Despite proven efficacy of pharmacotherapies targeting primarily global neurohormonal dysregulation, heart failure (HF) is a growing pandemic with increasing burden. Treatments mechanistically focusing at the cardiomyocyte level are lacking. MicroRNAs (miRNA) are transcriptional regulators and essential drivers of disease progression. We previously demonstrated that miR-132 is both necessary and sufficient to drive the pathological cardiomyocytes growth, a hallmark of adverse cardiac remodelling. Therefore, miR-132 may serve as a target for HF therapy. Here we report further mechanistic insight of the mode of action and translational evidence for an optimized, synthetic locked nucleic acid antisense oligonucleotide inhibitor (antimiR-132). We reveal the compound's therapeutic efficacy in various models, including a clinically highly relevant pig model of HF. We demonstrate favourable pharmacokinetics, safety, tolerability, dose-dependent PK/PD relationships and high clinical potential for the antimiR-132 treatment scheme.

Adverse left ventricular (LV) remodeling after myocardial infarction (MI) is a major cause for heart failure. Molecular modifiers of the remodeling process remain poorly defined. Patients with heart failure after MI have reduced LV expression levels of muscle LIM protein (MLP), a component of the sarcomeric Z-disk that is involved in the integration of stress signals in cardiomyocytes. By using heterozygous MLP mutant (MLP ؉/؊ ) mice, we explored the role of MLP in post-MI remodeling. LV dimensions and function were similar in sham-operated WT and MLP ؉/؊ mice. After MI, however, MLP ؉/؊ mice displayed more pronounced LV dilatation and systolic dysfunction and decreased survival compared with WT mice, indicating that reduced MLP levels predispose to adverse LV remodeling. LV dilatation in MLP ؉/؊ mice was associated with reduced thickening but enhanced elongation of cardiomyocytes. Activation of the stress-responsive, prohypertrophic calcineurinnuclear factor of activated T-cells (NFAT) signaling pathway was reduced in MLP ؉/؊ mice after MI, as shown by a blunted transcriptional activation of NFAT in cardiomyocytes isolated from MLP ؉/؊ ͞NFAT-luciferase reporter gene transgenic mice. Calcineurin was colocalized with MLP at the Z-disk in WT mice but was displaced from the Z-disk in MLP ؉/؊ mice, indicating that MLP is essential for calcineurin anchorage to the Z-disk. In vitro assays in cardiomyocytes with down-regulated MLP confirmed that MLP is required for stress-induced calcineurin-NFAT activation. Our study reveals a link between the stress sensor MLP and the calcineurin-NFAT pathway at the sarcomeric Z-disk in cardiomyocytes and indicates that reduced MLP-calcineurin signaling predisposes to adverse remodeling after MI.heart failure ͉ stress signaling C hronic heart failure is a worldwide epidemic. Recently, a fundamental shift in the underlying etiology of heart failure has occurred, in which the most common cause of heart failure is no longer hypertension or valvular disease, but myocardial infarction (MI) (1). MI induces profound alterations of left ventricular (LV) architecture with scar formation, ventricular dilatation, and hypertrophy of the noninfarcted (remote) myocardium (2). Biomechanical stress and humoral growth factors are important mediators of this remodeling process (3, 4). At the level of the single cardiomyocyte, post-MI LV remodeling is characterized by increases in cell diameter and cell length and alterations in gene expression levels (5-7).The Z-disk is a multiprotein complex located at the interface of the cytoskeleton, the contractile apparatus, and the sarcolemma in cardiomyocytes (8). Muscle LIM protein (MLP), which is tethered to the Z-disk via its interacting partners, ␣-actinin and telethonin, has been proposed to be an essential part of the mechanical stretch sensor machinery (9) and to be involved in the transmission of humoral growth signals in cardiomyocytes (10). Intriguingly, myocardial MLP levels are reduced by Ϸ50% in patients with heart failure after MI (11). However, ...

Fibroblast growth factor 2 (FGF-2) is an important modulator of cell growth and differentiation and a neurotrophic factor. FGF-2 occurs in isoforms, at a low molecular weight of 18,000 and at least two high molecular weight forms (21,000 and 23,000), representing alternative translation products from a single mRNA. In addition to its role as an extracellular ligand, FGF-2 localizes to the nuclei of cells. Here we show differential localization of the 18-and 23-kDa isoforms in the nuclei of rat Schwann cells. Whereas the 18-kDa isoform was found in the nucleoli, nucleoplasm, and Cajal bodies, the 23-kDa isoform localized in a punctuate pattern and associates with mitotic chromosomes suggesting different functional roles of the isoforms. Moreover, we show here that the 23-kDa FGF-2 isoform co-immunoprecipitates specifically with the survival of motor neuron protein (SMN). SMN is an assembly and recycling factor of the splicing machinery and locates to the cytoplasm, the nucleoplasm, and nuclear gems, where it co-localizes with 23-kDa FGF-2. Patients with spinal muscular atrophy suffer from fatal degeneration of motoneurons because of mutations and deletions of the gene for the SMN protein.A number of mitogenic growth factors, growth-regulatory proteins, and growth factor receptors have been reported to be localized in the cell nucleus and its substructures: e.g. plateletderived growth factor, fibroblast growth factor-1 (FGF-1), 1 FGF-2, and ciliary neurotrophic factor (1, 2). The data show that nuclear localization is a general phenomenon for some growth factors, suggesting nuclear functions independent of the function as extracellular factors.FGF-2 is a member of the FGF family, which has been shown to mediate a variety of biological processes during development and in the adult organism, including mitogenesis, angiogenesis, chemotaxis, mesoderm induction, and differentiation of various mesoderm-and neuroectoderm-derived cells. FGF-2 as an extracellular ligand is able to bind to high affinity tyrosine transmembrane receptors (FGF receptors, FGFR). FGF-2 has been shown to bind to all four known FGFR, however, with distinct affinities (3).FGF-2 exists in protein isoforms translated from a common messenger RNA by alternative use of AUG (18,000 isoform) and CUG (high molecular weight isoforms 21,000 and 23,000) start codons. The isoforms exert different biological effects when overexpressed in different cell types. Specific effects seen by the 23-kDa isoform but not the 18-kDa isoform after overexpression are reduced spreading of pancreatic cancer cells (4), the ability of NIH 3T3 cells to grow in low serum medium (5), increased radioresistance in HeLa cells (6), growing in serumfree medium of rat AR4 -2J cells (7), and differential effects on binucleation and nuclear morphology of neonatal rat cardiac myocytes (8). We have previously shown that in PC12 cells and rat immortalized Schwann cells, cell growth and morphology are altered after transfection with constructs coding for 18-and 23-kDa FGF-2, respectively (9, 1...

Rationale: We previously discovered the human 10T3 C (Trp4Arg) missense mutation in exon 2 of the muscle LIM protein (MLP, CSRP3) gene. Objective: We sought to study the effects of this single-nucleotide polymorphism in the in vivo situation. Methods and Results: We now report the generation and detailed analysis of the corresponding Mlp W4R/؉ and Mlp W4R/W4R knock-in animals, which develop an age-and gene dosage-dependent hypertrophic cardiomyopathy and heart failure phenotype, characterized by almost complete loss of contractile reserve under catecholamine induced stress. In addition, evidence for skeletal muscle pathology, which might have implications for human mutation carriers, was observed. Importantly, we found significantly reduced MLP mRNA and MLP protein expression levels in hearts of heterozygous and homozygous W4R-MLP knock-in animals. We also detected a weaker in vitro interaction of telethonin with W4R-MLP than with wild-type MLP. These alterations may contribute to an increased nuclear localization of W4R-MLP, which was observed by immunohistochemistry. Conclusions: Given the well-known high frequency of this mutation in Caucasians of up to 1%, our data suggest that W4R-MLP might contribute significantly to human cardiovascular disease. (Circ Res. 2010;106:695-704.)Key Words: genetics Ⅲ mechanosensation Ⅲ mechanotransduction Ⅲ cardiomyopathy Ⅲ heart failure Ⅲ circulation C ardiomyopathies are primary disorders of cardiac muscle and represent major causes of morbidity and mortality at all ages. 1 With a prevalence of 200 per 100 000 individuals, hypertrophic cardiomyopathy (HCM) is the most common cardiovascular disease inherited as an autosomal dominant trait and accounts for Ϸ36% of all sudden deaths in the United States in competitive athletes. Onset of disease is variable and can be late in adolescence. 1 In the majority of cases, the disease is caused by mutations in sarcomeric and/or structural components. 2 However, the underlying molecular mechanisms remain unclear.We cloned and sequenced previously the human MLP gene (CLP, CRP3, or CSRP3), which is encoded by Ϸ20 000 bp and organized into 6 exons, giving rise to a 194-aa LIM only protein. 3 Homozygous loss of Mlp in a genetically engineered mouse model results in cardiac hypertrophy followed by dilated cardiomyopathy (DCM), 4 the first model for this condition in a genetically manipulatable organism. In addition, we discovered and characterized the 10T3 C (Trp4Arg) missense mutation in exon 2 of the MLP gene, the first human MLP mutation (and together with a telethonin mutation, the first Z-disk mutation in general) linked to cardiomyopathy, and proposed a role for this gene in cardiac mechanosensation. 3 Because of the well-known limitations of human studies, owing in part to differences in environmental and epigenetic factors, it is often impossible to perform an in depth functional analysis of mutations in the human setting. Therefore, we generated Mlp W4R/ϩ and Mlp W4R/W4R knock-in (KI) animals and compared the phenotype with Mlp ϩ...

X-ray diffraction patterns were obtained from skinned rabbit psoas muscle under relaxing and rigor conditions over a wide range of ionic strengths (50-170 mM) and temperatures (1 degree C-30 degrees C). For the first time, an intensification of the first actin-based layer line is observed in the relaxed muscle. The intensification, which increases with decreasing ionic strength at various temperatures, including 30 degrees C, parallels the formation of weakly attached cross-bridges in the relaxed muscle. However, the overall intensities of the actin-based layer lines are low. Furthermore, the level of diffuse scattering, presumably a measure of disorder among the cross-bridges, is little affected by changing ionic strength at a given temperature. The results suggest that the intensification of the first actin layer line is most likely due to the cross-bridges weakly bound to actin, and that the orientations of the weakly attached cross-bridges are hardly distinguishable from the detached cross-bridges. This suggests that the orientations of the weakly attached cross-bridges are not precisely defined with respect to the actin helix, i.e., nonstereospecific. Intensities of the myosin-based layer lines are only marginally affected by changing ionic strength, but markedly by temperature. The results could be explained if in a relaxed muscle the cross-bridges are distributed between a helically ordered and a disordered population with respect to myosin filament structure. Within the disordered population, some are weakly attached to actin and others are detached. The fraction of cross-bridges in the helically ordered assembly is primarily a function of temperature, while the distribution between the weakly attached and the detached within the disordered population is mainly affected by ionic strength. Some other notable features in the diffraction patterns include a approximately 1% decrease in the pitch of the myosin helix as the temperature is raised from 4 degrees C to 20 degrees C.