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Biological sex is an important modifier of physiology and influences pathobiology in many diseases. While heart disease is the number one cause of death worldwide in both men and women, sex differences exist at the organ and cellular scales, affecting clinical presentation, diagnosis, and treatment. In this Review, we highlight baseline sex differences in cardiac structure, function, and cellular signaling and discuss the contribution of sex hormones and chromosomes to these characteristics. The heart is a remarkably plastic organ and rapidly responds to physiological and pathological cues by modifying form and function. The nature and extent of cardiac remodeling in response to these stimuli are often dependent on biological sex. We discuss organ- and molecular-level sex differences in adaptive physiological remodeling and pathological cardiac remodeling from pressure and volume overload, ischemia, and genetic heart disease. Finally, we offer a perspective on key future directions for research into cardiac sex differences.

Proline substitutions within the coiled-coil rod region of the β-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in β-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human β, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in β myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in β myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not β, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin's highly allosteric nature., Competing Interests: Competing interests statement:J.A.S. is cofounder and on the Scientific Advisory Board of Cytokinetics, Inc., a company developing small molecule therapeutics for treatment of HCM. J.A.S. is cofounder and CEO, and K.M.R. is cofounder and Research and Clinical Advisor, of Kainomyx, Inc., a company developing small molecule therapeutics targeting myosins in parasites. G.R.B. is cofounder and equity holder in Decrypt Biomedicine.

Left ventricular reverse remodeling in heart failure is associated with improved clinical outcomes. However, the molecular features that drive this process are poorly defined. Left ventricular assist devices (LVADs) are the therapy associated with the greatest reverse remodeling and lead to partial myocardial recovery in most patients. In this study, we examined whether autophagy may be implicated in post-LVAD reverse remodeling. We found expression of key autophagy factors increased post-LVAD, while autophagic substrates decreased. Autolysosome numbers increased post-LVAD, further indicating increased autophagy. These findings support the conclusion that mechanical unloading activates autophagy, which may underly the reverse remodeling observed., Competing Interests: The authors have received support from the National Institute of Health (R01HL117138 and R01GM029090 to Dr Leinwand, T32 HL007822 to Dr Martin, and T32 GM142607 to Dr Juarros). REDCap was provided by National Institutes of Health/NCATS Colorado CTSA Grant Number UL1 TR002535. The contents are the authors’ sole responsibility and do not necessarily represent the official views of the National Institutes of Health. Dr Leinwand is a cofounder of MyoKardia, acquired by Bristol Myers Squibb; and is a paid member of their Scientific Advisory Board. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes in response to mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a potentially novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.

For decades, sarcomeric myosin heavy chain proteins were assumed to be restricted to striated muscle where they function as molecular motors that contract muscle. However, MYH7b, an evolutionarily ancient member of this myosin family, has been detected in mammalian nonmuscle tissues, and mutations in MYH7b are linked to hereditary hearing loss in compound heterozygous patients. These mutations are the first associated with hearing loss rather than a muscle pathology, and because there are no homologous mutations in other myosin isoforms, their functional effects were unknown. We generated recombinant human MYH7b harboring the D515N or R1651Q hearing loss-associated mutation and studied their effects on motor activity and structural and assembly properties, respectively. The D515N mutation had no effect on steady-state actin-activated ATPase rate or load-dependent detachment kinetics but increased actin sliding velocity because of an increased displacement during the myosin working stroke. Furthermore, we found that the D515N mutation caused an increase in the proportion of myosin heads that occupy the disordered-relaxed state, meaning more myosin heads are available to interact with actin. Although we found no impact of the R1651Q mutation on myosin rod secondary structure or solubility, we observed a striking aggregation phenotype when this mutation was introduced into nonmuscle cells. Our results suggest that each mutation independently affects MYH7b function and structure. Together, these results provide the foundation for further study of a role for MYH7b outside the sarcomere., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least six of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 ms of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatin's binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R 2 =0.82). In a blind prediction for an isoform (Myh7b) whose blebbistatin sensitivity was unknown, we find good agreement between predicted and measured IC50s (0.67 μM vs. 0.36 μM). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets., Competing Interests: AM, JL, LS, BN, LL, CK, LG, LL, MG, GB No competing interests declared, (© 2023, Meller et al.)

Myosin heavy chain 7b (MYH7b) is an evolutionarily ancient member of the sarcomeric myosin family, which typically supports striated muscle function. However, in mammals, alternative splicing prevents MYH7b protein production in cardiac and most skeletal muscles and limits expression to a subset of specialized muscles and certain nonmuscle environments. In contrast, MYH7b protein is abundant in python cardiac and skeletal muscles. Although the MYH7b expression pattern diverges in mammals versus reptiles, MYH7b shares high sequence identity across species. So, it remains unclear how mammalian MYH7b function may differ from that of other sarcomeric myosins and whether human and python MYH7b motor functions diverge as their expression patterns suggest. Thus, we generated recombinant human and python MYH7b protein and measured their motor properties to investigate any species-specific differences in activity. Our results reveal that despite having similar working strokes, the MYH7b isoforms have slower actin-activated ATPase cycles and actin sliding velocities than human cardiac β-MyHC. Furthermore, python MYH7b is tuned to have slower motor activity than human MYH7b because of slower kinetics of the chemomechanical cycle. We found that the MYH7b isoforms adopt a higher proportion of myosin heads in the ultraslow, super-relaxed state compared with human cardiac β-MyHC. These findings are supported by molecular dynamics simulations that predict MYH7b preferentially occupies myosin active site conformations similar to those observed in the structurally inactive state. Together, these results suggest that MYH7b is specialized for slow and energy-conserving motor activity and that differential tuning of MYH7b orthologs contributes to species-specific biological roles., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Abnormalities in Ca 2+ homeostasis are associated with cardiac arrhythmias and heart failure. Triadin plays an important role in Ca 2+ homeostasis in cardiomyocytes. Alternative splicing of a single triadin gene produces multiple triadin isoforms. The cardiac-predominant isoform, mouse MT-1 or human Trisk32, is encoded by triadin exons 1 to 8. In humans, mutations in the triadin gene that lead to a reduction in Trisk32 levels in the heart can cause cardiac dysfunction and arrhythmias. Decreased levels of Trisk32 in the heart are also common in patients with heart failure. However, mechanisms that maintain triadin isoform composition in the heart remain elusive., Methods: We analyzed triadin expression in heart explants from patients with heart failure and cardiac arrhythmias and in hearts from mice carrying a knockout allele for Trdn-as , a cardiomyocyte-specific long noncoding RNA encoded by the antisense strand of the triadin gene, between exons 9 and 11. Catecholamine challenge with isoproterenol was performed on Trdn-as knockout mice to assess the role of Trdn-as in cardiac arrhythmogenesis, as assessed by ECG. Ca 2+ transients in adult mouse cardiomyocytes were measured with the IonOptix platform or the GCaMP system. Biochemistry assays, single-molecule fluorescence in situ hybridization, subcellular localization imaging, RNA sequencing, and molecular rescue assays were used to investigate the mechanisms by which Trdn-as regulates cardiac function and triadin levels in the heart., Results: We report that Trdn-as maintains cardiac function, at least in part, by regulating alternative splicing of the triadin gene. Knockout of Trdn-as in mice downregulates cardiac triadin, impairs Ca 2+ handling, and causes premature death. Trdn-as knockout mice are susceptible to cardiac arrhythmias in response to catecholamine challenge. Normalization of cardiac triadin levels in Trdn-as knockout cardiomyocytes is sufficient to restore Ca 2+ handling. Last, Trdn-as colocalizes and interacts with serine/arginine splicing factors in cardiomyocyte nuclei and is essential for efficient recruitment of splicing factors to triadin precursor mRNA., Conclusions: These findings reveal regulation of alternative splicing as a novel mechanism by which a long noncoding RNA controls cardiac function. This study indicates potential therapeutics for heart disease by targeting the long noncoding RNA or pathways regulating alternative splicing.

Aortic valve stenosis (AVS) is a progressive fibrotic disease that is caused by thickening and stiffening of valve leaflets. At the cellular level, quiescent valve interstitial cells (qVICs) activate to myofibroblasts (aVICs) that persist within the valve tissue. Given the persistence of myofibroblasts in AVS, epigenetic mechanisms have been implicated. Here, we studied changes that occur in VICs during myofibroblast activation by using a hydrogel matrix to recapitulate different stiffnesses in the valve leaflet during fibrosis. We first compared the chromatin landscape of qVICs cultured on soft hydrogels and aVICs cultured on stiff hydrogels, representing the native and diseased phenotypes respectively. Using assay for transposase-accessible chromatin sequencing (ATAC-Seq), we found that open chromatin regions in aVICs were enriched for transcription factor binding motifs associated with mechanosensing pathways compared to qVICs. Next, we used RNA-Seq to show that the open chromatin regions in aVICs correlated with pro-fibrotic gene expression, as aVICs expressed higher levels of contractile fiber genes, including myofibroblast markers such as alpha smooth muscle actin (αSMA), compared to qVICs. In contrast, chromatin remodeling genes were downregulated in aVICs compared to qVICs, indicating qVICs may be protected from myofibroblast activation through epigenetic mechanisms. Small molecule inhibition of one of these remodelers, CREB Binding Protein (CREBBP), prevented qVICs from activating to aVICs. Notably, CREBBP is more abundant in valves from healthy patients compared to fibrotic valves. Our findings reveal the role of mechanical regulation in chromatin remodeling during VIC activation and quiescence and highlight one potential therapeutic target for treating AVS., Competing Interests: The authors declare no conflicts of interests., (© 2022 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403 +/- ) with Lpar1 -ablated mice to create mice carrying both genetic changes (403 +/- LPAR1 -/- ) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403 +/- LPAR1 WT , 403 +/- LPAR1 -/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a . Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.

Pathological cardiac hypertrophy is associated with increased morbidity and mortality. Understanding the mechanisms whereby pathological cardiac growth can be reversed could be of therapeutic value. Here, we show that pathways leading to regression of pathological cardiac hypertrophy are strongly dependent on the hypertrophic trigger and are significantly modified by sex. Two pathological stimuli causing hypertrophy via distinct pathways were administered to male and female mice: angiotensin II (ANG II) or isoproterenol (Iso). Stimuli were removed after 7 days of treatment, and left ventricles (LVs) were studied at 1, 4, and 7 days. ANG II-treated females did not show regression after stimulus removal. Iso-treated males showed rapid LV hypertrophy regression. Somewhat surprisingly, RNAseq analysis at day 1 after removal of triggers revealed only 45 differentially regulated genes in common among all the groups, demonstrating distinct responses. Ingenuity pathway analysis predicted strong downregulation of the TGFβ1 pathway in all groups except for ANG II-treated females. Consistently, we found significant downregulation of Smad signaling after stimulus removal including in ANG II-treated females. In addition, the ERK1/2 pathway was significantly reduced in the groups showing regression. Finally, protein degradation pathways were significantly activated only in Iso-treated males 1 day after stimulus removal. Our data indicate that TGFβ1 downregulation may play a role in the regression of pathological cardiac hypertrophy via downregulation of the ERK1/2 pathway and activation of autophagy and proteasome activity in Iso-treated males. This work highlights that the reversal of pathological hypertrophy does not use universal signaling pathways and that sex potently modifies this process. NEW & NOTEWORTHY Pathological cardiac hypertrophy is a major risk factor for mortality and is thought to be largely irreversible in many individuals. Although cardiac hypertrophy itself has been studied extensively, very little is understood about its regression. It is important that we have a better understanding of mechanisms leading to regression, why this process is not reversible in some individuals and that sex differences need to be considered when contemplating therapies.

As an opportunistic predator, the Burmese python (Python molurus bivittatus) consumes large and infrequent meals, fasting for up to a year. Upon consuming a large meal, the Burmese python exhibits extreme metabolic responses. To define the pathways that regulate these postprandial metabolic responses, we performed a comprehensive profile of plasma metabolites throughout the digestive process. Following ingestion of a meal equivalent to 25% of its body mass, plasma lipoproteins and metabolites, such as chylomicra and bile acids, reach levels observed only in mammalian models of extreme dyslipidemia. Here, we provide evidence for an adaptive response to postprandial nutrient overload by the python liver, a critical site of metabolic homeostasis. The python liver undergoes a substantial increase in mass through proliferative processes, exhibits hepatic steatosis, hyperlipidemia-induced insulin resistance indicated by PEPCK activation and pAKT deactivation, and de novo fatty acid synthesis via FASN activation. This postprandial state is completely reversible. We posit that Burmese pythons evade the permanent hepatic damage associated with these metabolic states in mammals using evolved protective measures to inactivate these pathways. These include a transient activation of hepatic nuclear receptors induced by fatty acids and bile acids, including PPAR and FXR, respectively. The stress-induced p38 MAPK pathway is also transiently activated during the early stages of digestion. Taken together, these data identify a reversible metabolic response to hyperlipidemia by the python liver, only achieved in mammals by pharmacologic intervention. The factors involved in these processes may be relevant to or leveraged for remediating human hepatic pathology., (© 2022 Magida et al.)

Background: Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored., Methods: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors., Results: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling., Conclusions: Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.

Background Although the roles of alpha-myosin heavy chain (α-MyHC) and beta-myosin heavy chain (β-MyHC) proteins in cardiac contractility have long been appreciated, the biological contribution of another closely related sarcomeric myosin family member, MYH7b (myosin heavy chain 7b), has become a matter of debate. In mammals, MYH7b mRNA is transcribed but undergoes non-productive alternative splicing that prevents protein expression in a tissue-specific manner, including in the heart. However, several studies have recently linked MYH7b variants to different cardiomyopathies or have reported MYH7b protein expression in mammalian hearts. Methods and Results By analyzing mammalian cardiac transcriptome and proteome data, we show that the vast majority of MYH7b RNA is subject to exon skipping and cannot be translated into a functional myosin molecule. Notably, we discovered a lag in the removal of introns flanking the alternatively spliced exon, which could retain the non-coding RNA in the nucleus. This process could play a significant role in controlling MYH7b expression as well as the activity of other cardiac genes. Consistent with the negligible level of full-length protein coding mRNA, no MYH7b protein expression was detected in adult mouse, rat, and human hearts by Western blot analysis. Furthermore, proteome surveys including quantitative mass spectrometry analyses revealed only traces of cardiac MYH7b protein and even then, only in a subset of individual samples. Conclusions The comprehensive analysis presented here suggests that previous studies showing cardiac MYH7b protein expression were likely attributable to antibody cross-reactivity. More importantly, our data predict that the MYH7b disease-associated variants may operate through the alternately spliced RNA itself.

The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (β/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of β-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj < 0.05) but not with the clade (padj > 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human β-myosin to match the rat sequence. Biochemical analysis revealed that the rat-human β-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin-myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of β-myosin as species mass increased to enable a reduced contraction velocity and heart rate., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: LAL owns stock in MyoKardia, Inc. and has a Sponsored Research Agreement with MyoKardia, Inc.

Background Biological sex is an important modifier of cardiovascular disease and women generally have better outcomes compared with men. However, the contribution of cardiac fibroblasts (CFs) to this sexual dimorphism is relatively unexplored. Methods and Results Isoproterenol (ISO) was administered to rats as a model for chronic β-adrenergic receptor (β-AR)-mediated cardiovascular disease. ISO-treated males had higher mortality than females and also developed fibrosis whereas females did not. Gonadectomy did not abrogate this sex difference. To determine the cellular contribution to this phenotype, CFs were studied. CFs from both sexes had increased proliferation in vivo in response to ISO, but CFs from female hearts proliferated more than male cells. In addition, male CFs were significantly more activated to myofibroblasts by ISO. To investigate potential regulatory mechanisms for the sexually dimorphic fibrotic response, β-AR mRNA and PKA (protein kinase A) activity were measured. In response to ISO treatment, male CFs increased expression of β1- and β2-ARs, whereas expression of both receptors decreased in female CFs. Moreover, ISO-treated male CFs had higher PKA activity relative to vehicle controls, whereas ISO did not activate PKA in female CFs. Conclusions Chronic in vivo β-AR stimulation causes fibrosis in male but not female rat hearts. Male CFs are more activated than female CFs, consistent with elevated fibrosis in male rat hearts and may be caused by higher β-AR expression and PKA activation in male CFs. Taken together, our data suggest that CFs play a substantial role in mediating sex differences observed after cardiac injury.

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification. The test has two steps: (1) heat saliva with a stabilization solution and (2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections., Competing Interests: QY, NM, CP Some of the authors of this study (NRM, QY, CLP, SLS) are founders of Darwin Biosciences, who licenses the Saliva TwoStep assay described herein. SC is an employee of Darwin Biosciences. WF, AG, AB, BH, EW, SW, DM, CD, TS, EL, PG, MF, KT, CH, JD, CO, GB, MM, LL, RP No competing interests declared, SS Senior editor, eLife, (© 2021, Yang et al.)

Background: The impact of sex on phenotypic expression in hypertrophic cardiomyopathy (HCM) has not been well characterized in genotyped cohorts., Methods: Retrospective cohort study from an international registry of patients receiving care at experienced HCM centers. Sex-based differences in baseline characteristics and clinical outcomes were assessed., Results: Of 5873 patients (3788 genotyped), 2226 (37.9%) were women. At baseline, women were older (49.0±19.9 versus 42.9±18.4 years, P <0.001) and more likely to have pathogenic/likely pathogenic sarcomeric variants (HCM patients with a sarcomere mutation; 51% versus 43%, P <0.001) despite equivalent utilization of genetic testing. Age at diagnosis varied by sex and genotype despite similar distribution of causal genes. Women were 3.6 to 7.1 years older at diagnosis ( P <0.02) except for patients with MYH7 variants where age at diagnosis was comparable for women and men (n=492; 34.8±19.2 versus 33.3±16.8 years, P =0.39). Over 7.7 median years of follow-up, New York Heart Association III-IV heart failure was more common in women (hazard ratio, 1.87 [CI, 1.48-2.36], P <0.001), after controlling for their higher burden of symptoms and outflow tract obstruction at baseline, reduced ejection fraction, HCM patients with a sarcomere mutation, age, and hypertension. All-cause mortality was increased in women (hazard ratio, 1.50 [CI, 1.13-1.99], P <0.01) but neither implantable cardioverter-defibrillator utilization nor ventricular arrhythmia varied by sex., Conclusions: In HCM, women are older at diagnosis, partly modified by genetic substrate. Regardless of genotype, women were at higher risk of mortality and developing severe heart failure symptoms. This points to a sex-effect on long-term myocardial performance in HCM, which should be investigated further.

Myosin heavy chain 7b (MYH7b) is an ancient member of the myosin heavy chain motor protein family that is expressed in striated muscles. In mammalian cardiac muscle, MYH7b RNA is expressed along with two other myosin heavy chains, β-myosin heavy chain (β-MyHC) and α-myosin heavy chain (α-MyHC). However, unlike β-MyHC and α-MyHC, which are maintained in a careful balance at the protein level, the MYH7b locus does not produce a full-length protein in the heart due to a posttranscriptional exon-skipping mechanism that occurs in a tissue-specific manner. Whether this locus has a role in the heart beyond producing its intronic microRNA, miR-499, was unclear. Using cardiomyocytes derived from human induced pluripotent stem cells as a model system, we found that the noncoding exon-skipped RNA (lncMYH7b) affects the transcriptional landscape of human cardiomyocytes, independent of miR-499. Specifically, lncMYH7b regulates the ratio of β-MyHC to α-MyHC, which is crucial for cardiac contractility. We also found that lncMYH7b regulates beat rate and sarcomere formation in cardiomyocytes. This regulation is likely achieved through control of a member of the TEA domain transcription factor family (TEAD3, which is known to regulate β-MyHC). Therefore, we conclude that this ancient gene has been repurposed by alternative splicing to produce a regulatory long-noncoding RNA in the human heart that affects cardiac myosin composition., Competing Interests: Conflict of interest No conflicts of interest were reported., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background Cardiac fibroblasts (CFs) have the ability to sense stiffness changes and respond to biochemical cues to modulate their states as either quiescent or activated myofibroblasts. Given the potential for secretion of bioactive molecules to modulate the cardiac microenvironment, we sought to determine how the CF secretome changes with matrix stiffness and biochemical cues and how this affects cardiac myocytes via paracrine signaling. Methods and Results Myofibroblast activation was modulated in vitro by combining stiffness cues with TGFβ1 (transforming growth factor β 1) treatment using engineered poly (ethylene glycol) hydrogels, and in vivo with isoproterenol treatment. Stiffness, TGFβ1, and isoproterenol treatment increased AKT (protein kinase B) phosphorylation, indicating that this pathway may be central to myofibroblast activation regardless of the treatment. Although activation of AKT was shared, different activating cues had distinct effects on downstream cytokine secretion, indicating that not all activated myofibroblasts share the same secretome. To test the effect of cytokines present in the CF secretome on paracrine signaling, neonatal rat ventricular cardiomyocytes were treated with CF conditioned media. Conditioned media from myofibroblasts cultured on stiff substrates and activated by TGFβ1 caused hypertrophy, and one of the cytokines in that media was insulin growth factor 1, which is a known mediator of cardiac myocyte hypertrophy. Conclusions Culturing CFs on stiff substrates, treating with TGFβ1, and in vivo treatment with isoproterenol all caused myofibroblast activation. Each cue had distinct effects on the secretome or genes encoding the secretome, but only the secretome of activated myofibroblasts on stiff substrates treated with TGFβ1 caused myocyte hypertrophy, most likely through insulin growth factor 1.

Striated muscle is a highly specialized collection of tissues with contractile properties that vary according to functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-dependent adaptation. Functional adaptation requires molecular adaptation, which is partially provided by miRNA-mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-206 expression increases in both physiological (including female sex and endurance exercise) and pathological conditions (muscular dystrophy and adrenergic agonism) that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-206-knockout mice displays a faster phenotype than wild-type mice. Moreover, left ventricles of male miR-206 knockout mice have a faster myosin profile, accompanied by dilation and systolic dysfunction. Thus, miR-206 appears to be necessary to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Objective: Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ERα signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of this study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development., Methods: A novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKO ism ). ERαshRNA was also used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women., Results: Twelve weeks of HFD exposure had no differential effects on body composition, VO 2 , VCO 2 , RER, energy expenditure, and activity counts across genotypes. Although ERαKO ism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKO ism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKO ism , but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H 2 O 2 emission potential was not affected in the ERαKO ism mice. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity., Conclusions: Collectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete., (Copyright © 2019 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Background: Hypertrophic cardiomyopathy (HCM) is characterized by myocyte hypertrophy and fibrosis. Studies in two mouse models (R92W-TnT/R403Q-MyHC) at early HCM stage revealed upregulation of endothelin (ET1) signaling in both mutants, but TGFβ signaling only in TnT mutants. Dysregulation of miR-29 expression has been implicated in cardiac fibrosis. But it is unknown whether expression of miR-29a/b/c and profibrotic genes is commonly regulated in mouse and human HCM. Methods: In order to understand mechanisms underlying fibrosis in HCM, and examine similarities/differences in expression of miR-29a/b/c and several profibrotic genes in mouse and human HCM, we performed parallel studies in rat cardiac myocyte/fibroblast cultures, examined gene expression in two mouse models of ( non-obstructive ) HCM (R92W-TnT, R403Q-MyHC)/controls at early (5 weeks) and established (24 weeks) disease stage, and analyzed publicly available mRNA/miRNA expression data from obstructive -HCM patients undergoing septal myectomy/controls (unused donor hearts). Results: Myocyte cultures: ET1 increased superoxide/H 2 O 2 , stimulated TGFβ expression/secretion, and suppressed miR-29a expression in myocytes. The effect of ET1 on miR-29 and TGFβ expression/secretion was antagonized by N-acetyl-cysteine, a reactive oxygen species scavenger. Fibroblast cultures: ET1 had no effect on pro-fibrotic gene expression in fibroblasts. TGFβ1/TGFβ2 suppressed miR-29a and increased collagen expression, which was abolished by miR-29a overexpression. Mouse and human HCM: Expression of miR-29a/b/c was lower, and TGFB1 /collagen gene expression was higher in TnT mutant-LV at 5 and 24 weeks; no difference was observed in expression of these genes in MyHC mutant-LV and in human myectomy tissue. TGFB2 expression was higher in LV of both mutant mice and human myectomy tissue. ACE2 , a negative regulator of the renin-angiotensin-aldosterone system, was the most upregulated transcript in human myectomy tissue. Pathway analysis predicted upregulation of the anti-hypertrophic/anti-fibrotic liver X receptor/retinoid X receptor (LXR/RXR) pathway only in human myectomy tissue. Conclusions: Our in vitro studies suggest that activation of ET1 signaling in cardiac myocytes increases reactive oxygen species and stimulates TGFβ secretion, which downregulates miR-29a and increases collagen in fibroblasts, thus contributing to fibrosis. Our gene expression studies in mouse and human HCM reveal allele-specific differences in miR-29 family/profibrotic gene expression in mouse HCM, and activation of anti-hypertrophic/anti-fibrotic genes and pathways in human HCM., (Copyright © 2019 Liu, Afzal, Vakrou, Greenland, Talbot, Hebl, Guan, Karmali, Tardiff, Leinwand, Olgin, Das, Fukunaga and Abraham.)

Background: Myogenesis is driven by specific changes in the transcriptome that occur during the different stages of muscle differentiation. In addition to controlled transcriptional transitions, several other post-transcriptional mechanisms direct muscle differentiation. Both alternative splicing and miRNA activity regulate gene expression and production of specialized protein isoforms. Importantly, disruption of either process often results in severe phenotypes as reported for several muscle diseases. Thus, broadening our understanding of the post-transcriptional pathways that operate in muscles will lay the foundation for future therapeutic interventions., Methods: We employed bioinformatics analysis in concert with the well-established C2C12 cell system for predicting and validating novel miR-1 and miR-206 targets engaged in muscle differentiation. We used reporter gene assays to test direct miRNA targeting and studied C2C12 cells stably expressing one of the cDNA candidates fused to a heterologous, miRNA-resistant 3' UTR. We monitored effects on differentiation by measuring fusion index, myotube area, and myogenic gene expression during time course differentiation experiments., Results: Gene ontology analysis revealed a strongly enriched set of putative miR-1 and miR-206 targets associated with RNA metabolism. Notably, the expression levels of several candidates decreased during C2C12 differentiation. We discovered that the splicing factor Srsf9 is a direct target of both miRNAs during myogenesis. Persistent Srsf9 expression during differentiation impaired myotube formation and blunted induction of the early pro-differentiation factor myogenin as well as the late differentiation marker sarcomeric myosin, Myh8., Conclusions: Our data uncover novel miR-1 and miR-206 cellular targets and establish a functional link between the splicing factor Srsf9 and myoblast differentiation. The finding that miRNA-mediated clearance of Srsf9 is a key myogenic event illustrates the coordinated and sophisticated interplay between the diverse components of the gene regulatory network.

Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the β-cardiac myosin heavy chain gene (β- MyHC ) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any β -MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 K app along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations., (© 2019 Vera et al.)

Striated muscle myosins are encoded by a large gene family in all mammals, including humans. These isoforms define several of the key characteristics of the different striated muscle fiber types, including maximum shortening velocity. We have previously used recombinant isoforms of the motor domains of seven different human myosin isoforms to define the actin·myosin cross-bridge cycle in solution. Here, we present data on an eighth isoform, the perinatal, which has not previously been characterized. The perinatal is distinct from the embryonic isoform, appearing to have features in common with the adult fast-muscle isoforms, including weak affinity of ADP for actin·myosin and fast ADP release. We go on to use a recently developed modeling approach, MUSICO, to explore how well the experimentally defined cross-bridge cycles for each isoform in solution can predict the characteristics of muscle fiber contraction, including duty ratio, shortening velocity, ATP economy, and load dependence of these parameters. The work shows that the parameters of the cross-bridge cycle predict many of the major characteristics of each muscle fiber type and raises the question of what sequence changes are responsible for these characteristics., (© 2019 Johnson et al.)

Background In mammals, muscle contraction is controlled by a family of 10 sarcomeric myosin motors. The expression of one of its members, MYH7b, is regulated by alternative splicing, and while the protein is restricted to specialized muscles such as extraocular muscles or muscle spindles, RNA that cannot encode protein is expressed in most skeletal muscles and in the heart. Remarkably, birds and snakes express MYH7b protein in both heart and skeletal muscles. This observation suggests that in the mammalian heart, the motor activity of MYH7b may only be needed during development since its expression is prevented in adult tissue, possibly because it could promote disease by unbalancing myocardial contractility. Methods and Results We have analyzed MYH7b null mice to determine the potential role of MYH7b during cardiac development and also generated transgenic mice with cardiac myocyte expression of MYH7b protein to measure its impact on cardiomyocyte function and contractility. We found that MYH7b null mice are born at expected Mendelian ratios and do not have a baseline cardiac phenotype as adults. In contrast, transgenic cardiac MYH7b protein expression induced early cardiac dilation in males with significantly increased left ventricular mass in both sexes. Cardiac dilation is progressive, leading to early cardiac dysfunction in males, but later dysfunction in females. Conclusions The data presented show that the expression of MYH7b protein in the mammalian heart has been inhibited during the evolution of mammals most likely to prevent the development of a severe cardiomyopathy that is sexually dimorphic.

Striated muscles express an array of sarcomeric myosin motors that are tuned to accomplish specific tasks. Each myosin isoform found in muscle fibers confers unique contractile properties to the fiber in order to meet the demands of the muscle. The sarcomeric myosin heavy chain (MYH) genes expressed in the major cardiac and skeletal muscles have been studied for decades. However, three ancient myosins, MYH7b, MYH15, and MYH16, remained uncharacterized due to their unique expression patterns in common mammalian model organisms and due to their relatively recent discovery in these genomes. This article reviews the literature surrounding these three ancient sarcomeric myosins and the specialized muscles in which they are expressed. Further study of these ancient myosins and how they contribute to the functions of the specialized muscles may provide novel insight into the history of striated muscle evolution.

During pregnancy, the heart undergoes significant and numerous changes, including hypertrophy, that are usually described as physiological and reversible. Two aspects of the cardiac response to pregnancy are relatively understudied: advanced maternal age and multiple pregnancies (multiparity). Repeated breeder (RB) mice that have undergone five to seven consecutive pregnancies were euthanized 21 days after the weaning of their last pups and compared with age-matched primiparous, one-time pregnant (O1P) mice. The ages of the older mouse groups were similar (12 ± 1 mo). Pregnancy at a later age resulted in reduced fertility (40%); resorption was 29%, maternal mortality was 10%, and mortality of the pups was 17%. Contractile function as indicated by percent fractional shortening was significantly decreased in O1P and RB groups compared with the old nonpregnant control (ONP) group. There was no pathological induction of the fetal program of gene expression, with the exception of β-myosin heavy chain mRNA, which was induced in O1P compared with ONP mice ( P < 0.05) but not in RB mice. MicroRNA-208a was significantly increased in O1P compared with ONP mice ( P < 0.05) but significantly decreased in RB compared with ONP mice ( P < 0.05). mRNA of genes regulating angiogenesis (i.e., vascular endothelial growth factor-A) were significantly downregulated, whereas proinflammatory genes [i.e., interleukin-6, chemokine (C-C motif) ligand 2, and Cd36] were significantly upregulated in O1P ( P < 0.05) but not in RB mice. Overall, our results suggest that rather than multiparity, pregnancy in advanced age is a much more stressful event in both pregnant dams and fetuses, as evidenced by increased mortality, lower fertility, downregulation of angiogenesis, upregulation of inflammation, and cardiac dysfunction. NEW & NOTEWORTHY Pregnancy in older mice significantly decreases cardiac function, although repeated breeder mice demonstrated increased wall hypertrophy and dilated chamber size compared with one-time pregnant mice. Interestingly, many of the molecular changes were altered in one-time pregnant mice but not in repeated breeder mice, which may contribute to adverse pregnancy outcomes in a first pregnancy at a later age.

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human β-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M·D complex in the steady state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force-holding capacity due to the reduced occupancy of the force-holding state., (© 2018 Ujfalusi et al.)

Hypertrophic cardiomyopathy (HCM) stems from mutations in sarcomeric proteins that elicit distinct biophysical sequelae, which in turn may yield radically different intracellular signaling and molecular pathologic profiles. These signaling events remain largely unaddressed by clinical trials that have selected patients based on clinical HCM diagnosis, irrespective of genotype. In this study, we determined how two mouse models of HCM differ, with respect to cellular/mitochondrial function and molecular biosignatures, at an early stage of disease. We show that hearts from young R92W-TnT and R403Q-αMyHC mutation-bearing mice differ in their transcriptome, miRNome, intracellular redox environment, mitochondrial antioxidant defense mechanisms, and susceptibility to mitochondrial permeability transition pore opening. Pathway analysis of mRNA-sequencing data and microRNA profiles indicate that R92W-TnT mutants exhibit a biosignature consistent with activation of profibrotic TGF-β signaling. Our results suggest that the oxidative environment and mitochondrial impairment in young R92W-TnT mice promote activation of TGF-β signaling that foreshadows a pernicious phenotype in young individuals. Of the two mutations, R92W-TnT is more likely to benefit from anti-TGF-β signaling effects conferred by angiotensin receptor blockers and may be responsive to mitochondrial antioxidant strategies in the early stage of disease. Molecular and functional profiling may therefore serve as aids to guide precision therapy for HCM.

Because they are perceived as distinct from the biological sciences, entrepreneurial pursuits may be daunting to the average researcher. In this report, we explain why academic scientists and in particular translational researchers should be naturally as well as rationally attracted to entrepreneurial endeavors. We go into some detail of how entrepreneurial achievements are actually accomplished and offer a few caveats for consideration when embarking down entrepreneurial pathways. We conclude that, although not for everyone, for translational investigators in the biologic sciences, entrepreneurial pursuits are desirable, accomplishable, and professionally rewarding.

A number of signaling pathways underlying pathological cardiac hypertrophy have been identified. However, few studies have probed the functional significance of these signaling pathways in the context of exercise or physiological pathways. Exercise studies were performed on females from six different genetic mouse models that have been shown to exhibit alterations in pathological cardiac adaptation and hypertrophy. These include mice expressing constitutively active glycogen synthase kinase-3β (GSK-3βS9A), an inhibitor of CaMK II (AC3-I), both GSK-3βS9A and AC3-I (GSK-3βS9A/AC3-I), constitutively active Akt (myrAkt), mice deficient in MAPK/ERK kinase kinase-1 (MEKK1 -/- ), and mice deficient in cyclin D2 (cyclin D2 -/- ). Voluntary wheel running performance was similar to NTG littermates for five of the mouse lines. Exercise induced significant cardiac growth in all mouse models except the cyclin D2 -/- mice. Cardiac function was not impacted in the cyclin D2 -/- mice and studies using a phospho-antibody array identified six proteins with increased phosphorylation (greater than 150%) and nine proteins with decreased phosphorylation (greater than 33% decrease) in the hearts of exercised cyclin D2 -/- mice compared to exercised NTG littermate controls. Our results demonstrate that unlike the other hypertrophic signaling molecules tested here, cyclin D2 is an important regulator of both pathologic and physiological hypertrophy. Impact statement This research is relevant as the hypertrophic signaling pathways tested here have only been characterized for their role in pathological hypertrophy, and not in the context of exercise or physiological hypertrophy. By using the same transgenic mouse lines utilized in previous studies, our findings provide a novel and important understanding for the role of these signaling pathways in physiological hypertrophy. We found that alterations in the signaling pathways tested here had no impact on exercise performance. Exercise induced cardiac growth in all of the transgenic mice except for the mice deficient in cyclin D2. In the cyclin D2 null mice, cardiac function was not impacted even though the hypertrophic response was blunted and a number of signaling pathways are differentially regulated by exercise. These data provide the field with an understanding that cyclin D2 is a key mediator of physiological hypertrophy.

Exercise elicits coordinated multi-organ responses including skeletal muscle, vasculature, heart, and lung. In the short term, the output of the heart increases to meet the demand of strenuous exercise. Long-term exercise instigates remodeling of the heart including growth and adaptive molecular and cellular re-programming. Signaling pathways such as the insulin-like growth factor 1/PI3K/Akt pathway mediate many of these responses. Exercise-induced, or physiologic, cardiac growth contrasts with growth elicited by pathological stimuli such as hypertension. Comparing the molecular and cellular underpinnings of physiologic and pathologic cardiac growth has unveiled phenotype-specific signaling pathways and transcriptional regulatory programs. Studies suggest that exercise pathways likely antagonize pathological pathways, and exercise training is often recommended for patients with chronic stable heart failure or following myocardial infarction. Herein, we summarize the current understanding of the structural and functional cardiac responses to exercise as well as signaling pathways and downstream effector molecules responsible for these adaptations., (Copyright © 2017. Published by Elsevier Inc.)

Background: Human skeletal muscles express three major myosin heavy chain (MyHC) isoforms: MyHCIIx (MYH1) in fast type 2B muscle fibers, MyHCIIa (MYH2) in fast type 2A fibers and MyHCI/β-cardiac MyHC (MYH7) in slow type I skeletal fibers and cardiac ventricles. In line with its expression pattern, MYH7 mutations have been reported in association with hypertrophic or dilated cardiomyopathy, skeletal myopathies or a combination of both. We analyzed the clinical and molecular phenotype of two unrelated families of Jewish Moroccan ancestry that presented with apparently autosomal dominant inheritance of progressive Laing-like distal myopathy with non-specific myopathic changes, but uncommon marked contractures and wasting of the neck extensors., Methods: Clinical phenotyping, whole exome sequencing and restriction analysis, generation of mutants followed by cell culture transfection and imaging., Results: Using whole exome sequencing we identified in both families two novel heterozygous proline substitutions located in exon 31 of MYH7 within its rod domain: c.4309G>C (p.Ala1437Pro) and c.4301G>C (p.Arg1434Pro). Here we show that the phenotype caused by these mutations includes marked cervical muscle contracture, and report that the severity of the phenotype varies significantly, to the extent of non-penetrance in one of the families. Finally, we provide evidence that both proline substitutions impair myosin self-assembly in non-muscle cells transfected with β-myosin constructs carrying the mutations, but do not prevent incorporation of the mutant molecules into the sarcomere., Conclusions: This study expands our clinical and molecular knowledge of MYH7 rod mutations causing skeletal myopathies, and underscores the importance of discussing disease penetrance during genetic counseling.