Myofibril Organization Research Articles

Histone demethylase KDM5 regulates cardiomyocyte maturation by promoting fatty acid oxidation, oxidative phosphorylation, and myofibrillar organization.

Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs. KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.

Abstract PR16: Neutrophil-intrinsic tumor necrosis factor (TNF) is a novel driver of T-cell and cancer-associated fibroblast (CAF) dysfunction in pancreatic cancer

Abstract INTRODUCTION: We have recently shown that neutrophilic myeloid-derived suppressor cells (MDSC) are the dominant source of TNF in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). Moreover, neutrophil-derived TNF amplifies stromal fibrosis and immune tolerance via transmembraneTNF-TNFR2 interactions to promote therapeutic resistance in PDAC. Here, we develop a novel tumor-bearing murine model with efficient neutrophil lineage-restricted inactivation of Tnf to mechanistically dissect the impact of neutrophil-intrinsic TNF on stromal and T-cell dysfunction in PDAC. METHODS: We generated a neutrophil-lineage restricted MRP8Cre/+Tnfflox/flox model (Neu-TnfKO) by crossing Tnfflox/flox mice with mice bearing Cre-recombinase under control of MRP8 neutrophilic promoter, and confirmed >98% Tnf reduction in circulating neutrophils. Orthotopically implanted KPC tumors in Neu-TnfKO vs. littermates (MRP8Cre+/−Tnf+/+ or MRP8Cre-/−Tnffl/fl) were subjected to single-cell RNAseq (scRNAseq), flow cytometric immunophenotyping, and tissue histology. RESULTS: Compared with littermate controls, primary tumor weights and frequency of liver metastasis were significantly reduced in Neu-TnfKO mice. ScRNAseq in CD45− fraction of d21 tumors in Neu-TnfKO vs. control mice revealed downregulation of myofibroblastic features (i.e., Ccn2, Tgfb1), hypoxia signaling, and metabolic dysregulation in CAF single-cell transcriptomes. Tissue histology in Neu-TnfKO tumors revealed striking attenuation in fibrotic collagen deposition, and myofibril organization. Moreover, Lrrc15—implicated as a central regulator of CAF-driven immunotherapy resistance—was downregulated in Neu-TnfKO CAFs. This remodeling of stromal fibrosis manifested in increased trafficking of T-cells into Neu-TnfKO vs. littermate tumors via immunostaining and flow cytometry. ScRNAseq in CD45+ fractions of Neu-TnfKO mice corroborated enrichment in effector/helper T-cell and attenuation of MDSC and macrophage populations. In investigating if neutrophil-restricted Tnf silencing invokes reprogramming of T-cell single-cell transcriptomes, we observed global skewness towards effector vs exhausted programming in Neu-TnfKO tumors; enrichment of pathways related to ribosomal activation, interferon signaling, and negative regulation of T-cell apoptosis; and overexpression of multiple effector function genes—e.g., Adgre5, Cam1kd, Dgka—together indicating functional T-cell activation. Flow cytometry validated this activated CD8+ T-cell phenotype in Neu-TnfKO tumor-infiltrating by highlighting increased antigen-experienced (PD1+CD39neg) and short-lived effector populations (P<0.0001), and also revealed reduction in terminally exhausted (CD69+Ly108− in PD1hi) CD8+ T-cell subsets (P=0.028). CONCLUSIONS: Conditional silencing of neutrophilic-restricted TNF reprograms the immunotherapy-restrictive CAF and T-cell landscapes in the PDAC TME. Compartment-specific inhibition of TNF may be a provocative strategy to overcome immunotherapy resistance in PDAC. Citation Format: Anna Bianchi, Haleh Amirian, Iago De Castro Silva, Ifeanyichukwu Ogobuiro, Karthik Rajkumar, Andrew Mark Adams, Vanessa Garrido, Samara Singh, Siddharth Mehra, Christine Rafie, Nagarj Nagathihalli, Erietta Stelekati, Nipun Merchant, Jashodeep Datta. Neutrophil-intrinsic tumor necrosis factor (TNF) is a novel driver of T-cell and cancer-associated fibroblast (CAF) dysfunction in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR16.

Abstract 15271: Modification of Cardiomyocyte Lateral Membrane Composition Promotes Cardiac Function by Regulating Connexome Organization

Introduction: Loss of contacts between adjacent cardiomyocytes is detrimental to proper electromechanical function. For instance, connexome remodeling is a common feature of both acquired and inherited cardiac arrhythmias. We have shown that the lateral membrane-specific scaffolding protein CASK regulates the trafficking and targeting of certain connexome components, notably the sodium channel. Question: Does compartmentalization of the cardiomyocyte membrane involves lateral membrane proteins? Our hypothesis is that CASK, due to its association with costamere and its developmental expression gradient, may be involved in a more global control of connexome organization. Aims: To demonstrate that CASK, a lateral membrane-specific protein, is crucial for the development and maintenance of connexome integrity. Method: Viral constructs were designed to inhibit the expression of CASK in vivo (AAV-shCASK) and in vitro (Ad-shCASK) both in murine and human cardiomyocytes. Results: In vivo, echocardiography phenotyping of CASK invalidated rats at the neonatal stage revealed, at the adult stage, eccentric ventricular remodeling associated with increased stroke volume and increased cardiac output suggestive of athlete’s heart phenotype. Hemodynamic measurements (pressure-volume loops) confirmed that CASK invalidation increases both myocardium contractility and compliance. Proteomic analysis showed that CASK invalidation in vitro strongly modify immature cardiomyocyte proteome, notably junction signaling. Immunostaining experiments on cultured neonatal rat cardiomyocytes and adult rat hearts confirmed that CASK is required for the correct organization of myofibrils and favors connexome organization. Remarkably, CASK invalidation in a human in vitro model of arrhythmogenic cardiomyopathy (hiPSC-CM PKP2 +/- ) rescues desmosome assembly. Conclusion: CASK regulates the organization of myofibrils and connexome, with major functional consequences for cardiac performance. Our results suggest for the first time the existence of a crosstalk between lateral membrane and connexome proteins. Therefore, targeting CASK to promote electromechanical coupling of cardiomyocytes could be promising in the context of cardiac remodeling.

Biallelic variants in FLII cause pediatric cardiomyopathy by disrupting cardiomyocyte cell adhesion and myofibril organization.

Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, we identified biallelic variants in the highly conserved flightless-I (FLII) gene in 3 families with idiopathic, early-onset dilated CM. We demonstrated that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, resulted in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provided insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identified Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM and report biallelic variants as a genetic cause of pediatric CM.

Abstract P1130: Enhanced Electrophysiological Maturation Of Human Pluripotent Stem Cell Derived Cardiomyocytes By An Optimal Combination Of Metabolic Modulators

Human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) have emerged as ideal cell source for heart regeneration. Transplantation of hPSC-CMs to the damaged heart restores contractile function via electromechanical coupling between host and graft CMs. However, a few hurdles still remain in safety for clinical application, especially a transient period of ventricular arrhythmias after transplantation into large animals. Automaticity of graft cells, related to the electrical immaturity of hPSC-CMs, most likely triggers arrhythmic events. Here, we hypothesized that modulating metabolism toward an adult-like state would control ion channel gene expression and enhance electrophysiological maturation of hPSC-CMs in vitro. We systematically optimized combination of major metabolic components, including metabolic hormones, energy source and energy sensing, to achieve ion channel gene expression and electrophysiological phenotypes that more closely resemble adult cardiomyocytes. hPSC-CMs treated with optimized media displayed slower beat rates, longer field potentials, faster conduction velocities, and improved Ca 2+ kinetics. Moreover, optimized media increased mitochondrial content, respiratory capacity and fatty acid utilization for larger energy production, induced cell cycle exit, improved global transcriptome and sarcomere structure, and increased cell size. Subsequently, the matured cells were transplanted to mouse hearts, showing the engraftment comparable to immature hPSC-CMs. These results indicate that metabolic reprogramming can enhance many elements of maturation, including electrophysiology, myofibril organization, cell cycle, and capacity for oxidative phosphorylation.

Changes in myosin biomechanics influence growth and maturation of iPSC-cardiomyocytes.

Hypertrophic cardiomyopathy (HCM) is characterized by hypercontractility, cardiomyocyte hypertrophy, myofibril disarray, and altered energetics. Despite the identification of >1000 mutations in sarcomeric proteins, one-third of which are in β-cardiac myosin (MYH7), the mechanism by which altered force at the level of the sarcomere is transduced into cellular hypertrophy and other phenotypes is still incompletely understood. Our biochemical studies reveal surprising heterogeneity in the impact of different MYH7 mutations on myosin force generation and ATPase activity with some mutations reducing activity at the molecular level. Using CRISPR-edited hiPSC-cardiomyocytes (hiPSC-CMs), we studied the impact of myosin HCM mutations on the generation of contractile forces, hypertrophy, myofibril organization, and mitochondrial function, and compared cellular phenotypes with the effects of the mutations on myosin biomechanics. Our studies reveal a critical role for alterations in the myosin super-relaxed state (SRX) as a mechanism for cellular hypercontractility. To validate the fidelity of our findings to human disease, we compared hiPSC-CM results to those in 26 septal myectomy samples from HCM patients and 13 donor heart controls. In both hiPSC-CMs and myectomy samples we observed fiber disarray, altered mitochondrial metabolism, and widening of the Z-disc. A genome-wide key driver analysis revealed altered expression of 52 Z-disc-related genes in patient samples; 24 Z-disc genes in hiPSC-CMs; with 14 Z-disc genes shared between the two platforms. Activation of calcineurin pro-hypertrophic signaling provides a potential link between Z-disc mechano-sensation of sarcomeric forces and downstream hypertrophic signaling, which we explore using a vinculin FRET-tension sensor to directly measure intracellular force at the Z-disc. Our multi-scale approach, combined with both patient samples and CRISPR-edited hiPSC-CMs, confirms the fidelity of our in vitro model in recapitulating human disease and increases the potential translation of our findings to develop new precision-based HCM therapies.

Beyond Chaperoning: UCS Proteins Emerge as Regulators of Myosin-Mediated Cellular Processes.

The UCS (UNC-45/CRO1/She4p) family of proteins has emerged as chaperones specific for the folding, assembly, and function of myosin. UCS proteins participate in various myosin-dependent cellular processes including myofibril organization and muscle functions, cell differentiation, striated muscle development, cytokinesis, and endocytosis. Mutations in the genes that code for UCS proteins cause serious defects in myosin-dependent cellular processes. UCS proteins that contain an N-terminal tetratricopeptide repeat (TPR) domain are called UNC-45. Vertebrates usually possess two variants of UNC-45, the ubiquitous general-cell UNC-45 (UNC-45A) and the striated muscle UNC-45 (UNC-45B), which is exclusively expressed in skeletal and cardiac muscles. Except for the TPR domain in UNC-45, UCS proteins comprise of several irregular armadillo (ARM) repeats that are organized into a central domain, a neck region, and the canonical C-terminal UCS domain that functions as the chaperoning module. With or without TPR, UCS proteins form linear oligomers that serve as scaffolds that mediate myosin folding, organization into myofibrils, repair, and motility. This chapter reviews emerging functions of these proteins with a focus on UNC-45 as a dedicated chaperone for folding, assembly, and function of myosin at protein and potentially gene levels. Recent experimental evidences strongly support UNC-45 as an absolute regulator of myosin, with each domain of the chaperone playing different but complementary roles during the folding, assembly, and function of myosin, as well as recruiting Hsp90 as a co-chaperone to optimize key steps. It is becoming increasingly clear that UNC-45 also regulates the transcription of several genes involved in myosin-dependent cellular processes.

Caldesmon controls stress fiber force-balance through dynamic cross-linking of myosin II and actin-tropomyosin filaments

Contractile actomyosin bundles are key force-producing and mechanosensing elements in muscle and non-muscle tissues. Whereas the organization of muscle myofibrils and mechanism regulating their contractility are relatively well-established, the principles by which myosin-II activity and force-balance are regulated in non-muscle cells have remained elusive. We show that Caldesmon, an important component of smooth muscle and non-muscle cell actomyosin bundles, is an elongated protein that functions as a dynamic cross-linker between myosin-II and tropomyosin-actin filaments. Depletion of Caldesmon results in aberrant lateral movement of myosin-II filaments along actin bundles, leading to irregular myosin distribution within stress fibers. This manifests as defects in stress fiber network organization and contractility, and accompanied problems in cell morphogenesis, migration, invasion, and mechanosensing. These results identify Caldesmon as critical factor that ensures regular myosin-II spacing within non-muscle cell actomyosin bundles, and reveal how stress fiber networks are controlled through dynamic cross-linking of tropomyosin-actin and myosin filaments.

PPARdelta activation induces metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs.

Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of the longissimus thoracis of yaks

Yaks (Bos mutus) live in the Qinghai–Tibet plateau. The quality of yak meat is unique due to its genetic and physiological characteristics. Identification of the proteome of yak muscle could help to reveal its meat-quality properties. The common proteome, phosphoproteome, and N-glycoproteome of yak longissimus thoracis (YLT) were analyzed by liquid chromatography-tandem mass spectrometry-based shotgun analysis. A total of 1812 common proteins, 1303 phosphoproteins (3918 phosphorylation sites), and 204 N-glycoproteins (285 N-glycosylation sites) were identified in YLT. The common proteins in YLT were involved mainly in myofibril structure and energy metabolism; phosphoproteins were associated primarily with myofibril organization, regulation of energy metabolism, and signaling; N-glycoproteins were engaged mainly in extracellular-matrix organization, cellular immunity, and organismal homeostasis. We reported, for the first time, the “panorama” of the YLT proteome, specifically the N-glycoproteome of YLT. Our results provide essential information for understanding post mortem physiology (rigor mortis and aging) and the quality of yak meat.

Diallyl sulfide protects against dilated cardiomyopathy via inhibition of oxidative stress and apoptosis in mice

Cytochrome P450 family 2 subfamily E member 1 (CYP2E1) is a member of the cytochrome P450 enzyme family and catalyzes the metabolism of various substrates. CYP2E1 is upregulated in multiple heart diseases and causes damage mainly via the production of reactive oxygen species (ROS). In mice, increased CYP2E1 expression induces cardiac myocyte apoptosis, and knockdown of endogenous CYP2E1 can attenuate the pathological development of dilated cardiomyopathy (DCM). Nevertheless, targeted inhibition of CYP2E1 via the administration of drugs for the treatment of DCM remains elusive. Therefore, the present study aimed to investigate whether diallyl sulfide (DAS), a competitive inhibitor of CYP2E1, can be used to inhibit the development of the pathological process of DCM and identify its possible mechanism. Here, cTnTR141W transgenic mice, which developed typical DCM phenotypes, were used. Following treatment with DAS for 6 weeks, echocardiography, histological analysis and molecular marker detection were conducted to investigate the DAS-induced improvement on myocardial function and morphology. Biochemical analysis, western blotting and TUNEL assays were used to detected ROS production and myocyte apoptosis. It was found that DAS improved the typical DCM phenotypes, including chamber dilation, wall thinning, fibrosis, poor myofibril organization and decreased ventricular blood ejection, as determined using echocardiographic and histopathological analyses. Furthermore, the regulatory mechanisms, including inhibition both of the oxidative stress levels and the mitochondria-dependent apoptosis pathways, were involved in the effects of DAS. In particular, DAS showed advantages in terms of improved chamber dilation and dysfunction in model mice, and the improvement occurred in the early stage of the treatment compared with enalaprilat, an angiotensin-converting enzyme inhibitor that has been widely used in the clinical treatment of DCM and HF. The current results demonstrated that DAS could protect against DCM via inhibition of oxidative stress and apoptosis. These findings also suggest that inhibition of CYP2E1 may be a valuable therapeutic strategy to control the development of heart diseases, especially those associated with CYP2E1 upregulation. Moreover, the development of DAS analogues with lower cytotoxicity and metabolic rate for CYP2E1 may be beneficial.

Absence of Desmin Results in Impaired Adaptive Response to Mechanical Overloading of Skeletal Muscle

Background: Desmin is a muscle-specific protein belonging to the intermediate filament family. Desmin mutations are linked to skeletal muscle defects, including inherited myopathies with severe clinical manifestations. The aim of this study was to examine the role of desmin in skeletal muscle remodeling and performance gain induced by muscle mechanical overloading which mimics resistance training.Methods: Plantaris muscles were overloaded by surgical ablation of gastrocnemius and soleus muscles. The functional response of plantaris muscle to mechanical overloading in desmin-deficient mice (DesKO, n = 32) was compared to that of control mice (n = 36) after 7-days or 1-month overloading. To elucidate the molecular mechanisms implicated in the observed partial adaptive response of DesKO muscle, we examined the expression levels of genes involved in muscle growth, myogenesis, inflammation and oxidative energetic metabolism. Moreover, ultrastructure and the proteolysis pathway were explored.Results: Contrary to control, absolute maximal force did not increase in DesKO muscle following 1-month mechanical overloading. Fatigue resistance was also less increased in DesKO as compared to control muscle. Despite impaired functional adaptive response of DesKO mice to mechanical overloading, muscle weight and the number of oxidative MHC2a-positive fibers per cross-section similarly increased in both genotypes after 1-month overloading. However, mechanical overloading-elicited remodeling failed to activate a normal myogenic program after 7-days overloading, resulting in proportionally reduced activation and differentiation of muscle stem cells. Ultrastructural analysis of the plantaris muscle after 1-month overloading revealed muscle fiber damage in DesKO, as indicated by the loss of sarcomere integrity and mitochondrial abnormalities. Moreover, the observed accumulation of autophagosomes and lysosomes in DesKO muscle fibers could indicate a blockage of autophagy. To address this issue, two main proteolysis pathways, the ubiquitin-proteasome system and autophagy, were explored in DesKO and control muscle. Our results suggested an alteration of proteolysis pathways in DesKO muscle in response to mechanical overloading.Conclusion: Taken together, our results show that mechanical overloading increases the negative impact of the lack of desmin on myofibril organization and mitochondria. Furthermore, our results suggest that under these conditions, the repairing activity of autophagy is disturbed. Consequently, force generation is not improved despite muscle growth, suggesting that desmin is required for a complete response to resistance training in skeletal muscle.

1189-P: Ankyrin-B Modulates the Integrity and Bioenergetic Capacity of Skeletal Muscle Fibers

Ankyrin-B (AnkB) is a cytoskeletal protein that positions and stabilizes specialized membrane-associated protein complexes. AnkB also associates with organelles and facilitates their dynamics across cell types. AnkB variants have been linked to higher risk for obesity and type 2 diabetes (T2D) in humans and mice. In skeletal muscle (SKM), AnkB localizes to costameres and acts as a scaffold to β-dystroglycan/dystrophin complexes. This protein complex has been demonstrated to be essential for SKM integrity and exercise injury protection. However, besides this structural role, our findings suggest that AnkB associates with mitochondria in SKM and likely plays important roles in SKM bioenergetics. We found that non-obese young mice lacking AnkB solely in SKM (SKM-AnkB KO) develop early signs of myofibrillar disorganization and fibrosis and display reduced physical activity without changes in body weight, fasting glucose levels, or peripheral insulin response. In contrast, SKM-AnkB KO mice are more susceptible to the metabolic effects of a HFD, manifested as lower insulin sensitivity and further reductions in physical activity. Our findings suggest that loss of myofibril organization synergizes with changes in mitochondrial function to worsen SKM bioenergetics and insulin action. These physiological alterations are in agreement with our RNA-seq and proteomics studies, which implicate AnkB in the regulation of SKM oxidative capacity, protein translation, and cellular responses to stress and nutrient availability. We will discuss our ongoing efforts to uncover the mechanistic basis of these metabolic impairments cause by AnkB deficits. Disclosure K. Voos: None. J. Tzeng: None. D. Lorenzo: None. Funding American Diabetes Association (1-19-JDF-081 to D.L.)

Desmin Interacts Directly with Mitochondria

Desmin intermediate filaments (IFs) play an important role in maintaining the structural and functional integrity of muscle cells. They connect contractile myofibrils to plasma membrane, nuclei, and mitochondria. Disturbance of their network due to desmin mutations or deficiency leads to an infringement of myofibril organization and to a deterioration of mitochondrial distribution, morphology, and functions. The nature of the interaction of desmin IFs with mitochondria is not clear. To elucidate the possibility that desmin can directly bind to mitochondria, we have undertaken the study of their interaction in vitro. Using desmin mutant Des(Y122L) that forms unit-length filaments (ULFs) but is incapable of forming long filaments and, therefore, could be effectively separated from mitochondria by centrifugation through sucrose gradient, we probed the interaction of recombinant human desmin with mitochondria isolated from rat liver. Our data show that desmin can directly bind to mitochondria, and this binding depends on its N-terminal domain. We have found that mitochondrial cysteine protease can disrupt this interaction by cleavage of desmin at its N-terminus.

Fine structure of the cardiac muscle cells in the orb-web spider Nephila clavata

The fine structural characteristics of cardiac muscle cells and its myofibril organization in the orb web spider N. clavata were examined by transmission electron microscopy. Although myofibril striations are not remarkable as those of skeletal muscles, muscle fibers contain multiple myofibrils, abundant mitochondria, extensive sarcoplasmic reticulum and transverse tubules (T-tubules). Myofibrils are divided into distinct sarcomeres defined by Z-lines with average length of 2.0 μm, but the distinction between the A-band and the I-bands is not clear due to uniform striations over the length of the sarcomeres. Dyadic junction which consisted of a single T-tubule paired with a terminal cisterna of the sarcoplasmic reticulum is found mainly at the A-I level of sarcomere. Each cell is arranged to form multiple connections with neighboring cells through the intercalated discs. These specialized junctions include three types of intercellular junctions: gap junctions, fascia adherens and desmosomes for heart function. Our transmission electron microscopy (TEM) observations clearly show that spider’s cardiac muscle contraction is controlled by neurogenic rather than myogenic mechanism since each cardiac muscle fiber is innervated by a branch of motor neuron through neuromuscular junctions.

Progressive Myofibril Reorganization of Human Cardiomyocytes on a Dynamic Nanotopographic Substrate.

Cardiomyocyte (CM) alignment with striated myofibril organization is developed during early cardiac organogenesis. Previous work has successfully achieved in vitro CM alignment using a variety of biomaterial scaffolds and substrates with static topographic features. However, the cellular processes that occur during the response of CMs to dynamic surface topographic changes, which may provide a model of in vivo developmental progress of CM alignment within embryonic myocardium, remains poorly understood. To gain insights into these cellular processes involved in the response of CMs to dynamic topographic changes, we developed a dynamic topographic substrate that employs a shape memory polymer coated with polyelectrolyte multilayers to produce a flat-to-wrinkle surface transition when triggered by a change in incubation temperature. Using this system, we investigated cellular morphological alignment and intracellular myofibril reorganization in response to the dynamic wrinkle formation. Hence, we identified the progressive cellular processes of human-induced pluripotent stem cell-CMs in a time-dependent manner, which could provide a foundation for a mechanistic model of cardiac myofibril reorganization in response to extracellular microenvironment changes.

Desmin Forms Toxic, Seeding Competent Amyloid Aggregates that Persist in Muscle Fibers

Desmin-associated myofibrillar myopathy (MFM) has pathologic similarities to neurodegeneration-associated protein aggregate diseases. Desmin is an abundant muscle-specific intermediate filament, and disease mutations lead to its aggregation in cells, animals, and patients. We reasoned that similar to neurodegeneration-associated proteins, desmin itself may form amyloid. Desmin peptides corresponding to putative amyloidogenic regions formed seeding-competent amyloid fibrils. Amyloid formation was increased when disease-associated mutations were made within the peptide, and this conversion was inhibited by the anti-amyloid compound epigallocatechin-gallate. Moreover, a purified desmin fragment (aa 117 to 348) containing both amyloidogenic regions formed amyloid fibrils under physiologic conditions. Desmin fragment-derived amyloid coaggregated with full-length desmin and was able to template its conversion into fibrils in vitro. Desmin amyloids were cytotoxic to myotubes and disrupted their myofibril organization compared with desmin monomer or other nondesmin amyloids. Finally, desmin fragment amyloid persisted when introduced into mouse skeletal muscle. These data suggest that desmin forms seeding-competent amyloid that is toxic to myofibers. Moreover, small molecules known to interfere with amyloid formation and propagation may have therapeutic potential in MFM.

Cardiac microtubules in health and heart disease.

Cardiomyocytes are large (∼40,000 µm3), rod-shaped muscle cells that provide the working force behind each heartbeat. These highly structured cells are packed with dense cytoskeletal networks that can be divided into two groups—the contractile (i.e. sarcomeric) cytoskeleton that consists of filamentous actin-myosin arrays organized into myofibrils, and the non-sarcomeric cytoskeleton, which is composed of β- and γ-actin, microtubules, and intermediate filaments. Together, microtubules and intermediate filaments form a cross-linked scaffold, and these networks are responsible for the delivery of intracellular cargo, the transmission of mechanical signals, the shaping of membrane systems, and the organization of myofibrils and organelles. Microtubules are extensively altered as part of both adaptive and pathological cardiac remodeling, which has diverse ramifications for the structure and function of the cardiomyocyte. In heart failure, the proliferation and post-translational modification of the microtubule network is linked to a number of maladaptive processes, including the mechanical impediment of cardiomyocyte contraction and relaxation. This raises the possibility that reversing microtubule alterations could improve cardiac performance, yet therapeutic efforts will strongly benefit from a deeper understanding of basic microtubule biology in the heart. The aim of this review is to summarize the known physiological roles of the cardiomyocyte microtubule network, the consequences of its pathological remodeling, and to highlight the open and intriguing questions regarding cardiac microtubules.Impact statementAdvancements in cell biological and biophysical approaches and super-resolution imaging have greatly broadened our view of tubulin biology over the last decade. In the heart, microtubules and microtubule-based transport help to organize and maintain key structures within the cardiomyocyte, including the sarcomere, intercalated disc, protein clearance machinery and transverse-tubule and sarcoplasmic reticulum membranes. It has become increasingly clear that post translational regulation of microtubules is a key determinant of their sub-cellular functionality. Alterations in microtubule network density, stability, and post-translational modifications are hallmarks of pathological cardiac remodeling, and modified microtubules can directly impede cardiomyocyte contractile function in various forms of heart disease. This review summarizes the functional roles and multi-leveled regulation of the cardiac microtubule cytoskeleton and highlights how refined experimental techniques are shedding mechanistic clarity on the regionally specified roles of microtubules in cardiac physiology and pathophysiology.

Desmin forms toxic, seeding-competent amyloid aggregates that persist in muscle fibers

Desmin-associated myofibrillar myopathy (MFM) has pathologic similarities to neurodegeneration-associated protein aggregate diseases. Desmin is an abundant muscle-specific intermediate filament, and disease mutations lead to its aggregation in cells, animals, and patients. We reasoned that similar to neurodegeneration-associated proteins, desmin itself may form amyloid. Desmin peptides corresponding to putative amyloidogenic regions formed seeding-competent amyloid fibrils. Amyloid formation was increased when disease-associated mutations were made within the peptide, and this conversion was inhibited by the anti-amyloid compound epigallocatechin-gallate. Moreover, a purified desmin fragment (aa 117 to 348) containing both amyloidogenic regions formed amyloid fibrils under physiologic conditions. Desmin fragment-derived amyloid coaggregated with full-length desmin and was able to template its conversion into fibrils in vitro. Desmin amyloids were cytotoxic to myotubes and disrupted their myofibril organization compared with desmin monomer or other nondesmin amyloids. Finally, desmin fragment amyloid persisted when introduced into mouse skeletal muscle. These data suggest that desmin forms seeding-competent amyloid that is toxic to myofibers. Moreover, small molecules known to interfere with amyloid formation and propagation may have therapeutic potential in MFM.

Abstract 435: Post-transcriptional Regulation of Tropomyosin Isoforms Altered in Human Heart Diseases

Background: Tropomyosin 1 (TPM1) is a coiled coil protein that forms polymers along with the actin filament, and it is required for myofibril organization, myocardial contraction, and cardiac development. TPM1 has cardiac muscle and non-muscle specific protein isoforms that dictate its function in cardiac muscle contraction or in cytoskeleton. TPM1 mutations in exon 2b, 5, 6b or 8 are associated with cardiovascular diseases including familial hypertrophic cardiomyopathy, dilated cardiomyopathy and left ventricular non-compaction. Thus, it is of great importance to investigate the mechanisms that control tropomyosin isoforms in order to identify novel therapeutic targets for cardiovascular diseases in which TPM1 is mutated. Method: We used nanopore sequencing technology using MinION and poly(A)click seq (PAC-seq) to identify different TPM1 transcripts generated in rat heart derived H9c2 cells. RT-PCR and western blotting were conducted to measure mRNA and protein levels of TPM1 respectively. Results: We identified 10 different isoforms of TPM1 using nanopore sequencing and determined several exons that are extensively spliced and favored in cardiac cells. We further identified an RNA binding protein that controls cardiac specific isoforms of TPM1 via alternative splicing and polyadenylation. Importantly, we found that this RNA binding protein regulates alternative splicing of exon 6b of TPM1, which is mutated in patients with cardiomyopathies. Conclusions: We identified an RNA binding protein, which controls expression of cardiac specific TPM1 isoforms by alternative polyadenylation and alternative splicing.