Blockade of thromboxane A2 signaling attenuates ethanol-induced myocardial inflammatory response in mice.

BACKGROUND: Alcohol-associated cardiomyopathy (ACM) is a cardiac muscle disease characterized by inflammation and oxidative stress. Thromboxane-prostanoid receptor (TP-R) plays an important role in the pathogenesis of cardiovascular disease. Herein, we hypothesize that TP-R mediates alcohol-induced early cardiac injury. METHODS: Eight-week-old male C57BL/6 wild-type mice were fed a chronic ethanol (ET) or control diet (CON) for 10 days followed by a single binge of ethanol or maltose-dextrin through oral gavage. A cohort of ethanol-fed mice received SQ 29,548 (SQ), a TP-R antagonist. RNA sequencing, real-time PCR, and western blot analysis were performed on left ventricle to investigate alterations in genes and/or proteins mediating oxidative stress, inflammation, and cardiac remodeling. Sirius Red staining was performed to measure myocardial fibrosis. RESULTS: RNA-sequencing analysis of myocardium from CON and ET groups identified 142 genes that were significantly altered between the two groups. In particular, the gene expression of thioredoxin-interacting protein (TXNIP), a component of NLR family pyrin domain containing 3 (NLRP3) signaling, which mediates oxidative stress and inflammatory response, was upregulated in response to ethanol exposure. The myocardial protein levels of TP-R and thromboxane A2 synthase were increased upon alcohol exposure. Ethanol increased the levels of 4-hydroxynonenal, a marker of oxidative stress, with a concomitant increase in the protein levels of TXNIP and NLRP3, and administration of SQ attenuated these effects. Additionally, ethanol increased the protein levels of pro-inflammatory mediators, including tumor necrosis factor alpha and the NLRP3 downstream product, secretory interleukin 1 beta, and SQ blunted these effects. Finally, the Sirius red staining of the myocardium revealed an increase in collagen deposition in ethanol-fed mice which was attenuated by TP-R antagonism. CONCLUSION: This study demonstrates that ethanol promotes the NLRP3 signaling pathway within the myocardium, leading to a pro-inflammatory milieu that potentially initiates early myocardial remodeling, and TP-R antagonism attenuates this effect.

Why the diabetic heart is energy inefficient: a ketogenesis and ketolysis perspective.

Lack of glucose uptake compromises metabolic flexibility and reduces energy efficiency in the diabetes mellitus (DM) heart. Although increased use of fatty acid to compensate glucose substrate has been studied, less is known about ketone body metabolism in the DM heart. Ketogenic diet reduces obesity, a risk factor for T2DM. How ketogenic diet affects ketone metabolism in the DM heart remains unclear. At the metabolic level, the DM heart differs from the non-DM heart because of altered metabolic substrate and the T1DM heart differs from the T2DM heart because of insulin levels. How these changes affect ketone body metabolism in the DM heart are poorly understood. Ketogenesis produces ketone bodies by using acetyl-CoA, whereas ketolysis consumes ketone bodies to produce acetyl-CoA, showing their opposite roles in the ketone body metabolism. Cardiac-specific transgenic upregulation of ketogenesis enzyme or knockout of ketolysis enzyme causes metabolic abnormalities leading to cardiac dysfunction. Empirical evidence demonstrates upregulated transcription of ketogenesis enzymes, no change in the levels of ketone body transporters, very high levels of ketone bodies, and reduced expression and activity of ketolysis enzymes in the T1DM heart. Based on these observations, I hypothesize that increased transcription and activity of cardiac ketogenesis enzyme suppresses ketolysis enzyme in the DM heart, which decreases cardiac energy efficiency. The T1DM heart exhibits highly upregulated ketogenesis compared with the T2DM heart because of the lack of insulin, which inhibits ketogenesis enzyme.

MiR-133a Mimic Alleviates T1DM-Induced Systolic Dysfunction in Akita: An MRI-Based Study.

Diabetic cardiomyopathy is a leading cause of heart failure. Developing a novel therapeutic strategy for diabetic cardiomyopathy and characterizing animal models used for diabetes mellitus (DM) are important. Insulin 2 mutant (Ins2+/-) Akita is a spontaneous, genetic, mouse model for T1DM, which is relevant to humans. There are contrasting reports on systolic dysfunction and pathological remodeling (hypertrophy and fibrosis) in Akita heart. Here, we used magnetic resonance imaging (MRI) approach, a gold standard reference for evaluating cardiac function, to measure ejection fraction (indicator of systolic dysfunction) in Akita. Moreover, we performed Wheat Germ Agglutinin (WGA) and hematoxylin and Eosin stainings to determine cardiac hypertrophy, and Masson's Trichrome and picrosirius red stainings to determine cardiac fibrosis in Akita. MiR-133a, an anti-hypertrophy and anti-fibrosis miRNA, is downregulated in Akita heart. We determined if miR-133a mimic treatment could mitigate systolic dysfunction and remodeling in Akita heart. Our MRI results revealed decreased ejection fraction in Akita as compared to WT and increased ejection fraction in miR-133a mimic-treated Akita. We also found that miR-133a mimic treatment mitigates T1DM-induced cardiac hypertrophy and fibrosis in Akita. We conclude that Akita shows cardiac hypertrophy, fibrosis and systolic dysfunction and miR-133a mimic treatment to Akita could ameliorate them.

Isolation, Characterization and Differentiation of Mouse Cardiac Progenitor Cells.

Despite several strategies developed for replenishing the dead myocardium after myocardial infarction (MI), stem cell therapy remains the leading method to regenerate new myocardium. Although induced pluripotent stem cells (iPS) and transdifferentiation of the differentiated cells have been used as novel approaches for myocardial regeneration, these approaches did not yield very successful results for myocardial regeneration in in vivo studies. Asynchronous contractility of newly formed cardiomyocytes with the existing cardiomyocytes is the most important issue with iPS approach, while very low yield of transdifferentiated cardiomyocytes and their less chances to beat in the same rhythm as existing cardiomyocytes in the MI heart are important caveats with transdifferentiation approach. CSCs are present in the heart and they have the potential to differentiate into myocardial cells. However, the number of resident CSCs is very low. Therefore, it is important to get maximum yield of CSCs during isolation process from the heart. Increasing the number of CSCs and initiating their differentiation ex vivo are crucial for CSC-based stem cell therapy. Here, we present a better method for isolation, characterization and differentiation of CSCs from the mouse heart. We also demonstrated morphological changes in the CSCs after 2 days, 3 days, and 7 days in maintenance medium and a separate group of CSCs cultured for 12 days in differentiation medium using Phase-Contrast microscopy. We have used different markers for identification of CSCs isolated from the mouse heart such as marker for mouse CSC, Sca-1, cardiac-specific markers NKX2-5, MEF2C, GATA4, and stemness markers OCT4 and SOX2. To characterize the differentiated CSCs, we used CSCs maintained in differentiation medium for 12 days. To evaluate differentiation of CSCs, we determined the expression of cardiomyocyte-specific markers actinin and troponin I. Overall; we described an elegant method for isolation, identification, differentiation and characterization of CSCs from the mouse heart.

Targeting miRNA for Therapy of Juvenile and Adult Diabetic Cardiomyopathy.

Prevalence of diabetes mellitus (DM), a multifactorial disease often diagnosed with high blood glucose levels, is rapidly increasing in the world. Association of DM with multi-organ dysfunction including cardiomyopathy makes it a leading cause of morbidity and mortality. There are two major types of DM: type 1 DM (T1D) and type 2 DM (T2D). T1D is diagnosed by reduced levels of insulin and high levels of glucose in the blood. It is caused due to pancreatic beta cell destruction/loss, and mostly found in juveniles (juvenile DM). T2D is diagnosed by increased levels of insulin and glucose in the blood. It is caused due to insulin receptor dysfunction, and mostly found in the adults (adult DM). Both T1D and T2D impair cardiac muscle function, which is referred to as diabetic cardiomyopathy. We and others have reported that miRNAs, a novel class of tiny non-coding regulatory RNAs, are differentially expressed in the diabetic heart and they contribute to diabetic cardiomyopathy. Here, we elaborated the biogenesis of miRNA, how miRNA regulates a gene, cardioprotective roles of different miRNAs including miRNAs present in exosomes, underlying molecular mechanisms by which miRNA ameliorates diabetic cardiomyopathy, and the role of miRNA as a potential therapeutic target for juvenile and adult diabetic cardiomyopathy.

H2S and homocysteine control a novel feedback regulation of cystathionine beta synthase and cystathionine gamma lyase in cardiomyocytes.

Hydrogen sulfide (H2S), a cardioprotective gas, is endogenously produced from homocysteine by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE) enzymes. However, effect of H2S or homocysteine on CBS and CSE expression, and cross-talk between CBS and CSE are unclear. We hypothesize that homocysteine and H2S regulate CBS and CSE expressions in a dose dependent manner in cardiomyocytes, and CBS deficiency induces cardiac CSE expression. To test the hypothesis, we treated murine atrial HL1 cardiomyocytes with increasing doses of homocysteine or Na2S/GYY4137, a H2S donor, and measured the levels of CBS and CSE. We found that homocysteine upregulates CSE but downregulates CBS whereas Na2S/GYY4137 downregulates CSE but upregulates CBS in a dose-dependent manner. Moreover, the Na2S-treatment downregulates specificity protein-1 (SP1), an inducer for CSE, and upregulates miR-133a that targets SP1 and inhibits cardiomyocytes hypertrophy. Conversely, in the homocysteine-treated cardiomyocytes, CBS and miR-133a were downregulated and hypertrophy was induced. In vivo studies using CBS+/-, a model for hyperhomocysteinemia, and sibling CBS+/+ control mice revealed that deficiency of CBS upregulates cardiac CSE, plausibly by inducing SP1. In conclusion, we revealed a novel mechanism for H2S-mediated regulation of homocysteine metabolism in cardiomyocytes, and a negative feedback regulation between CBS and CSE in the heart.

Stem Cell-Derived Exosomes, Autophagy, Extracellular Matrix Turnover, and miRNAs in Cardiac Regeneration during Stem Cell Therapy.

Stem cell therapy (SCT) raises the hope for cardiac regeneration in ischemic hearts. However, underlying molecular mechanisms for repair of dead myocardium by SCT in the ischemic heart is poorly understood. Growing evidences suggest that cardiac matrix stiffness and differential expressions of miRNAs play a crucial role in stem cell survival and differentiation. However, their roles on transplanted stem cells, for myocardial repair of the ischemic heart, remain unclear. Transplanted stem cells may act in an autocrine and/or paracrine manner to regenerate the dead myocardium. Paracrine mediators such as stem cell-derived exosomes are emerging as a novel therapeutic strategy to overcome some of the limitations of SCT. These exosomes carry microRNAs (miRNAs) that may regulate stem cell differentiation into a specific lineage. MicroRNAs may also contribute to stiffness of surrounding matrix by regulating extracellular matrix (ECM) turnover. The survival of transplanted stem cell depends on its autophagic process that maintains cellular homeostasis. Therefore, exosomes, miRNAs, extracellular matrix turnover, and autophagy may have an integral role in improving the efficacy of SCT. This review elaborates the specific roles of these regulatory components on cardiac regeneration in the ischemic heart during SCT.

Harnessing fetal and adult genetic reprograming for therapy of heart disease.

Heart is the first organ formed during organogenesis. The fetal heart undergoes several structural and functional modifications to form the four-chambered mammalian heart. The adult heart shows different adaptations during compensatory and decompensatory heart failure. However, one common adaptation in the pathological heart is fetal reprogramming, where the adult heart expresses several genes and miRNAs which are active in the fetal stage. The fetal reprogramming in the failing heart raises several questions, such as whether the switch of adult to fetal genetic programming is an adaptive response to cope with adverse remodeling of the heart, does the expression of fetal genes protect the heart during compensatory and/or decompensatory heart failure, does repressing the fetal gene in the failing heart is protective to the heart? To answer these questions, we need to understand the expression of genes and miRNAs that are reprogrammed in the failing heart. In view of this, we provided an overview of differentially expressed genes and miRNAs, and their regulation in this review. Further, we elaborated novel strategies for a plausible future therapy of cardiovascular diseases.

Hydrogen sulfide mitigates homocysteine-mediated pathological remodeling by inducing miR-133a in cardiomyocytes.

An elevated level of homocysteine called hyperhomocysteinemia (HHcy) is associated with pathological cardiac remodeling. Hydrogen sulfide (H2S) acts as a cardioprotective gas; however, the mechanism by which H2S mitigates homocysteine-mediated pathological remodeling in cardiomyocytes is unclear. We hypothesized that H2S ameliorates HHcy-mediated hypertrophy by inducing cardioprotective miR-133a in cardiomyocytes. To test the hypothesis, HL1 cardiomyocytes were treated with (1) plain medium (control, CT), (2) 100 µM of homocysteine (Hcy), (3) Hcy with 30 µM of H2S (Hcy + H2S), and (4) H2S for 24 h. The levels of hypertrophy markers: c-fos, atrial natriuretic peptide (ANP), and beta-myosin heavy chain (ß-MHC), miR-133a, and its transcriptional inducer myosin enhancer factor-2C (MEF2C) were determined by Western blotting, RT-qPCR, and immunofluorescence. The activity of MEF2C was assessed by co-immunoprecipitation of MEF2C with histone deacetylase-1(HDAC1). Our results show that H2S ameliorates homocysteine-mediated up-regulation of c-fos, ANP, and ß-MHC, and down-regulation of MEF2C and miR-133a. HHcy induces the binding of MEF2C with HDAC1, whereas H2S releases MEF2C from MEF2C-HDAC1 complex causing activation of MEF2C. These findings elicit that HHcy induces cardiac hypertrophy by promoting MEF2C-HDAC1 complex formation that inactivates MEF2C causing suppression of anti-hypertrophy miR-133a in cardiomyocytes. H2S mitigates hypertrophy by inducing miR-133a through activation of MEF2C in HHcy cardiomyocytes. To our knowledge, this is a novel mechanism of H2S-mediated activation of MEF2C and induction of miR-133a and inhibition of hypertrophy in HHcy cardiomyocytes.

Generating double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans.

Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/-) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3-4 weeks and show robust hyperglycemia at the age of 10-12 weeks. It is a chronic model of T1D. In Ins2+/- Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/-/Mmp9-/- (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy.

Differential expression of dicer, miRNAs, and inflammatory markers in diabetic Ins2+/- Akita hearts.

Diabetic cardiomyopathy is a leading cause of morbidity and mortality, and Insulin2 mutant (Ins2+/-) Akita is a genetic mice model of diabetes relevant to humans. Dicer, miRNAs, and inflammatory cytokines are associated with heart failure. However, the differential expression of miRNAs, dicer, and inflammatory molecules are not clear in diabetic cardiomyopathy of Akita. We measured the levels of miRNAs, dicer, pro-inflammatory tumor necrosis factor alpha (TNFα), and anti-inflammatory interleukin 10 (IL-10) in C57BL/6J (WT) and Akita hearts. The results revealed increased heart to body weight ratio and robust expression of brain natriuretic peptide (BNP: a hypertrophy marker) suggesting cardiac hypertrophy in Akita. The multiplex RT-PCR, qPCR, and immunoblotting showed up regulation of dicer, whereas miRNA array elicited spread down regulation of miRNAs in Akita including dramatic down regulation of let-7a, miR-130, miR-142-3p, miR-148, miR-338, miR-345-3p, miR-384-3p, miR-433, miR-450, miR-451, miR-455, miR-494, miR-499, miR-500, miR-542-3p, miR-744, and miR-872. Conversely, miR-295 is induced in Akita. Cardiac TNFα is upregulated at mRNA (RT-PCR and qPCR), protein (immunoblotting), and cellular (immunohistochemistry and confocal microscopy) levels, and is robust in hypertrophic cardiomyocytes suggesting direct association of TNFα with hypertrophy. Contrary to TNFα, cardiac IL-10 is downregulated in Akita. In conclusion, induction of dicer and TNFα, and attenuation of IL-10 and majority of miRNA are associated with cardiomyopathy in Akita and could be used for putative therapeutic target for heart failure in diabetics.

Cardiac matrix: a clue for future therapy.

Cardiac muscle is unique because it contracts ceaselessly throughout the life and is highly resistant to fatigue. The marvelous nature of the cardiac muscle is attributed to its matrix that maintains structural and functional integrity and provides ambient micro-environment required for mechanical, cellular and molecular activities in the heart. Cardiac matrix dictates the endothelium myocyte (EM) coupling and contractility of cardiomyocytes. The matrix metalloproteinases (MMPs) and their tissue inhibitor of metalloproteinases (TIMPs) regulate matrix degradation that determines cardiac fibrosis and myocardial performance. We have shown that MMP-9 regulates differential expression of micro RNAs (miRNAs), calcium cycling and contractility of cardiomyocytes. The differential expression of miRNAs is associated with angiogenesis, hypertrophy and fibrosis in the heart. MMP-9, which is involved in the degradation of cardiac matrix and induction of fibrosis, is also implicated in inhibition of survival and differentiation of cardiac stem cells (CSC). Cardiac matrix is distinct because it renders mechanical properties and provides a framework essential for differentiation of cardiac progenitor cells (CPC) into specific lineage. Cardiac matrix regulates myocyte contractility by EM coupling and calcium transients and also directs miRNAs required for precise regulation of continuous and synchronized beating of cardiomyocytes that is indispensible for survival. Alteration in the matrix homeostasis due to induction of MMPs, altered expression of specific miRNAs or impaired signaling for contractility of cardiomyocytes leads to catastrophic effects. This review describes the mechanisms by which cardiac matrix regulates myocardial performance and suggests future directions for the development of treatment strategies in cardiovascular diseases.

Cardiac stem cell niche, MMP9, and culture and differentiation of embryonic stem cells.

Embryonic stem cells (ESC) are totipotent, self-renewing, and clonogenic, having potential to differentiate into a wide variety of cell types. Due to regenerative capability, it has tremendous potential for treating myocardial infarction (death of myocardial tissue) and type 1 diabetes (death of pancreatic beta cells). Understanding the components regulating ESC differentiation is the key to unlock the regenerative potential of ESC-based therapies. Both the stiffness of extracellular matrix (ECM) and surrounding niche/microenvironment play pivotal roles in ESC differentiation. Matrix metalloproteinase-9 (MMP9) induces fibrosis that causes stiffness of the ECM and impairs differentiation of cardiac stem cells into cardiomyocytes. Here, we describe the method of ESC culture and differentiation, and the expression of MMP9 and its inhibitor, tissue inhibitor of metalloproteinase-4 (TIMP4) in differentiating ESC.

Ablation of MMP9 induces survival and differentiation of cardiac stem cells into cardiomyocytes in the heart of diabetics: a role of extracellular matrix.

The contribution of extracellular matrix (ECM) to stem cell survival and differentiation is unequivocal, and matrix metalloproteinase-9 (MMP9) induces ECM turn over; however, the role of MMP9 in the survival and differentiation of cardiac stem cells is unclear. We hypothesize that ablation of MMP9 enhances the survival and differentiation of cardiac stem cells into cardiomyocytes in diabetics. To test our hypothesis, Ins2(+/-) Akita, C57 BL/6J, and double knock out (DKO: Ins2(+/-)/MMP9(-/-)) mice were used. We created the DKO mice by deleting the MMP9 gene from Ins2(+/-). The above 3 groups of mice were genotyped. The activity and expression of MMP9 in the 3 groups were determined by in-gel gelatin zymography, Western blotting, and confocal microscopy. To determine the role of MMP9 in ECM stiffness (fibrosis), we measured collagen deposition in the histological sections of hearts using Masson's trichrome staining. The role of MMP9 in cardiac stem cell survival and differentiation was determined by co-immunoprecipitation (co-IP) of MMP9 with c-kit (a marker of stem cells) and measuring the level of troponin I (a marker of cardiomyocytes) by confocal microscopy in the 3 groups. Our results revealed that ablation of MMP9 (i) reduces the stiffness of ECM by decreasing collagen accumulation (fibrosis), and (ii) enhances the survival (elevated c-kit level) and differentiation of cardiac stem cells into cardiomyocytes (increased troponin I) in diabetes. We conclude that inhibition of MMP9 ameliorates stem cell survival and their differentiation into cardiomyocytes in diabetes.

Exercise mitigates homocysteine - ß2-adrenergic receptor interactions to ameliorate contractile dysfunction in diabetes.

We tested the hypothesis that exercise ameliorates contractile dysfunction by interfering with homocysteine - ß2-adrenergic receptor (AR) interactions, inducing ß2-adrenergic response and Gs (stimulatory G adenylyl cyclase dependent protein kinase), and lowering homocysteine level in diabetes. The effect of homocysteine on ß2-AR was determined by (a) scoring the ß2-AR in the cardiomyocytes treated with high dose of homocysteine using flow cytometry, and (b) co-localizing homocysteine with Gs (an inducer of ß2-AR) in the cardiomyocytes obtained from C57BL/ 6J (WT) and db/ db mice using confocal microscopy. The effect of exercise on the protein-protein interactions of homocysteine and ß2-AR in diabetes was evaluated by co-immunoprecipitation in the four groups of db/db mice: (1) sedentary, (2) treated with salbutamol (a ß2-AR agonist), (3) swimming exercise, and (4) swimming + salbutamol treatment. The effect of exercise on ß2-AR was determined by RT-PCR and Western blotting while cardiac dysfunction was assessed by echocardiography, and contractility and calcium transient of cardiomyocytes from the above four groups. The results revealed that elevated level of homocysteine decreases the number of ß2-AR and inhibits Gs in diabetes. However, exercise mitigates the interactions of homocysteine with ß2-AR and induces ß2-AR. Exercise also ameliorates cardiac dysfunction by enhancing the calcium transient of cardiomyocytes. To our knowledge, this is the first report showing mechanism of homocysteine mediated attenuation of ß2-AR response in diabetes and effect of exercise on homocysteine - ß2-AR interactions.

Attenuation of beta2-adrenergic receptors and homocysteine metabolic enzymes cause diabetic cardiomyopathy.

Although adrenergic receptors (AR) and hyperhomocysteinemia (HHcy) are implicated in heart failure, their role in diabetic cardiomyopathy is not completely understood. We tested the hypothesis that glucose mediated depletion of beta2-AR and HHcy impair contractile function of cardiomyocytes leading to diabetic cardiomyopathy. To prove the hypothesis, cardiac function was assessed in 12week male diabetic Ins2+/- Akita and C57BL/6J mice by echocardiography, pressure-volume loop, and contractile function of cardiomyocytes. The results revealed cardiac dysfunction in Akita. To investigate the mechanism, the levels of beta2-AR, GLUT4, sarcoplasmic reticulum calcium ATP-ase-isoform 2 (SERCA-2) and homocysteine (Hcy) metabolic enzymes-cystathionine beta synthase (CBS), cystathionine gamma lyase (CTH), and methyl tetrahydrofolate reductase (MTHFR) were determined in the heart. It revealed down-regulation of beta2-AR, GLUT4, SERCA-2, CBS, CTH, and MTHFR in Akita. Attenuation of beta2-AR in hyperglycemic condition was also confirmed in cardiomyocytes at in vitro level. Interestingly, the ex vivo treatment of cardiomyocytes with beta2-AR antagonist deteriorated whereas beta-AR agonist ameliorated contractile function. It points to the involvement of beta2-AR in diabetic cardiomyopathy. We conclude that degradation of beta2-AR and impairment of Hcy metabolism is implicated in diabetic cardiomyopathy.