Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling. Increased TRPC6 activity from enhanced gene expression or gain-of-function mutations contribute to cardiac and/or renal disease. Despite evidence supporting a pathophysiological role, no orally bioavailable selective TRPC6 inhibitor has yet been developed and tested in vivo in disease models. Here, we report an orally bioavailable TRPC6 antagonist (BI 749327; IC 50 13 nM against mouse TRPC6, t 1/2 8.5-13.5 hours) with 85-and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration ∼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis. TRPC6 | ion channels | calcium | nuclear factor of activated T cells | fibrosis

Cardiomyocytes undergo significant structural and functional changes after birth, and these fundamental processes are essential for the heart to pump blood to the growing body. However, due to the challenges of isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of the mature phenotype remains poorly understood. Here we implement large-particle sorting and analyze single myocytes from neonatal to adult hearts. Early myocytes exhibit wide-ranging transcriptomic and size heterogeneity that is maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion reveals that peroxisome proliferator-activated receptor coactivator-1 signaling, which is active in vivo but inactive in pluripotent stem cell-derived cardiomyocytes, mediates the shift. This signaling simultaneously regulates key aspects of cardiomyocyte maturation through previously unrecognized proteins, including YAP1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and identifies a multifaceted regulator controlling cardiomyocyte maturation.

Muscle contraction, which is initiated by Ca2+, results in precise sliding of myosin-based thick and actin-based thin filament contractile proteins. The interactions between myosin and actin are finely tuned by three isoforms of myosin binding protein-C (MyBP-C): slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C and cMyBP-C, respectively), each with distinct N-terminal regulatory regions. The skeletal MyBP-C isoforms are conditionally coexpressed in cardiac muscle, but little is known about their function. Therefore, to characterize the functional differences and regulatory mechanisms among these three isoforms, we expressed recombinant N-terminal fragments and examined their effect on contractile properties in biophysical assays. Addition of the fragments to in vitro motility assays demonstrated that ssMyBP-C and cMyBP-C activate thin filament sliding at low Ca2+. Corresponding 3D electron microscopy reconstructions of native thin filaments suggest that graded shifts of tropomyosin on actin are responsible for this activation (cardiac > slow-skeletal > fast-skeletal). Conversely, at higher Ca2+, addition of fsMyBP-C and cMyBP-C fragments reduced sliding velocities in the in vitro motility assays and increased force production in cardiac muscle fibers. We conclude that due to the high frequency of Ca2+ cycling in cardiac muscle, cardiac MyBP-C may play dual roles at both low and high Ca2+. However, skeletal MyBP-C isoforms may be tuned to meet the needs of specific skeletal muscles.

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

Rationale: Stimulated protein kinase G-1α (PKG1α) phosphorylates tuberous sclerosis complex 2 (TSC2) at serine 1365, potently suppressing mTORC1 activation by neuro-hormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine 42 is also induced by these stressors, which blunts its cardioprotective effects. Objective: We tested the dependence of mTORC1 activation on PKG1α C42-oxidation, and its capacity to suppress such activation by soluble guanylyl cyclase-1 (GC-1) activation. Methods and Results: Cardiomyocytes expressing WT PKG1α (PKG1α WT ) or C42S redox-dead PKG1α CS/CS were exposed to endothelin-1 (ET-1). Cells expressing PKG1α WT exhibited substantial mTORC1 activation (p70 S6K, 4EBP1, and Ulk1 phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1α CS/CS expression. Mice with global knock-in of PKG1α CS/CS subjected to pressure-overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1α WT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2 S1365 phosphorylation increased in PKG1α CS/CS more than PKG1α WT myocardium following PO. TSC2 S1365A/S1365A KI mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1α CS/CS mice, protection against PO-induced mTORC1 activation, cardio-depression, and mortality in PKG1α CS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1α CS/CS after PO, and blocked ET-1 stimulated mTORC1 in TSC2 S1365A expressing myocytes. Conclusions: Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.

While pluripotent stem cell-derived cardiomyocytes (PSC-CMs) offer tremendous potential for a range of clinical applications, their use has been constrained by the failure to mature these cells to a fully adult-like phenotype. Extensive efforts are currently underway with the goal to mature PSC-CMs. However, comprehensive metrics to benchmark the maturation status and trajectory of PSC-CMs have not been established. Here, we developed a novel approach to quantify CM maturation through single cell transcriptomic entropy. We found that transcriptomic entropy is robust across datasets regardless of differences in isolation protocols, library preparation methods, and other potential batch effects. We analyzed over 40 single cell RNA-sequencing (scRNA-seq) datasets and over 45,000 CMs to establish a cross-study, cross-species reference of CM maturation based on transcriptomic entropy. We subsequently computed the maturation status of PSC-CMs by direct comparison to in vivo development. Our study presents a robust, interpretable, and easy-to-use metric for quantifying CM maturation. cardiomyocyte | transcriptomic | single cell

Striated cardiac and skeletal muscles play very different roles in the body, but they are similar at the molecular level. In particular, contraction, regardless of the type of muscle, is a precise and complex process involving the integral protein myofilaments and their associated regulatory components. The smallest functional unit of muscle contraction is the sarcomere. Within the sarcomere can be found a sophisticated ensemble of proteins associated with the thick filaments (myosin, myosin binding protein-C, titin, and obscurin) and thin myofilaments (actin, troponin, tropomyosin, nebulin, and nebulette). These parallel thick and thin filaments slide across one another, pulling the two ends of the sarcomere together to regulate contraction. More specifically, the regulation of both timing and force of contraction is accomplished through an intricate network of intra- and interfilament interactions belonging to each myofilament. This review introduces the sarcomere proteins involved in striated muscle contraction and places greater emphasis on the more recently identified and less well-characterized myofilaments: cardiac myosin binding protein-C, titin, nebulin, and obscurin. © 2017 American Physiological Society. Compr Physiol 7:675-692, 2017.

Rationale: Single cell RNA sequencing (scRNA-seq) has emerged as a powerful tool to profile the transcriptome at single cell resolution, enabling comprehensive analysis of cellular trajectories and heterogeneity during development and disease. However, the use of scRNA-seq remains limited in cardiac pathology owing to technical difficulties associated with the isolation of single adult cardiomyocytes (CMs). Objective:We investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs. Methods and Results:We found that LP-FACS readily outperforms conventional FACS for isolation of struturally competent CMs, including large CMs. Additionally, LP-FACS enables isolation of fluorescent CMs from mosaic models. Importantly, the sorted CMs allow generation of high-quality scRNA-seq libraries. Unlike CMs isolated via previously utilized fluidic or manual methods, LP-FACisolated CMs generate libraries exhibiting normal levels of mitochondrial transcripts. Moreover, LP-FACS isolated CMs remain functionally competent and can be studied for contractile properties. Conclusions:Our study enables high quality dissection of adult CM biology at single-cell resolution.To date, however, scRNA-seq of adult CMs has been limited owing to technical difficulties in the isolation of single CMs. Specialized techniques are required for dissociation of the heart without damaging cells, as CMs are highly sensitive to pH, buffer ionic concentrations, and mechanical disruption 7 . Moreover, adult CMs are large (approximately 125x25µm 8 ), rod-shaped cells whose structure precludes isolation by either fluorescence-activated cell sorting (FACS) or commercial single cell microfluidic platforms (such as the Fluidigm C1 or Chromium 10X) 2,3 . Previous studies that have attempted to isolate CMs using conventional FACS equipment have resulted in frequent clogging of the instrument 9 or generation and/of fragmented myocytes 10 . Moreover, scRNA-seq libraries generated from CMs following FACS 10 or isolation on the Fluidigm C1 4 have been dominated by mitochondrial reads, potentially suggesting that the cells were damaged prior to library generation. While one group has previously reported on isolation of rod-shaped CMs by FACS 11,12 , this approach only worked with fixed cells, and required significant instrument and sort parameter customization that may preclude its use in other labs. One approach that has been successful in generating high quality scRNA-seq libraries from single CMs has been single cell picking by micropipette 13 . However, this approach is technically challenging, low throughput, and time intensive, which may in turn lead to significant transcriptomic changes during isolation. In lieu of whole cell scRNA-seq, others have utilized single nuclear RNA-seq (snRNA-seq), which overcomes the challenges of isolating large CMs [14][15][16] . While this approach can broadly capture cellular heterogeneity, snRNA-seq inherently detects fewer molecules and may skew gene expressi...