Interactions between the Regulatory Subunit of Type I Protein Kinase A and p90 Ribosomal S6 Kinase1 Regulate Cardiomyocyte Apoptosis s

ABSTRACT Cardiomyocyte apoptosis contributes toward the loss of muscle mass in myocardial pathologies. Previous reports have implicated type I cAMP-dependent protein kinase (PKA) and p90 ribosomal S6 kinase (RSK) in cardiomyocyte apoptosis. However, the precise mechanisms and the isoform of RSK involved in this process remain undefined. Using adult rat ventricular myocytes and mouse-derived cardiac HL-1 cardiomyocytes, we demonstrate that hypoxia/reoxygenation (H/R)-induced apoptosis is accompanied by a decrease in the type I PKA regulatory subunit (PKARIa) and activation of RSK1. As previously described by us for other cell types, in cardiomyocytes, inactive RSK1 also interacts with PKARIa, whereas the active RSK1 interacts with the catalytic subunit of PKA. Additionally, small interfering (siRNA)-mediated silencing of PKARIa or disrupting the RSK1/PKARIa interactions with a small, cell-permeable peptide activates RSK1 and recapitulates the H/R-induced apoptosis. Inhibition of RSK1 or siRNA-mediated silencing of RSK1 attenuates H/R-induced apoptosis, demonstrating the role of RSK1 in cardiomyocyte apoptosis. Furthermore, silencing of RSK1 decreases the H/R-induced phosphorylation of sodium-hydrogen exchanger 1 (NHE1), and inhibition of NHE1 with 59-N-ethyl-N-isopropyl-amiloride blocks H/R induced apoptosis, indicating the involvement of NHE1 in apoptosis. Overall, our findings demonstrate that H/R-mediated decrease in PKARIa protein levels leads to activation of RSK1, which via phosphorylation of NHE1 induces cardiomyocyte apoptosis

MicroRNA-210-mediated proliferation, survival, and angiogenesis promote cardiac repair post myocardial infarction in rodents

The final publication is available at Springer via http://dx.doi.org/10.1007/s00109-017-1591-8.An innovative approach for cardiac regeneration following injury is to induce endogenous cardiomyocyte (CM) cell cycle re-entry. In the present study, CMs from adult rat hearts were isolated and transfected with cel-miR-67 (control) and rno-miR-210. A significant increase in CM proliferation and mono-nucleation were observed in miR-210 group, in addition to a reduction in CM size, multi-nucleation, and cell death. When compared to control, β-catenin and Bcl-2 were upregulated while APC (adenomatous polyposis coli), p16, and caspase-3 were downregulated in miR-210 group. In silico analysis predicted cell cycle inhibitor, APC, as a direct target of miR-210 in rodents. Moreover, compared to control, a significant increase in CM survival and proliferation were observed with siRNA-mediated inhibition of APC. Furthermore, miR-210 overexpressing C57BL/6 mice (210-TG) were used for short-term ischemia/reperfusion study, revealing smaller cell size, increased mono-nucleation, decreased multi-nucleation, and increased CM proliferation in 210-TG hearts in contrast to wild-type (NTG). Likewise, myocardial infarction (MI) was created in adult mice, echocardiography was performed, and the hearts were harvested for immunohistochemistry and molecular studies. Compared to NTG, 210-TG hearts showed a significant increase in CM proliferation, reduced apoptosis, upregulated angiogenesis, reduced infarct size, and overall improvement in cardiac function following MI. β-catenin, Bcl-2, and VEGF (vascular endothelial growth factor) were upregulated while APC, p16, and caspase-3 were downregulated in 210-TG hearts. Overall, constitutive overexpression of miR-210 rescues heart function following cardiac injury in adult mice via promoting CM proliferation, cell survival, and angiogenesis

Myonuclear accretion is a determinant of exercise-induced remodeling in skeletal muscle.

Skeletal muscle adapts to external stimuli such as increased work. Muscle progenitors (MPs) control muscle repair due to severe damage, but the role of MP fusion and associated myonuclear accretion during exercise are unclear. While we previously demonstrated that MP fusion is required for growth using a supra-physiological model (Goh and Millay, 2017), questions remained about the need for myonuclear accrual during muscle adaptation in a physiological setting. Here, we developed an 8 week high-intensity interval training (HIIT) protocol and assessed the importance of MP fusion. In 8 month-old mice, HIIT led to progressive myonuclear accretion throughout the protocol, and functional muscle hypertrophy. Abrogation of MP fusion at the onset of HIIT resulted in exercise intolerance and fibrosis. In contrast, ablation of MP fusion 4 weeks into HIIT, preserved exercise tolerance but attenuated hypertrophy. We conclude that myonuclear accretion is required for different facets of exercise-induced adaptive responses, impacting both muscle repair and hypertrophic growth

Cardiac myosin binding protein-C plays no regulatory role in skeletal muscle structure and function

Myosin binding protein-C (MyBP-C) exists in three major isoforms: slow skeletal, fast skeletal, and cardiac. While cardiac MyBP-C (cMyBP-C) expression is restricted to the heart in the adult, it is transiently expressed in neonatal stages of some skeletal muscles. However, it is unclear whether this expression is necessary for the proper development and function of skeletal muscle. Our aim was to determine whether the absence of cMyBP-C alters the structure, function, or MyBP-C isoform expression in adult skeletal muscle using a cMyBP-C null mouse model (cMyBP-C((t/t))). Slow MyBP-C was expressed in both slow and fast skeletal muscles, whereas fast MyBP-C was mostly restricted to fast skeletal muscles. Expression of these isoforms was unaffected in skeletal muscle from cMyBP-C((t/t)) mice. Slow and fast skeletal muscles in cMyBP-C((t/t)) mice showed no histological or ultrastructural changes in comparison to the wild-type control. In addition, slow muscle twitch, tetanus tension, and susceptibility to injury were all similar to the wild-type controls. Interestingly, fMyBP-C expression was significantly increased in the cMyBP-C((t/t)) hearts undergoing severe dilated cardiomyopathy, though this does not seem to prevent dysfunction. Additionally, expression of both slow and fast isoforms was increased in myopathic skeletal muscles. Our data demonstrate that i) MyBP-C isoforms are differentially regulated in both cardiac and skeletal muscles, ii) cMyBP-C is dispensable for the development of skeletal muscle with no functional or structural consequences in the adult myocyte, and iii) skeletal isoforms can transcomplement in the heart in the absence of cMyBP-C

ERK1/2 signaling induces skeletal muscle slow fiber-type switching and reduces muscular dystrophy disease severity

© 2019 American Society for Clinical Investigation. MAPK signaling consists of an array of successively acting kinases. ERK1 and -2 (ERK1/2) are major components of the greater MAPK cascade that transduce growth factor signaling at the cell membrane. Here, we investigated ERK1/2 signaling in skeletal muscle homeostasis and disease. Using mouse genetics, we observed that the muscle-specifc expression of a constitutively active MEK1 mutant promotes greater ERK1/2 signaling that mediates fber-type switching to a slow, oxidative phenotype with type I myosin heavy chain expression. Using a conditional and temporally regulated Cre strategy, as well as Mapk1 (ERK2) and Mapk3 (ERK1) genetically targeted mice, MEK1-ERK2 signaling was shown to underlie this fast-to-slow fber-type switching in adult skeletal muscle as well as during development. Physiologic assessment of these activated MEK1-ERK1/2 mice showed enhanced metabolic activity and oxygen consumption with greater muscle fatigue resistance. In addition, induction of MEK1-ERK1/2 signaling increased dystrophin and utrophin protein expression in a mouse model of limb-girdle muscle dystrophy and protected myofbers from damage. In summary, sustained MEK1-ERK1/2 activity in skeletal muscle produces a fast-to-slow fber-type switch that protects from muscular dystrophy, suggesting a therapeutic approach to enhance the metabolic effectiveness of muscle and protect from dystrophic disease

The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle

Myosin and actin filaments are highly organized within muscle sarcomeres. Myosin-binding protein C (MyBP-C) is a flexible, rod-like protein located within the C-zone of the sarcomere. The C-terminal domain of MyBP-C is tethered to the myosin filament backbone, and the N-terminal domains are postulated to interact with actin and/or the myosin head to modulate filament sliding. To define where the N-terminal domains of MyBP-C are localized in the sarcomere of active and relaxed mouse myocardium, the relative positions of the N terminus of MyBP-C and actin were imaged in fixed muscle samples using super-resolution fluorescence microscopy. The resolution of the imaging was enhanced by particle averaging. The images demonstrate that the position of the N terminus of MyBP-C is biased toward the actin filaments in both active and relaxed muscle preparations. Comparison of the experimental images with images generated in silico, accounting for known binding partner interactions, suggests that the N-terminal domains of MyBP-C may bind to actin and possibly the myosin head but only when the myosin head is in the proximity of an actin filament. These physiologically relevant images help define the molecular mechanism by which the N-terminal domains of MyBP-C may search for, and capture, molecular binding partners to tune cardiac contractility

Myofilament Ca2+ desensitization mediates positive lusitropic effect of neuronal nitric oxide synthase in left ventricular myocytes from murine hypertensive heart

AbstractNeuronal nitric oxide synthase (NOS1 or nNOS) exerts negative inotropic and positive lusitropic effects through Ca2+ handling processes in cardiac myocytes from healthy hearts. However, underlying mechanisms of NOS1 in diseased hearts remain unclear. The present study aims to investigate this question in angiotensin II (Ang II)-induced hypertensive rat hearts (HP). Our results showed that the systolic function of left ventricle (LV) was reduced and diastolic function was unaltered (echocardiographic assessment) in HP compared to those in shams. In isolated LV myocytes, contraction was unchanged but peak [Ca2+]i transient was increased in HP. Concomitantly, relaxation and time constant of [Ca2+]i decay (tau) were faster and the phosphorylated fraction of phospholamban (PLN-Ser16/PLN) was greater. NOS1 protein expression and activity were increased in LV myocyte homogenates from HP. Surprisingly, inhibition of NOS1 did not affect contraction but reduced peak [Ca2+]i transient; prevented faster relaxation without affecting the tau of [Ca2+]i transient or PLN-Ser16/PLN in HP, suggesting myofilament Ca2+ desensitization by NOS1. Indeed, relaxation phase of the sarcomere length–[Ca2+]i relationship of LV myocytes shifted to the right and increased [Ca2+]i for 50% of sarcomere shortening (EC50) in HP. Phosphorylations of cardiac myosin binding protein-C (cMyBP-C282 and cMyBP-C273) were increased and cardiac troponin I (cTnI23/24) was reduced in HP. Importantly, NOS1 or PKG inhibition reduced cMyBP-C273 and cTnI23/24 and reversed myofilament Ca2+ sensitivity. These results reveal that NOS1 is up-regulated in LV myocytes from HP and exerts positive lusitropic effect by modulating myofilament Ca2+ sensitivity through phosphorylation of key regulators in sarcomere

Contractile Dysfunction Irrespective of the Mutant Protein in Human Hypertrophic Cardiomyopathy With Normal Systolic Function

Background-Hypertrophic cardiomyopathy (HCM), typically characterized by asymmetrical left ventricular hypertrophy, frequently is caused by mutations in sarcomeric proteins. We studied if changes in sarcomeric properties in HCM depend on the underlying protein mutation. Methods and Results-Comparisons were made between cardiac samples from patients carrying a MYBPC3 mutation (MYBPC3(mut); n = 17), mutation negative HCM patients without an identified sarcomere mutation (HCM(mn); n = 11), and nonfailing donors (n = 12). All patients had normal systolic function, but impaired diastolic function. Protein expression of myosin binding protein C (cMyBP-C) was significantly lower in MYBPC3(mut) by 33 +/- 5%, and similar in HCM(mn) compared with donor. cMyBP-C phosphory Conclusions-Changes in sarcomere function reflect the clinical HCM phenotype rather than the specific MYBPC3 mutation. Hypocontractile sarcomeres are a common deficit in human HCM with normal systolic left ventricular function and may contribute to HCM disease progression. (Circ Heart Fail. 2012; 5: 36-46.

Inhibiting cardiac myeloperoxidase alleviates the relaxation defect in hypertrophic cardiomyocytes.

AIMS: Hypertrophic cardiomyopathy (HCM) is characterised by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM. METHODS AND RESULTS: Human cardiomyocytes derived from control induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy. CONCLUSION: This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing. TRANSLATIONAL PERSPECTIVE: There are currently no specific therapies for improving diastolic function in patients with HCM. We show for the first time that myeloperoxidase (MPO) is present in and is up-regulated in cardiomyocytes derived from human iPSCs obtained from HCM patients, where it impairs cardiomyocyte relaxation by reducing phosphorylation of cardiac MYBPC3. Treatment with the MPO inhibitor, AZD5904, restored MYBPC3 phosphorylation and alleviated the relaxation defect, demonstrating cardiomyocyte MPO to be a novel therapeutic target for improving diastolic function in HCM, a treatment strategy which can be evaluated in HCM patients given that MPO inhibitors are already available for clinical testing