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Leptin signaling in mediating the cardiac hypertrophy associated with obesity

Posted in Cardiomyopathy, Cell Biology, Signaling & Cell Circuits, Frontiers in Cardiology and Cardiovascular Disorders, Metabolomics, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Nutrition, Population Health Management, Genetics & Pharmaceutical, Population Health Management, Nutrition and Phytochemistry, Proteomics, Systemic Inflammatory Response Related Disorders, tagged , , , , , on November 3, 2013| Leave a Comment »

Leptin signaling in mediating the cardiac hypertrophy associated with obesity

Curators: Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

There has been a lot of interest in leptins and both insulin resistance and obesity for the last decade.  The association between obesity and cardiac hypertrophy is also known, but what drives this association.  We have covered heart disease from many aspects in a long series of articles.  The next is a pleasure to take in.

Importance of leptin signaling and signal transducer and activator of transcription-3 activation in mediating the cardiac hypertrophy associated with obesity

Maren Leifheit-Nestler12, Nana-Maria Wagner13, Rajinikanth Gogiraju1,Michael Didié14, Stavros Konstantinides15, Gerd Hasenfuss1 and Katrin Schäfer1*

1Department of Cardiology and Pulmonary Medicine, Heart Research Center, Georg August University Medicine Goettingen, Robert Koch Strasse 40, D-37075, Göttingen, Germany

2Current address: Department of Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Hannover, Germany

3Current address: Clinic for Anesthesiology and Intensive Care Medicine, University Medicine Rostock, Rostock, Germany

4Department of Pharmacology, Georg August University Medicine Goettingen, Goettingen, Germany

5Current address: Center for Thrombosis and Hemostasis, University Medicine Mainz, Mainz, Germany

J Translational Medicine: Cardiovascular, Metabolic and Lipoprotein Translation. 2013; 11:170.  http://www.translational-medicine.com/content/11/1/170

http://dx.doi.org/10.1186/1479-5876-11-170

This is an Open Access article distributed under the terms of the Creative Commons Attribution License 
http://creativecommons.org/licenses/by/2.0

Abstract

Background

The adipokine leptin and its receptor are expressed in the heart, and

  • leptin has been shown to promote cardiomyocyte hypertrophy in vitro.

Obesity is associated with

  • hyperleptinemia 
  • hypothalamic leptin resistance and
  • an increased risk to develop cardiac hypertrophy and heart failure.

However, the role of cardiac leptin signaling in mediating the cardiomyopathy associated with increased body weight is unclear, in particular, whether it develops subsequently to cardiac leptin resistance or overactivation of hypertrophic signaling pathways via elevated leptin levels.

Methods

The cardiac phenotype of high-fat diet (HFD)-induced obese wildtype (WT) mice was examined and compared to age-matched genetically obese leptin receptor (LepR)-deficient (LepRdb/db) or lean WT mice. To study the role of leptin-mediated STAT3 activation during obesity-induced cardiac remodeling,

  • mice in which tyrosine residue 1138 within LepR had been replaced with a serine (LepRS1138) were also analyzed.

Results

Obesity was associated with hyperleptinemia and elevated cardiac leptin expression in both diet-induced and genetically obese mice.

  • Enhanced LepR and STAT3 phosphorylation levels were detected in hearts of obese WT mice, but not in those with LepR mutations, and
  • exogenous leptin continued to induce cardiac STAT3 activation in diet-induced obese mice.

Although echocardiography revealed signs of cardiac hypertrophy in all obese mice,

  • the increase in left ventricular (LV) mass and diameter was significantly more pronounced in LepRS1138 animals.

LepRS1138 mice also exhibited an increased activation of signaling proteins downstream of LepR, including Jak2 (1.8-fold), Src kinase (1.7-fold), protein kinase B (1.3-fold) or C (1.6-fold). Histological analysis of hearts revealed that the inability of leptin to activate STAT3 in LepRdb/db and LepRS1138 mice

  • was associated with reduced cardiac angiogenesis as well as increased apoptosis and fibrosis.

Conclusions

Our findings suggest that hearts from obese mice continue to respond to elevated circulating or cardiac leptin, which

  • may mediate cardioprotection via LepR-induced STAT3 activation, whereas
  • signals distinct from LepR-Tyr1138 promote cardiac hypertrophy.

On the other hand, the presence of cardiac hypertrophy in obese mice with complete LepR signal disruption indicates that additional pathways also play a role.

Keywords:

Heart; Hypertrophy; Leptin; Obesity; Signal transduction; STAT3

Background

Obesity is frequently associated with elevated circulating leptin levels [1] and an increased risk to develop cardiac hypertrophy [2,3] or heart failure [4]. Clinical studies demonstrated a positive correlation between serum leptin levels and left ventricular (LV) mass or wall thickness [5,6], independent of blood pressure levels, suggesting a direct role for leptin in the pathogenesis of obesity-associated cardiomyopathy. Furthermore, leptin was shown to promote hypertrophy of isolated rat or human ventricular cardiomyocytes [7,8], and

  • this effect could be prevented using neutralizing antibodies [9].

Cardiac hypertrophy also develops in obese rodents fed high-fat diet (HFD)[10,11], and

  • studies in mice with (functional) leptin deficiency suggested that the cardiac hypertrophy developing in states of chronic hyperleptinemia
  • may result from the inability to transduce anti-hypertrophic and/or cardioprotective effects of the adipokine [12,13].

The effects of leptin on cell shortening and intracellular Ca2+ transients were abrogated in cardiomyocytes isolated from HFD-fed obese rats [14], but then others found

  • a preserved signal transduction in response to leptin in hyperleptinemic obese mice [15,16] or rats[17].

The leptin receptor (LepR) belongs to the family of cytokine type I receptors that signal via activation of

  • Janus kinase (Jak)-2 and
  • signal transducer and
  • activator of transcription (STAT)-3 [18].

Analysis of cardiomyocytes ex vivo revealed leptin promotes hypertrophy via activation of p38 and p42/44 MAP kinases as well as protein kinase B (Akt) [19,20]. But it is unknown whether STAT-3 activation downstream of LepR is required to transmit the cardiac effects of leptin and whether it may be involved in mediating protective (i.e. anti-apoptotic, anti-fibrotic or pro-angiogenic) signals, as previously reported in mice with cardiomyocyte-specific STAT-3 deletion [21,22].

In this study, we examined the cardiac phenotype of diet-induced (i.e. with hypothalamic leptin resistance) and genetically obese (i.e. with systemic leptin receptor deficiency) hyperleptinemic mice, developing with age or after continuous β-adrenergic stimulation. Moreover, we determined

  • the importance of leptin-mediated STAT-3 activation
  • for the development of cardiac hypertrophy in obesity
  • by analyzing mice with targeted mutation of the STAT3 binding site within LepR.

Methods

Animals

C57Bl6/J leptin receptor-deficient db/db (LepRdb/db; BKS.Cg Leprdb/Leprdb) mice and C57Bl6/J wildtype (WT) controls were obtained from Harlan Winkelmann, Germany. Mice heterozygous mutant for the LepRS1138 allele (on the congenic B6.129/J background; 98- > 99% homozygous for C57Bl/6; [23]) were obtained from Professor Martin Myers (University of Michigan Medical School, Ann Arbor, USA) and bred at the animal facility of the University of Goettingen, Germany, to generate homozygous mutant obese LepRS1138 mice. Age- and gender-matched WT (LepR+/+) and heterozygous (LepRS/+) littermates were used as controls. To induce obesity, 3 months-old mice were switched to high-fat diet (HFD; D12451) for 4 months, while controls were maintained on normal rodent chow (D12450B; both Research Diets Inc.). The composition of both diets is shown in Additional file 1: Table S1. To examine the cardiac response to hypertrophic stimuli other than leptin, osmotic minipumps (Alzet®; model 2002; Charles River Laboratories) were filled with isoprenaline hydrochloride (Sigma; 20 mg/kg body weight [BW] per day) and implanted for 14 days under the dorsal skinfold of 2 months-old, 2% isoflurane anesthetized mice. At the time of tissue harvest, mice were weighed followed by intraperitoneal anesthesia with a mixture of 2% xylazine (6 mg/kg BW) and 10% ketamine hydrochloride (100 mg/kg BW), and blood was drawn by cardiac puncture. Hearts were rapidly excised, the atria removed and ventricles immediately processed for protein isolation or cryoembedding, respectively. All animal care and experimental procedures had been approved by the institutional Animal Research Committee and complied with national guidelines for the care and use of laboratory animals.

Additional file 1: Table S1. Diet composition.
Format: DOC Size: 33KB

Serum analysis

Freshly drawn blood was allowed to clot at room temperature (RT) for 30 min, followed by centrifugation for 10 min at 3,000 rpm. The supernatant was stored at -80°C pending analysis of serum leptin levels using specific enzyme-linked immunoassays (ELISA; R&D Systems).

Echocardiography

Echocardiography was performed by a blinded examiner at the day before tissue harvest in mice under 1.5% isoflurane anesthesia using the VisualSonics Vevo 2100 system (Visualsonics) equipped with a 30 MHz center frequency ultrasound transducer, as previously described [24]. M-mode echocardiographical recordings were used to determine the end-diastolic and end-systolic LV diameter (EDD and ESD, respectively) and the ventricular wall thickness (WTh), corresponding to the mean of the anterior and posterior WTh. LV mass was calculated using the formula: 1.055 × ([AWTh + EDD + PWTh]3 – EDD3). Fractional shortening (FS) was calculated as (EDD – ESD)/EDD × 100. B-mode echocardiography images were used to calculate the heart weight, using the equation: 1.05 × (5/6) × ((Episyst × (Lsyst + ((AWThsyst + PWThsyst)/2))) – (Areasyst × Lsyst)).

Histology and immunohistochemistry

Histochemical analyses were performed on 5 μm-thick frozen cross sections through the LV. For each mouse, 4 sections (approx. 500 μm apart) and 4 randomly selected viewing fields (at 200-fold magnification) per section were analyzed and findings averaged. Cardiac fibrosis was determined after overnight incubation in Bouin’s fixative followed by Masson’s trichrome (MTC) stain. Monoclonal rat antibodies against mouse CD31 (Santa Cruz Biotechnology) were used to detect endothelial cells [24,25]. Their number was manually counted by a person blinded to the mouse genotype and expressed per mm2 or cardiomyocyte, respectively.

Single cardiomyocytes were visualized by incubation with fluorescein-labeled wheat germ agglutinin (WGA; Molecular Probes), followed by determination of the cardiomyocyte cross-sectional area (CSA) using image analysis software (Image ProPlus). Per cross section, at least 10 randomly selected cardiomyocytes were evaluated and results averaged. Apoptosis was analyzed using the ‘In Situ Cell Death Detection kit’ (Roche). Cell nuclei were visualized using 4′,6-diamidino-2-phenylindole (DAPI; Sigma).

Immunoprecipitation and western blot analysis

Membranes were blocked in 1% bovine serum albumine (in TBS, containing 0.1% Tween-20) prior to incubation with antibodies against phosphorylated (p)-Akt (S473) and total Akt, p-Jak2 (Y1007/1008) and total Jak2, p-p38 (T180/Y182) and total p38, p-p42/44 (T202/Y204) and total p42/44, p-Src (Y416) and total Src, p-STAT3 (Y705) and total STAT3, or p-PKC (pan), respectively (all Cell Signaling Technologies), or against leptin (R&D Systems) and GAPDH (Biotrend), respectively. Protein bands were visualized using HRP-conjugated secondary antibodies (Amersham Biosciences), followed by detection with SuperSignal® West Pico Substrate (Pierce). For the analysis of LepR phosphorylation, 100 μg total heart tissue lysates were immunoprecipitated under rotation at 4°C with 2 μg anti-LepR antibody (against an internal domain present in the short and long isoforms of murine LepR; Santa Cruz Biotechnology) plus 50 μL nProtein A Sepharose™ 4 Fast Flow beads (GE Healthcare) followed by detection of phosphorylated tyrosines (p-Tyr [PY20]; Santa Cruz Biotechnology) or LepR. For the analysis of STAT3 phosphorylation in response to acute elevations of circulating leptin, mice were fasted overnight, injected with recombinant murine leptin (1 mg/kg BW i.p.) and hearts harvested 30 min later.

Statistical analysis

Quantitative data are presented as mean ± standard error of the mean (SEM). Normal data distribution was tested using the D’Agostino & Pearson omnibus normality test. When three or more groups were compared, ANOVA was employed, if samples were normally distributed, or Kruskal-Wallis test, if not. For post-hoc comparisons, ANOVA was followed by Bonferroni’s and Kruskal-Wallis by Dunn’s multiple comparison test. Differences before and after isoprenaline infusion were tested using Student’s t-test for paired means. Statistical significance was assumed when P reached a value less than 0.05. All statistical analyses were performed using GraphPad PRISM software, version 4.01 (GraphPad Software Inc).

Results

Clinical and experimental studies revealed that obesity is associated with LV hypertrophy [10,11], an important risk factor for the development of heart failure. As shown in Tables 1 and 2, WT mice fed HFD for 4 months (WT + HFD; mean body weight [BW], 44±1.9 g) to induce obesity exhibited a non-significant trend towards an increased mean heart weight, LV mass and WTh compared to age-matched lean controls fed normal chow (BW, 29±1.0 g). Marked LV hypertrophy was observed in 7 months-old obese LepRdb/db mice (Table 1 and 2), consistent with a previous report [12]. Longitudinal sections through hearts of WT, WT + HFD and LepRdb/db mice are shown in Figure 1A, representative M-mode echocardiography recordings in Figure 1B and cardiac cross-sections after WGA staining to delineate cardiomyocyte borders in Figure 1C. Of note, adiposity in mice with LepR deficiency was more pronounced compared to age-matched WT + HFD mice (Table 1; P < 0.001), in which obesity develops as result of hypothalamic resistance to chronically elevated leptin levels[26].

1479-5876-11-170-1 F1 Cardiac phenotype of lean and obese WT

Figure 1.Cardiac phenotype of lean and obese WT, WT + HFD, LepRS1138 and LepRdb/db mice.
(A)Representative H&E-stained longitudinal sections through hearts of 7 months-old mice are shown. Magnification, ×10.(B) Representative M-mode echocardiographic recordings.(C) Representative images of wheat germ agglutinin (WGA)-stained myocardial cross sections. The mean cardiomyocyte cross-sectional areas are given in Table 1.

Table 1.Body, visceral fat and heart weights in 7 months-old mice

Table 2.Echocardiographic parameter in 7 months-old mice

The presence of cardiac hypertrophy in LepR-deficient and, to a lesser extent also in diet-induced obese mice, suggests that it develops as a result of the heart’s inability to respond to elevated systemic (Table 1) and/or cardiac (Figure 2A) leptin levels. In this regard, Western blot analysis revealed increased levels of phosphorylated (p-) LepR (Figure 2B) and STAT-3 (Figure 2C) protein in hearts of HFD-induced obese mice (P < 0.05 vs. WT for both), whereas findings in LepRdb/db mice did not differ from those in lean controls or were reduced compared to those in WT + HFD mice (P < 0.05 for differences in LepR phosphorylation). Moreover, both lean and HFD-induced obese WT mice responded to a single i.p. injection of recombinant murine leptin with a significant increase in the cardiac STAT-3 phosphorylation (Figure 2D), suggesting a preserved cardiac leptin signal transduction in hyperleptinemic, diet-induced obese mice.

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Figure 2.Cardiac leptin expression and signal transduction in lean and obese mice.
Protein was extracted from hearts of 7 months-old mice (n = 8 per group) and analyzed for the expression of (A) leptin, (B)phosphorylated LepR (using immunoprecipitation of LepR, followed by the detection of total phosphotyrosines and LepR) and (C) phosphorylated STAT3. (D) Cardiac STAT3 phosphorylation in response (30 min later) to a single injection of recombinant murine leptin (1 mg/kg BW i.p.) was examined in WT (n = 4) and WT + HFD (n = 6) mice. Results are expressed as -fold increase of controls (black bars) after normalization for total protein and GAPDH expression. The mean ± SEM as well as representative Western blot results are shown. *P < 0.05 and **P < 0.01 vs. WT mice; #P < 0.05 vs. WT + HFD mice.

To further study the role of leptin signaling in the development of cardiac hypertrophy and also to determine, whether the inability of leptin to activate STAT3 contributes to the cardiac maladaptation in obesity, we examined mice in which tyrosine (Tyr)1138 within LepR had been replaced by a serine (LepRS1138). In these mice, leptin cannot signal via STAT3, but continues to be able to activate Jak2 and SH2 domain-containing adapter proteins. Western blot analysis revealed that p-LepR (Figure 2B) and p-STAT3 (Figure 2C) levels in hearts of LepRS1138 mice did not significantly differ from those in WT and LepRdb/db mice. Similar to mice with complete LepR deficiency, lack of LepR-mediated STAT3 activation resulted in severe adiposity, although serum leptin levels were lower than those in LepRdb/db mice (P < 0.001; Table 1). Interestingly, obese LepRS1138 exhibited a more pronounced increase in mean heart weights not only compared to lean or diet-induced obese WT mice, but also compared to LepRdb/db mice (P < 0.001 for all comparisons; Table 1), and differences persisted after normalization for body weight (P < 0.001) or tibia length (P < 0.001). Echocardiography confirmed increased LV mass (P < 0.01) or heart weights (P < 0.001) in LepRS1138 mice compared to their LepRdb/dbcounterparts (Table 2; please also see Figure 1A-C). Moreover, hearts of LepRS1138 mice exhibited elevated levels of phosphorylated Jak2 (P < 0.001 vs. WT; Figure 3A), Src kinase (P < 0.05 vs. WT, WT + HFD and LepRdb/db; Figure 3B), Akt (P < 0.001 vs. LepRdb/db; Figure 3C), PKC (P < 0.05 vs. WT and LepRdb/db, P < 0.01 vs. WT + HFD; Figure 3D) and p38 MAPK (P < 0.01 vs. LepRdb/db; Figure 3E), suggesting that an intact, Tyr1138-independent LepR activation in the presence of elevated leptin levels may have contributed to the pronounced cardiac hypertrophy present in these mice. On the other hand, cardiac levels of p-p42/44 MAPK did not significantly differ between the mouse groups (Figure 3F).

Figure 3. Hypertrophic signal transduction

Figure 3.Hypertrophic signal transduction in hearts of lean and obese mice. Protein was isolated from hearts of 7 months-old WT (n = 15), WT + HFD (n = 12), LepRS1138(n = 15) and LepRdb/db (n = 15) mice and analyzed for the expression of phosphorylated Jak2 (A), Src kinase (B), Akt (C), PKC (D), p38 (E) and p42/44 MAPK (F). Results are expressed as -fold increase of lean control mice (after normalization for total protein [with the exception of PKC] and GAPDH expression). The mean ± SEM as well as representative findings are shown. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. WT mice; #P < 0.05 and ##P < 0.01 vs. WT + HFD mice; §P < 0.05, §§P < 0.01 and §§§P < 0.001 for the difference between LepRdb/db and LepRS1138 mice.

M-mode echocardiography also revealed significantly increased enddiastolic LV diameters in LepRS1138 mice (P < 0.01 vs. WT and LepRdb/db mice; Table 2; representative findings are shown in Figure 1B), suggesting that the observed (over-)activation of LepR signaling together with the inability to induce STAT3 may result in augmented hypertrophy and maladaptive cardiac remodeling. Of note, fractional shortening (FS) was not significantly altered in HFD-induced obese WT mice (P = n.s. vs. WT mice), but found to be increased in both LepRdb/db (P < 0.01 vs. WT and P < 0.001 vs. WT + HFD mice) and LepRS1138mice (P < 0.05 vs. WT and P < 0.001 vs. WT + HFD mice). Histological analyses revealed significantly reduced numbers of CD31-positive capillary endothelial cells in LepRdb/db, and to a lesser extent also in LepRS1138 mice (Figure 4A), whereas the number of TUNEL-positive apoptotic cells (Figure 4B) and the fibrotic tissue area (Figure 4C) were found to be significantly increased in hearts of both LepRS1138 and LepRdb/db mice compared to lean and diet-induced obese WT mice.

Figure 4.Histological analysis of angiogenesis, apoptosis and fibrosis in hearts of lean and obese mice.
Serial cross sections through the LV of WT, WT + HFD, LepRS1138 and LepRdb/db mice (n = 10 per group) were immunostained and the number of (A) CD31-positive endothelial cells and (B) TUNEL-positive apoptotic cell nuclei determined. Results are expressed per cardiomyocyte and/or mm2.(C) The degree of cardiac fibrosis was quantified after Masson’s trichrome (MTC) staining. Results are expressed as % of total tissue area (at 200-fold magnification). The mean ± SEM as well as representative findings are shown. **P < 0.01 and ***P < 0.001 vs. WT; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. WT + HFD mice.

Figure 4.Histological analysis  (unable to post)

To examine the specificity of leptin’s hypertrophic action in obesity, the cardiac response of young, i.e. 2 months-old WT (n = 12; body weight, 22 ± 0.9 g), LepRS1138 (n = 9; 34 ± 1.1 g, P < 0.001 vs. WT) and LepRdb/db mice (n = 7; 40 ± 1.3 g; P < 0.001 vs. WT and P < 0.01 vs. LepRS1138) to chronic isoprenaline infusion (20 mg/kg BW per day) was examined. Under basal conditions, similar findings as those in 7 months-old mice were observed, i.e. LepRS1138 mice exhibited an increased heart weight (P < 0.05 vs. LepRdb/db; Figure 5A), LV mass (P < 0.01 vs. WT; Figure 5B) and mean WTh (P < 0.05 vs. WT; Figure 5C), whereas other changes, such as differences in fractional shortening (Figure 5D), ESD (Figure 5E) and EDD (Figure 5F) were not (yet) detected. On the other hand, all mouse groups responded to chronic β-adrenergic stimulation with significant cardiac hypertrophy, and no differences (with the exception of heart weight; Figure 5A) were observed between LepRS1138 and LepRdb/db mice. Representative M-mode echocardiography tracings are shown in Figure 6 and summarized in Additional file 2: Table S2.

1479-5876-11-170-5 F5 Echocardiography findings

Figure 5.Echocardiography findings in young lean and obese mice before and after chronic β-adrenergic stimulation.
Isoprenaline-filled osmotic minipumps were subcutaneously implanted into 2 months-old WT (n = 12), LepRS1138 (n = 9) and LepRdb/db (n = 7) mice to examine the cardiac response to a hypertrophic stimulus other than leptin. Echocardiography (A-F) was performed immediately before (open bars) as well as at the time of tissue harvest 14 days later (dotted bars). *P < 0.05, *P < 0.01 and ***P < 0.001 for differences vs. WT mice; §P < 0.05 for differences between LepRdb/db and LepRS1138 mice. Significance levels for differences before and after isoprenaline stimulation (as determined using Student’s t test for paired means) are indicated within the graph.

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Figure 6.Representative M-mode echocardiography recordings.

Additional file 2: Table S2. Echocardiographic parameter in 2 months-old mice before and 14 days after isoprenaline infusion.

Format: DOCX Size: 22KB

Discussion

The adipocytokine leptin may link obesity with cardiac hypertrophy, an important risk factor for the development of heart failure. Studies in humans [2,3] and rodents [10,11] have shown that obesity is associated with LV hypertrophy, and body mass index was identified as a strong and independent predictor of LV mass [2,3]. Importantly, cardiac hypertrophy is also observed in normotensive obese subjects [6], and plasma leptin levels are associated with increased myocardial wall thickness independent of BW or blood pressure elevations [5], suggesting a causal role for leptin in the pathogenesis of cardiac hypertrophy.

Although the major source of leptin is adipose tissue, cardiomyocytes are also capable of synthesizing leptin [27], and increased cardiac leptin levels have been reported in mice or rats following coronary ligation [13,18] or in patients with heart failure [28]. In this study, elevated circulating as well as cardiac leptin levels were detected in both diet-induced and genetically obese mice, which may have acted on cardiomyocytes as well as other, non-cardiomyocyte cells expressing leptin receptors [29]. Although leptin serum levels were higher than in previous publications [30], we explain this findings with the higher age of the mice, a factor previously found to be associated with increased circulating leptin levels [31]. Leptin has been shown to stimulate the hypertrophy of cardiomyocytes isolated from rats [7,20] or humans [8,19]. Moreover, chronic leptin infusion increased cardiac ANP expression after myocardial infarction (MI) in mice [32], whereas neutralizing LepR antibodies abrogated the hypertrophy of the surviving myocardium after coronary artery ligation in rats [33]. On the other hand and as confirmed in our analysis, cardiac hypertrophy also develops in leptin- and LepR-deficient mice and may be reversed by leptin substitution[12]. Caloric restriction experiments suggested that the anti-hypertrophic effects of leptin had occurred in addition to weight loss [12], which itself may preserve heart function and attenuate LV remodeling [34]. Thus, it is unclear whether the cardiac hypertrophy in obesity is the consequence of pro-hypertrophic effects of the adipokine or rather the result of a resistance towards leptin’s preventive effects on hypertrophic cardiac remodeling. Of note, since body weight is markedly elevated in the diet-induced and particularly, the genetically obese mice, the heart-to-body weight ratio decreases, even though the absolute heart weight is increased (but to a relatively lesser extent).

Obesity is associated with elevated circulating leptin levels and hypothalamic resistance to the weight-reducing effects of the adipokine, whereas the existence of a peripheral (e.g. cardiac) leptin resistance is controversial. For example, reduced cardiac LepR expression has been reported in HFD-fed rats[14], whereas others demonstrated unaltered cardiac STAT3 phosphorylation in diet-induced obese rodents following acute leptin administration [15–17]. Our findings also suggest that hearts from diet-induced obese mice continue to respond to leptin in the presence of chronically elevated leptin levels and that the observed elevation of serum and cardiac leptin may thus contribute to the development of cardiac hypertrophy in obesity. For example, hearts of hyperleptinemic obese WT mice (i.e. those with intact leptin receptors) exhibited signs of activated leptin signaling, including elevated levels of phosphorylated LepR and STAT3, while they were unchanged or reduced in mice with mutated or truncated forms of LepR (i.e. LepRS1138 or LepRdb/db mice). Moreover, both lean and obese WT mice responded to a single leptin injection with increased cardiac STAT3 phosphorylation. Of note, we could not spatially dissect the cardiac responsiveness to leptin, since whole heart homogenates were examined. Possible explanations underlying the discrepancy between the present and some previous studies include the animal species, as the absence of a response to leptin in obesity has been so far primarily observed in rats[14]. In addition, age, sex and feeding status of the animals or the time of recombinant leptin administration may have influenced the results. Of note, previous studies in humans have reported the existence of individuals (up to 40%) exhibiting a blunted response to leptin [35], although it is unknown, whether such phenomenon also occurs in rodents.

Interestingly, hearts from LepRS1138 mice exhibited a marked overactivation of STAT3-independent leptin signaling pathways, including Jak2, Src kinase, Akt or p38 MAPK, i.e. factors previously shown to mediate the pro-hypertrophic effects of the adipokine in cardiomyocytes [19,20]. Importantly, overactivation of leptin signaling in hearts of LepRS1138 mice was accompanied by a pronounced cardiac hypertrophy, both at the organ and the single cardiomyocyte level, despite similar adiposity. Although leptin levels were found to be lower in LepRS1138compared to LepRdb/db mice, as previously reported [23], leptin continues to be able to activate LepR signal transduction in these mice, for example via LepR-Tyr985. Similar echocardiographical findings were obtained in young (i.e. 2 months-old) and older (i.e. 7 months-old) mice, arguing against the development of cardiac hypertrophy secondary to hemodynamic or other metabolic changes associated with obesity, although we cannot exclude the possible contribution of a more pronounced hyperinsulinemia [23] to the development of cardiac hypertrophy in LepRS1138 mice. On the other hand, hypertension had not been observed in ob/ob mice [12], and heart weight increase and concentric LV hypertrophy in obese mice and humans also occurs without systolic and diastolic blood pressure elevations [5,6,36].

Although a predominant cardiac expression of the short (i.e. without STAT3 binding site) over the long LepR isoform has been reported [7,29], previous studies have shown that stimulation of neonatal rat cardiomyocytes with leptin increased STAT3 phosphorylation, nuclear translocation and DNA binding activity [32]. Also, cardiac STAT3 activation after MI was blunted in leptin-deficient mice [13]. The observation that increased cardiac STAT3 phosphorylation in hyperleptinemic, diet-induced obese mice was reduced or almost completely abolished in LepRS1138 or LepRdb/db mice suggests that cardiac STAT3 activation in obesity largely occurs downstream of elevated leptin levels and that other cytokines, also elevated in obesity and known to signal via Jak2-STAT3, may be of minor importance. On the other hand, the importance of leptin-mediated STAT3 activation in the heart and its contribution to cardioprotective signaling pathways in vivo have not been directly examined so far.

STAT3 has been implicated in cardioprotection after various injuries. For example, cardiomyocyte-specific STAT3 deletion results in dilatative cardiomyopathy, characterized by increased apoptosis and interstitial fibrosis as well as reduced myocardial capillary density [21,22]. Previous studies suggested that leptin may exert beneficial effects on the heart. For example, administration of leptin was associated with smaller infarct size after ischemia/reperfusion injury [37], whereas ischemic postconditioning failed to induce cardioprotection in mice lacking leptin or its receptor [38]. Also, leptin deficiency was associated with a worsened cardiac function and survival after coronary artery ligation, which could be improved by leptin repletion [13]. Regarding possible mechanisms, increased cardiac myocyte apoptosis was observed in hearts from leptin (receptor)-deficient mice [39,40]. Similar findings were obtained in vitro, showing that leptin protects cardiomyocytes against apoptotic cell death induced by serum starvation [41]. Our analyses also revealed significantly elevated numbers of apoptotic cells in hearts of obese LepRS1138 and LepRdb/dbmice, consistent with a reduced activation of STAT3-responsive anti-apoptotic genes [40]. Although findings in mice with systemic defects in leptin signal transduction may have been confounded by the concomitant presence of obesity and associated metabolic and inflammatory alterations, adverse cardiac remodeling after MI [42] or lethal heart failure [43] were recently reported in mice with cardiomyocyte-specific LepR deletion. On the other hand, the beneficial effects of leptin-mediated STAT3 activation may not be restricted to cardiomyocytes. For example, we and others have shown that leptin promotes the angiogenic properties of endothelial (progenitor) cells [25,44], and cardiac angiogenesis was reduced in LepRS1138 and LepRdb/db mice. In addition, hearts of obese LepRS1138 and LepRdb/db mice exhibited increased interstitial fibrosis, which may have occurred secondary to increased cardiomyocyte loss, although previous studies have shown that leptin may also directly influence myocardial matrix metabolism [45]. On the functional level, the enhanced activation of pro-hypertrophic signaling pathways in the absence of STAT3-mediated cardioprotection may have contributed to the echocardiographic finding of LV cavity dilation in LepRS1138 compared to LepRdb/db mice.

Conclusions

Taken together, our findings suggest that hearts from diet-induced obese mice continue to respond to chronically elevated leptin levels and that increased systemic and/or local leptin and enhanced cardiac LepR activation contribute the development of cardiac hypertrophy. On the other hand, chronic overactivation of hypertrophic signaling mediators together with an inabilitity to activate STAT3-dependent cardioprotective pathways may promote maladaptive cardiac remodeling. Of note, our findings also indicate that leptin signaling is not a prerequisite to develop cardiac hypertrophy in obesity and that additional pathways also contribute to the increase in LV mass associated with higher body weight.

References

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    N Engl J Med 1996, 334:292-295. PubMed Abstract | Publisher Full Text OpenURL

  2. Gottdiener JS, Reda DJ, Materson BJ, Massie BM, Notargiacomo A, Hamburger RJ, Williams DW, Henderson WG: Importance of obesity, race and age to the cardiac structural and functional effects of hypertension. The department of veterans affairs cooperative study group on antihypertensive agents.

    J Am Coll Cardiol 1994, 24:1492-1498. PubMed Abstract |Publisher Full Text OpenURL

  3. Lauer MS, Anderson KM, Kannel WB, Levy D: The impact of obesity on left ventricular mass and geometry. The framingham heart study.

    JAMA 1991, 266:231-236. PubMed Abstract | Publisher Full Text OpenURL

  4. Kenchaiah S, Evans JC, Levy D, Wilson PW, Benjamin EJ, Larson MG, Kannel WB, Vasan RS: Obesity and the risk of heart failure.

    N Engl J Med 2002, 347:305-313. PubMed Abstract | Publisher Full Text OpenURL

  5. Paolisso G, Tagliamonte MR, Galderisi M, Zito GA, Petrocelli A, Carella C, de Divitiis O, Varricchio M: Plasma leptin level is associated with myocardial wall thickness in hypertensive insulin-resistant men.

    Hypertension 1999, 34:1047-1052. PubMed Abstract |Publisher Full Text OpenURL

  6. Perego L, Pizzocri P, Corradi D, Maisano F, Paganelli M, Fiorina P, Barbieri M, Morabito A, Paolisso G, Folli F, Pontiroli AE: Circulating leptin correlates with left ventricular mass in morbid (grade III) obesity before and after weight loss induced by bariatric surgery: a potential role for leptin in mediating human left ventricular hypertrophy.

    J Clin Endocrinol Metab 2005, 90:4087-4093. PubMed Abstract |Publisher Full Text OpenURL

  7. Rajapurohitam V, Gan XT, Kirshenbaum LA, Karmazyn M: The obesity-associated peptide leptin induces hypertrophy in neonatal rat ventricular myocytes.

    Circ Res 2003, 93:277-279. PubMed Abstract | Publisher Full Text OpenURL

  8. Madani S, De Girolamo S, Munoz DM, Li RK, Sweeney G: Direct effects of leptin on size and extracellular matrix components of human pediatric ventricular myocytes.

    Cardiovasc Res 2006, 69:716-725. PubMed Abstract | Publisher Full Text

 

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G Protein–Coupled Receptor and S-Nitrosylation in Cardiac Ischemia and Acute Coronary Syndrome

Posted in Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Frontiers in Cardiology and Cardiovascular Disorders, Metabolomics, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Nitric Oxide in Health and Disease, Population Health Management, Genetics & Pharmaceutical, Pre-Clinical Animal Model Development, Proteomics, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , , , , , on November 2, 2013| Leave a Comment »

G Protein–Coupled Receptor and S-Nitrosylation in Cardiac Ischemia

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

This recently published article delineates a role of G-protein-coupled receptor with S-nitrosylation in outcomes for acute coronary syndrome.

Convergence of G Protein–Coupled Receptor and S-Nitrosylation Signaling Determines the Outcome to Cardiac Ischemic Injury

Z. Maggie Huang1, Erhe Gao1, Fabio Vasconcelos Fonseca2,3, Hiroki Hayashi2,3, Xiying Shang1, Nicholas E. Hoffman1, J. Kurt Chuprun1, Xufan Tian4, Doug G. Tilley1, Muniswamy Madesh1, David J. Lefer5, Jonathan S. Stamler2,3,6, and Walter J. Koch1*
1 Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA
2 Institute for Transformative Molecular Medicine, Case Western Reserve Univ SOM, Cleveland, OH
3 Department of Medicine, Case Western Reserve University, Cleveland, OH
4 Department of Biochemistry, Thomas Jefferson University, Philadelphia, PA
5 Department  Surgery, Div of Cardiothoracic Surgery, Emory University School of Medicine, Atlanta, GA
6 University Hospitals Harrington Discovery Institute, Cleveland, OH

Sci. Signal., 29 Oct 2013; 6(299), p. ra95         http:dx.doi.org/10.1126/scisignal.2004225

Abstract

Heart failure caused by ischemic heart disease is a leading cause of death in the developed world. Treatment is currently centered on regimens involving

  • G protein–coupled receptors (GPCRs) or nitric oxide (NO).

These regimens are thought to target distinct molecular pathways. We showed that

  • these pathways are interdependent and converge on the effector GRK2 (GPCR kinase 2) to regulate myocyte survival and function.

Ischemic injury coupled to

  • GPCR activation, including GPCR desensitization and myocyte loss,
  • required GRK2 activation,

and we found that cardioprotection mediated by inhibition of GRK2 depended on

  • endothelial nitric oxide synthase (eNOS) and
  • was associated with S-nitrosylation of GRK2.

Conversely, the cardioprotective effects of NO bioactivity were absent in a knock-in mouse with a form of GRK2 that cannot be S-nitrosylated. Because GRK2 and eNOS inhibit each other,

the balance of the activities of these enzymes in the myocardium determined the outcome to ischemic injury. Our findings suggest new insights into

  • the mechanism of action of classic drugs used to treat heart failure and
  • new therapeutic approaches to ischemic heart disease.

* Corresponding author. E-mail: walter.koch@temple.edu
Citation: Z. M. Huang, E. Gao, F. V. Fonseca, H. Hayashi, X. Shang, N. E. Hoffman, J. K. Chuprun, X. Tian, D. G. Tilley, M. Madesh, D. J. Lefer, J. S. Stamler, W. J. Koch, Convergence of G Protein–Coupled Receptor and S-Nitrosylation Signaling Determines the Outcome t

 Editor’s Summary

Sci. Signal., 29 Oct 2013; 6(299), p. ra95 [DOI: 10.1126/scisignal.2004225]

NO More Heart Damage

Damage caused by the lack of oxygen and nutrients that occurs during myocardial ischemia can result in heart failure. A therapeutic strategy that helps to limit the effects of heart failure is to

  • increase signaling through G protein–coupled receptors (GPCRs)
  • by inhibiting GRK2 (GPCR kinase 2), a kinase that
    • desensitizes GPCRs.

Another therapeutic strategy provides S-nitrosothiols, such as nitric oxide, which can be

  • added to proteins in a posttranslational modification called S-nitrosylation.

Huang et al. found that the ability of S-nitrosothiols to enhance cardiomyocyte survival after ischemic injury required the S-nitrosylation of GRK2, a modification that inhibits this kinase. Mice bearing a form of GRK2 that could not be S-nitrosylated 

  • were more susceptible to cardiac damage after ischemia.

These results suggest that therapeutic strategies that promote the S-nitrosylation of GRK2 could be used to treat heart failure after myocardial ischemia.

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Sensors and Signaling in Oxidative Stress

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Prevention: Research & Programs, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Cytoskeleton, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Genomic Testing: Methodology for Diagnosis, Metabolomics, Molecular Genetics & Pharmaceutical, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Pharmacogenomics, Population Health Management, Genetics & Pharmaceutical, tagged , , , , , , , , , , , , , , , , , , , on November 1, 2013| Leave a Comment »

Sensors and Signaling in Oxidative Stress

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

Article XI Sensors and Signaling in Oxidative Stress

Image created by Adina Hazan 06/30/2021

This is article ELEVEN in the following series on Calcium Role in Cardiovascular Diseases

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-
that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-
skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease
Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-
exchange-mechanism-in-health-and-disease/

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-
involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-
post-ischemic-arrhythmia-similarities-and-differen/

Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
and
Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary
Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-
for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure –
Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-
and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells:
The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-
muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction
(Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-
and-calcium-release-related-contractile-dysfunction-ryanopathy/

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of
vesicles with cell membranes during Neurotransmission
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-
regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Part XI: Sensors and Signaling in Oxidative Stress
Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

This important article on oxidative stress was published in Free Radical Biol. and Med.

Nrf2:INrf2(Keap1) Signaling in Oxidative Stress

James W. Kaspar, Suresh K. Niture, and Anil K. Jaiswal*
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD

Free Radic Biol Med. 2009 Nov; 47(9): 1304–1309.           http://dx.doi.org/10.1016/j.freeradbiomed.2009.07.035

Nrf2:INrf2(Keap1) are cellular sensors of chemical and radiation induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor INrf2. INrf2 functions as an adapter for Cul3/Rbx1 mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2cysteine151 and/or PKC phosphorylation of Nrf2serine40 results in

  • the escape or release of Nrf2 from INrf2.

Nrf2 is stabilized and

  • translocates to the nucleus,
  • forms heterodimers with unknown proteins, and
  • binds antioxidant response element (ARE) that
  • leads to coordinated activation of gene expression.
  • It takes less than fifteen minutes from the time of exposure to switch on nuclear import of Nrf2. This is followed by activation of a delayed mechanism that controls switching off of Nrf2 activation of gene expression. GSK3β phosphorylates Fyn at unknown threonine residue(s) leading to nuclear localization of Fyn. Fyn phosphorylates Nrf2tyrosine568
  • resulting in nuclear export of Nrf2, binding with INrf2 and
  • degradation of Nrf2.

The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis and promotes cell survival.

Introduction

Oxidative stress is induced by a vast range of factors including xenobiotics, drugs, heavy metals and ionizing radiation. Oxidative stress leads to the generation of Reactive Oxygen Species (ROS) and electrophiles. ROS and electrophiles generated can have a profound impact on survival, growth development and evolution of all living organisms [1,2] ROS include

  • both free radicals, such as the superoxide anion and the hydroxyl radical, and
  • oxidants such as hydrogen peroxide [3].

ROS and electrophiles can cause diseases such as cancer, cardiovascular complications, acute and chronic inflammation, and neurodegenerative diseases [1]. Therefore, it is obvious that

  • cells must constantly labor to control levels of ROS, preventing them from accumulation.

Much of what we know about the mechanisms of protection against oxidative stress has come from the study of prokaryotic cells [4,5]. Prokaryotic cells utilize transcription factors OxyR and SoxRS to sense the redox state of the cell, and

  • during oxidative stress these factors induce the expression of nearly eighty defensive genes [5].

Eukaryotic cells have similar mechanisms to protect against oxidative stress [Fig. 1; ref. 3,6–9]. Initial effect of oxidative/electrophilic stress leads to activation of a battery of defensive gene expression that leads to detoxification of chemicals and ROS and prevention of free radical generation and cell survival [Fig. 1].

Fig 1. Chemical and radiation exposure and coordinated induction of defensive genes.

Fig. 1. Chemical and radiation exposure and coordinated induction of defensive genes.

Of these genes, some are enzymes such as NAD(P)H:quinine oxidoreductase 1 (NQO1), NRH:quinone oxidoreductase 2 (NQO2), glutathione S-transferase Ya subunit (GST Ya Subunit), heme oxygenase 1 (HO-1), and γ-glutamylcysteine synthetase (γ-GCS), also known as glutamate cysteine ligase (GCL). Other genes have end products that regulate a wide variety of cellular activities including

  • signal transduction,
  • proliferation, and
  • immunologic defense reactions.

There is a wide variety of factors associated with the cellular response to oxidative stress. For example,

  • NF-E2 related factor 2 (Nrf2),
  • heat shock response activator protein 1, and
  • NF-kappaB promote cell survival,

whereas activation of c-jun, N-terminal kinases (JNK), p38 kinase and TP53 may lead to cell cycle arrest and apoptosis [10]. The Nrf2 pathway is regarded as the most important in the cell to protect against oxidative stress. [3,6–9]. It is noteworthy that accumulation of ROS and/or electrophiles leads to oxidative/electrophile stress,

  • membrane damage,
  • DNA adducts formation and
  • mutagenicity [Fig. 1].

These changes lead to degeneration of tissues and premature aging, apoptotic cell death, cellular transformation and cancer.

Antioxidant Response Element and Nrf2

Promoter analysis identified a cis-acting enhancer sequence designated as the antioxidant response element (ARE) that

  • controls the basal and inducible expression of antioxidant genes in response to xenobiotics, antioxidants, heavy metals and UV light [11].

The ARE sequence is responsive to a broad range of structurally diverse chemicals apart from β-nafthoflavone and phenolic antioxidants [12]. Mutational analysis revealed GTGACA***GC to be the core sequence of the ARE [11,13–14]. This core sequence is present in all Nrf2 downstream genes that respond to antioxidants and xenobiotics [3,6–9]. Nrf2 binds to the ARE and regulates ARE-mediated antioxidant enzyme genes expression and induction in response to a variety of stimuli including antioxidants, xenobiotics, metals, and UV irradiation [6,15–21].

Nrf2 is ubiquitously expressed in a wide range of tissue and cell types [22–24] and belongs to a subset of basic leucine zipper genes (bZIP) sharing a conserved structural domain designated as a cap’n’collar domain which is highly conserved in Drosphila transcription factor CNC (Fig. 2; ref. 25].

Fig. 2. Schematic Presentation of Various Domains of Nrf (Nrf1, Nrf2, Nrf3) and INrf2

Fig. 2. Schematic Presentation of Various Domains of Nrf (Nrf1, Nrf2, Nrf3) and INrf2

Nrf, NF-E2 Related Factor; INrf2, Inhibitor of Nrf2; NTR, N-Terminal Region; BTB, Broad complex, Tramtrack, Bric-a-brac; IVR, Intervening/linker Region; DGR, Kelch domain/ diglycine repeats; CTR, C-Terminal Region.

The basic region, just upstream of the leucine zipper region,

  • is responsible for DNA binding [3] and
  • the acidic region is required for transcriptional activation.

ARE-mediated transcriptional activation requires heterodimerization of Nrf2 with other bZIP proteins including Jun (c-Jun, Jun-D, and Jun-B) and small Maf (MafG, MafK, MafF) proteins [18– 20,26–27].

Initial evidence demonstrating the role of Nrf2 in antioxidant-induction of detoxifying enzymes came from studies on

  • the role of Nrf2 in ARE-mediated regulation of NQO1 gene expression [17].

Nrf2 was subsequently shown to be involved in

  • the transcriptional activation of other ARE-responsive genes such as
    • GST Ya, γ-GCS, HO-1, antioxidants, proteasomes, and drug transporters [3,6–9,28–33].

Overexpression of Nrf2 cDNA was shown to upregulate the expression and induction of the NQO1 gene in response to antioxidants and xenobiotics [17]. In addition, Nrf2-null mice exhibited a marked

  • decrease in the expression and induction of NQO1,
  • indicating that Nrf2 plays an essential role in the in vivo regulation of NQO1 in response to oxidative stress [26].

The importance of this transcription factor in upregulating ARE-mediated gene expression has been demonstrated by several in vivo and in vitro studies [reviewed in ref. 3]. The results indicate that Nrf2 is an important activator of phase II antioxidant genes [3,8].

Negative Regulation of Nrf2 mediated by INrf2

A cytosolic inhibitor (INrf2), also known as Keap1 (Kelch-like ECH-associating protein 1), of Nrf2 was identified and reported [Fig. 2; ref. 34–35]. INrf2, existing as a dimer [36], retains Nrf2 in the cytoplasm. Analysis of the INrf2 amino acid sequence and domain structure-function analyses have revealed that

  • INrf2 has a BTB (broad complex, tramtrack, bric-a-brac)/ POZ (poxvirus, zinc finger) domain and
  • a Kelch domain [34–35] also known as the DGR domain (Double glycine repeat) [37].

Keap1 has three additional domains/regions:

  1. the N-terminal region (NTR),
  2. the invervening region (IVR), and
  3. the C-terminal region (CTR) [8].

The BTB/POZ domain has been shown to be

  • a protein-protein interaction domain.

In the Drosophila Kelch protein, and in IPP,

  • the Kelch domain binds to actin [38–39]
  • allowing the scaffolding of INrf2 to the actin cytoskeleton
    • which plays an important role in Nrf2 retention in the cytosol [40].

The main function of INrf2 is to serve as

  • an adapter for the Cullin3/Ring Box 1 (Cul3/Rbx1) E3 ubiquitin ligase complex [41–43].

Cul3 serves as a scaffold protein that forms the E3 ligase complex with Rbx1 and recruits a cognate E2 enzyme [8].

INrf2

  1. via its N-terminal BTB/POZ domain binds to Cul3 [44] and
  2. via its C-terminal Kelch domain binds to the substrate Nrf2
  • leading to the ubiquitination and degradation of Nrf2 through the 26S proteasome [45–49].

Under normal cellular conditions, the cytosolic INrf2/Cul3-Rbx1 complex is constantly degrading Nrf2. When a cell is exposed to oxidative stress Nrf2 dissociates from the INrf2 complex, stabilizes and translocates into the nucleus leading to activation of ARE-mediated gene expression [3,6–9]. An alternative theory is that Nrf2 in response to oxidative stress escapes INrf2 degradation, stabilizes and translocates in the nucleus [49–50]. We suggested the theory of escape of Nrf2 from INrf2 [49] and similar suggestion was also made in another report [50]. However, the follow up studies in our laboratory could not support the escape theory. Escape theory is a possibility but has to be proven by experiments before it can be adapted. Therefore, we will use the release of Nrf2 from INrf2 in the rest of this review.

Numerous reports have suggested that

  • any mechanism that modifies INrf2 and/or Nrf2 disrupting the Nrf2:INrf2 interaction will result in the upregulation of ARE-mediated gene expression.

A model Nrf2:INrf2 signaling from antioxidant and xenobiotic to activation of ARE-mediated defensive gene expression is shown in Fig. 3.

Fig. 3. Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Fig. 3. Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Since the metabolism of antioxidants and xenobiotics results in the generation of ROS and electrophiles [51], it is thought that these molecules might act as second messengers, activating ARE-mediated gene expression. Several protein kinases including PKC, ERK, MAPK, p38, and PERK [49,52– 56] are known to modify Nrf2 and activate its release from INrf2. Among these mechanisms,

  1. oxidative/electrophilic stress mediated phosphorylation of Nrf2 at serine40 by PKC is necessary for Nrf2 release from INrf2, but
  2. is not required for Nrf2 accumulation in the nucleus [49,52–53].

In addition to post-translational modification in Nrf2, several crucial residues in INrf2 have also been proposed to be important for activation of Nrf2. Studies based on

  • the electrophile mediated modification,
  • location and
  • mutational analyses revealed
    • that three cysteine residues, Cys151, Cys273 and Cys288 are crucial for INrf2 activity [50].

INrf2 itself undergoes ubiquitination by the Cul3 complex, via a proteasomal independent pathway,

  • which was markedly increased in response to phase II inducers such as antioxidants [57].

It has been suggested that normally INrf2 targets Nrf2 for ubiquitin mediated degradation but

  • electrophiles may trigger a switch of Cul3 dependent ubiquitination from Nrf2 to INrf2 resulting in ARE gene induction.

The redox modulation of cysteines in INrf2

  • might be a mechanism redundant to the phosphorylation of Nrf2 by PKC, or that
  • the two mechanisms work in concert.

In addition to cysteine151 modification,

  • phosphorylation of Nrf2 has also been shown to play a role in INrf2 retention and release of Nrf2.

Serine104 of INrf2 is required for dimerization of INrf2, and

  • mutations of serine104 led to the disruption of the INrf2 dimer leading to the release of Nrf2 [36].

Recently, Eggler at al. demonstrated that modifying specific cysteines of the electrophile-sensing human INrf2 protein is insufficient to disrupt binding to the Nrf2 domain Neh2 (58). Upon introduction of electrophiles, modification of INrf2C151 leads to a change in the conformation of the BTB domain by means of perturbing the homodimerization site, disrupting Neh2 ubiquitination, and causing ubiquitination of INrf2. Modification of INrf2 cysteines by electrophiles does not lead to disruption of the INrf2–Nrf2 complex. Rather, the switch of ubiquitination from Nrf2 to INrf2 leads to Nrf2 nuclear accumulation.

More recently, our laboratory demonstrated that phosphorylation and de-phosphorylation of tyrosine141 in INrf2 regulates its stability and degradation, respectively [59]. The de-phosphorylation of tyrosine141 caused destabilization and degradation of INrf2 leading to the release of Nrf2. Furthermore, we showed that prothymosin-α mediates nuclear import of the INrf2/Cul3-Rbx1 complex [60]. The INrf2/Cul3-Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2 resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α mediated nuclear import of INrf2/Cul3-Rbx1 complex leads to ubiquitination and degradation of nuclear Nrf2 presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression. An auto-regulatory loop also exists within the Nrf2 pathway [61]. An ARE was identified in the INrf2 promoter that facilitates Nrf2 binding causing induction of the INrf2 gene. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by serving as an adaptor for degradation.

Other Regulatory Mediators of Nrf2

Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1) is a transcription repressor [62] that is ubiquitously expressed in tissues [63–64] and distantly related to Nrf2 [8]. In the absence of cellular stress, Bach1 heterodimers with small Maf proteins [65] that bind to the (ARE) [66] repressing gene expression. In the presence of oxidative stress, Bach1 releases from the ARE and is replaced by Nrf2. Bach1 competes with Nrf2 for binding to the ARE leading to suppression of Nrf2 downstream genes [66].

Nuclear import of Nrf2, from time of exposure to stabilization, takes roughly two hours [67]. This is followed by activation of a delayed mechanism involving Glycogen synthase kinase 3 beta (GSK3f3) that controls switching off of Nrf2 activation of gene expression (Fig. 3). GSK3f3 is a multifunctional serine/threonine kinase, which plays a major role in various signaling pathways [68]. GSK3f3 phosphorylates Fyn, a tyrosine kinase, at unknown threonine residue(s) leading to nuclear localization of Fyn [69]. Fyn phosphorylates Nrf2 tyrosine 568 resulting in nuclear export of Nrf2, binding with INrf2 and degradation of Nrf2 [70].

The negative regulation of Nrf2 by Bach1 and GSK3f3/Fyn are important in repressing Nrf2 downstream genes that were induced in response to oxidative/electrophilic stress. The tight control of Nrf2 is vital for the cells against free radical damage, prevention of apoptosis and cell survival [3,6–9,70].

Nrf2 in Cytoprotection, Cancer and Drug Resistance

Nrf2 is a major protective mechanism against xenobiotics capable of damaging DNA and initiating carcinogenesis [71]. Inducers of Nrf2 function as blocking agents that prevents carcinogens from reaching target sites, inhibits parent molecules undergoing metabolic activation, or subsequently preventing carcinogenic species from interacting with crucial cellular macromolecules, such as DNA, RNA, and proteins [72]. A plausible mechanism by which blocking agents impart their chemopreventive activity is the induction of detoxification and antioxidant enzymes [73]. Oltipraz, 3H-1,2,-dithiole-3-thione (D3T), Sulforaphane, and Curcumin can be considered potential chemopreventive agents because

  • these compounds have all been shown to induce Nrf2 [74–81].

Studies have shown a role of Nrf2 in protection against cadmium and manganese toxicity [82]. Nrf2 also plays an important role in reduction of methyl mercury toxicity [83]. Methylmercury activates Nrf2 and the activation of Nrf2 is essential for reduction of methylmercury by facilitating its excretion into extracellular space. In vitro and in vivo studies have shown a role of Nrf2 in neuroprotection and protection against Parkinson’s disease [84– 86]. Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice [87]. Nrf2-knockout mice were more prone to

  • tumor growth when exposed to carcinogens such as benzo[a]pyrene, diesel exhaust, and N-nitrosobutyl (4-hydroxybutyl) amine [88–90].

INrf2/Nrf2 signaling is also shown to regulate oxidative stress tolerance and lifespan in Drosophila [91].

A role of Nrf2 in drug resistance is suggested based on its property to induce detoxifying and antioxidant enzymes (92–97). The loss of INrf2 (Keap1) function is shown to

  • lead to nuclear accumulation of Nrf2, activation of metabolizing enzymes and drug resistance (95).

Studies have reported mutations resulting in dysfunctional INrf2 in lung, breast and bladder cancers (96–100). A recent study reported that somatic mutations also occur in the coding region of Nrf2, especially in cancer patients with a history of smoking or suffering from squamous cell carcinoma (101). These mutations abrogate its interaction with INrf2 and nuclear accumulation of Nrf2. This gives advantage to

  • cancer cell survival and
  • undue protection from anti-cancer treatments.

However, the understanding of the mechanism of Nrf2 induced drug resistance remains in its infancy. In addition, the studies on Nrf2 regulated downstream pathways that contribute to drug resistance remain limited.

Future Perspectives

Nrf2 creates a new paradigm in cytoprotection, cancer prevention and drug resistance. Considerable progress has been made to better understand all mechanisms involved within the intracellular pathways regulating Nrf2 and its downstream genes. Preliminary studies demonstrate that

  • deactivation of Nrf2 is as important as activation of Nrf2.

Further studies are needed to better understand the negative regulation of Nrf2. Also better understanding of the negative regulation of Nrf2 could help design a new class of effective chemopreventive compounds not only targeting Nrf2 activation, but also

  • targeting the negative regulators of Nrf2.

Abbreviations: 

Nrf2    NF-E2 related factor 2;  INrf2   Inhibitor of Nrf2 also known as Keap1;   ROS    Reactive oxygen species.

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Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K – Oxidative Stress

Posted in Cardiovascular Pharmacogenomics, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Frontiers in Cardiology and Cardiovascular Disorders, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Pharmaceutical Industry Competitive Intelligence, tagged , , , , , on November 1, 2013| Leave a Comment »

Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K

Author and Curator: Larry H Bernstein, MD, FCAP

 

 

This report from Science Translational Medicine is about the finding that a cardiomyocyte-specific kinase limits reperfusion injury in acute coronary syndrome, a phenomenon driven by oxidative stress, protecting cardiac cells from further damage.

Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K Limits Oxidative Stress, Injury, and Adverse Remodeling in the Ischemic Heart

Ronald J. Vagnozzi1,2,  Gregory J. Gatto Jr.3, Lara S. Kallander3, Nicholas E. Hoffman2, Karthik Mallilankaraman2, Victoria L. T. Ballard3, Brian G. Lawhorn3, …, and Thomas Force2,6,*
+ Author Affiliations
1Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA
2Center for Translational Medicine, and 6Cardiology Division, Temple University School of Medicine, Philadelphia, PA
3Heart Failure Discovery Performance Unit, Metabolic Pathways and Cardiovascular Therapeutic Area Unit, GlaxoSmithKline, King of Prussia, PA
4Platform Technology and Sciences, GlaxoSmithKline, King of Prussia, PA
5Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya 663-8131, Japan.

Sci Transl Med 16 Oct 2013; 5(207), p. 207ra141     http://dx.doi.org/10.1126/scitranslmed.3006479
Percutaneous coronary intervention is first-line therapy for acute coronary syndromes (ACS) but can promote cardiomyocyte death and cardiac dysfunction via reperfusion injury, a phenomenon driven in large part by oxidative stress. Therapies to limit this progression have proven elusive, with no major classes of new agents since the development of anti-platelets/anti-thrombotics. We report that cardiac troponin I–interacting kinase (TNNI3K), a cardiomyocyte-specific kinase,
  1. promotes ischemia/reperfusion injury,
  2. oxidative stress,
  3. and myocyte death.
TNNI3K-mediated injury occurs
  • through increased mitochondrial superoxide production and
  • impaired mitochondrial function and is largely
  • dependent on p38 mitogen-activated protein kinase (MAPK) activation.

We developed a series of small-molecule TNNI3K inhibitors that

  1. reduce mitochondrial-derived superoxide generation,
  2. p38 activation, and
  3. infarct size
when delivered at reperfusion to mimic clinical intervention.
TNNI3K inhibition also preserves cardiac function and limits chronic adverse remodeling.
Our findings demonstrate that TNNI3K modulates reperfusion injury in the ischemic heart and is a tractable therapeutic target for ACS.  Pharmacologic TNNI3K inhibition would be cardiac-selective,
  • preventing potential adverse effects of systemic kinase inhibition.
Citation: R. J. Vagnozzi, G. J. Gatto, L. S. Kallander, N. E. Hoffman, K. Mallilankaraman, V. L. T. Ballard, B. G. Lawhorn, P. Stoy, J. Philp, A. P. Graves, Y. Naito, J. J. Lepore, E. Gao, M. Madesh, T. Force, Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K Limits Oxidative Stress, Injury, and Adverse Remodeling in the Ischemic Heart. Sci. Transl. Med. 5, 207ra141 (2013).

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