Intracellular sodium elevation reprograms cardiac metabolism

Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodeling occurs. We assessed whether acute (75μM ouabain 100nM blebbistatin) and chronic myocardial Naiload (PLM3SA mouse) are causally linked to metabolic remodeling and whether the hypertrophied failing heart shares a common Na-mediated metabolic ‘fingerprint’. Control (PLMWT), transgenic (PLM3SA), ouabain treated and hypertrophied Langendorff-perfused mouse hearts were studied by 23Na, 31P, 13C NMR followed by 1H NMR metabolomic profiling. Elevated Nai leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorated the metabolic changes. In silico modelling indicated altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Nai overload or inhibition of Na/Camitomay be a new approach to ameliorate metabolic dysregulation in heart failure

Inhibition of miR-199a-3p in a murine hypertrophic cardiomyopathy (HCM) model attenuates fibrotic remodeling

Background Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ∼1: 200–500 and predisposes individuals to heart failure and sudden death. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and results Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured murine primary cardiac fibroblasts and analyzed the conditioned media by proteomics, we found that several extracellular matrix (ECM) proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially secreted (data are available via ProteomeXchange with identifier PXD042904). We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac fibroblasts drives upregulation of ECM gene expression, including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac fibroblasts. Conclusions Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac fibroblasts. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes

Inhibition of profibrotic microRNA-21 affects platelets and their releasate.

Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21-null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21-null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors

Phosphorylation at Serines 157 and 161 Is Necessary for Preserving Cardiac Expression Level and Functions of Sarcomeric Z-Disc Protein Telethonin

Aims: In cardiac myocytes, the sarcomeric Z-disc protein telethonin is constitutively bis-phosphorylated at C-terminal residues S157 and S161; however, the functional significance of this phosphorylation is not known. We sought to assess the significance of telethonin phosphorylation in vivo, using a novel knock-in (KI) mouse model generated to express non-phosphorylatable telethonin (Tcap(S157/161A)). Methods and Results:Tcap(S157/161A) and wild-type (WT) littermates were characterized by echocardiography at baseline and after sustained β-adrenergic stimulation via isoprenaline infusion. Heart tissues were collected for gravimetric, biochemical, and histological analyses. At baseline, Tcap(S157/161A) mice did not show any variances in cardiac structure or function compared with WT littermates and mutant telethonin remained localized to the Z-disc. Ablation of telethonin phosphorylation sites resulted in a gene-dosage dependent decrease in the cardiac telethonin protein expression level in mice carrying the S157/161A alleles, without any alteration in telethonin mRNA levels. The proteasome inhibitor MG132 significantly increased the expression level of S157/161A telethonin protein in myocytes from Tcap(S157/161A) mice, but not telethonin protein in myocytes from WT mice, indicating a role for the ubiquitin–proteasome system in the regulation of telethonin protein expression level. Tcap(S157/161A) mice challenged with sustained β-adrenergic stimulation via isoprenaline infusion developed cardiac hypertrophy accompanied by mild systolic dysfunction. Furthermore, the telethonin protein expression level was significantly increased in WT mice following isoprenaline stimulation but this response was blunted in Tcap(S157/161A) mice. Conclusion: Overall, these data reveal that telethonin protein turnover in vivo is regulated in a novel phosphorylation-dependent manner and suggest that C-terminal phosphorylation may protect telethonin against proteasomal degradation and preserve cardiac function during hemodynamic stress. Given that human telethonin C-terminal mutations have been associated with cardiac and skeletal myopathies, further research on their potential impact on phosphorylation-dependent regulation of telethonin protein expression could provide valuable mechanistic insight into those myopathies

Detection of cell proliferation markers by immunofluorescence staining and microscopy imaging in paraffin-embedded tissue sections

This unit provides a step-by-step protocol to detect markers of cell proliferation (BrdU or Ki67) by immunofluorescent staining and confocal microscopy imaging in paraffin-embedded tissue sections

Barcoding bias in high-throughput multiplex sequencing of miRNA

Second-generation sequencing is gradually becoming the method of choice for miRNA detection and expression profiling. Given the relatively small number of miRNAs and improvements in DNA sequencing technology, studying miRNA expression profiles of multiple samples in a single flow cell lane becomes feasible. Multiplexing strategies require marking each miRNA library with a DNA barcode. Here we report that barcodes introduced through adapter ligation confer significant bias on miRNA expression profiles. This bias is much higher than the expected Poisson noise and masks significant expression differences between miRNA libraries. This bias can be eliminated by adding barcodes during PCR amplification of libraries. The accuracy of miRNA expression measurement in multiplexed experiments becomes a function of sample number

SHP-2 Positively Regulates Myogenesis by Coupling to the Rho GTPase Signaling Pathway

Myogenesis is an intricate process that coordinately engages multiple intracellular signaling cascades. The Rho family GTPase RhoA is known to promote myogenesis, however, the mechanisms controlling its regulation in myoblasts have yet to be fully elucidated. We show here that the SH2-containing protein tyrosine phosphatase, SHP-2, functions as an early modulator of myogenesis by regulating RhoA. When MyoD was expressed in fibroblasts lacking functional SHP-2, muscle-specific gene activity was impaired and abolition of SHP-2 expression by RNA interference inhibited muscle differentiation. By using SHP-2 substrate-trapping mutants, we identified p190-B RhoGAP as a SHP-2 substrate. When dephosphorylated, p190-B RhoGAP has been shown to stimulate the activation of RhoA. During myogenesis, p190-B RhoGAP was tyrosyl dephosphorylated concomitant with the stimulation of SHP-2's phosphatase activity. Moreover, overexpression of a catalytically inactive mutant of SHP-2 inhibited p190-B RhoGAP tyrosyl dephosphorylation, RhoA activity, and myogenesis. These observations strongly suggest that SHP-2 dephosphorylates p190-B RhoGAP, leading to the activation of RhoA. Collectively, these data provide a mechanistic basis for RhoA activation in myoblasts and demonstrate that myogenesis is critically regulated by the actions of SHP-2 on the p190-B Rho GAP/RhoA pathway

Age-related autocrine diabetogenic effects of transgenic resistin in spontaneously hypertensive rats: gene expression profile analysis

Increased circulating levels of resistin have been proposed as a possible link between obesity and insulin resistance; however, many of the potential metabolic effects of resistin remain to be investigated, including systemic versus local resistin action. We investigated potential autocrine effects of resistin on lipid and glucose metabolism in 2- and 16-mo-old transgenic spontaneously hypertensive rats (SHR) expressing a nonsecreted form of mouse resistin under control of the aP2 promoter. To search for possible molecular mechanisms, we compared gene expression profiles in adipose tissue in 6-wk-old transgenic SHR versus control rats, before development of insulin resistance, by digital transcriptional profiling using high-throughput sequencing. Both young and old transgenic rats showed moderate expression of the resistin transgene in adipose tissue but had serum resistin levels similar to control SHR and undetectable levels of transgenic resistin in the circulation. Young transgenic rats exhibited mild glucose intolerance. In contrast, older transgenic rats displayed marked glucose intolerance in association with near total resistance of adipose tissue to insulin-stimulated glucose incorporation into lipids (6 ± 2 vs. 77 ± 19 nmol glucose·g−1·2 h−1, P < 0.00001). Ingenuity Pathway Analysis of differentially expressed genes revealed calcium signaling, Nuclear factor-erythroid 2-related factor-2 (NRF2)-mediated oxidative stress response, and actin cytoskeletal signaling canonical pathways as those most significantly affected. Analysis using DAVID software revealed oxidative phosphorylation, glutathione metabolism, pyruvate metabolism, and peroxisome proliferator-activated receptor (PPAR) signaling as top Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. These results suggest that with increasing age autocrine effects of resistin in fat tissue may predispose to diabetes in part by impairing insulin action in adipose tissue

Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by non-myocyte proliferation and requires Tgf-β

Mutations in sarcomere protein genes can cause hypertrophic cardiomyopathy (HCM), a disorder characterized by myocyte enlargement, fibrosis, and impaired ventricular relaxation. Here, we demonstrate that sarcomere protein gene mutations activate proliferative and profibrotic signals in non-myocyte cells to produce pathologic remodeling in HCM. Gene expression analyses of non-myocyte cells isolated from HCM mouse hearts showed increased levels of RNAs encoding cell-cycle proteins, Tgf-β, periostin, and other profibrotic proteins. Markedly increased BrdU labeling, Ki67 antigen expression, and periostin immunohistochemistry in the fibrotic regions of HCM hearts confirmed the transcriptional profiling data. Genetic ablation of periostin in HCM mice reduced but did not extinguish non-myocyte proliferation and fibrosis. In contrast, administration of Tgf-β–neutralizing antibodies abrogated non-myocyte proliferation and fibrosis. Chronic administration of the angiotensin II type 1 receptor antagonist losartan to mutation-positive, hypertrophy-negative (prehypertrophic) mice prevented the emergence of hypertrophy, non-myocyte proliferation, and fibrosis. Losartan treatment did not reverse pathologic remodeling of established HCM but did reduce non-myocyte proliferation. These data define non-myocyte activation of Tgf-β signaling as a pivotal mechanism for increased fibrosis in HCM and a potentially important factor contributing to diastolic dysfunction and heart failure. Preemptive pharmacologic inhibition of Tgf-β signals warrants study in human patients with sarcomere gene mutations