Rationale: The heart undergoes dramatic developmental changes during the prenatal to postnatal transition, including maturation of cardiac myocyte energy metabolic and contractile machinery. Delineation of the mechanisms involved in cardiac postnatal development could provide new insight into the fetal shifts that occur in the diseased heart and unveil strategies for driving maturation of stem cell–derived cardiac myocytes. Objective: To delineate transcriptional drivers of cardiac maturation. Methods and Results: We hypothesized that ERR (estrogen-related receptor) α and γ, known transcriptional regulators of postnatal mitochondrial biogenesis and function, serve a role in the broader cardiac maturation program. We devised a strategy to knockdown the expression of ERRα and γ in heart after birth (pn-csERRα/γ [postnatal cardiac-specific ERRα/γ]) in mice. With high levels of knockdown, pn-csERRα/γ knockdown mice exhibited cardiomyopathy with an arrest in mitochondrial maturation. RNA sequence analysis of pn-csERRα/γ knockdown hearts at 5 weeks of age combined with chromatin immunoprecipitation with deep sequencing and functional characterization conducted in human induced pluripotent stem cell–derived cardiac myocytes (hiPSC-CM) demonstrated that ERRγ activates transcription of genes involved in virtually all aspects of postnatal developmental maturation, including mitochondrial energy transduction, contractile function, and ion transport. In addition, ERRγ was found to suppress genes involved in fibroblast activation in hearts of pn-csERRα/γ knockdown mice. Disruption of Esrra and Esrrg in mice during fetal development resulted in perinatal lethality associated with structural and genomic evidence of an arrest in cardiac maturation, including persistent expression of early developmental and noncardiac lineage gene markers including cardiac fibroblast signatures. Lastly, targeted deletion of ESRRA and ESRRG in hiPSC-CM derepressed expression of early (transcription factor 21 or TCF21) and mature (periostin, collagen type III) fibroblast gene signatures. Conclusions: ERRα and γ are critical regulators of cardiac myocyte maturation, serving as transcriptional activators of adult cardiac metabolic and structural genes, an.d suppressors of noncardiac lineages including fibroblast determination.

Background Biased agonism of the angiotensin receptor (AT1R) is known to promote cardiac contractility. Our laboratory indicated that these effects may be due to changes at the level of the myofilaments. However, these signaling mechanisms remain unknown. As a common finding in dilated cardiomyopathy (DCM) is a reduction in the myofilament-Ca2+-response, we hypothesized that β-arrestin signaling would increase myofilament-Ca2+-responsiveness in a model of familial DCM and improve cardiac function and morphology. Methods We treated a DCM-linked mouse model expressing a mutant tropomyosin (Tm-E54K), for three months with either TRV120067, a β-arrestin 2 biased ligand of the AT1R, or losartan, an AT1R blocker. At the end of the treatment protocol, we assessed cardiac function using echocardiography, the myofilament-Ca2+-response of detergent-extracted fiber bundles, and used proteomic approaches to understand changes in post-translational modifications of proteins that may explain functional changes. We also assessed signaling pathways altered in vivo and using isolated myocytes. Results TRV120067- treated Tm-E54K mice showed improved cardiac structure and function, whereas losartan-treated mice had no improvement. Myofilaments of TRV120067-treated Tm-E54K mice had significantly improved myofilament-Ca2+-responsiveness, which was depressed in untreated Tm-E54K mice. We attributed these changes to increased MLC2v and MYPT1/2 phosphorylation seen only in TRV120067-treated mice. We found that the functional changes were due to an activation of ERK1/2-RSK3 signaling, mediated through β-arrestin, which may have a novel role in increasing MLC2v phosphorylation through a previously unrecognized interaction of β-arrestin localized to the sarcomere. Conclusions Long-term β-arrestin 2 biased agonism of the AT1R may be a viable approach to the treatment of DCM by not only preventing maladaptive signaling, but also improving cardiac function by altering the myofilament-Ca2+-response via β-arrestin signaling pathways.

S-glutathionylation of cardiac myosin-binding protein C (cMyBP-C) induces Ca(2+) sensitization and a slowing of cross-bridge kinetics as a result of increased oxidative signaling. Although there is evidence for a role of oxidative stress in disorders associated with hypertrophic cardiomyopathy (HCM), this mechanism is not well understood. We investigated whether oxidative myofilament modifications may be in part responsible for diastolic dysfunction in HCM. We administered N-acetylcysteine (NAC) for 30 days to 1-mo-old wild-type mice and to transgenic mice expressing a mutant tropomyosin (Tm-E180G) and nontransgenic littermates. Tm-E180G hearts demonstrate a phenotype similar to human HCM. After NAC administration, the morphology and diastolic function of Tm-E180G mice was not significantly different from controls, indicating that NAC had reversed baseline diastolic dysfunction and hypertrophy in our model. NAC administration also increased sarco(endo)plasmic reticulum Ca(2+) ATPase protein expression, reduced extracellular signal-related kinase 1/2 phosphorylation, and normalized phosphorylation of phospholamban, as assessed by Western blot. Detergent-extracted fiber bundles from NAC-administered Tm-E180G mice showed nearly nontransgenic (NTG) myofilament Ca(2+) sensitivity. Additionally, we found that NAC increased tension cost and rate of cross-bridge reattachment. Tm-E180G myofilaments were found to have a significant increase in S-glutathionylation of cMyBP-C, which was returned to NTG levels upon NAC administration. Taken together, our results indicate that oxidative myofilament modifications are an important mediator in diastolic function, and by relieving this modification we were able to reverse established diastolic dysfunction and hypertrophy in HCM.

Apart from transplant, there are no satisfactory therapies for the severe depression in contractility in familial dilated cardiomyopathy (DCM). Current heart failure treatments that act by increasing contractility involve signaling cascades that alter calcium homeostasis and induce arrhythmias. Omecamtiv mecarbil is a promising new inotropic agent developed for heart failure that may circumvent such limitations. Omecamtiv is a direct cardiac myosin activator that promotes and prolongs the strong myosin–actin binding conformation to increase the duration of systolic elastance. We tested the effect of omecamtiv on Ca2+ sensitivity of myofilaments of a DCM mouse model containing a tropomyosin E54K mutation. We compared tension and ATPase activity of detergent-extracted myofilaments with and without treatment with 316 nM omecamtiv at varying pCa values. When transgenic myofilaments were treated with omecamtiv, the pCa50 for activation of tension increased from 5.70 ± 0.02 to 5.82 ± 0.02 and ATPase activity increased from 5.73 ± 0.06 to 6.07 ± 0.04. This significant leftward shift restored Ca2+ sensitivity to levels no longer significantly different from controls. Proteomic studies lacked changes in sarcomeric protein phosphorylation. Our data demonstrate that omecamtiv can potentially augment cardiac contractility in DCM by increasing Ca2+ sensitivity. The use of direct myosin activators addresses functional defects without incurring the adverse side effects of Ca2+-dependent treatments.

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Treatment of mice with an AT1R biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R biased ligands may prove to be a novel inotropic approach in familial DCM.

Background: Hypertrophic cardiomyopathy (HCM) is a genetic cardiovascular disorder, primarily involving mutations in sarcomeric proteins. HCM patients present with hypertrophy, diastolic dysfunction, and fibrosis, but there is no specific treatment. The sphingosine-1-phosphate receptor modulator, FTY720/fingolimod, is approved for treatment of multiple sclerosis. We hypothesize that modulation of the sphingosine-1-phosphate receptor by FTY720 would be of therapeutic benefit in sarcomere-linked HCM. Methods: We treated mice with an HCM-linked mutation in tropomyosin (Tm-E180G) and nontransgenic littermates with FTY720 or vehicle for 6 weeks. Compared with vehicle-treated, FTY720-treated Tm-E180G mice had a significant reduction in left atrial size (1.99±0.19 [n=7] versus 2.70±0.44 [n=6] mm; P <0.001) and improvement in diastolic function (E/A ratio: 2.69±0.38 [n=7] versus 5.34±1.19 [n=6]; P =0.004) as assessed by echocardiography. Results: Pressure-volume relations revealed significant improvements in the end-diastolic pressure volume relationship, relaxation kinetics, preload recruitable stroke work, and ejection fraction. Detergent-extracted fiber bundles revealed a significant decrease in myofilament Ca 2+ -responsiveness (pCa 50 =6.15±0.11 [n=13] versus 6.24±0.06 [n=14]; P =0.041). We attributed these improvements to a downregulation of S-glutathionylation of cardiac myosin binding protein-C in FTY720-treated Tm-E180G mice and reduction in oxidative stress by downregulation of NADPH oxidases with no changes in fibrosis. Conclusions: This is the first demonstration that modulation of S1PR results in decreased myofilament-Ca 2+ -responsiveness and improved diastolic function in HCM. We associated these changes with decreased oxidative modification of myofilament proteins via downregulation of NOX2. Our data support the hypothesis that modification of sphingolipid signaling may be a novel therapeutic approach in HCM.

HCM phenotype was not associated with differences in myofilament Ca 2+ sensitivity between TG/PLN and TG/PLNKO mice. Moreover, compared to standard systolic echo parameters, such as ejection fraction (EF), speckle strain measurements provided a more sensitive approach to detect early systolic dysfunction in TG/PLN mice. In summary, our results indicate that targeting diastolic dysfunction through altering Ca 2+ fluxes with no change in myofilament response to Ca 2+ was able to prevent the development of the HCM phenotype and should be considered as a potential additional treatment for HCM patients.