American Journal of Physiology-Cell Physiology

BackgroundNeprilysin (NEP) is a zinc-dependent metalloprotease targeted in heart failure therapy to prevent it degrading circulating cardioprotective vasoactive peptides. NEP can also cleave sarcolipin (SLN), the skeletal- and atrial muscle-specific micropeptide regulator of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). A direct pathophysiological role of NEP in ventricular muscle has not been established. MethodsProteomics and immunoblot analysis of human myocardial specimens were used to quantify NEP abundance in failing and non-failing hearts. Heterologous protein expression and biochemical binding assays assessed NEP-mediated cleavage of phospholamban (PLB) and its impact on PLB-SERCA interactions. Functional consequences of NEP expression or inhibition were evaluated in neonatal rat ventricular myocytes and in a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of heart failure. ResultsWe observed increased NEP abundance in failing human myocardium relative to non-failing controls. We demonstrated that NEP cleaves phospholamban (PLB), disrupting PLB-SERCA interactions. Mutation of PLB (V49A), prevented NEP cleavage and preserved PLB-SERCA binding, indicating V49 is critical for NEP substrate recognition. In neonatal rat ventricular myocytes, NEP expression was associated with faster Ca2+ transient decay kinetics and increased SR Ca2+ load, consistent with reduced SERCA inhibition. Inhibition of NEP in a hiPSC-CM heart failure model attenuated the hypertrophic transcriptional responses and reversed Ca2+-transport dysregulation. ConclusionsThese findings implicate increased NEP expression in the sarcoplasmic reticulum of cardiomyocytes as previously unrecognized maladaptive consequence of heart failure contributing to cardiac dysfunction. In this novel pathophysiological mechanism, increased NEP results in PLB cleavage and loss of regulation of SERCA. While this may relieve SERCA inhibition and augment cellular Ca2+ handling, loss of PLB chronically disrupts hearts dynamic response to adrenergic stress, changing heart rate, or other physiological challenges. The data provide new insight into the cardioprotective effects of pharmacological NEP inhibition in clinical practice, reveal a novel mechanism of action of neprilysin inhibition in cardiomyocytes and may help inform future therapeutic strategies for patients with heart failure. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/732949v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@4daf93org.highwire.dtl.DTLVardef@41ef8aorg.highwire.dtl.DTLVardef@d568daorg.highwire.dtl.DTLVardef@d6d213_HPS_FORMAT_FIGEXP M_FIG C_FIG

Skeletal muscle homeostasis depends on the coordinated regulation of protein turnover and mitochondrial quality control; however, the molecular mechanisms linking these processes remain unclear. In this study, we examined the physiological role of leucine aminopeptidase 3 (LAP3), a post-proteolytic aminopeptidase, using constitutive LAP3-deficient mice. LAP3 deficiency preferentially affected skeletal muscle, causing reduced muscle mass and mitochondrial enlargement in both sexes. Female LAP3-deficient mice also showed reduced myofiber size, impaired endurance capacity, increased energy expenditure, elevated lipid oxidation, and lipid droplet accumulation adjacent to the mitochondria. Proteomic analyses revealed remodeling of pathways related to lipid metabolism and protein homeostasis. Consistent with these findings, LAP3 deficiency increased the expression of Pink1 and Tax1bp1 and promoted the accumulation of ubiquitinated proteins, suggesting alterations in mitochondrial quality control and proteostatic regulation. In cultured myogenic cells, LAP3 localized to mitochondrial fractions, and both LAP3 knockdown and overexpression altered mitochondrial morphology. Taken together, these results identify LAP3 as a regulator of skeletal muscle homeostasis and support a role for LAP3 in linking intracellular peptide turnover to mitochondrial homeostasis, with female skeletal muscle showing greater susceptibility to LAP3 deficiency.

Skeletal muscles secrete a variety of cytokines in response to inflammatory stimuli such as lipopolysaccharide (LPS); however, the contributions of resident macrophages or other non-muscle cells to the secretory responses are not well understood. To determine the potential impact of resident macrophages to inflammatory cytokine production, we tested the LPS responsiveness of isolated mouse soleus muscle when a critical toll receptor adapter protein (Myd88) was knocked down only in myeloid-derived cells within the muscle (e.g. resident macrophages). The phenotype is referred to as LyzMyd88-/- ; the litter mate controls were Myd88fl/fl. In solei from LyzMyd88-/- mice, cytokine secretory rates for interleukin-6 (IL-6) and keratinocyte-derived cytokine (KC, CXCL1) were significantly reduced to 56.3%, and 60.6% of control, respectively, over the first hour of LPS exposure. In the second hour, secretion of granulocyte colony stimulating factor (G-CSF), IL-6, KC(CXCL1) and monocyte chemoattractant protein-1 (MCP-1, CCL2) were greatly elevated by 5-10-fold in both phenotypes compared to the first hour. However, only MCP-1 secretion was decreased to 70.6% of control in the second hour. We also tested the secretory response to buffer containing 1% sterile mouse plasma because dilute plasma is known to amplify the responses of macrophages to LPS. Treatment with 1% plasma alone affected baseline measures of some cytokines but resulted in no further increases in secretion during either hour of exposure. However, small and gradual increases in secretory rates were observed for several cytokines over the study period, with or without plasma, with the largest responses seen in IL-6 and KC. Overall, the results are consistent with a significant early contribution of myeloid-derived, resident immune cells to the cytokine secretory responses of intact oxidative skeletal muscle. In addition, small quantities of plasma in the buffer have no independent stimulatory effects on cytokine secretion

Regular exercise induces adaptations in skeletal muscle and other organ systems to improve physical performance and overall health. Exercise results in phosphorylation of 5 AMP-activated protein kinase (AMPK) at threonine 172 (T172) of the 2 subunit; however, the role of this activation in cellular and functional adaptations has not been elucidated. To this end, we subjected non-activatable Ampk2(T172A) knock-in (KI) adult mice and wild-type (WT) littermates to 4 weeks of voluntary wheel running (VWR). Exercise training led to significant improvements in endurance capacity, maximal oxygen consumption ([Formula]O2max), and glucose tolerance, as well as skeletal muscle IIb-to-IIa fiber type shift in both WT and KI mice. Contrastingly, VWR resulted in increased mitochondrial OxPhos protein expression, mitochondrial volume density, and capillary density in skeletal muscle of WT but not KI mice. Exercise-induced improvements of mitochondrial respiration and conductance revealed by high-resolution respirometry of isolated mitochondria were blunted in KI mice. Therefore, for the first time, we reveal that AMPK2 T172 activation is required for exercise training-induced mitochondrial biogenesis, improvement of mitochondrial respiratory function, and angiogenesis in skeletal muscle, but that these adaptations are not solely responsible for improved [Formula]O2max and exercise endurance capacity. Significance StatementExercise is the most effective lifestyle intervention for promoting health and preventing chronic diseases through adaptive changes in skeletal muscle and many other tissues/organs. AMPK is an energy sensor and signaling regulator for exercise-induced skeletal muscle adaptation, yet its functional role and the impact on exercise capacity have been studied in mouse genetic models wherein protein stoichiometry is disrupted. Using non-activatable Ampk2(T172A) knock-in mice, we ascertained that AMPK2 activation via T172 phosphorylation is required for endurance training-induced mitochondrial and angiogenic adaptations in skeletal muscle. Importantly, these adaptations are not required for improved exercise capacity, challenging the prevailing concept that increased mitochondrial content and function and microvasculature are the sole driving factors for the performance gains with endurance training.

ABSTRACT Background: Athletes who return to soccer after anterior cruciate ligament reconstruction (ACLR) remain at elevated risk of secondary injury despite meeting conventional discharge criteria, and neuromuscular deficits in the reconstructed limb are known to be exposed by fatigue. Objective: To determine whether match-play fatigue differentially affects muscle stiffness, countermovement jump (CMJ) force symmetry, and rate of force development (RFD) asymmetry between soccer players with a history of ACLR and uninjured teammates. Methods: A prospective, cross-sectional, matched-control study enrolled 128 competitive soccer players (64 ACLR, 6-22 months post-surgery; 64 uninjured controls) across five recruitment waves (February-June 2026). Bilateral CMJ peak vertical force, jump height, RFD, and myotonometric stiffness of the rectus femoris (RF), vastus medialis (VM), and biceps femoris (BF) were recorded immediately before and after a standardized competitive match. Fatigue was quantified from second-half heart rate (percentage of age-predicted maximum) and end-match rating of perceived exertion (RPE). Within-group pre-to-post changes were evaluated with paired t-tests, between-group differences in the magnitude of change with independent-samples t-tests, and associations between fatigue indices and asymmetry changes with Pearson correlations. Results: Match play reduced CMJ limb symmetry index (LSI) in both groups, but the decline was more than three-fold greater in the ACLR group, 92.6% (SD 5.4%) to 85.1% (SD 7.1%), than in control group, 97.3% (SD 3.9%) to 95.0% (SD 4.2%), group-by-time difference, p < 0.001, (d = 0.64). RFD asymmetry approximately doubled in the ACLR group, 10.6% (SD 4.1%) to 17.6% (SD 6.5%), compared with a smaller rise in control group, 4.6% (SD 2.4%) to 6.3% (SD 3.7%); p < 0.001, d = 0.77). Involved-limb stiffness losses in the ACLR group exceeded those of controls for the RF (-21.2 vs. -9.2 N/m, p < 0.001), VM (-17.7 vs. -6.1 N/m, p < 0.001), and BF (-13.3 vs. -6.6 N/m, p < 0.001), whereas uninvolved-limb stiffness losses did not differ between groups (all p > 0.05). Fatigue markers (heart rate, RPE) were not significantly correlated with the magnitude of individual asymmetry change (|r| [&le;] 0.18, p > 0.15). Conclusions: In competitive soccer players 6-22 months after ACLR, match-play fatigue selectively compromises stiffness and explosive force output of the reconstructed limb, widening inter-limb asymmetries beyond what is seen in uninjured teammates, even though global cardiovascular and perceptual fatigue were comparable between groups. These findings suggest that return-to-sport testing performed only in a rested state may underestimate residual neuromuscular deficits, and support fatigue-inclusive assessment protocols before athletes are cleared for unrestricted competition. Abbreviations: ACL: anterior cruciate ligament, ACLR: anterior cruciate ligament reconstruction, BF: biceps femoris, CMJ: countermovement jump, HRmax: maximum heart rate, LSI: limb symmetry index, RF: rectus femoris, RFD: rate of force development, RPE: rating of perceived exertion, RTS: return to sport, VM: vastus medialis, SD: standard deviation. Keywords: Anterior cruciate ligament reconstruction, muscle fatigue, muscle stiffness, countermovement jump, limb symmetry index, rate of force development, soccer, return to sport.

Background Ischemic heart failure (IHF) has been shown to impair contractility and disrupt sarcomere function in the left ventricle. Left ventricular failure can cause left atrial dysfunction, which is associated with a greater risk of patient mortality. Despite this, the biochemical and biomechanical characteristics of the left atrium in IHF remain obscure. Methods Myocardial mechanical properties were measured using permeabilized muscle isolated from the left ventricle (LV) and left atrium (LA) of donors and patients with IHF. Tissue homogenates from these samples were used to measure titin and myosin isoforms as well as the phosphorylation of sarcomeric regulatory proteins. Histology was used to quantify fibrosis in the patients' left ventricle and left atrium. Results Length-dependent changes in Ca2+-sensitivity were blunted in LV myocardium from patients with IHF. LA myocardium did not show robust length-dependence of Ca2+-dependent force. The calcium sensitivity of both LA and LV myocardium was increased in IHF. The maximum force generated by LV but not LA myocardium was decreased in IHF. LA myocardial samples exhibited faster contractile kinetics than LV samples, irrespective of disease. Troponin I phosphorylation decreased in both chambers with IHF. Conclusions Left atrial IHF myocardium maintained contractile force and displayed increases in calcium sensitivity, which may allow for increased LA contraction under pathological conditions. The increases in calcium sensitivity observed in ischemic myocardium of both chambers are likely driven by decreased phosphorylation of troponin I, which alters thin filament regulation. Conversely, thick filament properties of the left ventricle, such as thick filament protein isoforms and phosphorylation of myosin binding protein-C, displayed chamber-specific differences independent of disease state. These biochemical changes may explain the chamber-specific differences in kinetics and length-dependent properties. Collectively, these biophysical and biochemical data suggest LA remodeling in IHF may assist in increasing LV end-diastolic volume to maintain adequate cardiac output.

The neuromuscular junction (NMJ) is a specialised synapse between motor neurons and skeletal muscle, and its progressive deterioration contributes to age-related and metabolic disease-associated declines in muscle function. Advanced glycation end-products (AGEs) accumulate in tissues during ageing, diabetes, and chronic metabolic dysfunction and have been implicated in neuromuscular degeneration, yet their effects on the intact NMJ have not previously been examined in a human model system. This study employed a fully human, serum-free, and neural growth factor-free NMJ co-culture system, combining neural progenitor cells with immortalised human myoblasts derived from an 83-year-old donor, to investigate the effects of AGE exposure on neuromuscular integrity across structural, metabolic, functional, and secretory outcomes. AGE exposure induced significant reductions in motor neuron axonal length, myotube remodelling with centralised nuclear positioning, mitochondrial membrane depolarisation, elevated mitochondrial superoxide production, mitochondrial uncoupling, and reductions in spontaneous contraction intensity and frequency. Neurotrophic and myogenic growth factor signalling was significantly downregulated in AGE-treated co-cultures. These findings identify the NMJ as a sensitive target of glycation stress and establish this fully human co-culture platform as a physiologically relevant model for investigating glycation-related neuromuscular pathology and evaluating candidate therapeutic interventions.

Pedalling cadence during an acute eccentric cycling exercise altered physiological and perceptual responses. We examined the influence of cycling cadence on neuromuscular adaptation induced by a 6-week eccentric cycling training period. Eighteen participants performed training (eighteen sessions) at a cadence of 30 or 60 rpm over six weeks. Power output was the same between the two groups. Perceived effort and heart rate were recorded at each training session. Muscle pain and fatigue were reported the day after each session. Maximal voluntary contractions torque, as well as concentric and eccentric cycling efficiency, were assessed before and after training. Additionally, the loss of maximal voluntary isometric torque was assessed after the first and last training sessions. Heart rate and perceived effort increased in the second week of training and then plateaued, with no difference between groups. Muscle pain and fatigue remained low throughout the training, with no difference between groups. Isometric (+28%) and eccentric (+13%) maximal voluntary torque of knee extensor muscles increased regardless of training cadence. Concentric maximal voluntary torque increased for the group pedalling at 60 rpm only (+21%). Cycling efficiency was improved in eccentric mode only (+43%), with no difference between the two training groups. Finally, the voluntary isometric torque loss induced by the first and last sessions were similar. While six weeks of eccentric cycling training improved neuromuscular and functional capacities, cadence had no observable effect. This finding suggest that patients could choose their preferred cadence to obtain better adherence to the rehabilitation program without altering the adaptations.

Identification of signaling networks is an essential goal in systems biology. Here, we use CRISPR/Cas9 knockout screening (employing a whole kinome sgRNA library) to identify functionally critical protein kinases in a well-studied Gs-dependent G-protein coupled receptor (GPCR)-signaling model, namely the vasopressin V2 receptor (V2R) pathway. Screening was done using a specially-designed fluorescence-based reporter cell line with green-fluorescent protein (GFP) co-transcribed with Aqp2, a gene whose transcription is dependent on vasopressin-mediated activation of protein kinase A (PKA). Positive regulators (n=14) included PKA-catalytic subunit (Prkaca) and Dyrk1a (minibrain homolog). Negative regulators (n=12) included PKA-regulatory subunit type I, Stk11 (catalytic subunit of liver kinase B1 [LKB1] complex), and three TGF-{beta} receptor subunits (Tgfbr1, Tgfbr2, Tgfbr3) (see https://esbl.nhlbi.nih.gov/Databases/Kinome-CRISPR-screen/ for full list). Dyrk1a knockout cell lines failed to express AQP2 protein and exhibited a profound decrease in AQP2 mRNA. RNA-sequencing demonstrated widespread increases in cell-cycle transcripts, with a general defect in cell differentiation, accounting for AQP2 loss. TGF-{beta} exposure to un-transformed cells results in a profound decrease in V2R and AQP2 mRNA abundance along with multiple additional transcriptional targets of V2R-PKA signaling, consistent with prior findings in TGF-{beta}-mediated vasopressin escape. Stk11/LKB1 knockout lines displayed marked increases in AQP2 protein and mRNA, even in the absence of vasopressin. RNA-sequencing showed a marked similarity between the responses to Stk11/LKB1 deletion and vasopressin exposure in untransformed cells. Phospho-proteomic data point to opposing roles of Stk11/LKB1 and PKA in the regulation of cAMP-responsive transcriptional coactivator (CRTC) proteins in the transcriptional response to V2R-PKA signaling. Significance StatementCells throughout the body are regulated by extracellular signals like the hormone, vasopressin. Hormonal effects on cellular function are mediated by membrane receptors that trigger biochemical changes, often by inducing chemical modification of the amino acids making up individual proteins, such as addition of function-altering phosphate groups (phosphorylation). Protein phosphorylation is mediated by enzymes known as "protein kinases". Here, we have screened all known protein kinases using modern CRISPR/Cas9 technology to identify those involved in vasopressin action in the kidney. As expected from prior knowledge, the screen identified protein kinase A and one of its regulatory subunits, but also identified several protein kinases not previously implicated in vasopressin action in the kidney.

Obesity is associated with an elevated risk of pathological cardiac hypertrophy, whereas exercise confers cardioprotective effects; however, the cellular mechanisms underlying these opposing influences remain incompletely defined, particularly in females. We investigated how obesity and exercise affect cardiomyocyte ultrastructure, Ca{superscript 2} release, and contractility in female Zucker Diabetic Fatty rats and their lean littermates. Animals were assigned at 12 weeks to sedentary or aerobic exercise-trained groups and maintained on a standard diet. By 18 weeks, obese rats exhibited increased body mass and myocardial hypertrophy in the absence of diabetes. Sedentary obese animals showed a reduced fraction of compact dyads and diminished stimulated and caffeine-induced Ca{superscript 2} release, while contractility remained preserved. In lean rats, exercise increased dyad density but reduced Ca{superscript 2} release, whereas in obese rats, exercise enhanced both dyad compactness and Ca{superscript 2} release. Across all groups, global cardiomyocyte ultrastructure and contractile function were similar. Type III ANOVA revealed a significant obesity x exercise interaction for dyadic structure and Ca{superscript 2} release. These findings demonstrate that obesity itself, independent of diabetes, triggers early dyadic remodeling and altered Ca{superscript 2} handling in female myocardium before detectable impairment of global cardiomyocyte structure or contractile function. Furthermore, exercise exerts beneficial effects on dyadic ultrastructure and Ca{superscript 2} signaling in obese animals. New & NoteworthyUsing a female rat model of obesity without diabetes, we demonstrate that obesity induces early remodeling of the dyadic system and impairs Ca{superscript 2} release in cardiac myocytes. We further show that the effects of aerobic exercise on dyadic structure and function are obesity-dependent, improving both dyad organization and Ca{superscript 2} signaling. These findings identify the dyadic microdomain as a vulnerable cellular site in obesity and a potential target for exercise-induced recovery.

Glucagon-like peptide 1 receptor agonists (GLP-1RAs) have become defining therapies in the management of type 2 diabetes and obesity. Despite recent interest in the effects of GLP-1RA therapy on skeletal muscle, their influence on muscle repair after injury remains largely untested. Because GLP-1RA use is common in populations at heightened risk for diminished regenerative capacity, a critical unanswered question is whether GLP-1R agonism supports muscle regeneration or alters the normal course of recovery after injury. Using intramuscular glycerol injection as an adipogenic injury model, we assessed whether semaglutide, a widely prescribed GLP-1RA, alters the balance between myogenesis and adipogenesis during regeneration. Surprisingly, semaglutide treatment markedly increased the formation of intramuscular adipose tissue (IMAT) and inhibited the growth of regenerated fibers. These effects were injury-dependent, as uninjured muscle showed no detectable differences in IMAT or myofiber size. Together, these findings identify a previously underappreciated context in which GLP-1RA therapy may adversely affect muscle quality.

Laminin-2 (gene: LAMA2) is a key protein in the basement membrane of muscle and Schwann cells. A complete lack of this protein results in LAMA2-related congenital muscular dystrophy (LAMA2-RD), a severe muscle disease characterized by progressive muscle weakness, respiratory insufficiency, failure to thrive and shortened life span. One key signature of this disease is early onset of fibrosis coupled with poor muscle growth. We previously showed that TGF-{beta} and its activator, integrin-V, are elevated in dystrophic fibers of DyW mice, a mouse model of LAMA2- RD. Other than activating TGF-{beta}, integrin-V is also known to facilitate the transdifferentiation of various cell types to myofibroblasts. In this study we present evidence for transcriptional dysregulation of genes driving myofibroblast transdifferentiation and extracellular matrix (ECM) remodelling during the early development of DyW mice that is also reflected in muscle biopsies from young LAMA2-RD patients. We hypothesize that the early ECM remodelling, seen in both DyW mice and LAMA2-RD children, may explain the congenital onset of fibrosis with poor muscle growth seen in the disease.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold tremendous promise for disease modeling, drug discovery, and cardiac regenerative therapies. However, the immature phenotype of hPSC-CMs remains a major barrier limiting their translational utility. Here, we performed integrated multi-omic profiling to identify molecular pathways and regulatory programs associated with hPSC-CM maturation during long-term culture. hPSC-CMs were cultured for 113 days and analyzed using metabolomics, proteomics, and transcriptomics across progressive stages of maturation. Long-term culture induced widespread multi-omic remodeling, including significant changes in 142/934 metabolites, 550/3,556 proteins, and 2,892/23,309 transcripts from Day 30 to Day 113. Metabolomic analyses revealed early increases in phospholipid biosynthesis and mitochondrial beta oxidation of fatty acids from Day 30 to Day 60, suggesting metabolic priming precedes later maturation events. In contrast, proteomic remodeling was more prominent during later stages of maturation and was characterized by enhanced calcium handling and cell cycle exit. Transcriptomic analyses demonstrated progressive increases in ion channel expression, t-tubule organization, fatty acid metabolism, creatine shuttle pathways, and cell cycle arrest programs. Transcriptomic and integrative multi-omic pathway analyses identified coordinated suppression of TGF{beta}, MAPK, Wnt, and Hedgehog signaling together with activation of integrin-related, respiratory electron transport, muscle contraction, and Slit-Robo signaling pathways during maturation. Moreover, multi-omic transcription factor activity analysis prioritized a GATA4-centered network of putative cardiomyocyte maturation regulators including SOX7, SOX18, TBX2, and ZFPM2 (FOG2). Together, these findings elucidate the degree and pace of hPSC-CM maturation during long-term culture and establish an integrated multi-omic framework for identifying strategies to accelerate hPSC-CM maturation.

Hereditary spastic paraplegia type 7 (SPG7) is a neurodegenerative disorder characterized by progressive motor impairment and cerebellar dysfunction. Mutations in the SPG7 gene, encoding the mitochondrial metalloprotease paraplegin, disrupt mitochondrial homeostasis and lead to neuronal vulnerability and deficits in motor coordination. Recent studies have identified defective flickering of the mitochondrial permeability transition pore (mPTP) in SPG7 models, suggesting that altered pore dynamics may represent a functional biomarker of mitochondrial dysfunction. Here, we investigated whether pharmacological modulation of mPTP activity could improve mitochondrial function and motor performance in SPG7 models. Mitochondrial flickering was assessed in vitro, while motor behavior was evaluated in vivo following chronic treatment with berberine, a natural isoquinoline alkaloid known to modulate mitochondrial bioenergetics. Spg7-/- mice and age-matched Spg7+/ littermate controls received daily oral berberine administration for several weeks, and motor coordination was assessed using the accelerating rotarod test. Untreated Spg7-/- mice exhibited reduced rotarod performance compared with controls, indicating impaired motor coordination. Berberine treatment significantly improved motor performance in pre-symptomatic mutant mice. These findings indicate that pharmacological modulation of mitochondrial permeability transition pore dynamics can ameliorate motor dysfunction associated with SPG7 deficiency and highlight mPTP flickering as a functional readout of mitochondrial health.

Human skeletal muscle is the principal site of insulin-stimulated glucose disposal and a major mediator of exercise-induced metabolic benefits, yet human models that preserve metabolic and exercise responsiveness remain limited. We generated primary human skeletal muscle organoids from donor-derived CD56+ myoblasts using a collagen-based extracellular matrix and serum-free IGF1-guided differentiation. The organoids formed aligned contractile tissues containing oxidative and glycolytic fiber type-like myotubes, displayed enhanced mitochondrial respiration, insulin-stimulated glucose uptake, and reproducible force generation. Electrical pulse stimulation induced AMPK activation, increased glucose utilization and lactate production, and upregulated canonical exercise-responsive genes including NR4A3 and PPARGC1A. Notably, transcriptional responses to in vitro exercise overlapped with acute exercise responses observed in skeletal muscle biopsies from the same donors. The organoids further detected functional impairments of skeletal muscle performance induced by TGF-{beta}1 and metformin and increased speed generation by testosterone treatment. These findings establish a donor-specific human skeletal muscle platform that recapitulates key features of insulin action and exercise adaptation and may enable mechanistic studies of skeletal muscle metabolism, exercise responsiveness, and therapeutic interventions relevant to diabetes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/735246v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@2ea1c9org.highwire.dtl.DTLVardef@17fa8c1org.highwire.dtl.DTLVardef@2045d5org.highwire.dtl.DTLVardef@c8b059_HPS_FORMAT_FIGEXP M_FIG C_FIG Article highlightsWe generated primary human skeletal muscle organoids under serum-free IGF1-guided conditions to reproduce key metabolic and exercise-responsive features of skeletal muscle. The organoids were insulin-responsive, displayed enhanced mitochondrial function and force-generating contractility, reproduced hallmark molecular and metabolic responses to exercise, overlapping with exercise responses observed in the same donors in vivo. The organoids were suitable to detect functional alterations after treatment with endogenous hormones and cytokines and diabetes medication This platform provides a human donor-specific system for studying skeletal muscle mechanisms underlying insulin sensitivity, exercise benefits, and therapeutic responses relevant to diabetes and metabolic disease.

Neuromuscular contractures arising from neonatal brachial plexus injuries (NBPI) are highly disabling and currently incurable. We previously showed that contractures involve impaired longitudinal growth of denervated muscles, a defect mediated through myostatin (MSTN) signaling, a potent negative regulator of muscle size. However, MSTN-mediated contractures occur independent of canonical signaling pathways, including SMAD 2/3 and AKT/mTOR. Through a mouse model of NBPI, our present study extended these findings by revealing pharmacologic inhibition of JNK signaling, a noncanonical pathway downstream of MSTN, partially rescues contractures without restoring muscle length. Rather, JNK activation upregulates myofiber expression of the target gene Lmna, which encodes the nuclear envelope proteins Lamin A and Lamin C that are vital for nuclear stability, resulting in pervasive myonuclear displacement. These results suggest that other factors contribute to contracture pathology beyond deficits in longitudinal muscle growth. Further, while JNK inhibition does not restore length of denervated muscles, it impedes size and mass of normally innervated neonatal muscles, suggesting a requirement of JNK signaling for neonatal muscle growth. Our collective findings thereby establish new mechanistic insights into the molecular basis of aberrant muscle growth and neuromuscular contracture formation, potentially leading to novel targets for muscle restorative strategies and medical contracture prevention.

AimsWhile the muscle-enriched microRNA-378a (miR-378a) has been implicated in cardiac hypertrophy and stress responses, its role in maintaining cardiomyocyte metabolic homeostasis, mitochondrial function, and angiogenic paracrine signaling under physiological and post-injury conditions remains unclear. This study addresses these gaps by examining the molecular and functional consequences of miR-378a deficiency in murine heart and human cardiomyocytes. Methods and ResultsCardiac structure and function were analyzed in miR-378a-deficient (miR-378a-/-) and wild-type (miR-378a+/+) mice at 12 weeks and 17 months of age, revealing that miR-378a loss promoted myocardial fibrosis, altered IGF1R-AKT signaling, and impaired cardiac performance, with age-dependent effects. Integrated transcriptomic and proteomic analyses in miR-378a-/- and control mice, as well as in human iPSC-derived cardiomyocytes (hiPSC-CM) of both genotypes, revealed deregulated pathways related to translation, metabolism, and cardiomyopathy-associated signaling. In hiPSC-CM, miR-378a knockout (KO) impaired mitochondrial respiration, disrupted mitochondrial morphology, and reduced mitochondrial DNA content, accompanied by altered mitophagy and biogenesis. KO cells also showed increased glucose uptake but reduced glycogen storage, accompanied by changes in key metabolic regulators, and displayed diminished angiogenic potential. Finally, hiPSC-CM overexpressing miR-378a were delivered in a mouse model of acute myocardial infarction, but overexpression did not further enhance their therapeutic effect. ConclusionsThis study broadens our understanding of miR-378as physiological role in murine hearts and human cardiomyocytes, demonstrating its impact on contractility, mitochondrial integrity, glucose metabolism, and angiogenic paracrine signaling. However, overexpression of miR-378a in hiPSC-CM offers limited additional benefit in cell therapy for acute myocardial infarction.

The endoplasmic reticulum (ER) and its muscle-specialized form, the sarcoplasmic reticulum (SR), are crucial organelles in muscle cells, involved notably in protein synthesis, calcium regulation and muscle contraction. A well-known process involved in ER remodeling and homeostasis is ER-phagy, also called reticulophagy, a selective form of autophagic process in which ER-phagy receptors mediate the delivery of ER portions to lysosomes for degradation. SH3KBP1 is an adaptor protein involved in membrane trafficking. Recently, it was shown to control ER morphology and SR formation in striated skeletal muscle. In this study, we demonstrate that SH3KBP1 can bind to LC3B and CKAP4 proteins, bridging ER to autophagosome membranes, and is degraded by autophagy, in developing muscle fibers. Moreover, SH3KBP1 down-regulation impacts basal autophagy efficiency and ER-phagy stimulation; it also impairs the turnover of numerous ER-resident proteins. Our work highlights a new role for SH3KBP1 as a soluble ER-phagy receptor in striated skeletal muscle.

Myotonic Dystrophy type 1 (DM1) is an autosomal multisystem disorder manifested due to unstable CTG nucleotide repeat expansion within the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Although progress towards understanding of molecular pathogenesis in muscle and heart has been made, the pathways that affect the brain in DM1 is fundamentally unknown. In addition, the congenital DM1 manifest even more complicated brain abnormalities. Despite the wealth of existing cellular and animal models, iPSCs based studies are being fostered as they replicate the human model more closely to the disease. In view of this context, we set out to characterize the differentiation potential of congenital DM1 patient derived iPSC lines towards neuronal cells. Using neurogenin2 (NGN2) induced direct reprogramming of iPSCs into neurons and chemically defined media-induced neural induction protocol, we find that congenital DM1 mutant iPSC derived neurons exhibited precocious differentiation, as evidenced by their expression of pan-neuronal markers TUJ1 and Map2, along with increased processes extension and neurite length. Moreover, unbiased RNA sequencing analyses and qPCR validation revealed precocious and enhanced expression of several neurogenic transcription factors including, Ascl1, NeuroG2, and NeuroD1. Furthermore, immunofluorescence imaging of MBNL1 and MBNL2, RNA-splicing factors, displayed enhanced nuclear aggregations, a hallmark of the DM1 disease, in the mutant lines. Moreover, investigation of RNA splicing events identified mis-splicing in many important genes/transcripts including RMST, ANK3 and MBD1 during the neural conversion of congenital DM1 lines. These studies reveal novel paradigms that may contribute to neurological pathogenesis in CDM1 patients. These studies also provide a strong foundation for future mechanistic investigation aimed at understanding CDM1 pathology and may open new avenues for the development of gene therapy approaches for individuals with DM1.

Resistance training lasting a few weeks increases maximal force mainly through neural adaptations that enhance the drive from the nervous system to muscle. While these adaptations have been well documented at the motor unit (MU) level during submaximal force contractions, the mechanisms underlying force increases during maximal voluntary contractions are poorly understood. This is due to a classic technical limitation in tracking MUs longitudinally during maximal force tasks. Here, we solved this technical challenge, enabling the investigation of MU adaptations during MVCs in both the trained and untrained limbs following unilateral resistance training. High-density surface electromyography was recorded from the biceps brachii of both limbs before and after a 4-week unilateral resistance-training intervention, and the same MUs were longitudinally tracked across sessions during MVCs by concatenation of three MVC trials of ~5-s each.Unilateral training increased maximal force in the trained limb (+16%) and induced strength transfer to the untrained limb (+8%). In both limbs, maximal contractions after training were characterized by greater EMG amplitude, faster muscle-fiber conduction velocity, and higher MU discharge rates, indicating enhanced neural drive to the motoneuron pool. These adaptations were strongly associated with improvements in maximal force (R2 > 0.7 for all). Importantly, longitudinal MU tracking revealed a non-uniform adaptation across the MU pool: MUs with higher baseline conduction velocity, indicative of higher recruitment threshold, exhibited the largest pre-post increases in discharge rate, whereas lower-threshold units showed smaller changes. Collectively, these findings demonstrate that gains in maximal force and their transfer to the untrained limb are primarily mediated by enhanced rate coding of higher-threshold MUs during MVCs.