Posts Tagged ‘Cardiomyopathy’

Familial transthyretin amyloid polyneuropathy

Curator: Larry H. Bernstein, MD, FCAP


UPDATED on 6/3/2020

Treatment of Cardiac Transthyretin Amyloidosis

Emdin M, Aimo A, Rapezzi C, et al.
Treatment of Cardiac Transthyretin Amyloidosis: An Update. Eur Heart J 2019;40:3699-3706.

The following are key points to remember from this update on the treatment of cardiac transthyretin amyloidosis:

  1. Transthyretin (TTR) is a highly conserved protein involved in transportation of thyroxine (T4) and retinol-binding protein. TTR is synthesized mostly by the liver and is rich in beta strands with an intrinsic propensity to aggregate into insoluble amyloid fibers, which deposit within tissue leading to the development of TTR-related amyloidosis (ATTR). ATTR can follow the deposition of either variant TTR (ATTRv, previously known as mutant ATTR) or wild type TTR (ATTRwt).
  2. Cardiac ATTR has a favorable survival rate compared to light chain (AL) amyloidosis, with a median survival of 75 versus 11 months. However, ATTR cardiomyopathy is a progressive disorder but newer therapeutic options include tafamidis (positive phase 3 clinical trial), and possibly patisiran and inotersen.

Inhibition of the Synthesis of Mutated Transthyretin

  1. Liver transplantation removes the source of mutated TTR molecules and prolongs survival, with a 20-year survival of 55.3%. However, tissue accumulation of TTR can continue after liver transplantation because TTR amyloid fibers promote subsequent deposition of ATTRwt. Combined liver–heart transplantation is feasible in younger patients with ATTRv cardiomyopathy and a small series suggests better prognosis than cardiac transplantation.
  2. Inhibition of TTR gene expression: Patisiran is a small interfering RNA blocking the expression of both variant and wt TTR. On the basis of the APOLLO trial, it was approved for therapy of adults with ATTRv-related polyneuropathy both in the United States and European Union. In this trial, patisiran promoted favorable myocardial remodeling based on echocardiographic and N-terminal B-type natriuretic peptide (NT-BNP) changes (this effect was not demonstrated for inotersen) and is still under investigation for tafamidis.
  3. Antisense oligonucleotides inotersen inhibits the production of both variant and wt TTR. Based on the findings of the NEURO-TTR trial, the Food and Drug Administration (FDA) approved this agent for patients with ATTRv-related polyneuropathy. In the NEURO-TTR trial, cardiomyopathy was present in 63%, but the study was not powered to measure effects of inotersen on heart disease. Inotersen can cause thrombocytopenia and must be used cautiously with bleeding risk.

Tetramer Stabilization

  1. Selective stabilizers include tafamidis and AG10. Tafamidis is a benzoxazole and a small molecule that inhibits the dissociation of TTR tetramers by binding the T4-binding sites. The phase ATTR-ACT study showed that when comparing the pooled tafamidis arms (80 and 20 mg) with the placebo arm, tafamidis was associated with lower all-cause mortality than placebo (78 of 264 [29.5%] vs. 76 of 177 [42.9%]; hazard ratio, 0.70; 95% confidence interval, 0.51-0.96) and a lower rate of cardiovascular hospitalizations. Based on the results of the ATTR-ACT trial, it has received Breakthrough Therapy designation from the FDA for treatment of ATTR cardiomyopathy.
  2. Nonselective agents: Diflunisal, a nonsteroidal anti-inflammatory drug, is reported to stabilize TTR tetramers. More studies are needed to confirm its clinical efficacy.

Inhibition of Oligomer Aggregation and Oligomer Disruption

  1. Epigallocatechin gallate is the most abundant catechin in green tea. One single-center open-label 12-month study did not show survival benefits or any change in echocardiographic parameters or NT-BNP compared to baseline.

Degradation and Reabsorption of Amyloid Fibers

  1. Doxycycline-taurosodeoxycholic acid (TUDCA) has been evaluated in two small studies and the results appear to be modest. More data are needed to confirm its efficacy.
  2. Antibodies targeting serum amyloid P protein or amyloid fibrils: Patient enrollment for miridesap followed by anti-SAP antibodies was suspended, and this approach is not being evaluated currently. However, a monoclonal antibody designed to specifically target TTR amyloid deposits (PRX004) has entered clinical evaluation, with an ongoing phase 1 study on ATTRv.

Supportive Treatment of Cardiac Involvement

  1. Drug therapies: Although angiotensin-converting enzyme (ACE) inhibitors/angiotensin-receptor blockers (ARBs) and beta-blockers may have been poorly tolerated in the ATTR-ACT trial, 30% of the patients were on ACE inhibitors/ARBs. There are no data with digoxin in TTR amyloid, and non-dihydropyridine calcium channel blockers are contraindicated due to negative inotropy.
  2. Implantable cardioverter-defibrillators (ICDs): In one study, which included 53 patients with amyloid, ICD shocks occurred exclusively in the AL amyloid group and none in the TTR amyloid patients. Higher defibrillation thresholds and complication rates are of concern.
  3. Cardiac pacing: In a large series of ATTRv-related polyneuropathy (n = 262), a pacemaker was implanted in 110 patients with His ventricular interval >700 ms. The authors recommend that any conduction disturbance on 12-lead electrocardiogram (ECG) warrants further investigation with Holter monitoring to determine candidacy for a pacemaker.
  4. Left ventricular assist device (LVAD): Although an LVAD is technically feasible, it is associated with high short-term mortality and worse outcomes than in dilated cardiomyopathy.
  5. Cardiac transplantation: This is a valuable option for patients with end-stage heart failure when significant extracardiac disease is excluded. In one study with 10 patients, only episodes of amyloid recurrence occurred.

This is an outstanding overview of this topic and recommended reading for anyone who cares for patients with cardiac transthyretin amyloid.


First-Ever Evidence that Patisiran Reduces Pathogenic, Misfolded TTR Monomers and Oligomers in FAP Patients

We reported data from our ongoing Phase 2 open-label extension (OLE) study of patisiran, an investigational RNAi therapeutic targeting transthyretin (TTR) for the treatment of TTR-mediated amyloidosis (ATTR amyloidosis) patients with familial amyloidotic polyneuropathy (FAP). Alnylam scientists and collaborators from The Scripps Research Institute and Misfolding Diagnostics, Inc. were able to measure the effects of patisiran on pathogenic, misfolded TTR monomers and oligomers in FAP patients. Results showed a rapid and sustained reduction in serum non-native conformations of TTR (NNTTR) of approximately 90%. Since NNTTR is pathogenic in ATTR amyloidosis and the level of NNTTR reduction correlated with total TTR knockdown, these results provide direct mechanistic evidence supporting the therapeutic hypothesis that TTR knockdown has the potential to result in clinical benefit. Furthermore, complete 12-month data from all 27 patients that enrolled in the patisiran Phase 2 OLE study showed sustained mean maximum reductions in total serum TTR of 91% for over 18 months and a mean 3.1-point decrease in mNIS+7 at 12 months, which compares favorably to an estimated increase in mNIS+7 of 13 to 18 points at 12 months based upon analysis of historical data sets in untreated FAP patients with similar baseline characteristics. Importantly, patisiran administration continues to be generally well tolerated out to 21 months of treatment.

Read our press release

View the non-native TTR poster (480 KB PDF)

View the complete 12-month patisiran Phase 2 OLE data presentation (620 KB PDF)

We are encouraged by these new data that provide continued support for our hypothesis that patisiran has the potential to halt neuropathy progression in patients with FAP. If these results are replicated in a randomized, double-blind, placebo-controlled study, we believe that patisiran could emerge as an important treatment option for patients suffering from this debilitating, progressive and life-threatening disease.


Hereditary ATTR Amyloidosis with Polyneuropathy (hATTR-PN)

ATTR amyloidosis is a progressive, life-threatening disease caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in multiple organs, but primarily in the peripheral nerves and heart. ATTR amyloidosis can lead to significant morbidity, disability, and mortality. The TTR protein is produced primarily in the liver and is normally a carrier for retinol binding protein – one of the vehicles used to transport vitamin A around the body.  Mutations in the TTR gene cause misfolding of the protein and the formation of amyloid fibrils that typically contain both mutant and wild-type TTR that deposit in tissues such as the peripheral nerves and heart, resulting in intractable peripheral sensory neuropathy, autonomic neuropathy, and/or cardiomyopathy.

Click to Enlarge


ATTR represents a major unmet medical need with significant morbidity and mortality. There are over 100 reported TTR mutations; the particular TTR mutation and the site of amyloid deposition determine the clinical manifestations of the disease whether it is predominantly symptoms of neuropathy or cardiomyopathy.

Specifically, hereditary ATTR amyloidosis with polyneuropathy (hATTR-PN), also known as familial amyloidotic polyneuropathy (FAP), is an inherited, progressive disease leading to death within 5 to 15 years. It is due to a mutation in the transthyretin (TTR) gene, which causes misfolded TTR proteins to accumulate as amyloid fibrils predominantly in peripheral nerves and other organs. hATTR-PN can cause sensory, motor, and autonomic dysfunction, resulting in significant disability and death.

It is estimated that hATTR-PN, also known as FAP, affects approximately 10,000 people worldwide.  Patients have a life expectancy of 5 to 15 years from symptom onset, and the only treatment options for early stage disease are liver transplantation and TTR stabilizers such as tafamidis (approved in Europe) and diflunisal.  Unfortunately liver transplantation has limitations, including limited organ availability as well as substantial morbidity and mortality. Furthermore, transplantation eliminates the production of mutant TTR but does not affect wild-type TTR, which can further deposit after transplantation, leading to cardiomyopathy and worsening of neuropathy. There is a significant need for novel therapeutics to treat patients who have inherited mutations in the TTR gene.

Our ATTR program is the lead effort in our Genetic Medicine Strategic Therapeutic Area (STAr) product development and commercialization strategy, which is focused on advancing innovative RNAi therapeutics toward genetically defined targets for the treatment of rare diseases with high unmet medical need.  We are developing patisiran (ALN-TTR02), an intravenously administered RNAi therapeutic, to treat the hATTR-PN form of the disease.

Patisiran for the Treatment hATTR-PN

APOLLO Phase 3 Trial

In 2012, Alnylam entered into an exclusive alliance with Genzyme, a Sanofi company, to develop and commercialize RNAi therapeutics, including patisiran and revusiran, for the treatment of ATTR amyloidosis in Japan and the broader Asian-Pacific region. In early 2014, this relationship was extended as a significantly broader alliance to advance RNAi therapeutics as genetic medicines. Under this new agreement, Alnylam will lead development and commercialization of patisiran in North America and Europe while Genzyme will develop and commercialize the product in the rest of world.


Hereditary ATTR Amyloidosis with Cardiomyopathy (hATTR-CM)

ATTR amyloidosis is a progressive, life-threatening disease caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in multiple organs, but primarily in the peripheral nerves and heart. ATTR amyloidosis can lead to significant morbidity, disability, and mortality. The TTR protein is produced primarily in the liver and is normally a carrier for retinol binding protein – one of the vehicles used to transport vitamin A around the body.  Mutations in the TTR gene cause misfolding of the protein and the formation of amyloid fibrils that typically contain both mutant and wild-type TTR that deposit in tissues such as the peripheral nerves and heart, resulting in intractable peripheral sensory neuropathy, autonomic neuropathy, and/or cardiomyopathy.

Click to Enlarge                            http://www.alnylam.com/web/assets/tetramer.jpg

ATTR represents a major unmet medical need with significant morbidity and mortality. There are over 100 reported TTR mutations; the particular TTR mutation and the site of amyloid deposition determine the clinical manifestations of the disease, whether it is predominantly symptoms of neuropathy or cardiomyopathy.

Specifically, hereditary ATTR amyloidosis with cardiomyopathy (hATTR-CM), also known as familial amyloidotic cardiomyopathy (FAC), is an inherited, progressive disease leading to death within 2 to 5 years. It is due to a mutation in the transthyretin (TTR) gene, which causes misfolded TTR proteins to accumulate as amyloid fibrils primarily in the heart. Hereditary ATTR amyloidosis with cardiomyopathy can result in heart failure and death.

While the exact numbers are not known, it is estimated hATTR-CM, also known as FAC affects at least 40,000 people worldwide.  hATTR-CM is fatal within 2 to 5 years of diagnosis and treatment is currently limited to supportive care.  Wild-type ATTR amyloidosis (wtATTR amyloidosis), also known as senile systemic amyloidosis, is a nonhereditary, progressive disease leading to death within 2 to 5 years. It is caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in the heart. Wild-type ATTR amyloidosis can cause cardiomyopathy and result in heart failure and death. There are no approved therapies for the treatment of hATTR-CM or SSA; hence there is a significant unmet need for novel therapeutics to treat these patients.

Our ATTR program is the lead effort in our Genetic Medicine Strategic Therapeutic Area (STAr) product development and commercialization strategy, which is focused on advancing innovative RNAi therapeutics toward genetically defined targets for the treatment of rare diseases with high unmet medical need.  We are developing revusiran (ALN-TTRsc), a subcutaneously administered RNAi therapeutic for the treatment of hATTR-CM.

Revusiran for the Treatment of hATTR-CM

ENDEAVOUR Phase 3 Trial

In 2012, Alnylam entered into an exclusive alliance with Genzyme, a Sanofi company, to develop and commercialize RNAi therapeutics, including patisiran and revusiran, for the treatment of ATTR amyloidosis in Japan and the broader Asian-Pacific region. In early 2014, this relationship was extended as a broader alliance to advance RNAi therapeutics as genetic medicines. Under this new agreement, Alnylam and Genzyme have agreed to co-develop and co-commercialize revusiran in North America and Europe, with Genzyme developing and commercializing the product in the rest of world. This broadened relationship on revusiran is aimed at expanding and accelerating the product’s global value.

Pre-Clinical Data and Advancement of ALN-TTRsc02 for Transthyretin-Mediated Amyloidosis

We presented pre-clinical data with ALN-TTRsc02, an investigational RNAi therapeutic targeting transthyretin (TTR) for the treatment of TTR-mediated amyloidosis (ATTR amyloidosis).  In pre-clinical studies, including those in non-human primates (NHPs), ALN-TTRsc02 achieved potent and highly durable knockdown of serum TTR of up to 99% with multi-month durability achieved after just a single dose, supportive of a potentially once quarterly dose regimen. Results from studies comparing TTR knockdown activity of ALN-TTRsc02 to that of revusiran showed that ALN-TTRsc02 has a markedly superior TTR knockdown profile.  Further, in initial rat toxicology studies, ALN-TTRsc02 was found to be generally well tolerated with no significant adverse events at doses as high as 100 mg/kg.

Read our press release

View the presentation



Emerging Therapies for Transthyretin Cardiac Amyloidosis Could Herald a New Era for the Treatment of HFPEF

Oct 14, 2015   |  Adam Castano, MDDavid Narotsky, MDMathew S. Maurer, MD, FACC


Heart failure with a preserved ejection fraction (HFPEF) is a clinical syndrome that has no pharmacologic therapies approved for this use to date. In light of failed medicines, cardiologists have refocused treatment strategies based on the theory that HFPEF is a heterogeneous clinical syndrome with different etiologies. Classification of HFPEF according to etiologic subtype may, therefore, identify cohorts with treatable pathophysiologic mechanisms and may ultimately pave the way forward for developing meaningful HFPEF therapies.1

A wealth of data now indicates that amyloid infiltration is an important mechanism underlying HFPEF. Inherited mutations in transthyretin cardiac amyloidosis (ATTRm) or the aging process in wild-type disease (ATTRwt) cause destabilization of the transthyretin (TTR) protein into monomers or oligomers, which aggregate into amyloid fibrils. These insoluble fibrils accumulate in the myocardium and result in diastolic dysfunction, restrictive cardiomyopathy, and eventual congestive heart failure (Figure 1). In an autopsy study of HFPEF patients, almost 20% without antemortem suspicion of amyloid had left ventricular (LV) TTR amyloid deposition.2 Even more resounding evidence for the contribution of TTR amyloid to HFPEF was a study in which 120 hospitalized HFPEF patients with LV wall thickness ≥12 mm underwent technetium-99m 3,3-diphosphono-1,2-propranodicarboxylic acid (99mTc-DPD) cardiac imaging,3,4 a bone isotope known to have high sensitivity and specificity for diagnosing TTR cardiac amyloidosis.5,6 Moderate-to-severe myocardial uptake indicative of TTR cardiac amyloid deposition was detected in 13.3% of HFPEF patients who did not have TTR gene mutations. Therefore, TTR cardiac amyloid deposition, especially in older adults, is not rare, can be easily identified, and may contribute to the underlying pathophysiology of HFPEF.

Figure 1

As no U.S. Food and Drug Administration-approved drugs are currently available for the treatment of HFPEF or TTR cardiac amyloidosis, the development of medications that attenuate or prevent TTR-mediated organ toxicity has emerged as an important therapeutic goal. Over the past decade, a host of therapies and therapeutic drug classes have emerged in clinical trials (Table 1), and these may herald a new direction for treating HFPEF secondary to TTR amyloid.

Table 1

TTR Silencers (siRNA and Antisense Oligonucleotides)


Ribonucleic acid interference (RNAi) has surfaced as an endogenous cellular mechanism for controlling gene expression. Small interfering RNAs (siRNAs) delivered into cells can disrupt the production of target proteins.7,8 A formulation of lipid nanoparticle and triantennary N-acetylgalactosamine (GalNAc) conjugate that delivers siRNAs to hepatocytes is currently in clinical trials.9 Prior research demonstrated these GalNAc-siRNA conjugates result in robust and durable knockdown of a variety of hepatocyte targets across multiple species and appear to be well suited for suppression of TTR gene expression and subsequent TTR protein production.

The TTR siRNA conjugated to GalNAc, ALN-TTRSc, is now under active investigation as a subcutaneous injection in phase 3 clinical trials in patients with TTR cardiac amyloidosis.10 Prior phase 2 results demonstrated that ALN-TTRSc was generally well tolerated in patients with significant TTR disease burden and that it reduced both wild-type and mutant TTR gene expression by a mean of 87%. Harnessing RNAi technology appears to hold great promise for treating patients with TTR cardiac amyloidosis. The ability of ALN-TTRSc to lower both wild-type and mutant proteins may provide a major advantage over liver transplantation, which affects the production of only mutant protein and is further limited by donor shortage, cost, and need for immunosuppression.

Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) are under clinical investigation for their ability to inhibit hepatic expression of amyloidogenic TTR protein. Currently, the ASO compound, ISIS-TTRRx, is under investigation in a phase 3 multicenter, randomized, double-blind, placebo-controlled clinical trial in patients with familial amyloid polyneuropathy (FAP).11 The primary objective is to evaluate its efficacy as measured by change in neuropathy from baseline relative to placebo. Secondary measures will evaluate quality of life (QOL), modified body mass index (mBMI) by albumin, and pharmacodynamic effects on retinol binding protein. Exploratory objectives in a subset of patients with LV wall thickness ≥13 mm without a history of persistent hypertension will examine echocardiographic parameters, N-terminal pro–B-type natriuretic peptide (NT-proBNP), and polyneuropathy disability score relative to placebo. These data will facilitate analysis of the effect of antisense oligonucleotide-mediated TTR suppression on the TTR cardiac phenotype with a phase 3 trial anticipated to begin enrollment in 2016.

TTR Stabilizers (Diflunisal, Tafamidis)


Several TTR-stabilizing agents are in various stages of clinical trials. Diflunisal, a traditionally used and generically available nonsteroidal anti-inflammatory drug (NSAID), binds and stabilizes familial TTR variants against acid-mediated fibril formation in vitro and is now in human clinical trials.12,13 The use of diflunisal in patients with TTR cardiac amyloidosis is controversial given complication of chronic inhibition of cyclooxygenase (COX) enzymes, including gastrointestinal bleeding, renal dysfunction, fluid retention, and hypertension that may precipitate or exacerbate heart failure in vulnerable individuals.14-17 In TTR cardiac amyloidosis, an open-label cohort study suggested that low-dose diflunisal with careful monitoring along with a prophylactic proton pump inhibitor could be safely administered to compensated patients.18 An association was observed, however, between chronic diflunisal use and adverse changes in renal function suggesting that advanced kidney disease may be prohibitive in diflunisal therapy.In FAP patients with peripheral or autonomic neuropathy randomized to diflunisal or placebo, diflunisal slowed progression of neurologic impairment and preserved QOL over two years of follow-up.19 Echocardiography demonstrated cardiac involvement in approximately 50% of patients.20 Longer-term safety and efficacy data over an average 38 ± 31 months in 40 Japanese patients with hereditary ATTR amyloidosis who were not candidates for liver transplantation showed that diflunisal was mostly well tolerated.12 The authors cautioned the need for attentive monitoring of renal function and blood cell counts. Larger multicenter collaborations are needed to determine diflunisal’s true efficacy in HFPEF patients with TTR cardiac amyloidosis.


Tafamidis is under active investigation as a novel compound that binds to the thyroxine-binding sites of the TTR tetramer, inhibiting its dissociation into monomers and blocking the rate-limiting step in the TTR amyloidogenesis cascade.21 The TTR compound was shown in an 18-month double-blind, placebo-controlled trial to slow progression of neurologic symptoms in patients with early-stage ATTRm due to the V30M mutation.22 When focusing on cardiomyopathy in a phase 2, open-label trial, tafamidis also appeared to effectively stabilize TTR tetramers in non-V30M variants, wild-type and V122I, as well as biochemical and echocardiographic parameters.23,24 Preliminary data suggests that clinically stabilized patients had shorter disease duration, lower cardiac biomarkers, less myocardial thickening, and higher EF than those who were not stabilized, suggesting early institution of therapy may be beneficial. A phase 3 trial has completed enrollment and will evaluate the efficacy, safety, and tolerability of tafamidis 20 or 80 mg orally vs. placebo.25 This will contribute to long-term safety and efficacy data needed to determine the therapeutic effects of tafamidis among ATTRm variants.

Amyloid Degraders (Doxycycline/TUDCA and Anti-SAP Antibodies)


While silencer and stabilizer drugs are aimed at lowering amyloidogenic precursor protein production, they cannot remove already deposited fibrils in an infiltrated heart. Removal of already deposited fibrils by amyloid degraders would be an important therapeutic strategy, particularly in older adults with heavily infiltrated hearts reflected by thick walls, HFPEF, systolic heart failure, and restrictive cardiomyopathy. Combined doxycycline and tauroursodeoxycholic acid (TUDCA) disrupt TTR amyloid fibrils and appeared to have an acceptable safety profile in a small phase 2 open-label study among 20 TTR patients. No serious adverse reactions or clinical progression of cardiac or neuropathic involvement was observed over one year.26 An active phase 2, single-center, open-label, 12-month study will assess primary outcome measures including mBMI, neurologic impairment score, and NT-proBNP.27 Another phase 2 study is examining the tolerability and efficacy of doxycycline/TUDCA over an 18-month period in patients with TTR amyloid cardiomyopathy.28 Additionally, a study in patients with TTR amyloidosis is ongoing to determine the effect of doxycycline alone on neurologic function, cardiac biomarkers, echocardiographic parameters, modified body mass index, and autonomic neuropathy.29

Anti-SAP Antibodies

In order to safely clear established amyloid deposits, the role of the normal, nonfibrillar plasma glycoprotein present in all human amyloid deposits, serum amyloid P component (SAP), needs to be more clearly understood.30 In mice with amyloid AA type deposits, administration of antihuman SAP antibody triggered a potent giant cell reaction that removed massive visceral amyloid deposits without adverse effects.31 In humans with TTR cardiac amyloidosis, anti-SAP antibody treatments could be feasible because the bis-D proline compound, CPHPC, is capable of clearing circulating human SAP, which allow anti-SAP antibodies to reach residual deposited SAP. In a small, open-label, single-dose-escalation, phase 1 trial involving 15 patients with systemic amyloidosis, none of whom had clinical evidence of cardiac amyloidosis, were treated with CPHPC followed by human monoclonal IgG1 anti-SAP antibody.32 No serious adverse events were reported and amyloid deposits were cleared from the liver, kidney, and lymph node. Anti-SAP antibodies hold promise as a potential amyloid therapy because of their potential to target all forms of amyloid deposits across multiple tissue types.

Mutant or wild-type TTR cardiac amyloidoses are increasingly recognized as a cause of HFPEF. Clinicians need to be aware of this important HFPEF etiology because the diverse array of emerging disease-modifying agents for TTR cardiac amyloidosis in human clinical trials has the potential to herald a new era for the treatment of HFPEF.


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  10. U.S. National Institutes of Health. Phase 2 Study to Evaluate ALN-TTRSC in Patients With Transthyretin (TTR) Cardiac Amyloidosis (ClinicalTrials.gov website). 2014. Available at: https://www.clinicaltrials.gov/ct2/show/NCT01981837. Accessed 8/19/2015.
  11. U.S. National Institutes of Health. Efficacy and Safety of ISIS-TTRRx in Familial Amyloid Polyneuropathy (Clinical Trials.gov Website. 2013. Available at: http://www.clinicaltrials.gov/ct2/show/NCT01737398. Accessed 8/19/2015.
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The Acid-Mediated Denaturation Pathway of Transthyretin Yields a Conformational Intermediate That Can Self-Assemble into Amyloid

Zhihong Lai , Wilfredo Colón , and Jeffery W. Kelly *
Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255
Biochemistry199635 (20), pp 6470–6482   http://dx.doi.org:/10.1021/bi952501g
Publication Date (Web): May 21, 1996  Copyright © 1996 American Chemical Society

Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0−3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores. The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand−loop−D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS−PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly β-sheet structure having a well-defined tertiary structure.


Prevention of Transthyretin Amyloid Disease by Changing Protein Misfolding Energetics

Per Hammarström*, R. Luke Wiseman*, Evan T. Powers, Jeffery W. Kelly   + Author Affiliations

Science  31 Jan 2003; 299(5607):713-716   http://dx.doi.org:/10.1126/science.1079589

Genetic evidence suggests that inhibition of amyloid fibril formation by small molecules should be effective against amyloid diseases. Known amyloid inhibitors appear to function by shifting the aggregation equilibrium away from the amyloid state. Here, we describe a series of transthyretin amyloidosis inhibitors that functioned by increasing the kinetic barrier associated with misfolding, preventing amyloidogenesis by stabilizing the native state. The trans-suppressor mutation, threonine 119 → methionine 119, which is known to ameliorate familial amyloid disease, also functioned through kinetic stabilization, implying that this small-molecule strategy should be effective in treating amyloid diseases.


Rational design of potent human transthyretin amyloid disease inhibitors

Thomas Klabunde1,2, H. Michael Petrassi3, Vibha B. Oza3, Prakash Raman3, Jeffery W. Kelly3 & James C. Sacchettini1

Nature Structural & Molecular Biology 2000; 7: 312 – 321.                http://dx.doi.org:/10.1038/74082

The human amyloid disorders, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and senile systemic amyloidosis, are caused by insoluble transthyretin (TTR) fibrils, which deposit in the peripheral nerves and heart tissue. Several nonsteroidal anti-inflammatory drugs and structurally similar compounds have been found to strongly inhibit the formation of TTR amyloid fibrils in vitro. These include flufenamic acid, diclofenac, flurbiprofen, and resveratrol. Crystal structures of the protein–drug complexes have been determined to allow detailed analyses of the protein–drug interactions that stabilize the native tetrameric conformation of TTR and inhibit the formation of amyloidogenic TTR. Using a structure-based drug design approach ortho-trifluormethylphenyl anthranilic acid and N-(meta-trifluoromethylphenyl) phenoxazine 4,6-dicarboxylic acid have been discovered to be very potent and specific TTR fibril formation inhibitors. This research provides a rationale for a chemotherapeutic approach for the treatment of TTR-associated amyloid diseases.


First European consensus for diagnosis, management, and treatment of transthyretin familial amyloid polyneuropathy

Adams, Davida; Suhr, Ole B.b; Hund, Ernstc; Obici, Laurad; Tournev, Ivailoe,f; Campistol, Josep M.g; Slama, Michel S.h; Hazenberg, Bouke P.i; Coelho, Teresaj; from the European Network for TTR-FAP (ATTReuNET)

Current Opin Neurol: Feb 2016; 29 – Issue – p S14–S26      http://dx.doi.org:/10.1097/WCO.0000000000000289

Purpose of review: Early and accurate diagnosis of transthyretin familial amyloid polyneuropathy (TTR-FAP) represents one of the major challenges faced by physicians when caring for patients with idiopathic progressive neuropathy. There is little consensus in diagnostic and management approaches across Europe.

Recent findings: The low prevalence of TTR-FAP across Europe and the high variation in both genotype and phenotypic expression of the disease means that recognizing symptoms can be difficult outside of a specialized diagnostic environment. The resulting delay in diagnosis and the possibility of misdiagnosis can misguide clinical decision-making and negatively impact subsequent treatment approaches and outcomes.

Summary: This review summarizes the findings from two meetings of the European Network for TTR-FAP (ATTReuNET). This is an emerging group comprising representatives from 10 European countries with expertise in the diagnosis and management of TTR-FAP, including nine National Reference Centres. The current review presents management strategies and a consensus on the gold standard for diagnosis of TTR-FAP as well as a structured approach to ongoing multidisciplinary care for the patient. Greater communication, not just between members of an individual patient’s treatment team, but also between regional and national centres of expertise, is the key to the effective management of TTR-FAP.


Transthyretin familial amyloid polyneuropathy (TTR-FAP) is a highly debilitating and irreversible neurological disorder presenting symptoms of progressive sensorimotor and autonomic neuropathy [1▪,2▪,3]. TTR-FAP is caused by misfolding of the transthyretin (TTR) protein leading to protein aggregation and the formation of amyloid fibrils and, ultimately, to amyloidosis (commonly in the peripheral and autonomic nervous system and the heart) [4,5]. TTR-FAP usually proves fatal within 7–12 years from the onset of symptoms, most often due to cardiac dysfunction, infection, or cachexia [6,7▪▪].

The prevalence and disease presentation of TTR-FAP vary widely within Europe. In endemic regions (northern Portugal, Sweden, Cyprus, and Majorca), patients tend to present with a distinct genotype in large concentrations, predominantly a Val30Met substitution in the TTR gene [8–10]. In other areas of Europe, the genetic footprint of TTR-FAP is more varied, with less typical phenotypic expression [6,11]. For these sporadic or scattered cases, a lack of awareness among physicians of variable clinical features and limited access to diagnostic tools (i.e., pathological studies and genetic screening) can contribute to high rates of misdiagnosis and poorer patient outcomes [1▪,11]. In general, early and late-onset variants of TTR-FAP, found within endemic and nonendemic regions, present several additional diagnostic challenges [11,12,13▪,14].

Delay in the time to diagnosis is a major obstacle to the optimal management of TTR-FAP. With the exception of those with a clearly diagnosed familial history of FAP, patients still invariably wait several years between the emergence of first clinical signs and accurate diagnosis [6,11,14]. The timely initiation of appropriate treatment is particularly pertinent, given the rapidity and irreversibility with which TTR-FAP can progress if left unchecked, as well as the limited effectiveness of available treatments during the later stages of the disease [14]. This review aims to consolidate the existing literature and present an update of the best practices in the management of TTR-FAP in Europe. A summary of the methods used to achieve a TTR-FAP diagnosis is presented, as well as a review of available treatments and recommendations for treatment according to disease status.

Patients with TTR-FAP can present with a range of symptoms [11], and care should be taken to acquire a thorough clinical history of the patient as well as a family history of genetic disease. Delay in diagnosis is most pronounced in areas where TTR-FAP is not endemic or when there is no positive family history [1▪]. TTR-FAP and TTR-familial amyloid cardiomyopathy (TTR-FAC) are the two prototypic clinical disease manifestations of a broader disease spectrum caused by an underlying hereditary ATTR amyloidosis [19]. In TTR-FAP, the disease manifestation of neuropathy is most prominent and definitive for diagnosis, whereas cardiomyopathy often suggests TTR-FAC. However, this distinction is often superficial because cardiomyopathy, autonomic neuropathy, vitreous opacities, kidney disease, and meningeal involvement all may be present with varying severity for each patient with TTR-FAP.

Among early onset TTR-FAP with usually positive family history, symptoms of polyneuropathy present early in the disease process and usually predominate throughout the progression of the disease, making neurological testing an important diagnostic aid [14]. Careful clinical examination (e.g., electromyography with nerve conduction studies and sympathetic skin response, quantitative sensation test, quantitative autonomic test) can be used to detect, characterize, and scale the severity of neuropathic abnormalities involving small and large nerve fibres [10]. Although a patient cannot be diagnosed definitively with TTR-FAP on the basis of clinical presentation alone, symptoms suggesting the early signs of peripheral neuropathy, autonomic dysfunction, and cardiac conduction disorders or infiltrative cardiomyopathy are all indicators that further TTR-FAP diagnostic investigation is warranted. Late-onset TTR-FAP often presents as sporadic cases with distinct clinical features (e.g., milder autonomic dysfunction) and can be more difficult to diagnose than early-onset TTR-FAP (Table 2) [1▪,11,12,13▪,14,20].


Genetic testing is carried out to allow detection of specific amyloidogenic TTR mutations (Table 1), using varied techniques depending on the expertise and facilities available in each country (Table S2, http://links.lww.com/CONR/A39). A targeted approach to detect a specific mutation can be used for cases belonging to families with previous diagnosis. In index cases of either endemic and nonendemic regions that do not have a family history of disease, are difficult to confirm, and have atypical symptoms, TTR gene sequencing is required for the detection of both predicted and new amyloidogenic mutations [26,27].

Following diagnosis, the neuropathy stage and systemic extension of the disease should be determined in order to guide the next course of treatment (Table 4) [3,30,31]. The three stages of TTR-FAP severity are graded according to a patient’s walking disability and degree of assistance required [30]. Systemic assessment, especially of the heart, eyes, and kidney, is also essential to ensure all aspects of potential impact of the disease can be detected [10].

Table 4


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The goals of cardiac investigations are to detect serious conduction disorders with the risk of sudden death and infiltrative cardiomyopathy. Electrocardiograms (ECG), Holter-ECG, and intracardiac electrophysiology study are helpful to detect conduction disorders. Echocardiograms, cardiac magnetic resonance imaging, scintigraphy with bone tracers, and biomarkers (e.g., brain natriuretic peptide, troponin) can all help to diagnose infiltrative cardiomyopathy[10]. An early detection of cardiac abnormalities has obvious benefits to the patient, given that the prophylactic implantation of pacemakers was found to prevent 25% of major cardiac events in TTR-FAP patients followed up over an average of 4 years [32▪▪]. Assessment of cardiac denervation with 123-iodine meta-iodobenzylguanidine is a powerful prognostic marker in patients diagnosed with FAP [33].



Tafamidis is a first-in-class therapy that slows the progression of TTR amyloidogenesis by stabilizing the mutant TTR tetramer, thereby preventing its dissociation into monomers and amyloidogenic and toxic intermediates [55,56]. Tafamidis is currently indicated in Europe for the treatment of TTR amyloidosis in adult patients with stage I symptomatic polyneuropathy to delay peripheral neurological impairment [57].

In an 18-month, double-blind, placebo-controlled study of patients with early-onset Val30Met TTR-FAP, tafamidis was associated with a 52% lower reduction in neurological deterioration (P = 0.027), a preservation of nerve function, and TTR stabilization versus placebo [58▪▪]. However, only numerical differences were found for the coprimary endpoints of neuropathy impairment [neuropathy impairment score in the lower limb (NIS-LL) responder rates of 45.3% tafamidis vs 29.5% placebo; P = 0.068] and quality of life scores [58▪▪]. A 12-month, open-label extension study showed that the reduced rates of neurological deterioration associated with tafamidis were sustained over 30 months, with earlier initiation of tafamidis linking to better patient outcomes (P = 0.0435) [59▪]. The disease-slowing effects of tafamidis may be dependent on the early initiation of treatment. In an open-label study with Val30Met TTR-FAP patients with late-onset and advanced disease (NIS-LL score >10, mean age 56.4 years), NIS-LL and disability scores showed disease progression despite 12 months of treatment with tafamidis, marked by a worsening of neuropathy stage in 20% and the onset of orthostatic hypotension in 22% of patients at follow-up [60▪].

Tafamidis is not only effective in patients exhibiting the Val30Met mutation; it also has proven efficacy, in terms of TTR stabilization, in non-Val30Met patients over 12 months [61]. Although tafamidis has demonstrated safe use in patients with TTR-FAP, care should be exercised when prescribing to those with existing digestive problems (e.g., diarrhoea, faecal incontinence) [60▪].

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Diflunisal is a nonsteroidal anti-inflammatory drug (NSAID) that, similar to tafamidis, slows the rate of amyloidogenesis by preventing the dissociation, misfolding, and misassembly of the mutated TTR tetramer [62,63]. Off-label use has been reported for patients with stage I and II disease, although diflunisal is not currently licensed for the treatment of TTR-FAP.

Evidence for the clinical effectiveness of diflunisal in TTR-FAP derives from a placebo-controlled, double-blind, 24-month study in 130 patients with clinically detectable peripheral or autonomic neuropathy[64▪]. The deterioration in NIS scores was significantly more pronounced in patients receiving placebo compared with those taking diflunisal (P = 0.001), and physical quality of life measures showed significant improvement among diflunisal-treated patients (P = 0.001). Notable during this study was the high rate of attrition in the placebo group, with 50% more placebo-treated patients dropping out of this 2-year study as a result of disease progression, advanced stage of the disease, and varied mutations.

One retrospective analysis of off-label use of diflunisal in patients with TTR-FAP reported treatment discontinuation in 57% of patients because of adverse events that were largely gastrointestinal [65]. Conclusions on the safety of diflunisal in TTR-FAP will depend on further investigations on the impact of known cardiovascular and renal side-effects associated with the NSAID drug class [66,67].





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MYBPC3 gene and the heart

Larry H. Bernstein, MD, FCAP, Curator



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


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.



Aliases for MYBPC3 Gene


  • 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


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).


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.

  • 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
    European Molecular Biology Laboratory
    familial hypertrophic cardiomyopathy
    fibronectin III
    myosin binding protein C
    polymerase chain reaction
    single-strand conformation polymorphism analysis
  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
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MYBPC3 – Hypertrophic Cardiomyopathy Testing

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.
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.
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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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

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

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.

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.

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

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

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

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)


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).


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.


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

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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Diagnostic Value of Cardiac Biomarkers

Diagnostic Value of Cardiac Biomarkers

Author and Curator: Larry H Bernstein, MD, FCAP 

These presentations covered several views of the utilization of cardiac markers that have evolved for over 60 years.  The first stage was the introduction of enzymatic assays and isoenzyme measurements to distinguish acute hepatitis and acute myocardial infarction, which included lactate dehydrogenase (LD isoenzymes 1, 2) at a time that late presentation of the patient in the emergency rooms were not uncommon, with the creatine kinase isoenzyme MB declining or disappeared from the circulation.  The world health organization (WHO) standard definition then was the presence of two of three:

1. Typical or atypical precordial pressure in the chest, usually with radiation to the left arm

2. Electrocardiographic changes of Q-wave, not previously seen, definitive; ST- elevation of acute myocardial injury with repolarization;
T-wave inversion.

3. The release into the circulation of myocardial derived enzymes –
creatine kinase – MB (which was adapted to measure infarct size), LD-1,
both of which were replaced with troponins T and I, which are part of the actomyosin contractile apparatus.

The research on infarct size elicited a major research goal for early diagnosis and reduction of infarct size, first with fibrinolysis of a ruptured plaque, and this proceeded into the full development of a rapidly evolving interventional cardiology as well as cardiothoracic surgery, in both cases, aimed at removal of plaque or replacement of vessel.  Surgery became more imperative for multivessel disease, even if only one vessel was severely affected.

So we have clinical history, physical examination, and emerging biomarkers playing a large role for more than half a century.  However, the role of biomarkers broadened.  Patients were treated with antiplatelet agents, and a hypercoagulable state coexisted with myocardial ischemic injury.  This made the management of the patient reliant on long term followup for Warfarin with the international normalized ratio (INR) for a standardized prothrombin time (PT), and reversal of the PT required transfusion with thawed fresh frozen plasma (FFP).  The partial thromboplastin test (PPT) was necessary in hospitalization to monitor the heparin effect.

Thus, we have identified the use of traditional cardiac biomarkers for:

1. Diagnosis
2. Therapeutic monitoring

The story is only the beginning.  Many patients who were atypical in presentation, or had cardiovascular ischemia without plaque rupture were problematic.  This led to a concerted effort to redesign the troponin assays for high sensitivity with the concern that the circulation should normally be free of a leaked structural marker of myocardial damage. But of course, there can be a slow leak or a decreased rate of removal of such protein from the circulation, and the best example of this would be the patient with significant renal insufficiency, as TnT is clear only through the kidney, and TNI is clear both by the kidney and by vascular endothelium.  The introduction of the high sensitivity assay has been met with considerable confusion, and highlights the complexity of diagnosis in heart disease.  Another test that is used for the diagnosis of heart failure is in the class of natriuretic peptides (BNP, pro NT-BNP, and ANP), the last of which has been under development.

While there is an exponential increase in the improvement of cardiac devices and discovery of pharmaceutical targets, the laboratory support for clinical management is not mature.  There are miRNAs that may prove valuable, matrix metalloprotein(s), and potential endothelial and blood cell surface markers, they require

1. codevelopment with new medications
2. standardization across the IVD industry
3. proficiency testing applied to all laboratories that provide testing
4. the measurement  on multitest automated analyzers with high capability in proteomic measurement  (MS, time of flight, MS-MS)

nejmra1216063_f1   Atherosclerotic Plaques Associated with Various Presentations               nejmra1216063_f2     Inflammatory Pathways Predisposing Coronary Arteries to Rupture and Thrombosis.        atherosclerosis progression

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Stabilizers that prevent Transthyretin-mediated Cardiomyocyte Amyloidotic Toxicity

Reporter and curator: Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/12-2-2013/larryhbern/Stabilizers that prevent transthyretin-mediated cardiomyocyte amyloidotic toxicity

Transthyretin is a small protein with a half-life of < 48 hours, synthesized by the liver, and a major transport protein for thyroxin.  There are 80 variants known, and some variants that occur in the Portuguese, a small section of Japan, Sweden, and Brazil, are associated will primary amyloidosis, the only cure for which is liver transplantation.  It causes fibrillary inclusions in the heart, but also affects the autonomic nervous system.  Some of the major work on this has been done for many years in the laboratory of   Jeffery W. Kelly, at the Skaggs Institute for Chemical Biology, the Scripps Research Institute.  A recent publication is of considerable interest.

Potent Kinetic Stabilizers that Prevent Transthyretin-mediated Cardiomyocyte Proteotoxicity

 Mamoun M. Alhamadsheh1,6,7, Stephen Connelly2,7, Ahryon Cho1, Natàlia Reixach3, Evan T. Powers3,4,5, Dorothy W. Pan1, Ian A. Wilson2,5, Jeffery W. Kelly3,4,5, and Isabella A. Graef1,*
Sci Transl Med. Author manuscript; available in PMC 2012 August 24.
1Department of Pathology, Stanford University Medical School, Stanford, California, USA
2Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA
3Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
4Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA
5The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA
6Department of Pharmaceutics & Medicinal Chemistry, University of the Pacific, Stockton, California, USA


The V122I mutation that alters the stability of transthyretin (TTR) affects 3–4% of African Americans and leads to amyloidogenesis and development of cardiomyopathy. In addition, 10–15% of individuals over the age of 65 develop senile systemic amyloidosis (SSA) and cardiac
TTR deposits due to wild-type TTR amyloidogenesis. As no approved therapies for TTR amyloid cardiomyopathy are available, the development of drugs that prevent amyloid-mediated cardiotoxicity is desired. To this aim, we developed a fluorescence polarization-based HTS screen,
which identified several new chemical scaffolds targeting TTR. These novel compounds were potent kinetic stabilizers of TTR and
  • prevented tetramer dissociation,
  • unfolding and aggregation of both wild type and the most common cardiomyopathy-associated TTR mutant, V122I-TTR.
High-resolution co-crystal structures and characterization of the binding energetics revealed how these diverse structures bound to tetrameric TTR. Our study also showed that these compounds effectively inhibited the proteotoxicity of V122I-TTR towards human cardiomyocytes.
Several of these ligands stabilized TTR in human serum more effectively than diflunisal, which is one of the best known inhibitors of TTR aggregation, and may be promising leads for the treatment and/or prevention of TTR-mediated cardiomyopathy.

Author Contributions:

M.M.A. designed and performed most experiments, S.C. performed crystallographic structure determination, A.C peformed the serum TTR stabilization. N.R. performed the cell-based assays.   E.T.P. analyzed the ITC data. D.W.P. helped with probe synthesis. I.A.W. supervised the crystallographic work. J.W.K. supervised the work, S.C., N.R., I.A.W. and J.W.K. edited the paper. I.A.G supervised the work, M.M.A. and I.A.G prepared the manuscript.


The misassembly of soluble proteins into toxic amyloid aggregates underlies a large number of human degenerative diseases (1–3). TTR is one of more than 30 human amyloidogenic proteins whose misassembly can cause
  • a variety of degenerative gain-of-toxic-function diseases.
TTR is a tetrameric protein (54 kDa), secreted from the liver into the blood where, using orthogonal sites,
  • it transports thyroxine (T4) and
  • holo-retinol binding protein (4).
However, 99% of the TTR T4 binding sites remain unoccupied in humans
  • owing to the presence of two other T4 transport proteins in blood (3).
Familial TTR amyloid diseases, which are associated with one of more than 80 mutations in the TTR gene, include
  • the systemic neuropathies (familial amyloid polyneuropathy [FAP]),
  • cardiomyopathies (familial amyloid cardiomyopathy [FAC]), and
  • central nervous system amyloidoses (CNSA) (5–8).
Cardiac amyloidosis is most commonly caused by
  • deposition of immunoglobulin light chains or
  • TTR in the cardiac interstitium and conducting system.
It is a chronic and progressive condition, which can lead to arrhythmias, biventricular heart failure, and death (8–10). Two types of TTR-associated amyloid cardiomyopathies are clinically important.
  1. Wild-type (WT) TTR aggregation underlies the development of senile systemic amyloidosis (SSA). Cardiac TTR deposits can be found in 10 to 15% of the population over the age of 65 at autopsy (10,11). Many of these patients are asymptomatic, but there is little doubt that SSA is an underdiagnosed disease.
  2. In addition, a number of TTR mutations, including V122I, lead to amyloidogenesis and familial amyloid cardiomyopathy (FAC) (12–15). Population studies show that the V122I mutation is found in 3–4% of African Americans (~1.3 million people) and contributes to the increased prevalence of heart failure among this population segment (14,15).

The mutant TTR allele behaves as an autosomal dominant allele with age-dependent penetrance and

  • the frequency of cardiac amyloidosis from TTR in African-American individuals above age 60 is four times that seen in Caucasian-Americans of comparable age.
All of the TTR mutations associated with familial amyloidosis decrease tetramer stability, and
  • some decrease the kinetic barrier for tetramer dissociation (3, 16).
  • The latter is important because tetramer dissociation is the rate-limiting step in the TTR amyloidogenesis cascade (3).

Kinetic stabilization of the native, tetrameric structure of TTR by

  • interallelic trans suppression (incorporation of mutant subunits that raise the dissociative transition state energy) prevents
    1. post-secretory dissociation and aggregation, as well as the related disease 
    2. familial amyloid polyneuropathy (FAP), by slowing TTR tetramer dissociation (17).
Occupancy of the TTR T4 binding sites with rationally designed small molecules is known to stabilize the native tetrameric state of TTR over the dissociative transition state,
  • raising the kinetic barrier,
  • imposing kinetic stabilization on the tetramer and
  • preventing amyloidogenesis (3, 16, 18).
Previous studies have focused on rational ligand design and as a result
  • most of the TTR stabilizers reported to date are halogenated biaryl analogues of T4,
  • many resembling non-steroidal anti-inflammatory drugs (NSAIDs).
Some of these compounds, such as the NSAID diflunisal, which is currently tested in clinical trials in FAP patients for its efficacy to ameliorate
  • peripheral neuropathy resulting from TTR deposition, (19) have anti-inflammatory activity (20, 21).
The pharmacological effects of NSAIDs are due to inhibition of cyclo-oxygenase (COX) enzymes (22). Inhibition of COX-1 can produce side effects such as
  • gastrointestinal irritation, leading to ulcers and bleeding (23).
Inhibition of COX-2 has been associated with an
  • increased risk of severe cardiovascular events, including heart failure,
  • particularly in patients with preexisting cardiorenal dysfunction (20, 21, 24, 25).
Therefore, heart and kidney impairment are exclusion criteria for participation of patients in the diflunisal clinical trials to treat TTR-mediated FAP (19). Genomic variations can
  • increase the sensitivity of individuals to adverse side effects of NSAIDs.
Serum concentrations of NSAIDs depend on CYP2C9 and/or CYP2C8 activity. CYP2C9 polymorphism might play a significant role in the profile of adverse side effects of NSAID and alleles that affect the activity of CYP2C9 are found at different frequency in subjects of Caucasian, African or Asian descent (26, 27). Hence, the long-term therapy with drugs that have inhibitory effect on COX activity to prevent TTR aggregation is especially problematic in patients who suffer from TTR-mediated cardiomyopathy. The design and development of drugs to treat/prevent FAC or SSA thus presents the challenge
  1. not only to find compounds with a greater variety of chemical scaffolds that accomplish stabilization, but
  2. do so without the adverse side effects due to inhibition of COX activity.
 For these reasons, the development of a rapid and robust screen for compounds that bind to and stabilize TTR could be useful. To date, no high-throughput screening (HTS) methodology is available for the discovery of TTR ligands (28,29). Therefore, we developed a versatile
  • fluorescence polarization (FP) based HTS assay that can detect
  • binding of small molecules to the T4 binding pocket of TTR under physiological conditions.


Design and synthesis of the TTR FP probe

FP is used to study molecular interactions by monitoring changes in the apparent size of a fluorescently labeled molecule. Binding is measured by an increase in the FP signal, which is proportional to the decrease in the rate of tumbling of a fluorescent ligand upon association with macromolecules such as proteins (Fig. 1A). To synthesize a fluorescent TTR ligand 1, we initially started with the NSAID diflunisal analogue 2 (Fig. 1B) (30). The product of attaching a linker to 2, compound 3, had very low binding affinity to TTR (Kd1 >3290 nM, fig. S1A and fig. S1B).
The crystal structure of the diclofenac analog 4 showed that
  • the phenolic hydroxyl flanked by the two chlorine atoms is oriented out of the binding pocket into the solvent (31).
  • We reasoned that attaching a PEG amine linker to the phenol group of 4 would generate compound 5 which would bind to TTR (Fig. 1B and fig. S1C)

5 was coupled to fluorescein isothiocyanate (FITC) to produce the FITC-coupled TTR FP probe (1, Fig. 1B). The binding characteristics of the probe (Kd1 = 13 nM and Kd2 = 100 nM) were assessed with ITC (Fig. 2A).

Evaluation of the FP assay

The binding of 1 to TTR was evaluated to test its suitability for the FP assay with a standard saturation binding experiment. A fixed concentration of probe 1 (0.1 μM) was incubated with increasing concentrations of TTR (0.005 μM to 10 μM) and the formation of 1•TTR complex was quantified by the increase in FP signal (excitation λ 485 nm, emission λ 525 nm) relative to the concentration of TTR (Fig. 2B). The fluorescence polarization increased with the concentration of TTR until saturation was reached. A large dynamic range (70 – 330 mP) was measured for the assay. To validate the FP assay, we tested known TTR binders in a displacement assay (for detailed information see Supplemental Material). Compound 2 (Kapp = 231 nM, R2 = 0.997), Thyroxine (T4) (Kapp = 186 nM, R2 = 0.998) and diclofenac (Kapp = 4660 nM, R2 = 0.999) decreased the FP signal in a dose- dependent  manner  (Fig. 2C,  fig. S2B and S2C). The FP assay is a competitive displacement assay and therefore it provides apparent binding constants (Kapp). However, these apparent binding constants correlate well with the data obtained by ITC which measures direct interactions in solution and gives an actual (Kd) value.

 Adaptation of the FP assay for HTS

Next, we optimized the FP assay for HTS and screened a ~130,000 small molecule library for compounds that displaced probe 1 from the T4 binding sites of TTR. The FP assay was performed in 384-well plates with low concentrations of probe 1 (1.5 nM) and TTR (50 nM) in a 10  μL assay volume.  Detergent (0.01% Triton X-100) was added to the assay buffer to avoid false positive hits from aggregation of the small molecules. The assay demonstrated robust performance, with a, large dynamic range (~70–230 mP) and a Z′ factor (32, 33) in the range of 0.57–0.78 (fig. S3A and S3B).

Hits were defined as compounds, which resulted in at least 50% decrease in FP and demonstrated relative fluorescence between 70 and 130%. Many fluorescence quenchers and enhancers, which have less than 70% and greater than 130% total fluorescence relative to a control (compound without TTR), were excluded from the hit list. The excluded compounds have native fluorescence that is similar to fluorescein, which would interfere with the FP measurements and result in false positive hits. Two hundred compounds were designated as positive hits (0.167% hit rate). The top 33 compounds (compounds with lowest FP IC50) were assayed in a 10-point duplicate dose-response FP assay and displayed an IC50 (concentration that resulted in 50% decrease in the FP signal) between 0.277 and 10.957 μM (table S2).

Validation of the HTS hits

The top 33 compounds were retested with the FP assay (table S2) and with surface plasmon resonance (SPR) as another independent biophysical method. Solutions of the 33 hits were passed over immobilized, biotinylated TTR on a streptavidin coated chip. The binding of a small molecule to TTR on the sensor chip produces a SPR response signal (RU). The RU signal after addition of the top 33 compounds was measured and compared to a negative, solvent only, control. All compounds identified by the screen as hits were confirmed as TTR binders using SPR (fig. S4). We also found known TTR binders, such as NSAIDs (diclofenac, meclofenamic acid, and niflumic acid) and isoflavones (apigenin) in our screen (3, 34) (table S2). Among the best ligands (Fig. 2D) were the NSAID, niflumic acid, two catechol-O-methyl-tranferase (COMT) inhibitors, 3,5-dintrocatechol and Ro 41-0960 (35) and a number  of compounds   not previously known to bind to TTR. The chemical structures of these ligands were confirmed by 1H NMR and high-resolution mass spectrometry(HRMS) and the chemical purity was determined to be >95% (fig. S5).

Inhibition of TTR amyloidogenesis by the HTS hits

To test whether the new TTR ligands (7.2 μM) could function as kinetic stabilizers, we measured their ability to inhibit TTR (3.6 μM) amyloidogenesis at 72 hrs at pH 4.4 (fig. S6) (29). All 33 compounds inhibited TTR aggregation (<50% fibril formation, table S2). Of these, 23 were very good (<20% fibril formation) and 11 were excellent (<2% fibril formation) TTR kinetic stabilizers (Fig. 3A). All of the potent TTR stabilizers, except niflumic acid, and the two COMT inhibitors 3,5-dintrocatechol and Ro 41-0960, were chemical entities with no previously reported biological activity. Since occupancy of only
one T4 binding site within TTR is sufficient for kinetic stabilization of the tetramer (3), we tested the most potent ligands at substoichiometric concentrations (2.4 fold molar excess of TTR relative to ligand) in a kinetic aggregation assay monitored over 5 days (Fig. 3B). Under these conditions ligands 7, 14, 15 and Ro 41-0960 dramatically slowed fibril formation and outperformed the known TTR stabilizer, diclofenac, which blocked only ~55% of TTR aggregation.

Evaluating the TTR ligands for COX-1 enzymatic inhibition and binding to thyroid hormone receptor

A successful clinical candidate against TTR amyloid cardiomyopathy should have minimal off-target toxicity due to the potential need for life-long use of these drugs. Specifically, the TTR ligands should exhibit minimal binding to COX and the nuclear thyroid hormone receptor (THR). Inhibition of COX is contraindicated for treating FAC patients, since COX inhibition can not only lead to renal dysfunction and blood pressure elevation, but may precipitate heart failure in vulnerable individuals (20, 21, 24, 25). Therefore, the most potent TTR ligands were evaluated for their ability to inhibit COX-1 activity, as well as, for binding to THR, in comparison with the NSAID niflumic acid. Although niflumic acid exhibited substantial (94%) COX-1 inhibition, three of the 12 new compounds evaluated (7, 6 and 10) displayed less than 1% inhibition of COX-1. Only one ligand (compound 8) showed significant (58%) and two compounds (6 and 10) minor (5%) binding to THR (Fig. 3C).

Characterization of the binding energetics to TTR

Many reported TTR ligands, including T4, bind TTR with negative cooperativity, which appears to arise from subtle conformational changes in TTR upon ligand binding to the first T4 site (3, 16, 36). We used ITC to determine the binding constants and to evaluate cooperativity between the two TTR T4 sites (Fig. 2A, Fig. 4A, Fig. 4B and fig. S1 and fig. S7). The ITC data for compounds 1, 7, 14, and Ro 41-0906 binding to TTR were fit to a two-site binding model and show that these potent ligands bind TTR with low nanomolar affinity. The dissociation constants for these ligands indicated that they bound TTR with negative cooperativity (table S3). Analysis of the free energies associated with ligand binding to TTR indicates that binding was driven both by burial of the hydrophobic ligand in the TTR binding site (which leads to the favorable binding entropies) and specific ligand-TTR interactions (which leads to the favorable binding enthalpies) (Fig. 2A, Fig. 4A, Fig.4B, and fig. S7B) (37). The binding of compounds 7 (Kd1 = 58 nM and Kd2 = 500 nM) and 14 (Kd1 = 26 nM and Kd2 = 1800 nM) to TTR did not cause major conformational changes to the TTR tetramer structure (Fig. 5).
Remainder of document is found at publication site, including Figures.

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Cardiotoxicity and Cardiomyopathy Related to Drugs Adverse Effects

Curator: Larry H Bernstein, MD, FCAP

This is the second part of a series on toxicities of therapeutic medications, the first being on the impact on drug development of early phase failure to identify toxicities that are found in late stage trials and result in withdrawal.  This portion will go into details of identifying the effects clinically and give some examples.  In the future, the design of therapies, the identification of high probability successful genomic targets, and more accurate patient selection will transform the approach to development, clinical trials, and clinical use of pharmaceuticals in patients.

Cardiotoxicity and Cardiomyopathy

refer to: What are cardiotoxicity and cardiomyopathy?

The Scott Hamilton Cares Initiative
Cardiotoxicity is a condition of heart muscle functional impairment from toxicity as an
  • adverse secondary effect of taking an essential medication, or
  • as a result of interactions between prescribed medications that result in heart damage,
  • usually dose and time related.
If severe, the adverse effect of chemotherapy may lead to cardiomyopathy.  While cardiomyopathy might be a result of treatments, such as chemotherapeutic medications, it may also caused by a group of diseases or disorders, leading to
  • damaged myocardiocytes, and the injury leads to
  • insufficient cardiac output, referred to as
  • heart failure.
Cardiomyopathy has many causes, singly or in combination:
  • viruses – such as,
    • coxsackie B,
    • human immunodeficiency virus (HIV)
  • systemic inflammatory disorder
    • systemic lupus erythematosis
  • Amyloidosis – amyloid protein deposits in the myocardium alone, and/or other organs
  • Infection –
    • bacterial (tetanus),
    • parasitic (Chaga’s disease)
    • Rheumatic fever
  •  high blood pressure
  • Chronic or long-term alcohol use (B vitamin deficiency)
  • Endocrine disease, such as hyperthyroidism
  • Thiamine and Vitamin B deficiency
  • Radiation therapy
  • Medications – anthracyclines.

Anthracyclines may be used to treat leukemia, lymphoma, multiple myeloma, breast cancer, and sarcoma. A commonly used anthracycline is called doxorubicin (Adriamycin®).

  • cardiomyopathy may also result from genetic defects
  • illegal drugs and toxic substances, cocaine, may also produce serious myocardial damage

With certain drugs, such as doxorubicin, there is a dose at which these cardiotoxic effects on the heart may occur.
An echocardiogram, or a radionuclide ventriculography scan, is performed

  • prior to initiating a cardiotoxic medication
  • to determine baseline cardiac function., and
  • repeated at intervals to monitor heart function while receiving cardiotoxic medications.

The ejection fraction (EF) is a percentage of blood pumped out into the body during each heartbeat. An EF of 50%-75% is considered normal.

  • The lower the ejection fraction, the more severe the heart failure may be.
This may determine if the cardiotoxic drug has caused cardiomyopathy.

Symptoms of cardiomyopathy:

  • fatigue
  •  shortness of breath
  •  fever and aching of the joints,
      • all characteristic of a flu-like illness.
  • Or, sudden heart failure or sudden cardiac death without any prior symptoms.
    • swollen feet and ankles
    • distended neck veins
    • tachycardia
    • dyspnea while reclining


  • history & physical examination
  • laboratory tests
  • EKG
  • Chest x-ray
  • Echocardiography
  • Cardiac cath
  • Angiography


  • Dexraoxane HCL –  doxorubicin
  • ACE inhibitors
  • Beta-blockers
  • Diuretics
  • Digoxin

 Biomolecular Screening for Drug Toxicity

Multiparameter In Vitro Assessment of Compound Effects on Cardiomyocyte Physiology Using iPSC Cells

O Sirenko, C Crittenden, N Callamaras, J Hesley, Yen-Wen Chen, et al.
 A sufficient percentage of drugs fail in clinical studies due to cardiac toxicity that the development of new, sensitive in vitro assays that can evaluate potential adverse effects on cardiomyocytes is needed. Cell-based models are more clinically relevant than those used in practice. Human-induced pluripotent stem cell–derived cardiomyocytes are especially attractive because
  • they express ion channels and
  • demonstrate spontaneous mechanical and electrical activity
    • similar to adult cardiomyocytes.
This study introduces techniques for measuring the impact of pharmacologic compounds on the beating rate of cardiomyocytes with ImageXpress Micro and FLIPR Tetra systems. The assays employ
calcium-sensitive dyes to monitor changes in Ca2+ fluxes
  • synchronous with cell beating,
This method allows monitoring of the
  • beat rate
  • amplitude, and
  • other parameters.
The system detects
  • concentration-dependent atypical patterns caused by
  • hERG inhibitors and other ion channel blockers.
In addition,
  • both positive and negative chronotropic effects on cardiac rate can be observed and
  • IC50 values determined.
This methodology is well suited for safety testing and can be used to estimate efficacy and dosing of drug candidates prior to clinical studies.
J Biomol Screen Jan 2013;18(1): 39-53  http://dx.doi.org/10.1177/1087057112457590

Estimating the risk of drug-induced proarrhythmia using human induced pluripotent stem cell-derived cardiomyocytes.

L Guo, RMC Abrams, JE Babiarz, JD Cohen, S Kameoka, et al.
 Early prediction of drug-induced toxicity is needed in the pharmaceutical and biotechnology industries to decrease late-stage drug attrition.
  • Cardiotoxicity accounts for about one third of safety-based withdrawn pharmaceuticals.
This study reports a high-throughput functional assay,  detailing a model that accurately detects
  • drug-induced cardiac abnormalities.
It employs
  • induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).
  • Using 96-well plates with interdigitated electrode arrays  detect
    • assess impedance,
    • the rhythmic, synchronous contractions of the iPSC-CMs
Treatment of the iPSC-CMs with 28 different compounds with known cardiac effects resulted in
  • compound-specific changes in the beat rate and/or
  • the amplitude of the impedance measurement.
Changes in impedance for the compounds tested were comparable with the results from a related technology,
  • electric field potential assessment obtained from microelectrode arrays.
Using the results from the set of compounds,
  • an index of drug-induced arrhythmias was calculated,
  • which enabled the determination of a drug’s proarrhythmic potential.
This system of interrogating human cardiac function in vitro opens new opportunities for predicting cardiac toxicity and studying cardiac biology.
Toxicol Sci. Sep 2011; 123 (1):281-9  21693436

Determination of the Human Cardiomyocyte mRNA and miRNA Differentiation Network by Fine-Scale Profiling.

JE Babiarz, M Ravon, S Sridhar, P Ravindran, B Swanson,  et al.
This study is a detailed comparison of the mRNA and miRNA transcriptomes
  • across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and
  • biopsies from fetal, adult, and hypertensive human hearts
Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes
revealed 3 distinct groups of genes:
  • pluripotent specific,
  • transitional cardiac specification, and
  • mature cardiomyocyte specific.
Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes
  • stabilizes 20 days after initiation of differentiation.
But analysis of cells continuously cultured for 120 days indicated that
  • the cardiomyocytes continued to mature toward a more adult-like gene expression pattern.
Analysis of cardiomyocyte-specific miRNAs (miR-1, miR-133a/b, and miR-208a/b) revealed
  • an miRNA pattern indicative of stem cell to cardiomyocyte specification.
A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a
  • cardiomyocyte differentiation miRNA network and
  • identified putative mRNAs targeted by multiple miRNAs.
Together, these data reveal the
miRNA network in human heart development and
  • support the notion that overlapping miRNA networks
  • re-enforce transcriptional control during developmental specification.
Stem Cells Dev. 2012 Jul 20;21 (11):1956-65  22050602

Comparative Gene Expression Profiling in Human Induced Pluripotent Stem Cell Derived Cardiocytes and Human and Cynomolgus Heart Tissue.

D Puppala, LP Collis, SZ Sun, V Bonato, X Chen, B Anson, et al.  Compound Safety Prediction.
Cardiotoxicity is one of the leading causes of drug attrition. Current in vitro models insufficiently predict cardiotoxicity.  The authors describe
  • the gene expression profile of human induced pluripotent stem cell derived cardiocytes (iCC)
  • post-thaw over a period of 42 days in culture and
  • compare this profile to human fetal and adult as well as
  • adult cynomolgus nonhuman primate (NHP: Macaca fascicularis) heart tissue.
The results indicate that iCC express relevant cardiac markers such as
  • ion channels (SCN5A, KCNJ2, CACNA1C, KCNQ1 and KCNH2),
  • tissue specific structural markers (MYH6, MYLPF, MYBPC3, DES, TNNT2 and TNNI3),
  • transcription factors (NKX2.5, GATA4 and GATA6), and
  • lack the expression of stem cell markers (FOXD3, GBX2, NANOG, POU5F1, SOX2, and ZFP42).

A functional evaluation of contractility of the iCC showed

  • functional and pharmacological correlations with myocytes isolated from adult NHP hearts.
The results suggest that stem cell derived cardiocytes may represent
  • a novel in vitro model to study human cardiac toxicity with potential ex vivo and in vivo translation.
Toxicol Sci. Sep 14 2012;:   22982684

Characterization of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes:
Bioenergetics and Utilization in Safety Screening.

P Rana, B Anson, S Engle, Y Will.   Compound Safety Prediction. Pfizer Global R&D, Groton CT.
Cardiotoxicity remains the number one reason for drug withdrawal from the market and FDA issued black box warnings; thus
  • demonstrating the need for more predictive preclinical safety screening,
  • especially early in the drug discovery process.
Whereas hERG screening has become routine to mitigate proarrhythmic risk,
  • the development of in vitro assays predicting additional on- and off-target biochemical toxicities
  • will benefit from cellular models exhibiting true cardiomyocyte characteristics
    • such as, native tissue-like mitochondrial activity.
An hypothesis was tested  for using human stem cell derived tissue cells by using a combination of
  • flux analysis,
  • gene and protein expression, and
  • toxicity-profiling techniques
    • to characterize mitochondrial function
    • in induced pluripotent stem cell (iPSC)-derived human cardiomyocytes
      • in the presence of differing carbon sources
      • over extended periods in cell culture.
Functional analyses demonstrate that iPSC-derived cardiomyocytes:
1) are capable of utilizing anaerobic or aerobic respiration depending upon the available carbon substrate,
2) are bioenergetically closest to adult heart tissue cells when cultured in galactose or galactose supplemented with fatty acids, and
3) show a dose dependent toxicity profile to a variety of kinase inhibitors with known clinical cardiac liabilities.
Furthermore, gene and protein expression analyses revealed that in comparison to adult cardiac tissue,
  • iPSCs-derived cardiomyocytes possess a qualitatively similar expression pattern of mitochondrial genes,
  • an up-regulation of apoptotic and antioxidant genes, and
  • a mitochondrial transcription pattern that is similar across different carbon substrates
  • despite showing changes in protein levels and functional bioenergetic adaptation.
Toxicol Sci. 2012 Jul 27;:   22843568

Decreasing cardiac chamber sizes and associated heart dysfunction in COPD – role of hyperinflation.

H Watz, B Waschki, T Meyer, G Kretschmar, A Kirsten, M Claussen, H Magnussen
This study examined the relationship of
  • lung function with heart size and heart dysfunction and
  • associated consequences for 6-minute walk distance (6MWD)
    • in patients with COPD of different severity.
METHODS:   138 patients with COPD (GOLD I-IV)
  • the size of all cardiac chambers,
  • left ventricular diastolic dysfunction (relaxation and filling), and
  • global right ventricular dysfunction (Tei-index)
    • were measured by echocardiography .
  • lung function (spirometry, bodyplethysmography, diffusion capacity) and
  • 6MWD …. were measured.
RESULTS: Size of all cardiac chambers decreased with GOLD stages. Overall,
moderate relationships existed between
  • variables of lung function and cardiac chamber sizes.
  • Static hyperinflation (inspiratory-to-total lung capacity ratio [IC/TLC],
  • functional residual capacity, and residual volume)
showed stronger associations with
  • cardiac chamber sizes than
  • airway obstruction or diffusion capacity.
IC/TLC ratio correlated best with cardiac chamber sizes and was
  • an independent predictor of cardiac chamber sizes
    • after adjustment for body surface area.
Patients with an IC/TLC ratio <!–= 0.25 had a significantly–>
  • impaired left ventricular diastolic filling pattern and
  • a significantly impaired Tei-index
    • compared to patients with an IC/TLC ratio > 0.25.
An impaired left ventricular diastolic filling pattern was independently associated with
  • a reduced 6MWD.
An increasing severity of COPD is associated with a decreasing heart size.
Hyperinflation in patients with COPD might have an important role with respect to
  • heart size and
  • cardiac dysfunction
Chest. Feb 26 2010;:   20190002  Cit:4

Cardiovascular Events After Clarithromycin Use in Lower Respiratory Tract Infections
Analysis of Two Prospective Cohort Studies

S Schembri, PA Williamson, PM Short, A Singanayagam, A Akram, et al. British Medical Journal
Acute exacerbations of chronic obstructive pulmonary disease and community acquired pneumonia are common causes of admission to a hospital.
Antibiotics, including clarithromycin, are commonly prescribed during acute exacerbations of chronic obstructive pulmonary disease, especially
  • in the presence of increased breathlessness,
  • sputum volume, and
  • purulence.
 Use of macrolide antibiotics in community acquired pneumonia has been consistently associated with improved short term mortality in observational studies,
and national and international guidelines therefore recommend their use in combination with β lactams for patients admitted to hospital.

Widespread use of macrolide antibiotics has been accompanied by concerns about adverse effects on cardiovascular morbidity and mortality.
A retrospective study of erythromycin use in 1,249,943 patients identified an increase in deaths from cardiovascular disease.
Azithromycin was shown to have a similar association with increased cardiovascular deaths

  • during the time of administration.

CLARICOR (Effect of Clarithromycin on Mortality and Morbidity in Patients with Ischemic Heart Disease trial) was a double blind, placebo controlled trial
showing that a two week course of clarithromycin administered to patients with coronary heart disease 

  • increased cardiovascular and all cause mortality
The increased mortality rate (clear of pulmonary infection)
  • persisted for three years after discontinuation of the drug.

A recent meta-analysis of 17 trials of antibiotics in coronary heart disease showed

  • increased long term mortality after macrolides, primarily due
  • to increased deaths from cardiovascular disease.

However, no studies have examined the long term effect of clarithromycin on cardiovascular events and mortality in patients

  • after acute exacerbations of chronic obstructive pulmonary disease or community acquired pneumonia.

Therefore, this prospective cohort study was undertaken to examine the association of clarithromycin with cardiovascular events

  • in the setting of acute exacerbations of chronic obstructive pulmonary disease and community acquired pneumonia.


  • 1343 patients admitted to hospital with acute exacerbations of chronic obstructive pulmonary disease
  • and 1631 patients admitted with community acquired pneumonia.

Main Outcome Measures.

Hazard ratios for cardiovascular events at one year (defined as hospital admissions with

  • acute coronary syndrome, decompensated cardiac failure, serious arrhythmia, or sudden cardiac death) and
  • admissions for acute coronary syndrome (acute ST elevation myocardial infarction, non-ST elevation myocardial infarction, and unstable angina).

Secondary outcomes were all cause and cardiovascular mortality at one year.


  •  268 cardiovascular events occurred in the acute exacerbations of chronic obstructive pulmonary disease cohort and
  • 171 in the community acquired pneumonia cohort over one year.

After multivariable adjustment, clarithromycin use in acute exacerbations of chronic obstructive pulmonary disease

  • was associated with an increased risk of cardiovascular events and acute coronary syndrome—
  • hazard ratios 1.50 (95% confidence interval 1.13 to 1.97) and 1.67 (1.04 to 2.68).
After multivariable adjustment, clarithromycin use in community acquired pneumonia
  •  was associated with increased risk of cardiovascular events (hazard ratio 1.68, 1.18 to 2.38)
  • but not acute coronary syndrome (1.65, 0.97 to 2.80).
This association was found between clarithromycin use in acute exacerbations of COPD and
  • cardiovascular mortality (adjusted hazard ratio 1.52, 1.02 to 2.26)
  • but not all cause mortality (1.16, 0.90 to 1.51) .

No association was found between clarithromycin use in community acquired pneumonia and all cause mortality or cardiovascular mortality.
Use of β lactam antibiotics or doxycycline was not associated with increased cardiovascular events in patients with

  • acute exacerbations of chronic obstructive pulmonary disease, suggesting an effect specific to clarithromycin.

 Timing of Cardiovascular Events

The study found no significantly increased risk of cardiovascular events while patients were taking clarithromycin in the COPD cohort
(hazard ratio 1.73, 0.71 to 4.25), but
  • an increased risk was present after the clarithromycin course ended (1.41, 1.05 to 1.89).
In the community acquired pneumonia cohort, the hazard ratio for association between clarithromycin use and cardiovascular events
was 1.84 (0.75 to 4.51) during clarithromycin use and 1.66 (1.14 to 2.43) after the antibiotic was stopped.

Association With Duration of Antibiotic Use

Longer courses of clarithromycin were associated with more cardiovascular events. The median duration of treatment was seven days in both cohorts.
Less than three days of clarithromycin treatment was not associated with cardiovascular events in the chronic obstructive pulmonary disease cohort
(hazard ratio 0.89, 0.50 to 1.57) or the community acquired pneumonia cohort (0.63, 0.15-2.65), compared with patients who did not receive clarithromycin.

Effect of Age and Cardiovascular Risk Status

The hazard ratios of the effect of clarithromycin on cardiovascular events in such patients were
  • 1.35 (0.94 to 1.95) in those with a high cardiovascular risk and 0.88 (0.20 to 3.96) in those with a low risk.

The lowest hazard ratios for cardiovascular events were in patients aged 60 or below (1.01, 0.36 to 2.91).
The hazard ratio was 1.47 (1.01 to 2.14) for patients aged 60-79, and a higher risk was associated with

  • clarithromycin use in patients aged over 80 (hazard ratio 1.68, 1.05 to 2.69).

Use of Other Antibiotics

Use of β lactam or doxycycline was not associated with increased cardiovascular events
  • (hazard ratios 1.06 (0.83 to 1.37) and 0.96 (0.61 to 1.51), respectively)
in the chronic obstructive pulmonary disease cohort compared with patients not receiving antibiotics.

Possible Explanations for Findings

There was a strong association between prolonged (more than seven days) courses of clarithromycin and
  • cardiovascular events,
    • which strengthens the case for a true biological cause.

The association between duration of antibiotic treatment and cardiovascular events

  • could also represent residual confounding by severity of illness.

How do the results point to the effect on outcome after cessation of the drug?  The authors support an ischaemic mechanism.
Clarithromycin may activate macrophages, leading to an inflammatory cascade resulting in more vulnerable plaques that

  • over time may lead to acute coronary syndromes or sudden cardiac death by plaque rupture.


Prolonged courses of clarithromycin (more than seven days) may be associated with

  • increased risk of cardiovascular events,
  • especially in patients with a pre-existing history of coronary heart disease.

This may be of particular importance given recent data supporting long term macrolide use

  • to prevent exacerbations of chronic obstructive pulmonary disease.

Biomarkers Role in Drug Development

Biomarkers: An indispensible addition to the drug development toolkit

Biomarkers are becoming an essential part of clinical development. In this white paper, Thomson Reuters explores
the role of biomarkers as evaluative tools in improving clinical research and the challenges this presents.
The potential of biomarkers to

  • improve decision making,
  • accelerate drug development and
  • reduce development costs

is discussed with insights into a faster alternative to the conventional drug development approach and the promise of

  • safer drugs,
  • in greater numbers,
  • approved more quickly.
The attrition rate for drugs in clinical development is high: the percentage of tested products
  • entering phase I trials that eventually gain regulatory approval has been estimated at a paltry 8%.
Many of these failures happen late in clinical trials, with the consequence that expenditure in clinical drug development is increasing.
One study calculated that the cost of developing a drug increased by over 50% between 2002 and 2007. The related concern is that
very few drugs are making it out of the clinical research pipeline.
In 2007, the FDA approved just 17 new molecular entities and 2 biologic licenses, the lowest number since 1983.
The problem is mainly due to a gap in the industry’s ability to predict a drug candidate’s performance early, and with a large degree of certainty.
The convention in clinical research has been to measure the performance of novel therapies using clinical outcomes. This approach is
laborious, inexact and, as the US Food and Drug Administration (FDA ) puts it, decades old.

Why and what kinds of biomarkers do we determine are ESSENTIAL?

Biomarkers — a measure of
  • a normal biological process in the body,
  • a pathological process, or
  • the response of the body to a therapy —
may offer information about
  • the mechanism of action of the drug,
  • its efficacy, its safety and
  • its metabolic profile.
They feature heavily in the FDA ’s Critical Path Opportunities List for their potential
  • to speed the development and approval of medical products.
  • Moreover, they can predict drug efficacy more quickly than conventional clinical endpoints.
The first three examples are measures of drug efficacy and treatment response, but are not indicators of TOXICITY.

In 1960, researchers discovered that some patients with chronic myelogenous leukemia (CML), a form of adult leukemia

  • in which there is a proliferation of myeloid cells in the bone marrow,
  • have a specific genetic change associated with their cancer, a shortened version of chromosome.

The Philadelphia chromosome is caused by a translocation between chromosomes 9 and 22. The consequence of this genetic swap

  • is the creation of the BCR-ABL ‘oncogene’;
  • this cancer-causing gene produces a protein with elevated tyrosine kinase activity
  • that induces the onset of leukemia.

Researchers were able to use the Philadelphia chromosome as a biomarker

  • to indicate which patients would benefit from drug candidates (tyrosine-kinase inhibitors)
    • specifically targeting the rogue protein.

The drug imatinib (Gleevec) is a Tyr kinase inhibitor and

  • decreases the proliferation of Philadelphia chromosome+ cells,
  • slowing the progression of the disease.

Specific mutations in the BCR–ABL gene were biomarkers that predicted resistance to imatinib,

  • leading to the development of newer tyrosine-kinase inhibitors dasatinib and nilotinib.

In the late 1980’s, scientists discovered that HIV viral load could be used as a marker of disease progression
Viral load was used to show that patients receiving combination therapy had

  • a higher reduction in viral load than those on monotherapy.

Eventually, the viral load biomarker was used in the development and assessment of Highly Active Antiretroviral Therapy (HAART)
treatment regimens taken by many people living with HIV today.

The HER-2 gene and receptor was also discovered in the mid 1980’s. Between 20–30% of breast cancer patients show an

  • over-expression of the HER-2 receptor on their cancer cells (usually postmenopausal).

This biomarker indicates a higher risk of adverse outcomes, but it gave clinicians a new target for novel therapies, and

  • the antibody trastuzumab (Herceptin) was developed
  • to target HER-2 receptors in these ‘overexpressing’ patients.

 Preventing Drug Development Disasters

The need for biomarkers to guide clinical research is perhaps best highlighted in the stories of recent drug development failures.
Between 1995 and 2005, at least 34 drugs were withdrawn from the market, mainly as a result of hepatotoxic or cardiotoxic effects.

Many of us are familiar with the withdrawal in 2004 of the anti-inflammatory drug rofecoxib (Vioxx) due to concerns about its

  • increased risk of heart attack and stroke, and more recently with
  • the extremely serious adverse effects in the phase I clinical trial and subsequent failure of the monoclonal antibody, TGN1412.

TG N1412, a ‘superagonist’, produced by the firm TeGenero, stimulates an immune response. While originally intended to treat B cell
chronic lymphocytic leukemia and rheumatoid arthritis, it had been tested pre-clinically with no toxic or pro-inflammatory effects.
In 2006, six healthy male volunteers took part in a phase I clinical trial to test the safety of the candidate. Within 90 minutes of receiving the drug,

  • all six men were experiencing the beginnings of a ‘cytokine storm’, a term that describes
  • a cascade of proinflammatory cytokine release
  • leading to organ failure due to hypotension.

Although all the men survived, they required weeks of hospitalization. The cost of a failure, such as TGN1412,

  • in terms of patient health and lost resources is huge.

The TGN1412 trial failure highlighted a need for improved preclinical safety testing. It has been suggested that had procedures using safety biomarkers to

  • guide dosing and predict the toxicity of this drug been used, the disaster may not have occurred.

Biomarkers today

Today you “would not even conceive” of developing a new drug without simultaneously looking for biomarkers for
  • efficacy,
  • safety, and
  • to measure the pharmacodynamics of the drug,
says Dr Jeffrey Ross, Head of Pathology at the Albany Medical Center in New York, involved in the original work on HER-2.
The field of oncology is leading the way in the use of biomarkers in drug development. “Clinical trials are designed upon biomarker assays,”
“abstracts of phase II and III cancer trials talk about what biomarkers were selected.
  • In vivo biomarkers,
  • imaging biomarkers,
  • blood and tissue based biomarkers,

One example of a biomarker in use in oncology is circulating tumor cells (CTCs), a biomarker present in the blood of cancer patients.
At the moment, CTCs are used in the development of anti-cancer drugs as

  • an objective and direct measurement of the response of the cancer to a novel agent.

The way that clinical trials had been done previously was to enroll all patients

  • with a given disease independent of gene or phenotypic makers.
  • By selecting a population with the particular gene which is predicted
  • to be important for response to a novel therapeutic, then
    • a smaller clinical trial should be sufficient to see whether it works or not.

The chemotherapy drug irinotecan (Camptosar) is an example of personalized medicine,

  • using a biomarker to guide both clinical practice and subsequent clinical trials.

Irinotecan is used to treat advanced colorectal cancer. Once administered, irinotecan is

  • activated to the metabolite SN-38, and then
  • eventually inactivated in the body by the UGT1A1 enzyme.

In 2005, the US Food and Drug Administration added a warning to the label of the drug, stating that patients

  • homozygous for a particular a version of the UGT1A1 gene — the UGT1A1*28 allele,
  • associated with decreased UGT1A1 enzyme activity —
    • should be given a reduced dose.
Because patients with this allele clear the drug less quickly from their body than the rest of the population,
  • they effectively receive a greater exposure to the drug from the same dose.
As a consequence, they are at higher risk of potentially life-threatening side effects such as neutropenia (a decrease in white blood cells) and diarrhea.
The toxicity of irinotecan has long been a concern, and this biomarker now allows clinicians to better identify those patients who are at high risk of
  • serious side-effects (about 10% of the population are homozygous for UGT1A1*28).
And while this pharmacogenomics information has helped improve the clinical use and efficacy of irinotecan, it has also fed back into
the development of other drugs; this new understanding prompted the use of the UGT1A1 biomarker to guide other studies,
including several new irinotecan and oxaliplatin-based chemotherapy regimens.

Using preclinical biomarkers as evidence of efficacy

  • biomarkers can accelerate research by substituting for clinical symptoms as a measure of efficacy.
  • biomarkers can also replace clinical symptoms when it comes to measuring drug safety
  • an efficacy biomarker plus a safety biomarker will define not just whether a drug will work, but also what kind of dose might be relevant in humans

 Improving efficacy in cardiology

Consider the role of inflammatory marker C-reactive-protein (CRP) in cardiovascular disease. CRP is released by inflamed atherosclerotic plaques in the arteries
of individuals with coronary heart disease, and increased levels of CRP are associated with a greater risk of plaque rupture, but also of a silent heart attack.
CRP is being used as a biomarker to measure drug efficacy, in particular whether rosuvastatin (Crestor)

  • reduces the risk of cardiovascular morbidity and mortality
  • in apparently healthy individuals with low LDL-cholesterol levels but elevated CRP.

The JUPITER study (Justification for the Use of Statins in Primary Prevention: an Intervention Trial Evaluating Rosuvastatin) was halted in March 2008

  • due to firm evidence that the drug is indeed more beneficial than placebo and
  • improves the prognosis of individuals with high CRP levels.
A related biomarker of cardiovascular risk called neopterin. Just as CRP is produced by inflamed atherosclerotic plaques at risk of rupture,
neopterin is produced by activated macrophages in this inflammatory process. Circulating neopterin levels are higher in patients with ACS and may be
a marker of coronary disease activity. In addition, “Neopterin could also potentially be a marker of drug efficacy because
if you reduce the number of active macrophages in the plaque or the circulation, the levels of neopterin also decrease,” says Dr Juan Carlos Kaski,
Professor of Cardiovascular Science and Director of the Cardiovascular Biology Research Centre at St George’s University of London.

Other uses of biomarkers

These types of biomarkers can be used to drive critical ‘go/no go’ decision in drug development
Mechanistic or ‘target’ biomarkers can be used in pre-clinical or phase I trials to measure the pharmacological effect of the drug, i.e.
  • whether the drug interacts with its receptor, enzyme, or protein target,
  • whether it is distributed to the site where it needs to act,whether there is some
    • form of downstream pharmacology, and
  • the dose ranges in which the drug is pharmacologically active.
Drugs such as 5-HT4 receptor agonists (e.g. cisapride, mosapride), used in gastro-esophageal reflux disease (GERD), stimulate
  • the secretion of aldosterone as a side-effect.
Although aldosterone is not linked to GERD (and can’t be used as a biomarker of the disease), the hormone can be used
  • as a mechanistic biomarker in drug development to assess whether
    • novel 5-HT4 agonists in development have a pharmacological effect.

Discovering new biomarkers

The fundamental issue we have to deal with, both with target selection and developing better biomarkers,
  • is a better understanding of pathophysiology.
The clinical need is huge, not least in diseases like chronic obstructive pulmonary disease (COPD), an illness about which we know very little.

“COPD has very few markers to indicate severity and disease progression,” says Dr Trevor Hansel, Medical Director of the National Heart &
Lung Institute Clinical Studies Unit in London. Many pharmaceutical companies have begun to invest in ‘omics’ —

  • genomics,
  • proteomics,
  • metabonomics —

to begin to sort through this mountain of molecules and characterize biomarkers based on a molecular understanding of disease.

The ‘omics’ approach enables
  • the detection of small changes in tissue composition through protein profiling technologies such as
  • mass spectrometry and gel electrophoresis.

Essentially, it is about capturing a molecular profile from a clinical sample and converting this into

  • information about a clinical condition — for example the stage of disease or
  • what players are involved in the disease pathways.
“We will be able to look at diseases and catagorize them based on
  • biochemical or physiological findings, rather than just on symptoms” …  David Roblin, Pfizer

Companion Diagnostics and the Drug–Diagnostic Codevelopment Model

 Jan Trøst Jørgensen   Drug Development Research Nov 2012; 73(7):390-397.   http://dx.doi.org/10.1002/ddr.21029

The concept of using a predictive or selective diagnostic assay in relation to drug development goes back to the 1970s when

  • the selective estrogen receptor modulator, tamoxifen (AstraZeneca) was developed for metastatic breast cancer.
Clinical data showed that the estrogen receptor status correlated well with the clinical outcome when the patients were treated with tamoxifen.
It is only within the last decade that this model has gained widespread acceptance. The drug and the diagnostic are interdependent, and
if the development project proves successful, the companion diagnostic assay (CoDx) will end up determining the conditions for the use of the drug.
This gatekeeper role obviously requires that the CoDx assays adhere to the same strict rules and regulations that are known from the development of drugs.
For any CoDx assay, it must be documented that it is robust and reliable and that it possesses clinical utility. The article focus on some of the most important
aspects of the CoDx development process with emphasis on the clinical validation and clinical utility but also other critical issues, such as,
  • the biomarker selection process,
  • determination of the cut-off value, and
  • the analytical validation.

 Detecting Potential Toxicity in Mitochondria

 Brad Larson, Principal Scientist; Peter Banks, Scientific Director; BioTek Instruments, Winooski, Vt.
Mitochondrial dysfunction may be
  • inherited,
  • arise spontaneously, or
  • develop as a result of drug toxicity.
Mitochondrial toxicity as a result of pharmaceutical use may damage key organs, such as the liver and heart. For example,
  • nefazodone—a depression treatment—was withdrawn from the U.S. market after it was shown to
significantly inhibit mitochondrial respiration in liver cells, leading to liver failure. Troglitazone, an anti-diabetic and anti-inflammatory,
was withdrawn from all markets after research concluded that it caused acute mitochondrial membrane depolarization, also leading to liver failure.
Drug recalls are costly to a manufacturer’s bottom line and reputation, and more importantly, can be harmful or even fatal to users. As drug
discovery continues to evolve, much lead compound research now includes careful review of its interaction and potential toxicity with mitochondria.
Cytotoxicity and ATP production are measured in cancerous and normal hepatocytes using a known inducer of cellular necrosis. (All figures: BioTek Instruments)
Cell-based mitochondrial assays in microplate format may include
  • mitochondrial membrane potential,
  • total energy metabolism,
  • oxygen consumption, and
  • metabolic activity;
and offer a truer environment for mitochondrial function in the presence of drug compounds compared to isolated mitochondria-based tests.
Combining more than one assay in a multiplex format increases the amount of data per well while decreasing data variability arising from running the assays separately.
The aggregated data also provides a more encompassing analysis of the drug’s effect on mitochondria than a single test.
Human cardiac muscle

Human cardiac muscle (Photo credit: Carolina Biological Supply Company)

English: Non-sustained run of ventricular tach...

English: Non-sustained run of ventricular tachycardia on telemonitoring from a patient with chemotherapy-induced cardiomyopathy. (Photo credit: Wikipedia)

English: Doxorubicin 3D model Русский: Трёхмер...

English: Doxorubicin 3D model Русский: Трёхмерная модель молекулы доксорубицина (Photo credit: Wikipedia)

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Mitochondrial Dynamics and Cardiovascular Diseases

Author and Curator: Ritu Saxena, Ph.D.


Morphological changes in mitochondria have been observed in several human diseases including myopathies, diabetes mellitus, liver diseases, neurodegeneration, aging, and cancer. Ong et al (2010) studied neonatal rat ventricular myocytes as an experimental model of aging and concluded that the interplay between mitochondrial fission and autophagy controls the rate of mitochondrial turnover. A disturbance in the balance is observed in aging heart cells resulting in giant mitochondria. This observation is an indication that mitochondrial morphology is connected to pathogenesis of cardiac disease. http://www.ncbi.nlm.nih.gov/pubmed/20631158 Thus, it is important to understand the mechanism of mitochondrial dynamics in order to correlate it with the development of cardiovascular diseases.

Mitochondrial dynamics

The shape of mitochondria is very dynamic in living cells, constantly interchanging between thread-like and grain-like morphology through what we know now as the fusion and fission processes, respectively. The fusion and fission processes together with the mitochondrial movement have been termed “mitochondrial dynamics”.  Nucleoids, the assemblies of mitochondrial DNA (mtDNA) with its associated proteins, are distributed during fission in such a way that each mitochondrion contains at least one nucleoid.

Mitochondrial fusion is a complex process that involves the fusing together of four lipid bilayers. Proteins involved in the mitochondrial fission and fusion have been discussed in an earlier post published on October 31, 2012. Mitochondrial fusion requires two 85kD-GTPase isoforms mitofusin1 (Mfn1) and mitofusin2 (Mfn2). Mfn1 and Mfn1 are both anchored to the outer mitochondrial membrane. They contain – two transmembrane domains connected by a small intermembrane-space loop, a cytosolic N-terminal GTPase domain and two cytosolic hydrophobic heptad-repeat coiled-coil domains. The coiled-coil domains of Mfn1 and Mfn2 help in tethering adjacent mitochondria in both homo-oligomeric and hetero-oligomeic fashion. The fusion process requires GTP hydrolysis and the cells where Mfn2 had a GTPase mutation; mitochondria were not able to undergo fusion even after tethering. Mitochondrial fission and fusion have been illustrated in Figure 1.

Mitochondrial fission is opposite of the fusion process. Mammalian mitochondria undergo fission by the interaction of two proteins: dynamin-like protein 1 or dynamin-related protein 1 (DLP1/Drp1), an 80–85-kD cytosolic GTPase, and human fission protein 1 (hFis1), a 17-kD outer mitochondrial membrane anchored protein. Mitochondrial fission too requires GTP hydrolysis. DLP1 mainly localizes in the cytosol and with the help of hFis1, DLP1 is recruited to the constriction sites of the membrane. DLP1 translocation depends on actin and microtubules and once inside, DLP1 oligomerizes into a ring around the mitochondrion. The self-assembly of DLP1 stimulates the final step of fission which is disassembly and it requires GTP hydrolysis.

Figure 1: Model of mammalian mitochondrial fission and fusion (Hom et al, J Mol Cell Cardiol, 2009)


Additional information on different aspects of mitochondria could be found articles published earlier in the Pharmaceutical Intelligence webpage.

Mitochondrial dynamics in the heart

In cultured cardiovascular cell line the mitochondria are arranged in a filamentous network and are highly dynamic, constantly undergoing fusion and fission. Similar mitochondrial network is observed in vascular smooth muscle cells, cardiac stem cells, and neonatal cardiomyocytes. Thus, these cell types have been used to study mitochondrial dynamics.

However, in the adult cardiomyocyte, there are three distinct populations of mitochondria:

(i)           peri-nuclear mitochondria,

(ii)         subsarcolemmal (SSC) mitochondria, and

(iii)       interfibrillar (IF) mitochondria

Electron micrographs of adult cardiac muscle cells, especially ventricular myocytes, show that mitochondria are numerous, making up about 35% of the cell volume, and that mitochondria are highly organized and compacted between contractile filaments and next to T-tubules. This crystal-like pattern of mitochondria in adult ventricular myocytes raises an interesting question- Do the mitochondria in these cells also undergo physiological fission, fusion, and movement just like other cell types? Whether the crystal-like lattice arrangement restricts their movements and prevents them from undergoing fusion or fission is unclear. It has been speculated that the fission and fusion processes might occur at a slower rate because of the tight packing. A four-dimensional (x, y, z axis and time) live-cell imaging is needed to detect possible movements like mitochondria winding slowly through the myofibrils in the third dimension.

Figure 2. Representative electron micrograph of adult murine heart depicting the three subpopulations of mitochondria: perinuclear (PN) mitochondria; interfibrillar (IF) mitochondria; and subsarcolemmal mitochondria (SSM). Photo credit: Ong et al, Cardiovascular Research (2010).

Expression of fission/fusion proteins in adult heart: Interestingly, it has been observed that proteins required for mitochondrial dynamics including fission and fusion proteins is abundantly present in the adult heart and would have been active during cardiomyocyte differentiation to ensure the unique spatial organization of the three different subpopulations of cardiac mitochondria.

Several studies suggest the existence of fission and fusion proteins in the adult heart.

  • Mfn1 and Mfn2 fusion proteins have been found to be expressed in highest amounts in the heart compared to that in human tissues of pancreas, skeletal muscle, brain, liver, placenta, lung, and kidney using both Northern and Western blot analysis. Infact, Mfn2 mRNA was found to be abundantly expressed in heart and muscle tissue but expressed only at low levels in other tissues. Mfn1 and Mfn2 expression has also been confirmed in heart tissue of rat and mouse by RT-PCR.
  • hFis1, a fission protein, has been shown to be ubiquitously expressed in isolated rat mitochondria in heart tissue apart from several other tissues.
  • DLP1 mRNA, coding for a fusion protein, have been detected in high levels in several adult tissues including heart, skeletal muscle, kidney and brain.
  • OPA1 codes for another fusion protein and four transcripts of OPA1 have been detected in adult mouse hearts.

Mitochondria in cardiac diseases:

Morphological changes in mitochondria have been observed in several human diseases including myopathies, diabetes mellitus, liver diseases, neurodegeneration, aging, and cancer. Ong et al (2010) studied neonatal rat ventricular myocytes as an experimental model of aging and concluded that the interplay between mitochondrial fission and autophagy controls the rate of mitochondrial turnover. A disturbance in the balance is observed in aging heart cells resulting in giant mitochondria. This observation is an indication that mitochondrial morphology is connected to pathogenesis of cardiac disease. http://www.ncbi.nlm.nih.gov/pubmed/20631158

Abnormal mitochondrial morphology corresponding to various cardiac diseases has been listed as follows:

  • Abnormally small and disorganized mitochondria – observed in endstage dilated cardiomyopathy, myocardial hibernation, cardiac rhabdomyoma, and ventricular-associated congenital heart diseases.
  • Disorganized clusters of fragmented mitochondria – observed in Tetralogy of Fallot and are located away from contractile filaments, along with having a very small diameter measured to be 0.1 μm as observed in the electron micrographs.
  • Big and defective mitochondria – observed in senescent cardiomyocytes.



Condition Cell type Change in mitochondrial morphology Other findings Study
Ischemia-perfusion injury HL-1 cells Fission P38 inhibition at reperfusion allows mitochondrial re-fusion Brady et al
β – Adrenergic stimulation by isoproterenol or exercise Adult murine heart Not investigated Phosphorylation and inhibiton of Drp1 at Ser656 Cribbs and Strack et al
Cardiac differentiation Embryonic stem cells Fusion Fusion is required to support Oxidative phosphorylation Chung et al
Hyperglycemia H9C2 rat myoblast Fission Yu et al
Post-MI heart failure and dilated cardiomyopathy Adult rat and human heart Fragmentation Decrease in OPA1 Chen et al
Diabetes Murine coronary endothelial cell Fission Decreased OPA1, increased Drp1 Makino et al
Diabetes Adult murine diabetic heart Fission Lower mitochondrial membrane potential Williamson et al
Ischaemia-reperfusion injury and cardioprotection HL-1 cells, adult heart Fission Inhibiting fission cardioprotective Ong et al
Cytosolic calcium overload Neonatal cardiomyocytes and adult heart Fission Hom et al

Table 1: Studies implicating changes in mitochondrial morphology in cardiovascular diseases, Adapted from Ong et al, Cardiovascular Research (2010).

Mitochondrial dynamics in heart failure

Fission and Fusion in Heart Failure

Mutation or abnormal expression of fission and fusion proteins have been implicated in several diseases including neuropathies, Parkinson’s disease, type 2 diabetes and so on. However, few studies have addressed the involvement of mitochondrial dynamics in heart failure. Research groups have used cardiac-like cell lines, neonatal and adult cardiomyocytes, and animal models to demonstrate the importance of fission and fusion proteins. Observations from some studies have been listed below:

  • Mitochondria are highly organized and compacted between contractile filaments (interfibrillar) or adjacent to the sarcolemma (subsarcolemmal) in adult mammalian cardiomyocytes. However, during heart failure, interfibrillar mitochondria may lose their normal organization.
  • There is also a reduction in size and density of interfibrillar mitochondria in rodent models of heart failure.
  • It was recently reported that OPA1 is decreased in both human and rat heart failure.
  • Electron microscopic data showed an increase in the number and decrease in the size of the mitochondria in a coronary artery ligation rat heart failure model.
  • Inhibition of fission in cultured neonatal ventricular myocytes by overexpression of dominant negative mutant form of Drp1, Drp1-K38A, prevents overproduction of ROS, mitochondrial permeability transient pore formation and ultimately cell death under high glucose conditions.
  • In cultured neonatal and adult cardiomyocytes, cytosolic Ca2+ overload induced by thapsigargin (Tg) or potassium chloride (KCl) resulted in rapid mitochondrial fragmentation. Calcium overload is a common feature in heart failure, which might lead to increase in fission contributing to decrease in energy production in the failing heart.
  • In H9c2 cells, reduction in OPA1 increased apoptosis both at baseline and after simulated ischemia, via cytochrome c release from mitochondria.
  • Drosophila heart tube-specific silencing of OPA1 and mitochondrial assembly regulatory factor (MARF) increased mitochondrial morphometric heterogeneity and induced heart tube dilation with profound contractile impairment. In this model, human MFN1/2 was rescued MARF RNAi induced cardiomyopathy.
  • MFN-2-deficient mice have mild cardiac hypertrophy and mild depression of cardiac function. Also, mitochondria of cardiac myocytes lacking MFN-2 are pleiotropic and larger.
  • In rat hearts, decreased MFN2, increased Fis1 and no change in OPA1 expression was observed 12–18 weeks after myocardial infarction. http://www.ncbi.nlm.nih.gov/pubmed/22848903

However, further research is needed to accurately and fully define the role of abnormal mitochondrial morphology in heart failure. Those researches might lead to developing new interventions for treating abnormal mitochondrial function based diseases.


Related reading:

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Percutaneous Endocardial Ablation of Scar-Related Ventricular Tachycardia

Reporter: Aviva Lev-Ari, PhD, RN

UPDATED on 11/15/2013

Atrial Fibrillation Catheter Ablation Versus Surgical Ablation Treatment (FAST)

A 2-Center Randomized Clinical Trial

  1. Lucas V.A. Boersma, MD, PhD, FESC;
  2. Manuel Castella, MD, PhD;
  3. WimJan van Boven, MD;
  4. Antonio Berruezo, MD;
  5. Alaaddin Yilmaz, MD;
  6. Mercedes Nadal, MD;
  7. Elena Sandoval, MD;
  8. Naiara Calvo, MD;
  9. Josep Brugada, MD, PhD, FESC;
  10. Johannes Kelder, MD;
  11. Maurits Wijffels, MD, PhD;
  12. Lluís Mont, MD, PhD, FESC

+Author Affiliations

  1. From the Departments of Cardiology (L.V.A.B., J.K., M.W.) and Cardiothoracic Surgery (W.J.v.B., A.Y.), St. Antonius Hospital, Nieuwegein, the Netherlands, and Thorax Institute, Hospital Clinic, University of Barcelona, Barcelona, Catalonia, Spain (M.C., A.B., M.N., E.S., N.C., J.B., L.M.).
  1. Correspondence to Lucas V.A. Boersma, MD, PhD, FESC, Cardiology Department, St. Antonius Hospital, PO 2500, Nieuwegein, Netherlands. E-maill.boersma@antoniusziekenhuis.nl


Background—Catheter ablation (CA) and minimally invasive surgical ablation (SA) have become accepted therapy for antiarrhythmic drug–refractory atrial fibrillation. This study describes the first randomized clinical trial comparing their efficacy and safety during a 12-month follow-up.

Methods and Results—One hundred twenty-four patients with antiarrhythmic drug–refractory atrial fibrillation with left atrial dilatation and hypertension (42 patients, 33%) or failed prior CA (82 patients, 67%) were randomized to CA (63 patients) or SA (61 patients). CA consisted of linear antral pulmonary vein isolation and optional additional lines. SA consisted of bipolar radiofrequency isolation of the bilateral pulmonary vein, ganglionated plexi ablation, and left atrial appendage excision with optional additional lines. Follow-up at 6 and 12 months was performed by ECG and 7-day Holter recording. The primary end point, freedom from left atrial arrhythmia >30 seconds without antiarrhythmic drugs after 12 months, was 36.5% for CA and 65.6% for SA (P=0.0022). There was no difference in effect for subgroups, which was consistent at both sites. The primary safety end point of significant adverse events during the 12-month follow-up was significantly higher for SA than for CA (n=21 [34.4%] versus n=10 [15.9%]; P=0.027), driven mainly by procedural complications such as pneumothorax, major bleeding, and the need for pacemaker. In the CA group, 1 patient died at 1 month of subarachnoid hemorrhage.

Conclusion—In atrial fibrillation patients with dilated left atrium and hypertension or failed prior atrial fibrillation CA, SA is superior to CA in achieving freedom from left atrial arrhythmias after 12 months of follow-up, although the procedural adverse event rate is significantly higher for SA than for CA.

Clinical Trial Registration—URL: http://clinicaltrials.gov. Unique identifier:NCT00662701.

Key Words:



 A single-center experience of clinical and electrophysiologic outcomes of patients undergoing percutaneous endocardial ablation of scar-related Ventricular Tachycardia (VT) is reported in

IMAJ Isr Med Assoc J 2010; 12: 667-670.


 Catheter ablation can control or prevent recurrent ischemic VT and reduce incidence of implantable cardioverter defibrillator (ICD) therapy. The ablation can be done during VT in patients with stable VTs or during sinus rhythm in patients with unstable unmappable VTs by targeting the scar border using electroanatomic substrate mapping. VT ablation should be offered to ischemic patients with recurrent uncontrolled VT. Radiofrequency ablation of VT in patients with ischemic cardiomyopathy was proposed to treat and control recurrent VT

J Cardiovasc Electrophys 2005; 16(Suppl 1): S59-70.

Curr Opin Cardiol 2005; 20: 42-7.

An experience with VT ablation in patients with ischemic cardiomyopathy using the electroanatomic mapping system (CARTO) was presented in

Circulation 1997; 95: 1611-22

There are several reasons for the limited success of ischemic VT ablation. Ventricular scars are not electrically homogenous. They are composed of variable regions of dense fibrosis that create conduction block and surviving myocyte bundles with interstitial fibrosis and diminished coupling, which produce circuitous slow conduction paths that promote reentry

Circulation 1993; 88: 915-26.

Repeated programmed stimulation typically induces more than one monomorphic VT. Multiple VTs can be due to different circuits in widely disparate areas of scar, different axis from the same region of the scar, or changes in activation remote from the circuit due to functional regions of block

Circulation 2007; 115: 2750-60.

Catheter ablation using conventional techniques are suitable for stable VT. VT reentry circuit can be defined using electroanatomic mapping (CARTO) only during stable and tolerable tachycardia. However, many patients with reduced ejection fraction secondary to coronary heart disease have unstable VTs. These patients do not tolerate sustained VT or rapid pacing. Thus, electric or pace mapping is not available in most cases (unmappable VT with catheter technique). In these cases, scar mapping and ablation can be done only during sinus rhythm using the CARTO system

IMAJ Isr Med Assoc J 2007; 9: 260-4.

Radiofrequency catheter ablation of ventricular tachycardia in the setting of ischemic cardiomyopathy has emerged recently as a useful adjunctive therapy to ICD. Scar related reentrant ventricular tachycardia is the most common underlying mechanism of sustained monomorphic VT in patients with ischemic heart disease.

Limitations of Alternative Treatment Methods:

1.            Recurrent ICD shocks have had physiological and psychological side effects.

2.            Antiarrythmic drugs are used to reduce incidence of ICD therapy, but their role in reducing mortality is not proven. In addition, these drugs have important side effects including pro-arrythmic effect and worsening of heart failure status.

Conclusions: Ablation of ischemic VT using electroanatomic scar mapping is feasible, has an acceptable success rate and should be offered for ischemic patients with recurrent uncontrolled VT.


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fibrillation ablation in the setting of ischemic heart disease. J Cardiovasc

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2. Stevenson WG. Catheter ablation of monomorphic ventricular tachycardia.

Curr Opin Cardiol 2005; 20: 42-7.

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Circulation 2007; 115: 2750-60.

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nonischemic cardiomyopathy. Circulation 2000; 101: 1288-96.

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of ventricular tachycardia on left ventricular function in patients with prior

myocardial infarction. J Interv Card Electrophysiol 2002; 7: 243-7.

8. Stevenson WG, Wilber DJ, Natale A, et al; for the Multicenter Thermocool

VT Ablation Trial Investigators. Irrigated radiofrequency catheter ablation

guided by electroanatomic mapping for recurrent ventricular tachycardia

after myocardial infarction. The Multicenter Thermocool Ventricular

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9. Reddy VY, Reynolds MR, Neuzil P, et al. Prophylactic catheter ablation for

the prevention of defibrillator therapy. N Engl J Med 2007; 357: 2657-65.

10. Szumowski L, Sanders P, Walczak F, et al. Mapping and ablation of

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12. de Bakker JM, van Capelle FJ, Janse MJ, et al. Slow conduction in the infarcted

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13. Suleiman M, Gepstein L, Roguin A, et al. Catheter ablation of cardiac

arrhythmias guided by electroanatomic imaging (CARTO): a single-center

experience. IMAJ Isr Med Assoc J 2007; 9: 260-4.

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