American Journal of Physiology-Cell Physiology

Store-operated calcium entry (SOCE) is a ubiquitous calcium (Ca2+) signalling mechanism that replenishes endoplasmic and sarcoplasmic reticulum Ca2+ stores. We previously identified STIM1L, a skeletal muscle-enriched STIM1 splice variant that enables rapid SOCE activation in human myotubes. To determine its physiological role in vivo, we generated a constitutive knockout mouse model that selectively abolishes STIM1L expression while preserving the canonical STIM1 isoform. Stim1l-KO mice developed normally and exhibited no overt abnormalities, but both sexes showed reduced voluntary wheel-running activity, suggesting neuromuscular perturbation. Skeletal muscle deprived of STIM1L did not show any histological alterations or changes in fibre- type and size. Likewise, force-frequency relationships and fatigue resistance measured ex vivo in isolated extensor digitorum longus and soleus muscles were indistinguishable from control muscles. This lack of difference may be explained by the increased expression of STIM1 in several skeletal muscles and suggests that changes in the voluntary running activity of Stim1l-KO mice may arise from STIM1L loss in non-muscular tissues, including the central nervous system. Notwithstanding, primary myotubes derived from Stim1l-KO mice displayed a marked delay in SOCE activation kinetics, whereas SOCE amplitude remained comparable to controls. This was consistent with a compensatory STIM1 upregulation maintaining overall Ca2+ entry capacity. Interestingly, electrically evoked Ca2+ transients in intact Stim1l-KO flexor digitorum brevis fibres exhibited significantly faster decay kinetics than controls. This revealed another role of STIM1L in Ca2+ handling in skeletal muscle that appears independent of its contribution to SOCE activation. Together, these findings establish STIM1L as a regulator of SOCE activation kinetics and indicate an additional role in myofibre Ca2+ clearance, while suggesting that compensatory upregulation of canonical STIM1 may preserve muscle structure and contractile function in its absence in mice.

Skeletal muscle is a dynamic tissue able to regenerate in response to injury. This regeneration relies primarily on the activation of precursor cells, known as muscle stem cells (MuSC). Upon activation, skeletal MuSC re-enter the cell cycle and proliferate as myoblasts, which subsequently either differentiate and fuse to form new fibers or return to quiescence to maintain long-term regenerative capacity. From primary human myoblasts, we generated reserve cells (RCs), quiescent cells similar to MuSC, and investigated their activation after stimulation with a growth medium containing serum. Here, we show that Orai3 calcium channel is primarily expressed in RC, where it promotes RC activation in a calcium-independent manner. We demonstrate that Orai3 does not contribute to store-operated calcium entry (SOCE) or calcium response induced by serum stimulation in RC cells, supporting the hypothesis that Orai3 plays a unique role in RC, distinct from its calcium channel activity. Using a protein proximity assay (BioID), we identified the large scaffold protein AHNAK2 as a potential partner of Orai3. AHNAK2 is expressed at high level in myotubes, and has a similar effect on RC activation than Orai3. Both Orai3 and AHNAK2 are necessary for proper myoblast differentiation and RC activation, while acting through different signaling pathways. Specifically, we show that Orai3 directly influences the RC fate, whereas AHNAK2 facilitates RC activation via signals derived from myotubes. These findings provide new insights into the molecular mechanisms underlying the activation and quiescence of human MuSC.

Longitudinal muscle growth occurs through the addition of sarcomeres in series, a process termed serial sarcomerogenesis. To investigate contraction mode dependence of sarcomerogenesis in Sprague-Dawley rats (n=36, 14 to18 weeks, ~468g), we compared the effects of isometric, eccentric, and concentric training (4 to 6 weeks; 3 times/week) on morphological and mechanical adaptations of the plantar flexors. Isometric training was performed at a joint angle corresponding to the plateau nearing the descending limb of the force length relationship, eccentric contractions began at a shorter muscle length and lengthened to this same joint position, and concentric contractions started at this lengthened position but then actively shortened. Owing to the rapid regulation of serial sarcomere number the 4-and 6-week timepoints were collapsed. While force output increased across all training modes (+16%; p<0.05), longitudinal muscle growth was regulated by contraction type. Both isometric and eccentric training were highly effective at increasing serial sarcomere number when optimal loading conditions (high forces at longer lengths) are provided (+7-9%; p<0.05). Concentric training, even when initiated at long lengths with high forces was not an effective stimulus for longitudinal remodelling (+2%; p>0.05). Therefore, these findings identify the mechanical loading environment associated with isometric and eccentric training as a key driver of serial sarcomerogenesis.

Axonal action potential propagation critically depends on tightly regulated intra and extracellular electrolyte concentrations and acid base homeostasis. Experimental studies have demonstrated that changes in extracellular sodium, potassium, and calcium concentrations alter axonal excitability and conduction properties. However, it remains unknown whether small variations in electrolyte concentrations within the physiological range are associated with measurable changes in nerve conduction velocity in humans. In this study, data from neurologically healthy children who underwent both nerve conduction studies and blood gas analysis were analyzed to investigate the relationship between electrolyte concentrations and median motor nerve conduction velocity. Nerve conduction velocity was significantly negatively correlated with ionized calcium concentration and positively correlated with sodium concentration, whereas no significant correlation was observed for potassium. In an a priori defined multiple linear regression model, ionized calcium, sodium, and bicarbonate remained significantly associated with nerve conduction velocity after correction for multiple testing. These findings indicate that even small variations in electrolyte concentrations within the physiological range are associated with measurable differences in peripheral nerve conduction velocity and suggest an additional contribution of acid base status to nerve conduction.

Background: Human induced pluripotent stem cell-derived cardiomyocytes are valuable for cardiac disease modelling and drug testing but remain limited by immature structural and electrophysiological properties in conventional two-dimensional cultures. Three-dimensional cardiac models, including cardioids, provide a more physiological environment through multicellular organization and enable tissue-level functional analysis. Their electrophysiological properties and maturation, however, remain incompletely characterized. Methods: We performed an integrated electrophysiological characterization of human cardioids over the first three weeks of maturation using sharp microelectrode recordings, calcium transient measurements, whole-cell patch clamp recordings, immunofluorescence, transcriptomic analysis, and pharmacological interventions. Results: During maturation, cardioids exhibited progressive electrophysiological maturation, characterized by more physiological resting membrane potentials (-54.0{+/-}3.1mV to -68.1{+/-}1.2mV), increased action potential amplitude (73.5{+/-}4.8mV to 101.7{+/-}2.1mV) and upstroke velocity (44.3{+/-}5mV/ms to 86.6{+/-}8.4mV/ms), and shorter action potential duration (178.9{+/-}8.7ms to 144.0{+/-}4.8ms at 1Hz stimulation). These changes were associated with progressively increased INa density and enhanced SCN5A mRNA and Nav1.5 protein expression, consistent with progressive maturation of cardiac excitability. IKr current and KCNH2/hERG expression were already present at early maturation stages and remained stable, as evidenced by preserved sensitivity to dofetilide-blockade. Pharmacological inhibition of barium- and HMR-sensitive currents altered resting membrane and/or action potential characteristics in intact cardioids, whereas in isolated cardiomyocytes classical IK1 remained undetectable and IKs was small, highlighting a certain degree of persisting immaturity of the model. Conclusions: These findings demonstrate that human cardioids undergo progressive electrophysiological maturation and provide complementary tissue- and cellular-level insights into cardiac function, establishing their suitability for investigating INa and IKr-related cardiac pathologies.

Weekly training volume is the primary driver of resistance-training hypertrophy, but the independent role of frequency can be isolated only when weekly volume is equated. We quantified the effect of training frequency on hypertrophy across one to three volume-equated sessions per muscle per week. Following PRISMA 2020, five databases were searched to October 2026 for trials of at least six weeks that equated weekly volume between per-muscle frequencies and measured muscle size. One change-score standardized mean difference per trial (Hedges g; negative favoring higher frequency) was pooled with a random-effects model, and certainty was rated with GRADE. Sixteen trials (380 participants) were pooled. The frequency effect was trivial and non-significant (g = -0.02; 95% CI -0.21 to 0.16; I-squared = 0%; prediction interval -0.23 to 0.18); the interval admits at most a small effect favoring the higher frequency. Contrast-specific estimates were similarly trivial, and no moderator was significant, although the moderator analyses could detect only large effects. Certainty was low to very low. Within this range, the data give no basis for preferring one, two or three sessions at a fixed weekly volume. Most trials measured muscle thickness, which does not track whole-muscle growth reliably.

Capacitation is a post-ejaculatory maturation process by which sperm become hyperactive and undergo the acrosome reaction, both of which are required to fertilize an oocyte. Two key features of capacitation are increased intracellular calcium via the calcium channel CatSper and changes in mitochondrial function. In mouse spermatozoa, CatSper-dependent calcium entry promotes mitochondrial calcium uptake and mitochondrial activation, contributing to hyperactivated motility and fertilization ability. Whether this mechanism is conserved in human spermatozoa was unknown. Here, we assessed mitochondrial function and mitochondrial calcium in paired non-capacitated and capacitated human spermatozoa from normozoospermic donors. Capacitation increased mitochondrial respiratory control ratio, coupling efficiency, mitochondrial membrane potential, and mitochondrial calcium. The increase in mitochondrial calcium required extracellular calcium and was prevented by pharmacological inhibition of CatSper or the mitochondrial calcium uniporter (MCU). Inhibition of CatSper or mitochondrial calcium uniporter also attenuated the capacitation-associated increase in mitochondrial membrane potential and reduced hyperactivated motility without affecting progressive or total motility. Pharmacological disruption of mitochondrial function reduced capacitation-associated hyperactivated motility without affecting the acrosome reaction. Together, these findings support a conserved pathway in human and mouse spermatozoa in which CatSper-mediated calcium entry promotes MCU-dependent mitochondrial calcium uptake and mitochondrial activation, contributing to the acquisition of hyperactivated motility during capacitation. These findings identify mitochondrial calcium handling as a potential target for modulating human spermatozoa function.

Background: Loss- and gain-of-function variants in SCN5A, encoding the cardiac sodium channel Nav1.5, are major causes of inherited cardiac channelopathies. Their pathophysiology has almost exclusively been attributed to altered cardiomyocyte excitability and cardiac conduction. However, growing evidence indicates that autonomic dysfunction contributes to these disorders, raising the possibility that Nav1.5 dysfunction may also affect the intrinsic cardiac nervous system. Objectives: We investigated whether Nav1.5 is functionally expressed in murine intrinsic cardiac neurons and evaluated its contribution to neuronal excitability. Methods: Scn5a expression was investigated by digital PCR and immunocytochemistry in isolated murine intracardiac neurons. Whole-cell patch-clamp recordings were used to characterize sodium currents and action potential properties in wild-type and Scn5a+/- mice. Results: Scn5a transcripts and Nav1.5 protein were detected in intrinsic cardiac neurons. Voltage-clamp recordings identified a tetrodotoxin-resistant sodium current accounting for approximately 8% of the total sodium current and displaying pharmacological and biophysical properties consistent with Nav1.5. Activation and inactivation analyses indicated that Nav1.5 is the predominant sodium conductance available at most polarized membrane potentials. Accordingly, Scn5a haploinsufficiency selectively altered action potential threshold, maximal upstroke velocity, overshoot and duration in neurons resting below -60 mV, whereas more depolarized neurons were essentially unaffected. No structural or neurochemical remodeling of intrinsic cardiac ganglia was detected. Conclusion: Nav1.5 is a functional determinant of intrinsic cardiac neuronal excitability. These findings identify the intrinsic cardiac nervous system as a new cellular target of SCN5A dysfunction and suggest that inherited cardiac sodium channelopathies could involve alterations in both myocardial and intrinsic neuronal electrophysiology.

Objective: Mitochondrial oxidative capacity in striated muscle is governed by the PGC-1/ERR transcriptional axis, which controls not only steady-state respiration but the capacity to expand energy production when demand rises. Whether this reserve function requires dedicated downstream effectors, and whether its loss is detectable at rest, remains unclear. We asked whether Perm1, a striated muscle-specific PGC-1/ERR-induced regulator, is required cell-autonomously for cardiac bioenergetic reserve. Methods: We generated cardiomyocyte-specific Perm1 knockout mice using alphaMHC-nCre, complemented by an adult tamoxifen-inducible alphaMHC-MerCreMer model, and assessed cardiac development, structure, and function longitudinally through 12 months of age in both sexes. We evaluated mitochondrial function by high-resolution respirometry, transmission electron microscopy, and molecular profiling. We tested energetic capacity under load by treadmill exercise with tissue ATP quantification and by beta-adrenergic stimulation of cultured neonatal cardiomyocytes. Results: Perm1 deletion did not perturb embryonic cardiac development, and neither constitutive nor adult-onset deletion impaired cardiac structure, systolic function, or diastolic function through 12 months. Despite this preserved resting phenotype, mitochondrial respiration was reduced by 20 - 25% across complex I- and complex I+II-supported conditions, without changes in citrate synthase activity, mtDNA copy number, or OXPHOS subunit abundance. Instead, the MICOS components IMMT (MIC60) and CHCHD3 (MIC19) were selectively reduced, and mitochondrial subpopulations shifted toward fewer subsarcolemmal and more interfibrillar mitochondria, with unchanged total mitochondrial density. Functionally, Perm1-deficient mice of both sexes showed reduced treadmill capacity at 6 and 12 months and failed to maintain cardiac ATP during exercise at a submaximal workload, while skeletal muscle ATP and plasma lactate were unaffected. This deficit was cardiomyocyte-autonomous: beta-adrenergic stimulation increased ATP content in control cardiomyocytes, but Perm1 knockdown reduced this response. Conclusions. Perm1 is a cardiomyocyte-intrinsic determinant of bioenergetic reserve, dispensable for resting cardiac function but required to expand ATP production under increased demand. These findings identify a mode of metabolic regulation that is invisible to resting functional phenotyping.

Aged skeletal muscle has decreased ability to rebound from physiological stressors such as disuse atrophy. We previously observed that older adults during recovery following muscle disuse were characterized by rapid skeletal muscle immune cell expansion, cellular senescence and collagen deposition, responses that were biased toward older women. Here we used single-nucleus RNA sequencing and complementary in vitro muscle primary cell experiments in young (YF; 22[plusmn]3y; n=8) and older females (OF; 66[plusmn]5y; n=9) to investigate age-mediated cellular function and intercellular communication during recovery from disuse atrophy. Compared with YF, OF exhibited a markedly greater transcriptional response at 7d-recovery (YF: 1,548 DEGs, OF: 7,999 DEGs), driven primarily by slow- (YF: 96 DEGs, OF: 984 DEGs) and fast-twitch myonuclei (YF: 194 DEGs, OF: 1,234 DEGs), satellite cells (YF: 548 DEGs, OF: 2,784 DEGs) and FAPs (YF: 326 DEGs, OF: 2,102 DEGs). Satellite cells from OF demonstrated collagen signatures including elevated THBS1 expression and enrichment of TGF-[beta] signaling. Concurrently, OF FAPs exhibited increased expression of fibroblast activation marker ADAMTS14. Furthermore, NicheNet analyses unmasked altered FAP-satellite cell communication in OF. Complementary In vitro experiments revealed that myogenic progenitor cells collected at 7d-recovery from OF (vs YF) donors displayed impaired myogenic differentiation and a cellular senescence-associated phenotype, while fibroblasts exhibited greater myofibroblast-like activation. Conditioned media derived from OF fibroblasts at 7d-recovery further increased cellular senescence and impaired myogenic potential (vs YF fibroblast conditioned media). Collectively, these findings suggest that recovery from disuse atrophy in older females is characterized by altered FAP-satellite cell communication and intrinsic function which may contribute to poor muscle remodeling.

Methylmalonic aciduria (MMA) is caused by deficiency of methylmalonyl-CoA mutase (MMUT), which catalyses the final step of propionate catabolism and fuels the tricarboxylic acid cycle for energy production. Previous studies reported disrupted mitochondrial homeostasis in MMA, including reduced mitochondrial membrane potential and increased oxidative stress, especially in energetically demanding and chronically affected tissues like brain and kidneys. However, how these changes impact mitochondrial energy production remains unclear. Here, we systematically investigated mitochondrial energy production using extracellular flux analysis in cellular models of MMA, including 293T cells, patient-derived fibroblasts, urine-derived epithelial kidney cells and induced pluripotent stem cells (iPSCs) as well as iPSC-derived neurons harbouring either complete loss (knockout, KO) or pathogenic missense variants in MMUT. We found no impact on mitochondrial energy production in MMUT-KO 293T cells and fibroblasts compared to controls. In contrast, fibroblasts and 293T cells expressing the pathogenic MMUT-p.N219Y variant showed decreased energy production. This corresponds to the altered mitochondrial membrane potential in 293T cells, but contrasts with their unchanged mitochondrial abundance. Depletion of glucose, glutamine and pyruvate from the media or provision of each individually as sole fuel source exacerbated the phenotype of MMUT-p.N219Y clones, but did not induce a phenotype in MMUT-KO 293T cells. Finally, we confirmed reduced mitochondrial energy production in patient-derived kidney cells but found no evidence in MMUT-p.N219Y iPSCs and their derived neurons. Overall, our work suggests that the impact of MMUT-deficiency on mitochondrial energy production is cell type- and variant-dependent. Further investigations should clarify molecular mechanisms and their clinical impact.

Fibro-adipogenic progenitors (FAPs) have emerged as central regulators of the skeletal muscle homeostasis and the muscle microenvironment. However, the role of FAPs in the context of exercise-induced muscle hypertrophy remains largely unexplored. Here, we utilized six weeks of blood flow restricted resistance exercise (BFRRE) to study cellular adaptations within the muscle microenvironment accompanying muscle hypertrophy in healthy older individuals. Using flow cytometry, we characterized global changes of key cell populations within the skeletal muscle microenvironment, including FAPs, muscle stem cells (MuSCs), and immune cells. BFRRE induced significant enlargement of both FAPs and MuSCs, consistent with cellular adaptation to exercise. Notably, exercise shifted the FAP pool toward an increased predominance of the CD90high FAP phenotype, without altering total FAP abundance. In addition to matrix and collagen-related genes, transcriptional analysis of genes associated with secretory proteins revealed enrichment of promyogenic factors in CD90high versus CD90low FAPs. Conditioned media experiments of freshly isolated FAPs demonstrated that CD90high FAPs promote myotube growth in vitro compared to CD90low counterparts, suggesting that this phenotypic shift may facilitate muscle hypertrophy. In parallel, BFRRE increased the proportion of pro-inflammatory (CD11c+) macrophages within the skeletal muscle niche, highlighting a dynamic immune response during adaptation. Finally, we show identified possible link between pro-inflammatory macrophages and FAPs, as TNF markedly reduced the proliferation of human primary FAPs ex vivo, suggesting that macrophage-derived signals may attenuate excessive FAP expansion during tissue remodelling. Together, these findings provide new insight into how the remodelling of the cellular niche may support muscle hypertrophy in response to exercise. The coordinated expansion and phenotypic remodeling of FAP and immune cell populations may represent an important mechanism through which exercise supports hypertrophy in older individuals.

Maternal hyperglycemia can affect offspring development and metabolism, but how its effects on skeletal muscle change during postnatal life remains unclear. We investigated skeletal muscle phenotypes in male offspring of streptozotocin-induced hyperglycemic mice at 4 and 8 weeks of age, representing early post-weaning and later postnatal stages, respectively. Maternal hyperglycemia resulted in lower offspring body and skeletal muscle weights at both time points. Myofiber cross-sectional area was reduced without a reduction in total myofiber number, indicating impaired myofiber growth rather than myofiber loss. At 4 weeks, skeletal muscle showed type I/IIA hybrid myofibers and increased expression of fetal, myogenic, and proliferation-associated genes. This immature profile was not maintained at 8 weeks; instead, the transcriptional profile shifted toward reduced mitochondrial and oxidative metabolism. DNA methylation changes at 4 weeks showed limited correspondence with transcriptional alterations. Taken together, maternal hyperglycemia impaired postnatal myofiber growth at both time points, with distinct molecular profiles at 4 and 8 weeks.

Aldosterone is a key regulator of systemic sodium, potassium, and fluid homeostasis. Chronic elevation of aldosterone, as observed in primary aldosteronism, chronic heart and kidney diseases, promotes cardiac hypertrophy, inflammation, fibrosis, and heart failure, which are further enhanced in aging. Although these pathological effects are primarily attributed to the mineralocorticoid receptor (MR), emerging evidence suggests roles for nonclassical G protein-coupled receptor (GPCR) signaling. We identified lysophosphatidic acid receptor 2 (LPAR2) as a novel GPCR candidate that mediates aldosterone signaling in cardiomyocytes. Using AC16 human cardiomyocyte cells and reporter gene assays, we demonstrate that aldosterone activates a Gi/o-dependent GPCR pathway. Pharmacological inhibition of LPAR2 internalization confirms its specific reaction to aldosterone. Functional studies revealed that aldosterone-induced cardiomyocyte injury requires both MR and LPAR2, where specific antagonists to both receptors reduced the ability of aldosterone to induce collagen expression and cell death markers. These findings uncover a previously unrecognized non-classical mechanism of aldosterone action in the heart and highlight LPAR2 as a promising therapeutic target for aldosterone-driven cardiovascular disease.

Fibroblasts are readily obtained and expanded and may provide an alternative cell source for cultivated seafood if their latent myogenic potential can be accessed. We examined whether transient exposure to 5-azacytidine (AZA), a cytidine analogue known to convert mouse embryonic fibroblasts into myoblasts, elicits a persistent muscle gene program in embryo-derived zebrafish (Danio rerio) Z3 fibroblasts. Cells were exposed to 0, 2.5, 5 or 10 M AZA for 48 h and maintained in drug-free medium for 3 weeks. RNA-seq identified 744 genes that increased and 670 that decreased consistently across the concentration series. Increased genes encompassed myogenic regulators, fusion factors, sarcomeric components and excitation-contraction machinery, whereas decreased genes were enriched for extracellular-matrix and adhesion functions. In a separate six-dose RT-qPCR experiment (0-20 M AZA) with three biological replicates per concentration, all 19 selected muscle genes were significantly increased at 1.25 M, the lowest concentration tested, whereas 9 of 12 extracellular-matrix/fibroblast genes were significantly decreased. Several muscle genes plateaued or declined at higher concentrations, and multinucleated myotubes were not observed. A separate 5-aza-2'-deoxycytidine (decitabine) series showed a comparable muscle-associated response at 0.25 M. These findings indicate that zebrafish fibroblasts retain access to a myogenic-like transcriptional program, and they provide a model for identifying food-compatible approaches to control fish-cell lineage plasticity for cultivated seafood.

Mitochondrial transplantation has recently emerged as an alternative treatment for cardiovascular disease (CVD), aimed at increasing mitochondrial number and improving mitochondrial function. Though mitochondrial dysfunction is a key factor in the development of right ventricular hypertrophy/failure, the effects of mitochondrial transplantation on disease mitigation have been largely unexplored. Therefore, this in vitro study aimed to determine whether the transplantation of exogenous L6 myotube mitochondria mitigates negative outcomes in H9C2 cardiomyocytes that were stimulated to hypertrophy with phenylephrine. Control (CTL), control with mitochondrial transplantation (MitoTx), Phenylephrine only (Phe-only), and phenylephrine with mitochondrial transplantation (Phe+MitoTx) treatments were evaluated. Pilot experiments indicated mitochondrial transplantation into healthy cardiomyocytes acutely increased Complex I-linked oxidative phosphorylation (OXPHOS) capacity (p=0.005) and maximal respiratory capacity (p=0.022) within 24 hours, and these data alongside microscopic evidence of fluorescently labeled L6 mitochondria in H9C2 cardiomyocytes suggested successful transplantation. Regarding treatment comparisons, Phe-only showed a significant increase in cell area (p<0.05), while Phe+MitoTx blunted the hypertrophic cardiomyocyte response. Consistent with these results, proteomic analysis of 4,806 proteins showed that transplantation enriched the mitochondrial proteome, impacting pathways including OXPHOS, respiration, fatty acid and amino acid metabolism, while suppressing extracellular matrix remodeling and de-differentiation signatures. This response was observed in healthy and phenylephrine-stressed cardiomyocytes. In conclusion, our in vitro data indicates that transplantation of L6 skeletal muscle mitochondria mitigates negative effects induced by phenylephrine in H9C2 cardiomyocytes. However, more rigorous in vivo studies are needed to determine if this is a suitable approach for disease mitigation.

Background: Chronic kidney disease (CKD) is a major risk factor for cardiovascular morbidity, promoting left ventricular hypertrophy (LVH) prior to overt heart failure. The mechanisms underlying early cardiac remodeling in CKD remain incompletely understood. The IL-23/Th17/IL-17A inflammatory axis has emerged as a key mediator of cardiovascular pathology; however, its role in CKD-associated cardiac remodeling remains unclear. This study aimed to elucidate the contribution of this pathway to the early cardiac changes in CKD. Methods: Using a 5/6 nephrectomy (5/6Nx) mouse model of CKD, we evaluated cardiac structure, mitochondrial function, and inflammatory signaling. Cardiomyocyte transcriptomes were analyzed by RamDA-seq to identify molecular alterations. Circulating cytokines were profiled longitudinally, and IL-17A neutralization experiments were performed to assess causality. In addition, serum IL-12p40 concentrations were measured in patients with CKD and correlated with their renal function and cardiac remodeling indices. Results: At 16 weeks post-5/6Nx, mice developed LVH with preserved systolic function, accompanied by increased oxidative stress and mitochondrial structural abnormalities, but without significant myocardial fibrosis. Transcriptomic analysis identified oxidative phosphorylation as the most significantly downregulated pathway in cardiomyocytes. Despite increased mitochondrial DNA copy number and elevated PGC-1 expression, ATP production and mitochondrial respiratory chain proteins were reduced, indicating mitochondrial dysfunction. Circulating IL-12p40 and IL-23 levels increased progressively, concurrent with expansion of splenic Th17 cells and elevated cardiac IL-17A expression. Recombinant IL-17A directly impaired mitochondrial respiration in cultured cardiomyocytes. In vivo, IL-17A neutralization attenuated LVH, reduced oxidative stress, and partially restored mitochondrial protein expression without improving renal function. In patients with CKD, serum IL-12p40 levels correlated inversely with estimated glomerular filtration rate and positively with left ventricular mass index, supporting clinical relevance. Conclusions: Activation of the systemic IL-23/Th17/IL-17A axis contributes to early CKD-associated cardiac remodeling by promoting oxidative stress and mitochondrial dysfunction. Circulating IL-12p40 may serve as a biomarker of cardiorenal remodeling, and the IL-23/IL-17A pathway represents a potential therapeutic target.

Background: Barth syndrome (BTHS) is an X-linked mitochondrial disorder caused by loss-of-function mutations in TAFAZZIN (TAZ), resulting in defective cardiolipin (CL) remodeling, cardiomyopathy, and premature death. The mechanisms linking TAZ deficiency to impaired mitochondrial quality control remain incompletely understood, and there are no disease-modifying therapies available for BTHS. Here, we investigated the role of Acyl-CoA Lysocardiolipin Acyltransferase-1 (ALCAT1), a stress-inducible phospholipid-remodeling enzyme, in BTHS cardiomyopathy. Methods: Inducible TAZ knockdown (TAZKD) and cardiomyocyte-specific TAZ knockout (TAZcKO) mice were used to determine the effects of genetic ALCAT1 deletion or pharmacological inhibition with Juvenatin, a highly selective small molecule ALCAT1 inhibitor. Cardiac function, exercise capacity, mitochondrial function, lipid remodeling, lysosomal function, and mitophagic flux were assessed. Results: ALCAT1 was markedly upregulated in TAZ-deficient hearts. Genetic ALCAT1 deletion attenuated cardiac dysfunction in TAZKD mice. Juvenatin similarly improved cardiac function in TAZKD mice and reversed established cardiomyopathy in TAZcKO mice. Genetic or pharmacological ALCAT1 inhibition improved mitochondrial ultrastructure and respiration and reduced mitochondrial oxidative stress. These benefits occurred without correcting the characteristic CL abnormalities caused by TAZ deficiency. Instead, ALCAT1 deletion or inhibition corrected pathological phosphatidylinositol remodeling, normalized phosphoinositide signaling, and restored lysosomal function and mitophagic flux, leading to improved mitochondrial quality control in TAZ-deficient cells. Conclusions: ALCAT1 contributes to BTHS cardiomyopathy through a CL-independent pathway involving dysregulated phosphatidylinositol/phosphoinositide signaling, lysosomal dysfunction, and impaired mitophagy. Genetic and pharmacological targeting of ALCAT1 restores mitochondrial and cardiac function despite persistent CL abnormalities, establishing ALCAT1 as a promising disease-modifying therapeutic target for BTHS.

Introduction: Knee muscle coactivation is elevated after anterior cruciate ligament reconstruction (ACLR), contributing to altered joint loading and post-traumatic osteoarthritis. Existing methods average coactivation within predefined gait phases and require priori designation of muscles roles, limiting the resolution for when differences occur. Objective: Characterize knee joint coactivation and dynamics throughout gait in individuals with ACLR using statistical parametric mapping. Methods: Ten individuals with unilateral ACLR and ten healthy matched controls performed overground walking at their preferred speed, while kinematics, kinetics, and electromyography of the quadriceps, hamstring, and gastrocnemius muscles were recorded. Coactivation was quantified using time-varying, multi-muscle coactivation, and was compared using a one-tailed statistical parametric mapping (SPM) t-test. Knee joint angle, angular speed, and moment were compared using SPM Hotelling's T2 tests with Bonferroni-adjusted post hoc comparisons (= 0.05). Results: Coactivation was greater in the ACLR group at 0.01%-6.14%, 35.23%-40.94%, 69.73-71.99%, 72.12-77.31%, 77.89-78.05%, and 94.63%-95.87% (p < 0.0001) of stride. The ACLR group also demonstrated greater knee internal-external rotation angle at 0.01%-47.34%, 52.14%-61.87%, and 66.47%-100% (p <0.0001) and lower knee flexion angle at 55.79%-65.93% (p = 0.004) of stride, than healthy controls. Finally, a smaller knee flexion-extension moment was observed in the ACLR group at 57.43%-62.18% (p = 0.005) of stride. Conclusions: Coactivation after ACLR is elevated at loading response, late mid-stance, terminal stance and early push-off phase. Greater coactivation was observed during weight-acceptance phases, whereas lower knee moments characterized initial swing. These alterations are localized rather than uniformly distributed, supporting phase-targeted rehabilitation.

Oxidative phosphorylation (OxPhos) and substrate-level phosphorylation (SLP) are the primary biochemical mechanisms for ATP generation in mammalian cells. Increased dependency on SLP can arise from either transient (e.g., Crabtree effect) or chronic (e.g., Warburg effect) suppression of OxPhos. We evaluated the steady-state bioenergetics of quiescent and proliferating mouse cells matched in genetic background and cell biology, including primary thioglycollate-elicited peritoneal macrophages, malignant VM-M3 glioma with macrophage characteristics, and malignant CT-2A astrocytoma. Data are presented showing: 1) OxPhos insufficiency was the primary bioenergetic hallmark of proliferating malignant cells. 2) Dysregulated proliferation was associated with lower metabolic flexibility under nutrient stress than was growth arrest or regulated proliferation. 3) The Crabtree effect was present in macrophages and involved a glucose- and time-dependent suppression of OxPhos coupled to ATP production through SLP and resistance to mitochondrial inhibitors; lowering glucose concentration or substituting glucose with respiratory fuels increased OxPhos and restored sensitivity to mitochondrial inhibitors. 4) The Warburg effect in tumor cells involved a persistent SLP dependency and resistance to mitochondrial inhibitors under equivalent glucose-replete conditions. 5) OxPhos could sustain the regulated proliferation of non-tumor cells in the absence of a Warburg effect. Collectively, our findings emphasize that comparisons between normal and cancer cell metabolism require long-term experimental endpoints that ensure steady-state ATP sufficiency.