Journal of Cellular Physiology

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.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

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.

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.

Background. Cancer cachexia is a multifactorial syndrome characterized by substantial metabolic disturbances including systemic inflammation, and muscle atrophy, which may affect the regulation of iron metabolism in patients. This study aimed to characterize this regulation in two murine models of colorectal and pancreatic cancer, both associated with a high prevalence of cachexia. Methods. Histological, biochemical and molecular analyses were performed in serum, skeletal muscle, liver, spleen and tumor from UN-KC-6141 pancreatic tumor bearing mice and from ApcMin/+ mice (colorectal cancer model), both models exhibiting variable cachexia severity. Results. Both models induce cachexia, but diverge markedly in their regulation of iron metabolism. UNKC mice display reduced plasma iron availability (-9.6% transferrin saturation, p<0.001) without concomitant anemia, while hepatic hepcidin synthesis is upregulated (+100.6%, p<0.001) by inflammation, promoting ferroportin (FPN) downregulation (-42.4%, p<0.001) and iron sequestration in the spleen (+134% total iron content, p<0.001). In the atrophied gastrocnemius, iron concentration is preserved through increased ferritin (+86.6%, p=0.010) and hemoproteins (i.e., myoglobin, CYSC, COXIV) expression. Conversely, ApcMin/+ mice exhibit anemia together with hepatic and splenic iron depletion (-48.4%, p<0.001 and -84.9%, p<0.001, respectively). Hepcidin synthesis is downregulated (-99.9%, p<0.001) despite persistent inflammation, indicating that hypoxia and/or iron-deficiency signals probably predominate over the inflammatory pathway. FPN is markedly decreased (-94.2%, p<0.001) in the iron-depleted spleen despite low hepcidin. In the atrophied quadriceps of ApcMin/+ mice, iron deficiency is associated with decreased FPN and ferritin (-87.9%, p<0.001, and -38.4%, p=0.016, respectively), and increased TfR1 expression (+305%, p=0.107). Finally, tumor iron import may play a significant role in cachexia progression, as suggested by the association between tumor weight, tumor TfR1 protein levels and body weight loss in UNKC mice. Conclusion. These findings reveal cancer type-specific signatures of iron regulation in cachexia, which appear to be more driven by cancer type specific features than by cachexia severity itself, with skeletal muscle iron metabolism impaired mainly when cachexia is associated with anemia. This suggests that the clinical management of iron metabolism in cachectic patients should be guided by the underlying cancer type.

Protein misfolding in the endoplasmic reticulum (ER) of podocytes/glomerular epithelial cells (GECs) induces ER stress and contributes to the pathogenesis of glomerular disease. Activation of IRE1 and consequently the unfolded protein response (UPR) and autophagy sustains proteostasis. The present study examines the role of Surf4 and collagen IV-5 (COL4A5) trafficking in podocyte proteostasis. Glomerular Surf4 protein expression increased in IRE1-replete mice with experimental focal segmental glomerulosclerosis (FSGS) compared to podocyte-IRE1 knockout mice with FSGS and control mice. Glomerular Surf4 gene expression was greater in human FSGS and membranous nephropathy compared to healthy controls. In cultured GECs, basal Surf4 expression was greater in IRE1-replete (control) compared to IRE1 knockout GECs, although ER stress (tunicamycin treatment) did not increase Surf4 despite activating the UPR. Tunicamycin enhanced autophagy (LC3-II) in an IRE1-dependent manner, and the amount of Surf4 localized in autophagosomes. In GECs, tunicamycin increased the amount and colocalization of endogenous COL4A5 or ectopically-expressed COL4A5-wild type (WT) and Surf4 in autophagosomes. Compared to COL4A5-WT, the human COL4A5-C1567R mutant (which is misfolded) showed enhanced basal colocalization with Surf4 in autophagosomes; colocalization was similar to COL4A5-WT after tunicamycin treatment. Degradation of COL4A5-C1567R was more rapid compared to COL4A5-WT and was comparable to degradation of COL4A5-WT in the presence of tunicamycin. Thus, during ER stress, subsets of Surf4 and COL4A5 are redirected from the secretory pathway to autophagy. Surf4 may help deliver misfolded ER cargo, such as COL4A5 for autosomal degradation, thereby sustaining proteostasis in podocytes.

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.

Myocardin (MYOCD) is the principal trigger-switch for vascular smooth muscle cell (VSMC) growth arrest and the cyto-contractile program of differentiation. While much is known about MYOCD-dependent VSMC differentiation, limited information exists as to its transcriptional regulation. Here, we report moderate mRNA expression of Myeloid Ecotropic viral Integration Site 1 (MEIS1) in both human and mouse VSMCs. MEIS1 mRNA is reduced in parallel with myocardin mRNA and protein under conditions of VSMC de-differentiation. Loss- and gain-of-Meis1 expression studies in cultured human VSMCs and the mouse carotid artery support MEIS1 as an activator of myocardin and VSMC differentiation. CRISPR-mediated knockout of the endogenous Meis1 gene in vitro and elicited lower levels of VSMC contractile markers and an increase in growth rate. Similarly, Femoral artery wire injury, in mice lacking Meis1 showed and increased of neointima formation. Mechanistically, we identified a conserved MEIS1 response element in the proximal promoter of myocardin, that appears to contribute to the promoter activity, suggesting a potential regulatory interaction. These results suggest a previously unrecognized role for the homeodomain protein MEIS1 in the activation of myocardin and the quiescent, VSMC differentiated state.

Uterine leiomyomas, commonly known as fibroids, are benign tumors of the female reproductive tract. They are the most common tumor of the female reproductive system and the leading cause of hysterectomy in the United States. Progesterone, estrogen, and their receptors are important drivers for the development and growth of leiomyomas. How estrogen drives leiomyoma growth remains unclear. We have previously shown that simvastatin can inhibit leiomyoma growth by modulating intracellular calcium. This suggests that calcium signaling is important for leiomyoma growth. We therefore hypothesized that estrogen may drive leiomyoma growth by stimulating calcium release through the membrane estrogen receptor GPR30 (also known as GPER). We found that estrogen dose-dependently induced calcium release in leiomyoma cells. The GPR30-specific agonist G-1 also induced calcium release, indicating that GPR30 mediates estrogen-mediated calcium release. The GPR30 antagonist G-15 blocked estrogen-induced calcium release and significantly reduced leiomyoma proliferation. Our results demonstrate that GPR30 is a major driver of estrogen-dependent growth in leiomyoma cells by modulating intracellular calcium. These findings may have high therapeutic relevance to leiomyomas and other estrogen-dependent tumors.

Hepatic stellate cells (HSCs) are the key cellular drivers of liver fibrosis. During liver injury and chronic inflammation HSCs adopt an activated phenotype and secrete fibrotic extracellular matrix (ECM) components such as collagen 1. Mechanical cues derived from the fibrotic ECM drive and support the activation of HSCs, via mechanisms that involve integrins and the mechano-sensitive transcriptional regulator YAP1. It is not yet well understood how external mechanical cues are translated into a molecular response that alters YAP1 nuclear shuttling. There is evidence that suggests the PDZ and LIM domain protein (PDLIM) 5 can regulate YAP1 shuttling in human epithelial cells. We therefore investigated whether PDLIM5 is expressed in HSCs and contributes to YAP1 associated HSC mechano-activation. PDLIM5 protein was detected in HSCs in fibrotic human and mouse liver. PDLIM5 transcript and protein were expressed by primary human and mouse HSCs and by the immortalised HSC LX-2 cell line. PDLIM5 localised with actin stress fibres suggesting a role in HSC adhesion. Co-immunoprecipitation and proximity ligation in LX-2 cells support an association between PDLIM5 and YAP1. We used pharmacological (paclitaxel) and genetic (siRNA and CRISPRi) approaches to inhibit PDLIM5 in HSCs. Inhibiting PDLIM5 reduced YAP1 nuclear localisation and fibrotic gene (COL1A1, ACTA2) expression in LX-2 cells. Overall, these data support a role for PDLIM5 in regulating YAP1 localisation and fibrogenic gene expression in HSCs.

Type 2 diabetes mellitus (T2DM) is a chronic disorder associated with obesity and hyperglycemia. T2DM is linked to significant clinical complications such as delayed wound healing and diabetic foot ulcers. Tumor necrosis factor-stimulated gene 6 (TSG-6) is a protein with multiple functions including regulation of cytokine binding to glycosaminoglycans in extracellular matrices and on vascular endothelial walls. Loss of TSG-6 in knockout mice increases skin inflammation and delays wound healing. Here, in both a genetic model (db/db mice) and a dietary model that simulates human T2DM (mice treated with a high-fat diet [HFD] and streptozotocin [STZ]), we observed sustained hyperglycemia and a reproducible delay in wound closure. TSG-6 expression was markedly reduced in diabetic skin in these models, both before injury and post wounding. HFD+STZ diabetic mouse wounds showed elevated levels of pro-inflammatory cytokines (IL-6, TNF-, and MIP-1) and lower levels of the anti-inflammatory cytokine IL-10, relative to normoglycemic mice. Re-introduction of TSG-6 protein into the wounds normalized healing rates, corrected abnormal cytokine expression levels, and increased macrophage accumulation in dermal and adipose compartments. Thus, T2DM is associated with reduced levels of TSG-6 in diabetic skin, which contributes to the pro-inflammatory milieu and the delay in wound closure.

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.

Retinal neurons rely on continuous oxygen supply to maintain function, yet some species, like goldfish, withstand prolonged hypoxia due to adaptations that remain unclear. Horizontal cells (HCs) are inhibitory interneurons of the retina that express ionotropic glutamate receptors (iGluRs) and secrete gama-aminobutyric acid (GABA), allowing them to shape visual signalling by feeding back to photoreceptors. We investigated whether iGluRs in dissociated goldfish HCs are suppressed during hypoxia, and whether this process is mediated by oxygen-sensitive activation of mitochondrial ATP-sensitive K+ (mKATP) channels. Through perforated patch-clamp electrophysiology, we determined that 20 min of hypoxia reduced peak inward current density associated with glutamate application by ~25% (P = 0.0059). When mKATP channels were selectively blocked with 100 microM glibenclamide, hypoxic suppression of iGluRs was abolished. Inhibiting mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter with ruthenium red, or blocking Ca2+ release through ryanodine receptors in the endoplasmic reticulum, also abolished the hypoxia-induced suppression. Similarly, inhibition of calmodulin with calmidazolium, or of protein phosphatase 1 and 2A (PP1/PP2A) activity with calyculin A, prevented the hypoxia-induced suppression of iGluRs. Taken together, these results indicate that mKATP activation is required to mediate an intracellular Ca2+ signal that downregulates iGluR activity during hypoxia. Through the reduction of iGluR activity, HCs may lower their metabolic demand, allowing them to maintain cell function during hypoxia. Additionally, GABA release from HCs, as measured by mass spectrometry, was not affected by hypoxia, and we propose that downregulation of iGluR may account for hypoxic suppression of the retina in hypoxia-tolerant organisms.

Objectives: The bile acid nuclear receptor Farnesoid X Receptor (FXR, NR1H4) is a major regulator of metabolism and energy homeostasis in peripheral organs. It modulates bile acid, glucose, and lipid metabolism, as well as fat mass and body weight. However, FXR is also expressed in the brain, particularly in the hypothalamus, a key center for the regulation of energy homeostasis. Although one study has demonstrated a role for brain FXR activation in energy balance, its specific hypothalamic role is still unknown. Here, we examined the role of FXR in the mediobasal hypothalamus in the regulation of energy balance. Methods: We used a genetic approach combined with metabolic phenotyping to determine the effect of FXR invalidation in the mediobasal hypothalamus on metabolic parameters involved in the central regulation of energy homeostasis. Results: Our results demonstrate that hypothalamic FXR deficiency induces a positive energy balance, resulting in a reduction in energy expenditure due to alterations in glucose metabolism accompanied by structural changes in white adipose tissues. Conclusion: This study uncovers a previously unrecognized role for hypothalamic FXR in the central homeostatic control of energy balance, providing new insights into its contribution to peripheral glucose metabolism and adipose tissue structural remodeling.

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: 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.

Background and aims: Cyclin E1 is a canonical regulator of cell cycle progression known to promote liver fibrosis and hepatocarcinogenesis. However, its precise contribution to hepatic immune responses remains poorly understood. We investigated whether Cyclin E1 regulates macrophage-associated inflammation and acute immune-mediated liver injury. Methods: Hepatic immune populations were analyzed in untreated and concanavalin A-treated wildtype and constitutive Ccne1-/- mice by flow cytometry. Liver injury was assessed by survival, histopathology, and caspase activation. Inflammatory mediators were quantified by qPCR and ELISA. Bone marrow-derived macrophages were examined during differentiation and pro-inflammatory polarization using flow cytometry, cytokine measurements, and RNA-sequencing. Lipid nanoparticle-mediated delivery of Ccne1-specific siRNA was evaluated in J774A.1 macrophage-like cells. Results: Under homeostatic conditions, Ccne1-/- mice exhibited reduced hepatic immune cell numbers, including Kupffer cells and CD4+ and CD8+ lymphoid cells. Following concanavalin A administration, Ccne1-/- mice showed improved survival, reduced hepatic necrosis, and diminished caspase-3 and caspase-8 activation. Protection was associated with reduced Kupffer and CD8+ lymphoid cell abundance and attenuated inflammatory mediator expression. Cyclin E1 deficiency did not substantially alter bone marrow-derived macrophage differentiation or cell cycle distribution. However, pro-inflammatory Ccne1-/- macrophages showed reduced interleukin-6 expression, attenuated inflammatory gene programs, and lower inferred IRF1 and NF-{kappa}B1 activity. Ccne1 knockdown similarly reduced interleukin-6 expression in pro-inflammatory J774A.1 cells. Conclusions: Cyclin E1 contributes to hepatic immune homeostasis and promotes macrophage-associated inflammatory signaling during immune-mediated hepatitis. Its inflammatory function is uncoupled from overt changes in macrophage cell cycle progression and may involve regulation of IRF1- and NF-{kappa}B-associated transcriptional programs. Targeting Cyclin E1 may therefore provide a strategy to attenuate selected inflammatory responses in liver disease.

Interleukin-15 (IL-15) trans-presentation (TP) by B cells is an important mechanism of T-cell activation; however, the spatial organisation of interleukin-15 receptor (IL-15R) relative to major histocompatibility complex (MHC) molecules on B-cell microvilli remains poorly understood. As microvilli protrude from the B-cell surface and may serve as sites of initial B cell-T-cell contact, the distribution of IL-15R and MHC molecules within these structures may be important during the earliest stages of T-cell recognition and activation. Here, we investigated the spatial association and molecular proximity of IL-15R with MHC class I and class II molecules on B-cell microvilli before immunological synapse formation, using confocal microscopy, stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), and fluorescence lifetime imaging microscopy-based Forster resonance energy transfer (FLIM-FRET). Both MHC class I and class II molecules showed significant spatial association with IL-15R; however, the extent of colocalisation decreased as spatial resolution increased. STED microscopy revealed significant colocalisation between IL-15R and MHC class I, whereas STORM did not detect this association. In contrast, IL-15R and MHC class II remained significantly colocalised at both resolutions. FLIM-FRET further demonstrated molecular proximity between IL-15R and both MHC class I and class II molecules, with higher FRET efficiency observed for MHC class II. Collectively, these findings indicate that IL-15R is spatially organised in proximity to both MHC class I and class II molecules on B-cell microvilli before immunological synapse formation. This arrangement at potential sites of initial B-cell-T-cell contact may facilitate the coordination of IL-15 trans-presentation and antigen presentation during the earliest stages of B-cell-T-cell interactions.

Background: KCNH2 encodes Kv11.1 channel proteins that conduct the rapidly activating delayed-rectifier K+ current (IKr), which is critical for cardiac repolarization. KCNH2 encodes two functional isoforms, Kv11.1a and Kv11.1b, via alternative transcription start sites. Kv11.1a is the principal determinant of cardiac IKr and ventricular repolarization. The circadian clock, a transcriptional-translational feedback loop that cycles with a period of ~24 hours and drives the circadian expression of many genes, including Kcnh2 in the mouse heart. Because daily body temperature rhythms provide a systemic signal that synchronizes cardiac circadian clocks, we tested whether physiological temperature cycles drive the circadian promoter activity of the cloned human KCNH2 (hKCNH2) promoter. Hypothesis: hKCNH2 is a direct transcriptional target of the circadian clock, with temperature driving its promoter activity through BMAL1:CLOCK acting at a conserved tandem E-box. Methods: We cloned the conserved proximal promoter of KCNH2 (-1631 bp upstream of Kv11.1a exon 1) to generate hKCNH2 promoter luciferase reporter constructs. Constructs were transfected into C2C12 myotubes and synchronized by serum shock (static 37{degrees}C) or temperature cycling (36.5-38.5{degrees}C). Bioluminescence was recorded and assessed for period, phase, and amplitude. BMAL1:CLOCK dependence was tested via dominant-negative CLOCK{Delta}19 co-expression. Results: Temperature cycling did not exhibit the rapid damping characteristic of serum-shock-synchronized oscillations, consistent with continuous entrainment by an external zeitgeber rather than a free-running oscillator. Deletion analysis identified a conserved tandem E-box required for oscillation under both serum shock and temperature cycling, and for BMAL1:CLOCK-dependent transactivation (1.75 {+/-} 0.21 vs. 0.86 {+/-} 0.06 RLU, p = 0.0038). CLOCK{Delta}19 reduced hKCNH2 promoter amplitude under temperature cycling without altering period. Conclusion: The circadian clock regulates KCNH2 promoter activity through daily temperature rhythms.

Background: Excessive inflammation in early phases post-ischemic stroke is associated with poor long-term outcome and increased stroke recurrence. Interventions capable of downregulating persistent states of inflammation could improve recovery and prevent secondary stroke. Cardiovascular exercise (CE) has well-established anti-inflammatory effects but its capacity to attenuate the exaggerated inflammatory response triggered by ischemic stroke is unclear. Methods: We investigated the impact of eight weeks of CE on the serum concentration of inflammatory mediators in the early subacute phase post-stroke. Seventy-one patients with firstever ischemic stroke were randomly assigned to moderate-to-vigorous CE (n=45) or standard care (SC) (n=26). Concentrations of CRP, IL-6, IL-8, IL-10, TNF- and IL-1{beta} were measured at baseline and after the interventions. Differences between groups were analyzed with linear mixed models adjusted for age and sex. Results: Compared with the CE group, the SC group showed significant increases in IL-6 ({beta}=0.585, 95% CI=0.164-1.006, p=0.0073), IL-8 ({beta}=0.160, 95% CI=0.004-0.317, p=0.0447), and TNF- ({beta}=0.0845, 95% CI=0.0137-0.1553, p=0.0206). The SC group also showed greater increases in CRP ({beta}=0.250, 95% CI=-0.407, 0.906, p=0.4499) and IL-10 ({beta}=0.251, 95% CI=-0.024, 0.527, p=0.0729) but group differences were not significant. IL-1{beta} levels remained stable and differences between groups were not significant ({beta}=-0.008, 95% CI=-0.058, 0.042, p=0.7618). Conclusions: When introduced in the early subacute phase of stroke recovery, moderate-tovigorous CE mitigates increases in inflammation, but the effect differs across mediators. To what extent reducing the inflammatory response in the early stages post-stroke with CE can impact longterm recovery and stroke recurrence needs to be determined.