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Hypertrophic/dilated cardiomyopathy, often a prequel to heart failure, is accompanied by maladaptive transcriptional changes that contribute to arrythmias and contractile misfunction. Transgenic mice constitutively expressing high levels of calcineurin are known to develop extreme heart hypertrophy, which progresses to dilated cardiomyopathy, and to die several weeks after birth. Here, we characterized aberrant transcriptional and epigenetic pathways in this mouse model and established a pharmacological approach to treat established cardiomyopathy. We found that H3K4me3 (trimethyl histone 3 lysine 4) and H3K9me3 (trimethyl histone 3 lysine 9) Jumonji histone demethylases are markedly increased at the protein level and show enhanced enzymatic activity in diseased hearts. These epigenetic regulators continued to increase with time, further affecting cardiac gene expression. Our findings parallel the lower H3K4me3 and H3K9me3 levels seen in human patients. Inhibition of Jumonji demethylase activities in vivo results in lower histone demethylase enzymatic function in the heart and higher histone methylation levels and leads to partial reduction of heart size, reversal of maladaptive transcriptional programs, improved heart function, and prolonged survival. At the molecular level, target genes of transcription factor myocyte enhancer factor 2 are specifically regulated in response to pharmacological or genetic inhibition of Jumonji demethylases. Similar transcriptional reversal of disease-associated genes is seen in a second disease model based on cardiac mechanical overload. Our findings validate pharmacological inhibitors of Jumonji demethylases as potential therapeutics for the treatment of cardiomyopathies across disease models and provide evidence of the reversal of maladaptive transcriptional reprogramming leading to partial restoration of cardiac function. In addition, this study defines pathways of therapeutic resistance upregulated with disease progression.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a transcriptional coactivator that binds to a diverse range of transcription factors. PPAR gamma coactivator 1 (PGC-1) coactivators possess an extensive range of biological effects in different tissues, and play a key part in the regulation of the oxidative metabolism, consequently modulating the production of reactive oxygen species, autophagy, and mitochondrial biogenesis. Owing to these findings, a large body of studies, aiming to establish the role of PGC-1 in the neuromuscular system, has shown that PGC-1 could be a promising target for therapies targeting neuromuscular diseases. Among these, some evidence has shown that various signaling pathways linked to PGC-1 alpha are deregulated in muscular dystrophy, leading to a reduced capacity for mitochondrial oxidative phosphorylation and increased reactive oxygen species (ROS) production. In the light of these results, any intervention aimed at activating PGC-1 could contribute towards ameliorating the progression of muscular dystrophies. PGC-1 alpha is influenced by different patho-physiological/pharmacological stimuli. Natural products have been reported to display modulatory effects on PPAR gamma activation with fewer side effects in comparison to synthetic drugs. Taken together, this review summarizes the current knowledge on Duchenne muscular dystrophy, focusing on the potential effects of natural compounds, acting as regulators of PGC-1 alpha., This work was supported by the crowd funding #Sport4Therapy to Giuseppe D'Antona (Italy). A. Sureda was supported by Instituto de Salud Carlos III, Grant Number: CIBEROBN CB12/03/30038.

Objective The peroxisome proliferator-activated receptor-γ coactivator-1α1 (PGC-1α1) regulates genes involved in energy metabolism. Increasing adipose tissue energy expenditure through PGC-1α1 activation is potentially beneficial for systemic metabolism. Pharmacological PGC-1α1 activators could be valuable tools in the fight against obesity and metabolic disease. Finding such compounds has been challenging partly because PGC-1α1 is a transcriptional coactivator with no known ligand-binding properties. While, PGC-1α1 activation is regulated by several mechanisms, protein stabilization is a crucial limiting step due to its short half-life under unstimulated conditions. Methods We designed a cell-based high-throughput screening system to identify PGC-1α1 protein stabilizers. Positive hits were tested for their ability to induce endogenous PGC-1α1 protein accumulation and activate target gene expression in brown adipocytes. Select compounds were analyzed for their effects on global gene expression and cellular respiration in adipocytes. Results Among 7,040 compounds screened, we highlight four small molecules with high activity as measured by: PGC-1α1 protein accumulation, target gene expression, and uncoupled mitochondrial respiration in brown adipocytes. Conclusions We identify compounds that induce PGC-1α1 protein accumulation and show that this increases uncoupled respiration in brown adipocytes. This screening platform establishes the foundation for a new class of therapeutics with potential use in obesity and associated disorders., Graphical abstract, Highlights • A high-throughput platform to identify PGC-1α1 activators. • PGC-1α1 protein stabilizers work as activators in brown adipocytes. • Small molecule PGC-1α1 activators induce Ucp1 expression and cellular respiration.

PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP–PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α1-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming., Protein kinase D has multiple roles in cardiac myocytes, where its regulatory mechanisms remain incompletely defined. In the present study we identify four-and-a-half LIM domains proteins 1 and 2 as novel binding partners and regulators of protein kinase D in this cell type.

The Myocyte Enhancer Factor 2C (MEF2C) transcription factor plays a critical role in skeletal muscle differentiation, promoting muscle-specific gene transcription. Here we report that in proliferating cells MEF2C is degraded in mitosis by the Anaphase Promoting Complex/Cyclosome (APC/C) and that this downregulation is necessary for an efficient progression of the cell cycle. We show that this mechanism of degradation requires the presence on MEF2C of a D-box (R-X-X-L) and 2 phospho-motifs, pSer98 and pSer110. Both the D-box and pSer110 motifs are encoded by the ubiquitous alternate α1 exon. These two domains mediate the interaction between MEF2C and CDC20, a co-activator of APC/C. We further report that in myoblasts, MEF2C regulates the expression of G2/M checkpoint genes (14-3-3γ, Gadd45b and p21) and the sub-cellular localization of CYCLIN B1. The importance of controlling MEF2C levels during the cell cycle is reinforced by the observation that modulation of its expression affects the proliferation rate of colon cancer cells. Our findings show that beside the well-established role as pro-myogenic transcription factor, MEF2C can also function as a regulator of cell proliferation.

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4–6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24 hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4–6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4–5–6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity., Highlights • mef2ca and mef2cb gene products are alternatively spliced in zebrafish. • Inclusion of exon 5 in mef2ca transcripts is regulated during zebrafish development. • Exon 5 confers on Mef2ca the ability to activate early patterning genes. • Mef2cb includes an extra octapeptide encoded by a region of intron 5. • Inclusion of the extra-octapeptide confers on Mef2cb pro-myogenic activity.