Model Of Dilated Cardiomyopathy Research Articles

Production of TRPV2-targeting functional antibody ameliorating dilated cardiomyopathy and muscular dystrophy in animal models

Abnormal Ca2+ handling is essential in the pathophysiology of degenerative muscle disorders, such as dilated cardiomyopathy (DCM) and muscular dystrophy (MD). Transient receptor potential cation channel, subfamily V, member 2 (TRPV2) is a candidate for Ca2+ entry and a potential therapeutic target for degenerative muscle disorders, there are few specific inhibitors for TRPV2. In this study, we produced a monoclonal antibody (designated mAb88-2) and two polyclonal antibodies (pAb591 and pAb592) that selectively recognize TRPV2 from the outside of cells and interact with the turret region of the pore-forming outer gate. These antibodies inhibited Ca2+ influx via TRPV2 in cultured cells and substantially reduced TRPV2 in the plasma membrane via cellular internalization. We evaluated the therapeutic efficacy of the functional antibody in δ-sarcoglycan-deficient hamster (J2N-k) models of DCM and MD and in the 4C30 DCM model of murine heart failure. The intraperitoneal administration of the functional antibody (0.5 mg/kg) for 2 weeks (once a week) prevented the progression of cardiac dysfunction, as evaluated by echocardiography and histological staining, and improved the abnormal Ca2+ handling (high diastolic Ca2+ level and small Ca2+ transient peak) in cardiomyocytes isolated from J2N-k hamsters and prevented skeletal muscle damage. Further, the antibody effectively prevented heart failure in the 4C30 mouse model with end-stage DCM. Interestingly, endogenous TRPV2 that accumulated in the cardiac and skeletal muscle sarcolemma disappeared upon antibody administration. Thus, the newly produced antibodies are capable of ameliorating DCM and MD by promoting the cellular internalization of TRPV2; antibodies specific to human TRPV2 may substantially improve the treatment of patients with degenerative muscle diseases.

Cardioprotective effects of α-cardiac actin on oxidative stress in a dilated cardiomyopathy mouse model.

The expression of α-cardiac actin, a major constituent of the cytoskeleton of cardiomyocytes, is dramatically decreased in a mouse model of dilated cardiomyopathy triggered by inducible cardiac-specific serum response factor (Srf) gene disruption that could mimic some forms of human dilated cardiomyopathy. To investigate the consequences of the maintenance of α-cardiac actin expression in this model, we developed a new transgenic mouse based on Cre/LoxP strategy, allowing together the induction of SRF loss and a compensatory expression of α-cardiac actin. Here, we report that maintenance of α-cardiac actin within cardiomyocytes temporally preserved cytoarchitecture from adverse cardiac remodeling through a positive impact on both structural and transcriptional levels. These protective effects were accompanied in vivo by the decrease of ROS generation and protein carbonylation and the downregulation of NADPH oxidases NOX2 and NOX4. We also show that ectopic expression of α-cardiac actin protects HEK293 cells against oxidative stress induced by H2 O2 . Oxidative stress plays an important role in the development of cardiac remodeling and contributes also to the pathogenesis of heart failure. Taken together, these findings indicate that α-cardiac actin could be involved in the regulation of oxidative stress that is a leading cause of adverse remodeling during dilated cardiomyopathy development.

Cardiac-Specific Overexpression of Catalytically Inactive Corin Reduces Edema, Contractile Dysfunction, and Death in Mice with Dilated Cardiomyopathy.

Humans with dilated cardiomyopathy (DCM) and heart failure (HF) develop low levels of corin, a multi-domain, cardiac-selective serine protease involved in natriuretic peptide cleavage and sodium and water regulation. However, experimental restoration of corin levels markedly attenuates HF progression. To determine whether the beneficial effects of corin in HF require catalytic activity, we engineered cardiac overexpression of an enzymatically inactive corin transgene (corin-Tg(i)). On a wild-type (WT) background, corin-Tg(i) had no evident phenotypic effects. However, in a well-established genetic model of DCM, corin-Tg(i)/DCM mice had increased survival (p < 0.01 to 0.001) vs. littermate corin-WT/DCM controls. Pleural effusion (p < 0.01), lung edema (p < 0.05), systemic extracellular free water (p < 0.01), and heart weight were decreased (p < 0.01) in corin-Tg(i)/DCM vs. corin-WT/DCM mice. Cardiac ejection fraction and fractional shortening improved (p < 0.01), while ventricular dilation decreased (p < 0.0001) in corin-Tg(i)/DCM mice. Plasma atrial natriuretic peptide, cyclic guanosine monophosphate, and neprilysin were significantly decreased. Cardiac phosphorylated glycogen synthase kinase-3β (pSer9-GSK3β) levels were increased in corin(i)-Tg/DCM mice (p < 0.01). In summary, catalytically inactive corin-Tg(i) decreased fluid retention, improved contractile function, decreased HF biomarkers, and diminished cardiac GSK3β activity. Thus, the protective effects of cardiac corin on HF progression and survival in experimental DCM do not require the serine protease activity of the molecule.

Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs

There is increasing momentum toward the development of gene therapy for heart failure (HF) that is defined by impaired calcium (Ca2+) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca2+ pump) and PLB (phospholamban, ventricular peptide inhibitor of SERCA) to test directly the effectiveness of loss-of-inhibition/gain-of-binding (LOI/GOB) PLB mutants (PLBM) that were engineered to compete with the binding of inhibitory wild-type PLB (PLBWT). Our therapeutic strategy is to relieve PLBWT inhibition of SERCA2a by using the reserve adrenergic capacity mediated by PLB to enhance cardiac contractility. Using a FRET assay, we determined that the combination of a LOI PLB mutation (L31A) and a GOB PLB mutation (I40A) results in a novel engineered LOI/GOB PLBM (L31A/I40A) that effectively competes with PLBWT binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of PLBM enhances SERCA Ca-ATPase activity by increasing enzyme Ca2+ affinity (1/KCa) in PLBWT-inhibited HEK293 cell homogenates. For an initial assessment of PLBM physiological effectiveness, we used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual. In this system, we observed that adeno-associated virus 2 (rAAV2)-driven expression of PLBM enhances the amplitude of SR Ca2+ release and the rate of SR Ca2+ re-uptake. To assess therapeutic potential, we used a hiPSC-CM model of dilated cardiomyopathy (DCM) containing PLB mutation R14del, where we observed that rAAV2-driven expression of PLBM rescues arrhythmic Ca2+ transients and alleviates decreased Ca2+ transport. Thus, we propose that PLBM transgene expression is a promising gene therapy strategy that directly targets the underlying pathophysiology of abnormal Ca2+ transport and thus contractility in underlying systolic heart failure.

LTBP2 knockdown by siRNA reverses myocardial oxidative stress injury, fibrosis and remodelling during dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterised by left ventricular dilation and associated with systolic dysfunction. Recent evidence has reported the high expression of latent transforming growth factor beta binding protein 2 (LTBP2) in heart diseases, which may play a role in regulating multiple biological functions of myocardial cells. Thus, this study set out to investigate the molecular mechanism and effects of LTBP2 in myocardial oxidative stress injury, fibrosis and remodelling in a rat model of DCM, with the involvement of NF-κB signalling pathway. The rat model of DCM was treated with si-LTBP2 and/or activator of NF-κB signalling pathway to examine the haemodynamic indexes, cardiac functions, oxidative stress injury, fibrosis and remodelling. Moreover, in vitro experiments were conducted to verify the regulatory role of LTBP2 and NF-κB signalling pathway in DCM. LTBP2 was up-regulated in DCM rats. After LTBP2 was knocked down, haemodynamic indexes, HW/BW ratio, collagen volume fraction (CVF) level, positive expression of LTBP2, levels of reactive oxygen species (ROS), malondialdehyde (MDA), interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), tumour necrosis factor beta 1 (TGF-β1) and brain natriuretic peptide (BNP) were all decreased. Meanwhile, levels of LTBP2, Col-I, Col-III, p65 and p52 were also reduced, while HW, BW and levels of SOD and TAOC were increased. In contrast, activation of NF-κB signalling pathway reversed effects of LTBP2 gene silencing. These findings were confirmed by in vivo experiments. LTBP2 silencing can attenuate myocardial oxidative stress injury, myocardial fibrosis and myocardial remodelling in DCM rats by down-regulating the NF-κB signalling pathway.

Wisteria floribunda agglutinin staining for the quantitative assessment of cardiac fibrogenic activity in a mouse model of dilated cardiomyopathy

Cardiac fibrosis is a typical phenomenon in failing hearts for most cardiac diseases, including dilated cardiomyopathy (DCM), and its specific detection and quantification are crucial for the analysis of cardiac remodeling. Since cardiac fibrosis is characterized by extensive remodeling of the myocardial extracellular matrix (ECM), in which glycoproteins are the major components, we assumed that fibrosis-related alterations in the cardiac glycome and glycoproteome would be suitable targets for the detection of cardiac fibrosis. Here, we compared protein glycosylation between heart tissues of normal and DCM model mice by laser microdissection-assisted lectin microarray. Among 45 lectins, Wisteria floribunda agglutinin (WFA) was selected as the most suitable lectin for staining cardiac fibrotic tissues. Although the extent of WFA staining was highly correlated (r > 0.98) with that of picrosirius red staining, a common collagen staining method, WFA did not bind to collagen fibers. Further histochemical analysis with N-glycosidase revealed that WFA staining of fibrotic tissues was attributable to the binding of WFA to N-glycoproteins. Using a mass spectrometry-based approach, we identified WFA-binding N-glycoproteins expressed in DCM hearts, many of which were fibrogenesis-related ECM proteins, as expected. In addition, the identified glycoproteins carrying WFA-binding N-glycans were detected only in DCM hearts, suggesting their cooperative glycosylation alterations with disease progression. Among these WFA-binding ECM N-glycoproteins, co-localization of the collagen α6(VI) chain protein and WFA staining in cardiac tissue sections was confirmed with a double-staining analysis. Collectively, these results indicate that WFA staining is more suitable for the quantitative assessment of cardiac fibrogenic activity than current collagen staining methods. Furthermore, given that plasma WFA-binding glycoprotein levels were significantly correlated with the echocardiographic parameters for left ventricular remodeling, cardiac WFA-binding glycoproteins are candidate circulating glyco-biomarkers for the quantification and monitoring of cardiac fibrogenesis.

3071Peptidomimetic targeting of CavBeta2 improves contractility in models of senescence- or genetically (MYBPC3 KI)-induced heart failure

Abstract Background L-type calcium channel (LTCC) trafficking controls LTCC density at T-tubule levels for optimal Excitation-Coupling (EC) and resultant adaptive heart work. In some forms of heart failure (HF), abnormalities in calcium-induced calcium-release have been proposed to arise from alteration of T-tubular dyad architecture (LTCC-RyRs) associated with impaired LTCC density. Recently, the R7W-MP peptide, working as a binder of the LTCC Cavβ2 chaperone, was shown to restore the altered density of LTCC current by both promoting forward and reducing reverse trafficking, which consequently improved cellular calcium homeostasis. Accordingly, R7W-MP improved the impaired cardiomyocyte calcium current density and the reduced Ejection Fraction (EF%) in a pharmacologically-induced diabetes model (STZ mice). We aimed to investigate further the benefit to improve LTCC trafficking pathway with R7W-MP in a more physiological model of HF (senescent mice) and in a Dilated Cardiomyopathy (DCM) model (HO MYBPC3 targeted KI mutant). Methods Senescent male C57Bl/6J mice (26 months) or HO MYBPC3 KI male mice (2 months) were treated with R7W-MP (3 mg/kg/d IP for 3 days). Echocardiographies (echo) were conducted before treatment and 4-hours after the last injection. When applied, Pressure-Volume (PV)-loop investigations were conducted one day post-echo 4 hours following an additional R7W-MP injection. Results In senescent mice population, HF was characterized by a midrange ejection fraction (EF%= 43±2 vs 55±1 for young adult mice) associated with enlarged ventricles and decreased cardiac contractility. In contrast to a scrambled peptide (scrP), R7W-MP markedly increased EF% monitored by echo (+38%, 63±3 vs 45±1 for scrP, p≤0.001, n=6–7) without modification of heart rate. EF% improvement was confirmed by PV-loop analysis (78±3 vs 51±4 for scrP (+54%), p≤0.001, n=5), associated with a marked, although not significant, 2.5-fold increase in myocardial contractility [end systolic pressure volume relationship (ESPVR) = 12.1±3.6 vs 4.9±1.3 for scrP, p=0.10, n=4]. Stroke volume, cardiac output and end diastolic volume tended to decrease suggesting an impaired LV filling at this dose regimen. In the DCM model, HF was more severe with a dramatically low EF% (26±1, n=8), impaired myocardial contractility and a pronounced left ventricle enlargement. R7W-MP significantly increased EF% (+17%, reaching 31±1, p≤0.01, n=8) without altering heart rate. Stroke volume was significantly increased by 36% (32±3 vs 24±3 mL at baseline, p≤0.01), without any impairment of diastolic function. All parameters returned to baseline after a 2 week-washout period. Conclusions R7W-MP displays potent positive inotrope properties in senescent or DCM mice models. Although further asses tsments of diastolic function are needed (different dosing and duration), these data underline the potential benefit brought by LTCC trafficking modulation to treat severe dilated cardiomyopathy.

Haploinsufficiency of mechanistic target of rapamycin ameliorates bag3 cardiomyopathy in adult zebrafish.

ABSTRACTThe adult zebrafish is an emerging vertebrate model for studying human cardiomyopathies; however, whether the simple zebrafish heart can model different subtypes of cardiomyopathies, such as dilated cardiomyopathy (DCM), remains elusive. Here, we generated and characterized an inherited DCM model in adult zebrafish and used this model to search for therapeutic strategies. We employed transcription activator-like effector nuclease (TALEN) genome editing technology to generate frame-shift mutants for the zebrafish ortholog of human BCL2-associated athanogene 3 (BAG3), an established DCM-causative gene. As in mammals, the zebrafish bag3 homozygous mutant (bag3e2/e2) exhibited aberrant proteostasis, as indicated by impaired autophagy flux and elevated ubiquitinated protein aggregation. Through comprehensive phenotyping analysis of the mutant, we identified phenotypic traits that resembled DCM phenotypes in mammals, including cardiac chamber enlargement, reduced ejection fraction characterized by increased end-systolic volume/body weight (ESV/BW), and reduced contractile myofibril activation kinetics. Nonbiased transcriptome analysis identified the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling in bag3e2/e2 mutant hearts. Further genetic studies showed that mtorxu015/+, an mTOR haploinsufficiency mutant, repaired abnormal proteostasis, improved cardiac function and rescued the survival of the bag3e2/e2 mutant. This study established the bag3e2/e2 mutant as a DCM model in adult zebrafish and suggested mtor as a candidate therapeutic target gene for BAG3 cardiomyopathy.

P6274Role of the nicotinamide riboside kinase 2 in NAD metabolism in the heart in basal and pathological condition

Abstract Background NAD is a major coenzyme in energy metabolism and a substrate for SIRT1 and PARP1 enzymes involved in the response to energy and oxidative stress. We have shown the beneficial effects of nicotinamide riboside (NR), a new type of vitamin B3, on cardiac function and remodelling in a mouse model of dilated cardiomyopathy (DCM) triggered by deletion of the SRF transcription factor in the heart (Srf-HKO) (1). This functional improvement correlated with protection of NAD metabolism and a robust increase in cardiac expression of the Nicotinamide Riboside Kinase 2 (NMRK2) that phosphorylates NR to generate nicotinamide mononucleotide (NMN), an immediate precursor of NAD. Purpose We aim to understand the role of the NMRK2-mediated NAD biosynthetic pathway in the heart at baseline and in the DCM context. Methods We generated Nmrk2-KO mice that we bred with Srf-HKO to generate double KO mice (db-KO). We analysed cardiac function and remodelling by echocardiography and quantified myocardial NAD levels at baseline and following NR supplementation in food. Results Nmrk2KO mice developed a progressive eccentric remodelling of LV and decline in EF with aging. At 24-mo, we observed a reduction of myocardial NAD levels (−40% compared to wild type, p&lt;0.05) and of LVEF (61%, SD 6.3% in Nmrk2-KO vs 78%, SD 1.5% in WT, p&lt;0.05). To assess the contribution of cardiac Nmrk2 induction to NR response in DCM, we compared SrfH-KO and db-KO mice fed with control diet (CD) or NR supplemented diet for 40 days starting at young age (2-mo). NR reduced the extent of LV eccentric remodelling and drop in EF as well as the thinning of the LV posterior wall in both genotypes (2-way ANOVA, diet effect, p&lt;0.01). Myocardial NAD levels were more reduced in db-KO mice under CD diet (−22% compared to control mice, p&lt;0.05) than in Srf-HKO mice (−11%, non-significant), when we previously showed a 25% drop in myocardial NAD in aged SrfHKO mice (1). NR partially preserved cardiac NAD pool in db-KOmice (−10% compared to controls, non-significant). Parallel pathways for NMN synthesis were studied. Nampt gene expression was significantly repressed in db-KO mice fed with CD or NR diet compared to control mice (−50% in average, p&lt;0.01), when there was only a trend toward lower expression in SrfHKO mice (−40% in average, p&gt;0.05). Nmrk1 gene expression trended to increase in all groups compared to wild-type control mice. Conclusion We show that NMRK2 pathway plays a role in the maintenance of basal cardiac function and NAD levels when relying on the endogenous myocardial NR pool. In contrast, the beneficial effect of a therapeutic dose of NR is not affected by the lack of NMRK2 suggesting compensation by NMRK1 in the heart and/or that NR beneficial effects on cardiac function could be mediated through its action on systemic metabolism. Aging appears as an aggravating factor for the loss of myocardial NAD coenzyme in DCM. Acknowledgement/Funding Agence Nationale pour la Recherche, Fondation de France

P318Exosomes mediate myocardial regeneration of cardiac progenitor cells in a swine model of dilated cardiomyopathy

Abstract Introduction Stem cell therapy has been shown to improve cardiac function. The mechanisms of therapeutic efficacy are considered the secretion of paracrine factors but the details are still unknown. Hypothesis Exosomes are extracellular vesicles containing bioactive substances such as proteins, messenger RNAs and micro RNAs. We hypothesized that exosomes may be the main paracrine factors to mediate therapeutic efficacy of cardiosphere-derived cells (CDCs). Methods Farm pigs (30 kg, n=10) were treated by intracoronary administration of 10,000 microspheres (100–300 μm) into three vessels. Two weeks later, 9.0×106 CDCs pretreated by exosome inhibitor (EI; 20μM of GW4869) or DMSO as controls were selectively infused into three coronary arteries. Evaluation of ejection fraction (EF) was performed before cell infusion and 1 month after protocol treatment. Results Pigs developed diffuse hypokinetic heart failure (baseline EF 37.1%±2.1%) and randomly assigned into two groups (CDCs with EI: n=5, CDCs with DMSO: n=5). No serious adverse events were found during the CDCs infusion. Significant improvement of EF was observed in CDCs with DMSO group (37.1%±2.1% to 42.5%±3.0%; P=0.01), whereas no change was found in CDCs with EI group (37.1%±2.4% to 36.2%±2.9%; P=0.58). Myocardial fibrosis stained by picrosirius red was significantly reduced in CDCs with DMSO group compared with CDCs with EI group (9.5±3.6% versus 17.3±5.3%; P&lt;0.01). Conclusions We confirmed the therapeutic efficacy of CDCs and these effects were mainly mediated by exosomes.

4968Increased Gao expression underlies cardiac dysfunction and lethal arrhythmias accompanied with abnormal Ca2+ handling

Abstract Background We previously demonstrated that a transcriptional repressor, neuron restrictive silencer factor (NRSF), maintains normal cardiac function and electrical stability. Transgenic mice expressing a dominant-negative mutant of NRSF in their hearts (dnNRSF-Tg) exhibit systolic dysfunction with cardiac dilation and premature death due to lethal arrhythmias like human dilated cardiomyopathy (DCM). Underlining mechanisms remain to be elucidated, however. Purpose We studied underling mechanisms by which NRSF maintains normal cardiac function to identify novel therapeutic targets for heart failure. Methods and results We generated cardiac-specific NRSF knockout mice (NRSFcKO) and confirmed that cardiac phenotypes of NRSFcKO are similar to those of dnNRSF-Tg. cDNA microarray analysis revealed that cardiac gene expression of GNAO1 that encodes Gαo, a member of inhibitory G protein Gαi family, is increased in both dnNRSF-Tg and NRSFcKO ventricles. We confirmed that GNAO1 is a direct target of NRSF through ChIP-seq analysis, reporter assay and electrophoretic mobility shift assay. In dnNRSF-Tg, pharmacological inhibition of Gαo with pertussis toxin improved systolic dysfunction and knockdown of Gαo by crossing with GNAO1 knockout mice improved not only systolic function but also frequency of ventricular arrhythmias and survival rates. Electrophysiological and biochemical analysis in ventricular myocytes obtained from dnNRSF-Tg demonstrated that genetic reduction of Gαo ameliorated abnormalities in Ca2+ handling, which include increased current density in surface sarcolemmal L-type Ca2+ channel, reduced content of sarcoplasmic reticulum Ca2+ and lowered peak of Ca2+ transient. Furthermore, genetic reduction of Gαo attenuated increased phosphorylation levels of CAMKII in dnNRSF-Tg ventricles, which presumably underlies the improvement in Ca2+ handling. In addition, we identified increased Gαo expression in ventricles of heart failure model mice induced by transverse aortic constriction and cardiac troponin T mutant DCM model mice, in both of which, genetic reduction of Gαo ameliorated cardiac dysfunction. Figure 1 Conclusions We found that increased expression of Gαo, induced by attenuation of NRSF-mediated repression, plays a crucial role in the progression of cardiac dysfunction and lethal arrhythmias by evoking Ca2+ handling abnormality. These data demonstrate that Gαo is a potential therapeutic target for heart failure.

Effect of bilateral sympathectomy in a rat model of dilated cardiomyopathy induced by doxorubicin

ObjectiveThe study objective was to evaluate the effect of bilateral sympathectomy on ventricular remodeling and function in a rat model of dilated cardiomyopathy induced by doxorubicin. MethodsDilated cardiomyopathy was induced in male Wistar rats by weekly intraperitoneal injection of doxorubicin (2 mg/kg) for 9 weeks. Animals were divided into 4 groups: dilated cardiomyopathy; bilateral sympathectomy, submitted on day 15 of the protocol to bilateral sympathectomy; angiotensin-converting enzyme inhibitor, treated with enalapril through day 15 until the end of the experimental protocol; and sham, nonsubmitted through doxorubicin protocol, with weekly intraperitoneal injections of saline solution (0.9%). The left ventricular function was assessed, and the heart was collected for posterior analyses. ResultsThe dilated cardiomyopathy group presented a significant decrease in the myocardial efficiency when compared with the sham group (33.4% vs 71.2%). Only the bilateral sympathectomy group was able to preserve it (57.5%; P = .0001). A significant dilatation in the left ventricular chamber was observed in the dilated cardiomyopathy group (15.9 μm2) compared with the sham group (10.2 μm2; P = .0053). Sympathectomy and enalapril prevented ventricular remodeling (9.5 and 9.6 μm2, respectively; P = .0034). There was a significant increase in interstitial myocardial fibrosis in the dilated cardiomyopathy group (14.8%) when compared with the sham group (2.4%; P = .0001). This process was significantly reduced with sympathectomy and enalapril (8.7 and 3.9%, respectively; P = .0001). ConclusionsBilateral sympathectomy was effective in preventing remodeling and left ventricular dysfunction in a rat model of dilated cardiomyopathy induced by doxorubicin.

Normalizing Plasma Renin Activity in Experimental Dilated Cardiomyopathy: Effects on Edema, Cachexia, and Survival.

Heart failure (HF) patients frequently have elevated plasma renin activity. We examined the significance of elevated plasma renin activity in a translationally-relevant model of dilated cardiomyopathy (DCM), which replicates the progressive stages (A–D) of human HF. Female mice with DCM and elevated plasma renin activity concentrations were treated with a direct renin inhibitor (aliskiren) in a randomized, blinded fashion beginning at Stage B HF. By comparison to controls, aliskiren treatment normalized pathologically elevated plasma renin activity (p < 0.001) and neprilysin levels (p < 0.001), but did not significantly alter pathological changes in plasma aldosterone, angiotensin II, atrial natriuretic peptide, or corin levels. Aliskiren improved cardiac systolic function (ejection fraction, p < 0.05; cardiac output, p < 0.01) and significantly reduced the longitudinal development of edema (extracellular water, p < 0.0001), retarding the transition from Stage B to Stage C HF. The normalization of elevated plasma renin activity reduced the loss of body fat and lean mass (cachexia/sarcopenia), p < 0.001) and prolonged survival (p < 0.05). In summary, the normalization of plasma renin activity retards the progression of experimental HF by improving cardiac systolic function, reducing the development of systemic edema, cachexia/sarcopenia, and mortality. These data suggest that targeting pathologically elevated plasma renin activity may be beneficial in appropriately selected HF patients.

Abstract 652: Increased Gα o Expression Plays a Pivotal Role in the Progression of Heart Failure by Impairing Ca 2+ Handling

Background: A transcriptional repressor, neuron restrictive silencer factor (NRSF), maintains normal cardiac function and electrical stability. Transgenic mice expressing a dominant-negative mutant of NRSF in their hearts (dnNRSF-Tg) exhibit systolic dysfunction and premature death due to lethal arrhythmias like human dilated cardiomyopathy(DCM). Underlining mechanisms remain to be elucidated, however. Purpose: To clarify how NRSF maintains normal cardiac homeostasis. Methods and Results: cDNA microarray analysis of dnNRSF-Tg and newly generated cardiac-specific NRSF knockout mice (NRSFcKO), which show similar cardiac phenotypes to those of dnNRSF-Tg, revealed that cardiac gene expression of GNAO1 that encodes Gα o , a member of inhibitory G protein Gα i family, is increased in both dnNRSF-Tg and NRSFcKO ventricles. ChIP-seq analysis, reporter assay and electrophoretic mobility shift assay identified that GNAO1 is a direct target of NRSF. In dnNRSF-Tg, pharmacological inhibition of Gα o by pertussis toxin improved systolic dysfunction and knockdown of Gα o by crossing with GNAO1 knockout mice improved not only systolic function but also frequency of ventricular arrhythmias and survival rates. Electrophysiological analysis in ventricular myocytes obtained from dnNRSF-Tg demonstrated that genetic reduction of Gα o ameliorated abnormalities in Ca 2 + handling, which include increased current density in surface sarcolemmal L-type Ca 2 + channel, reduced content of sarcoplasmic reticulum Ca 2+ and lowered peak of Ca 2+ transient. In addition, genetic reduction of Gα o attenuated increased phosphorylation levels of CAMKII and RyR2 in dnNRSF-Tg ventricles, which presumably underlies the improvement in Ca 2 + handling. Furthermore, we identified increased Gα o expression in ventricles of heart failure model mice induced by transverse aortic constriction and cardiac troponin T mutant DCM model mice, in both of which, genetic reduction of Gα o ameliorated cardiac dysfunction. Conclusions: We found that increased expression of Gα o , induced by attenuation of NRSF-mediated repression, plays a pivotal role in the progression of heart failure by evoking Ca 2+ handling abnormality. Gα o is a potential therapeutic target for heart failure.

Reduced hybrid/complex N-glycosylation disrupts cardiac electrical signaling and calcium handling in a model of dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is the third most common cause of heart failure, with ~70% of DCM cases considered idiopathic. We showed recently, through genetic ablation of the MGAT1 gene, which encodes an essential glycosyltransferase (GlcNAcT1), that prevention of cardiomyocyte hybrid/complex N-glycosylation was sufficient to cause DCM that led to heart failure and early death. Our findings are consistent with increasing evidence suggesting a link between aberrant glycosylation and heart diseases of acquired and congenital etiologies. However, the mechanisms by which changes in glycosylation contribute to disease onset and progression remain largely unknown. Activity and gating of voltage-gated Na+ and K+ channels (Nav and Kv respectively) play pivotal roles in the initiation, shaping and conduction of cardiomyocyte action potentials (APs) and aberrant channel activity was shown to contribute to cardiac disease. We and others showed that glycosylation can impact Nav and Kv function; therefore, here, we investigated the effects of reduced cardiomyocyte hybrid/complex N-glycosylation on channel activity to investigate whether chronic aberrant channel function can contribute to DCM. Ventricular cardiomyocytes from MGAT1 deficient (MGAT1KO) mice display prolonged APs and pacing-induced aberrant early re-activation that can be attributed to, at least in part, a significant reduction in Kv expression and activity that worsens over time suggesting heart disease-related remodeling. MGAT1KO Nav demonstrate no change in expression or maximal conductance but show depolarizing shifts in voltage-dependent gating. Together, the changes in MGAT1KO Nav and Kv function likely contribute to observed anomalous electrocardiograms and Ca2+ handling. These findings provide insight into mechanisms by which altered glycosylation contributes to DCM through changes in Nav and Kv activity that impact conduction, Ca2+ handling and contraction. The MGAT1KO can also serve as a useful model to study the effects of aberrant electrical signaling on cardiac function and the remodeling events that can occur with heart disease progression.

Association of intronic DNA methylation and hydroxymethylation alterations in the epigenetic etiology of dilated cardiomyopathy.

In this study, we investigated the role of DNA methylation [5-methylcytosine (5mC)] and 5-hydroxymethylcytosine (5hmC), epigenetic modifications that regulate gene activity, in dilated cardiomyopathy (DCM). A MYBPC3 mutant mouse model of DCM was compared with wild type and used to profile genomic 5mC and 5hmC changes by Chip-seq, and gene expression levels were analyzed by RNA-seq. Both 5mC-altered genes (957) and 5hmC-altered genes (2,022) were identified in DCM hearts. Diverse gene ontology and KEGG pathways were enriched for DCM phenotypes, such as inflammation, tissue fibrosis, cell death, cardiac remodeling, cardiomyocyte growth, and differentiation, as well as sarcomere structure. Hierarchical clustering of mapped genes affected by 5mC and 5hmC clearly differentiated DCM from wild-type phenotype. Based on these data, we propose that genomewide 5mC and 5hmC contents may play a major role in DCM pathogenesis. NEW & NOTEWORTHY Our data demonstrate that development of dilated cardiomyopathy in mice is associated with significant epigenetic changes, specifically in intronic regions, which, when combined with gene expression profiling data, highlight key signaling pathways involved in pathological cardiac remodeling and heart contractile dysfunction.

Reversion of cardiac dysfunction by a novel orally available calcium/calmodulin-dependent protein kinase II inhibitor, RA306, in a genetic model of dilated cardiomyopathy.

Despite improvements in patient identification and management, heart failure (HF) remains a major public health burden and an important clinical challenge. A variety of animal and human studies have provided evidence suggesting a central role of calcium/calmodulin-dependent protein kinase II (CaMKII) in the development of pathological cardiac remodelling and HF. Here, we describe a new potent, selective, and orally available CaMKII inhibitor. Chemical optimization led to the identification of RA306 as a selective CaMKII inhibitor. This compound was found potent on the cardiac CaMKII isoforms delta and gamma (IC50 in the 10 nM range), with pharmacokinetic properties allowing oral administration in animal models of HF. RA306 was administered to diseased mice carrying a mutation in alpha-actin that is responsible for dilated cardiomyopathy (DCM) in humans. In two separate studies, RA306 was orally administered at 30 mg/kg either for 2 weeks (twice a day) or for 2 months (once a day). Echocardiography monitoring showed that RA306 significantly improved cardiac function (ejection fraction and cardiac output) as compared to vehicle. These disease modifying effects of RA306 were associated with inhibition of cardiac phosphorylation of phospholamban (PLN) at threonine-17, indicating reduced cardiac CaMKII activity. This work supports the feasibility of identifying potent orally available CaMKII inhibitors suitable for clinical use to treat heart disease.

Effect of IL-6-mediated STAT3 signaling pathway on myocardial apoptosis in mice with dilated cardiomyopathy.

To investigate the effect of interleukin-6 (IL-6) gene knockout on apoptosis of myocardial cells in mice with Coxsackievirus B3 (CVB3)-induced dilated cardiomyopathy (DCM) and its potential mechanism, so as to provide certain references for the clinical prevention and treatment of DCM. A total of 40 male C57 mice were randomly divided into Sham group (n=20) and DCM group (n=20) using a random number table. Another 20 mice with IL-6 gene knockout were enrolled into DCM+IL-6 KO group (n=20). The DCM model was established via CVB3 repeated incremental infection. After 9 months, the heart weight/body weight (HW/BW) ratio of mice in each group was detected. The ejection fraction [EF (%)] and fraction shortening [FS (%)] of mice in each group were detected via two-dimensional ultrasonography. The cross-sectional area and pathological changes in myocardial cells in the heart in each group were determined using hematoxylin-eosin (HE) staining. The collagen content in myocardial tissues in each group was detected via Masson staining and picrosirius red (PSR) staining, and the expressions of Collagen I and Collagen III in myocardial tissues in each group were detected via immunohistochemistry. In addition, the myocardial apoptosis in myocardial tissues in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Finally, the protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and total STAT3 (t-STAT3) were detected via Western blotting. The expression of IL-6 messenger ribonucleic acids (mRNAs) in myocardial tissues in DCM group was significantly increased compared with that in Sham group (p<0.05). After IL-6 knockout, the HW/BW ratio of DCM mice significantly declined (p<0.05), and the cross-sectional area of myocardial cells was significantly reduced (p<0.05). According to the results of echocardiography, the cardiac function of mice in DCM+IL-6 KO group was significantly superior to that in DCM group, manifested as the significant increase in FS (%) and EF (%) (p<0.05). The results of Masson staining, PSR staining, and immunohistochemical staining showed that IL-6 knockout could reduce the collagen content and Collagen I and Collagen III expressions in myocardial tissues of DCM mice (p<0.05). Furthermore, it was found via TUNEL staining that the number of apoptotic myocardial cells in DCM+IL-6 KO group was markedly smaller than that in DCM group (p<0.05). At the same time, the Bax/Bcl-2 ratio in myocardial tissues in DCM+IL-6 KO group was lower (p<0.05). Finally, the results of Western blotting revealed that DCM+IL-6 KO group had a lower phosphorylation level of STAT3 than DCM group (p<0.05). Inhibiting IL-6 gene may improve the DCM-induced myocardial remodeling through reducing myocardial apoptosis.

Metformin Enhances Autophagy and Provides Cardioprotection in δ-Sarcoglycan Deficiency-Induced Dilated Cardiomyopathy.

Metformin is a popular antidiabetic agent that is also used to treat heart failure patients with type 2 diabetes mellitus. Several reports suggest that metformin may also have cardioprotective effects in patients without diabetes mellitus. In the present study, we investigated the possible therapeutic effect of metformin in heart failure and its underlying molecular mechanisms using a δ-sarcoglycan-deficient mouse model of dilated cardiomyopathy. Thirty-two-week-old δ-sarcoglycan-deficient mice exhibiting established cardiomyopathy with extensive left ventricular dilatation and dysfunction were administered saline or metformin (200 mg/kg per day) for 4 weeks using osmotic mini-pumps. Metformin partially reversed the left ventricular dilatation (reverse remodeling) and significantly improved cardiac function. The hearts of metformin-treated mice showed less fibrosis, less cardiomyocyte hypertrophy, and fewer degenerative subcellular changes than saline-treated mice. These effects were accompanied by restored expression of the sarcomeric proteins myosin heavy chain and troponin I, and their transcription factor, GATA-4. Autophagy was enhanced in the hearts from metformin-treated mice, as indicated by increase of myocardial microtubule-associated protein-1 LC-3 (light chain 3)-II levels and LC3-II/-I ratios as well as levels of cathepsin D and ATP. In addition, increased numbers of autophagic vacuoles and lysosomes were accompanied increased AMP-activated protein kinase activity and suppression of mammalian target of rapamycin phosphorylation. Finally, autophagic flux assays using short-term chloroquine treatment revealed that autophagy was activated in δ-sarcoglycan-deficient hearts and was further augmented by metformin treatment. Metformin is a beneficial pharmacological tool that mitigates heart failure caused by δ-sarcoglycan deficiency in association with enhanced autophagy.

Metabolic regulation of heart failure caused by imbalanced mitochondrial dynamics

Dilated Cardiomyopathy (DCM) is characterized by an expansion of the left ventricle having as a consequence inefficient oxygenated blood pumping to the body. DCM can result in heart failure and is the most common cause of heart transplantation. The healthy adult heart relies mainly on free fatty acids as its main fuel source and rather than glucose but in the failing heart, a metabolic switch away from fatty acid oxidation and towards glucose uptake has been observed and has been proposed to represent an adaptive bioenergetic mechanism. We developed a mouse model of DCM caused my mitochondrial fragmentation in cardiomyocytes due to the deletion of the mitochondrial protease YME1L. This mouse model recapitulates all the clinical features of DCM as well as the metabolic features, such as cardiac glucose overload, progressing into chronic heart failure and middle-aged death. Using our DCM mouse model, we will test whether reducing cardiac glucose overload to the heart diminishes or exacerbates cardiac and metabolic dysfunction. To his end, we will ablate the primary glucose transporter GLUT 4 either partially or completely in DCM mice to diminish glucose uptake to the heart. We generated cardiac specific deletions of YME1L and GLUT4 by crossing the cardiomyocyte specific Cre-recombinase drive Myh6-Cre to conditional YME1LLoxp/LoxP and SLC2A4Loxp/LoxP mice. Cardiac function will be assessed using echocardiography and mitochondrial activity using high resolution oxygraphy (Oroboros) in isolated cardiac mitochondria. Our studies will allow us to determine the importance of glucose uptake and usage in the failing adult heart.