17 research outputs found

    Opposing actions of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) in regulating microtubule stabilization during cardiac hypertrophy

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    Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an a-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glutubulin) stable MT’s. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy

    The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

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    UNC-45 is a UCS (UNC-45/CRO1/She4P) class chaperone necessary for myosin folding and/or accumulation, but its requirement for maintaining cardiac contractility has not been explored. Given the prevalence of myosin mutations in eliciting cardiomyopathy, chaperones like UNC-45 are likely to be equally critical in provoking or modulating myosin-associated cardiomyopathy. Here, we used the Drosophila heart model to examine its role in cardiac physiology, in conjunction with RNAi-mediated gene silencing specifically in the heart in vivo. Analysis of cardiac physiology was carried out using high-speed video recording in conjunction with movement analysis algorithms. unc-45 knockdown resulted in severely compromised cardiac function in adults as evidenced by prolonged diastolic and systolic intervals, and increased incidence of arrhythmias and extreme dilation; the latter was accompanied by a significant reduction in muscle contractility. Structural analysis showed reduced myofibrils, myofibrillar disarray, and greatly decreased cardiac myosin accumulation. Cardiac unc-45 silencing also dramatically reduced life-span. In contrast, third instar larval and young pupal hearts showed mild cardiac abnormalities, as severe cardiac defects only developed during metamorphosis. Furthermore, cardiac unc-45 silencing in the adult heart (after metamorphosis) led to less severe phenotypes. This suggests that UNC-45 is mostly required for myosin accumulation/folding during remodeling of the forming adult heart. The cardiac defects, myosin deficit and decreased life-span in flies upon heart-specific unc-45 knockdown were significantly rescued by UNC-45 over-expression. Our results are the first to demonstrate a cardiac-specific requirement of a chaperone in Drosophila, suggestive of a critical role of UNC-45 in cardiomyopathies, including those associated with unfolded proteins in the failing human heart. The dilated cardiomyopathy phenotype associated with UNC-45 deficiency is mimicked by myosin knockdown suggesting that UNC-45 plays a crucial role in stabilizing myosin and possibly preventing human cardiomyopathies associated with functional deficiencies of myosin

    MELD-GRAIL and MELD-GRAIL-Na Are Not Superior to MELD or MELD-Na in Predicting Liver Transplant Waiting List Mortality at a Single-center Level

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    BACKGROUND: Controversy exists regarding the best predictive model of liver transplant waiting list (WL) mortality. Models for end-stage liver disease-glomerular filtration rate assessment in liver disease (MELD-GRAIL) and MELD-GRAIL-Na were recently described to provide better prognostication, particularly in females. We evaluated the performance of these scores compared to MELD and MELD-Na. METHODS: Consecutive patients with cirrhosis waitlisted for liver transplant from 1998 to 2017 were examined in this single-center study. The primary outcome was 90-d WL mortality. MELD, MELD-Na, MELD-GRAIL, and MELD-GRAIL-Na at the time of WL registration were compared. Model discrimination was assessed with area under the receiver operating characteristic curves and Harrell's C-index after fitting Cox models. Model calibration was examined with Grønnesby and Borgan's modification of the Hosmer-Lemeshow formula and by comparing predicted/observed outcomes across model strata. RESULTS: The study population comprised 1108 patients with a median age of 53.5 (interquartile range 48-59) y and male predominance (74.9%). All models had excellent areas under the receiver operating characteristic curves for the primary outcome (MELD 0.89, MELD-Na 0.91, MELD-GRAIL 0.89, MELD-GRAIL-Na 0.89; all comparisons P > 0.05). Youden index cutoffs for 90-d mortality were as follows: MELD, 19; MELD-Na, 22; MELD-GRAIL, 18; and MELD-GRAIL-Na, 17. Variables associated with 90-d mortality on multivariable Cox regression were sodium, bilirubin, creatinine, and international normalized ratio. There were no differences in model discrimination using Harrell's C-index. All models were well calibrated; however, divergence between observed and predicted mortality was noted with scores ≥25. CONCLUSION: There were no demonstrable differences in discrimination or calibration of GRAIL-based models compared with MELD or MELD-Na in our cohort. This suggests that GRAIL-based models may not have meaningful improvements in discriminatory ability when applied to other settings

    Heterogeneous myocyte enhancer factor-2 (Mef2) activation in myocytes predicts focal scarring in hypertrophic cardiomyopathy

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    Unknown molecular responses to sarcomere protein gene mutations account for pathologic remodeling in hypertrophic cardiomyopathy (HCM), producing myocyte growth and increased cardiac fibrosis. To determine if hypertrophic signals activated myocyte enhancer factor-2 (Mef2), we studied mice carrying the HCM mutation, myosin heavy-chain Arg403Gln, (MHC403/+) and an Mef2-dependent β-galactosidase reporter transgene. In young, prehypertrophic MHC403/+ mice the reporter was not activated. In hypertrophic hearts, activation of the Mef2-dependent reporter was remarkably heterogeneous and was observed consistently in myocytes that bordered fibrotic foci with necrotic cells, MHC403/+ myocytes with Mef2-dependent reporter activation reexpressed the fetal myosin isoform (βMHC), a molecular marker of hypertrophy, although MHC403/+ myocytes with or without βMHC expression were comparably enlarged over WT myocytes. To consider Mef2 roles in severe HCM, we studied homozygous MHC403/403 mice, which have accelerated remodeling, widespread myocyte necrosis, and neonatal lethality. Levels of phosphorylated class II histone deacetylases that activate Mef2 were substantially increased in MHC403/403 hearts, but Mef2-dependent reporter activation was patchy. Sequential analyses showed myocytes increased Mef2-dependent reporter activity before death. Our data dissociate myocyte hypertrophy, a consistent response in HCM, from heterogeneous Mef2 activation and reexpression of a fetal gene program. The temporal and spatial relationship of Mef2-dependent gene activation with myocyte necrosis and fibrosis in MHC403/+ and MHC403/403 hearts defines Mef2 activation as a molecular signature of stressed HCM myocytes that are poised to die
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