary artery systolic pressure and pulmonary capillary wedge pressure. Most important, pulmonary artery systolic pressure, rather than other echocardiography-derived measures of diastolic dysfunction, was the only significant multivariate predictor of mortality, a finding that was confirmed even when combined comorbid diseases potentially contributing to PH development, such as chronic obstructive pulmonary disease, were taken into account.

In patients with systolic heart failure, a major determinant of PH development is mitral regurgitation. Whether mitral regurgitation could be a putative factor in the pathogenesis of PH in HFpEF patients remains an open and intriguing question.

Accordingly, it would be of interest if the authors could provide some details on how many HFpEF patients exhibited mitral regurgitation, especially in comparison with control hypertensive patients without HFpEF.

Lam CSP, Roger VL, Rodeheffer RJ, Borlaug BA, Enders FT, Redfield MM. Pulmonary hypertension in heart failure with preserved ejection fraction: a community-based study. J Am Coll Cardiol 2009; 53:1119–23.

Midregion Prohormone Adrenomedullin and Prognosis in Patients Presenting with Acute Dyspnea Results from the BACH (Biomarkers in Acute Heart Failure) Trial

Alan Maisel, MD, Christian Mueller, Richard M. Nowak,W. Frank Peacock, et al.
J Am Coll Cardiol 2011; 58(10):1057–67
http://dx.doi.org:/10.1016/j.jacc.2011.06.006

Objectives The aim of this study was to determine the prognostic utility of midregion proadrenomedullin (MR-proADM) in all patients, cardiac and noncardiac, presenting with acute shortness of breath.
Background
The recently published BACH (Biomarkers in Acute Heart Failure) study demonstrated that MR-proADM had superior accuracy for predicting 90-day mortality compared with B-type natriuretic peptide (area under the curve: 0.674 vs. 0.606, respectively, p < 0.001) in acute heart failure.
Methods The BACH trial was a prospective, 15-center, international study of 1,641 patients presenting to the emergency department with dyspnea. Using this dataset, the prognostic accuracy of MR-proADM was evaluated in all patients enrolled for predicting 90-day mortality with respect to other biomarkers, the added value in addition to clinical variables, as well as the added value of additional measurements during hospital admission.
Results Compared with B-type natriuretic peptide or troponin, MR-proADM was superior for predicting 90-day all-cause mortality in patients presenting with acute dyspnea (c index = 0.755, p < 0.0001). Furthermore, MR-proADM added significantly to all clinical variables (all adjusted hazard ratios: HR=3.28), and it was also superior to all other biomarkers. MRproADM added significantly to the best clinical model (bootstrap-corrected c index increase: 0.775 to 0.807; adjusted standardized hazard ratio: 2.59; 95% confidence interval: 1.91 to 3.50; p < 0.0001). Within the model, MR-proADM was the biggest contributor to the predictive performance, with a net reclassification improvement of 8.9%. Serial evaluation of MR-proADM performed in patients admitted provided a significant added value compared with a model with admission values only (p< 0.0005). More than one-third of patients originally at high risk could be identified by the biomarker evaluation at discharge as low-risk patients. Conclusions MR-proADM identifies patients with high 90-day mortality and adds prognostic value to natriuretic peptides in patients presenting with acute shortness of breath. Serial measurement of this biomarker may also prove useful for monitoring, although further studies will be required. (Biomarkers in Acute Heart Failure [BACH]; NCT00537628)

Invasive Hemodynamic Characterization of Heart Failure with Preserved Ejection Fraction

Mads J. Andersen, Barry A. Borlaug
Heart Failure Clin 10 (2014) 435–444
http://dx.doi.org/10.1016/j.hfc.2014.03.001

KEY POINTS

  • Invasive hemodynamic assessment in heart failure with preserved ejection fraction (HFpEF) was originally a primary research tool to advance the understanding of the pathophysiology of HFpEF.
  • The role of invasive hemodynamic assessment in HFpEF is expanding to the diagnostic arena where invasive assessment offers a robust, sensitive, and specific way to diagnose or exclude HFpEF in patients with unexplained dyspnea and normal ejection fraction.
  • In future years, invasive hemodynamic profiling may more rigorously phenotype patients to individualized therapy and, potentially, deliver novel device-based structural interventions.

The circulatory system serves to deliver substrates to the body via the bloodstream while removing the byproducts of cellular metabolism. Hemodynamics broadly refers to the study of the forces involved in the circulation of blood, which are governed by to the physical properties of the heart and vasculature and their dynamic regulation by the autonomic nervous system.

Afterload represents the forces opposing ventricular ejection and can be quantified by systolic left ventricular (LV) wall stress and aortic input impedance or its individual components (resistance, compliance, characteristic impedance). Wall stress is inconvenient because it depends on heart size and geometry, whereas impedance is cumbersome because it is a frequency-domain parameter that cannot be easily coupled with time-domain measures of ventricular function. Effective arterial elastance (Ea), defined by the ratio of LV end-systolic pressure (ESP) to stroke volume, provides a robust measure of total arterial load. Ea is not a directly measured parameter but, instead, a net or lumped stiffness of the vasculature that incorporates both mean and oscillatory components of afterload (Fig. 1). Preload reflects the degree of myofiber stretch before the onset of contraction, which, in turn, dictates the force and velocity of contraction according to the Frank-Starling principle. In everyday practice, preload is often conceptualized as equivalent to LV filling pressures. However, in fact, preload is most accurately reflected by the LV volume at end-diastole volume (EDV). Filling pressures are related to EDV by the LV diastolic chamber stiffness, which differs in healthy volunteers and subjects with HFpEF.

Fig. 1. Not shown. Ventricular-arterial coupling in the pressure-volume plane. Pressure volume loop at steady state is shown in dark black. The area subtended by the loop (shaded) represents the stroke work. Stroke volume is the difference between end-diastolic volume (EDV) and end-systolic volume (ESV). Ea is defined by the negative slope connecting the ESP and ESV coordinates with EDV and pressure = 0. With acute preload reduction (dotted line loops) there is progressive reduction in EDV, ESV, and ESP. The linear slope of the endsystolic pressure volume relationship (ESPVR) is LV end-systolic elastance (Ees). The curvilinear slope of the end diastolic pressure–volume relationship (EDVPR) is derived by fitting pressure volume coordinates measured during diastasis to the equation shown. The exponential power or stiffness constant (b) obtained is a measure of LV diastolic stiffness. (Adapted from Borlaug BA, Kass DA. Invasive hemodynamic assessment in heart failure. Heart Fail Clin 2009;5(2):217–28; with permission.)

Fig. 3. Not shown. Left ventricular diastolic reserve in HFpEF. In the normal healthy adult, the rate of LV pressure decay during isovolumic contraction (t) is rapid and increases markedly during exercise in association with a reduction in LVmin, allowing for suction of blood into the LV, with no increase in left atrial pressure or LV end-diastolic pressure (LVEDP) despite an increase in LV end-diastolic volume and marked shortening of the cycle length. In HFpEF, relaxation is prolonged at baseline (increased t) with inadequate hastening (shortening of t) during exercise, contributing to an inability to reduce LVmin and, consequently, a complete lack of suction effects. LV filling then completely depends on left atrial hypertension, which develops in tandem with marked elevation in LVEDP. (Data from Borlaug BA, Jaber WA, Ommen SR, et al. Diastolic relaxation and compliance reserve during dynamic exercise in heart failure with preserved ejection fraction. Heart 2011;97(12):964–9.)

Fig. 4. Preload and filling pressures in HFpEF. (A) Cumulative distribution plot shows that acute changes in stroke volume with nitroprusside infusion are lower in HFpEF (black) compared with HFrEF (red). Because afterload (Ea) is lowered, any acute reduction in SV must be related to reduction in preload volume (EDV) and nearly 40% of HFpEF patients experienced stroke volume reduction with nitroprusside, despite high filling pressures (PCWP 20–25 mm Hg), indicating increased reliance on high pressures to achieve adequate EDV. *p<0.0001 compared with HFrEF. (B) LVEDP in a healthy adult (blue) and in a HFpEF patient with increased LV diastolic stiffness (green). At the same preload (EDV), pressure is more than twofold higher in HFpEF. In contrast, at the same LV diastolic pressure (15 mm Hg), LV volume is much lower in HFpEF, indicating decreased LV diastolic capacitance. V15, volume at end-diastolic pressure = 15 mm Hg; LVEDP. (Adapted from Schwartzenberg S, Redfield MM, From AM, et al. Effects of vasodilation in heart failure with preserved or reduced ejection fraction implications of distinct pathophysiologies on response to therapy. J Am Coll Cardiol 2012;59(5):442–51; with permission.)

Updated Clinical Classification of Pulmonary Hypertension

Gérald Simonneau, Ivan M. Robbins, Maurice Beghetti, et al.
J Am Coll of Cardiol   2009; 54(1), Suppl S
http://dx.doi.org:/10.1016/j.jacc.2009.04.012

The aim of a clinical classification of pulmonary hypertension (PH) is to group together different manifestations of disease sharing similarities in pathophysiologic mechanisms, clinical presentation, and therapeutic approaches. In 2003, during the 3rd World Symposium on Pulmonary Hypertension, the clinical classification of PH initially adopted in 1998 during the 2nd World Symposium was slightly modified. During the 4th World Symposium held in 2008, it was decided to maintain the general architecture and philosophy of the previous clinical classifications. The modifications adopted during this meeting principally concern Group 1, pulmonary arterial hypertension (PAH). This subgroup includes patients with PAH with a family history or patients with idiopathic PAH with germline mutations (e.g., bone morphogenetic protein receptor-2, activin receptor-like kinase type 1, and endoglin). In the new classification, schistosomiasis and chronic hemolytic anemia appear as separate entities in the subgroup of PAH associated with identified diseases. Finally, it was decided to place pulmonary venoocclusive disease and pulmonary capillary hemangiomatosis in a separate group, distinct from but very close to Group 1 (now called Group 1=). Thus, Group 1 of PAH is now more homogeneous. (J Am Coll Cardiol 2009; 54: S43–54)
Updated Evidence-Based Treatment Algorithm in Pulmonary Arterial Hypertension

Robyn J. Barst,  J. Simon R. Gibbs, Hossein A. Ghofrani, et al.
J Am Coll Cardiol 2009; 54(1), Suppl S,

Uncontrolled and controlled clinical trials with different compounds and procedures are reviewed to define the risk benefit profiles for therapeutic options in pulmonary arterial hypertension (PAH). A grading system for the level of evidence of treatments based on the controlled clinical trials performed with each compound is used to propose an evidence-based treatment algorithm. The algorithm includes drugs approved by regulatory agencies for the treatment of PAH and/or drugs available for other indications. The different treatments have been evaluated mainly in idiopathic PAH, heritable PAH, and in PAH associated with the scleroderma spectrum of diseases or with anorexigen use. Extrapolation of these recommendations to other PAH subgroups should be done with caution. Oral anticoagulation is proposed for most patients; diuretic treatment and supplemental oxygen are indicated in cases of fluid retention and hypoxemia, respectively. High doses of calcium-channel blockers are indicated only in the minority of patients who respond to acute vasoreactivity testing. Nonresponders to acute vasoreactivity testing or responders who remain in World Health Organization (WHO) functional class III, should be considered candidates for treatment with either an oral phosphodiesterase-5 inhibitor or an oral endothelin-receptor antagonist. Continuous intravenous administration of epoprostenol remains the treatment of choice in WHO functional class IV patients. Combination therapy is recommended for patients treated with PAH monotherapy who remain in WHO functional class III. Atrial septostomy and lung transplantation are indicated for refractory patients or where medical treatment is unavailable. (J Am Coll Cardiol 2009;54:S78–84)

Inhibition and down-regulation of gene transcription and guanylyl cyclase activity of NPRA by angiotensin II involving protein kinase C

Kiran K. Arise, Kailash N. Pandey
Biochem and Biophys Res Commun 349 (2006) 131–135
http://dx.doi.org:/10.1016/j.bbrc.2006.08.003

The objective of this study was to investigate the role of protein kinase C (PKC) in the angiotensin II (Ang II)-dependent repression of Npr1 (coding for natriuretic peptide receptor-A, NPRA) gene transcription. Mouse mesangial cells (MMCs) were transfected with Npr1 gene promoter-luciferase construct and treated with Ang II and PKC agonist or antagonist. The results showed that the treatment of MMCs with 10 nM Ang II produced a 60% reduction in the promoter activity of Npr1 gene. MMCs treated with 10 nM Ang II exhibited 55% reduction in NPRA mRNA levels, and subsequent stimulation with 100 nM ANP resulted in 50% reduction in guanylyl cyclase (GC) activity. Furthermore, the treatment of MMCs with Ang II in the presence of PKC agonist phorbol ester (100 nM) produced an almost 75% reduction in NPRA mRNA and 70% reduction in the intracellular accumulation of cGMP levels. PKC antagonist staurosporine completely reversed the effect of Ang II and phorbol ester. This is the first report to demonstrate that ANG II-dependent transcriptional repression of Npr1 gene promoter activity and down-regulation of GC activity of translated protein, NPRA is regulated by PKC pathways.

Transcriptional regulation of guanylyl cyclase/natriuretic peptide receptor-A gene

Prerna Kumar, Kiran K. Arise, Kailash N. Pandey
peptides 27 (2006) 1762–1769
http://dx.doi.org:/10.1016/j.peptides.2006.01.004

Activation of natriuretic peptide receptor-A (NPRA) produces the second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. In the present study, we have examined the role of trans-acting factor Ets-1 in transcriptional regulation of Npr1 gene (coding for NPRA).Using deletional analysis of the Npr1 promoter, we have defined a 400 base pair (bp) region as the core promoter, which contains consensus binding sites for transcription factors including: Ets-1, Lyf-1, and GATA-1/2. Over-expression of Ets-1 in mouse mesangial cells (MMCs) enhanced Npr1 gene transcription by 12-fold. However, overexpression of GATA-1 or Lyf-1 repressed Npr1 basal promoter activity by 50% and 80%, respectively. The constructs having a mutant Ets-1 binding site or lacking this site failed to respond to Ets-1 activation of Npr1 gene transcription. Collectively, the present results demonstrate that Ets-1 greatly stimulates Npr1 gene promoter activity, implicating its critical role in the regulation and function of NPRA at the molecular level.

Several agents that are known to upregulate Ets-1 transcription, include RA, TNF-alpha, VEGF, and TPA. Ets-1 is upregulated at exposure to agonists such as serum in vitro and is expressed in injured vasculature. MAPK-mediated phosphorylation positively regulates the transcriptional activation functions of Ets-1 by recruiting CBP/p300. Not much is known about Ets-1 expression or regulation in mesangial cells. A temporal increase of mesangial cell Ets-1 expression has been reported which correlates with mesangial cell activation
in mesangioproliferative glomerulonephritis suggesting involvement of PDGF-B. There might be a possibility that during glomerulonephritis increased Ets-1 expression upregulates Npr1 gene as a protective mechanism. Npr1 gene has been shown to negatively regulate mitogen-activated protein kinase and proliferation of mesangial cells.

In conclusion, our results demonstrate that the precise control of Npr1 gene transcriptional activity is achieved through a synergy of activators and repressors in which Ets-1 plays an integral role as a transcriptional activator. Comparatively, Lyf-1 and GATA-1 act as repressors, inhibiting and regulating the transcriptional activity of Npr1 gene promoter. The present findings suggest that Ets-1 plays a critical role in enhancing Npr1 gene transcription and may have an important influence in hypertension and cardiovascular homeostasis at the molecular level.

Krüppel-like transcription factor 11 (KLF11) overexpression inhibits cardiac hypertrophy and fibrosis in mice

Yue Zheng, Ye Kong, Feng Li
Biochem and Biophys Res Commun 443 (2014) 683–688
http://dx.doi.org/10.1016/j.bbrc.2013.12.024

The Krüppel-like factors (KLFs) belong to a subclass of Cys2/His2 zinc-finger DNA-binding proteins. The KLF family member KLF11 is originally identified as a transforming growth factor b (TGF-b)-inducible gene and is one of the most studied in this family. KLF11 is expressed ubiquitously and participates  in diabetes and regulates hepatic lipid metabolism. However, the role of KLF11 in cardiovascular system is largely unknown. Here in this study, we reported that KLF11 expression is down-regulated in failing human hearts and hypertrophic murine hearts. To evaluate the roles of KLF11 in cardiac hypertrophy, we generated cardiac-specific KLF11 transgenic mice. KLF11 transgenic mice do not show any difference from their littermates at baseline. However, cardiac-specific KLF11 overexpression protects mice from TAC-induced cardiac hypertrophy, with reduced radios of heart weight (HW)/body weight (BW), lung weight/BW and HW/tibia length, decreased left ventricular wall thickness and increased fractional shortening. We also observe lower expression of hypertrophic fetal genes in TAC-challenged KLF11 transgenic mice compared with WT mice. In addition, KLF11 reduces cardiac fibrosis in mice underwent hypertrophy. The expression of fibrosis markers are also down-regulated when KLF11 is overexpressed in TAC-challenged mice. Taken together, our findings identify a novel anti-hypertrophic and anti-fibrotic role of KLF11, and KLF11 activator may serve as candidate drug for heart failure patients.

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Biomarker Guided Therapy

Writer and Curator: Larry H. Bernstein, MD, FCAP

Novel serum protein biomarker panel revealed by mass spectrometry and its prognostic value in breast cancer

Liping Chung, K Moore, L Phillips, FM Boyle, DJ Marsh and RC Baxter
Breast Cancer Research 2014, 16:R63
http://breast-cancer-research.com/content/16/3/R63

Introduction: Serum profiling using proteomic techniques has great potential to detect biomarkers that might improve diagnosis and predict outcome for breast cancer patients (BC). This study used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) to identify differentially expressed  proteins in sera from BC and healthy volunteers (HV), with the
goal  of developing a new prognostic biomarker panel.
Methods: Training set serum samples from 99 BC and 51 HV subjects were applied to four adsorptive chip surfaces (anion-exchange, cation-exchange, hydrophobic, and metal affinity) and analyzed by time-of-flight MS. For validation, 100 independent BC serum samples and 70 HV samples were analyzed similarly. Cluster analysis of protein spectra was performed to identify protein patterns related to BC and HV groups. Univariate and multivariate statistical analyses were used to develop a protein panel to distinguish breast cancer sera from healthy sera, and its prognostic potential was evaluated.
Results: From 51 protein peaks that were significantly up- or downregulated in BC patients by univariate analysis, binary logistic regression yielded five protein peaks that together classified BC and HV with a receiver operating characteristic (ROC) area-under-the-curve value of 0.961. Validation on an independent patient cohort confirmed the five-protein parameter (ROC value 0.939). The five-protein parameter showed positive association with large tumor size (P = 0.018) and lymph node involvement (P = 0.016). By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, immunoprecipitation and western blotting the proteins were identified as a fragment of apolipoprotein H (ApoH), ApoCI, complement C3a, transthyretin, and ApoAI. Kaplan-Meier analysis on 181 subjects after median follow-up of >5 years demonstrated that the panel significantly predicted disease-free survival (P = 0.005), its efficacy apparently greater in women with estrogen receptor (ER)-negative tumors (n = 50, P = 0.003) compared to ER-positive (n = 131, P = 0.161), although the influence of ER status needs to be confirmed after longer follow-up.
Conclusions: Protein mass profiling by MS has revealed five serum proteins which, in combination, can distinguish between serum from women with breast cancer and healthy control subjects with high sensitivity and specificity. The five-protein panel significantly predicts recurrence-free survival in women with ER-negative tumors and may have value in the management of these patients.

Variants of uncertain significance in BRCA: a harbinger of ethical and policy issues to come?

Jae Yeon Cheon, Jessica Mozersky and Robert Cook-Deegan
Genome Medicine 2014, 6:121
http://genomemedicine.com/content/6/12/121

After two decades of genetic testing and research, the BRCA1 and BRCA2 genes are two of the most well-characterized genes in the human genome. As a result, variants of uncertain significance (VUS; also called variants of unknown significance) are reported less frequently than for genes that have been less thoroughly studied. However, VUS continue to be uncovered, even for BRCA1/2. The increasing use of multi-gene panels and whole-genome and whole-exome sequencing will lead to higher rates of VUS detection because more genes are being tested, and most genomic loci have been far less intensively characterized than BRCA1/2. In this article, we draw attention to ethical and policy-related issues that will emerge. Experience garnered from BRCA1/2 testing is a useful introduction to the challenges of detecting VUS in other genetic testing contexts, while features unique to BRCA1/2 suggest key differences between the BRCA experience and the current challenges of multi-gene panels in clinical care. We propose lines of research and policy development, emphasizing the importance of pooling data into a centralized open-access database for the storage of gene variants to improve VUS interpretation. In addition, establishing ethical norms and regulated practices for sharing and curating data, analytical algorithms, interpretive frameworks and patient re-contact are important policy areas.

The Significance of Normal Pretreatment Levels of CA125 (<35 U/mL) in Epithelial Ovarian Carcinoma

Joseph Menczer,  Erez Ben-Shem,  Abraham Golan, and Tally Levy
Rambam Maimonides Med J 2015;6 (1):e0005. http://dx.doi.org:/10.5041/RMMJ.10180

Objective: To assess the association between normal CA125 levels at diagnosis of epithelial ovarian carcinoma (EOC) with prognostic factors and with outcome.
Methods: The study group consisted of histologically confirmed EOC patients with normal pretreatment CA125 levels, and the controls consisted of EOC patients with elevated (≥35 U/mL) pretreatment CA125 levels, diagnosed and treated between 1995 and 2112. Study and control group patients fulfilled the following criteria: 1) their pretreatment CA125 levels were assessed; 2) they had full standard primary treatment, i.e. cytoreductive surgery and cisplatin-based chemotherapy; and 3) they were followed every 2–4 months during the first two years and every 4–6 months thereafter.
Results: Of 114 EOC patients who fulfilled the inclusion criteria, 22 (19.3%) had normal pretreatment CA125 levels. The control group consisted of the remaining 92 patients with ≥35 U/mL serum CA125 levels pretreatment. The proportion of patients with early-stage and low-grade disease, with optimal cytoreduction, and with platin-sensitive tumors was significantly higher in the study group than in the control group. The progression-free survival (PFS) and overall survival (OS) were significantly higher in the study group than in the control group on univariate analysis but not on multivariate analysis.

Higher gene expression variability in the more aggressive subtype of chronic lymphocytic leukemia

Simone Ecker, Vera Pancaldi, Daniel Rico and Alfonso Valencia
Genome Medicine (2015) 7:8 http://dx.doi.org:/10.1186/s13073-014-0125-z

Background: Chronic lymphocytic leukemia (CLL) presents two subtypes which have drastically different clinical outcomes, IgVH mutated (M-CLL) and IgVH unmutated (U-CLL). So far, these two subtypes are not associated to clear differences in gene expression profiles. Interestingly, recent results have highlighted important roles for heterogeneity, both at the genetic and at the epigenetic level in CLL progression.
Methods: We analyzed gene expression data of two large cohorts of CLL patients and quantified expression variability across individuals to investigate differences between the two subtypes using different measures and statistical tests. Functional significance was explored by pathway enrichment and network analyses. Furthermore, we implemented a random forest approach based on expression variability to classify patients into disease subtypes.
Results: We found that U-CLL, the more aggressive type of the disease, shows significantly increased variability of gene expression across patients and that, overall, genes that show higher variability in the aggressive subtype are related to cell cycle, development and inter-cellular communication. These functions indicate a potential relation between gene expression variability and the faster progression of this CLL subtype. Finally, a classifier based on gene expression variability was able to correctly predict the disease subtype of CLL patients.
Conclusions: There are strong relations between gene expression variability and disease subtype linking significantly increased expression variability to phenotypes such as aggressiveness and resistance to therapy in CLL.

The Emerging Roles of Thyroglobulin

Yuqian Luo, Yuko Ishido, Naoki Hiroi, Norihisa Ishii, and Koichi Suzuki
Advances in Endocrinology 2014, Article ID 189194, 7 pages http://dx.doi.org/10.1155/2014/189194

Thyroglobulin (Tg), the most important and abundant protein in thyroid follicles, is well known for its essential role in thyroid hormone synthesis. In addition to its conventional role as the precursor of thyroid hormones, we have uncovered a novel function of Tg as an endogenous regulator of follicular function over the past decade. The newly discovered negative feedback effect of Tg on follicular function observed in the rat and human thyroid provides an alternative explanation for the observation of follicle heterogeneity. Given the essential role of the regulatory effects of Tg, we consider that dysregulation of normal Tg function is associated with multiple human thyroid diseases including autoimmune thyroid disease and thyroid cancer. Additionally, extrathyroid Tg may serve a regulatory function in other organs. Further exploration of Tg action, especially at the molecular level, is needed to obtain a better understanding of both the physiological and pathological roles of Tg.

The GUIDE-IT trial will help doctors find a new standard of care for heart failure.

Heart failure affects more than 25 million people worldwide, including 5.8 million in the United States and 6.9 million in Europe. About one to two percent of adults in developed countries have been diagnosed with heart failure; this increases to more than 10 percent in people over age 70. Moreover, heart failure accounts for more than 17 percent of Medicare spending and about 5 percent of total US healthcare spending. The cost to society in the US is about 30 billion dollars a year—and rising.

For people hospitalized due to heart failure, the outlook isn’t encouraging. Following discharge, one in four patients is likely to be back in the hospital in less than a month. With every acute heart failure event that requires readmission, the chances of dying from the disease increase.

Heart failure occurs when the heart is unable to fill with or pump sufficient blood to meet the needs of the body. Some heart failure symptoms—shortness of breath, fatigue and fluid buildup—which are present in other health problems. Heart failure may develop from coronary artery disease, high blood pressure, cardiomyopathy, heart valve disease, arrhythmias, viral or bacterial infections, and congenital heart defects. As a consequence, these patients often have additional diseases (comorbidities) and managing heart failure can be extremely challenging.

There have been no new drugs for heart failure in more than a decade. The last breakthrough was cardiac resynchronization therapy, a device and not a drug. The goals of therapy are to treat heart failure’s underlying causes, reduce symptoms, improve the patient’s quality of life and keep the disease from getting worse.

More than a pump

The heart isn’t just a muscle pumping blood through the body. It is also an endocrine gland that secretes peptides and hormones. When the heart is failing, its stressed cells release larger amounts of substances known as natriuretic peptides, including N-terminal prohormone brain natriuretic peptide, or NT-proBNP.

Roche’s NT-proBNP test measures the levels of this peptide and helps doctors to determine whether patients are suffering from heart failure and to assess their prognosis. Most recently, NT-proBNP has also been shown to help physicians guide and adjust the patient’s drug therapy. The objective of the pivotal GUIDE-IT trial is to demonstrate the efficacy and safety of NT-proBNP guided heart failure therapy.

Sponsored by the National Institutes of Health (NIH), the GUIDE-IT trial will help doctors answer important questions about NT-proBNP’s impact on medical care. About 1100 patients are enrolled in this robustly powered, randomized controlled trial comparing NT-proBNP guided therapy on top of standard care versus standard care alone in high-risk heart failure patients. Its primary endpoint is time to cardiovascular death or first heart failure hospitalization.

With the NT-proBNP biomarker, doctors can create personalized treatment plans for patients to substantially reduce mortality and morbidity. It can be viewed as a companion diagnostic that works with all the drugs recommended by the major guidelines.

Finding new answers

GUIDE-IT will last five years and involve approximately 45 trial sites in the United States. The first group of patients will be enrolled by the end of 2012.

“We need to take a more strategic approach if we are going to meet the AHA/ASA’s 2020 goal of reducing heart failure hospitalizations by 20 percent,” Dr. O’Connor, Chief of the Division of Cardiovascular Medicine at Duke Heart Center in Durham, North Carolina, said at a media briefing held in October at Roche Diagnostics International in Rotkreuz, Switzerland.
The relative and combined ability of: high-sensitivity cardiac troponin T, and N-terminal pro-B-type natriuretic Peptide – to predict cardiovascular events and death in patients with type 2 diabetes.

Hillis GS; Welsh P; Chalmers J; Perkovic V; Chow CK; Li Q; Jun M; Neal B; et al.
http://reference.medscape.com/medline/abstract/24089534?src=wnl_ref_prac_diab

OBJECTIVE Current methods of risk stratification in patients with type 2 diabetes are suboptimal. The current study assesses the ability of N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin T (hs-cTnT) to improve the prediction of cardiovascular events and death in patients with type 2 diabetes.
RESEARCH DESIGN AND METHODS A nested case-cohort study was performed in 3,862 patients who participated in the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial. RESULTS Seven hundred nine (18%) patients experienced a major cardiovascular event (composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke) and 706 (18%) died during a median of 5 years of follow-up. In Cox regression models, adjusting for all established risk predictors, the hazard ratio for cardiovascular events for NT-proBNP was 1.95 per 1 SD increase (95% CI 1.72, 2.20) and the hazard ratio for hs-cTnT was 1.50 per 1 SD increase (95% CI 1.36, 1.65). The hazard ratios for death were 1.97 (95% CI 1.73, 2.24) and 1.52 (95% CI 1.37, 1.67), respectively. The addition of either marker improved 5-year risk classification for cardiovascular events (net reclassification index in continuous model, 39% for NT-proBNP and 46% for hs-cTnT). Likewise, both markers greatly improved the accuracy with which the 5-year risk of death was predicted. The combination of both markers provided optimal risk discrimination.
CONCLUSIONS NT-proBNP and hs-cTnT appear to greatly improve the accuracy with which the risk of cardiovascular events or death can be estimated in patients with type 2 diabetes.

Genetics and Heart Failure: A Concise Guide for the Clinician

Cécile Skrzynia, Jonathan S. Berg, Monte S. Willis and Brian C. Jensen
Current Cardiology Reviews, 2013; 9.

Abstract: The pathogenesis of heart failure involves a complex interaction between genetic and environmental factors. Genetic factors may influence the susceptibility to the underlying etiology of heart failure, the rapidity of disease progression, or the response to pharmacologic therapy. The genetic contribution to heart failure is relatively minor in most multifactorial cases, but more direct and profound in the case of familial dilated cardiomyopathy. Early studies of genetic risk for heart failure focused on polymorphisms in genes integral to the adrenergic and renin-angiotensin-aldosterone system. Some of these variants were found to increase the risk of developing heart failure, and others appeared to affect the therapeutic response to neurohormonal antagonists. Regardless, each variant individually confers a relatively modest increase in risk and likely requires complex interaction with other variants and the environment for heart failure to develop. Dilated cardiomyopathy frequently leads to heart failure, and a genetic etiology increasingly has been recognized in cases previously considered to be “idiopathic”. Up to 50% of dilated cardiomyopathy cases without other cause likely are due to a heritable genetic mutation. Such mutations typically are found in genes encoding sarcomeric proteins and are inherited in an autosomal dominant fashion. In recent years, rapid advances in sequencing technology have improved our ability to diagnose familial dilated cardiomyopathy and those diagnostic tests are available widely. Optimal care for the expanding population of patients with heritable heart failure involves counselors and physicians with specialized training in genetics, but numerous online genetics resources are available to practicing clinicians.

Cardiac Troponin Testing Is Overused after the Rule-In or Rule-Out of Myocardial Infarction

Olaia Rodriguez Fraga, Y Sandoval, SA Love, ZJ McKinney, MAM Murakami, SW Smith, FS Apple
Clinical Chemistry 2015; 61:2 http://dx.doi.org:/10.1373/clinchem.2014.232694

No good studies have systematically evaluated appropriate clinical utilization of cardiac troponin testing in the clinical setting of the rule-in and rule-out of myocardial infarction (MI). Our collective 100-plus years of clinical and laboratory experience suggested that provider test ordering and use of cardiac troponin has been excessive after a diagnosis of MI or no MI has been determined. There is no evidence that supports continuation of cardiac troponin testing after a diagnosis is made.

Number of cTnI results demonstrating excessive orders by diagnosis

Number of cTnI results demonstrating excessive orders by diagnosis

Time and Frequency Domain Analysis of Heart Rate Variability and their orrelations in Diabetes Mellitus
T. Ahamed Seyd, V. I. Thajudin Ahamed, Jeevamma Jacob, Paul Joseph K
Intl J Biolog and Life Sciences 2008; 4(1)

Diabetes mellitus (DM) is frequently characterized by autonomic nervous dysfunction. Analysis of heart rate variability (HRV) has become a popular noninvasive tool for assessing the activities of autonomic nervous system (ANS). In this paper, changes in ANS
activity are quantified by means of frequency and time domain analysis of R-R interval variability. Electrocardiograms (ECG) of 16 patients suffering from DM and of 16 healthy volunteers were recorded. Frequency domain analysis of extracted normal to normal interval (NN interval) data indicates significant difference in very low frequency (VLF) power, low frequency (LF) power and high frequency (HF) power, between the DM patients and control group. Time domain measures, standard deviation of NN interval (SDNN), root mean square of successive NN interval differences (RMSSD), successive NN intervals differing more than 50 ms (NN50 Count), percentage value of NN50 count (pNN50), HRV triangular index and triangular interpolation of NN intervals (TINN) also show significant difference between the DM patients and control group.

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

Power Spectral Density of the RR interval of a 62 year old woman suffering

Power Spectral Density of the RR interval of a 62 year old woman suffering

Power Spectral Density of the RR interval of a 62 year old woman suffering
from diabetes for the last 15 years

HRV analysis has gained much importance in recent years, as a technique employed to explore the activity of ANS, and as an important early marker for identifying different pathological conditions. DM is a disease in which the cardiac autonomic activity is progressively compromised. Our investigation indicates that different time domain and frequency domain measures of HRV would be able to provide valuable information regarding the autonomic dysfunction to DM.

Time domain and frequency domain analysis of the RR interval variability of diabetic and normal subjects shows that there is significant difference in these measures for DM patients with respect to normal subjects. Variation of the HRV parameters indicates changes in ANS activity of DM patients. This can provide valid information regarding autonomic neuropathy in people with diabetes. It may be noted that these methods can detect changes before clinical signs appear. So we can expect that these measures enable early detection and treatment/subsequent management of patients and thus can avoid acute and chronic complications.

Multiparametric diagnostics of cardiomyopathies by microRNA signatures

Christine S. Siegismund & Maria Rohde & Uwe Kühl & Dirk Lassner
Microchim Acta 2014   http://dx.doi.org:/10.1007/s00604-014-1249-y

The diagnosis of cardiomyopathies by endomyocardial biopsy analysis is the gold standard for confirmation of causative reasons but is failing if a sample does not contain the area of interest due to focal pathology. Biopsies are revealing an extract of the current situation of the heart muscle only, and the need for global organ-specific or systemic markers is obvious in order to minimize sampling errors. Global markers like specific gene expression signatures in myocardial tissue may therefore reflect the focal situation or condition of the whole myocardium. Besides gene expression profiles, microRNAs (miRNAs) represent a new group of stable biomarkers that are detectable both in tissue and body fluids. Such miRNAs may serve as cardiological biomarkers to characterize inflammatory processes, to confirm viral infections, and to differentiate various forms of infection.
The predictive power of single miRNAs for diagnosis of complex diseases may be further increased if several distinctly deregulated candidates are combined to form a specific miRNA signature. Diagnostic systems that generate disease related miRNA profiles are based on microarrays, bead-based oligo sorbent assays, or on assays based on real-time polymerase chain reactions and placed on microfluidic cards or nanowell plates. Multiparametric diagnostic systems that can measure differentially expressed miRNAs may become the diagnostic tool of the future due to their predictive value with respect to clinical course, therapeutic decisions, and therapy monitoring. We discuss here specific merits, limitations and the potential of currently available analytical platforms for diagnostics of heart muscle diseases based on miRNA profiling.

Predictive value of plasma galectin-3 levels in heart failure with reduced and preserved ejection fraction

Rudolf A. de Boer, DJA Lok, T Jaarsma, P van der Meer, AA Voors, et al.
Annals Med, 2011; 43: 60–68 http://dx.doi.org:/10.3109/07853890.2010.538080

We studied the prognostic value of base-line galectin-3 in a large HF cohort, with preserved and reduced left ventricular ejection fraction (LVEF), and compared this to other biomarkers.
Methods. We studied 592 HF patients who had been hospitalized for HF and were followed for 18 months. The primary end-point was a composite of all-cause mortality and HF hospitalization.
Results. A doubling of galectin-3 levels was associated with a hazard ratio (HR) of 1.97 (1.62–2.42) for the primary outcome (P= 0.001). After correction for age, gender, BNP, eGFR, and diabetes the HR was 1.38 (1.07–1.78; P= 0.015). Galectin-3 levels were correlated with higher IL -6 and CRP levels (P= 0.002). Changes of galectin-3 levels after 6 months did not add prognostic information to the base-line value (n= 291); however, combining plasma galectin-3 and BNP levels increased prognostic value over either biomarker alone (ROC analysis, P = 0.05). The predictive value of galectin-3 was stronger in patients with preserved LVEF (n= 114) compared to patients with reduced LVEF (P= 0.001).
Conclusions. Galectin-3 is an independent marker for outcome in HF and appears to be particularly useful in HF patients with preserved LVEF.

Criteria for the use of omics-based predictors in clinical trials

Lisa M. McShane, MM Cavenagh, TG Lively, DA Eberhard, et al.
Nature  17 Oct 2013; 502: 317-320. http://dx.doi.org:/10.1038/nature12564

The US National Cancer Institute (NCI), in collaboration with scientists representing multiple areas of expertise relevant to ‘omics’-based test development, has developed a checklist of criteria that can be used to determine the readiness of omics-based tests for guiding patient care in clinical trials. The checklist criteria cover issues relating to specimens, assays, mathematical modelling, clinical trial design, and ethical, legal and regulatory aspects. Funding bodies and journals are encouraged to consider the checklist, which they may find useful for assessing study quality and evidence strength. The checklist will be used to evaluate proposals for NCI-sponsored clinical
trials in which omics tests will be used to guide therapy.

M-Atrial Natriuretic Peptide and Nitroglycerin in a Canine Model of Experimental Acute Hypertensive Heart Failure: Differential Actions of 2 cGMP Activating Therapeutics.

Paul M McKie, Alessandro Cataliotti, Tomoko Ichiki, S Jeson Sangaralingham, Horng H Chen, John C Burnett
J Am Heart Assoc 01/2014; 3(1):e000206. http://dx.doi.org:/10.1161/JAHA.113.000206

Systemic hypertension is a common characteristic in acute heart failure (HF). This increasingly recognized phenotype is commonly associated with renal dysfunction and there is an unmet need for renal enhancing therapies. In a canine model of HF and acute vasoconstrictive hypertension we characterized and compared the cardiorenal actions of M-atrial natriuretic peptide (M-ANP), a novel particulate guanylyl cyclase (pGC) activator, and nitroglycerin, a soluble guanylyl cyclase (sGC) activator.
HF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II infusion. We characterized the cardiorenal and humoral actions prior to, during, and following intravenous M-ANP (n=7), nitroglycerin (n=7), and vehicle (n=7) infusion. Mean arterial pressure (MAP) was reduced by M-ANP (139±4 to 118±3 mm Hg, P<0.05) and nitroglycerin (137±3 to 116±4 mm Hg, P<0.05); similar findings were recorded for pulmonary wedge pressure (PCWP) with M-ANP (12±2 to 6±2 mm Hg, P<0.05) and nitroglycerin (12±1 to 6±1 mm Hg, P<0.05). M-ANP enhanced renal function with significant increases (P<0.05) in glomerular filtration rate (38±4 to 53±5 mL/min), renal blood flow (132±18 to 236±23 mL/min), and natriuresis (11±4 to 689±37 mEq/min) and also inhibited aldosterone activation (32±3 to 23±2 ng/dL, P<0.05), whereas nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation.
Our results advance the differential cardiorenal actions of pGC (M-ANP) and sGC (nitroglycerin) mediated cGMP activation. These distinct renal and aldosterone modulating actions make M-ANP an attractive therapeutic for HF with concomitant hypertension, where renal protection is a key therapeutic goal.

Genome-Wide Association Study of a Heart Failure Related Metabolomic Profile Among African Americans in the Atherosclerosis Risk in Communities (ARIC) Study

Bing Yu, Y Zheng, D Alexander, TA Manolio, A Alonso, JA Nettleton, & E Boerwinkle
Genet Epidemiol 2013; 00:1–6, http://dx.doi.org:/10.1002/gepi.21752

Both the prevalence and incidence of heart failure (HF) are increasing, especially among African Americans, but no large-scale, genome-wide association study (GWAS) of HF-related metabolites has been reported. We sought to identify novel genetic variants that are associated with metabolites previously reported to relate to HF incidence. GWASs of three metabolites identified previously as risk factors for incident HF (pyroglutamine, dihydroxy docosatrienoic acid, and X-11787, being either hydroxy-leucine or hydroxy-isoleucine) were performed in 1,260 African Americans free of HF at the baseline examination of the Atherosclerosis Risk in Communities (ARIC) study. A significant association on chromosome 5q33 (rs10463316, MAF = 0.358, P-value = 1.92 × 10−10) was identified for pyroglutamine. One region on chromosome 2p13 contained a nonsynonymous substitution in N-acetyltransferase 8 (NAT8) was associated with X-11787 (rs13538, MAF = 0.481, P-value = 1.71 × 10−23). The smallest P-value for dihydroxy docosatrienoic acid was rs4006531 on chromosome 8q24 (MAF = 0.400, P-value = 6.98 × 10−7). None of the above SNPs were individually associated with incident HF, but a genetic risk score (GRS) created by summing the most significant risk alleles from each metabolite detected 11% greater risk of HF per allele. In summary, we identified three loci associated with previously reported HF-related metabolites. Further use of metabolomics technology will facilitate replication of these findings in independent samples.

Global Left Atrial Strain Correlates with CHADS2 Risk Score in Patients with Atrial Fibrillation

SK Saha, PL Anderson, G Caracciolo, A Kiotsekoglou, S Wilansky, S Govind, et al.
J Am Soc Echocardiogr 2011; 24(5): 506-512.
http://dx.doi.org:/10.1016/j.echo.2011.02.012

Background: The aim of this cross-sectional study was to explore the association between echocardiographic parameters and CHADS2 score in patients with nonvalvular atrial fibrillation (AF).
Methods: Seventy-seven subjects (36 patients with AF, 41 control subjects) underwent standard twodimensional, Doppler, and speckle-tracking echocardiography to compute regional and global left atrial (LA) strain.
Results: Global longitudinal LA strain was reduced in patients with AF compared with controls (P < .001) and was a predictor of high risk for thromboembolism (CHADS2 score $ 2; odds ratio, 0.86; P = .02). LA strain indexes showed good interobserver and intraobserver variability. In sequential Cox models, the prediction of hospitalization and/or death was improved by addition of global LA strain and indexed LA volume to CHADS2 score (P = .003).
Conclusions: LA strain is a reproducible marker of dynamic LA function and a predictor of stroke risk and cardiovascular outcomes in patients with AF.

Gene Expression and Genetic Variation in Human Atria

Honghuang Lin, EV Dolmatova, MP Morley, KL Lunetta, et al.
Heart Rhythm, HRTHM5533. PII: S1547-5271(13)01226-5
http://dx.doi.org/10.1016/j.hrthm.2013.10.051

Background— The human left and right atria have different susceptibilities to develop atrial fibrillation (AF). However, the molecular events related to structural and functional changes that enhance AF susceptibility are still poorly understood.
Objective— To characterize gene expression and genetic variation in human atria.
Methods— We studied the gene expression profiles and genetic variations in 53 left atrial and 52 right atrial tissue samples collected from the Myocardial Applied Genomics Network (MAGNet) repository. The tissues were collected from heart failure patients undergoing transplantation and from unused organ donor hearts with normal ventricular function. Gene expression was profiled using the Affymetrix GeneChip Human Genome U133A Array. Genetic variation was profiled using the Affymetrix Genome-Wide Human SNP Array 6.0.
Results— We found that 109 genes were differentially expressed between left and right atrial tissues. A total of 187 and 259 significant cis-associations between transcript levels and genetic variants were identified in left and right atrial tissues, respectively. We also found that a SNP at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right atrial tissues. Conclusion— We found a distinct transcriptional profile between the right and left atrium, and extensive cis-associations between atrial transcripts and common genetic variants. Our results implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF.

Atrial Natriuretic Peptide Single Nucleotide Polymorphisms in Patients with Nonfamilial Structural Atrial Fibrillation

Pietro Francia, A Ricotta, A Frattari, R Stanzione, A Modestino, et al.
Clinical Medicine Insights: Cardiology 2013:7 153–159
http://dx.doi.org:/10.4137/CMC.S12239

Background: Atrial natriuretic peptide (ANP) has antihypertrophic and antifibrotic properties that are relevant to AF substrates. The −G664C and rs5065 ANP single nucleotide polymorphisms (SNP) have been described in association with clinical phenotypes, including hypertension and left ventricular hypertrophy. A recent study assessed the association of early AF and rs5065 SNPs in low-risk subjects. In a Caucasian population with moderate-to-high cardiovascular risk profile and structural AF, we conducted a case-control study to assess whether the ANP −G664C and rs5065 SNP associate with nonfamilial structural AF.
Methods: 168 patients with nonfamilial structural AF and 168 age- and sex-matched controls were recruited. The rs5065 and −G664C ANP SNPs were genotyped.
Results: The study population had a moderate-to-high cardiovascular risk profile with 86% having hypertension, 23% diabetes, 26% previous myocardial infarction, and 23% left ventricular systolic dysfunction. Patients with AF had greater left atrial diameter (44 ± 7
vs. 39 ± 5 mm; P , 0.001) and higher plasma NTproANP levels (6240 ± 5317 vs. 3649 ± 2946 pmol/mL; P , 0.01). Odds ratios (ORs)
for rs5065 and −G664C gene variants were 1.1 (95% confidence interval [CI], 0.7–1.8; P = 0.71) and 1.2 (95% CI, 0.3–3.2; P = 0.79), respectively, indicating no association with AF. There were no differences in baseline clinical characteristics among carriers and noncarriers of the −664C and rs5065 minor allele variants.
Conclusions: We report lack of association between the rs5065 and −G664C ANP gene SNPs and AF in a Caucasian population of patients with structural AF. Further studies will clarify whether these or other ANP gene variants affect the risk of different subphenotypes of AF driven by distinct pathophysiological mechanisms.

N-terminal proBNP and mortality in hospitalized patients with heart failure and preserved vs. reduced systolic function: data from the prospective Copenhagen Hospital Heart Failure Study (CHHF)

Kirk, M. Bay, J. Parnerc, K. Krogsgaard, T.M. Herzog, S. Boesgaard, et al.
Eur Journal Heart Failure 6 (2004) 335–341
http://dx.doi.org:/10.1016/j.ejheart.2004.01.002

Preserved systolic function among heart failure patients is a common finding, a fact that has only recently been fully appreciated. The aim of the present study was to examine the value of NT-proBNP to predict mortality in relation to established risk factors among consecutively hospitalised heart failure patients and secondly to characterise patients in relation to preserved and reduced systolic function. Material: At the time of admission 2230 consecutively hospitalised patients had their cardiac status evaluated through determinations of NT-proBNP, echocardiography, clinical examination and medical history. Follow-up was performed 1 year later in all patients. Results: 161 patients fulfilled strict diagnostic criteria for heart failure (HF). In this subgroup of patients 1-year mortality was approximately 30% and significantly higher as compared to the remaining non-heart failure population (approx. 16%). Using univariate analysis left ventricular ejection fraction (LVEF), New York Heart Association classification (NYHA) and plasma levels of NT-proBNP all predicted mortality independently. However, regardless of systolic function, age and NYHA class, risk-stratification was provided by measurements of NT-proBNP. Having measured plasma levels of NT-proBNP, LVEF did not provide any additional prognostic information on mortality among heart failure patients (multivariate analysis).
Conclusion: The results show that independent of LVEF, measurements of NT-proBNP add additional prognostic information. It is concluded that NT-proBNP is a strong predictor of 1-year mortality in consecutively hospitalised patients with heart failure with preserved as well as reduced systolic function.

N-terminal pro-B-type natriuretic peptide and the prediction of primary cardiovascular events: results from 15-year follow-up of WOSCOPS

Paul Welsh, Orla Doolin, Peter Willeit, Chris Packard, Peter Macfarlane, et al.
Eur Heart Journal 2014. http://eurheartj.oxfordjournals.org/

Aims: To test whether N-terminal pro-B-type natriuretic peptide (NT-proBNP) was independently associated with, and improved the prediction of, cardiovascular disease (CVD) in a primary prevention cohort.
Methods and results:  In the West of Scotland Coronary Prevention Study (WOSCOPS), a cohort of middle-aged men with hypercholesterolemia at a moderate risk of CVD, we related the baseline NT-proBNP (geometric mean 28 pg/mL) in 4801 men to the risk of CVD over 15 years during which 1690 experienced CVD events. Taking into account the competing risk of non-CVD death, NT-proBNP was associated with an increased risk of all CVD [HR: 1.17 (95% CI: 1.11–1.23) per standard deviation increase in log NT-proBNP] after adjustment for classical and clinical cardiovascular risk factors plus C-reactive protein. N-terminal pro-B-type natriuretic peptide was more strongly related to the risk of fatal [HR: 1.34 (95% CI: 1.19–1.52)] than non-fatal CVD [HR: 1.17 (95% CI: 1.10–1.24)] (P ¼ 0.022). The addition of NT-proBNP to traditional risk factors improved the C-index (+0.013; P , 0.001). The continuous net reclassification index improved with the addition of NT-proBNP by 19.8% (95% CI: 13.6–25.9%) compared with 9.8% (95% CI: 4.2–15.6%) with the addition of C-reactive protein. N-terminal pro-B-type natriuretic peptide correctly reclassified 14.7% of events, whereas C-reactive protein correctly reclassified 3.4% of events. Results were similar in the 4128 men without evidence of angina, nitrate prescription, minor ECG abnormalities, or prior cerebrovascular disease.
Conclusion: N-terminal pro-B-type natriuretic peptide predicts CVD events in men without clinical evidence of CHD, angina, or history of stroke, and appears related more strongly to the risk for fatal events. N-terminal pro-B-type natriuretic peptide also provides moderate risk discrimination, in excess of that provided by the measurement of C-reactive protein.

Effect of B-type natriuretic peptide-guided treatment of chronic heart failure on total mortality and hospitalization: an individual patient meta-analysis

Richard W. Troughton, Christopher M. Frampton, Hans-Peter Brunner-La Rocca,
Matthias Pfisterer, Luc W.M. Eurlings, Hans Erntell, Hans Persson, et al.
Eur Heart J 2014; 35: 1559–1567 http://dx.doi.org:/10.1093/eurheartj/ehu090

Aims Natriuretic peptide-guided (NP-guided) treatment of heart failure has been tested against standard clinically guided care in multiple studies, but findings have been limited by study size. We sought to perform an individual patient data metaanalysis to evaluate the effect of NP-guided treatment of heart failure on all-cause mortality.
Methods and results
Eligible randomized clinical trials were identified from searches of Medline andEMBASEdatabases and the Cochrane Clinical
Trials Register. The primary pre-specified outcome, all-cause mortality was tested using a Cox proportional hazards regression model that included study of origin, age (< 75 or ≥75 years), and left ventricular ejection fraction (LVEF, ≤45 or .45%) as covariates. Secondary endpoints included heart failure or cardiovascular hospitalization. Of 11 eligible studies, 9 provided individual patient data and 2 aggregate data. For the primary endpoint individual data from 2000 patients were included, 994 randomized to clinically guided care and 1006 to NP-guided care. All-cause mortality was significantly reduced by NP-guided treatment [hazard ratio = 0.62 (0.45–0.86);
P = 0.004] with no heterogeneity between studies or interaction with LVEF. The survival benefit from NP-guided therapy was seen in younger ( <75 years) patients [0.62 (0.45–0.85); P = 0.004] but not older (≥75 years) patients [0.98 (0.75–1.27); P = 0.96]. Hospitalization due to heart failure [0.80 (0.67–0.94); P = 0.009] or cardiovascular disease [0.82 (0.67–0.99); P = 0.048]was significantly lower in NP-guided patients with no heterogeneity between studies and no interaction with age or LVEF.
Conclusion: Natriuretic peptide-guided treatment of heart failure reduces all-cause mortality in patients aged < 75 years and overall reduces heart failure and cardiovascular hospitalization.

Diagnostic and prognostic evaluation of left ventricular systolic heart failure by plasma N-terminal pro-brain natriuretic peptide concentrations in a large sample of the general population

B A Groenning, I Raymond, P R Hildebrandt, J C Nilsson, M Baumann, F Pedersen
Heart 2004;90:297–303. http://dx.doi.org:/10.1136/hrt.2003.026021

Objective: To evaluate N-terminal pro-brain natriuretic peptide (NT-proBNP) as a diagnostic and prognostic marker for systolic heart failure in the general population.
Design: Study participants, randomly selected to be representative of the background population, filled in a heart failure questionnaire and underwent pulse and blood pressure measurements, electrocardiography, echocardiography, and blood sampling and were followed up for a median (range) period of 805 (6021171) days.
Setting: Participants were recruited from four randomly selected general practitioners and were examined in a Copenhagen university hospital.
Patients: 382 women and 290 men in four age groups (50259 (n = 174); 60269 (n = 204); 70279 (n = 174); > 80 years (n = 120)).
Main outcome measures: Value of NT-proBNP in evaluating patients with symptoms of heart failure and impaired left ventricular (LV) systolic function; prognostic value of NT-proBNP for mortality and hospital admissions.
Results: In 38 (5.6%) participants LV ejection fraction (LVEF) was (40%. NT-proBNP identified patients with symptoms of heart failure and LVEF (40% with a sensitivity of 0.92, a specificity of 0.86, positive and negative predictive values of 0.11 and 1.00, and area under the curve of 0.94. NT-proBNP was the strongest independent predictor of mortality (hazard ratio (HR) = 5.70, p = 0.0001), hospital admissions for heart failure (HR = 13.83, p = 0.0001), and other cardiac admissions (HR = 3.69, p = 0.0001). Mortality (26 v 6, p = 0.0003), heart failure admissions (18 v 2, p = 0.0002), and admissions for other cardiac causes (44 v 13, p = 0.0001) were significantly higher in patients with NTproBNP above the study median (32.5 pmol/l). Conclusions: Measurement of NT-proBNP may be useful as a screening tool for systolic heart failure in the general population.

Copeptin—Marker of Acute Myocardial Infarction

Martin Möckel & Julia Searle
Curr Atheroscler Rep 2014; 16:421 http://dx.doi.org:/10.1007/s11883-014-0421-5

The concentration of copeptin, the C-terminal part of pro-arginine vasopressin, has been shown to increase early after acute and severe events. Owing to complementary pathophysiology and kinetics, the unspecific marker copeptin, in combination with highly cardio-specific troponin, has been evaluated as an early-rule-out strategy for acute myocardial infarction in patients presenting with signs and symptoms of acute coronary syndrome. Overall, most studies have reported a negative predictive value between 97 and 100 % for the diagnosis of acute myocardial infarction in low- to intermediate-risk patients with suspected acute coronary syndrome. Additionally, a recent multicenter, randomized process study, where patients who tested negative for copeptin and troponin were discharged from the emergency department, showed that the safety of the new process was comparable to that of the current standard process. Further interventional trials and data from registries are needed to ensure the effectiveness and patient benefit of the new strategy.

The role of copeptin as a diagnostic and prognostic biomarker for risk stratification in the emergency department

Christian H Nickel1, Roland Bingisser and Nils G Morgenthaler
BMC Medicine 2012, 10:7 http://www.biomedcentral.com/1741-7015/10/7

The hypothalamic-pituitary-adrenal axis is activated in response to stress. One of the activated hypothalamic hormones is arginine vasopressin, a hormone involved in hemodynamics and osmoregulation. Copeptin, the C-terminal part of the arginine vasopressin precursor peptide, is a sensitive and stable surrogate marker for arginine vasopressin release. Measurement of copeptin levels has been shown to be useful in a variety of clinical scenarios, particularly as a prognostic marker in patients with acute diseases such as lower respiratory tract infection, heart disease and stroke. The measurement of copeptin levels may provide crucial information for risk stratification in a variety of clinical situations. As such, the emergency department appears to be the ideal setting for its potential use. This review summarizes the recent progress towards determining the prognostic and diagnostic value of copeptin in the emergency department.

Variability of the Transferrin Receptor 2 Gene in AMD

Daniel Wysokinski, Janusz Blasiak, Mariola Dorecka, Marta Kowalska, et al.
Disease Markers 2014, Article ID 507356, 8 pages http://dx.doi.org/10.1155/2014/507356

Oxidative stress is a major factor in the pathogenesis of age-related macular degeneration (AMD). Iron may catalyze the Fenton reaction resulting in overproduction of reactive oxygen species. Transferrin receptor 2 plays a critical role in iron homeostasis and variability in its gene may influence oxidative stress and AMD occurrence. To verify this hypothesis we assessed the association between  polymorphisms of the TFR2 gene and AMD. A total of 493AMDpatients and 171matched controls were genotyped for the two polymorphisms of the TFR2 gene: c.1892C>T (rs2075674) and c.−258+123T>C (rs4434553). We also assessed the modulation of some AMD risk factors by these polymorphisms.The CC and TT genotypes of the c.1892C>T were associated with AMD occurrence but the latter only in obese patients. The other polymorphism was not associated with AMD occurrence, but the CC genotype was correlated with an increasing AMD frequency in subjects with BMI < 26. The TT genotype and the T allele of this polymorphism decreased AMD occurrence in subjects above 72 years, whereas the TC genotype and the C allele increased occurrence of AMD in this group.The c.1892C>T and c.−258+123T>C polymorphisms of the TRF2 gene may be associated with AMD occurrence, either directly or by modulation of risk factors.

Urinary N-Acetyl-beta-D-glucosaminidase as an Early Marker for Acute Kidney Injury in Full-Term Newborns with Neonatal Hyperbilirubinemia

Bangning Cheng, Y Jin, G Liu, Z Chen, H Dai, and M Liu
Disease Markers 2014, Article ID 315843, 6 pages http://dx.doi.org/10.1155/2014/315843

Purpose. To investigate renal function estimated by markers in full-term newborns with hyperbilirubinemia.
Methods. A total of 332 full-term newborns with hyperbilirubinemia and 60 healthy full-term newborns were enrolled. Total serum bilirubin, serum creatinine (Cr), serum blood urea nitrogen (BUN), serum cystatin C (Cys-C), urinary beta-2-microglobulin (𝛽2MG) index, and urinary N-acetyl-beta-D-glucosaminidase (NAG) index were measured before and after treatment. All newborns were divided into three groups according to total serum bilirubin levels: group 1 (221-256), group 2 (256-342), and group 3 (>342). Results. The control group and group 1 did not differ significantly in regard to serum Cr, serum BUN, serum Cys-C, urinary 𝛽2MG index, and urinary NAG index. Urinary NAG index in group 2 was significantly higher than that in control group (𝑃 < 0.001). Between control group and group 3, serum Cys-C, urinary 𝛽2MG index, and urinary NAG index differed significantly. The significant positive correlation between total serum bilirubin and urinary NAG index was found in newborns when total serum bilirubin level was more than 272 𝜇mol/L.
Conclusions. High unconjugated bilirubin could result in acute kidney injury in full-term newborns. Urinary NAG might be the suitable marker for predicting acute kidney injury in full-term newborns with hyperbilirubinemia.

Urinary C-peptide creatinine ratio detects absolute insulin deficiency in Type 2 diabetes.

S V Hope, A G Jones, E Goodchild, M Shepherd, R E J Besser, B Shields, T McDonald, B A Knight, A Hattersley

Department of Geriatrics, Royal Devon and Exeter NHS Foundation Trust; NIHR Exeter Clinical Research Facility, University of Exeter.

Diabetic Medicine (impact factor: 2.9). 05/2013; http://dx.doi.org:/10.1111/dme.12222

Source: PubMed

ABSTRACT AIMS: To determine the prevalence and clinical characteristics of absolute insulin deficiency in long-standing Type 2 diabetes, using a strategy based on home urinary C-peptide creatinine ratio measurement.
METHODS: We assessed the urinary C-peptide creatinine ratios, from urine samples taken at home 2 h after the largest meal of the day, in 191 insulin-treated subjects with Type 2 diabetes (diagnosis age ≥45 years, no insulin in the first year). If the initial urinary C-peptide creatinine ratio was ≤0.2 nmol/mmol (representing absolute insulin deficiency), the assessment was repeated. A standardized mixed-meal tolerance test with 90-min stimulated serum C-peptide measurement was performed in nine subjects with a urinary C-peptide creatinine ratio ≤ 0.2 nmol/mmol (and in nine controls with a urinary C-peptide creatinine ratio >0.2 nmol/mmol) to confirm absolute insulin deficiency.
RESULTS: A total of 2.7% of participants had absolute insulin deficiency confirmed by a mixed-meal tolerance test. They were identified initially using urinary C-peptide creatinine ratio: 11/191 subjects (5.8%) had two consistent urinary C-peptide creatinine ratios ≤ 0.2 nmol/mmol; 9/11 subjects completed a mixed-meal tolerance test and had a median stimulated serum C-peptide of 0.18nmol/l. Five out of nine subjects had stimulated serum C-peptide <0.2 nmol/l and 9/9 subjects with urinary C-peptide creatinine ratio >0.2 had endogenous insulin secretion confirmed by the mixed-meal tolerance test. Compared with subjects with a urinary C-peptide creatinine ratio >0.2 nmol/mmol, those with confirmed absolute insulin deficiency had a shorter time to insulin treatment (median 2.5 vs. 6 years, P=0.005) and lower BMI (25.1 vs. 29.1kg/m(2) , P=0.04). Two out of five patients were glutamic acid decarboxylase autoantibody-positive.
CONCLUSIONS: Absolute insulin deficiency may occur in long-standing Type 2 diabetes, and cannot be reliably predicted by clinical features or autoantibodies. Its recognition should help guide treatment, education and management. The urinary C-peptide creatinine ratio is a practical non-invasive method to aid detection of absolute insulin deficiency, with a urinary C-peptide creatinine ratio > 0.2 nmol/mmol being a reliable indicator of retained endogenous insulin secretion.

Unlocking Biomarkers’ Full Potential

David Daniels, Ph.D.     genengnews  Feb 1, 2013 (Vol. 33, No. 3)

http://www.genengnews.com/gen-articles/unlocking-biomarkers-full-potential/4700/

Biomarker research and development has evolved over the past years from looking for a single marker (e.g., PSA) linked to a disease state to looking for a panel of markers that can capture the heterogeneity inherent in both the disease and the impacted patient population.

That is one of the key messages to be delivered at GTC’s “Biomarkers Summit” next month. Across the board, resources are being focused on the delivery of more precise, quantifiable biomarkers with predictive value in therapeutic decisions and for the prognosis of illness.

“Our focus on biomarker development is the recognition that the new products need to provide cost savings for the already strapped healthcare systems rather than just be cost effective,” shares Paul Billings, M.D., Ph.D., CMO at Life Technologies.

“We have built a new medical sciences group to address the needs of the multiple delivery systems in the world—from the sophisticated medical clinics in the developed world to the nurse-run shanty clinic in the third world. Providing tools for equitable access to quality diagnosis, on assay platforms that can provide care for all patients, is our goal.”

Life Tech’s medical sciences division has been built by acquisition of Pinpoint Genomics, Navigenics, and Compendia, and collaborations with partners such as Ingenuity Systems and CollabRx. The division is focused on taking the tools that have been used in the life science laboratories and providing molecular diagnostic data to the clinic. The intent is to deliver data in a valuable format that can be used by the molecular pathologist or the treating physician.

The division is developing the Pervenio™ Lung RS assay, a 14-gene expression profile that serves as a risk stratifier that uses a weighted algorithm for the expressed biomarkers within the tumor biopsy, a first-of-its-kind prognostic test for lung cancer, the firm reports.

Initially, tests will be offered as a service through Life Tech’s CLIA laboratory. Then, from the performance lessons learned, Life Tech’s will develop a simpler assay platform, with FDA approval, that can be dispersed globally without reduction of the essential content in the biomarker panel. The focus is on the workflow—screening for known mutations using established easy-to-use assay platforms, like RT-PCR. Should the screen not produce useful results, clinicians can search for new mutations via discovery platforms like next-gen sequencing (NGS).

http://www.genengnews.com/Media/images/Article/thumb_Sequenom_LungCartaPanel1722631391.jpg

Sequenom’s LungCarta panel of 214 somatic mutations in 26 tumor suppressors and oncogenes covers highly mutated pathways in lung adenocarcinomas.

At Sequenom, the company provides both the tools (DNA mass spectrometry and reagents) for confirmatory biomarker development as well as serving on the front lines as a diagnostic service provider (CLIA lab). The beauty of DNA mass spec is that it can process multiplexed PCR samples (10–60 loci) in a method that is quantitative when used for profiling tumor biopsies that are either archival or fresh tissue.

Given a tumor sample with multiple somatic mutations, the instrument enables the determination of the homogeneity of the cells, in which case the mutations will have the same allele frequency. Accuracy, as measured by coefficient of variance, is less than 2%. Despite this level of sensitivity, the mass spec can only be used as a confirmatory tool looking for known mutations. Discovery is best done using DNA sequencing. DNA mass spec can also be used to study methylation in tumor samples.

“In the not-too-distant future, we will be looking for mutations in plasma samples rather than biopsies,” predicts Charles Cantor, Ph.D., CSO at Sequenom.

“The key is to look noninvasively for mutations within plasma samples such that we can potentially catch the disease state earlier, rather than after tumor formation. Regardless of the tumor type, this approach will enable us to monitor therapeutic response and metastatic potential noninvasively. DNA mass spec is an ultrasensitive detection product that can detect somatic mutations at levels of 1 per 1,000. This level of sensitivity is critical for the future of plasma screening. NGS technology is not that sensitive.”

Sequenom’s CLIA lab is using automated DNA mass spec to provide three different test protocols: (1) carrier screening for cystic fibrosis looking at more than 100 different mutations, (2) adult macular degeneration progression using an SNP test with 13 loci, and (3) a noninvasive test for Rh compatibility between a mother and her unborn fetus.

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Scientists are using Illumina’s HiSeq system to discover molecular biomarkers that may provide opportunities for early detection of a range of diseases.

Sequenom has also set up an NGS facility within a CLIA lab in San Diego using Illumina’s HiSEQ platform. The NGS platform has been set up for noninvasive aneuploidy detection of maternal plasma (10 cc sample) looking at chromosomes 13, 18, and 21. The lab says it has analyzed more than 40,000 samples this year and is planning to increase that volume up to 100,000 samples per year. Most of these samples come from the U.S., but given the development of a new blood collection tube that allows for 72-hour ambient shipping, the lab is looking to increase the number of samples from outside the U.S.

Drug Development

During drug development, biomarkers function as pharmacodynamic markers to help assess the mechanism of action of a drug candidate, to define the downstream biological pathway, and to determine whether the drug is engaging the target with the anticipated biological effect. Later, biomarkers help determine whether a drug is effective using the tested regime (route of delivery, dosage level, and length of exposure time).

Following early development, the second stage is to use biomarkers to help segment patients for clinical trials. Part of the consideration here is how heterogeneous the disease is; are there homogeneous subsets of patients that will respond differentially to the drug based on different mechanisms of the disease?

“Biomarker research is focused on on- target effects,” says Nick Dracopoli, Ph.D., vp, head of oncology biomarkers at Janssen Research and Development, a J&J company.

“We look at indications and at patients with those indications that are most likely to respond to the drug candidates we’re developing. For oncology biomarkers, germ-line effects are weaker indicators than somatic changes in the tumor. As a consequence, SNP-based, genome-wide association studies are not very useful. It is better to focus on molecular changes within the tumor and define gene expression profiles and epigenetic modifications that correlate with the tumor phenotype. We are increasingly tracking patient immune response, particularly as more immuno-oncology products are moving into the drug development pipeline.”

The number of biomarkers being developed varies from project to project. But it is very clear that to be successful in the clinic, the biomarkers and the assays need to be of low complexity. Of the 10 to 12 companion diagnostics that have been approved by the FDA to date, all measure the status of the drug target (on-target markers). For example, EGFR measures the level of receptor expression; Braf and Kras markers measure the presence of the mutation and translocation in the ALK gene measures gene knockout.

It is important to realize that molecular profiles for first-in-class drugs are not optimal because they are based on only a few patients. Consequently they have weak predictive value overall.

“Aside from that rule of thumb, if you have a greater than 50% response rate for your drug, it is unlikely that you need a biomarker to predict response. Biomarker utility is best for drugs that would have a difficult road to approval, where it is critical to enrich for the subpopulation of responders. For example, Pfizer’s crizontinib was approved for non-small-cell lung patients but is only effective for 5% of all patients. If Pfizer was unable to demonstrate the relationship between activation of the ALK gene and disease, this inhibitor would not have been approved,” says Dr. Dracopoli.

“Drugs that are more broadly active can come to market without a companion diagnostic test. There is always a balance between the predictive values of the biomarker test and the response rates to treatment. That is, we should not treat if the chance of response is only 3–5%, rather than if it were 50% where the patient would want to take the chance if the drug were safe.”

An important take-home message is that mutations are not unique to an indication. So if you find a driver mutation in indications for which the drug has not been approved, you could discover new indications for the drug.

“At the end of the day, this is what cancer is—heterogeneous,” says Dr. Dracopoli. “We’d all love to treat one cancer with one drug and at one dose, but the story is more complex. The future of oncology is around understanding the molecular heterogeneity or underlying molecular pathology of the disease and the diversity of it, and then treat each patient accordingly.”

Clinical Considerations

“Given the complexity of biology,” says Achim Plum, Ph.D., principal consultant, Siemens, “whether is it cancer, metabolic disease, or any other disease state, we have been forced to move away from the idea that a single biomarker can capture the entire ‘story’ or mechanistic view of any disease. Hence newly developed biomarkers will be made up of a panel of markers that serve as a profile. In addition, with the sheer volume of DNA and protein analytics data, the clinic will need to employ software tools and algorithms to help the decision making.”

The task of getting broad profiling technologies that are analytical into a clinical setting and making them routine is difficult but not insurmontable. This will take a collaborative effort, something that Siemens among others are looking to develop. The key is to avoid technology hype and to establish good reliable software to process the data for decision making. “Data is not knowledge, and knowledge is not automatically decision making.”

As an academic, Daniel Chan, Ph.D., has a view of the whole value chain for biomarkers from discovery to development to use in the clinic. Dr. Chan holds the titles of professor in pathology, oncology, radiology, and urology, and is the director of the clinical chemistry division lab at Johns Hopkins Hospital.

Given his perspective from discovery to clinical use, Dr. Chan indicated that from the clinical point of view, “we need more markers.” He oversees the discovery of new biomarkers in his research lab, their validation in his translational research lab, and finally their utility in practice in his clinical chemistry lab. He is a strong advocate for collaboration of biomarker development from discovery to verification and validation to incorporation within the clinical practice.

Beyond the use of biomarkers for patient stratification and correlation between marker and therapeutic choice, as is the focus of the biopharma industry, for the clinic the use of biomarkers is for prevention and early detection. The earlier the detection, the better the outcome. That is, provide the “cure” before you need to initiate treatment.

To be successful in the future of biomarkers, we need to look beyond the biopharma focus and expand the horizon for early detection and monitor therapy later, says Dr. Chan. He describes a roadmap of developing bridges (to bridge the knowledge gaps), gates (decision gates for go/no go decisions as to whether a development path is viable), and partnerships (to collaborate with different points of view) for efficient new biomarker development.

According to Dr. Chan, we must define the intended use of the biomarker, which identifies the specific application and sets up the clinical study and study population to meet the clinical needs. We need to define specific assays to monitor biomarkers that will work within a clinical setting, not a research lab setting that uses disease models (tissue culture cells or small animals) and not real patient samples.

“The days when single markers are sufficient (PSA for prostate cancer or troponin for cardiovascular disease) are behind us. We need to develop a panel of markers or a profile pattern to address patient population heterogeneity and disease complexity that will guide our decision-making process,” remarks Dr. Chan. “Molecular biomarkers are giving way to protein biomarkers,” he adds.

Prevention and early detection will require the use of whole-body scans, so the sampling technology and analytical tools to be developed are critical to realize this goal. Assay ease of use, automation, and analytical performance that is suitable for the clinical lab are fundamental.

“An important future goal for biomarkers,” says Dr. Billings, “is to sample circulating tumor cells or circulating DNA in blood or plasma samples as a noninvasive measure of patient status. A decline in tumor biomarkers during chemotherapy, for example, could reflect the efficacy of the therapy. In contrast, an increase in tumor biomarkers, in a patient who had previously undergone surgery and therapy, might indicate disease recurrence, and is likely to do so before a tumor mass is detectable by imaging methods.”

STAT4 Gene Polymorphisms Are Associated with Susceptibility and ANA Status in Primary Biliary Cirrhosis

Satoru Joshita, T Umemura, M Nakamura, Y Katsuyama, S Shibata, et al.
Disease Markers  2014, Article ID 727393, 8 pages http://dx.doi.org/10.1155/2014/727393

Recent genome-wide association studies suggest that genetic factors contribute to primary biliary cirrhosis (PBC) susceptibility. Although several reports have demonstrated that the interleukin (IL) 12 signaling pathway is involved in PBC pathogenesis, its precise genetic factors have not been fully clarified. Here, we performed an association analysis between IL12A, IL12RB, and signal transducer and activator of transcription 4 (STAT4) genetic variations and susceptibility to PBC. Single nucleotide polymorphisms (SNPs) were genotyped in 395 PBC patients and 458 healthy subjects of Japanese ethnicity and evaluated for associations with PBC susceptibility, anti-nuclear antibody (ANA) status, and anti-mitochondrial antibody (AMA) status. We detected significant associations with PBC susceptibility for several STAT4 SNPs (rs10168266; p = 9.4 × 10−3, rs11889341; p = 1.2 × 10−3, rs7574865; p = 4.0 × 10−4, rs8179673; p = 2.0 × 10−4, and rs10181656; p = 4.2 × 10−5). Three risk alleles (rs7574865; p = 0.040, rs8179673; p = 0.032, and rs10181656; p = 0.031) were associated with ANA status, but not with AMA positivity. Our findings confirm that STAT4 is involved in PBC susceptibility and may play a role in ANA status in the Japanese population.

Serum Omentin-1 as a Disease Activity Marker for Crohn’s Disease

Yan Lu, Li Zhou, L Liu, Yan Feng, Li Lu, X Ren, X Dong, & W Sang
Disease Markers  2014, Article ID 162517, 5 pages   http://dx.doi.org/10.1155/2014/162517

Background and Aim. It remains challenging to determine the inflammatory activity in Crohn’s disease (CD) for lack of specific laboratory markers. Recent studies suggest that serum omentin-1 is associated with inflammatory response. We aimed to assess the potential of serum omentin-1 as a marker of disease activity in CD patients.
Methods. Serum omentin-1 concentrations were determined by enzyme-linked immunosorbent assay (ELISA) in patients with CD (n = 240), functional gastrointestinal disorders (FGDs, n = 120), and healthy controls (HC, n = 60) and evaluated for correlation with disease activity. Expression of omentin-1 in colonic tissues from patients with CD was also analyzed by real-time PCR and Western blotting. Serum omentin-1 levels as an activity index were evaluated using a receiver operating characteristic (ROC) curve.
Results. Serum omentin-1 concentrations were significantly decreased in active CD patients compared with patients in remission, FGDs, and HC (all p < 0.001). Expression of omentin-1 was decreased at mRNA and protein levels in inflamed colonic tissues in active CD than that in noninflamed colonic tissues. Serum omentin-1 levels were negatively correlated with disease activity in CD, better than C-reactive protein (CRP).
Conclusion. Our results indicate that serum and colonic omentin-1 expressions are decreased in active CD patients. The correlation of serum omentin-1 with disease activity in CD is superior to that of CRP. Serum omentin-1 is a potential marker for CD disease activity.
Serum Levels of Resistin, Adiponectin, and Apelin in Gastroesophageal Cancer Patients

Dorota Diakowska, K Markocka-Mdczka, P Szelachowski, and K Grabowski
Disease Markers 2014, Article ID 619649, 8 pages   http://dx.doi.org/10.1155/2014/619649

The aim of the study was the investigation of relationship between cachexia syndrome and serum resistin, adiponectin, and apelin in patients with gastroesophageal cancer (GEC).
Material and Methods. Adipocytokines concentrations were measured in sera of 85 GEC patients and 60 healthy controls. They were also evaluated in tumor tissue and appropriate normal mucosa of 38 operated cancer patients.
Results. Resistin and apelin concentrations were significantly higher in GEC patients than in the controls. The highest resistin levels were found in cachectic patients and in patients with distant metastasis. Serum adiponectin significantly decreased in GEC patients with regional and distant metastasis. Serum apelin was significantly higher in cachectic patients than in the controls. Apelin was positively correlated with hsCRP level. Resistin and apelin levels increased significantly in tumor tissues. Weak positive correlations between adipocytokines levels in serum and in tumor tissue were observed.
Conclusions. Resistin is associated with cachexia and metastasis processes of GEC. Reduction of serum adiponectin reflects adipose tissue wasting in relation to GEC progression. Correlation of apelin with hsCRP can reflect a presumable role of apelin in systemic inflammatory response in esophageal and gastric cancer.

Serum Level of HER-2 Extracellular Domain in Iranian Patients with Breast Cancer: A Follow-up Study

Mehrnoosh Doroudchi, Abdolrasoul Talei, Helmout Modjtahedi, et al.
IJI 2005; 2(4): 191-200

Background: A soluble form of HER-2/neu extracellular domain (sHER-2) is reported to be released in the sera of metastatic breast cancer patients.
Objective: To measure the level of sHER-2 in sera of 115 breast cancer patients. Methods: Serial samples of 27 patients with metastasis, 18 non-metastatic patients, 15 patients in stage 0/I and 14 patients with accompanying benign breast disease were also included in this study.
Results: No significant difference was observed between sHER-2 level in the pre-operative sera of breast cancer patients and that of healthy individuals. Only 8 out of 27 patients whom later developed metastasis showed elevated levels of sHER-2 in their first serum sample. However, a trend of increase in the level of sHER-2 was observed in 14 (51.8%) of 27 metastatic sera before clinical diagnosis of the metastasis. A significant association between sHER-2 positive status and vascular invasion of the tumor was observed (P = 0.02). In addition, significant correlation of sHER-2 level with CEA (highest r = 0.74) and CA 15.3 (highest r = 0.74) tumor marker levels in the serial sera were observed. The mean time from sHER-2 positivity to tumor metastasis was calculated to be 98 days (range = 29-174).  Conclusion: Our results indicate that a relatively high percentage of Iranian patients with breast cancer show an elevated level of sHER-2 in their sera before clinical diagnosis of the tumor metastasis. Therefore, measuring the level of this oncoprotein, not only helps physicians in monitoring the patients during HERCEPTIN therapy, but also can be helpful in choosing more aggressive treatments at the early satges of tumor metastasis.
B-type natriuretic peptide is a biomarker for pulmonary hypertension in preterm infants with bronchopulmonary dysplasia

Alain Cuna, Jegen Kandasamy, Naomi Fineberg, Brian Sims
Research and Reports in Neonatology 2013:3 33–36
http://dx.doi.org/10.2147/RRN.S42236

Background: B-type natriuretic peptide (BNP) is a cardiac biomarker useful in screening for pulmonary hypertension (PH) in adults. It is possible that BNP may also be useful in detecting PH among preterm infants with bronchopulmonary dysplasia (BPD).
Objective: To determine the utility of BNP for identification of PH among preterm infants with BPD.
Methods: We retrospectively identified preterm infants with BPD who underwent screening echocardiography for suspected PH and had serum BNP levels measured within 10 days before or after echocardiography. Eligible infants were classified based on echocardiographic diagnosis of either PH or no PH. Median and interquartile ranges (IQR) of BNP values were compared, and area under the curve (AUC) of receiver operator characteristic (ROC) analysis was used to determine the optimum threshold value for detection of PH.
Results: Twenty-five preterm infants with BPD (mean gestational age 26.5 ± 1.7 weeks, mean birth weight 747 ± 248 g) were identified. The median difference in days between echocardiography and BNP measurement was 1 day (IQR 0–3, range 0–10 days). Based on echocardiography, 16 were diagnosed with PH and nine without PH. No significant difference in terms of gestational age, birth weight, sex, race, or respiratory support was found between the two groups. Median (IQR) BNP values of those with PH were higher than those without PH (413 [212–1178] pg/mL versus 55 [21–84] pg/mL, P , 0.001). AUC of ROC analysis showed that a BNP value of 117 pg/mL had 93.8% sensitivity and 100% specificity for detecting PH.
Conclusion: BNP estimation may be useful for screening of PH in infants with BPD.

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