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.

Age-related differences in the loss and recovery of serial sarcomere number following disuse atrophy in rats.

BACKGROUND: Older adults exhibit a slower recovery of muscle mass following disuse atrophy than young adults. At a smaller scale, muscle fibre cross-sectional area (i.e., sarcomeres in parallel) exhibits this same pattern. Less is known, however, about age-related differences in the recovery of muscle fibre length, driven by increases in serial sarcomere number (SSN), following disuse. The purpose of this study was to investigate age-related differences in SSN adaptations and muscle mechanical function during and following muscle immobilization. We hypothesized that older adult rats would experience a similar magnitude of SSN loss during immobilization, however, take longer to recover SSN than young following cast removal, which would limit the recovery of muscle mechanical function. METHODS: We casted the plantar flexors of young (8 months) and old (32 months) male rats in a shortened position for 2 weeks, and assessed recovery during 4 weeks of voluntary ambulation. Following sacrifice, legs were fixed in formalin for measurement of soleus SSN and physiological cross-sectional area (PCSA) with the un-casted soleus acting as a control. Ultrasonographic measurements of pennation angle (PA) and muscle thickness (MT) were conducted weekly. In-vivo active and passive torque-angle relationships were constructed pre-cast, post-cast, and following 4 weeks of recovery. RESULTS: From pre- to post-cast, young and older adult rats experienced similar decreases in SSN (-20%, P < 0.001), muscle wet weight (-25%, P < 0.001), MT (-30%), PA (-15%, P < 0.001), and maximum isometric torque (-40%, P < 0.001), but there was a greater increase in passive torque in older (+ 180%, P < 0.001) compared to young adult rats (+ 68%, P = 0.006). Following cast removal, young exhibited quicker recovery of SSN and MT than old, but SSN recovered sooner than PA and MT in both young and old. PCSA nearly recovered and active torque fully recovered in young adult rats, whereas in older adult rats these remained unrecovered at ∼ 75%. CONCLUSIONS: This study showed that older adult rats retain a better ability to recover longitudinal compared to parallel muscle morphology following cast removal, making SSN a highly adaptable target for improving muscle function in elderly populations early on during rehabilitation.

Keeping it fresh: ribosomal protein SA sustains sarcomeric function via localized translation.

Mechanical stress from cardiomyocyte contraction causes misfolded sarcomeric protein replacement. Sarcomeric maintenance utilizes localized pools of mRNAs and translation machinery, yet the importance of localized translation remains unclear. In this issue of the JCI, Haddad et al. identify the Z-line as a critical site for localized translation of sarcomeric proteins, mediated by ribosomal protein SA (RPSA). RPSA localized ribosomes at Z-lines and was trafficked via microtubules. Cardiomyocyte-specific loss of RPSA in mice resulted in mislocalized protein translation and caused structural dilation from myocyte atrophy. These findings demonstrate the necessity of RPSA-dependent spatially localized translation for sarcomere maintenance and cardiac structure and function.

Imaging of Existing and Newly Translated Proteins Elucidates Mechanisms of Sarcomere Turnover.

BACKGROUND: How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres. METHODS: We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands. RESULTS: We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells. CONCLUSIONS: Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles.

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.

ELM2-SANT Domain-Containing Scaffolding Protein 1 Regulates Differentiation and Maturation of Cardiomyocytes Derived From Human-Induced Pluripotent Stem Cells.

BACKGROUND: ELMSAN1 (ELM2-SANT domain-containing scaffolding protein 1) is a newly identified scaffolding protein of the MiDAC (mitotic deacetylase complex), playing a pivotal role in early embryonic development. Studies on Elmsan1 knockout mice showed that its absence results in embryo lethality and heart malformation. However, the precise function of ELMSAN1 in heart development and formation remains elusive. To study its potential role in cardiac lineage, we employed human-induced pluripotent stem cells (hiPSCs) to model early cardiogenesis and investigated the function of ELMSAN1. METHODS AND RESULTS: We generated ELMSAN1-deficient hiPSCs through knockdown and knockout techniques. During cardiac differentiation, ELMSAN1 depletion inhibited pluripotency deactivation, decreased the expression of cardiac-specific markers, and reduced differentiation efficiency. The impaired expression of genes associated with contractile sarcomere structure, calcium handling, and ion channels was also noted in ELMSAN1-deficient cardiomyocytes derived from hiPSCs. Additionally, through a series of structural and functional assessments, we found that ELMSAN1-null hiPSC cardiomyocytes are immature, exhibiting incomplete sarcomere Z-line structure, decreased calcium handling, and impaired electrophysiological properties. Of note, we found that the cardiac-specific role of ELMSAN1 is likely associated with histone H3K27 acetylation level. The transcriptome analysis provided additional insights, indicating maturation reduction with the energy metabolism switch and restored cell proliferation in ELMSAN1 knockout cardiomyocytes. CONCLUSIONS: In this study, we address the significance of the direct involvement of ELMSAN1 in the differentiation and maturation of hiPSC cardiomyocytes. We first report the impact of ELMSAN1 on multiple aspects of hiPSC cardiomyocyte generation, including cardiac differentiation, sarcomere formation, calcium handling, electrophysiological maturation, and proliferation.

Filamin protects myofibrils from contractile damage through changes in its mechanosensory region.

Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton in a variety of shapes and tissues. In muscles, filamin crosslinks actin filaments from opposing sarcomeres, the smallest contractile units of muscles. This happens at the Z-disc, the actin-organizing center of sarcomeres. In flies and vertebrates, filamin mutations lead to fragile muscles that appear ruptured, suggesting filamin helps counteract muscle rupturing during muscle contractions by providing elastic support and/or through signaling. An elastic region at the C-terminus of filamin is called the mechanosensitive region and has been proposed to sense and counteract contractile damage. Here we use molecularly defined mutants and microscopy analysis of the Drosophila indirect flight muscles to investigate the molecular details by which filamin provides cohesion to the Z-disc. We made novel filamin mutations affecting the C-terminal region to interrogate the mechanosensitive region and detected three Z-disc phenotypes: dissociation of actin filaments, Z-disc rupture, and Z-disc enlargement. We tested a constitutively closed filamin mutant, which prevents the elastic changes in the mechanosensitive region and results in ruptured Z-discs, and a constitutively open mutant which has the opposite elastic effect on the mechanosensitive region and gives rise to enlarged Z-discs. Finally, we show that muscle contraction is required for Z-disc rupture. We propose that filamin senses myofibril damage by elastic changes in its mechanosensory region, stabilizes the Z-disc, and counteracts contractile damage at the Z-disc.

Loss of connectin novex-3 leads to heart dysfunction associated with impaired cardiomyocyte proliferation and abnormal nuclear mechanics.

Connectin (also known as titin) is a giant striated muscle protein that functions as a molecular spring by providing elasticity to the sarcomere. Novex-3 is a short splice variant of connectin whose physiological function remains unknown. We have recently demonstrated using in vitro analyses that in addition to sarcomere expression, novex-3 was also expressed in cardiomyocyte nuclei exclusively during fetal life, where it provides elasticity/compliance to cardiomyocyte nuclei and promotes cardiomyocyte proliferation in the fetus, suggesting a non-sarcomeric function. Here, we analyzed novex-3 knockout mice to assess the involvement of this function in cardiac pathophysiology in vivo. Deficiency of novex-3 compromised fetal cardiomyocyte proliferation and induced the enlargement of individual cardiomyocytes in neonates. In adults, novex-3 deficiency resulted in chamber dilation and systolic dysfunction, associated with Ca2+ dysregulation, resulting in a reduced life span. Mechanistic analyses revealed a possible association between impaired proliferation and abnormal nuclear mechanics, including stiffer nuclei positioned peripherally with stabilized circumnuclear microtubules in knockout cardiomyocytes. Although the underlying causal relationships were not fully elucidated, these data show that novex-3 has a vital non-sarcomeric function in cardiac pathophysiology and serves as an early contributor to cardiomyocyte proliferation.

Integrating Clinical Phenotype With Multiomics Analyses of Human Cardiac Tissue Unveils Divergent Metabolic Remodeling in Genotype-Positive and Genotype-Negative Patients With Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by sarcomere gene mutations (genotype-positive HCM) in ≈50% of patients and occurs in the absence of mutations (genotype-negative HCM) in the other half of patients. We explored how alterations in the metabolomic and lipidomic landscape are involved in cardiac remodeling in both patient groups. METHODS: We performed proteomics, metabolomics, and lipidomics on myectomy samples (genotype-positive N=19; genotype-negative N=22; and genotype unknown N=6) from clinically well-phenotyped patients with HCM and on cardiac tissue samples from sex- and age-matched and body mass index-matched nonfailing donors (N=20). These data sets were integrated to comprehensively map changes in lipid-handling and energy metabolism pathways. By linking metabolomic and lipidomic data to variability in clinical data, we explored patient group-specific associations between cardiac and metabolic remodeling. RESULTS: HCM myectomy samples exhibited (1) increased glucose and glycogen metabolism, (2) downregulation of fatty acid oxidation, and (3) reduced ceramide formation and lipid storage. In genotype-negative patients, septal hypertrophy and diastolic dysfunction correlated with lowering of acylcarnitines, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines. In contrast, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines were positively associated with septal hypertrophy and diastolic impairment in genotype-positive patients. CONCLUSIONS: We provide novel insights into both general and genotype-specific metabolic changes in HCM. Distinct metabolic alterations underlie cardiac disease progression in genotype-negative and genotype-positive patients with HCM.

Levosimendan's Effects on Length-Dependent Activation in Murine Fast-Twitch Skeletal Muscle.

Levosimendan's calcium sensitizing effects in heart muscle cells are well established; yet, its potential impact on skeletal muscle cells has not been evidently determined. Despite controversial results, levosimendan is still expected to interact with skeletal muscle through off-target sites (further than troponin C). Adding to this debate, we investigated levosimendan's acute impact on fast-twitch skeletal muscle biomechanics in a length-dependent activation study by submersing single muscle fibres in a levosimendan-supplemented solution. We employed our MyoRobot technology to investigate the calcium sensitivity of skinned single muscle fibres alongside their stress-strain response in the presence or absence of levosimendan (100 µM). While control data are in agreement with the theory of length-dependent activation, levosimendan appears to shift the onset of the 'descending limb' of active force generation to longer sarcomere lengths without notably improving myofibrillar calcium sensitivity. Passive stretches in the presence of levosimendan yielded over twice the amount of enlarged restoration stress and Young's modulus in comparison to control single fibres. Both effects have not been described before and may point towards potential off-target sites of levosimendan.

Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission.

Cofilin, an actin-severing protein, plays key roles in muscle sarcomere addition and maintenance. Our previous work found that Drosophila cofilin (DmCFL) knockdown in muscle causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA-sequencing analysis that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown resulted in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand our understanding of the roles of cofilin in muscle to include NMJ structural development and suggest that NMJ defects may contribute to the pathophysiology of nemaline myopathy.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance, often treated via electrical cardioversion. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs, suggesting that defects in contractility occur in AFib and are revealed upon restoration of rhythm. This project aims to define the contractile remodeling that occurs in AFib. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared with sinus rhythm atria. Atrial cardiomyocytes show reduced force of contraction, decreased resting tension, and increased calcium sensitivity in skinned single cardiomyocyte studies. These alterations correlated with degradation of myofilament proteins including myosin heavy chain altering force of contraction, titin altering resting tension, and troponin I altering calcium sensitivity. We measured degradation of other myofilament proteins, including cardiac myosin binding protein C and actinin, that show degradation products in the AFib samples that are absent in the sinus rhythm atria. Many of the degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. These results provide an understanding of the contractile remodeling that occurs in AFib and provide insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.NEW & NOTEWORTHY Atrial fibrillation is the most common cardiac rhythm disorder, and remodeling during atrial fibrillation is highly variable between patients. This study has defined the biophysical changes in contractility that occur in atrial fibrillation along with identifying potential molecular mechanisms that may drive this remodeling. This includes proteolysis of several myofilament proteins including titin, troponin I, myosin heavy chain, myosin binding protein C, and actinin, which is consistent with the observed contractile deficits.

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function.

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

PHF2 regulates sarcomeric gene transcription in myogenesis.

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.

The myosin chaperone UNC-45 has an important role in maintaining the structure and function of muscle sarcomeres during adult aging.

C. elegans undergo age-dependent declines in muscle organization and function, similar to human sarcopenia. The chaperone UNC-45 is required to fold myosin heads after translation and is likely used for refolding after thermally- or chemically-induced unfolding. UNC-45's TPR region binds HSP-90 and its UCS domain binds myosin heads. We observe early onset sarcopenia when UNC-45 is reduced at the beginning of adulthood. There is sequential decline of HSP-90, UNC-45, and MHC B myosin. A mutation in age-1 delays sarcopenia and loss of HSP-90, UNC-45, and myosin. UNC-45 undergoes age-dependent phosphorylation, and mass spectrometry reveals phosphorylation of six serines and two threonines, seven of which occur in the UCS domain. Additional expression of UNC-45 results in maintenance of MHC B myosin and suppression of A-band disorganization in old animals. Our results suggest that increased expression or activity of UNC-45 might be a strategy for prevention or treatment of sarcopenia.

Localized translation and sarcomere maintenance requires ribosomal protein SA in mice.

Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using proximity labeling, we identified ribosomal protein SA (RPSA) as a Z-line protein. In cultured cardiomyocytes, the loss of RPSA led to impaired local protein translation and reduced sarcomere integrity. By employing CAS9-expressing mice, along with adeno-associated viruses expressing CRE recombinase and single-guide RNAs targeting Rpsa, we knocked out Rpsa in vivo and observed mislocalization of ribosomes and diminished local translation. These genetic mosaic mice with Rpsa knockout in a subset of cardiomyocytes developed dilated cardiomyopathy, featuring atrophy of RPSA-deficient cardiomyocytes, compensatory hypertrophy of unaffected cardiomyocytes, left ventricular dilation, and impaired contractile function. We demonstrated that RPSA C-terminal domain is sufficient for localization to the Z-lines and that if the microtubule network is disrupted RPSA loses its sarcomeric localization. These findings highlight RPSA as a ribosomal factor essential for ribosome localization to the Z-line, facilitating local translation and sarcomere maintenance.

Fast myosin binding protein C knockout in skeletal muscle alters length-dependent activation and myofilament structure.

In striated muscle, the sarcomeric protein myosin-binding protein-C (MyBP-C) is bound to the myosin thick filament and is predicted to stabilize myosin heads in a docked position against the thick filament, which limits crossbridge formation. Here, we use the homozygous Mybpc2 knockout (C2-/-) mouse line to remove the fast-isoform MyBP-C from fast skeletal muscle and then conduct mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers present deficits in force production and calcium sensitivity. Structurally, passive C2-/- fibers present altered sarcomere length-independent and -dependent regulation of myosin head conformations, with a shift of myosin heads towards actin. At shorter sarcomere lengths, the thin filament is axially extended in C2-/-, which we hypothesize is due to increased numbers of low-level crossbridges. These findings provide testable mechanisms to explain the etiology of debilitating diseases associated with MyBP-C.

Differences in thick filament activation in fast rodent skeletal muscle and slow porcine cardiac muscle.

There is a growing appreciation that regulation of muscle contraction requires both thin filament and thick filament activation in order to fully activate the sarcomere. The prevailing mechano-sensing model for thick filament activation was derived from experiments on fast-twitch muscle. We address the question whether, or to what extent, this mechanism can be extrapolated to the slow muscle in the hearts of large mammals, including humans. We investigated the similarities and differences in structural signatures of thick filament activation in porcine myocardium as compared to fast rat extensor digitorum longus (EDL) skeletal muscle under relaxed conditions and sub-maximal contraction using small angle X-ray diffraction. Thick and thin filaments were found to adopt different structural configurations under relaxing conditions, and myosin heads showed different changes in configuration upon sub-maximal activation, when comparing the two muscle types. Titin was found to have an X-ray diffraction signature distinct from those of the overall thick filament backbone, and its spacing change appeared to be positively correlated to the force exerted on the thick filament. Structural changes in fast EDL muscle were found to be consistent with the mechano-sensing model. In porcine myocardium, however, the structural basis of mechano-sensing is blunted suggesting the need for additional activation mechanism(s) in slow cardiac muscle. These differences in thick filament regulation can be related to their different physiological roles where fast muscle is optimized for rapid, burst-like, contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned, graded response to allow for their substantial functional reserve. KEY POINTS: Both thin filament and thick filament activation are required to fully activate the sarcomere. Thick and thin filaments adopt different structural configurations under relaxing conditions, and myosin heads show different changes in configuration upon sub-maximal activation in fast extensor digitorum longus muscle and slow porcine cardiac muscle. Titin has an X-ray diffraction signature distinct from those of the overall thick filament backbone and this titin reflection spacing change appeared to be directly proportional to the force exerted on the thick filament. Mechano-sensing is blunted in porcine myocardium suggesting the need for additional activation mechanism(s) in slow cardiac muscle. Fast skeletal muscle is optimized for rapid, burst-like contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned graded response to allow for their substantial functional reserve.

The effect of muscle ultrastructure on the force, displacement and work capacity of skeletal muscle.

Skeletal muscle powers animal movement through interactions between the contractile proteins, actin and myosin. Structural variation contributes greatly to the variation in mechanical performance observed across muscles. In vertebrates, gross structural variation occurs in the form of changes in the muscle cross-sectional area : fibre length ratio. This results in a trade-off between force and displacement capacity, leaving work capacity unaltered. Consequently, the maximum work per unit volume-the work density-is considered constant. Invertebrate muscle also varies in muscle ultrastructure, i.e. actin and myosin filament lengths. Increasing actin and myosin filament lengths increases force capacity, but the effect on muscle fibre displacement, and thus work, capacity is unclear. We use a sliding-filament muscle model to predict the effect of actin and myosin filament lengths on these mechanical parameters for both idealized sarcomeres with fixed actin : myosin length ratios, and for real sarcomeres with known filament lengths. Increasing actin and myosin filament lengths increases stress without reducing strain capacity. A muscle with longer actin and myosin filaments can generate larger force over the same displacement and has a higher work density, so seemingly bypassing an established trade-off. However, real sarcomeres deviate from the idealized length ratio suggesting unidentified constraints or selective pressures.

Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.