MEK1 Inhibits Cardiac PPARα Activity by Direct Interaction and Prevents Its Nuclear Localization

BACKGROUND: The response of the postnatal heart to growth and stress stimuli includes activation of a network of signal transduction cascades, including the stress activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK) and the extracellular signal-regulated kinase (ERK1/2) pathways. In response to increased workload, the mitogen-activated protein kinase kinase (MAPKK) MEK1 has been shown to be active. Studies embarking on mitogen-activated protein kinase (MAPK) signaling cascades in the heart have indicated peroxisome-proliferators activated-receptors (PPARs) as downstream effectors that can be regulated by this signaling cascade. Despite the importance of PPARα in controlling cardiac metabolism, little is known about the relationship between MAPK signaling and cardiac PPARα signaling. METHODOLOGY/PRINCIPAL FINDING: Using co-immunoprecipitation and immunofluorescence approaches we show a complex formation of PPARα with MEK1 and not with ERK1/2. Binding of PPARα to MEK1 is mediated via a LXXLL motif and results in translocation from the nucleus towards the cytoplasm, hereby disabling the transcriptional activity of PPARα. Mice subjected to voluntary running-wheel exercise showed increased cardiac MEK1 activation and complex formation with PPARα, subsequently resulting in reduced PPARα activity. Inhibition of MEK1, using U0126, blunted this effect. CONCLUSION: Here we show that activation of the MEK1-ERK1/2 pathway leads to specific inhibition of PPARα transcriptional activity. Furthermore we show that this inhibitory effect is mediated by MEK1, and not by its downstream effector kinase ERK1/2, through a mechanism involving direct binding to PPARα and subsequent stimulation of PPARα export from the nucleus

Loss of miR-132/212 Has No Long-Term Beneficial Effect on Cardiac Function After Permanent Coronary Occlusion in Mice

Background: Myocardial infarction (MI) is caused by occlusion of the coronary artery and induces ischemia in the myocardium and eventually a massive loss in cardiomyocytes. Studies have shown many factors or treatments that can affect the healing and remodeling of the heart upon infarction, leading to better cardiac performance and clinical outcome. Previously, miR-132/212 has been shown to play an important role in arteriogenesis in a mouse model of hindlimb ischemia and in the regulation of cardiac contractility in hypertrophic cardiomyopathy in mice. In this study, we explored the role of miR-132/212 during ischemia in a murine MI model. Methods and Results: miR-132/212 knockout mice show enhanced cardiac contractile function at baseline compared to wild-type controls, as assessed by echocardiography. One day after induction of MI by permanent occlusion, miR-132/212 knockout mice display similar levels of cardiac damage as wild-type controls, as demonstrated by infarction size quantification and LDH release, although a trend toward more cardiomyocyte cell death was observed in the knockout mice as shown by TUNEL staining. Four weeks after MI, miR-132/212 knockout mice show no differences in terms of cardiac function, expression of cardiac stress markers, and fibrotic remodeling, although vascularization was reduced. In line with these in vivo observation, overexpression of miR-132 or miR-212 in neonatal rat cardiomyocyte suppress hypoxia induced cardiomyocyte cell death. Conclusion: Although we previously observed a role in collateral formation and myocardial contractility, the absence of miR-132/212 did not affect the overall myocardial performance upon a permanent occlusion of the coronary artery. This suggests an interplay of different roles of this miR-132/212 before and during MI, including an inhibitory effect on cell death and angiogenesis, and a positive effect on cardiac contractility and autophagic response. Thus, spatial or tissue specific manipulation of this microRNA family may be essential to fully understand the roles and to develop interventions to reduce infarct size

MiR-337-3p Promotes Adipocyte Browning by Inhibiting TWIST1

The prevalence of metabolic syndrome (MetS) and obesity is an alarming health issue worldwide. Obesity is characterized by an excessive accumulation of white adipose tissue (WAT), and it is associated with diminished brown adipose tissue (BAT) activity. Twist1 acts as a negative feedback regulator of BAT metabolism. Therefore, targeting Twist1 could become a strategy for obesity and metabolic disease. Here, we have identified miR-337-3p as an upstream regulator of Twist1. Increased miR-337-3p expression paralleled decreased expression of TWIST1 in BAT compared to WAT. Overexpression of miR-337-3p in brown pre-adipocytes provoked a reduction in Twist1 expression that was accompanied by increased expression of brown/mitochondrial markers. Luciferase assays confirmed an interaction between the miR-337 seed sequence and Twist1 3 0UTR. The inverse relationship between the expression of TWIST1 and miR-337 was finally validated in adipose tissue samples from non-MetS and MetS subjects that demonstrated a dysregulation of the miR-337-Twist1 molecular axis in MetS. The present study demonstrates that adipocyte miR-337-3p suppresses Twist1 repression and enhances the browning of adipocytes

The transverse aortic constriction heart failure animal model: a systematic review and meta-analysis

The transverse aortic constriction (TAC) model is frequently used to study adverse cardiac remodeling upon pressure overload. We set out to define the most important characteristics that define the degree of cardiac remodeling in this model. A systematic review and meta-analyses were performed on studies using the TAC mouse/rat model and reporting echocardiographic outcome parameters. We included all animal studies in which a constriction around the transverse aorta and at least one of the predefined echocardiography or MRI outcome parameters were assessed. A total of 502 articles and > 3000 wild-type, untreated animals undergoing TAC were included in this study and referenced to a control group. The duration of aortic constriction correlated to the degree of adverse remodeling. However, the mouse data is strongly biased by the preferential use of male C57Bl/6 mice (66% of studies). Furthermore, mostly ketamine/xylazine anesthetics, 27G needle constriction, and silk sutures are used. Nonetheless, despite the homogeneity in experimental design, the model contained a substantial degree of heterogeneity in the functional outcome measures. When looking at study quality, only 12% reported randomization, 23% mentioned any sort of blinding, 25% adequately addressed the outcomes, and an amazingly low percentage (2%) showed sample size calculation. Meta-analyses did not detect specific study characteristics that explained the heterogeneity in the reported outcome measures, however this might be related to the strong bias towards the use of specific mouse lines, sex as well as age or to poor reporting of characteristics of study quality

MicroRNA-132/212 family enhances arteriogenesis after hindlimb ischaemia through modulation of the Ras-MAPK pathway

Arteriogenesis is a complicated process induced by increased local shear-and radial wall-stress, leading to an increase in arterial diameter. This process is enhanced by growth factors secreted by both inflammatory and endothelial cells in response to physical stress. Although therapeutic promotion of arteriogenesis is of great interest for ischaemic diseases, little is known about the modulation of the signalling cascades via microRNAs. We observed that miR-132/212 expression was significantly upregulated after occlusion of the femoral artery. miR-132/212 knockout (KO) mice display a slower perfusion recovery after hind-limb ischaemia compared to wildtype (WT) mice. Immunohistochemical analysis demonstrates a clear trend towards smaller collateral arteries in KO mice. Although Ex vivo aortic ring assays score similar number of branches in miR-132/212 KO mice compared to WT, it can be stimulated with exogenous miR-132, a dominant member of the miR-132/212 family. Moreover, in in vitro pericyte-endothelial co-culture cell assays, overexpression of miR-132 and mir-212 in endothelial cells results in enhanced vascularization, as shown by an increase in tubular structures and junctions. Our results suggested that miR-132/212 may exert their effects by enhancing the Ras-Mitogen-activated protein kinases MAPK signalling pathway through direct inhibition of Rasa1, and Spred1. The miR-132/212 cluster promotes arteriogenesis by modulating Ras-MAPK signalling via direct targeting of its inhibitors Rasa1 and Spred1

H3K27ac acetylome signatures reveal the epigenomic reorganization in remodeled non-failing human hearts

BACKGROUND: H3K27ac histone acetylome changes contribute to the phenotypic response in heart diseases, particularly in end-stage heart failure. However, such epigenetic alterations have not been systematically investigated in remodeled non-failing human hearts. Therefore, valuable insight into cardiac dysfunction in early remodeling is lacking. This study aimed to reveal the acetylation changes of chromatin regions in response to myocardial remodeling and their correlations to transcriptional changes of neighboring genes. RESULTS: We detected chromatin regions with differential acetylation activity (DARs; Padj. < 0.05) between remodeled non-failing patient hearts and healthy donor hearts. The acetylation level of the chromatin region correlated with its RNA polymerase II occupancy level and the mRNA expression level of its adjacent gene per sample. Annotated genes from DARs were enriched in disease-related pathways, including fibrosis and cell metabolism regulation. DARs that change in the same direction have a tendency to cluster together, suggesting the well-reorganized chromatin architecture that facilitates the interactions of regulatory domains in response to myocardial remodeling. We further show the differences between the acetylation level and the mRNA expression level of cell-type-specific markers for cardiomyocytes and 11 non-myocyte cell types. Notably, we identified transcriptome factor (TF) binding motifs that were enriched in DARs and defined TFs that were predicted to bind to these motifs. We further showed 64 genes coding for these TFs that were differentially expressed in remodeled myocardium when compared with controls. CONCLUSIONS: Our study reveals extensive novel insight on myocardial remodeling at the DNA regulatory level. Differences between the acetylation level and the transcriptional level of cell-type-specific markers suggest additional mechanism(s) between acetylome and transcriptome. By integrating these two layers of epigenetic profiles, we further provide promising TF-encoding genes that could serve as master regulators of myocardial remodeling. Combined, our findings highlight the important role of chromatin regulatory signatures in understanding disease etiology