American Journal of Physiology-Cell Physiology

GLP-1 and GIP/GLP-1 receptor agonists produce substantial weight loss in clinical trials but significant loss of lean body mass is reported. Whether this reflects a direct pharmacological effect on skeletal muscle or an indirect consequence of caloric restriction and reduced mechanical loading is unknown. Primary myoblasts were isolated from skeletal muscle of older adults with obesity undergoing orthopaedic surgery. GIPR and GLP-1R expression was characterised by RT- qPCR and flow cytometry. Differentiated myotubes were treated with semaglutide or GIP peptide and assessed for atrophy-related gene expression (qPCR), secretome perturbation (Olink Reveal), mitochondrial and glycolytic bioenergetics (Seahorse XF Real-Time ATP Rate Assay, glucose uptake, lactate secretion) and myotube morphology and myogenesis (immunofluorescence). GIPR mRNA was consistently detected across all donors; GLP-1R mRNA was undetectable by PCR, though LUXendin645 flow cytometry identified low-level surface GLP-1R protein in 51-66% of myoblasts. Neither semaglutide nor GIP altered atrophy-related gene expression or the secretome, with no proteins reaching significance. Semaglutide reduced glycolytic and total ATP production rates, accompanied by reduced lactate secretion, suggesting modest suppression of glycolytic flux; mitochondrial parameters were unaffected. Neither treatment impaired myotube thickness or differentiation; GIP increased myotube thickness after 8 days. Direct GLP-1 and GIP receptor activation does not substantively perturb atrophic signalling, myogenesis, or the secretome of primary human skeletal muscle myotubes. These findings suggest that lean mass loss with incretin-based therapies is unlikely to be driven by direct pharmacological action on skeletal muscle - particularly relevant as these agents are increasingly used in older adults at risk of sarcopenia.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent inherited muscular dystrophies, for which there are no disease-modifying therapies. Metabolic perturbation, mitochondrial dysfunction, and oxidative stress are key contributors to FSHD pathology. Here, the effects of the metabolic regulator and anti-diabetic drug Metformin on myogenesis and muscle function in human and murine models of FSHD were investigated. Metformin did not affect the proliferation rate of human control or patient-derived FSHD myoblasts but promoted their myogenic differentiation, increasing myotube formation and maturation. Metformin also enhanced the metabolic health and viability of myotubes. Mechanistic interrogation revealed reduced levels of mitochondrial reactive oxygen species and modified mitochondrial turnover. These cellular investigations were complemented with in vivo functional assessment in a murine model of FSHD, in which Metformin treated mice exhibited significantly improved muscle strength. Collectively, these findings identify metabolic regulation as a therapeutically tractable feature of FSHD and demonstrate that Metformin improves muscle function in multiple models of FSHD via reduction of oxidative stress and augmentation of cellular metabolic fitness. These results provide insight into the therapeutic actions of Metformin and pre-clinical data to support its testing for repurposing in FSHD.

Introduction. Skeletal muscle loss in sarcopenia and neuromuscular disorders remains a major unmet medical need. AAV9-delivered follistatin (FST), a myostatin/activin antagonist, induces muscle hypertrophy; however, fibre growth without adequate vascular adaptation may limit therapeutic efficacy. We evaluated whether co-administration of a VEGF-A165 plasmid enhances the hypertrophic and angiogenic effects of intramuscular AAV-FST gene transfer in C57BL/6 mice. Methods. Thirty-six C57BL/6 mice (18 males, 18 females) were assigned to PBS vehicle (n=10), AAV-FST (1 x 10^11 vg; n=10), VEGF plasmid (100 ug; n=6), or combination treatment (VEGF plus AAV-FST; n=10). The contralateral hindlimb served as an internal control. Endpoints at Day 115 included hindlimb muscle mass ratio (R/L), transgene expression, FST protein levels, muscle fibre morphometry, capillary density, and safety assessments. Results. Combination therapy produced the highest R/L ratio (1.176 +/- 0.091; p=0.004; d=2.04), whereas AAV-FST alone showed a borderline effect (R/L=1.113; p=0.050). Compared with AAV-FST monotherapy, combination treatment increased muscle FST mRNA approximately 2.1-fold, protein levels approximately 2.0-fold, and the muscle-to-liver expression ratio 2.6-fold. It also induced larger muscle fibres and doubled CD31+ vessel counts versus AAV-FST alone, indicating simultaneous hypertrophy and angiogenesis. No adverse haematological, biochemical, or histopathological findings were observed. Discussion. Combined AAV-FST and VEGF therapy enhanced local muscle hypertrophy, increased capillary density, and improved the muscle-to-liver transgene expression profile compared with AAV-FST monotherapy. The regimen was well tolerated and supports further evaluation of angiogenic preconditioning as a strategy to improve muscle-directed gene therapy for muscle-wasting disorders.

The mitochondrial fatty acid synthesis (mtFAS) pathway is a highly conserved process in mitochondria implicated in metabolic state sensing. Aberrant functioning of this pathway leads to neurodegenerative diseases in humans. Animal experiments indicates that the mtFAS pathway is essential in mammals, and mtFAS inactivation leads to neuronal cell death. Nuclear encoded mitochondrial 3-ketoacyl-acyl carrier protein reductase (KAR) is a heterotetrameric enzyme in this process, consisting of two CBR4 and two HSD17B8 polypeptides. CBR4 works as the catalytic subunit of the enzyme. Here, we provide evidence that CBR4 function is essential in mammals. In contrast, a skeletal muscle-specific Cbr4 KO in mice did not result in any measurable defects in muscle strength and endurance, and the overall structure of the muscle remained unchanged. The Cbr4 KO did not affect the lipoylation process in quadriceps muscle samples, and high-resolution respirometry analysis of soleus muscle samples showed no defects in mitochondrial respiration capacity. The lack of a phenotype of a muscle-specific Cbr4 KO is consistent with previous reports on a lack of effects of mtFAS inactivation in muscle and re-iterates the question about the existence of bypass mechanisms that can alleviate mtFAS deficiencies in non-neuronal cell types.

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.

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.

Transverse (t)-tubules enable rapid, synchronous Ca release required for efficient cardiac contraction by bringing L-type Ca channels into close apposition with ryanodine receptors. In heart failure with reduced ejection fraction (HFrEF), t-tubule disorganisation and loss occur alongside cardiac microtubule remodelling, contributing to impaired Ca handling and contractile dysfunction. Despite their canonical function in contraction, how t-tubules develop is unknown. Microtubules support delivery of L-type Ca channels to t-tubules via Amphiphysin-II/BIN1, yet whether microtubules directly regulate t-tubule formation and maintenance is unclear. Here, we investigated a role for microtubules in t-tubule development and homeostasis. Neonatal rat ventricular myocytes (NRVMs), which lack endogenous t-tubules, were used as a reductionist model in which BIN1 overexpression induces nascent membrane tubules. Microtubule depolymerisation with nocodazole before BIN1 overexpression impaired BIN1-driven tubule formation, reducing tubule density and length. Dynein inhibition with EHNA produced similar effects, indicating a requirement for microtubule-based motor activity during tubule elongation. Knockdown of the microtubule +TIP tracking protein CLIP-170 also reduced BIN1-driven tubule density, implicating BIN1-CLIP-170-dependent microtubule capture in tubule initiation. Microtubules were also required to maintain existing tubules. In NRVMs with established BIN1-driven tubules, microtubule depolymerisation, microtubule stabilisation or dynein inhibition each reduced tubule density and length. Consistent with this, acute microtubule depolymerisation or stabilisation disrupted native t-tubule networks in isolated adult sheep left atrial myocytes. Together, these findings identify cardiac microtubules as active regulators of t-tubule architecture. We propose that BIN1-dependent tubule formation requires CLIP-170-mediated microtubule plus-end capture and dynein-dependent elongation, while ongoing microtubule dynamics are necessary to preserve mature t-tubule structure.

Although microtubules (MT) are established regulators of striated muscle mechanics, how MT lattice organization and post-translational modifications (PTM) individually shape contractility in healthy skeletal muscle fibers remains incompletely understood. We used an ex vivo single muscle fiber culture under unloading, examining acetylation and detyrosination (deTyr). During long-term 2D culture, the MT lattice was disrupted by transverse MT depletion without changes in MT abundance. In individual fibers, MT structure, but not abundance, positively correlated with sarcomere shortening non-linearly. When fibers were cultured in 3D hydrogels, the MT lattice was similarly disrupted yet shortening was preserved, with increased longitudinal MTs and decreased deTyr-MTs. Pharmacologically, contractility increased with either a decrease (parthenolide) or an increase (Taxol) in deTyr-MTs, and Taxol further rescued the MT lattice. Our findings identify MT organization and detyrosination, rather than MT abundance, as key determinants of muscle fiber contractility, positioning deTyr-MT as a load-responsive, bidirectional marker relevant to disuse atrophy and aging.

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.

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.

BackgroundDuchenne muscular dystrophy (DMD) cardiomyopathy is characterized by progressive microtubule remodeling, connexin-43 (Cx43) dysregulation, and ventricular arrhythmias. We previously demonstrated phospho-mimic knock-in of {beta}III-tubulin S172E preserves microtubule organization and attenuates cardiac pathology in mdx mice. However, whether these protective effects can be reproduced using a clinically relevant gene-delivery strategy remains unknown. Methods and ResultsWe generated a cardiomyocyte-specific adeno-associated virus serotype 9 (AAV9) vector expressing phospho-mimic {beta}III-tubulin (Tubb3-S172E) under the cardiac troponin T promoter and delivered it to 4-5-month-old wild-type and mdx mice. Cardiac Tubb3-S172E expression was confirmed by quantitative qPCR and immunoblotting. In mdx mice, AAV9-mediated Tubb3-S172E expression significantly reduced mononuclear inflammatory infiltration, restored Cx43 localization at intercalated discs, and attenuated isoproterenol-induced arrhythmia susceptibility. In contrast, cardiac fibrosis, Nav1.5 protein expression, and peak sodium current density were not significantly improved. Overexpression of wild-type {beta}III-tubulin in healthy hearts increased Cx43 lateralization and arrhythmia susceptibility, indicating that {beta}III-tubulin phosphorylation state rather than protein abundance determines its protective function. ConclusionsCardiomyocyte-targeted delivery of phospho-mimic {beta}III-tubulin partially recapitulates the protective effects observed in the genetic S172E knock-in model. These findings identify {beta}III-tubulin Ser172 phosphorylation as a critical regulator of microtubule-dependent electrical remodeling and support therapeutic modulation of this pathway in Duchenne muscular dystrophy cardiomyopathy. Research PerspectiveO_LICardiomyocyte-targeted AAV9 delivery of phospho-mimic aIII-tubulin improves Cx43 organization, inflammatory remodeling, and arrhythmia susceptibility in dystrophic hearts, demonstrating that therapeutic modulation of {beta}III-tubulin Ser172 phosphorylation partially recapitulates the protective effects observed in the genetic S172E model. C_LIO_LIThe dissociation between improved electrical remodeling and persistent Nav1.5 and fibrotic abnormalities suggests that {beta}III-tubulin Ser172 phosphorylation selectively regulates specific microtubule-dependent pathological pathways in dystrophic cardiomyopathy. C_LIO_LIFuture studies should define the molecular mechanisms linking {beta}III-tubulin Ser172 phosphorylation to cardiomyocyte-immune cell communication and determine how this pathway coordinates electrical and inflammatory remodeling in dystrophic hearts. C_LI

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 Limited heart rate rise contributes to reduced exercise tolerance for people who have heart failure with reduced ejection fraction (HFrEF), yet rate-adaptive pacing does not improve functional capacity due to an attenuated force-frequency relationship (FFR). How the FFR relates to total peripheral resistance and sympathetic tone in HFrEF is unknown. Methods In a prospective, observational study, participants with HFrEF and controls underwent an incremental pacing protocol, during which heart rate was increased from 50 to 140 beats per minute. At each heart rate increment LV contractility was measured by echocardiography to determine the FFR, as well as continuous beat-to-beat measurement of systolic and diastolic blood pressures with a plethysmography device to determine cardiac output, total peripheral resistance and blood pressure variability (BPV). A microneurography study was then conducted to measure muscle sympathetic nerve activity (MSNA) during incremental pacing. Results A total of 157 participants with HFrEF and 55 controls (mean age 71.1{+/-}1.4 years, 172 (81.1%) male) underwent the pacing protocol. We observed single units in seven of 11 participants who participated in the microneurography study. In both groups, LV contractility and cardiac output increased until the peak of the FFR, after which these declined. We observed a reduction in total peripheral resistance, blood pressure variability, MSNA frequency and incidence coinciding with the peak of the FFR, beyond which these increased. Whilst these relationships were present in both groups, they were more evident in participants with HFrEF. Conclusions For people with HFrEF there is a bidirectional relationship between heart rate and sympathetic activation, with a nadir of sympathetic tone occurring at the peak of the FFR. Both excessively low and high heart rates are accompanied by greater sympathetic activation. Taken together, these data suggest that optimal heart rate targets for HFrEF are likely to be individual.

Cerebrospinal fluid (CSF) provides a specialized extracellular environment for the central nervous system, which is predominantly produced by the choroid plexus, a highly vascularized epithelial structure whose ion transport processes are fundamental to CSF secretion, composition, and homeostasis. The mechanisms of Na+ entry into choroid plexus epithelial cells (CPECs) from the interstitial side remain disputed. The slc4a10 gene product encoding the Na+-dependent Cl-/HCO3- exchanger, Ncbe, was suggested as a key transport mechanism based on its impact on the cell's Na+-dependent regulation of intracellular pH and its basolateral membrane expression. The current study was undertaken to directly assess the contribution of Ncbe to the Na+ uptake into CPECs. Intracellular Na+ was recorded by fluorometry using the Na+ probe Sodium Binding Fluorescent Indicator in clusters of CPECs with access to both the luminal and basolateral membranes. Removal of extracellular Na+ reduced the apparent ex vivo intracellular [Na+] to ~5 mM from a baseline of ~43 mM in the absence of CO2/HCO3- and ~54 mM in the presence of CO2/HCO3-. Flame photometry estimated the intracellular [Na+] ex vivo to ~28 mM. The CO2/HCO3--dependent rate of [Na+] recovery amounted to ~53% of the total recovery rate upon re-addition of Na+. Experiments with access to only the luminal membrane show a [Na+] recovery of a similar rate as observed in the absence of CO2/HCO3- in the clusters. The CO2/HCO3--independent [Na+] recovery was inhibited to ~50% by the NKCC1 inhibitor bumetanide and to ~30% by the TRPv4 inhibitor RN1734. NHE contributed to a minor extent to the CO2/HCO3--independent transport. The HCO3- transport inhibitor DIDS, however, inhibited the total [Na+] recovery rate to ~50%, indicating a role for Ncbe rather than NBCn1 in the cellular [Na+] recovery. Indeed, docking of DIDS into Ncbe and NBCn1 indicated that both proteins can accommodate the binding of DIDS. However, the orientation of the DIDS poses in Ncbe suggests a binding mode more similar to that found in the Anion Exchangers (SLC4A1-3), which seems to accommodate the covalent-type docking more than NBCn1. The Ncbe inhibition by DIDS was supported by the rate of [Na+] recovery that was significantly higher in CPECs from Ncbe-wt than Ncbe-ko mice in the presence of CO2/HCO3-. As both NKCC1 and TRPv4 are localized to the luminal membrane, the findings collectively suggest that Ncbe is the most prominent mechanism for Na+ entry into CPECs expressed at the basolateral side. We suggest Ncbe as the rate-limiting mechanism in the vectorial Na+ transport driving CSF secretion.

Age-related reductions in muscle fiber size and contractile function, particularly in fibers expressing fast myosin heavy chains, contribute to declines in whole-muscle power. However, methodological limitations in estimating fiber size during contractile experiments have likely contributed to conflicting findings regarding whether reduced single-fiber force and power in older adults reflects their smaller size and/or impaired intrinsic contractile function. To address this, we coupled single-fiber contractile experiments with 3D-imaging in 7 young (19-40yrs) and 6 older (69-84yrs) males to assess intrinsic contractile function and compare agreement between 3D-derived cross-sectional area (CSA) and CSA estimates obtained either in air or solution. Fast fiber CSA from older males were [~]28-45% smaller across measurement conditions compared with young, whereas slow fiber CSA did not differ. Accordingly, absolute force and power of fast fibers were 41% and 37% lower. When normalized to CSA from measurements in air or 3D-imaging, size-specific force and power either did not differ or were greater in older adults, indicating preserved intrinsic contractile function in both fiber types. This was supported by no age-related differences in the rate of tension redevelopment (ktr), a size-independent measure of intrinsic contractile function. In contrast, size-specific force and power calculated using solution-based CSA estimates were lower in older compared with young adults, and Bland-Altman analyses demonstrated the poorest agreement between solution-based and 3D CSA measurements. These findings indicate that intrinsic contractile function is preserved with aging and suggest that methodological differences in CSA measurement contributes to the disparate findings in the literature.

The urinary bladder functions to store and release urine, yet how the sensation of bladder fullness is conveyed and perceived to the central nervous system is not understood. During bladder filling, the detrusor smooth muscle (DSM) generates phasic contractions, resulting in pressure fluctuations within the bladder. These transient pressure events drive bursts of afferent nerve activity, yet the underlying mechanism leading to rhythmic contractions remains unclear. Here, we examined the role of Gq protein-coupled receptor (GqPCR) activity on DSM excitability and contractility. Using ex vivo pressurized urinary bladder preparations and sharp microelectrode experiments on bladder strips from mice, we evaluated whole bladder transient pressure events, whole bladder DSM Ca2+ activity, and membrane potential in bladder strips. We found that global inhibition of urinary bladder GqPCR activity with YM-254890 abates phasic contractility and transient pressure events through a reduction in DSM Ca2+ activity and propagation of Ca2+ waves. Further, we found inhibition of GqPCR significantly hyperpolarizes DSM, reducing action potentials and decreasing excitability, and activation of protein kinase C restores membrane potential to baseline levels. These findings highlight that GqPCR activity mediates DSM excitability and contractility in such a way as to result in phasic detrusor contractions and transient pressure events.

Micro and nano plastics (MNPs) have become ubiquitous contaminants in the environment with their occurrence being detected in air, water and food. They can cross biological barriers and slowly build up in different organs, including the placenta, raising concerns about possible impacts on maternal and fetal health. Placenta, a highly metabolically active organ composed primarily of trophoblast cells, requires substantial energy for proper development and function. However, the effects of MNPs exposure on trophoblast biology and mitochondrial health remain poorly understood. This study investigated the in vivo systemic accumulation of MNP in different organs of pregnant mice and their localization within various organelles in vitro. These effects influenced trophoblast energy metabolism and led to reduced migration. Mice received fluorescent polystyrene MNPs via their drinking water. Biodistribution was evaluated in vivo using IVIS whole-body imaging, while ex vivo fluorescence imaging confirmed accumulation of these particles in multiple organs and cells. In parallel, human HTR-8/SVneo trophoblast cells were exposed to MNPs, demonstrating rapid cellular uptake and mitochondrial and nuclear localization via fluorescence microscopy. TEM analysis uncovered mitochondrial structural alterations and the localization of MNPs. Seahorse analysis revealed impaired mitochondrial respiration and oxygen consumption rates, indicating compromised cellular bioenergetics in MNPs-treated cells, which led to inflammation, altered mtDNA copy number, and impaired trophoblast migration. Overall, these findings indicate that pregnant mice exposed to MNPs undergo systemic transfer, with trophoblast uptake marked by mitochondrial dysfunction, inflammation, and reduced migration. Our study identifies mitochondrial dysfunction as a central mechanism underlying MNP-mediated placental toxicity and underscores the potential role of environmental microplastic exposure in adverse pregnancy outcomes.

Infantile cardiomyopathies are rare, life-threatening disorders for which genetic diagnosis has been accelerated by next-generation sequencing approaches, including gene panel, exome, and genome sequencing. However, determining the functional consequences of identified variants remains a major challenge. Variants in SMYD1, a striated muscle-specific lysine methyltransferase critical for cardiac development and mitochondrial function, have only recently been linked to human cardiomyopathy. Here, we functionally characterize a homozygous SMYD1 variant (c.302A>G; p.Asn101Ser) identified in a patient with severe early-onset cardiomyopathy requiring cardiac transplantation. Structural modeling predicts that the N101S substitution perturbs a highly conserved residue near the cofactor binding pocket within SMYD1s catalytic domain, disrupting local interactions and modestly destabilizing the protein. Consistent with these predictions, in vitro studies demonstrate that the N101S variant impairs mitochondrial respiratory capacity in myocytes. Quantification of SMYD1 protein levels in patient cardiac tissue revealed increased SMYD1 abundance, suggesting that the N101S variant results in functional impairment rather than protein instability and may trigger compensatory upregulation of SMYD1 expression. Together, these findings support a hypomorphic mechanism in which the N101S variant disrupts SMYD1 activity, leading to mitochondrial dysfunction and cardiomyopathy. This study provides mechanistic insight into SMYD1-associated cardiomyopathy and highlights the importance of integrating genetic, structural, and functional analyses to establish the pathogenicity of rare variants. New & NoteworthyThis study provides the first functional characterization of the cardiomyopathy-associated SMYD1 N101S variant identified in a child with severe infantile cardiomyopathy. Structural modeling predicts reduced protein stability, while cellular assays demonstrate impaired mitochondrial respiratory function, supporting a hypomorphic effect. These findings establish a mechanistic link between SMYD1 dysfunction and infantile cardiomyopathy and highlight the importance of integrating genomic and functional approaches in rare cardiovascular disease.

Spinal cord injury (SCI) disrupts innervation to the lower urinary tract, resulting in bladder dysfunction that predisposes to urinary infections and renal impairment. While inflammation is central to bladder pathology after SCI, the molecular events linking acute to chronic remodeling are poorly defined. We hypothesized that early treatment with pirfenidone, an anti-inflammatory and anti-fibrotic drug, would attenuate bladder pathology after SCI. Adult female C57BL/6J mice underwent contusive SCI or sham laminectomy, and bladders were collected at 2, 7, 16, and 45 days later. SCI induced bladder hypertrophy, edema, hemorrhage, neutrophil infiltration, cell proliferation and loss of voiding function in the first 48 hours. Transcriptomic profiling at this timepoint was characterized by activation of inflammatory and cytokine pathways including TNFalpha, IL-6, the complement cascade, and TGFbeta. Although bladder function partially recovered by day 7, inflammatory pathways persisted and extracellular matrix (ECM) remodeling programs emerged. By day 16, robust activation of ECM-remodeling pathways was evident in all bladders. Treatment with pirfenidone during the acute inflammatory phase (day 2-7) reduced bladder hypertrophy and suppressed expression of pro-fibrotic, inflammatory, and neuroplasticity-associated genes including Bdnf and Chrm2 that encodes muscarinic receptor 2 (M2). Mechanistically, pirfenidone attenuated TGFbeta signaling as shown by downregulation of phosphoSmad2 protein in whole bladders and decreased M2 receptor expression in the urothelium. These molecular changes correlated with improved function in pirfenidone-treated mice as shown by fewer voiding events with larger urine volumes up until 45 days after SCI. Early treatment with pirfenidone limits inflammation and fibrosis, normalizes neural signaling, and improves bladder function after SCI.

Parkinsons disease (PD) is the second most common neurodegenerative disorder worldwide, and mutations in the LRRK2 and PRKN genes are among the most common familial causes of the disease. In neurodegenerative diseases such as PD, disturbances in Ca{superscript 2} homeostasis and cellular bioenergetics impair the function of neurons and glial cells, contributing to disease progression. These changes are not limited to neurons; mitochondrial dysfunction and disrupted Ca2+ homeostasis in astrocytes are increasingly recognized as key contributors to PD, impairing bioenergetics, redox balance, neuroinflammatory responses, and metabolic support essential for dopaminergic neuron survival. In this study, we investigated mitochondrial calcium homeostasis, mitochondrial oxidative phosphorylation, morphology and distribution in human induced pluripotent stem cell (hiPSC)-derived astrocytes with mutations in the PD genes LRRK2 (G2019S) and PRKN (c.155delA; Ex3-4del) and wild-type controls. Intracellular calcium dynamics were assessed using Fura-2 AM. Compared with control astrocytes, LRRK2-related PD patient-derived mutant astrocytes exhibited lower intracellular calcium levels, and slower calcium extrusion following stimulation with ATP. Mitochondrial morphology was analyzed using MitoTracker Deep Red, revealing increased mitochondrial fragmentation and redistribution of mitochondria toward the cell periphery in both PD mutant cell types. Because oxidative phosphorylation is tightly regulated by mitochondrial morphology and calcium homeostasis, we next assessed oxygen consumption rates using a continuous metabolic monitoring system (Resipher) and quantified the expression of genes (RT-qPCR) and proteins (capillary electrophoresis-based western detection) involved in mitochondrial calcium transport and bioenergetics. These analyses showed that PRKN mutant astrocytes exhibit a more oxidative bioenergetic phenotype than LRRK2 mutant astrocytes, while both mutant lines displayed altered phosphorylation of mitochondrial morphology regulator DRP1 as well as decreased levels of respiratory complexes relative to control astrocytes. In summary, this study identifies astrocyte-specific mitochondrial dysfunctions and calcium dysregulation as key features of LRRK2- and PRKN-related pathology, providing new insights into how glial metabolic alterations contribute to neurodegeneration in PD.