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Background The coronavirus disease 2019 pandemic spread to >200 countries in Methods Given high residence hall student density, the University of Colorado Boulder established a mandatory weekly screening test program. We analyzed longitudinal data from 6408 students and identified 116 likely transmission events in which a second roommate tested positive within 14 days of the index roommate. Results Although the infection rate was lower in single-occupancy rooms (10%) than in multiple-occupancy rooms (19%), interroommate transmission occurred only about 20% of the time. Cases were usually asymptomatic at the time of detection. Notably, individuals who likely transmitted had an average viral load approximately 6.5-fold higher than individuals who did not (mean quantification cycle [Cq], 26.2 vs 28.9). Although students with diagnosed SARS-CoV-2 infection moved to isolation rooms, there was no difference in time to isolation between cases with or without interroommate transmission. Conclusions This analysis argues that interroommate transmission occurs infrequently in residence halls and provides strong correlative evidence that viral load is proportional to transmission probability.

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 (ERαKOism). 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, VO2, VCO2, RER, energy expenditure, and activity counts across genotypes. Although ERαKOism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKOism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKOism, 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 H2O2 emission potential was not affected in the ERαKOism 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., Highlights • Induced skeletal muscle specific ERαKO (ERαKOism) examines the role of ERα without confounding factors of development. • Skeletal muscle glucose uptake is not impaired in ERαKOism. • Skeletal muscle mitochondrial function is not impaired in ERαKOism. • ERαKD in human myotubes does not severely affect mitochondrial respiratory capacity.

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., Heart and skeletal muscles of larger mammals contract more slowly than smaller ones. This study identifies amino acid changes in myosin isoforms that correlate with species size; mutating the residues in human β-myosin to match the rat sequence at these positions increased its in vitro velocity to that of the rat protein.

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.

Significance We analyzed data from saliva-based COVID-19 screening deployed on the University of Colorado Boulder campus. Our dataset is unique in that all SARS-CoV-2−positive individuals reported no symptoms at the time of saliva collection, and therefore were infected but asymptomatic or presymptomatic. We found that 1) the distribution of viral loads observed in our asymptomatic college population was indistinguishable from what has been reported in hospitalized populations; 2) regardless of symptomatic status, approximately 50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the infection; and 3) just 2% of infected individuals carry 90% of the virions circulating within communities, serving as viral “supercarriers” and likely also superspreaders., We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral “supercarriers” and possibly also superspreaders.

In 2019-2020, the COVID-19 pandemic spread to over 200 countries in less than six months. To understand the basis of this aggressive spread, it is essential to determine the transmission rate and define the factors that increase the risk of transmission. One complication is the large fraction of asymptomatic cases, particularly in young populations: these individuals have viral loads indistinguishable from symptomatic people and do transmit the SARS-CoV-2 virus, but they often go undetected. As university students living in residence halls commonly share a small living space with roommates, some schools established regular, high density testing programs to mitigate on-campus spread. In this study, we analyzed longitudinal testing data of residence hall students at the University of Colorado Boulder. We observed that students in single rooms were infected at a lower rate than students in multiple occupancy rooms. However, this was not due to high rates of transmission between roommates, which only occurred approximately 20% of the time. Since these cases were usually asymptomatic at the time of diagnosis, this provides further evidence for asymptomatic transmission. Notably, individuals who likely transmitted to their roommates had an average viral load ∼6.5 times higher than individuals who did not. Although students were moved to separate isolation rooms after diagnosis, there was no difference in time to isolation between these cases with or without transmission. This analysis argues that inter-roommate transmission occurs in a minority of cases in university residence halls and provides strong correlative evidence that viral load can be proportional to the probability of transmission.

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 P P 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 P 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.

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.

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). 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.

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.

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/H2O2, 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.

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.

Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass and insulin resistance, suggesting muscle growth may be restricted by molecular events that occur during fetal development. To explore the basis of restricted fetal muscle growth, we used a sheep model of progressive placental insufficiency-induced IUGR to assess myoblast proliferation within intact skeletal musclein vivoand isolated myoblasts stimulated with insulinin vitro. Gastrocnemius and soleus muscle weights were reduced by 25% in IUGR fetuses compared to those in controls (CON). The ratio of PAX7+ nuclei (a marker of myoblasts) to total nuclei was maintained in IUGR muscle compared to CON, but the fraction of PAX7+ myoblasts that also expressed Ki-67 (a marker of cellular proliferation) was reduced by 23%. Despite reduced proliferationin vivo, fetal myoblasts isolated from IUGR biceps femoris and cultured in enriched mediain vitroresponded robustly to insulin in a dose- and time-dependent manner to increase proliferation. Similarly, insulin stimulation of IUGR myoblasts upregulated key cell cycle genes and DNA replication. There were no differences in the expression of myogenic regulatory transcription factors that drive commitment to muscle differentiation between CON and IUGR groups. These results demonstrate that the molecular machinery necessary for transcriptional control of proliferation remains intact in IUGR fetal myoblasts, indicating thatin vivofactors such as reduced insulin and IGF1, hypoxia and/or elevated counter-regulatory hormones may be inhibiting muscle growth in IUGR fetuses.

The transcatheter aortic valve replacement (TAVR) procedure has emerged as a minimally invasive treatment for patients with aortic valve stenosis (AVS). However, alterations in serum factor composition and biological activity after TAVR remain unknown. Here, we quantified the systemic inflammatory effects of the TAVR procedure and hypothesized that alterations in serum factor composition would modulate valve and cardiac fibrosis. Serum samples were obtained from patients with AVS immediately before their TAVR procedure (pre-TAVR) and about 1 month afterward (post-TAVR). Aptamer-based proteomic profiling revealed alterations in post-TAVR serum composition, and ontological analysis identified inflammatory macrophage factors implicated in myofibroblast activation and deactivation. Hydrogel biomaterials used as valve matrix mimics demonstrated that post-TAVR serum reduced myofibroblast activation of valvular interstitial cells relative to pre-TAVR serum from the same patient. Transcriptomics and curated network analysis revealed a shift in myofibroblast phenotype from pre-TAVR to post-TAVR and identified p38 MAPK signaling as one pathway involved in pre-TAVR–mediated myofibroblast activation. Post-TAVR serum deactivated valve and cardiac myofibroblasts initially exposed to pre-TAVR serum to a quiescent fibroblast phenotype. Our in vitro deactivation data correlated with patient disease severity measured via echocardiography and multimorbidity scores, and correlations were dependent on hydrogel stiffness. Sex differences in cellular responses to male and female sera were also observed and may corroborate clinical observations regarding sex-specific TAVR outcomes. Together, alterations in serum composition after TAVR may lead to an antifibrotic fibroblast phenotype, which suggests earlier interventions may be beneficial for patients with advanced AVS to prevent further disease progression., One-sentence summary: Transcatheter aortic valve replacement alters a patient’s serum proteome, reversing valvular interstitial cell and cardiac myofibroblast activation., Editor’s Summary: Responding to replacement Aortic valve stenosis (narrowing of the aortic valve) contributes to inadequate blood flow, fibrosis, hypertrophy, and, ultimately, heart failure. Transcatheter aortic valve replacement (TAVR) improves blood flow, but little is known about cardiac remodeling after the procedure. Aguado and colleagues performed proteomics on serum samples collected from patients before and after TAVR and studied the effects of serum on valve and cardiac cells using hydrogel culture platforms. A role for p38 MAPK signaling in activating cells was identified using pre-TAVR serum, whereas post-TAVR serum returned cells to a quiescent state. Along with preliminary insights into sex-specific differences, the authors’ research supports a role for TAVR-induced alteration of circulating inflammatory cytokines in regulating valve cell phenotype.

IntroductionMore than 400 mutations in β-myosin, a slow myosin motor, can cause both cardiac and skeletal myopathy in humans. A small subset of these mutations, mostly located in the myosin rod, leads to a progressive skeletal muscle disease known as Laing distal myopathy (MPD1). While this disease has previously been studied using a variety of systems, it has never been studied in the mammalian muscle environment. Here, we describe a mouse model for the MPD1-causing mutation R1500P to elucidate disease pathogenesis and to act as a future platform for testing therapeutic interventions.MethodsBecause mice have very few slow skeletal muscles compared to humans, we generated mice expressing the β-myosin R1500P mutation or WT β-myosin in fast skeletal muscle fibers and determined the structural and functional consequences of the R1500P mutation.ResultsThe mutant R1500P myosin affects both muscle histological structure and function and the mice exhibit a number of the histological hallmarks that are often identified in patients with MPD1. Furthermore, R1500P mice show decreased muscle strength and endurance, as well as ultrastructural abnormalities in the SR & t-tubules. Somewhat surprisingly because of its location in the rod, the R1500P mutation weakens acto-myosin binding by affecting cross-bridge detachment rate.ConclusionsWhile each group of MPD1-causing mutations most likely operates through distinct mechanisms, our model provides new insight into how a mutation in the rod domain impacts muscle structure and function and leads to disease.

Striated muscle is a highly specialized collection of tissues that have different contractile properties depending on their functional roles. These properties derive from differential gene expression, including sarcomeric genes such as myosin heavy chains (MyHCs) and metabolic genes that coordinate energy demands with the contractile machinery. Although muscle fiber types are established shortly after birth, there is lifelong plasticity that facilitates adaptation to both physiologic and pathologic stimuli. Functional adaptation requires molecular adaptation, and this is partially provided by miRNA-mediated post-transcriptional regulation. Several miRNAs are specific to muscle, including miR-206, which is particularly associated with slow oxidative muscles and is up-regulated in conditions that shift fast muscles towards a slower phenotype. We sought to determine whether miR-206 is a driver of the slow muscle phenotype or merely a terminal outcome. We found that miR-206 expression increases in both physiologic (female sex and endurance exercise) and pathologic conditions (muscular dystrophy and response to acute injury) that promote the slow phenotype. We also found that the predominantly slow soleus displays a faster phenotype in miR-206 knockout mice. Left ventricles of these animals also have a faster MyHC profile that is accompanied by dilation and systolic dysfunction. Interestingly, these phenotypes were male-specific. Our data indicate that miR-206 is necessary to enforce the slow straited muscle phenotype and that miR-206 plays a key role in sexual dimorphisms in cardiac and skeletal muscle.

Background In mammals, muscle contraction is controlled by a family of 10 sarcomeric myosin motors. The expression of one of its members, MYH 7b, 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 MYH 7b protein in both heart and skeletal muscles. This observation suggests that in the mammalian heart, the motor activity of MYH 7b 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 MYH 7b null mice to determine the potential role of MYH 7b during cardiac development and also generated transgenic mice with cardiac myocyte expression of MYH 7b protein to measure its impact on cardiomyocyte function and contractility. We found that MYH 7b null mice are born at expected Mendelian ratios and do not have a baseline cardiac phenotype as adults. In contrast, transgenic cardiac MYH 7b 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 MYH 7b 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.

AimsPathological cardiac hypertrophy is the result of increased cardiomyocyte size, leading to thickening of the left ventricular walls and a decrease in the left ventricular chamber. With early treatment of the underlying cause, cardiac hypertrophy can be reversed in some individuals, while it persists in others. Here, we investigate mechanisms leading to regression of pathological cardiac hypertrophy in two mouse models, in addition to defining the sex differences associated with hypertrophy and regression.Methods and ResultsTwo pathological hypertrophic stimuli were used in male and female mice (Isoproterenol or Angiotensin II). The stimulus was removed after 7 days of treatment, then the left ventricle was studied at intervals up to 7 days following the removal of the stimulus. Following Isoproterenol removal, male hearts returned to baseline sizes in 4 days while it took 7 days for female hearts to regress. After Angiotensin II removal, the left ventricular masses of males and females did not regress. ERK1/2 was activated in response to both Isoproterenol and Angiotensin II in males, then decreased back to baseline one day after stimulus removal. Expression of ECM genes was greater in response to Angiotensin II and remained elevated longer after Angiotensin II removal, compared to Isoproterenol. Further, collagen content may be playing a role in the irreversible state of Angiotensin II induced hypertrophy as hydroxyproline content was increased following the removal of Angiotensin II in both males and females.ConclusionsRegression of pathological cardiac hypertrophy is possible in some people and in some mouse models; however, the ability for the heart to regress is dependent on the stimulus and biological sex. Further, molecular changes including cellular signaling, protein degradation pathways and the formation of a fibrotic network may contribute to the ability to reverse pathological cardiac hypertrophy and are stimulus- and sex-dependent.Translational PerspectivePathological cardiac hypertrophy is a major risk factor for mortality. If cardiac hypertrophy persists for an extended time, there can be many maladaptive changes to the myocardium. With early treatment of the underlying cause, cardiac hypertrophy can be reversed in some individuals, but not in others. While cardiac hypertrophy has been studied extensively, very little is understood about regression of cardiac hypertrophy. It is important that we have a better understanding of mechanisms leading to regression and why this process might not be reversible in some individuals.

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.

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.

The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/β cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5-9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2-35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function.

Powering down yields a healthier heart In hypertrophic cardiomyopathy (HCM), the heart muscle enlarges and becomes progressively less efficient at pumping blood. HCM can be caused by mutations in components of the sarcomere (the heart's contractile unit), most notably myosin. Hypercontractility is among the earliest heart disturbances seen in mice carrying these myosin mutations, implying that the mutations inflict their damage by increasing myosin's power production. Green et al. identified a small molecule that binds to myosin and inhibits its activity (see the Perspective by Warshaw). When orally administered to young mice, the molecule prevented the development of several hallmark features of HCM without adversely affecting skeletal muscle. Science , this issue p. 617 ; see also p. 556

Introduction: Biological sex is an important modifier of cardiovascular disease (CVD). However, women and female animals have traditionally been excluded from clinical trials and most preclinical research studies, resulting in therapeutics that are not as effective, or with different side effects, in women relative to men. Methods and Results: We show here that expression of α- and β-adrenergic receptors, cardiac stiffness, myofibroblast proliferation, and expression of fibrotic markers are sexually dimorphic at baseline. To determine whether pathogenesis was different between the sexes, the β-adrenergic agonist, isoproterenol (ISO), was administered to rats. In response to 7-days of ISO treatment, female animals were more likely to survive than their male counterparts (percent survival: 44% in males versus 77% in females). Both sexes developed significant cardiac hypertrophy, activation of cardiac fetal genes (ANF and β-MHC) compared to vehicle controls. Cardiac myocytes from ISO treated animals took less time to reach peak shortening and displayed increased departure velocity; however, only male ISO treated cells exhibited reduced relaxation times and increased return velocities. Importantly, ISO treated animals developed significant fibrosis prompting us to analyze isolated cardiac fibroblasts at baseline and in response to ISO. We observed significantly increased expression of α-SMA and Col1a, two indicators of myofibroblast activation, only in fibroblasts isolated from ISO treated males. Both ISO treated male and female fibroblasts showed significant increases in periostin and decreases in TCF21, indicative of increases in myofibroblasts and decreases in quiescent fibroblasts, respectively. In addition, both sexes revealed increased fibroblast proliferation in vivo , as measured by EdU incorporation, in response to ISO. Conclusions: Chronic in vivo β-adrenergic receptor stimulation revealed dramatic sex differences in mortality, cardiac hypertrophy, myocyte contractility, and cardiac fetal gene expression. Increases in myofibroblast differentiation and fibroblast proliferation should lead to a robust fibrotic response in both male and female ISO treated animals.

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.

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.

Aims To define the molecular mechanisms of cardiotoxicity induced by Sunitinib and to identify the role of biological sex in modulating toxicity. Methods and results Exposure of isolated cardiomyocytes to plasma-relevant concentrations of Sunitinib and other tyrosine kinase inhibitors produces a broad spectrum of abnormalities and cell death via apoptosis downstream of sexually dimorphic kinase inhibition. Phosphorylation of protein kinase C and phospholipase γ abrogates these effects for most tyrosine kinase inhibitors tested. Female sex and estradiol cause increased cardiotoxicity, which is mediated by reduced expression of a drug efflux transporter and a metabolic enzyme. Female but not male mice exposed to a 28-day course of oral Sunitinib exhibit similar abnormalities as well as functional deficits and their hearts exhibit differential expression of genes responsible for transport and metabolism of Sunitinib. Conclusion We identify the specific pathways affected by tyrosine kinase inhibitors in mammalian cardiomyocytes, interactions with biological sex, and a role for oestrogen in modulating drug efflux and metabolism. These findings represent a critical step toward reducing the incidence of cardiotoxicity with tyrosine kinase inhibitor chemotherapeutics.

Use of soy supplements to inhibit cancer cell growth is increasing among patients due to the perception that phytoestrogens in soy inhibit carcinogenesis via induction of apoptosis. Genistein, the most prevalent phytoestrogen in soy, is a potent endocrine disruptor and tyrosine kinase inhibitor (TKI) that causes apoptosis in many cells types. Chemotherapeutic TKIs limit cancer cell growth via the same mechanisms. However, TKIs such as Sunitinib cause cardiotoxicity in a significant number of patients. Molecular interactions between Sunitinib and dietary TKIs like genistein have not been examined in cardiomyocytes. Significant lethality occurred in mice treated with Sunitinib and fed a phytoestrogen-supplemented diet. Isolated cardiomyocytes co-treated with genistein and Sunitinib exhibited additive inhibition of signaling molecules important for normal cardiac function and increased apoptosis compared with Sunitinib alone. Thus, dietary soy supplementation should be avoided during administration of Sunitinib due to exacerbated cardiotoxicity, despite evidence for positive effects in cancer.

Previous studies have identified over 3,000 genes that are differentially expressed in male and female skeletal muscle. Here, we review the sex-based differences in skeletal muscle fiber composition, myosin heavy chain expression, contractile function, and the regulation of these physiological differences by thyroid hormone, estrogen, and testosterone. The findings presented lay the basis for the continued work needed to fully understand the skeletal muscle differences between males and females.

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.

Introduction: The postprandial Burmese python displays a remarkable level of cardiac adaptation to high levels of circulating triglycerides and an increased metabolic rate without any evidence of cardiac pathology. To challenge the level of cardioprotection, we designed an experiment to attempt to induce metabolic syndrome in the Burmese python. Metabolic syndrome is typically defined as obesity concomitant with other metabolic abnormalities which contribute to an increased risk for cardiovascular disease. Methods and Results: Unlike mice or rats who readily undergo hypertrophy in response to pressure-overload or β-adrenergic receptor stimulation, inducing pathological hypertrophy in the Burmese python is challenging. In the current study, we attempted to induce metabolic syndrome through overfeeding. We offered Burmese pythons a meal equivalent to 25% of the python’s body mass every 4 days for a period of 2 months. Two similar groups of pythons were maintained on our standard python feeding schedule (one meal equivalent to 25% of their body weight every 28 days) over the same time course. At the end of 2 months, the weight of the overfed pythons (6197g ± 180g) dramatically increased when compared to fasted (2352g ± 23g) or normal fed pythons (3044g ± 62g). In addition to an overall increase in body weight, the ventricle weight/brain weight ratio increased significantly in the overfed pythons (19.5 ± 1.1) compared to fasted (7.5 ± 0.2) or normal fed pythons (8.8 ± 0.2) indicating considerable hypertrophy. However, despite developing hypertrophy, there was no evidence of aberrant gene expression or impaired glucose tolerance when compared to normal fed pythons. Conclusions: Despite repeatedly consuming a large meal, nearly tripling in body weight, and developing significant hypertrophy, the Burmese python is protected from developing metabolic abnormalities such as impaired glucose tolerance. Fetal gene expression markers of pathological cardiac hypertrophy were not induced in overfed pythons. Thus, studying the mechanisms of cardiac adaptation in the Burmese python may potentially provide insights into therapeutic targets for combating metabolic syndrome in patients.

Introduction: Cardiac hypertrophy is initially, a compensatory mechanism to maintain cardiac output when there is an increased load on the heart. However, if cardiac hypertrophy persists for an extended time, there can be maladaptive changes to the myocardium. Even when the underlying cause of hypertrophy is treated, regression is often minimal or absent. Clinical cases of cardiac regression do exist, including patients receiving bariatric surgery or a left ventricular assist device. While many of the mechanisms leading to cardiac hypertrophy are well understood, little is known about the mechanisms of reversal of hypertrophy and why it is sometimes irreversible. We hypothesized that a reversal of isoproterenol (Iso) induced cardiac hypertrophy in the mouse will be observed within 7 days following the removal of the stimulus and we will be able to identify alterations in signaling pathways. Methods: We induced pathological cardiac hypertrophy with Iso for 7 days, at which peak hypertrophy is achieved. To identify if/when regression occurs, the Iso treatment was stopped and the mice were monitored for 7 days. Heart weights were measured at peak hypertrophy, post-drug days 1, 2, 3 & 7, along with vehicle treated mice (8/group). We used left ventricle tissue for protein analysis and protein degradation activity assays. Results: Regression from cardiac hypertrophy occurs by post-drug day 7 (p=0.016) in the Iso mouse model. p-Akt is increased with Iso treatment and returns to vehicle control levels by post-drug day 7. There is a decrease in p-mTOR and an increase in LC3-II levels at post-drug day 7, indicating a possible role of autophagy in cardiac regression. In addition, there was a decrease in cell size when neonatal rat ventricular myocytes were treated with the Akt inhibitor, Wortmannin, following phenylephrine induced hypertrophy. Conclusion: Regression of Iso-induced cardiac hypertrophy occurs in the mouse after 7 days following the removal of the stimulus. The Akt pathway is activated with Iso treatment and when this pathway is inactivated during regression, autophagy is activated, which may be an important mechanism to degrade proteins and lead to a decrease in cardiac hypertrophy. Finally, when the Akt pathway is inhibited in vitro , hypertrophic cells regress.

In cardiovascular disease research, studies often include measuring cardiac function and performing histological examination of heart tissue. After measuring contractility, hearts from animals such as mice and rats are often frozen or fixed, sliced, and stained to quantify the morphology of various structures such as extracellular matrix proteins, cell nuclei, and F-actin. Traditional scoring methods have largely consisted of assessing sections of images for the presence or absence of myocyte disarray. These approaches require unbiased manual assessment, which can require extra personnel, and are not scalable to the quantity of data that can be generated by modern automated experimental techniques. Here, we describe an automated image analysis approach for unbiased numerical measurement of myocyte disarray. We provide step-by-step instructions for image preparation as well as a basic Matlab script for measurements. © 2017 by John Wiley & Sons, Inc.

Pediatric heart failure (HF) is the inability of the heart of an infant, child, or adolescent to meet the body’s metabolic demands. It involves circulatory, neurohumoral, and molecular abnormalities that manifest as edema, respiratory distress, growth failure, and exercise intolerance. The myriad causes include inherited and acquired myocardial anomalies (cardiomyopathy [CM]), volume overload (intracardiac shunts, valvular regurgitation), and the unique hemodynamics predicated by a functional single ventricle (palliated complex congenital heart disease [CHD]). Although the societal and financial costs of adult HF are well known, the burden of pediatric HF is less familiar, but no less onerous. New-onset HF requiring hospital admission occurs in 0.87 per 100 000 children,1 yet that does not include the growing population with CHD-related HF. In 2006, there were nearly 14 000 pediatric hospitalizations for HF from all causes in the United States.2 The rate of HF-related admissions was nearly 18 per 100 000 children,2 which is comparable to severe sepsis.3 The mortality for pediatric HF hospitalizations is significant. The 7% overall hospital mortality rate exceeds the 4% mortality of adult HF admissions4 and represents a 20-fold increase over children without HF.2 With comorbidities like renal failure, sepsis, or stroke, hospital mortality in pediatric HF can exceed 20%,2 yet the risk does not end with discharge. After an initial HF hospitalization, only 21% of children in 1 study avoided readmission, death, or transplantation.5 Pediatric HF treatment is resource intensive. Although the total healthcare costs for pediatric HF are lower than for adults, per-patient costs are higher. The estimated hospital charge per pediatric HF admission in 2006 was >$135 000, with aggregate charges exceeding $1.8 billion.6 Certain subpopulations of pediatric HF incurred disproportionally higher costs. For example, single-ventricle CHD averaged >$200 000 per hospitalization,7 whereas adult …

Familial hypertrophic cardiomyopathy (HCM) is largely caused by dominant mutations in genes encoding cardiac sarcomeric proteins, and it is etiologically distinct from secondary cardiomyopathies resulting from pressure/volume overload and neurohormonal or inflammatory stimuli. Here, we demonstrate that decreased left ventricular contractile function in male, but not female, HCM mice is associated with reduced fatty acid translocase (CD36) and AMP-activated protein kinase (AMPK) activity. As a result, the levels of myocardial ATP and triglyceride (TG) content are reduced, while the levels of oleic acid and TG in circulating very low density lipoproteins (VLDLs) and liver are increased. With time, these metabolic changes culminate in enhanced glucose production in male HCM mice. Remarkably, restoration of ventricular TG and ATP deficits via AMPK agonism as well as inhibition of gluconeogenesis improves ventricular architecture and function. These data underscore the importance of the systemic effects of a primary genetic heart disease to other organs and provide insight into potentially novel therapeutic interventions for HCM.

Matrix elasticity regulates proliferation, apoptosis, and differentiation of many cell types across various tissues. In particular, stiffened matrix in fibrotic lesions has been shown to promote pathogenic myofibroblast activation. To better understand the underlying pathways by which fibroblasts mechano-sense matrix elasticity, we cultured primary valvular interstitial cells (VICs) isolated from porcine aortic valves on poly(ethylene glycol)-based hydrogels with physiologically relevant and tunable elasticities. We show that soft hydrogels preserve the quiescent fibroblast phenotype of VICs much better than stiff plastic plates. We demonstrate that the PI3K/AKT pathway is significantly up-regulated when VICs are cultured on stiff gels or tissue culture polystyrene compared with freshly isolated VICs. In contrast, myofibroblasts de-activate and pAKT/AKT decreases as early as 2 h after reducing the substrate modulus. When PI3K or AKT is inhibited on stiff substrates, myofibroblast activation is blocked. When constitutively active PI3K is overexpressed, the myofibroblast phenotype is promoted even on soft substrates. These data suggest that valvular fibroblasts are sensing the changes in matrix elasticity through the PI3K/AKT pathway. This mechanism may be used by other mechano-sensitive cells in response to substrate modulus, and this pathway may be a worthwhile target for treating matrix stiffness-associated diseases. Furthermore, hydrogels can be designed to recapitulate important mechanical cues in native tissues to preserve aspects of the native phenotype of primary cells for understanding basic cellular responses to biophysical and biochemical signals, and for tissue-engineering applications.

Aims Calcium fluctuations and cardiac hypertrophy occur during pregnancy, but the role of the well-studied calcium-activated phosphatase, calcineurin, has not been studied in this setting. The purpose of this study was to determine whether calcineurin signalling is required for cardiac remodelling during pregnancy in mice. Methods and results We first examined calcineurin expression in the heart of mice during pregnancy. We found both calcineurin levels and activity were significantly increased in early-pregnancy and decreased in late-pregnancy. Since progesterone levels start to rise in early-pregnancy, we investigated whether progesterone alone was sufficient to modulate calcineurin levels in vivo . After implantation of progesterone pellets in non-pregnant female mice, cardiac mass increased, whereas cardiac function was maintained. In addition, calcineurin levels increased, which is also consistent with early-pregnancy. To determine whether these effects were occurring in the cardiac myocytes, we treated neonatal rat ventricular myocytes (NRVMs) with pregnancy-associated sex hormones. We found that progesterone treatment, but not oestradiol, increased calcineurin levels. To obtain a functional read-out of increased calcineurin activity, we measured the activity of the transcription factor NFAT, a downstream target of calcineurin. Progesterone treatment significantly increased NFAT activity in NRVMs, and this was blocked by the calcineurin inhibitor cyclosporine A (CsA), showing that the progesterone-mediated increase in NFAT activity requires calcineurin activity. Importantly, CsA treatment of mice completely blocked pregnancy-induced cardiac hypertrophy. Conclusion Our results show that calcineurin is required for pregnancy-induced cardiac hypertrophy, and that calcineurin activity in early-pregnancy is due at least in part to increased progesterone.

PGC-1α is a transcriptional coactivator induced by exercise that gives muscle many of the best known adaptations to endurance-type exercise, but has no effects on muscle strength or hypertrophy. We have identified a novel form of PGC-1α (PGC-1α4) that results from alternative promoter usage and splicing of the primary transcript. PGC-1α4 is highly expressed in exercised muscle but does not regulate most known PGC-1α targets such as the mitochondrial OXPHOS genes. Rather, it specifically induces IGF1 and represses myostatin, and expression of PGC-1α4 in vitro and in vivo induces robust skeletal muscle hypertrophy. Importantly, mice with skeletal muscle-specific transgenic expression of PGC-1α4 show increased muscle mass and strength, and dramatic resistance to the muscle wasting of cancer cachexia. Expression of PGC-1α4 is preferentially induced in mouse and human muscle during resistance exercise. These studies identify a novel PGC-1α protein that regulates and coordinates factors involved in skeletal muscle hypertrophy.

Maintaining skeletal muscle mass is essential for general health and prevention of disease progression in various neuromuscular conditions. Currently, no treatments are available to prevent progressive loss of muscle mass in any of these conditions. Hibernating mammals are protected from muscle atrophy despite prolonged periods of immobilization and starvation. Here, we describe a mechanism underlying muscle preservation and translate it to non-hibernating mammals. Although Akt has an established role in skeletal muscle homeostasis, we find that serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates muscle mass maintenance via downregulation of proteolysis and autophagy as well as increased protein synthesis during hibernation. We demonstrate that SGK1 is critical for the maintenance of skeletal muscle homeostasis and function in non-hibernating mammals in normal and atrophic conditions such as starvation and immobilization. Our results identify a novel therapeutic target to combat loss of skeletal muscle mass associated with muscle degeneration and atrophy.

Chronic kidney disease (CKD) is characterized by increased serum levels of the phosphaturic hormone fibroblast growth factor 23 (FGF23), the development of left ventricular hypertrophy, and increased risk of mortality due to cardiovascular disease. The FGF receptors (FGFRs) responsible for facilitating the effect of FGF23 on the heart and the expression patterns of FGFRs in the different cell types in the heart remain largely uncharacterized. We hypothesized that cardiomyocytes express a different FGFR profile than cardiac fibroblasts and that this differential expression profile could contribute to cell type-specific responses to FGF23. We measured FGFR mRNA levels at baseline and following pro-hypertrophic growth factor stimulation in isolated adult rat ventricular cardiomyocytes (ARVMs), neonatal rat ventricular cardiomyocytes (NRVMs), and neonatal rat ventricular fibroblasts (NRVFs). At baseline, NRVMs expressed predominantly FGFR1 but also expressed FGFRs 2 and 3. In ARVMs, FGFRs 1 and 3 predominated while FGFRs 2 and 4 were expressed at very low levels. FGFRs in ARVMs and NRVMs were unchanged by growthfactor stimulation. At baseline, NRVFs expressed equal amounts of FGFRs 1, 2 and 3 which were all downregulated with FGF23 stimulation. To determine whether these results translated to human hearts, we examined the FGFR profile in a cohort of non-failing, hypertrophic cardiomyopathy (HCM), and ischemic cardiomyopathy (ICM) human patient samples. Compared to non-failing patients, we found FGFR2 was significantly increased in HCM patients and trended towards an increase in ICM patients. The cardiac FGFR2 profile matched the FGF23 profile. In addition, we found that FGFR4 was significantly down regulated in both ICM and HCM compared to non-failing patients. In conclusion, different cell types in the heart differ in their FGFR profiles at baseline. The FGFR profile does not change in NRVMs or ARVMs in response to FGF23 treatment, but the expression of FGFRs 1, 2, and 3 decrease in NRVFs in response to FGF23. HCM and ICM in humans is characterized by an increase in FGFR2 and FGF23 expression accompanied with a downregulation of FGFR4 expression. Modulating FGFRs in the myocardium may provide a novel therapeutic target in CKD patients.

Estrogen signaling appears critical in the heart. However a mechanistic understanding of the role of estrogen in the cardiac myocyte is lacking. Moreover, there are multiple cell types in the heart and multiple estrogen receptor (ER) isoforms. Therefore, we studied expression, localization, transcriptional and signaling activity of ERs in isolated cardiac myocytes. We found only ERα RNA (but no ERβ RNA) in cardiac myocytes using two independent methods. The vast majority of full-length ERα protein (ERα66) localizes to cardiac myocyte nuclei where it is competent to activate transcription. Alternate isoforms of ERα encoded by the same genomic locus (ERα46 and ERα36) have differential transcriptional activity in cardiac myocytes but also primarily localize to nuclei. In contrast to other reports, no ERα isoform is competent to activate MAPK or PI3K signaling in cardiac myocytes. Together these data support a role for ERα at the level of transcription in cardiac myocytes.

Cardiac hypertrophy is a major risk factor for heart failure, and it has been shown that this increase in size occurs at the level of the cardiac myocyte. Cardiac myocyte model systems have been developed to study this process. Here we focus on cell culture tools, including primary cells, immortalized cell lines, human stem cells, and their morphological and molecular responses to pathological stimuli. For each cell type, we discuss commonly used methods for inducing hypertrophy, markers of pathological hypertrophy, advantages for each model, and disadvantages to using a particular cell type over other in vitro model systems. Where applicable, we discuss how each system is used to model human disease and how these models may be applicable to current drug therapeutic strategies. Finally, we discuss the increasing use of biomaterials to mimic healthy and diseased hearts and how these matrices can contribute to in vitro model systems of cardiac cell biology.

Nearly one-third of deaths in the United States are caused by cardiovascular disease (CVD) each year. In the past, CVD was thought to mainly affect men, leading to the exclusion of women and female animals from clinical studies and preclinical research. In light of sexual dimorphisms in CVD, a need exists to examine baseline cardiac differences in humans and the animals used to model CVD. In humans, sex differences are apparent at every level of cardiovascular physiology from action potential duration and mitochondrial energetics to cardiac myocyte and whole-heart contractile function. Biological sex is an important modifier of the development of CVD with younger women generally being protected, but this cardioprotection is lost later in life, suggesting a role for estrogen. Although endogenous estrogen is most likely a mediator of the observed functional differences in both health and disease, the signaling mechanisms involved are complex and are not yet fully understood. To investigate how sex modulates CVD development, animal models are essential tools and should be useful in the development of therapeutics. This review will focus on describing the cardiovascular sexual dimorphisms that exist both physiologically and in common animal models of CVD.

Hypertrophic cardiomyopathy (HCM) is a genetic disease that affects 1 in 500 people. In infants it is particularly severe and is the leading cause of sudden cardiac death in pediatric populations. A high percentage of HCM is attributed to mutations in beta cardiac myosin, the motor protein responsible for ventricular contraction. This study explores how pediatric-specific HCM mutations in beta cardiac myosin (D239N, H251N, and P710R) alter the biochemical and biomechanical properties of beta cardiac myosin at the molecular and cellular levels. Because these mutations manifest early in life, we hypothesize that they will be more severe than mutations that are not pediatric-specific. We investigated the biomechanical properties of these mutations at the molecular level using a purified S1 domain of human beta cardiac myosin. Specifically, we looked at the effects of these mutations on the ATPase activity and the unloaded velocity (in vitro motility) of beta cardiac S1. Upon comparison with WT beta cardiac S1, we find significant differences between the biomechanical properties of the WT and the pediatric-specific myosin mutations, which are more drastic as compared to mutations that are not pediatric-specific. To fully characterize the system, we are currently performing single molecule optical trap force measurements to determine the intrinsic force of these mutations. Future studies will involve creating iPSC-derived cardiomyocytes to study the mechanics of these mutations at the cellular level. The results from this study will increase the understanding of how genetic mutations that cause HCM lead to the presentation of the disease, and will uncover potential targets for therapeutic design to treat the cause of the disease instead of the symptoms.

Hypertrophic Cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young athletes and has an incidence of 1 in 500 people. Dilated cardiomyopathy (DCM) has a prevalence of 1 in 2000 people. Both diseases can be inherited, with HCM being autosomal dominant, and DCM having different genotypic profiles. There are over 400 mutations in cardiac myosin (MYH7) that have been linked to both diseases. Also noteworthy, early-onset HCM and DCM patients typically have a worse prognosis than adult-onset patients. The only intervention for these pediatric patients is a heart transplant. Here we describe the kinetic analysis of several cardiac myosin mutations that have been identified to be unique to early-onset patients. One set of mutations has been identified in pediatric HCM and the other set is DCM. Steady-state characterization of ATP hydrolysis for both mutant sets using an NADH coupled ATPase assay is reported. Additionally, transient kinetic analysis for several mutants was conducted illustrating differences in the cross-bridge kinetics between HCM and DCM mutants, namely ATP-induced dissociation, ADP release, and the binding constants of actin and the different nucleotide states. This data suggests that the motor activity is different among mutants and may provide insight into the mechanistic defects that can lead to both diseases.

The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.