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S100A1 is a small EF-type Ca2+sensor protein that belongs to the multigenic S100 protein family. It is abundantly expressed in cardiomyocytes (CMs) and has been described as a key regulator of CM performance due to its unique ability to interact with structural contractile proteins, regulators of cardiac Ca2+cycling, and mitochondrial proteins. However, our understanding of the molecular mechanisms regulating S100A1 protein levels is limited. We used the bioinformatics tool GPS-SUMO2.0 to identify a putative SUMO interacting motif (SIM) on S100A1. Consistently, a S100A1:SUMO interaction assay showed a Ca2+-dependent interaction of S100A1 with SUMO proteins. In neonatal rat ventricular myocytes (NRVM) and COS1 cells, S100A1 protein abundance increased in the presence of overexpressed SUMO1 without affecting the S100A1 mRNA transcript. We then generated S100A1 truncation mutants, where the SIM motif was removed by truncation or in which the core residues of the SIM motif (residues 77-79) were deleted or replaced by alanine. In COS1 cells and NRVM, overexpression of these S100A1 mutants led to elevated S100A1 mutant mRNA levels but failed to produce respective protein levels. Protein expression of these mutants could be rescued from degradation by addition of the proteasome inhibitor MG-132. By using an information-driven approach to dock the three-dimensional structures of S100A1 and SUMO, we predict a novel interaction mode between the SIM in S100A1 and SUMO. This study shows an important role of SUMO:SIM-mediated protein:protein interaction in the regulation of post-translational protein stability, and provides mechanistic insights into the indispensability of the core SIM for S100A1 post-translational stability.

Catecholamines stimulate the first step of lipolysis by PKA-dependent release of the lipid droplet-associated protein ABHD5 from perilipin to co-activate the lipase ATGL. Here, we unmask a yet unrecognized proteolytic and cardioprotective function of ABHD5. ABHD5 acts in vivo and in vitro as a serine protease cleaving HDAC4. Through the production of an N-terminal polypeptide of HDAC4 (HDAC4-NT), ABHD5 inhibits MEF2-dependent gene expression and thereby controls glucose handling. ABHD5-deficiency leads to neutral lipid storage disease in mice. Cardiac-specific gene therapy of HDAC4-NT does not protect from intra-cardiomyocyte lipid accumulation but strikingly from heart failure, thereby challenging the concept of lipotoxicity-induced heart failure. ABHD5 levels are reduced in failing human hearts and murine transgenic ABHD5 expression protects from pressure-overload induced heart failure. These findings represent a conceptual advance by connecting lipid with glucose metabolism through HDAC4 proteolysis and enable new translational approaches to treat cardiometabolic disease.

The stress-responsive epigenetic repressor histone deacetylase 4 (HDAC4) regulates cardiac gene expression. Here we show that the levels of an N-terminal proteolytically derived fragment of HDAC4, termed HDAC4-NT, are lower in failing mouse hearts than in healthy control hearts. Virus-mediated transfer of the portion of the Hdac4 gene encoding HDAC4-NT into the mouse myocardium protected the heart from remodeling and failure; this was associated with decreased expression of Nr4a1, which encodes a nuclear orphan receptor, and decreased NR4A1-dependent activation of the hexosamine biosynthetic pathway (HBP). Conversely, exercise enhanced HDAC4-NT levels, and mice with a cardiomyocyte-specific deletion of Hdac4 show reduced exercise capacity, which was characterized by cardiac fatigue and increased expression of Nr4a1. Mechanistically, we found that NR4A1 negatively regulated contractile function in a manner that depended on the HBP and the calcium sensor STIM1. Our work describes a new regulatory axis in which epigenetic regulation of a metabolic pathway affects calcium handling. Activation of this axis during intermittent physiological stress promotes cardiac function, whereas its impairment in sustained pathological cardiac stress leads to heart failure.

Background: S100A1 is an EF-hand calcium (Ca 2+ ) sensor protein with predominant expression in the mammalian heart. Although its outstanding influence on cardiac performance is well described, only few target molecules are known so far, such as the sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a). Objective and Results: The discrepancy between the known interactors and the diversity of S100A1`s functions brings up the question which key targets are undetected so far and can, in addition to the known ones, explain the importance of S100A1 for the heart function. S100A1 knock-out (SKO -/- ) mouse heart extracts and lysates of isolated SKO -/- cardiomyocytes were subjected to HPLC S100A1 affinity-purification (AP) columns coupled to tandem mass spectrometry (MS) followed by network modeling of the interactome, to identify novel and to confirm previously known targets in the heart. Human recombinant S100A1 protein AP columns were used to trap EGTA- and Ca 2+ -dependent interactions. Subsequent MS and computational analyses revealed more than 90 novel cardiac interactors for S100A1 in a strict Ca 2+ -dependent manner, while previously known targets were confirmed. We showed that helix 4 of S100A1 (S100A1ct) alone can improve main parameters for the heart performance and might thereby function as a therapeutic biologic. Investigating the molecular mechanisms of S100A1`s interactions with its target molecules might help to understand this effect and unveil relevant regions for other therapeutic effects. In order to uncover the mechanisms of the various interactions, docking experiments were performed to gain an understanding, which areas of S100A1 and S100A1ct might be relevant for the biological functions on selected targets. Thus we detected a region on SERCA2a where S100A1 as well as S100A1ct might compete with the SERCA2a inhibitor phospholamban and by that may improve the SERCA2a activity. These in silico findings will be combined with experimental investigations of the mode of interaction by Cross-link MS experiments. Conclusion: Combining target screening via AP-MS/MS with computer-supported epitope mapping provides a powerful strategy to understand the molecular action of known therapies and by that helps to develop new treatments against heart failure.

Introduction: S100A1 is a type of S100 protein with two EF-hand motif. It has a unique characteristic of interacting with multiple proteins of contractile machinery & mitochondria. S100A1 protein able to govern proper systolic & diastolic performance of cardiomyocyte interacting, respectively, with SERCA2A & RYR2. S100A1 level decreases dramatically as a consequence of cellular cues such as oxidative-stress or activation of cellular proteasomes. Selective reconstitution of reduced S100A1 level by AAV9-gene transfer rescued failing heart. Because of the unique ability of S100A1 to interact with myriads of proteins with multiple function, we aimed to dissect the structure-function relationship of the domains of S100A1. Methods & results: The C-terminus domain of S100A1 (S100A1ct) equally recapitulated the inotropic & other benefits of the S100A1-FL in experimental model of heart failure & identified as the bioactive lead domain of S100A1. To elucidate the function of the S100A1 without the C-terminus, here after, referred as S100A1ΔCT, we adenoviraly expressed S100A1ΔCT, & as a control, either expressing GFP or S100A1-FL. We, inadvertently, discovered that S100A1ΔCT could not be detected at protein level. PCR analysis & sequencing proved the presence S100A1ΔCT mRNA transcript. Bioinformatics revealed the presence of SUMO interaction motif (SIM) with in the C-terminus & multiple ubiquitination residues on S100A1ΔCT, suggesting the SIM domain of the S100A1 is crucial for stability. Inhibition of cellular proteasome lead to the detection of the S100A1ΔCT protein in cells over expressing S100A1ΔCT. We overexpressed S100a1ΔCT, deletion mutants having part of the SIM motif (S100a1-SIM - ), the entire motif (S100a1-SIM + ) & the S100a1-FL. Whereas the overexpression of S100A1-SIM + & S100A1-FL were detected, S100A1ΔCT & S100A1-SIM - could not be detected at protein level. Conclusion: The C-terminus of S100A1 is essential for stability of S100A1, besides, its potent inotropic function. Taken together, we deciphered the previously unrecognized posttranslational modification governed S100A1 regulation, thus providing new insight into the development of novel therapeutic strategies for heart failure via posttranslational manipulation of S100A1.

Histone deacetylase 4 (HDAC4) regulates numerous gene expression programs through its signal-dependent repression of the transcription factor myocyte enhancer factor 2 (MEF2). Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling promotes pathological cardiac remodeling by phosphorylating HDAC4, with consequent stimulation of MEF2 activity. Recently, we described a novel mechanism whereby protein kinase A (PKA) overcomes CaMKII-mediated activation of MEF2 by regulated proteolysis of HDAC4. PKA induces the generation of an N-terminal HDAC4 cleavage product (HDAC4-NT). HDAC4-NT inhibits MEF2 activity, thereby antagonizing the pro-hypertrophic actions of CaMKII signaling. However, the individual protease mediating HDAC4 proteolysis remains unknown. Using a number of group specific chemical protease inhibitors, we found that the PKA-dependent HDAC4 protease belongs to the family of serine proteases. To further identify the individual serine protease, we screened a human serine protease siRNA library and found abhydrolase domain containing 5 (ABHD5) to be required for PKA-induced HDAC4 proteolysis. The screening result was validated using three different siRNAs. Remarkably, forced adenoviral expression of ABHD5 led to robust production of HDAC4-NT in isolated neonatal rat ventricular myocytes (NRVMs) even in the absence of PKA. Furthermore, adenoviral expression of ABHD5 in NRVMs inhibited MEF2 transcriptional activity and attenuated cardiomyocyte hypertrophy. Interestingly, ABHD5 was initially described to be involved in lipid metabolism. Taken together, we show that ABHD5 is required for PKA-dependent HDAC4 proteolysis and sufficient for HDAC4-NT production as well as inhibition of cardiomyocyte hypertrophy. These data imply a so far unknown link between lipid metabolism and epigenetic regulation of cardioprotection.