Sarcomeric Organization Research Articles

In vitro cardiac modeling of desmin-related myofibrillar myopathy and deep learning-based image analysis to develop a high-content screening assay

Mutations of DES, the gene encoding desmin, the main intermediate filament of muscle cells, are one of the major cause of Myofibrillar myopathy(MFM). This disease is mainly characterized by a muscular dystrophy but also, in many cases, by the progressive emergence of cardiac dysfunctions evolving frequently from dilated cardiomyopathy to heart failure. For the moment, no specific treatment can revert this disease. The following study relied on the development of an automated tool base on deep-learning to discriminate efficiently mutant and control cardiomyocytes in the perspective of drug screening. We use cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) obtained from control or patients carrying a DES mutation (E439K). Phenotyping of hiPSC-CMs was performed by immunocytostaining of the sarcomeres or the desmin network. After imaging, the morphological perturbations were evaluated manually based either on the sarcomeric organization or on the aspect of the desmin network at single cell level. Then an automated classification model was generated by using machine-learning method trained with the previous images annotated with the results of the manual classifications. In parallel, another automated classification model based on cell profiling from sarcomeric, desmin and mitochondrial staining by using the deep learning EfficientNet B0 model was setting up in order to distinguish Control cell and DES mutated cell. Manual classification based on sarcomeric organization was established on 4 classes ranging from cells with disorganized sarcomeres to cells with a well-organized sarcomeric network on 1500 single cells. We observed that the distribution of DES mutated cells is shifted toward the class of disorganized sarcomere compared to control cells. Identically, we also manually classified 1200 single cells based on the organization of the desmin network (4 classes). We also observed a shift of DES mutant cells to be classified as cells with aggregated desmin network compared to control cells. Finally, the various validation tests of the two automated classification methods showed significant correlation with the manual classification methods. Here we demonstrate the efficacy of our automated classification software to be able to discriminates mutant and control cells. This analysis platform will then be used to perform high throughput drug screening assays to discover new therapeutic molecules.

Electrophysiological Properties of Tetraploid Cardiomyocytes Derived from Murine Pluripotent Stem Cells Generated by Fusion of Adult Somatic Cells with Embryonic Stem Cells.

Most cardiomyocytes (CMs) in the adult mammalian heart are either binucleated or contain a single polyploid nucleus. Recent studies have shown that polyploidy in CMs plays an important role as an adaptive response to physiological demands and environmental stress and correlates with poor cardiac regenerative ability after injury. However, knowledge about the functional properties of polyploid CMs is limited. In this study, we generated tetraploid pluripotent stem cells (PSCs) by fusion of murine embryonic stem cells (ESCs) and somatic cells isolated from bone marrow or spleen and performed a comparative analysis of the electrophysiological properties of tetraploid fusion-derived PSCs and diploid ESC-derived CMs. Fusion-derived PSCs exhibited characteristics of genuine ESCs and contained a near-tetraploid genome. Ploidy features and marker expression were also retained during the differentiation of fusion-derived cells. Fusion-derived PSCs gave rise to CMs, which were similar to their diploid ESC counterparts in terms of their expression of typical cardiospecific markers, sarcomeric organization, action potential parameters, response to pharmacologic stimulation with various drugs, and expression of functional ion channels. These results suggest that the state of ploidy does not significantly affect the structural and electrophysiological properties of murine PSC-derived CMs. These results extend our knowledge of the functional properties of polyploid CMs and contribute to a better understanding of their biological role in the adult heart.

HSPB8 frameshift mutant aggregates weaken chaperone-assisted selective autophagy in neuromyopathies

ABSTRACT Chaperone-assisted selective autophagy (CASA) is a highly selective pathway for the disposal of misfolding and aggregating proteins. In muscle, CASA assures muscle integrity by favoring the turnover of structural components damaged by mechanical strain. In neurons, CASA promotes the removal of aggregating substrates. A crucial player of CASA is HSPB8 (heat shock protein family B (small) member 8), which acts in a complex with HSPA, their cochaperone BAG3, and the E3 ubiquitin ligase STUB1. Recently, four novel HSPB8 frameshift (fs) gene mutations have been linked to neuromyopathies, and encode carboxy-terminally mutated HSPB8, sharing a common C-terminal extension. Here, we analyzed the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. We demonstrated that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Notably, all HSPB8 frameshift mutants retain their ability to interact with CASA members but sequester them into the HSPB8-positive aggregates together with two autophagy receptors SQSTM1/p62 and TAX1BP1. This copartitioning process negatively affects the CASA capability to remove its clients and causes a general failure in proteostasis response. Further analyses revealed that the aggregation of the HSPB8_fs mutants occurs independently of the other CASA members or from the autophagy receptors interaction, but it is an intrinsic feature of the mutated amino acid sequence. HSPB8_fs mutants aggregation alters the differentiation capacity of muscle cells and impairs sarcomere organization. Collectively, these results shed light on a potential pathogenic mechanism shared by the HSPB8_fs mutants described in neuromuscular diseases. Abbreviations: ACD: α-crystallin domain; ACTN: actinin alpha; BAG3: BAG cochaperone 3; C: carboxy; CASA: chaperone-assisted selective autophagy; CE: carboxy-terminal extension; CLEM: correlative light and electron microscopy; CMT2L: Charcot-Marie-Tooth type 2L; CTR: carboxy-terminal region; dHMNII: distal hereditary motor neuropathy type II; EV: empty vector; FRA: filter retardation assay; fs: frameshift; HSPA/HSP70: heat shock protein family A (Hsp70); HSPB1/Hsp27: heat shock protein family B (small) member 1; HSPB8/Hsp22: heat shock protein family B (small) member 8; HTT: huntingtin; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MD: molecular dynamics; MTOC: microtubule organizing center; MYH: myosin heavy chain; MYOG: myogenin; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; NSC34: Neuroblastoma X Spinal Cord 34; OPTN: optineurin; polyQ: polyglutamine; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1: Tax1 binding protein 1; TUBA: tubulin alpha; WT: wild-type.

Effect of Ethanolic Extract of Vernonia amygdalina on the Proliferation, Viability and Function of Mouse Induced Pluripotent Stem Cells and Cardiomyocytes

Vernonia amygdalina (V. amygdalina) leaves are commonly used in traditional medicine around the world for the treatment of a plethora disorders, including heart disease. The aim of this study was to examine and evaluate the cardiac effect of V. amygdalina leaf extracts using mouse induced pluripotent stem cells (miPSCs) and their cardiomyocytes' (CMs) derivatives. We used a well-established stem cell culture to assess the effect of V. amygdalina extract on miPSC proliferation, EB formation and the beating activity of miPS cell-derived CMs. To study the cytotoxic effect of our extract, undifferentiating miPSCs were exposed to different concentrations of V. amygdalina. Cell colony formation and EB morphology were assessed using microscopy, whereas the cell viability was accessed with an impedance-based method and immunocytochemistry following treatment with different concentrations of V. amygdalina. Ethanolic extract of V. amygdalina induced toxicity in miPSCs, as revealed by a decrease in cell proliferation and colony formation, and an increase in cell death at a concentration of ≥20 mg/mL. At a concentration of 10 mg/mL, the rate of beating EBs was observed with no significant difference regarding the yield of cardiac cells. In addition, V. amygdalina did not affect the sarcomeric organization, but induced positive or negative effects on miPS cell-derived CMs' differentiation in a concentration-dependent manner. Taken together, our findings demonstrate that the ethanolic extract of V. amygdalina affected cell proliferation, colony forming and cardiac beating capacities in a concentration-dependent manner.

Effect of 11-Deoxycorticosterone in the Transcriptomic Response to Stress in Rainbow Trout Skeletal Muscle.

In aquaculture, many stressors can negatively affect growth in teleosts. It is believed that cortisol performs glucocorticoid and mineralocorticoid functions because teleosts do not synthesize aldosterone. However, recent data suggest that 11-deoxycorticosterone (DOC) released during stress events may be relevant to modulate the compensatory response. To understand how DOC modifies the skeletal muscle molecular response, we carried out a transcriptomic analysis. Rainbow trout (Oncorhynchus mykiss) were intraperitoneally treated with physiological doses of DOC in individuals pretreated with mifepristone (glucocorticoid receptor antagonist) or eplerenone (mineralocorticoid receptor antagonist). RNA was extracted from the skeletal muscles, and cDNA libraries were constructed from vehicle, DOC, mifepristone, mifepristone plus DOC, eplerenone, and eplerenone plus DOC groups. The RNA-seq analysis revealed 131 differentially expressed transcripts (DETs) induced by DOC with respect to the vehicle group, mainly associated with muscle contraction, sarcomere organization, and cell adhesion. In addition, a DOC versus mifepristone plus DOC analysis revealed 122 DETs related to muscle contraction, sarcomere organization, and skeletal muscle cell differentiation. In a DOC versus eplerenone plus DOC analysis, 133 DETs were associated with autophagosome assembly, circadian regulation of gene expression, and regulation of transcription from RNA pol II promoter. These analyses indicate that DOC has a relevant function in the stress response of skeletal muscles, whose action is differentially modulated by GR and MR and is complementary to cortisol.

Sarcomere dynamics simulations to uncover mechanisms in hypertrophic cardiomyopathy.

Mutations in the beta myosin heavy chain (MYH7) can cause hypercontractility and cardiac remodeling in hypertrophic cardiomyopathy (HCM), but the mechanisms of specific mutations can be difficult to predict. Many mutations change intrinsic rates of myosin activity and their force sensitivity, which can dynamically alter chemo-mechanical cycle events and resultant force generation during cardiac contraction. Our team's multiscale approach enables measurement of the kinetics and force generation in purified myosin proteins, isolated myofibrils, and single and multicellular human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) platforms. Computational modeling can integrate results across these scales and provide additional insights into the individual and combinatorial effects of different changes in myosin kinetics. We have previously used a continuum model to highlight the significance of dysregulation of the super relaxed state of myosin driving hypercontractility in hiPSC-CMs with the P710R mutation. Sensitivity analysis also supported variable myofibril density and sarcomeric organization as a source of population heterogeneity in the force generating capacity of individual hiPSC-CMs. In our ongoing studies, we are leveraging computational modeling to compare the predicted and measured effects of HCM mutations in our different experiments. We have incorporated the measured changes in unloaded myosin to predict force generation after isometric maximal calcium activation (myofibril measurements) and twitch stimulation in single hiPSC-CMs on gels of physiologic stiffness. Additional simulations with spatially explicit models enable more direct prediction of the impact of heterogeneous mixtures of myosins with different molecular profiles and on cooperativity effects associated with the transmission of tension along the length of a sarcomere. Finally, we have visualized the strain profiles of individual sarcomeres in contracting myofibrils in live cells and are correlating these measurements to our simulations. These studies will give new insights into the key biophysical drivers of HCM.

Alpha kinase 3 signaling at the M-band maintains sarcomere integrity and proteostasis in striated muscle

Muscle contraction is driven by the molecular machinery of the sarcomere. As phosphorylation is a critical regulator of muscle function, the identification of regulatory kinases is important for understanding sarcomere biology. Pathogenic variants in alpha kinase 3 (ALPK3) cause cardiomyopathy and musculoskeletal disease, but little is known about this atypical kinase. Here we show that ALPK3 is an essential component of the M-band of the sarcomere and define the ALPK3-dependent phosphoproteome. ALPK3 deficiency impaired contractility both in human cardiac organoids and in the hearts of mice harboring a pathogenic truncating Alpk3 variant. ALPK3-dependent phosphopeptides were enriched for sarcomeric components of the M-band and the ubiquitin-binding protein sequestosome-1 (SQSTM1) (also known as p62). Analysis of the ALPK3 interactome confirmed binding to M-band proteins including SQSTM1. In human pluripotent stem cell-derived cardiomyocytes modeling cardiomyopathic ALPK3 mutations, sarcomeric organization and M-band localization of SQSTM1 were abnormal suggesting that this mechanism may underly disease pathogenesis.

The polyamine spermidine enhances contractile function and sarcomere dynamics post-doxorubicin treatment.

Doxorubicin (DOX) is a chemotherapy used to treat childhood and adult malignancies. However, despite being a cornerstone for oncological care, the use of DOX is limited due to dose-dependent cardiotoxicity, which can lead to contractile dysfunction and heart failure. The exact mechanism of DOX-induced cardiotoxicity is not fully understood. However, proposed mechanisms include sarcomere degradation and impairment of autophagy. To determine whether enhancing autophagy can restore contractile function, we treated µ-patterned single-cell human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), displaying sarcomere disarray and reduced contractile force post-DOX treatment with spermidine, an autophagy activator. The contractile force was quantified using traction force microscopy. There were five conditions (untreated control, DOX, maintenance medium post-DOX, spermidine post-DOX, and spermidine-only). We found that hiPSC-CMs treated with spermidine post-DOX produce 67% of the contractile force generated by control cells (0.22 µN vs. 0.33 µN, not significant) and significantly (0.22 µN vs. 0.11 µN, p < 0.0001) higher contractile force compared to cells in maintenance medium post-DOX. In the spermidine-only group, our results demonstrate that spermidine significantly (0.57 µN vs. 0.33 µN, p < 0.0001) increases contractile force compared to control. Spermidine-only treated cells were also bigger (spread area = ∼1716 µm2 vs. ∼1353 µm2, p < 0.00001) than the control and had a more robust and organized sarcomere network. Preliminary qPCR results suggest that spermidine upregulates genes that control sarcomere function (e.g., MYH7) and organization (e.g., ANKD1). These data demonstrate that spermidine can enhance contractile force post-DOX treatment and increase hiPSC-CMs size and sarcomere network. The data further suggests that sarcomere homeostasis is one of the mechanisms spermidine regulates in hiPSC-CMs. This work opens the door for developing therapies that target autophagy and sarcomere proteostasis to treat cardiac dysfunction.

Genome-Wide Analysis of Left Ventricular Maximum Wall Thickness in the UK Biobank Cohort Reveals a Shared Genetic Background With Hypertrophic Cardiomyopathy.

Left ventricular maximum wall thickness (LVMWT) is an important biomarker of left ventricular hypertrophy and provides diagnostic and prognostic information in hypertrophic cardiomyopathy (HCM). Limited information is available on the genetic determinants of LVMWT. We performed a genome-wide association study of LVMWT measured from the cardiovascular magnetic resonance examinations of 42 176 European individuals. We evaluated the genetic relationship between LVMWT and HCM by performing pairwise analysis using the data from the Hypertrophic Cardiomyopathy Registry in which the controls were randomly selected from UK Biobank individuals not included in the cardiovascular magnetic resonance sub-study. Twenty-one genetic loci were discovered at P<5×10-8. Several novel candidate genes were identified including PROX1, PXN, and PTK2, with known functional roles in myocardial growth and sarcomere organization. The LVMWT genetic risk score is predictive of HCM in the Hypertrophic Cardiomyopathy Registry (odds ratio per SD: 1.18 [95% CI, 1.13-1.23]) with pairwise analyses demonstrating a moderate genetic correlation (rg=0.53) and substantial loci overlap (19/21). Our findings provide novel insights into the genetic underpinning of LVMWT and highlight its shared genetic background with HCM, supporting future endeavours to elucidate the genetic etiology of HCM.

Age-dependent Lamin changes induce cardiac dysfunction via dysregulation of cardiac transcriptional programs.

As we age, structural changes contribute to progressive decline in organ function, which in the heart act through poorly characterized mechanisms. Taking advantage of the short lifespan and conserved cardiac proteome of the fruit fly, we found that cardiomyocytes exhibit progressive loss of Lamin C (mammalian Lamin A/C homologue) with age, coincident with decreasing nuclear size and increasing nuclear stiffness. Premature genetic reduction of Lamin C phenocopies aging's effects on the nucleus, and subsequently decreases heart contractility and sarcomere organization. Surprisingly, Lamin C reduction downregulates myogenic transcription factors and cytoskeletal regulators, possibly via reduced chromatin accessibility. Subsequently, we find a role for cardiac transcription factors in regulating adult heart contractility and show that maintenance of Lamin C, and cardiac transcription factor expression, prevents age-dependent cardiac decline. Our findings are conserved in aged non-human primates and mice, demonstrating that age-dependent nuclear remodeling is a major mechanism contributing to cardiac dysfunction.

Automated Sarcomere Structure Analysis for Studying Cardiotoxicity in Human Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract Drug-induced cardiotoxicity is one of the main causes of heart failure (HF), a worldwide major and growing public health issue. Extensive research on cardiomyocytes has shown that two crucial features of the mechanisms involved in HF are the disruption of striated sarcomeric organization and myofibril deterioration. However, most studies that worked on extracting these sarcomere features have only focused on animal models rather than the more representative human pluripotent stem cells (hPSCs). Currently, there are limited established image analysis systems to specifically assess and quantify the sarcomeric organization of hPSC-derived cardiomyocytes (hPSC-CMs). Here, we report a fully automated and robust image analysis pipeline to detect z-lines and myofibrils from hPSC-CMs with a high-throughput live-imaging setup. Phenotype measurements were further quantified to evaluate the cardiotoxic effect of the anticancer drug Doxorubicin. Our findings show that this pipeline is able to capture z-lines and myofibrils. The pipeline filters out disrupted sarcomere structures and irrelevant noisy signals, which allows us to perform automated high-throughput imaging for accurate quantification of cardiomyocyte injury.

Three-dimensional heart extracellular matrix enhances chemically induced direct cardiac reprogramming

Direct cardiac reprogramming has emerged as a promising therapeutic approach for cardiac regeneration. Full chemical reprogramming with small molecules to generate cardiomyocytes may be more amenable than genetic reprogramming for clinical applications as it avoids safety concerns associated with genetic manipulations. However, challenges remain regarding low conversion efficiency and incomplete cardiomyocyte maturation. Furthermore, the therapeutic potential of chemically induced cardiomyocytes (CiCMs) has not been investigated. Here, we report that a three-dimensional microenvironment reconstituted with decellularized heart extracellular matrix can enhance chemical reprogramming and cardiac maturation of fibroblasts to cardiomyocytes. The resultant CiCMs exhibit elevated cardiac marker expression, sarcomeric organization, and improved electrophysiological features and drug responses. We investigated the therapeutic potential of CiCMs reprogrammed in three-dimensional heart extracellular matrix in a rat model of myocardial infarction. Our platform can facilitate the use of CiCMs for regenerative medicine, disease modeling, and drug screening.

Differential gene expression analysis in the scallop Argopecten purpuratus exposed to altered pH and temperature conditions in an upwelling-influenced farming area

Increased carbon dioxide in the atmosphere and its absorption across the ocean surface will alter natural variations in pH and temperature levels, occurring in coastal upwelling ecosystems. The scallop Argopecten purpuratus, one of the most economically important species farmed in northern Chile, has been shown to be vulnerable to these environmental drivers. However, the regulatory responses at the gene-level of scallops to these climate stressors remain almost unknown. Consequently, we used an orthogonal experimental design and RNAseq approach to analyze the acute effects of variability in pH and temperature on gene expression in the muscle tissue of A. purpuratus. In respect to control conditions (pH ~ 8.0/ 14 °C), the influence of low pH (~ 7.7) and temperature (14 °C) induced the activation of several genes associated with apoptotic signaling pathways and protein localization to plasma membrane. Elevated temperature (18 °C) and pH (~8.0) conditions increased the expression of transcripts associated with the activation of muscle contraction, regulation, and sarcomere organization effects on muscle tissue. In scallops exposed to low pH and elevated temperature, the genes expressed were differentially associated with the oxidation-reduction process, signal translation, and positive regulation of GTPase activity. These results indicated that the differentially expressed genes under the experimental conditions tested are mainly related to the mitigation of cellular damage and homeostasis control. Our results add knowledge about the function of the adductor muscle in response to stressors in scallops. Furthermore, these results could help in the identification of molecular biomarkers of stress necessary to be integrated into the aquaculture programs for the mitigation of climate change.

Inducible deletion of raptor and mTOR from adult skeletal muscle impairs muscle contractility and relaxation.

Skeletal muscle weakness has been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and is accompanied by altered mammalian target of rapamycin (mTOR) signalling. We wanted to elucidate the functional role of mTOR in muscle contractility. Most loss-of-function studies for mTOR signalling have used the drug rapamycin to inhibit some of the signalling downstream of mTOR. However, given that rapamycin does not inhibit all mTOR signalling completely, we generated a double knockout for mTOR and for the scaffold protein of mTORC1, raptor, in skeletal muscle. We found that double knockout in mice results in a more severe phenotype compared with deletion of raptor or mTOR alone. Indeed, these animals display muscle weakness, increased fibre denervation and a slower muscle relaxation following tetanic stimulation. This is accompanied by a shift towards slow-twitch fibres and changes in the expression levels of calcium-related genes, such as Serca1 and Casq1. Double knockout mice show a decrease in calcium decay kinetics after tetanus in vivo, suggestive of a reduced calcium reuptake. In addition, RNA sequencing analysis revealed that many downregulated genes, such as Tcap and Fhod3, are linked to sarcomere organization. These results suggest a key role for mTOR signalling in maintaining proper fibre relaxation in skeletal muscle. KEY POINTS: Skeletal muscle wasting and weakness have been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and are accompanied by altered mammalian target of rapamycin (mTOR) signalling. Mammalian target of rapamycin plays a crucial role in the maintenance of muscle mass and functionality. We found that the loss of both mTOR and raptor results in contractile abnormalities, with severe muscle weakness and delayed relaxation following tetanic stimulation. These results are associated with alterations in the expression of genes involved in sarcomere organization and calcium handling and with an impairment in calcium reuptake after contraction. Taken together, these results provide a mechanistic insight into the role of mTOR in muscle contractility.

Abstract 14213: The Polyamine Spermidine Enhances the Contractile Force of μ-Patterned Single-Cell Stem-Cell-Derived Cardiomyocytes Post Doxorubicin Treatment

Doxorubicin (DOX) is a chemotherapy used to treat childhood and adult malignancies, including pediatric leukemia and breast cancer. However, despite being a cornerstone for oncological care, the use of DOX is limited due to dose-dependent cardiotoxicity, which can lead to contractile dysfunction and heart failure. Yet, Dox is still widely used due to the lack of suitable replacements. The exact mechanism of Dox-induced cardiotoxicity is not fully understood. However, proposed mechanisms include sarcomere degradation and impairment of autophagy. To determine whether enhancing autophagy can restore contractile function, we treated μ-patterned single-cell human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), displaying sarcomere disarray and reduced contractile force post-Dox treatment with spermidine, an autophagy activator. The contractile force was quantified using traction force microscopy. There were five conditions (untreated control, Dox, maintenance medium post-Dox, spermidine post-Dox, and spermidine-only). We found that hiPSC-CMs treated with spermidine post-Dox produce 67% of the contractile force generated by control cells (.22 μN vs. .33 μN, not significant) and significantly (.22 μN vs. .11 μN, p &lt; .0001) higher contractile force compared to cells in maintenance medium post-Dox. In the spermidine-only group, our results demonstrate that spermidine significantly (.57 μN vs. .33 μN, p &lt; .0001) increases contractile force compared to control. Spermidine-only treated cells were also bigger (spread area = ~1716 μm 2 vs. ~1353 μm 2 , p &lt; .00001) than the control and had a more robust and organized sarcomere network. Preliminary qPCR results suggest that spermidine upregulates genes that control sarcomere function (e.g., MYH7) and organization (e.g., ANKD1). These data demonstrate that spermidine can enhance contractile force post-Dox treatment and increase cellular size and sarcomere network in hiPSC-CMs. The data further suggests that sarcomere homeostasis is one of the cellular mechanisms spermidine regulates in hiPSC-CMs. This work opens the door for developing therapies that target autophagy and sarcomere proteostasis to treat cardiac dysfunction.

SENP7 deSUMOylase-governed transcriptional program coordinates sarcomere assembly and is targeted in muscle atrophy.

Disorganization of the basic contractile unit of muscle cells, i.e., the sarcomeres, leads to suboptimal forcegeneration and is a hallmark of muscle atrophy. Here, we demonstrate that the nuclear role of SENP7 deSUMOylase is pivotal for sarcomere organization. SENP7 expression is temporally upregulated in mature muscle cells and directly regulates transcription of the myosin heavy chain (MyHC-IId) gene. We identify SENP7-dependent deSUMOylation of flightless-1 (Fli-I) as a signal for Fli-I association with scaffold attachment factor b1 (Safb1). SENP7 deficiency leads to higher Fli-I SUMOylation and lower chromatin residency of Safb1, thus generating transcriptionally incompetent chromatin conformation on MyHC-IId. Consequently, lower expression of MyHC-IId causes sarcomere disorganization and disrupted muscle cell contraction. Remarkably, cachexia signaling impedes the SENP7-governed transcriptional program, leading to muscle atrophy, with profound loss of motor protein MyHC-IId. We propose a SENP7-driven distinct transcription program as paramount for muscle cell function, which was found targeted in cachexia.

Transcriptome Response of Differentiating Muscle Satellite Cells to Thermal Challenge in Commercial Turkey.

Early muscle development involves the proliferation and differentiation of stem cells (satellite cells, SCs) in the mesoderm to form multinucleated myotubes that mature into muscle fibers and fiber bundles. Proliferation of SCs increases the number of cells available for muscle formation while simultaneously maintaining a population of cells for future response. Differentiation dramatically changes properties of the SCs and environmental stressors can have long lasting effects on muscle growth and physiology. This study was designed to characterize transcriptional changes induced in turkey SCs undergoing differentiation under thermal challenge. Satellite cells from the pectoralis major (p. major) muscle of 1-wk old commercial fast-growing birds (Nicholas turkey, NCT) and from a slower-growing research line (Randombred Control Line 2, RBC2) were proliferated for 72 h at 38 °C and then differentiated for 48 h at 33 °C (cold), 43 °C (hot) or 38 °C (control). Gene expression among thermal treatments and between turkey lines was examined by RNAseq to detect significant differentially expressed genes (DEGs). Cold treatment resulted in significant gene expression changes in the SCs from both turkey lines, with the primary effect being down regulation of the DEGs with overrepresentation of genes involved in regulation of skeletal muscle tissue regeneration and sarcomere organization. Heat stress increased expression of genes reported to regulate myoblast differentiation and survival and to promote cell adhesion particularly in the NCT line. Results suggest that growth selection in turkeys has altered the developmental potential of SCs in commercial birds to increase hypertrophic potential of the p. major muscle and sarcomere assembly. The biology of SCs may account for the distinctly different outcomes in response to thermal challenge on breast muscle growth, development, and structure of the turkey.

I.09 Induced pluripotent stem cells for modeling neuromuscular disorders: development of disease-specific assays, live cells functional testing and drug design

Induced pluripotent stem cells (iPSC) represent an innovative, easily obtained and renewable stem cell source. This somatic cell-derived model that can be derived without considerable ethical issues has generated significant enthusiasm for its potential application in basic and translational research or development of new therapeutics. In particular, patient-specific iPS-derived tissues and latterly, organoids offer attractive platform to model a wide range of diseases but also personalized or regenerative medicine. Compared to other tissues, only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human iPSCs (hiPSCs). We developed a procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization and formation of neuromuscular junction. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be maintained and regenerates on the long-term. These in vitro muscle constructs can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling and drug discovery. Our “muscle in a dish” model has been used to successfully model a number of neuromuscular disorders. By combining transcriptomics with artificial intelligence, we were able to design specific pipelines for a comprehensive understanding of the molecular, cell biological and functional properties of hiPSC-derived muscle tissue in diseases. Overall, this strategy opens a wide rage of opportunities for disease modeling, personalized medicine for patients in diagnosis deadlocks, drug design or tissue bioengineering and bioprinting. Induced pluripotent stem cells (iPSC) represent an innovative, easily obtained and renewable stem cell source. This somatic cell-derived model that can be derived without considerable ethical issues has generated significant enthusiasm for its potential application in basic and translational research or development of new therapeutics. In particular, patient-specific iPS-derived tissues and latterly, organoids offer attractive platform to model a wide range of diseases but also personalized or regenerative medicine. Compared to other tissues, only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human iPSCs (hiPSCs). We developed a procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization and formation of neuromuscular junction. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be maintained and regenerates on the long-term. These in vitro muscle constructs can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling and drug discovery. Our “muscle in a dish” model has been used to successfully model a number of neuromuscular disorders. By combining transcriptomics with artificial intelligence, we were able to design specific pipelines for a comprehensive understanding of the molecular, cell biological and functional properties of hiPSC-derived muscle tissue in diseases. Overall, this strategy opens a wide rage of opportunities for disease modeling, personalized medicine for patients in diagnosis deadlocks, drug design or tissue bioengineering and bioprinting.

Promoting maturation and contractile function of neonatal rat cardiomyocytes by self-powered implantable triboelectric nanogenerator

Cardiomyocyte-based therapeutic strategy is a promising approach to treat myocardial injury; however, the prognostic power of this approach is currently limited by the immaturity of cardiomyocytes. Here, a flexible self-powered implantable electrical stimulator based on the triboelectric nanogenerator (TENG) was proposed to induce the maturation of cardiomyocytes by generating an electric field on the interdigitated electrode. The results showed that the TENG-based self-powered stimulator significantly promoted the maturation of neonatal rat cardiomyocytes (NRCMs) in vitro by increasing the expression of connexin 43, α-actinin, and c-troponin T. In addition, electrical stimulation also improved sarcomere organization and fracture formation, and significantly increased the intracellular Ca2+ levels, Ca2+ transient rate, and Ca2+ peak amplitudes of cardiomyocytes. TENG was also shown to be driven by the breath of rats and the heartbeat of rabbits, suggesting it could be used as an implantable medical electronic device for electrically promoting the maturation of neonatal cardiomyocytes. This work develops a TENG-based self-powered implantable medical device, which provides important technical support for clinical treatment of myocardial defects and restoration of the physiological function of cardiac tissue.

Investigating and Resolving Cardiotoxicity Induced by COVID‐19 Treatments using Human Pluripotent Stem Cell‐Derived Cardiomyocytes and Engineered Heart Tissues

Coronavirus disease 2019 continues to spread worldwide. Given the urgent need for effective treatments, many clinical trials are ongoing through repurposing approved drugs. However, clinical data regarding the cardiotoxicity of these drugs are limited. Human pluripotent stem cell‐derived cardiomyocytes (hCMs) represent a powerful tool for assessing drug‐induced cardiotoxicity. Here, by using hCMs, it is demonstrated that four antiviral drugs, namely, apilimod, remdesivir, ritonavir, and lopinavir, exhibit cardiotoxicity in terms of inducing cell death, sarcomere disarray, and dysregulation of calcium handling and contraction, at clinically relevant concentrations. Human engineered heart tissue (hEHT) model is used to further evaluate the cardiotoxic effects of these drugs and it is found that they weaken hEHT contractile function. RNA‐seq analysis reveals that the expression of genes that regulate cardiomyocyte function, such as sarcomere organization (TNNT2, MYH6) and ion homeostasis (ATP2A2, HCN4), is significantly altered after drug treatments. Using high‐throughput screening of approved drugs, it is found that ceftiofur hydrochloride, astaxanthin, and quetiapine fumarate can ameliorate the cardiotoxicity of remdesivir, with astaxanthin being the most prominent one. These results warrant caution and careful monitoring when prescribing these therapies in patients and provide drug candidates to limit remdesivir‐induced cardiotoxicity.