Sarcomere Assembly Research Articles

Mechanoresponsive regulation of myogenesis by the force-sensing transcriptional regulator Tono

Muscle morphogenesis is a multi-step program, starting with myoblast fusion, followed by myotube-tendon attachment and sarcomere assembly, with subsequent sarcomere maturation, mitochondrial amplification, and specialization. The correct chronological order of these steps requires precise control of the transcriptional regulators and their effectors. How this regulation is achieved during muscle development is not well understood. In a genome-wide RNAi screen in Drosophila, we identified the BTB-zinc-finger protein Tono (CG32121) as a muscle-specific transcriptional regulator. tono mutant flight muscles display severe deficits in mitochondria and sarcomere maturation, resulting in uncontrolled contractile forces causing muscle rupture and degeneration during development. Tono protein is expressed during sarcomere maturation and localizes in distinct condensates in flight muscle nuclei. Interestingly, internal pressure exerted by the maturing sarcomeres deforms the muscle nuclei into elongated shapes and changes the Tono condensates, suggesting that Tono senses the mechanical status of the muscle cells. Indeed, external mechanical pressure on the muscles triggers rapid liquid-liquid phase separation of Tono utilizing its BTB domain. Thus, we propose that Tono senses high mechanical pressure to adapt muscle transcription, specifically at the sarcomere maturation stages. Consistently, tono mutant muscles display specific defects in a transcriptional switch that represses early muscle differentiation genes and boosts late ones. We hypothesize that a similar mechano-responsive regulation mechanism may control the activity of related BTB-zinc-finger proteins that, if mutated, can result in uncontrolled force production in human muscle.

Abstract Tu092: Role of Z-disc as a Driver of Hypertrophy and Sarcomere Assembly in Hypertrophic Cardiomyopathy

The Z-disc is an electron-dense structure demarcating the lateral boundaries of the sarcomere and postulated to play a key role in both cell signaling and sarcomere assembly. We performed EM on both HCM patient septal myectomies and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with 3 different HCM MYH7 mutations, finding marked Z-disc widening along with a hallmark feature of HCM, myofibrillar disarray. Mutations in MYH7 that cause HCM do so by increasing force at the sarcomere level, leading to the proposal that the Z-disc may act as a mechanosensor that converts myosin-generated force into the intracellular signaling that drives hypertrophy. We focused on understanding the mechanisms of Z-disc mechanotransduction of both hypertrophy and sarcomere disarray. RNA-seq from HCM myectomies demonstrated 52 Z-disc differentially expressed genes (DEGs). scRNA-seq from HCM hiPSC-CMs identified 24 Z-disc DEGs. 14 Z-disc DEGs were shared across both datasets, most notably 5 upstream regulators of calcineurin signalling. To correlate changes in gene expression with altered force we transfected hiPSC-CMs with a FRET-based tension sensor in the Z-disc protein vinculin allowing the direct measurement of force experienced by the Z-disc. Live-cell confocal imaging of hiPSC-CMs expressing EGFP-α-actinin-2 showed that indistinct α-actinin-2 foci coalesced into sarcomeres over the course of 24-48 h after replating. Imaging at nm scale using cryo-electron tomography revealed marked disorder in individual thick filaments in HCM hiPSC-CMs, distinct from the classical disarray at the level of whole myofibrils and which would not be visible on conventional EM. Our data suggest that early alterations in sarcomeric micro-structure and signaling induced by altered myosin force may precede hypertrophy and could occur decades before clinical signs (hypertrophy on ECG or echo) are present, with potential implications for early preventive intervention.

Distinct functional and molecular profiles between physiological and pathological atrial enlargement offer potential new therapeutic opportunities for atrial fibrillation.

Atrial fibrillation (AF) remains challenging to prevent and treat. A key feature of AF is atrial enlargement. However, not all atrial enlargement progresses to AF. Atrial enlargement in response to physiological stimuli such as exercise is typically benign and reversible. Understanding the differences in atrial function and molecular profile underpinning pathological and physiological atrial remodelling will be critical for identifying new strategies for AF. The discovery of molecular mechanisms responsible for pathological and physiological ventricular hypertrophy has uncovered new drug targets for heart failure. Studies in the atria have been limited in comparison. Here, we characterised mouse atria from (1) a pathological model (cardiomyocyte-specific transgenic (Tg) that develops dilated cardiomyopathy [DCM] and AF due to reduced protective signalling [PI3K]; DCM-dnPI3K), and (2) a physiological model (cardiomyocyte-specific Tg with an enlarged heart due to increased insulin-like growth factor 1 receptor; IGF1R). Both models presented with an increase in atrial mass, but displayed distinct functional, cellular, histological and molecular phenotypes. Atrial enlargement in the DCM-dnPI3K Tg, but not IGF1R Tg, was associated with atrial dysfunction, fibrosis and a heart failure gene expression pattern. Atrial proteomics identified protein networks related to cardiac contractility, sarcomere assembly, metabolism, mitochondria, and extracellular matrix which were differentially regulated in the models; many co-identified in atrial proteomics data sets from human AF. In summary, physiological and pathological atrial enlargement are associated with distinct features, and the proteomic dataset provides a resource to study potential new regulators of atrial biology and function, drug targets and biomarkers for AF.

Styxl2 regulates de novo sarcomere assembly by binding to non-muscle myosin IIs and promoting their degradation.

Styxl2, a poorly characterized pseudophosphatase, was identified as a transcriptional target of the Jak1-Stat1 pathway during myoblast differentiation in culture. Styxl2 is specifically expressed in vertebrate striated muscles. By gene knockdown in zebrafish or genetic knockout in mice, we found that Styxl2 plays an essential role in maintaining sarcomere integrity in developing muscles. To further reveal the functions of Styxl2 in adult muscles, we generated two inducible knockout mouse models: one with Styxl2 being deleted in mature myofibers to assess its role in sarcomere maintenance, and the other in adult muscle satellite cells (MuSCs) to assess its role in de novo sarcomere assembly. We find that Styxl2 is not required for sarcomere maintenance but functions in de novo sarcomere assembly during injury-induced muscle regeneration. Mechanistically, Styxl2 interacts with non-muscle myosin IIs, enhances their ubiquitination, and targets them for autophagy-dependent degradation. Without Styxl2, the degradation of non-muscle myosin IIs is delayed, which leads to defective sarcomere assembly and force generation. Thus, Styxl2 promotes de novo sarcomere assembly by interacting with non-muscle myosin IIs and facilitating their autophagic degradation.

Styxl2 regulates de novo sarcomere assembly by binding to non-muscle myosin IIs and promoting their degradation

Styxl2, a poorly characterized pseudophosphatase, was identified as a transcriptional target of the Jak1-Stat1 pathway during myoblast differentiation in culture. Styxl2 is specifically expressed in vertebrate striated muscles. By gene knockdown in zebrafish or genetic knockout in mice, we found that Styxl2 plays an essential role in maintaining sarcomere integrity in developing muscles. To further reveal the functions of Styxl2 in adult muscles, we generated two inducible knockout mouse models: one with Styxl2 being deleted in mature myofibers to assess its role in sarcomere maintenance, and the other in adult muscle satellite cells (MuSCs) to assess its role in de novo sarcomere assembly. We find that Styxl2 is not required for sarcomere maintenance but functions in de novo sarcomere assembly during injury-induced muscle regeneration. Mechanistically, Styxl2 interacts with non-muscle myosin IIs, enhances their ubiquitination, and targets them for autophagy-dependent degradation. Without Styxl2, the degradation of non-muscle myosin IIs is delayed, which leads to defective sarcomere assembly and force generation. Thus, Styxl2 promotes de novo sarcomere assembly by interacting with non-muscle myosin IIs and facilitating their autophagic degradation.

Characterization of a new arrhythmogenic cardiomyopathy mouse model due to filamin c haploinsufficiency

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Spanish Ministry of Universities FPU fellowship (FPU19/04044), Spanish Ministry of Science and Innovation Digital Transition call (TED2021-129774B-C22), EU HORIZON EIC Pathfinder Challenges 2022 Cardiogenomics call (Project 101115416). Background Filamin C (FLNC) is widely expressed in heart and skeletal muscle. It participates in cell junction and sarcomere assembly and function. We have previously shown that heterozygous FLNC truncating variants (FLNCtv) cause arrhythmogenic/dilated cardiomyopathy (ACM/DCM) in patients. Diverse studies have suggested haploinsufficiency as the potential mechanism of this disease. However, animal models of FLNCtv have only been reported to develop ACM/DCM in homozygosity. Purpose To develop a new model of ACM/DCM caused by a FLNCtv and study the progression of the disease in heterozygosity. Methods Two gRNAs flanking exon 15 of the Flnc gene and SpCas9 protein were microinjected into mouse zygotes to generate a mouse line with a constitutive deletion of Flnc exon 15 (FlncWt/Ex15del). mRNA and protein stability were determined by qRT-PCR and western blot, respectively. Cardiac function was analysed by ECG and echocardiography every 2 months, starting at 8 weeks of age (Fig. 1B). The mean ECG values were calculated using 3 time intervals and standard ECG waves. Results FlncWt/Ex15del mice showed a reduction in Flnc mRNA and protein similar to that observed in a human ACM/DCM patient bearing a similar mutation, suggesting that this FLNCtv causes haploinsufficiency. Both male and female FlncWt/Ex15del mice developed cardiac electrophysiological defects, including reduced amplitude of the QRS complex and the P-wave, notched R and/or S waves, and increased QT interval times, and these defects worsened with time. At 40 weeks of age, FlncWt/Ex15del mice showed some initial signs of left ventricular dilatation. Conclusions Mice carrying FLNCtv in heterozygosity develop electrophysiological changes resembling pathological features of ACM/DCM patients due to haploinsufficiency. The development of this pathological phenotype in heterozygosity, as opposed to previous models, paves the way for the development of new gene therapies using this model.

IGF1 promotes human myotube differentiation toward a mature metabolic and contractile phenotype.

Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal muscle models in vitro are incapable of fully recapitulating its physiological functions especially muscle contractility. By supplementation of insulin-like growth factor 1 (IGF1), a growth factor secreted by myofibers in vivo, we aimed to overcome these limitations. We monitored the differentiation process starting from primary human CD56-positive myoblasts in the presence/absence of IGF1 in serum-free medium in daily collected samples for 10 days. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon electrical pulse stimulation (EPS) following day 6. Myotubes without IGF1 were almost incapable of contraction. IGF1 treatment shifted the proteome toward skeletal muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7, and reduced MYH1/2 suggest a more oxidative phenotype further demonstrated by higher abundance of proteins of the respiratory chain and elevated mitochondrial respiration. IGF1-treatment also upregulated glucose transporter (GLUT)4 and increased insulin-dependent glucose uptake compared with myotubes differentiated without IGF1. To conclude, addition of IGF1 to serum-free medium significantly improves the differentiation of human myotubes that showed enhanced myofibril formation, response to electrical pulse stimulation, oxidative respiratory capacity, and glucose metabolism overcoming limitations of previous standards. This novel protocol enables investigation of muscular exercise on a molecular level.NEW & NOTEWORTHY Human skeletal muscle models are highly valuable to study how exercise prevents type 2 diabetes without invasive biopsies. Current models did not fully recapitulate the function of skeletal muscle especially during exercise. By supplementing insulin-like growth factor 1 (IGF1), the authors developed a functional human skeletal muscle model characterized by inducible contractility and increased oxidative and insulin-sensitive metabolism. The novel protocol overcomes the limitations of previous standards and enables investigation of exercise on a molecular level.

Bruno 1/CELF regulates splicing and cytoskeleton dynamics to ensure correct sarcomere assembly in Drosophila flight muscles.

Muscles undergo developmental transitions in gene expression and alternative splicing that are necessary to refine sarcomere structure and contractility. CUG-BP and ETR-3-like (CELF) family RNA-binding proteins are important regulators of RNA processing during myogenesis that are misregulated in diseases such as Myotonic Dystrophy Type I (DM1). Here, we report a conserved function for Bruno 1 (Bru1, Arrest), a CELF1/2 family homolog in Drosophila, during early muscle myogenesis. Loss of Bru1 in flight muscles results in disorganization of the actin cytoskeleton leading to aberrant myofiber compaction and defects in pre-myofibril formation. Temporally restricted rescue and RNAi knockdown demonstrate that early cytoskeletal defects interfere with subsequent steps in sarcomere growth and maturation. Early defects are distinct from a later requirement for bru1 to regulate sarcomere assembly dynamics during myofiber maturation. We identify an imbalance in growth in sarcomere length and width during later stages of development as the mechanism driving abnormal radial growth, myofibril fusion, and the formation of hollow myofibrils in bru1 mutant muscle. Molecularly, we characterize a genome-wide transition from immature to mature sarcomere gene isoform expression in flight muscle development that is blocked in bru1 mutants. We further demonstrate that temporally restricted Bru1 rescue can partially alleviate hypercontraction in late pupal and adult stages, but it cannot restore myofiber function or correct structural deficits. Our results reveal the conserved nature of CELF function in regulating cytoskeletal dynamics in muscle development and demonstrate that defective RNA processing due to misexpression of CELF proteins causes wide-reaching structural defects and progressive malfunction of affected muscles that cannot be rescued by late-stage gene replacement.

Reorganization of mitochondria-organelle interactions during postnatal development in skeletal muscle.

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.

Mechanisms of Sarcomere Assembly in Muscle Cells Inferred from Sequential Ordering of Myofibril Components

Voluntary movement in animals is driven by the contraction of myofibrils in striated muscle cells, which are linear chains of regular cytoskeletal units termed sarcomeres. While the ordered structure of mature sarcomeres, key to muscle contraction, is well established, the physical mechanism governing sarcomere assembly is still under debate. Here we put forward a theory of sarcomere self-assembly based on data. We measured the sequential ordering of sarcomere components in developing flight muscles, using a novel tracking-free algorithm. We find that myosin molecular motors, α-actinin cross-linkers, and the titin homologue Sallimus form periodic patterns before actin filaments become polarity sorted. Based on these, we propose that myosin, Sallimus, and sarcomere Z-disk proteins including α-actinin dynamically bind and unbind to an unordered bundle of actin to establish an initial periodic pattern. Periodicity of actin filaments is established only later. Our model proposes that nonlocal interactions between spatially extended myosin and titin/Sallimus containing complexes, and possibly tension-dependent feedback mediated by an α-actinin catch bond, drive this ordering process. We probe this hypothesis using mathematical models and derive predictive conditions for sarcomere pattern formation to guide future experiments. Published by the American Physical Society 2024

Cypher/ZASP drives cardiomyocyte maturation via actin-mediated MRTFA-SRF signalling.

Rationale: Cardiomyocytes (CMs) undergo dramatic structural and functional changes in postnatal maturation; however, the regulatory mechanisms remain greatly unclear. Cypher/Z-band alternatively spliced PDZ-motif protein (ZASP) is an essential sarcomere component maintaining Z-disc stability. Deletion of mouse Cypher and mutation in human ZASP result in dilated cardiomyopathy (DCM). Whether Cypher/ZASP participates in CM maturation and thereby affects cardiac function has not been answered. Methods: Immunofluorescence, transmission electron microscopy, real-time quantitative PCR, and Western blot were utilized to identify the role of Cypher in CM maturation. Subsequently, RNA sequencing and bioinformatics analysis predicted serum response factor (SRF) as the key regulator. Rescue experiments were conducted using adenovirus or adeno-associated viruses encoding SRF, both in vitro and in vivo. The molecular mechanisms were elucidated through G-actin/F-actin fractionation, nuclear-cytoplasmic extraction, actin disassembly assays, and co-sedimentation assays. Results: Cypher deletion led to impaired sarcomere isoform switch and morphological abnormalities in mitochondria, transverse-tubules, and intercalated discs. RNA-sequencing analysis revealed significant dysregulation of crucial genes related to sarcomere assembly, mitochondrial metabolism, and electrophysiology in the absence of Cypher. Furthermore, SRF was predicted as key transcription factor mediating the transcriptional differences. Subsequent rescue experiments showed that SRF re-expression during the critical postnatal period effectively rectified CM maturation defects and notably improved cardiac function in Cypher-depleted mice. Mechanistically, Cypher deficiency resulted in the destabilization of F-actin and a notable increase in G-actin levels, thereby impeding the nuclear localisation of myocardin-related transcription factor A (MRTFA) and subsequently initiating SRF transcription. Conclusion: Cypher/ZASP plays a crucial role in CM maturation through actin-mediated MRTFA-SRF signalling. The linkage between CM maturation abnormalities and the late-onset of DCM is suggested, providing further insights into the pathogenesis of DCM and potential treatment strategies.

Independent regulation of Z-lines and M-lines during sarcomere assembly in cardiac myocytes revealed by the automatic image analysis software sarcApp

Sarcomeres are the basic contractile units within cardiac myocytes, and the collective shortening of sarcomeres aligned along myofibrils generates the force driving the heartbeat. The alignment of the individual sarcomeres is important for proper force generation, and misaligned sarcomeres are associated with diseases, including cardiomyopathies and COVID-19. The actin bundling protein, α-actinin-2, localizes to the ‘Z-Bodies” of sarcomere precursors and the ‘Z-Lines’ of sarcomeres, and has been used previously to assess sarcomere assembly and maintenance. Previous measurements of α-actinin-2 organization have been largely accomplished manually, which is time-consuming and has hampered research progress. Here, we introduce sarcApp, an image analysis tool that quantifies several components of the cardiac sarcomere and their alignment in muscle cells and tissue. We first developed sarcApp to utilize deep learning-based segmentation and real space quantification to measure α-actinin-2 structures and determine the organization of both precursors and sarcomeres/myofibrils. We then expanded sarcApp to analyze ‘M-Lines’ using the localization of myomesin and a protein that connects the Z-Lines to the M-Line (titin). sarcApp produces 33 distinct measurements per cell and 24 per myofibril that allow for precise quantification of changes in sarcomeres, myofibrils, and their precursors. We validated this system with perturbations to sarcomere assembly. We found perturbations that affected Z-Lines and M-Lines differently, suggesting that they may be regulated independently during sarcomere assembly.

Independent regulation of Z-lines and M-lines during sarcomere assembly in cardiac myocytes revealed by the automatic image analysis software sarcApp.

Sarcomeres are the basic contractile units within cardiac myocytes, and the collective shortening of sarcomeres aligned along myofibrils generates the force driving the heartbeat. The alignment of the individual sarcomeres is important for proper force generation, and misaligned sarcomeres are associated with diseases, including cardiomyopathies and COVID-19. The actin bundling protein, α-actinin-2, localizes to the 'Z-Bodies" of sarcomere precursors and the 'Z-Lines' of sarcomeres, and has been used previously to assess sarcomere assembly and maintenance. Previous measurements of α-actinin-2 organization have been largely accomplished manually, which is time-consuming and has hampered research progress. Here, we introduce sarcApp, an image analysis tool that quantifies several components of the cardiac sarcomere and their alignment in muscle cells and tissue. We first developed sarcApp to utilize deep learning-based segmentation and real space quantification to measure α-actinin-2 structures and determine the organization of both precursors and sarcomeres/myofibrils. We then expanded sarcApp to analyze 'M-Lines' using the localization of myomesin and a protein that connects the Z-Lines to the M-Line (titin). sarcApp produces 33 distinct measurements per cell and 24 per myofibril that allow for precise quantification of changes in sarcomeres, myofibrils, and their precursors. We validated this system with perturbations to sarcomere assembly. We found perturbations that affected Z-Lines and M-Lines differently, suggesting that they may be regulated independently during sarcomere assembly.

Phosphoproteomic insight into the changes in structural proteins of muscle architecture associated with texture softening of grass carp (Ctenopharyngodon idella) fillets during chilling storage.

Texture is an important sensory attribute of fish affected by modifications of structural proteins in muscle architecture. To investigate the changes in protein phosphorylation during texture softening of fish, the proteins of grass carp muscle after chilling storage of 0day and 6days were compared by phosphoproteomics, and their association with texture was analyzed. Totally 1026 unique phosphopeptides on 656 phosphoproteins were identified as differential. They were mainly classified as intracellular myofibril and cytoskeleton, and extracellular matrix, of which the molecular function and biological process were binding into supramolecular assembly and myofilament contraction. The concomitant dephosphorylation of kinases and assembly regulators indicated dephosphorylation and disassembly tendency of sarcomeric architecture. Correlation analysis defined the relation between texture and dephosphorylation of myosin light chain, actin, collagen and cytoskeleton. This study revealed that protein phosphorylation may affect the texture of fish muscle through regulating sarcomeric assembly of structural proteins in muscle architecture.

The α-arrestin SUP-13/ARRD-15 promotes isoform turnover of actin-interacting protein 1 in Caenorhabditis elegans striated muscle.

Precise arrangement of actin, myosin, and other regulatory components in a sarcomeric pattern is critical for producing contractile forces in striated muscles. Actin-interacting protein 1 (AIP1), also known as WD-repeat protein 1 (WDR1), is one of essential factors that regulate sarcomeric assembly of actin filaments. In the nematode Caenorhabditis elegans, mutation in unc-78, encoding one of the two AIP1 isoforms, causes severe disorganization of sarcomeric actin filaments and near paralysis, but mutation in sup-13 suppresses the unc-78-mutant phenotypes to restore nearly normal sarcomeric actin organization and worm motility. Here, we identified that sup-13 is a nonsense allele of arrd-15 encoding an α-arrestin. The sup-13/arrd-15 mutation suppressed the phenotypes of unc-78 null mutant but required aipl-1 that encodes a second AIP1 isoform. aipl-1 was normally expressed highly in embryos and downregulated in mature muscle. However, in the sup-13/arrd-15 mutant, the AIPL-1 protein was maintained at high levels in adult muscle to compensate for the absence of the UNC-78 protein. The sup-13/arrd-15 mutation caused accumulation of ubiquitinated AIPL-1 protein, suggesting that a normal function of sup-13/arrd-15 is to enhance degradation of ubiquitinated AIPL-1, thereby promoting transition of AIP1 isoforms from AIPL-1 to UNC-78 in developing muscle. These results suggest that α-arrestin is a novel factor to promote isoform turnover by enhancing protein degradation.

Cardiomyocyte external mechanical unloading activatesmodifications of α-actinin differently fromsarcomere-originated unloading.

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano-signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano-signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1 μm mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α-actinin by 6 h. In myosin inhibition, this was accompanied by redistribution of intracellular poly-ubiquitin K48 to the cellular periphery relative to the poly-ubiquitin K48 reservoir at the I-band. Moreover, loading and unloading of the cellular substrate led to a three-fold increase in post-translational modifications (PTMs) when compared to the myosin-specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve extracellular signal-regulated kinase 1/2 and focal adhesion kinase activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α-actinin to increase its propensity to bind F-actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α-actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α-actinin are discussed with respect to cardiac atrophy and heart failure.

Cardiac myofibrillogenesis is spatiotemporally modulated by the molecular chaperone UNC45B.

Sarcomeres are fundamental to cardiac muscle contraction. Their impairment can elicit cardiomyopathies, leading causes of death worldwide. However, the molecular mechanism underlying sarcomere assembly remains obscure. We used human embryonic stem cell (hESC)-derived cardiomyocytes (CMs) to reveal stepwise spatiotemporal regulation of core cardiac myofibrillogenesis-associated proteins. We found that the molecular chaperone UNC45B is highly co-expressed with KINDLIN2 (KIND2), a marker of protocostameres, and later its distribution overlaps with that of muscle myosin MYH6. UNC45B-knockout CMs display essentially no contractility. Our phenotypic analyses further reveal that (1) binding of Z line anchor protein ACTN2 to protocostameres is perturbed because of impaired protocostamere formation, resulting in ACTN2 accumulation; (2) F-ACTIN polymerization is suppressed; and (3) MYH6 becomes degraded, so it cannot replace non-muscle myosin MYH10. Our mechanistic study demonstrates that UNC45B mediates protocostamere formation by regulating KIND2 expression. Thus, we show that UNC45B modulates cardiac myofibrillogenesis by interacting spatiotemporally with various proteins.

Drosophila Tropomodulin is required for multiple actin-dependent processes within developing myofibers.

Proper muscle contraction requires the assembly and maintenance of sarcomeres and myofibrils. Although the protein components of myofibrils are generally known, less is known about the mechanisms by which they individually function and together synergize for myofibril assembly and maintenance. For example, it is unclear how the disruption of actin filament (F-actin) regulatory proteins leads to the muscle weakness observed in myopathies. Here, we show that knockdown of Drosophila Tropomodulin (Tmod), results in several myopathy-related phenotypes, including reduction of muscle cell (myofiber) size, increased sarcomere length, disorganization and misorientation of myofibrils, ectopic F-actin accumulation, loss of tension-mediating proteins at the myotendinous junction, and misshaped and internalized nuclei. Our findings support and extend the tension-driven self-organizing myofibrillogenesis model. We show that, like its mammalian counterpart, Drosophila Tmod caps F-actin pointed-ends, and we propose that this activity is crucial for cellular processes in different locations within the myofiber that directly and indirectly contribute to the maintenance of muscle function. Our findings provide significant insights to the role of Tmod in muscle development, maintenance and disease.

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.

Target formation in muscle fibres indicates reinnervation - A proteomic study in muscle samples from peripheral neuropathies.

Target skeletal muscle fibres - defined by different concentric areas in oxidative enzyme staining - can occur in patients with neurogenic muscular atrophy. Here, we used our established hypothesis-free proteomic approach with the aim of deciphering the protein composition of targets. We also searched for potential novel interactions between target proteins. Targets and control areas were laser microdissected from skeletal muscle sections of 20 patients with neurogenic muscular atrophy. Samples were analysed by a highly sensitive mass spectrometry approach, enabling relative protein quantification. The results were validated by immunofluorescence studies. Protein interactions were investigated by yeast two-hybrid assays, coimmunoprecipitation experiments and bimolecular fluorescence complementation. More than 1000 proteins were identified. Among these, 55 proteins were significantly over-represented and 40 proteins were significantly under-represented in targets compared to intraindividual control samples. The majority of over-represented proteins were associated with the myofibrillar Z-disc and actin dynamics, followed by myosin and myosin-associated proteins, proteins involved in protein biosynthesis and chaperones. Under-represented proteins were mainly mitochondrial proteins. Functional studies revealed that the LIM domain of the over-represented protein LIMCH1 interacts with isoform A of Xin actin-binding repeat-containing protein 1 (XinA). In particular, proteins involved in myofibrillogenesis are over-represented in target structures, which indicate an ongoing process of sarcomere assembly and/or remodelling within this specific area of the muscle fibres. We speculate that target structures are the result of reinnervation processes in which filamin C-associated myofibrillogenesis is tightly regulated by the BAG3-associated protein quality system.