Abhinav Diwan

MicroRNA-133a Protects Against Myocardial Fibrosis and Modulates Electrical Repolarization Without Affecting Hypertrophy in Pressure-Overloaded Adult Hearts

Rationale: MicroRNA (miR)-133a regulates cardiac and skeletal muscle differentiation and plays an important role in cardiac development. Because miR-133a levels decrease during reactive cardiac hypertrophy, some have considered that restoring miR-133a levels could suppress hypertrophic remodeling. Objective: To prevent the “normal” downregulation of miR-133a induced by an acute hypertrophic stimulus in the adult heart. Methods and Results: miR-133a is downregulated in transverse aortic constriction (TAC) and isoproterenol-induced hypertrophy, but not in 2 genetic hypertrophy models. Using MYH6 promoter-directed expression of a miR-133a genomic precursor, increased cardiomyocyte miR-133a had no effect on postnatal cardiac development assessed by measures of structure, function, and mRNA profile. However, increased miR-133a levels increased QT intervals in surface electrocardiographic recordings and action potential durations in isolated ventricular myocytes, with a decrease in the fast component of the transient outward K+ current, Ito,f, at baseline. Transgenic expression of miR-133a prevented TAC-associated miR-133a downregulation and improved myocardial fibrosis and diastolic function without affecting the extent of hypertrophy. Ito,f downregulation normally observed post-TAC was prevented in miR-133a transgenic mice, although action potential duration and QT intervals did not reflect this benefit. miR-133a transgenic hearts had no significant alterations of basal or post-TAC mRNA expression profiles, although decreased mRNA and protein levels were observed for the Ito,f auxiliary KChIP2 subunit, which is not a predicted target. Conclusions: These results reveal striking differences between in vitro and in vivo phenotypes of miR expression, and further suggest that mRNA signatures do not reliably predict either direct miR targets or major miR effects.

Reciprocal Regulation of Myocardial microRNAs and Messenger RNA in Human Cardiomyopathy and Reversal of the microRNA Signature by Biomechanical Support

Background— Much has been learned about transcriptional control of cardiac gene expression in clinical and experimental congestive heart failure (CHF), but less is known about dynamic regulation of microRNAs (miRs) in CHF and during CHF treatment. We performed comprehensive microarray profiling of miRs and messenger RNAs (mRNAs) in myocardial specimens from human CHF with (n=10) or without (n=17) biomechanical support from left ventricular assist devices in comparison to nonfailing hearts (n=11). Methods and Results— Twenty-eight miRs were upregulated >2.0-fold (P<0.001) in CHF, with nearly complete normalization of the heart failure miR signature by left ventricular assist device treatment. In contrast, of 444 mRNAs that were altered by >1.3-fold in failing hearts, only 29 mRNAs normalized by as much as 25% in post-left ventricular assist device hearts. Unsupervised hierarchical clustering of upregulated miRs and mRNAs with nearest centroid analysis and leave-1-out cross-validation revealed that combining the miR and mRNA signatures increased the ability of RNA profiling to serve as a clinical biomarker of diagnostic group and functional class. Conclusions— These results show that miRs are more sensitive than mRNAs to the acute functional status of end-stage heart failure, consistent with important functions for regulated miRs in the myocardial response to stress. Combined miR and mRNA profiling may have superior potential as a diagnostic and prognostic test in end-stage cardiomyopathy.

Impaired Autophagosome Clearance Contributes to Cardiomyocyte Death in Ischemia-Reperfusion Injury

Background— In myocardial ischemia, induction of autophagy via the AMP-induced protein kinase pathway is protective, whereas reperfusion stimulates autophagy with BECLIN-1 upregulation and is implicated in causing cell death. We examined flux through the macroautophagy pathway as a determinant of the discrepant outcomes in cardiomyocyte cell death in this setting. Methods and Results— Reversible left anterior descending coronary artery ligation was performed in mice with cardiomyocyte-restricted expression of green fluorescent protein–tagged microtubule–associated protein light chain-3 to induce ischemia (120 minutes) or ischemia/reperfusion (30–90 minutes) with saline or chloroquine pretreatment (n=4 per group). Autophagosome clearance, assessed as the ratio of punctate light chain-3 abundance in saline to chloroquine-treated samples, was markedly impaired with ischemia/reperfusion compared with sham controls. Reoxygenation increased cell death in neonatal rat cardiomyocytes compared with hypoxia alone, markedly increased autophagosomes but not autolysosomes (assessed as punctate dual fluorescent mCherry-green fluorescent protein tandem-tagged light chain-3 expression), and impaired clearance of polyglutamine aggregates, indicating impaired autophagic flux. The resultant autophagosome accumulation was associated with increased reactive oxygen species and mitochondrial permeabilization, leading to cell death, which was attenuated by cyclosporine A pretreatment. Hypoxia-reoxygenation injury was accompanied by reactive oxygen species–mediated BECLIN-1 upregulation and a reduction in lysosome-associated membrane protein-2, a critical determinant of autophagosome-lysosome fusion. Restoration of lysosome-associated membrane protein-2 levels synergizes with partial BECLIN-1 knockdown to restore autophagosome processing and to attenuate cell death after hypoxia-reoxygenation. Conclusion— Ischemia/reperfusion injury impairs autophagosome clearance mediated in part by reactive oxygen species–induced decline in lysosome-associated membrane protein-2 and upregulation of BECLIN-1, contributing to increased cardiomyocyte death.

Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of Bnip3 restrains postinfarction remodeling in mice

Following myocardial infarction, nonischemic myocyte death results in infarct expansion, myocardial loss, and ventricular dysfunction. Here, we demonstrate that a specific proapoptotic gene, Bnip3, minimizes ventricular remodeling in the mouse, despite having no effect on early or late infarct size. We evaluated the effects of ablating Bnip3 on cardiomyocyte death, infarct size, and ventricular remodeling after surgical ischemia/reperfusion (IR) injury in mice. Immediately following IR, no significant differences were observed between Bnip3(-/-) and WT mice. However, at 2 days after IR, apoptosis was diminished in Bnip3(-/-) periinfarct and remote myocardium, and at 3 weeks after IR, Bnip3(-/-) mice exhibited preserved LV systolic performance, diminished LV dilation, and decreased ventricular sphericalization. These results suggest myocardial salvage by inhibition of apoptosis. Forced cardiac expression of Bnip3 increased cardiomyocyte apoptosis in unstressed mice, causing progressive LV dilation and diminished systolic function. Conditional Bnip3 overexpression prior to coronary ligation increased apoptosis and infarct size. These studies identify postischemic apoptosis by myocardial Bnip3 as a major determinant of ventricular remodeling in the infarcted heart, suggesting that Bnip3 may be an attractive therapeutic target.

Cardiac-Specific Ablation of G-Protein Receptor Kinase 2 Redefines Its Roles in Heart Development and β-Adrenergic Signaling

G-protein receptor kinase 2 (GRK2) is 1 of 7 mammalian GRKs that phosphorylate ligand-bound 7-transmembrane receptors, causing receptor uncoupling from G proteins and potentially activating non–G-protein signaling pathways. GRK2 is unique among members of the GRK family in that its genetic ablation causes embryonic lethality. Cardiac abnormalities in GRK2 null embryos implicated GRK2 in cardiac development but prevented studies of the knockout phenotype in adult hearts. Here, we created GRK2-loxP–targeted mice and used Cre recombination to generate germline and cardiac-specific GRK2 knockouts. GRK2 deletion in the preimplantation embryo with EIIa-Cre (germline null) resulted in developmental retardation and embryonic lethality between embryonic day 10.5 (E10.5) and E11.5. At E9.5, cardiac myocyte specification and cardiac looping were normal, but ventricular development was delayed. Cardiomyocyte-specific ablation of GRK2 in the embryo with Nkx2.5-driven Cre (cardiac-specific GRK2 knockout) produced viable mice with normal heart structure, function, and cardiac gene expression. Cardiac-specific GRK2 knockout mice exhibited enhanced inotropic sensitivity to the &bgr;-adrenergic receptor agonist isoproterenol, with impairment of normal inotropic and lusitropic tachyphylaxis, and exhibited accelerated development of catecholamine toxicity with chronic isoproterenol treatment. These findings show that cardiomyocyte autonomous GRK2 is not essential for myocardial development after cardiac specification, suggesting that embryonic developmental abnormalities may be attributable to extracardiac effects of GRK2 ablation. In the adult heart, cardiac GRK2 is a major factor regulating inotropic and lusitropic tachyphylaxis to &bgr;-adrenergic agonist, which likely contributes to its protective effects in catecholamine cardiomyopathy.

Nix-Mediated Apoptosis Links Myocardial Fibrosis, Cardiac Remodeling, and Hypertrophy Decompensation

Background— Pathological cardiac hypertrophy inevitably remodels, leading to functional decompensation. Although modulation of apoptosis-regulating genes occurs in cardiac hypertrophy, a causal role for programmed cardiomyocyte death in left ventricular (LV) remodeling has not been established. Methods and Results— We targeted the gene for proapoptotic Nix, which is transcriptionally upregulated in pressure overload and Gq-dependent hypertrophies, in the mouse germ line or specifically in cardiomyocytes (knockout [KO]) and conditionally overexpressed it in the heart (transgenic [TG]). Conditional forced Nix expression acted synergistically with the prohypertrophic Gq transgene to increase cardiomyocyte apoptosis (0.8±0.1% in GqTG versus 7.8±0.6% in GqTG+NixTG; P<0.001), causing lethal cardiomyopathy with LV dilation and depressed systolic function (percent fractional shortening, 39±4 versus 23±4; P=0.042). In the reciprocal experiment, germ-line Nix ablation significantly reduced cardiomyocyte apoptosis (4.8±0.2% in GqTG+Nix KO versus 8.4±0.5% in GqTG; P=0.001), which improved percent fractional shortening (43±3% versus 27±3%; P=0.017), attenuated LV remodeling, and largely prevented lethality in the Gq peripartum model of apoptotic cardiomyopathy. Cardiac-specific (Nkx2.5-Cre) Nix KO mice subjected to transverse aortic constriction developed significantly less LV dilation by echocardiography and magnetic resonance imaging, maintained concentric remodeling, and exhibited preserved LV ejection fraction (61±2% in transverse aortic constriction cardiac Nix KO versus 36±6% in transverse aortic constriction wild-type mice; P=0.003) at 9 weeks, with reduced cardiomyocyte apoptosis at day 4 (1.70±0.21% versus 2.73±0.35%; P=0.032). Conclusions— Nix-induced cardiomyocyte apoptosis is a major determinant of adverse remodeling in pathological hypertrophies, a finding that suggests therapeutic value for apoptosis inhibition to prevent cardiomyopathic decompensation.

Endoplasmic reticulum–mitochondria crosstalk in NIX-mediated murine cell death

Transcriptional upregulation of the proapoptotic BCL2 family protein NIX limits red blood cell formation and can cause heart failure by inducing cell death, but the requisite molecular events are poorly defined. Here, we show complementary mechanisms for NIX-mediated cell death involving direct and ER/sarcoplasmic reticulum-mediated (ER/SR-mediated) mitochondria disruption. Endogenous cardiac NIX and recombinant NIX localize both to the mitochondria and to the ER/SR. In genetic mouse models, cardiomyocyte ER/SR calcium stores are proportional to the level of expressed NIX. Whereas Nix ablation was protective in a mouse model of apoptotic cardiomyopathy, genetic correction of the decreased SR calcium content of Nix-null mice restored sensitivity to cell death and reestablished cardiomyopathy. Nix mutants specific to ER/SR or mitochondria activated caspases and were equally lethal, but only ER/SR-Nix caused loss of the mitochondrial membrane potential. These results establish a new function for NIX as an integrator of transcriptional and calcium-mediated signals for programmed cell death.

Dual autonomous mitochondrial cell death pathways are activated by Nix/BNip3L and induce cardiomyopathy

Dysregulation of programmed cell death due to abnormal expression of Bcl-2 proteins is implicated in cancer, neurodegenerative diseases, and heart failure. Among Bcl-2 family members, BNip proteins uniquely stimulate cell death with features of both apoptosis and necrosis. Localization of these factors to mitochondria and endoplasmic reticulum (ER) provides additional complexity. Previously, we observed regulation of intracellular calcium stores by reticular Nix. Here, we report effects of Nix targeting to mitochondria or ER on cell death pathways and heart failure progression. Nix-deficient fibroblasts expressing mitochondrial-directed or ER-directed Nix mutants exhibited similar cytochrome c release, caspase activation, annexin V and TUNEL labeling, and cell death. ER-Nix cells, but not mitochondrial-Nix cells, showed dissipation of mitochondrial inner membrane potential, Δψm, and were protected from cell death by cyclosporine A or ppif ablation, implicating the mitochondrial permeability transition pore (MPTP). ER-Nix cells were not protected from death by caspase inhibition or combined ablation of Bax and Bak. Combined inhibition of caspases and the MPTP fully protected against Nix-mediated cell death. To determine the role of the dual pathways in heart failure, mice conditionally overexpressing Nix or Nix mutants in hearts were created. Cardiomyocte death caused by mitochondrial- and ER-directed Nix was equivalent, but ppif ablation fully protected only ER-Nix. Thus, Nix stimulates dual autonomous death pathways, determined by its subcellular localization. Mitochondrial Nix activates Bax/Bak- and caspase-dependent apoptosis, whereas ER-Nix activates Bax/Bak-independent, MPTP-dependent necrosis. Complete protection against programmed cell death mediated by Nix and related factors can be achieved by simultaneous inhibition of both pathways.

Clinical and Genetic Modifiers of Long-Term Survival in Heart Failure

OBJECTIVES This study sought to identify genetic modifiers of beta-blocker response and long-term survival in heart failure (HF). BACKGROUND Differences in beta-blocker treatment effect between Caucasians and African Americans with HF have been reported. METHODS This was a prospective cohort study of 2,460 patients (711 African American, 1,749 Caucasian) enrolled between 1999 and 2007; 2,039 patients (81.7%) were treated with a beta-blocker. Each was genotyped for beta1-adrenergic receptor (ADRB1) Arg389>Gly and G-protein receptor kinase 5 (GRK5) Gln41>Leu polymorphisms, which are more prevalent among African Americans than Caucasians. The primary end point was survival time from HF onset. RESULTS There were 765 deaths during follow-up (median 46 months). beta-blocker treatment increased survival in Caucasians (log-rank p = 0.00038) but not African Americans (log-rank p = 0.327). Among patients not taking beta-blockers, ADRB1 Gly389 was associated with decreased survival in Caucasians (hazard ratio [HR]: 1.98, 95% confidence interval [CI]: 1.1 to 3.7, p = 0.03) whereas GRK5 Leu41 was associated with improved survival in African Americans (HR: 0.325, CI: 0.133 to 0.796, p = 0.01). African Americans with ADRB1 Gly389Gly GRK5 Gln41Gln derived a similar survival benefit from beta-blocker therapy (HR: 0.385, 95% CI: 0.182 to 0.813, p = 0.012) as Caucasians with the same genotype (HR: 0.529, 95% CI: 0.326 to 0.858, p = 0.0098). CONCLUSIONS These data show that differences caused by beta-adrenergic receptor signaling pathway gene polymorphisms, rather than race, are the major factors contributing to apparent differences in the beta-blocker treatment effect between Caucasians and African Americans; proper evaluation of treatment response should account for genetic variance.

Cardiomyocyte Degeneration With Calpain Deficiency Reveals a Critical Role in Protein Homeostasis

Regulating the balance between synthesis and proteasomal degradation of cellular proteins is essential for tissue growth and maintenance, but the critical pathways regulating protein ubiquitination and degradation are incompletely defined. Although participation of calpain calcium-activated proteases in post-necrotic myocardial autolysis is well characterized, their importance in homeostatic turnover of normal cardiac tissue is controversial. Hence, we evaluated the consequences of physiologic calpain (calcium-activated protease) activity in cultured cardiomyocytes and unstressed mouse hearts. Comparison of in vitro proteolytic activities of cardiac-expressed calpains 1 and 2 revealed calpain 1, but not calpain 2, activity at physiological calcium concentrations. Physiological calpain 1 activation was evident in adenoviral transfected cultured cardiomyocytes as proteolysis of specific substrates, generally increased protein ubiquitination, and accelerated protein turnover, that were each inhibited by coexpression of the inhibitor protein calpastatin. Conditional forced expression of calpain 1, but not calpain 2, in mouse hearts demonstrated substrate-specific proteolytic activity under basal conditions, with hyperubiquitination of cardiac proteins and increased 26S proteasome activity. Loss of myocardial calpain activity by forced expression of calpastatin diminished ubiquitination of 1 or more specific myocardial proteins, without affecting overall ubiquitination or proteasome activity, and resulted in a progressive dilated cardiomyopathy characterized by accumulation of intracellular protein aggregates, formation of autophagosomes, and degeneration of sarcomeres. Thus, calpain 1 is upstream of, and necessary for, ubiquitination and proteasomal degradation of a subset of myocardial proteins whose abnormal accumulation produces autophagosomes and degeneration of cardiomyocytes with functional decompensation.