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.

Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs.

There is increasing momentum toward the development of gene therapy for heart failure (HF) that is defined by impaired calcium (Ca) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca pump) and PLB (phospholamban, ventricular peptide inhibitor of SERCA) to test directly the effectiveness of loss-of-inhibition/gain-of-binding (LOI/GOB) PLB mutants (PLB) that were engineered to compete with the binding of inhibitory wild-type PLB (PLB). Our therapeutic strategy is to relieve PLB inhibition of SERCA2a by using the reserve adrenergic capacity mediated by PLB to enhance cardiac contractility.

Targeting protein-protein interactions for therapeutic discovery via FRET-based high-throughput screening in living cells.

We have developed a structure-based high-throughput screening (HTS) method, using time-resolved fluorescence resonance energy transfer (TR-FRET) that is sensitive to protein-protein interactions in living cells. The membrane protein complex between the cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) and phospholamban (PLB), its Ca-dependent regulator, is a validated therapeutic target for reversing cardiac contractile dysfunction caused by aberrant calcium handling. However, efforts to develop compounds with SERCA2a-PLB specificity have yet to yield an effective drug.

Ischemic Model of Heart Failure in Rats and Mice.

Temporary or permanent left coronary artery (LCA) ligation is the most widely used model of heart failure. In the present protocol, we describe the materials necessary for the procedure, key steps of the LCA ligation, triphenyl tetrazolium chloride (TTC) staining, and calculation of myocardial infarction (MI) size after ischemia-reperfusion (I/R) injury (30 min/24 h) in rats and mice. We discuss precautions and tips regarding the operation before and after surgery, both in vivo and ex vivo.

Cardiac Tissue Engineering Models of Inherited and Acquired Cardiomyopathies.

The lack of biomimetic in vitro models of the human heart has posed a critical barrier to progress in the field of modeling cardiac disease. Human engineered cardiac tissues (hECTs)-autonomous, beating structures that recapitulate key aspects of native cardiac muscle physiology-offer an attractive alternative to traditional in vitro models. Here we describe the use of hECTs to advance our understanding and modeling of cardiac diseases in order to test therapeutic interventions, with a focus on contractile dysfunction in the setting of inherited and acquired forms of cardiomyopathies.

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 β-agonist response and defective calcium handling.

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.

Physiologic, Pathologic, and Therapeutic Paracrine Modulation of Cardiac Excitation-Contraction Coupling.

Cardiac excitation-contraction coupling (ECC) is the orchestrated process of initial myocyte electrical excitation, which leads to calcium entry, intracellular trafficking, and subsequent sarcomere shortening and myofibrillar contraction. Neurohumoral β-adrenergic signaling is a well-established mediator of ECC; other signaling mechanisms, such as paracrine signaling, have also demonstrated significant impact on ECC but are less well understood. For example, resident heart endothelial cells are well-known physiological paracrine modulators of cardiac myocyte ECC mainly via NO and endothelin-1.

CXCR4 and CXCR7 play distinct roles in cardiac lineage specification and pharmacologic β-adrenergic response.

CXCR4 and CXCR7 are prominent G protein-coupled receptors (GPCRs) for chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). This study demonstrates that CXCR4 and CXCR7 induce differential effects during cardiac lineage differentiation and β-adrenergic response in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using lentiviral vectors to ablate CXCR4 and/or CXCR7 expression, hiPSC-CMs were tested for phenotypic and functional properties due to gene knockdown.

Experimental and Computational Insight Into Human Mesenchymal Stem Cell Paracrine Signaling and Heterocellular Coupling Effects on Cardiac Contractility and Arrhythmogenicity.

Rationale: Myocardial delivery of human mesenchymal stem cells (hMSCs) is an emerging therapy for treating the failing heart. However, the relative effects of hMSC-mediated heterocellular coupling (HC) and paracrine signaling (PS) on human cardiac contractility and arrhythmogenicity remain unresolved.

Objective: The objective is to better understand hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity by integrating experimental and computational approaches.

Variability in coronary artery anatomy affects consistency of cardiac damage after myocardial infarction in mice.

Low reliability and reproducibility in heart failure models are well established. The purpose of the present study is to explore factors that affect model consistency of myocardial infarction (MI) in mice. MI was induced by left coronary artery (LCA) ligation.

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 Probability of Inconstancy in Assessment of Cardiac Function Post-Myocardial Infarction in Mice.

In the present study, we explore the inherent variability that leads to overlaps in cardiac functional parameters between control and post-myocardial infarction (MI) mice. Heart failure was induced by Left Coronary Artery (LCA) ligation in mice. Average Ejection Fraction (EF) measured by echocardiography was lower in MI mice compared to control, but exhibited higher Standard Deviation (SD) and Standard Error (SEM), notably in 2D mode.

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.

Deception in simplicity: hereditary phospholamban mutations in dilated cardiomyopathy.

The sarcoplasmic reticulum (SR) calcium pump (SERCA) and its regulator phospholamban are required for cardiovascular function. Phospholamban alters the apparent calcium affinity of SERCA in a process that is modulated by phosphorylation via the β-adrenergic pathway. This regulatory axis allows for the dynamic control of SR calcium stores and cardiac contractility.

Phospholamban C-terminal residues are critical determinants of the structure and function of the calcium ATPase regulatory complex.

To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively.

Lethal, hereditary mutants of phospholamban elude phosphorylation by protein kinase A.

The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by β-adrenergic stimulation and phosphorylation of phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) deletion, alter regulation of SERCA.

Hydrophobic imbalance in the cytoplasmic domain of phospholamban is a determinant for lethal dilated cardiomyopathy.

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) and its regulatory partner phospholamban (PLN) are essential for myocardial contractility. Arg(9) → Cys (R9C) and Arg(14) deletion (R14del) mutations in PLN are associated with lethal dilated cardiomyopathy in humans. To better understand these mutations, we made a series of amino acid substitutions in the cytoplasmic domain of PLN and tested their ability to inhibit SERCA.

Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump.

Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We studied this interaction using electron microscopy of 2D crystals of SERCA in complex with phospholamban. In earlier studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a 3D model was constructed to show interactions with SERCA.

Peptide inhibitors use two related mechanisms to alter the apparent calcium affinity of the sarcoplasmic reticulum calcium pump.

The primary sequence of phospholamban (PLB) has provided a template for the rational design of peptide inhibitors of the sarcoplasmic reticulum calcium ATPase (SERCA). In the transmembrane domain of PLB, there are few polar residues and only one is essential (Asn (34)). Using synthetic peptides, we have previously investigated the role of Asn (34) in the context of simple hydrophobic transmembrane peptides.