Journal of Cellular Physiology

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

White, beige and brown adipocytes store energy as lipids, secrete hormones and produce heat, playing an important role in the regulation of energy balance through not completely defined mechanisms. We investigate herein the impact of the almost complete absence of mature adipocytes (severe lipoatrophy) in the determination of energy balance (energy intake and expenditure) and homeothermy in mice. For this, mice with severe lipoatrophy induced by adipocyte deletion of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) (PPAR{gamma} flox adiponectin-Cre) and littermate controls (PPAR{gamma} flox) were evaluated for energy balance, thermoneutral zone, core body temperature, locomotor activity, and gene expression profiles at different ambient temperatures. Severely lipoatrophic mice are heavier, hypermetabolic and hyperphagic and feature a widened thermoneutral zone, lower ambulatory activity, and metabolic inflexibility at both 23 and 17{degrees}C, along with unstable thermal behavior characterized by hyperthermia at 30{degrees}C, normothermia at 23{degrees}C, and bouts of hypothermia at 17{degrees}C. Noteworthy, lipoatrophic mice hypermetabolism at 30{degrees}C is not due to thyroid hormones, impaired insulation or increased body and lean masses and is not altered by pharmacological blockade of either {beta}-adrenergic receptor signaling with propranolol or skeletal muscle sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) and sarcolipin (SLN)-mediated calcium cycling with dantrolene, but is partially attenuated by pharmacological inhibition of acetyl-CoA carboxylase (ACC) and de novo lipogenesis with ND-630. In conclusion, severe lipoatrophy causes hypermetabolism and hyperthermia at 30{degrees}C partly through the activation of liver de novo fatty acid synthesis.

Cyclooxygenase-1 (COX-1) is classically regarded as a constitutive enzyme that produces eicosanoids with housekeeping functions, but recent evidence indicates that it may also be involved in the acute phase of severe systemic inflammation. There is evidence indicating that COX-1 is selectively activated in the spleen via post-translational mechanisms early the course of LPS-induced systemic inflammation. However, the mechanistic link between COX-1 and the spleen has not yet been demonstrated in direct experiments. The present study was conducted to fill this gap. The effects of the COX-1 inhibitor SC-560 on the LPS-induced severity triad (hypotension, hypothermia and acidosis) were evaluated in rats subjected to splenectomy or in sham-operated controls. In the sham-operated group, SC-560 significantly attenuated the severity triad independently of changes in plasma cytokines (TNF and IL-1{beta}). In the splenectomized rats, SC-560 completely lost its ability to attenuate the hypotension and the acidosis induced by LPS. The effect of SC-560 on LPS-induced hypothermia was also impaired by splenectomy, though not completely. We then conducted a lipidomic screening to identify which COX-1-derived eicosanoids might be responsible for mediating the severity triad. Based on spleen-blood correlations, the screening identified PGE2 and PGD2 as putative candidates. In conclusion, the present study provides direct evidence for a mechanistic link between the spleen and COX-1 in the mediation of severity in systemic inflammation, and identifies PGE2 and PGD2 as putative candidates involved.

Cell division cycle 42 (Cdc42) is a member of the Rho family of small GTPases, which plays crucial roles in regulating cytoskeletal remodeling, and membrane trafficking. While previous studies implicated Cdc42 in controlling intestinal epithelial homeostasis, the involvement of this small GTPase in the process of intestinal fibrogenesis remains unexplored. Our study was designed to determine whether Cdc42 regulates the fibrogenic activation of intestinal myofibroblasts in vitro. The study was conducted using a CCD-18Co normal human colonic fibroblast cell line, and primary human intestinal myofibroblasts (HIMF) isolated from Crohns disease (CD) patients. CCD-18Co and HIMF cells were stimulated by transforming growth factor-{beta}1 (TGF-{beta}1). Cdc42 was inhibited either genetically, using siRNA-mediated knockdown, or pharmacologically using specific Cdc42 inhibitors, ML141 and CASIN. Genetic and pharmacologic inhibition of Cdc42 markedly reduced TGF-{beta}1 induced expression of the major contractile cytoskeletal proteins, -smooth muscle actin, calponin 1 and L-caldesmon. Furthermore, Cdc42 inhibition significantly attenuated expression of key extracellular matrix (ECM) proteins, fibronectin and collagen I, in activated CCD-18Co cells and HIMF. Interestingly, decreased expression of contractile and ECM proteins in Cdc42-depleted myofibroblasts was not due to downregulation of the TGF-{beta}1 signaling, decreased mRNA transcription or increased lysosomal or proteasomal degradation of these proteins. Such suppressed pro-fibrotic activation of Cdc42-deficient CCD-18Co cells and HIMF involved a selective inhibition of protein translation due to inactivation of the AKT-mammalian target of rapamycin (mTOR) signaling module. These findings highlight Cdc42 as a key regulator of intestinal fibrosis that controls mTOR activation to enhance ECM production and contractile actomyosin cytoskeleton in intestinal myofibroblasts.

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

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

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

Sedentary behavior promotes chronic low-grade inflammation in adipose tissue, contributing to metabolic dysfunction and insulin resistance. High-intensity interval training (HIIT) is a time-efficient exercise strategy with potent anti-inflammatory and metabolic benefits; however, its effects on adipose tissue inflammatory signaling and microRNA (miRNA) regulation remain incompletely understood. This study investigated the effects of eight weeks of HIIT on inflammatory and epigenetic markers in interscapular white adipose tissue (iWAT) of male Wistar rats. Fourteen rats were randomly assigned to either a sedentary (SED; n = 7) or HIIT (n = 7) group. The HIIT protocol consisted of treadmill running five days per week for eight weeks. Body weight and iWAT mass were assessed, and molecular adaptations were evaluated at multiple regulatory levels using RT-qPCR for mRNA targets (NLRP3, TNF-, PPAR-{gamma}, and IL-10) and miRNAs (miR-21 and miR-30d-5p), while protein levels of NLRP3 and PPAR-{gamma} were assessed using Western blotting. Compared with the SED group, HIIT significantly reduced body weight (p < 0.001) and iWAT mass (p = 0.002). Furthermore, HIIT downregulated the expression of pro-inflammatory mediators, including NLRP3 (gene: p = 0.001; protein: p < 0.001) and TNF- (p = 0.025), while upregulating anti-inflammatory regulators PPAR-{gamma} (gene: p = 0.026; protein: p = 0.020) and IL-10 (p = 0.010). In parallel, inflammation-associated miRNAs, including miR-21 (p = 0.004) and miR-30d-5p (p = 0.002), were markedly downregulated. These coordinated transcriptional, post-transcriptional, and translational adaptations suggest that HIIT attenuates adipose tissue inflammation and promotes a favorable immunometabolic phenotype through integrated molecular and epigenetic mechanisms.

Background: Endothelial progenitor cells (EPCs) contribute to endothelial repair and neovascularization, and EPC dysfunction is closely associated with oxidative stress-related vascular injury. Forkhead box O3a (FoxO3a) regulates cellular stress responses, whereas miR-34a has been implicated in endothelial dysfunction, senescence, and apoptosis. However, the relationship between FoxO3a and miR-34a-3p in oxidatively injured EPCs remains incompletely defined. Objective: This study investigated the role of FoxO3a in H2O2-induced EPC dysfunction and examined whether miR-34a-3p directly interacts with the FoxO3a 3' untranslated region (3'UTR). Methods: Human umbilical cord blood-derived EPCs were identified by DiI-ac-LDL uptake, FITC-UEA-1 binding, and the expression of EPC-related markers. Oxidative stress was induced by H2O2. Cell viability, apoptosis, and angiogenic capacity were evaluated using CCK-8 assay, Annexin V/7-AAD flow cytometry, and Matrigel tube formation assay, respectively. FoxO3a expression was modulated using adenoviral overexpression or knockdown vectors, and miR-34a was modulated using mimics or antagomir. FoxO3a and miR-34a expression levels were detected by Western blot and qPCR. A dual-luciferase reporter assay was used to verify the interaction between hsa-miR-34a-3p and the FoxO3a 3'UTR. Results: H2O2 reduced EPC viability, increased apoptosis, and impaired tube formation in a concentration-dependent manner. H2O2 increased FoxO3a protein abundance and miR-34a expression, whereas FoxO3a mRNA did not change markedly. FoxO3a overexpression aggravated, whereas FoxO3a knockdown partially alleviated, H2O2-induced EPC dysfunction. Similarly, miR-34a mimics further suppressed EPC viability and tube formation, while miR-34a antagomir exerted a protective effect. Dual-luciferase reporter analysis showed that hsa-miR-34a-3p significantly reduced the activity of the wild-type FoxO3a 3'UTR reporter, while mutation of the predicted binding site abolished this suppression. Conclusion: FoxO3a and miR-34a participate in oxidative stress-induced EPC dysfunction. The dual-luciferase data demonstrate that hsa-miR-34a-3p directly targets the FoxO3a 3'UTR, suggesting the presence of miR-34a-3p-mediated post-transcriptional feedback within the FoxO3a-related stress-response network in EPCs.

Cellular material in apoptotic cells must be efficiently cleared by phagocytes to maintain tissue homeostasis. Defects in this process can lead to the onset of secondary necrosis and the release of intracellular contents such as damage associated molecular patterns (DAMPs) and autoantigens that are often derived from the nucleus. Therefore, appropriate handling and clearance of apoptotic material is vital to prevent unwanted inflammatory response and the onset of autoimmune disorders. However, how nuclear material is packaged by apoptotic cells for effective clearance by phagocytes is not well understood. By utilising murine models of apoptosis, we observed that a distinct subset of large extracellular vesicles generated from apoptotic thymocytes, known as apoptotic bodies (ApoBDs), can harbour the majority of nuclear contents. Mechanistically, we discovered that apoptotic cells can asymmetrically partition the nucleus into a single large membrane bleb located at one side of the cell, with other cellular contents such as mitochondria and acid organelles distributed to the opposite side. Whilst this newly observed apoptotic morphology, coined as asymmetric cell death morphology (AsyCDM), is morphologically similar to the process of erythroblast enucleation, pharmacological compounds that could interfere with erythroblast enucleation did not block the establishment of AsyCDM during apoptosis. Notably, AsyCDM was reliant on the contractile forces generated by ROCK1-dependent plasma membrane blebbing. Taken together, this study suggests that intracellular contents are partitioned into different ApoBD subsets during apoptosis through a regulated process driven by ROCK1-dependent actomyosin contraction.

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.

Background: Age is the most important risk factor for coronary artery disease (CAD) independent of traditional risk factors. Aging induces classic pro-inflammatory and prothrombotic vascular phenotypic changes whose molecular mediators remain poorly understood. Medin is a common cleavage product protein that accumulates in vasculature with aging and shown to cause endothelial dysfunction. Its role in CAD is unknown. The study aimed to evaluate the effects of medin on human coronary artery endothelial cell (HCAEC) pro-inflammatory and prothrombotic activation and establish the relationship between medin and coronary atherosclerosis in human decedents. Methods: HCAECs were exposed to physiologic dose of medin (5 M) for 20 hours and ribonucleic acid sequencing (RNAseq) with signaling pathway analyses and reverse transcription polymerase chain reaction of select pro-inflammatory and prothrombotic genes performed. Corresponding protein expression was measured by Western blot or enzyme linked immunosorbent assay in HCAECs exposed to medin (5 M) without or with nuclear factor-{kappa}B (NF{kappa}B) inhibitor RO106-9920 (10 M). Coronary arteries from 40 deceased individuals underwent immunohistochemistry and medin and plaque burden were quantified and their relationship evaluated. Results: RNAseq showed predominant pro-inflammatory gene expression changes induced by medin. HCAECs treated with medin showed increased phosphorylated NF{kappa}B, elevated protein expression of interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and plasminogen activator inhibitor (PAI)-1 and reduced protein expression of thrombomodulin; these changes were reversed by RO106-9920 co-treatment. In human tissues, coronary artery medin strongly correlated with plaque burden (R=0.76, p<0.0001) and coronary macrophage content (R=0.72, p<0.0001). Coronary arteries from decedents with myocardial infarction had higher medin than those without (5.53{+/-}2.67% versus 0.02{+/-}0.02%, p=0.0005). Conclusions: Medin induced NF{kappa}B-mediated endothelial cell pro-inflammatory and prothrombotic activation and was strongly associated with coronary plaque burden and inflammation. Medin is a novel candidate mediator linking aging and coronary atherosclerosis.

Ferroptosis, an intracellular iron-catalyzed form of programmed cell death (PCD) driven by lipid reactive oxygen species induced membrane damage, is mechanistically uncharacterized in its execution process. Here, we investigated ferroptosis execution in mesenchymal-like ovarian cancer cells treated with ferroptosis inducers ML162 and erastin. We showed that YVAD (a pyroptosis-associated inflammatory caspase inhibitor) and disulfiram (preventing gasdermin pore formation on plasma membrane) deterred ferroptotic cell death. Moreover, we also observed LDH release and IL-1{beta} secretion from ferroptotic cells, suggesting that ferroptosis involves a pore-forming process. Intriguingly, ferroptosis is independent of the canonical inflammasome pathway because caspase-1 is dispensable and not activated upon ferroptosis induction. In contrast, we found that caspase-5 was activated while caspase-4 was not during ferroptosis. In addition, depletion of caspase-5 rendered cells not responding to ferroptosis inducers. Also intriguingly, GSDMD, the well-established caspase-5 substrate, was not involved in ferroptosis. We instead detected GSDME cleavage upon ferroptosis induction and knockdown of GSDME reduced cell death induced by ferroptosis inducers. As caspase-5 activity was necessary for ferroptosis and caspase-5 directly cleaved GSDME, we conclude that the axis of caspase-5/GSDME executes ferroptosis in ovarian cancer cells.

Cellular senescence is characterized by stable cell-cycle arrest, cytoskeletal remodeling, and altered secretion of senescence-associated secretory phenotype (SASP) factors, including SERPINE1/plasminogen activator inhibitor-1 (PAI-1). Although extracellular matrix (ECM) stiffening has been linked to fibroblast mechanotransduction and SERPINE1-associated remodeling, the molecular pathway connecting substrate stiffness to SERPINE1 regulation in senescent fibroblasts remains incompletely understood. Here, we investigated how defined substrate stiffness affects fibroblast morphology, mechanical phenotype, and SERPINE1 expression, and examined whether the clathrin adaptor AP2A1 participates in this response in replicative senescent human fibroblasts. Using tunable polyacrylamide hydrogels, we found that increasing substrate stiffness enhanced fibroblast spreading, stress fiber thickening, focal adhesion maturation, cellular stiffness, and senescence-associated marker expression. Stiff substrates also increased SERPINE1 expression and its colocalization with actin fibers, with stronger responses observed in senescent than in young fibroblasts. Functional perturbation experiments further suggested that SERPINE1 contributes to stress fiber organization in senescent cells. In addition, AP2A1 colocalized with SERPINE1, and modulation of AP2A1 under knockdown and overexpression conditions altered SERPINE1 signal intensity. Conversely, perturbation of SERPINE1 also affected AP2A1, supporting a potential bidirectional relationship between these two components. Together, these findings identify SERPINE1 as a stiffness-responsive factor associated with senescence-linked cytoskeletal remodeling and support a functional relationship between AP2A1 and SERPINE1 in senescent fibroblasts. These results suggest that the AP2A1-SERPINE1 axis may contribute to the link between extracellular mechanical cues and senescence-associated fibroblast remodeling.

Thymosin {beta}4 (T{beta}4) is a conserved acidic polypeptide with 43-amino acids participating in multiple pathophysiological processes. In this study in vivo effects of T{beta}4 on liver regeneration are investigated in carbon-tetrachloride (CCL4) induced rodent animal liver jury models. Results illustrate that exogenous T{beta}4 treatment significantly reduced CCL4-rendered liver necrosis around central vein. At 48 hours after CCL4 insults hepatocytes proliferation occur mainly around the periportal area, while hepatocytes proliferation around the necrosis area is prominently increased by exogenous T{beta}4 treatment. The holistic proliferation level of liver tissues are also enhanced by exogenous T{beta}4. Hepatocyte proliferation activities negatively correlate with the necrosis extent of the liver tissue. These results suggested firstly exogenous T{beta}4 treatment could enhance liver regeneration and exhibit prosperous potential for application in clinical conditions such as liver transplantation.

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

Background: Hormone therapy (HT) has not consistently reduced atherosclerotic cardiovascular disease (ASCVD) events in post-menopausal women, yet the underlying mechanisms remain poorly understood. Methods: Female Ldlr-/- mice with established atherosclerosis were subjected to surgical menopause and treated with 17{beta}-estradiol (E2) following lipid normalization. Studies were performed in aging and young mice. To determine whether inflammation mediates the age-dependent response to HT, a cohort of aging mice underwent transplantation with Ifn{gamma}-/-bone marrow (BM) before hormone treatments. Metabolic parameters, HDL function, systemic inflammation, atherosclerotic burden, liver metabolic and oxidative stress signaling, and hepatic estrogen receptor signaling were evaluated. Results: In aging mice, menopause E2 treatment failed to reduce established atherosclerosis as shown in sham operated mice during lipid normalization. Instead, E2 treatment increased circulating IFN{gamma} and IL-6, impaired HDL antioxidant and cholesterol efflux functions, and promoted inflammatory and vulnerable plaque phenotypes. Suppression of inflammation through Ifn{gamma}-/-BM transplantation restored HDL function and significantly reduced atherosclerosis in E2-treated aging mice. In contrast to aging mice, young mice exhibited reduced systemic and plaque inflammation, improved HDL functions, and regression of atherosclerosis following E2 treatment. Liver RNA sequencing and qPCR validation identified activation of inflammatory, oxidative stress, and lipid metabolic pathways in aging E2-treated mice, which were largely attenuated following Ifn{gamma}-/- bone marrow transplantation as well as in young mice. Compared to young mice, aging mice presented hepatic estrogen receptor remodeling characterized by reduced estrogen receptor (ER) expression and increased G-protein coupled estrogen receptor (GPER) expression. Constitutive GPER activation was accompanied by induction of NOX1-dependent oxidative stress, which was further exacerbated by E2 treatment, leading to persistent inflammation. Conclusions: The cardiovascular effects of estrogen therapy are fundamentally age dependent. Aging shifts estrogen signaling toward hepatic oxidative stress and inflammation through increased GPER. While E2 treatment preserves both metabolic and cardiovascular protection in young mice, aging exacerbates GPER-NOX1-mediated oxidative stress, resulting in impaired HDL function and persistent residual ASCVD risk. These findings identify inflammation-driven, non-lipid mechanisms as potential therapeutic targets to improve cardiovascular outcomes during hormone therapy in postmenopausal women.

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.

Osteoarthritis (OA), a degenerative joint disease, is associated with increased systemic inflammation, chronic pain, and cardiovascular dysfunction. Epidemiological evidence establishes that OA increases the risk of cardiovascular disease (CVD) threefold, yet the causal role of OAs contributions remains underexamined. We assessed cardiac function longitudinally following destabilization of the medial meniscus (DMM) surgery to induce osteoarthritis in mice. DMM-mice exhibited significant, sexually dimorphic alterations in echocardiographic parameters. Female DMM mice developed impaired relaxation with altered E/A ratios, increased E/e ratios, and prolonged intraventricular relaxation time with no change in ejection fraction, while male DMM mice showed progressive systolic dysfunction with decreasing ejection fraction, increased E/e ratio, and prolonged intraventricular contraction time. Transcriptomic profiles and biochemical analyses demonstrated divergent cellular responses involving fibrosis and oxidative stress in female mice, whereas autophagic and apoptotic responses were observed in male mice. Using a tumor necrosis factor 2 (TNFR2) agonist shown to reduce systemic inflammation, we investigated its potential therapeutic role in the context of OA-induced cardiovascular dysfunction. TNFR2 agonism proved to be effective both prophylactically and therapeutically for female diastolic dysfunction. While prophylactic and therapeutic administration delayed male systolic dysfunction, the efficacy declined over time. Our findings demonstrate evidence of a novel sexually dimorphic model of OA-induced CVD that recapitulates the sexually dimorphic pattern of patient phenotypes and a promising new therapeutic approach to CVD. Translational RelevanceOsteoarthritis patients have higher, often unrecognized, cardiovascular risk, yet preclinical models linking joint disease to cardiac dysfunction remain unexplored. Using a murine preclinical model of OA reveals the key findings. First, OA alone drives sex-specific cardiac phenotypes - females develop diastolic dysfunction, whereas males develop progressive systolic impairment. Second, selective TNFR2 agonism prevents and reverses OA-induced diastolic dysfunction in female mice and delays systolic decline in males. These findings suggest sex-dependent cardiac monitoring in OA patients and indicate that TNFR2-targeted therapy will likely be a sex-informed intervention to provide cardioprotective benefit. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/736778v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1f41661org.highwire.dtl.DTLVardef@1e94cb7org.highwire.dtl.DTLVardef@1abbac0org.highwire.dtl.DTLVardef@171cc86_HPS_FORMAT_FIGEXP M_FIG C_FIG

Investigating futile recanalization indicators is very important. Here, we explored plasma endothelial microvesicles (EMVs) as biomarkers for recombinant tissue plasminogen activator (rtPA)-treated acute ischemic stroke. This study prospectively enrolled 195 acute IS patients who underwent rtPA and measured plasma EMVs levels via fluorescence nanoparticle tracking analysis at baseline, 24 h and 90 days. Early futile recanalization was assessed by transcranial Doppler and the National Institutes of Health Stroke Scale. The ROC curves and corresponding areas under the curve (AUC) of the EMVs were analysed. The plasma EMVs levels at baseline and 24 h were positively related to both early and late futile recanalization. In both the late recanalization and futile recanalization groups, the plasma levels of EMVs significantly increased at 24 h but decreased at 90 days. For early futile recanalization, the baseline and 24-h EMVs AUCs were 0.7 and 0.67, respectively. For late futile recanalization, the AUCs for baseline and 24-h EMVs levels were 0.52 and 0.66, respectively. Collectively, the results imply that the plasma level of EMVs could serve as a surrogate indicator of futile recanalization (both early and late) following rtPA administration in acute IS.