Identification and Characterization of p300-Mediated Lysine Residues in Cardiac SERCA2a.

Impaired calcium uptake resulting from reduced expression and activity of the cardiac sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2a) is a hallmark of heart failure (HF). Recently, new mechanisms of SERCA2a regulation, including post-translational modifications (PTMs), have emerged. Our latest analysis of SERCA2a PTMs has identified lysine acetylation as another PTM which might play a significant role in regulating SERCA2a activity. SERCA2a is acetylated, and that acetylation is more prominent in failing human hearts. In this study, we confirmed that p300 interacts with and acetylates SERCA2a in cardiac tissues. Several lysine residues in SERCA2a modulated by p300 were identified using in vitro acetylation assay. Analysis of in vitro acetylated SERCA2a revealed several lysine residues in SERCA2a susceptible to acetylation by p300. Among them, SERCA2a Lys514 (K514) was confirmed to be essential for SERCA2a activity and stability using an acetylated mimicking mutant. Finally, the reintroduction of an acetyl-mimicking mutant of SERCA2a (K514Q) into SERCA2 knockout cardiomyocytes resulted in deteriorated cardiomyocyte function. Taken together, our data demonstrated that p300-mediated acetylation of SERCA2a is a critical PTM that decreases the pump's function and contributes to cardiac impairment in HF. SERCA2a acetylation can be targeted for therapeutic aims for the treatment of HF.

Role of SIRT1 in Modulating Acetylation of the Sarco-Endoplasmic Reticulum Ca2+-ATPase in Heart Failure.

RATIONALE: SERCA2a, sarco-endoplasmic reticulum Ca2+-ATPase, is a critical determinant of cardiac function. Reduced level and activity of SERCA2a are major features of heart failure. Accordingly, intensive efforts have been made to develop efficient modalities for SERCA2a activation. We showed that the activity of SERCA2a is enhanced by post-translational modification with SUMO1 (small ubiquitin-like modifier 1). However, the roles of other post-translational modifications on SERCA2a are still unknown. OBJECTIVE: In this study, we aim to assess the role of lysine acetylation on SERCA2a function and determine whether inhibition of lysine acetylation can improve cardiac function in the setting of heart failure. METHODS AND RESULTS: The acetylation of SERCA2a was significantly increased in failing hearts of humans, mice, and pigs, which is associated with the reduced level of SIRT1 (sirtuin 1), a class III histone deacetylase. Downregulation of SIRT1 increased the SERCA2a acetylation, which in turn led to SERCA2a dysfunction and cardiac defects at baseline. In contrast, pharmacological activation of SIRT1 reduced the SERCA2a acetylation, which was accompanied by recovery of SERCA2a function and cardiac defects in failing hearts. Lysine 492 (K492) was of critical importance for the regulation of SERCA2a activity via acetylation. Acetylation at K492 significantly reduced the SERCA2a activity, presumably through interfering with the binding of ATP to SERCA2a. In failing hearts, acetylation at K492 appeared to be mediated by p300 (histone acetyltransferase p300), a histone acetyltransferase. CONCLUSIONS: These results indicate that acetylation/deacetylation at K492, which is regulated by SIRT1 and p300, is critical for the regulation of SERCA2a activity in hearts. Pharmacological activation of SIRT1 can restore SERCA2a activity through deacetylation at K492. These findings might provide a novel strategy for the treatment of heart failure.

Interaction of a Sarcolipin Pentamer and Monomer with the Sarcoplasmic Reticulum Calcium Pump, SERCA.

The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn4-Ala and Thr5-Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer.

miR-146a Suppresses SUMO1 Expression and Induces Cardiac Dysfunction in Maladaptive Hypertrophy.

RATIONALE: Abnormal SUMOylation has emerged as a characteristic of heart failure (HF) pathology. Previously, we found reduced SUMO1 (small ubiquitin-like modifier 1) expression and SERCA2a (sarcoplasmic reticulum Ca2+-ATPase) SUMOylation in human and animal HF models. SUMO1 gene delivery or small molecule activation of SUMOylation restored SERCA2a SUMOylation and cardiac function in HF models. Despite the critical role of SUMO1 in HF, the regulatory mechanisms underlying SUMO1 expression are largely unknown. OBJECTIVE: To examine miR-146a-mediated SUMO1 regulation and its consequent effects on cardiac morphology and function. METHODS AND RESULTS: In this study, miR-146a was identified as a SUMO1-targeting microRNA in the heart. A strong correlation was observed between miR-146a and SUMO1 expression in failing mouse and human hearts. miR-146a was manipulated in cardiomyocytes through AAV9 (adeno-associated virus serotype 9)-mediated gene delivery, and cardiac morphology and function were analyzed by echocardiography and hemodynamics. Overexpression of miR-146a reduced SUMO1 expression, SERCA2a SUMOylation, and cardiac contractility in vitro and in vivo. The effects of miR-146a inhibition on HF pathophysiology were examined by transducing a tough decoy of miR-146a into mice subjected to transverse aortic constriction. miR-146a inhibition improved cardiac contractile function and normalized SUMO1 expression. The regulatory mechanisms of miR-146a upregulation were elucidated by examining the major miR-146a-producing cell types and transfer mechanisms. Notably, transdifferentiation of fibroblasts triggered miR-146a overexpression and secretion through extracellular vesicles, and the extracellular vesicle-associated miR-146a transfer was identified as the causative mechanism of miR-146a upregulation in failing cardiomyocytes. Finally, extracellular vesicles isolated from failing hearts were shown to contain high levels of miR-146a and exerted negative effects on the SUMO1/SERCA2a signaling axis and hence cardiomyocyte contractility. CONCLUSIONS: Taken together, our results show that miR-146a is a novel regulator of the SUMOylation machinery in the heart, which can be targeted for therapeutic intervention.

Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility.

RATIONALE: The promising clinical benefits of delivering human mesenchymal stem cells (hMSCs) for treating heart disease warrant a better understanding of underlying mechanisms of action. hMSC exosomes increase myocardial contractility; however, the exosomal cargo responsible for these effects remains unresolved. OBJECTIVE: This study aims to identify lead cardioactive hMSC exosomal microRNAs to provide a mechanistic basis for optimizing future stem cell-based cardiotherapies. METHODS AND RESULTS: Integrating systems biology and human engineered cardiac tissue (hECT) technologies, partial least squares regression analysis of exosomal microRNA profiling data predicted microRNA-21-5p (miR-21-5p) levels positively correlate with contractile force and calcium handling gene expression responses in hECTs treated with conditioned media from multiple cell types. Furthermore, miR-21-5p levels were significantly elevated in hECTs treated with the exosome-enriched fraction of the hMSC secretome (hMSC-exo) versus untreated controls. This motivated experimentally testing the human-specific role of miR-21-5p in hMSC-exo-mediated increases of cardiac tissue contractility. Treating hECTs with miR-21-5p alone was sufficient to recapitulate effects observed with hMSC-exo on hECT developed force and expression of associated calcium handling genes (eg, SERCA2a and L-type calcium channel). Conversely, knockdown of miR-21-5p in hMSCs significantly diminished exosomal procontractile and associated calcium handling gene expression effects on hECTs. Western blots supported miR-21-5p effects on calcium handling gene expression at the protein level, corresponding to significantly increased calcium transient amplitude and decreased decay time constant in comparison to miR-scramble control. Mechanistically, cotreating with miR-21-5p and LY294002, a PI3K inhibitor, suppressed these effects. Finally, mathematical simulations predicted the translational capacity for miR-21-5p treatment to restore calcium handling in mature ischemic adult human cardiomyocytes. CONCLUSIONS: miR-21-5p plays a key role in hMSC-exo-mediated effects on cardiac contractility and calcium handling, likely via PI3K signaling. These findings may open new avenues of research to harness the role of miR-21-5p in optimizing future stem cell-based cardiotherapies.

Functional and transcriptomic insights into pathogenesis of R9C phospholamban mutation using human induced pluripotent stem cell-derived cardiomyocytes.

Dilated cardiomyopathy (DCM) can be caused by mutations in the cardiac protein phospholamban (PLN). We used CRISPR/Cas9 to insert the R9C PLN mutation at its endogenous locus into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. R9C PLN hiPSC-CMs display a blunted ß-agonist response and defective calcium handling. In 3D human engineered cardiac tissues (hECTs), a blunted lusitropic response to ß-adrenergic stimulation was observed with R9C PLN. hiPSC-CMs harboring the R9C PLN mutation showed activation of a hypertrophic phenotype, as evidenced by expression of hypertrophic markers and increased cell size and capacitance of cardiomyocytes. RNA-seq suggests that R9C PLN results in an altered metabolic state and profibrotic signaling, which was confirmed by gene expression analysis and picrosirius staining of R9C PLN hECTs. The expression of several miRNAs involved in fibrosis, hypertrophy, and cardiac metabolism were also perturbed in R9C PLN hiPSC-CMs. This study contributes to better understanding of the pathogenic mechanisms of the hereditary R9C PLN mutation in the context of human cardiomyocytes.

Structure-Function Relationship of the SERCA Pump and Its Regulation by Phospholamban and Sarcolipin.

Calcium is a universal second messenger involved in diverse cellular processes, including excitation-contraction coupling in muscle. The contraction and relaxation of cardiac muscle cells are regulated by the cyclic movement of calcium primarily between the extracellular space, the cytoplasm and the sarcoplasmic reticulum (SR). The rapid removal of calcium from the cytosol is primarily facilitated by the sarco(endo)plasmic reticulum calcium ATPase (SERCA) which pumps calcium back into the SR lumen and thereby controls the amount of calcium in the SR. The most studied member of the P-type ATPase family, SERCA has multiple tissue- and cell-specific isoforms and is primarily regulated by two peptides in muscle, phospholamban and sarcolipin. The multifaceted regulation of SERCA via these peptides is exemplified in the biological fine-tuning of their independent oligomerization and regulation. In this chapter, we overview the structure-function relationship of SERCA and its peptide modulators, detailing the regulation of the complexes and summarizing their physiological and disease relevance.

Regulation of the sarcoplasmic reticulum calcium pump by divergent phospholamban isoforms in zebrafish.

The sarcoplasmic reticulum calcium pump (SERCA) is regulated by the small integral membrane proteins phospholamban (PLN) and sarcolipin (SLN). These regulators have homologous transmembrane regions, yet they differ in their cytoplasmic and luminal domains. Although the sequences of PLN and SLN are practically invariant among mammals, they vary in fish. Zebrafish (zf) appear to harbor multiple PLN isoforms, one of which contains 18 sequence variations and a unique luminal extension. Characterization of this isoform (zfPLN) revealed that SERCA inhibition and reversal by phosphorylation were comparable with human PLN. To understand the sequence variations in zfPLN, chimeras were created by transferring the N terminus, linker, and C terminus of zfPLN onto human PLN. A chimera containing the N-terminal domain resulted in a mild loss of function, whereas a chimera containing the linker domain resulted in a gain of function. This latter effect was due to changes in basic residues in the linker region of PLN. Removing the unique luminal domain of zfPLN ((53)SFHGM) resulted in loss of function, whereas adding this domain to human PLN had a minimal effect on SERCA inhibition. We conclude that the luminal extension contributes to SERCA inhibition but only in the context of zfPLN. Although this domain is distinct from the SLN luminal tail, zfPLN appears to use a hybrid PLN-SLN inhibitory mechanism. Importantly, the different zebrafish PLN isoforms raise the interesting possibility that sarcoplasmic reticulum calcium handling and cardiac contractility may be regulated by the differential expression of PLN functional variants.

Post-translational Modifications in Heart Failure: Small Changes, Big Impact.

Heart failure is a complex disease process with various aetiologies and is a significant cause of morbidity and death world-wide. Post-translational modifications (PTMs) alter protein structure and provide functional diversity in terms of physiological functions of the heart. In addition, alterations in protein PTMs have been implicated in human disease pathogenesis. Small ubiquitin-like modifier mediated modification (SUMOylation) pathway was found to play essential roles in cardiac development and function. Abnormal SUMOylation has emerged as a new feature of heart failure pathology. In this review, we will highlight the importance of SUMOylation as a regulatory mechanism of SERCA2a function, and its therapeutic potential for the treatment of heart failure.

Sarco(endo)plasmic reticulum calcium ATPase (SERCA) inhibition by sarcolipin is encoded in its luminal tail.

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is regulated in a tissue-dependent manner via interaction with the short integral membrane proteins phospholamban (PLN) and sarcolipin (SLN). Although defects in SERCA activity are known to cause heart failure, the regulatory mechanisms imposed by PLN and SLN could have clinical implications for both heart and skeletal muscle diseases. PLN and SLN have significant sequence homology in their transmembrane regions, suggesting a similar mode of binding to SERCA. However, unlike PLN, SLN has a conserved C-terminal luminal tail composed of five amino acids ((27)RSYQY), which may contribute to a distinct SERCA regulatory mechanism. We have functionally characterized alanine mutants of the C-terminal tail of SLN using co-reconstituted proteoliposomes of SERCA and SLN. We found that Arg(27) and Tyr(31) are essential for SLN function. We also tested the effect of a truncated variant of SLN (Arg(27)stop) and extended chimeras of PLN with the five luminal residues of SLN added to its C terminus. The Arg(27)stop form of SLN resulted in loss of function, whereas the PLN chimeras resulted in superinhibition with characteristics of both PLN and SLN. Based on our results, we propose that the C-terminal tail of SLN is a distinct, essential domain in the regulation of SERCA and that the functional properties of the SLN tail can be transferred to PLN.

Transmembrane helix 11 is a genuine regulator of the endoplasmic reticulum Ca2+ pump and acts as a functional parallel of ß-subunit on α-Na+,K+-ATPase.

The housekeeping sarco(endo)plasmic reticulum Ca(2+) ATPase SERCA2b transports Ca(2+) across the endoplasmic reticulum membrane maintaining a vital Ca(2+) gradient. Compared with the muscle-specific isoforms SERCA2a and SERCA1a, SERCA2b houses an 11th transmembrane segment (TM11) and a short luminal extension (LE) at its C terminus (2b-tail). The 2b-tail imposes a 2-fold higher apparent Ca(2+) affinity and lower V(max). Previously, we assumed that LE is the sole functional region of the 2b-tail and that TM11 is a passive element providing an additional membrane passage. However, here we show that peptides corresponding to the TM11 region specifically modulate the activity of the homologous SERCA1a in co-reconstituted proteoliposomes and mimic the 2b-tail effect (i.e. lower V(max) and higher Ca(2+) affinity). Using truncated 2b-tail variants we document that TM11 regulates SERCA1a independently from LE, confirming that TM11 is a second, previously unrecognized functional region of the 2b-tail. A phylogenetic analysis further indicates that TM11 is the oldest and most conserved feature of the 2b-tail, found in the SERCA pump of all Bilateria, whereas LE is only present in Nematoda and vertebrates. Considering remarkable similarities with the Na(+),K(+)-ATPase α-ß interaction, we now propose a model for interaction of TM11 with TM7 and TM10 in the anchoring subdomain of the Ca(2+) pump. This model involves a TM11-induced helix bending of TM7. In conclusion, more than just a passive structural feature, TM11 acts as a genuine regulator of Ca(2+) transport through interaction with the pump.

Histone deacetylase 9 promotes endothelial-mesenchymal transition and an unfavorable atherosclerotic plaque phenotype.

Endothelial-mesenchymal transition (EndMT) is associated with various cardiovascular diseases and in particular with atherosclerosis and plaque instability. However, the molecular pathways that govern EndMT are poorly defined. Specifically, the role of epigenetic factors and histone deacetylases (HDACs) in controlling EndMT and the atherosclerotic plaque phenotype remains unclear. Here, we identified histone deacetylation, specifically that mediated by HDAC9 (a class IIa HDAC), as playing an important role in both EndMT and atherosclerosis. Using in vitro models, we found class IIa HDAC inhibition sustained the expression of endothelial proteins and mitigated the increase in mesenchymal proteins, effectively blocking EndMT. Similarly, ex vivo genetic knockout of Hdac9 in endothelial cells prevented EndMT and preserved a more endothelial-like phenotype. In vivo, atherosclerosis-prone mice with endothelial-specific Hdac9 knockout showed reduced EndMT and significantly reduced plaque area. Furthermore, these mice displayed a more favorable plaque phenotype, with reduced plaque lipid content and increased fibrous cap thickness. Together, these findings indicate that HDAC9 contributes to vascular pathology by promoting EndMT. Our study provides evidence for a pathological link among EndMT, HDAC9, and atherosclerosis and suggests that targeting of HDAC9 may be beneficial for plaque stabilization or slowing the progression of atherosclerotic disease.

Measuring Cardiomyocyte Contractility and Calcium Handling In Vitro.

In vitro measurements of cardiomyocyte contractility and Ca2+ handling have been used as a platform for determining physiological consequence of various genetic manipulations and identifying potential therapeutic targets for the treatment of heart failure. The Myocyte Calcium and Contractility System (IonOptix) offers a simultaneous trace of sarcomere movements and changes of intracellular Ca2+ levels in a single cardiomyocyte. Herein, we describe a modified protocol for the isolation of adult cardiomyocytes from murine hearts and provide a step-by-step description on how to analyze cardiomyocyte Ca2+ transient and contractility data collected using the IonOptix system. In our modified protocol, we recommend a novel cannulation technique which simplifies this difficult method and leads to improved viability of isolated cardiomyocytes. In addition, a comprehensive analysis of intracellular Ca2+ handling, SR Ca2+ load, myofilament Ca2+ sensitivity, and cardiomyocyte contractility is described in order to provide important insights into myocardial mechanics.

Altered myocardial calcium cycling and energetics in heart failure--a rational approach for disease treatment.

Cardiomyocyte function depends on coordinated movements of calcium into and out of the cell and the proper delivery of ATP to energy-utilizing enzymes. Defects in calcium-handling proteins and abnormal energy metabolism are features of heart failure. Recent discoveries have led to gene-based therapies targeting calcium-transporting or -binding proteins, such as the cardiac sarco(endo)plasmic reticulum calcium ATPase (SERCA2a), leading to improvements in calcium homeostasis and excitation-contraction coupling. Here we review impaired calcium cycling and energetics in heart failure, assessing their roles from both a mutually exclusive and interdependent viewpoint, and discuss therapies that may improve the failing myocardium.

Small-molecule activation of SERCA2a SUMOylation for the treatment of heart failure.

Decreased activity and expression of the cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a), a critical pump regulating calcium cycling in cardiomyocyte, are hallmarks of heart failure. We have previously described a role for the small ubiquitin-like modifier type 1 (SUMO-1) as a regulator of SERCA2a and have shown that gene transfer of SUMO-1 in rodents and large animal models of heart failure restores cardiac function. Here, we identify and characterize a small molecule, N106, which increases SUMOylation of SERCA2a. This compound directly activates the SUMO-activating enzyme, E1 ligase, and triggers intrinsic SUMOylation of SERCA2a. We identify a pocket on SUMO E1 likely to be responsible for N106's effect. N106 treatment increases contractile properties of cultured rat cardiomyocytes and significantly improves ventricular function in mice with heart failure. This first-in-class small-molecule activator targeting SERCA2a SUMOylation may serve as a potential therapeutic strategy for treatment of heart failure.

Distinct morphological and electrophysiological properties of an elk prion peptide.

A key event in prion diseases is the conversion of the prion protein (PrP) from its native α-helical conformation to a misfolded, ß-sheet rich conformation. Thus, preventing or reversing PrP misfolding could provide a means to disrupt prion disease progression and transmission. However, determining the structure of misfolded PrP has been notoriously difficult due to its inherent heterogeneity and aggregation behavior. For these reasons, simplified peptide fragments have been used as models that recapitulate characteristics of full-length PrP, such as amyloid-like aggregation and fibril formation, and in vitro toxicity. We provide a biochemical and structural comparison of PrP(127-147) peptides from elk, bovine and hamster using electrophysiology, electron microscopy and fluorescence. Our results demonstrate that the PrP(127-147) peptides adopt distinct populations of fibril structures. In addition, the elk PrP(127-147) peptide is unique in its ability to enhance Thioflavin T fluorescence and its ability to modulate neuronal ion channel conductances.