Extracellular signal-regulated kinase (ERK) activation preserves cardiac function in pressure overload induced hypertrophy.

BACKGROUND: Chronic pressure overload and a variety of mediators induce concentric cardiac hypertrophy. When prolonged, cardiac hypertrophy culminates in decreased myocardial function and heart failure. Activation of the extracellular signal-regulated kinase (ERK) is consistently observed in animal models of hypertrophy and in human patients, but its role in the process is controversial. METHODS: We generated transgenic mouse lines with cardiomyocyte restricted overexpression of intrinsically active ERK1, which similar to the observations in hypertrophy is phosphorylated on both the TEY and the Thr207 motifs and is overexpressed at pathophysiological levels. RESULTS: The activated ERK1 transgenic mice developed a modest adaptive hypertrophy with increased contractile function and without fibrosis. Following induction of pressure-overload, where multiple pathways are stimulated, this activation did not further increase the degree of hypertrophy but protected the heart through a decrease in the degree of fibrosis and maintenance of ventricular contractile function. CONCLUSIONS: The ERK pathway acts to promote a compensated hypertrophic response, with enhanced contractile function and reduced fibrosis. The activation of this pathway may be a therapeutic strategy to preserve contractile function when the pressure overload cannot be easily alleviated. The inhibition of this pathway, which is increasingly being used for cancer therapy on the other hand, should be used with caution in the presence of pressure-overload.

Analysis of rat cardiac myocytes and fibroblasts identifies combinatorial enhancer organization and transcription factor families.

Cardiac fibroblasts play key roles in both health and disease. Their regulatory elements, transcription factors (TFs), and mechanisms of expression control have not been fully elucidated. We used a differential open chromatin approach, coupled with active enhancer mark, transcriptomic, and computational TFs binding analysis to map cell-type-specific active enhancers in cardiac fibroblasts and cardiomyocytes, and outline the TFs families that control them. This approach was validated by its ability to uncover the known cardiomyocyte TF biology in an unbiased manner, and was then applied to cardiac fibroblasts. We identified Tead, Sox9, Smad, Tcf, Meis, Rbpj, and Runx1 as the main cardiac fibroblasts TF families. Our analysis shows that in both cell types, distal enhancers, containing concentrated combinatorial clusters of multiple tissue expressed TFs recognition motifs, are combinatorically clustered around tissue specific genes. This model for tissue specific gene expression in the heart supports the general "billboard" model for enhancer organization.

Localization of transcripts, translation, and degradation for spatiotemporal sarcomere maintenance.

The mechanisms responsible for maintaining macromolecular protein complexes, with their proper localization and subunit stoichiometry, are incompletely understood. Here we studied the maintenance of the sarcomere, the basic contractile macromolecular complex of cardiomyocytes. We performed single-cell analysis of cardiomyocytes using imaging of mRNA and protein synthesis, and demonstrate that three distinct mechanisms are responsible for the maintenance of the sarcomere: mRNAs encoding for sarcomeric proteins are localized to the sarcomere, ribosomes are localized to the sarcomere with localized sarcomeric protein translation, and finally, a localized E3 ubiquitin ligase allow efficient degradation of excess unincorporated sarcomeric proteins. We show that these mechanisms are distinct, required, and work in unison, to ensure both spatial localization, and to overcome the large variability in transcription. Cardiomyocytes simultaneously maintain all their sarcomeres using localized translation and degradation processes where proteins are continuously and locally synthesized at high rates, and excess proteins are continuously degraded.

Salt-inducible kinase induces cytoplasmic histone deacetylase 4 to promote vascular calcification.

A pathologic osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) promotes arterial calcifications, a process associated with significant morbidity and mortality. The molecular pathways promoting this pathology are not completely understood. We studied VSMCs, mouse aortic rings, and human aortic valves and showed here that histone deacetylase 4 (HDAC4) is upregulated early in the calcification process. Gain- and loss-of-function assays demonstrate that HDAC4 is a positive regulator driving this pathology. HDAC4 can shuttle between the nucleus and cytoplasm, but in VSMCs, the cytoplasmic rather than the nuclear activity of HDAC4 promotes calcification, and a nuclear-localized mutant of HDAC4 fails to promote calcification. The cytoplasmic location and function of HDAC4 is controlled by the activity of salt-inducible kinase (SIK). Pharmacologic inhibition of SIK sends HDAC4 to the nucleus and inhibits the calcification process in VSMCs, aortic rings, and in vivo In the cytoplasm, HDAC4 binds and its activity depends on the adaptor protein ENIGMA (Pdlim7) to promote vascular calcification. These results establish a cytoplasmic role for HDAC4 and identify HDAC4, SIK, and ENIGMA as mediators of vascular calcification.

Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination.

The "canonical" proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established-in both human and yeast cells-a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes.