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Volumetric muscle loss (VML) injuries characterized by critical loss of skeletal muscle tissues result in severe functional impairment. Current treatments involving use of muscle grafts are limited by tissue availability and donor site morbidity. In this study, we designed and synthesized an implantable glycosaminoglycan-based hydrogel system consisting of thiolated hyaluronic acid (HA) and thiolated chondroitin sulfate (CS) cross-linked with poly(ethylene glycol) diacrylate to promote skeletal muscle regeneration of VML injuries in mice. The HA-CS hydrogels were optimized with suitable biophysical properties by fine-tuning degree of thiol group substitution to support C2C12 myoblast proliferation, myogenic differentiation and expression of myogenic markers MyoD, MyoG and MYH8. Furthermore, in vivo studies using a murine quadriceps VML model demonstrated that the HA-CS hydrogels supported integration of implants with the surrounding host tissue and facilitated migration of Pax7+ satellite cells, de novo myofiber formation, angiogenesis, and innervation with minimized scar tissue formation during 4-week implantation. The hydrogel-treated and autograft-treated mice showed similar functional improvements in treadmill performance as early as 1-week post-implantation compared to the untreated groups. Taken together, our results demonstrate the promise of HA-CS hydrogels as regenerative engineering matrices to accelerate healing of skeletal muscle injuries., Graphical abstract Image 1, Highlights • Synthesized a biomimetic HA-CS hydrogel system with properties tailored for skeletal muscle. • HA-CS hydrogels supported in vitro myoblast cell behavior. • Implanted cell-free HA-CS hydrogels promoted de novo skeletal muscle regeneration. • HA-CS hydrogels enhanced in vivo angiogenesis and innervation. • HA-CS hydrogels promoted functional recovery comparable to autograft transplants.

Visual Abstract, Highlights • FYCO1, a component of the autophagic machinery, is highly expressed in the heart and a potent inducer of cardiomyocyte autophagy. • Loss of FYCO1 in vivo inhibits adaptation to starvation or biomechanical stress of the heart by an abrogated increase of autophagic flux and results in contractile dysfunction. • Heart specific overexpression of FYCO1 improves autophagic flux and rescues contractile dysfunction following pressure overload., Summary Autophagy is a cellular degradation process that has been implicated in diverse disease processes. The authors provide evidence that FYCO1, a component of the autophagic machinery, is essential for adaptation to cardiac stress. Although the absence of FYCO1 does not affect basal autophagy in isolated cardiomyocytes, it abolishes induction of autophagy after glucose deprivation. Likewise, Fyco1-deficient mice subjected to starvation or pressure overload are unable to respond with induction of autophagy and develop impaired cardiac function. FYCO1 overexpression leads to induction of autophagy in isolated cardiomyocytes and transgenic mouse hearts, thereby rescuing cardiac dysfunction in response to biomechanical stress.

Hypertrophic/dilated cardiomyopathy, often a prequel to heart failure, is accompanied by maladaptive transcriptional changes that contribute to arrythmias and contractile misfunction. Transgenic mice constitutively expressing high levels of calcineurin are known to develop extreme heart hypertrophy, which progresses to dilated cardiomyopathy, and to die several weeks after birth. Here, we characterized aberrant transcriptional and epigenetic pathways in this mouse model and established a pharmacological approach to treat established cardiomyopathy. We found that H3K4me3 (trimethyl histone 3 lysine 4) and H3K9me3 (trimethyl histone 3 lysine 9) Jumonji histone demethylases are markedly increased at the protein level and show enhanced enzymatic activity in diseased hearts. These epigenetic regulators continued to increase with time, further affecting cardiac gene expression. Our findings parallel the lower H3K4me3 and H3K9me3 levels seen in human patients. Inhibition of Jumonji demethylase activities in vivo results in lower histone demethylase enzymatic function in the heart and higher histone methylation levels and leads to partial reduction of heart size, reversal of maladaptive transcriptional programs, improved heart function, and prolonged survival. At the molecular level, target genes of transcription factor myocyte enhancer factor 2 are specifically regulated in response to pharmacological or genetic inhibition of Jumonji demethylases. Similar transcriptional reversal of disease-associated genes is seen in a second disease model based on cardiac mechanical overload. Our findings validate pharmacological inhibitors of Jumonji demethylases as potential therapeutics for the treatment of cardiomyopathies across disease models and provide evidence of the reversal of maladaptive transcriptional reprogramming leading to partial restoration of cardiac function. In addition, this study defines pathways of therapeutic resistance upregulated with disease progression.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has the characteristic accessory protein ORF8. Although clinical reports indicate that ORF8 variant strains (Δ382 and L84S variants) are less likely to cause severe illness, functional differences between wild-type and variant ORF8 are unknown. Furthermore, the physicochemical properties of the ORF8 protein have not been analyzed. In this study, the physicochemical properties of the wild-type ORF8 and its L84S variant were analyzed and compared. Using the tobacco BY-2 cell production system, which has been successfully used to produce the wild-type ORF8 protein with a single conformation, was used to successfully produce the ORF8 L84S variant protein at the same level as wild-type ORF8. The produced proteins were purified, and their temperature and pH dependencies were examined using nuclear magnetic resonance spectra. Our data suggested that the wild-type and L84S variant ORF8 structures are highly stable over a wide temperature range. Both proteins displayed an aggregated conformation at higher temperature that reverted when the temperature was decreased to room temperature. Moreover, ORF8 precipitated at acidic pH and this precipitation was reversed when the solution pH was shifted to neutral. Interestingly, the L84S variant exhibited greater solubility than wild-type ORF8 under acidic conditions. Thus, the finding indicated that conformational stability and reversibility of ORF8 are key properties related to function in oppressive environments.

Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.

Natural products generally fall into the biologically relevant chemical space and always possess novel biological activities, thus making them a rich source of lead compounds for new drug discovery. With the recent technological advances, natural product-based drug discovery is now reaching a new era. Natural products have also shown promise in epigenetic drug discovery, some of them have advanced into clinical trials or are presently being used in clinic. The histone lysine specific demethylase 1 (LSD1), an important class of histone demethylases, has fundamental roles in the development of various pathological conditions. Targeting LSD1 has been recognized as a promising therapeutic option for cancer treatment. Notably, some natural products with different chemotypes including protoberberine alkaloids, flavones, polyphenols, and cyclic peptides have shown effectiveness against LSD1. These natural products provide novel scaffolds for developing new LSD1 inhibitors. In this review, we mainly discuss the identification of natural LSD1 inhibitors, analysis of the co-crystal structures of LSD1/natural product complex, antitumor activity and their modes of action. We also briefly discuss the challenges faced in this field. We believe this review will provide a landscape of natural LSD1 inhibitors., Graphical abstract In this review, the identification of natural LSD1 inhibitors, analysis of the co-crystal structures of LSD1/natural product complex, antitumor activity, their modes of action, and the challenges faced in this field were mainly discussed. This review will provide a landscape of natural LSD1 inhibitors.Image 1

Visual Abstract, Highlights • Nitroxyl (HNO) enhances SR calcium uptake and release in cardiomyocytes, and improves myofilament calcium sensitivity without impacting L-type calcium channel or total SR calcium content. • The effects of the nitroxyl donor BMS-986231 on hemodynamics, left ventricular function, and pro-arrhythmic potential were assessed using canine models of HF. • BMS-986231 was associated with increased cardiac contractility and relaxation, as well as moderate vasodilatory effects; there was also a significant reduction in MVO2. • There were no clinically significant changes in HR, and no de novo arrhythmias were detected; BMS-986231 was also not associated with venotoxicity. • Thus, BMS-986231 has beneficial inotropic, lusitropic, and vasodilatory effects; clinical studies are ongoing., Summary The effects of the nitroxyl donor BMS-986231 on hemodynamics, left ventricular (LV) function, and pro-arrhythmic potential were assessed using canine heart failure models. BMS-986231 significantly (p < 0.05) increased LV end-systolic elastance, pre-load-recruitable stroke work, ejection fraction, stroke volume, cardiac output, ratio of early-to-late filling time integrals, and early mitral valve inflow velocity deceleration time. BMS-986231 significantly decreased LV filling pressures, end-diastolic stiffness, the time-constant of relaxation, end-diastolic wall stress, systemic vascular resistance, and myocardial oxygen consumption. BMS-986231 had little effect on heart rate and did not induce de novo arrhythmias. Thus, BMS-986231 has beneficial inotropic, lusitropic, and vasodilatory effects.

Visual Abstract, Highlights • The extent of myocardial fibrosis and the degree of isoform-expression and phosphorylation changes in cardiomyocyte titin were unknown in different hemodynamic subgroups of AS, including “paradoxical” low-flow, low-gradient AS with preserved ejection fraction. • Hemodynamic subtypes of AS were found to exhibit increased cardiac fibrosis, titin-isoform transition toward more compliant N2BA variants, and both total and site-specific titin (N2Bus) hypophosphorylation compared with donor heart controls. • A significant shift toward N2BA titin appeared in “paradoxical” AS, whereas alterations in total-titin phosphorylation and cardiac fibrosis were similar in all hemodynamic subtypes of AS, suggesting increased myocardial passive stiffness. • The unfavorable prognosis of “paradoxical” AS could be explained by the pronounced myocardial remodeling, which is no less severe than in other AS subtypes., Summary Titin-isoform expression, titin phosphorylation, and myocardial fibrosis were studied in 30 patients with severe symptomatic aortic stenosis (AS). Patients were grouped into “classical” high-gradient, normal-flow AS with preserved ejection fraction (EF); “paradoxical” low-flow, low-gradient AS with preserved EF; and AS with reduced EF. Nonfailing donor hearts served as controls. AS was associated with increased fibrosis, titin-isoform switch toward compliant N2BA, and both total and site-specific titin hypophosphorylation compared with control hearts. All AS subtypes revealed titin and matrix alterations. The extent of myocardial remodeling in “paradoxical” AS was no less severe than in other AS subtypes, thus explaining the unfavorable prognosis.

Cardiac myosin is the molecular motor that powers heart contraction by converting chemical energy from ATP hydrolysis into mechanical force. The power output of the heart is tightly regulated to meet the physiological needs of the body. Recent multiscale studies spanning from molecules to tissues have revealed complex regulatory mechanisms that fine-tune cardiac contraction, in which myosin not only generates power output but also plays an active role in its regulation. Thus, myosin is both shaped by and actively involved in shaping its mechanical environment. Moreover, these studies have shown that cardiac myosin-generated tension affects physiological processes beyond muscle contraction. Here, we review these novel regulatory mechanisms, as well as the roles that myosin-based force generation and mechanotransduction play in development and disease. We describe how key intra- and intermolecular interactions contribute to the regulation of myosin-based contractility and the role of mechanical forces in tuning myosin function. We also discuss the emergence of cardiac myosin as a drug target for diseases including heart failure, leading to the discovery of therapeutics that directly tune myosin contractility. Finally, we highlight some of the outstanding questions that must be addressed to better understand myosin's functions and regulation, and we discuss prospects for translating these discoveries into precision medicine therapeutics targeting contractility and mechanotransduction.

Embryonic and adult stem cells possess the capability of self-renewal and lineage-specific differentiation. The intricate balance between self-renewal and differentiation is governed by developmental signals and cell-type-specific gene regulatory mechanisms. A perturbed intra/extracellular environment during lineage specification could affect stem cell fate decisions resulting in pathology. Growing evidence demonstrates that metabolic pathways govern epigenetic regulation of gene expression during stem cell fate commitment through the utilization of metabolic intermediates or end products of metabolic pathways as substrates for enzymatic histone/DNA modifications. UDP-GlcNAc is one such metabolite that acts as a substrate for enzymatic mono-glycosylation of various nuclear, cytosolic, and mitochondrial proteins on serine/threonine amino acid residues, a process termed protein O-GlcNAcylation. The levels of GlcNAc inside the cells depend on the nutrient availability, especially glucose. Thus, this metabolic sensor could modulate gene expression through O-GlcNAc modification of histones or other proteins in response to metabolic fluctuations. Herein, we review evidence demonstrating how stem cells couple metabolic inputs to gene regulatory pathways through O-GlcNAc-mediated epigenetic/transcriptional regulatory mechanisms to govern self-renewal and lineage-specific differentiation programs. This review will serve as a primer for researchers seeking to better understand how O-GlcNAc influences stemness and may catalyze the discovery of new stem-cell-based therapeutic approaches.

p53 family members, p63 and p73, play a role in controlling early stage of myogenic differentiation. We demonstrated that TAp63gamma, unlike the other p53 family members, is markedly up-regulated during myogenic differentiation in murine C2C7 cell line. We also found that myotubes formation was inhibited upon TAp63gamma knock-down, as also indicated by atrophyic myotubes and reduction of myoblasts fusion index. Analysis of TAp63gamma-dependend transcripts identified several target genes involved in skeletal muscle contractility energy metabolism, myogenesis and skeletal muscle autocrine signaling. These results indicate that TAp63gamma is a late marker of myogenic differentiation and, by controlling different sub-sets of target genes, it possibly contributes to muscle growth, remodeling, functional differentiation and tissue homeostasis.

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative Gi-coupled C1P receptor as pertussis toxin, which maintains Gi protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury., Highlights ► In this study we show that C1P stimulates C2C12 myoblast proliferation. ► C1P does not affect myogenic differentiation or survival of myoblasts. ► The mitogenic action of C1P is mediated by PI3K/Akt, ERK1/2 and mTOR activation. ► C1P effects are pertussis toxin insensitive.

Hypoxic injury to cardiomyocytes is a stress that causes cardiac pathology through cardiac-restricted gene expression. SRF (serum-response factor) and myocardin are important for cardiomyocyte growth and differentiation in response to myocardial injuries. Previous studies have indicated that AngII (angiotensin II) stimulates both myocardin expression and cardiomyocyte hypertrophy. In the present study, we evaluated the expression of myocardin and AngII after hypoxia in regulating gene transcription in neonatal cardiomyocytes. Cultured rat neonatal cardiomyocytes were subjected to hypoxia, and the expression of myocardin and AngII were evaluated. Different signal transduction pathway inhibitors were used to identify the pathway(s) responsible for myocardin expression. An EMSA (electrophoretic mobility-shift assay) was used to identify myocardin/SRF binding, and a luciferase assay was used to identify transcriptional activity of myocardin/SRF in neonatal cardiomyocytes. Both myocardin and AngII expression increased after hypoxia, with AngII appearing at an earlier time point than myocardin. Myocardin expression was stimulated by AngII and ERK (extracellular-signal-regulated kinase) phosphorylation, but was suppressed by an ARB (AngII type 1 receptor blocker), an ERK pathway inhibitor and myocardin siRNA (small interfering RNA). AngII increased both myocardin expression and transcription in neonatal cardiomyocytes. Binding of myocardin/SRF was identified using an EMSA, and a luciferase assay indicated the transcription of myocardin/SRF in neonatal cardiomyocytes. Increased BNP (B-type natriuretic peptide), MHC (myosin heavy chain) and [3H]proline incorporation into cardiomyocytes was identified after hypoxia with the presence of myocardin in hypertrophic cardiomyocytes. In conclusion, hypoxia in cardiomyocytes increased myocardin expression, which is mediated by the induction of AngII and the ERK pathway, to cause cardiomyocyte hypertrophy. Myocardial hypertrophy was identified as an increase in transcriptional activities, elevated hypertrophic and cardiomyocyte phenotype markers, and morphological hypertrophic changes in cardiomyocytes.

Summary The present study tested the hypothesis that thrombin participates in formation of left atrial remodeling and that direct oral anticoagulants, such as direct thrombin inhibitors (DTIs), can prevent its progression. In a rat model of heart failure associated with left atrial dilation, we found that chronic treatment with DTIs reduces the atrial remodeling and the duration of atrial fibrillation (AF) episodes induced by burst pacing by inhibiting myocardial hypertrophy and fibrosis. In addition to the prevention of thromboembolism complicating AF, DTIs may be of interest to slow down the progression of the arrhythmogenic substrate., Visual Abstract, Highlights • Oral direct anticoagulants such as DTIs, but not vitamin K antagonist, diminish the progression of atrial dilation associated with heart failure in a rat model. • Prevention of atrial dilation by DTI is associated with decreased duration of atrial arrhythmia induced by burst pacing. • DTI inhibits interstitial fibrosis, extracellular matrix remodeling, and hypertrophy of atrial myocardium. • Plasma DTI concentrations inhibiting atrial remodeling are lower than those required for therapeutic anticoagulant activity. • DTIs may be of interest to prevent the progression of atrial fibrillation substrate in addition to their anticoagulant activity.

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as beta-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin-ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant K(D)) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (K(AD)/K(D) approximately 5 instead of K(AD)/K(D) approximately 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s(-1)) than reported previously (138 s(-1)). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.

Disturbances in acid-base balance, such as acidosis and alkalosis, have potential to alter the pharmacologic and toxicologic outcomes of statin therapy. Statins are commonly prescribed for elderly patients who have multiple comorbidities such as diabetes mellitus, cardiovascular, and renal diseases. These patients are at risk of developing acid-base imbalance. In the present study, the effect of disturbances in acid-base balance on the interconversion of simvastatin and pravastatin between lactone and hydroxy acid forms have been investigated in physiological buffers, human plasma, and cell culture medium over pH ranging from 6.8–7.8. The effects of such interconversion on cellular uptake and myotoxicity of statins were assessed in vitro using C2C12 skeletal muscle cells under conditions relevant to acidosis, alkalosis, and physiological pH. Results indicate that the conversion of the lactone forms of simvastatin and pravastatin to the corresponding hydroxy acid is strongly pH dependent. At physiological and alkaline pH, substantial proportions of simvastatin lactone (SVL; ∼87% and 99%, respectively) and pravastatin lactone (PVL; ∼98% and 99%, respectively) were converted to the active hydroxy acid forms after 24 hours of incubation at 37°C. At acidic pH, conversion occurs to a lower extent, resulting in greater proportion of statin remaining in the more lipophilic lactone form. However, pH alteration did not influence the conversion of the hydroxy acid forms of simvastatin and pravastatin to the corresponding lactones. Furthermore, acidosis has been shown to hinder the metabolism of the lactone form of statins by inhibiting hepatic microsomal enzyme activities. Lipophilic SVL was found to be more cytotoxic to undifferentiated and differentiated skeletal muscle cells compared with more hydrophilic simvastatin hydroxy acid, PVL, and pravastatin hydroxy acid. Enhanced cytotoxicity of statins was observed under acidic conditions and is attributed to increased cellular uptake of the more lipophilic lactone or unionized hydroxy acid form. Consequently, our results suggest that comorbidities associated with acid-base imbalance can play a substantial role in the development and potentiation of statin-induced myotoxicity.

Highlights • Myostatin signals via SMADs to promote muscle wasting. • FHL1 normally promotes hypertrophy but can activate SMAD signalling. • FHL1 promoted myostatin signalling in vitro. • FHL1 promoted hypertrophy in the absence of myostatin but atrophy in its presence., Myostatin is a TGFβ family ligand that reduces muscle mass. In cancer cells, TGFβ signalling is increased by the protein FHL1. Consequently, FHL1 may promote signalling by myostatin. We therefore tested the ability of FHL1 to regulate myostatin function. FHL1 increased the myostatin activity on a SMAD reporter and increased myostatin dependent myotube wasting. In mice, independent expression of myostatin reduced fibre diameter whereas FHL1 increased fibre diameter, both consistent with previously identified effects of these proteins. However, co-expression of FHL1 and myostatin reduced fibre diameter to a greater extent than myostatin alone. Together, these data suggest that the expression of FHL1 may exacerbate muscle wasting under the appropriate conditions.