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.

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 (Ca2+) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca2+ 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 (PLBM) that were engineered to compete with the binding of inhibitory wild-type PLB (PLBWT). Our therapeutic strategy is to relieve PLBWT inhibition of SERCA2a by using the reserve adrenergic capacity mediated by PLB to enhance cardiac contractility. Using a FRET assay, we determined that the combination of a LOI PLB mutation (L31A) and a GOB PLB mutation (I40A) results in a novel engineered LOI/GOB PLBM (L31A/I40A) that effectively competes with PLBWT binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of PLBM enhances SERCA Ca-ATPase activity by increasing enzyme Ca2+ affinity (1/KCa) in PLBWT-inhibited HEK293 cell homogenates. For an initial assessment of PLBM physiological effectiveness, we used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual. In this system, we observed that adeno-associated virus 2 (rAAV2)-driven expression of PLBM enhances the amplitude of SR Ca2+ release and the rate of SR Ca2+ re-uptake. To assess therapeutic potential, we used a hiPSC-CM model of dilated cardiomyopathy (DCM) containing PLB mutation R14del, where we observed that rAAV2-driven expression of PLBM rescues arrhythmic Ca2+ transients and alleviates decreased Ca2+ transport. Thus, we propose that PLBM transgene expression is a promising gene therapy strategy that directly targets the underlying pathophysiology of abnormal Ca2+ transport and thus contractility in underlying systolic heart failure.

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. We co-expressed GFP-SERCA2a (donor) in the endoplasmic reticulum membrane of HEK293 cells with RFP-PLB (acceptor), and measured FRET using a fluorescence lifetime microplate reader. We screened a small-molecule library and identified 21 compounds (Hits) that changed FRET by >3SD. 10 of these Hits reproducibly alter SERCA2a-PLB structure and function. One compound increases SERCA2a calcium affinity in cardiac membranes but not in skeletal, suggesting that the compound is acting specifically on the SERCA2a-PLB complex, as needed for a drug to mitigate deficient calcium transport in heart failure. The excellent assay quality and correlation between structural and functional assays validate this method for large-scale HTS campaigns. This approach offers a powerful pathway to drug discovery for a wide range of protein-protein interaction targets that were previously considered "undruggable".

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. Four major procedures are discussed in this chapter: (1) preparation of hECTs from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on single-tissue and multitissue bioreactors; (2) data acquisition of hECT contractile function on both of these platforms; (3) hECT modeling of hereditary phospholamban-R14 deletion-dilated cardiomyopathy; and (4) cryo-injury and doxorubicin-induced hECT models of acquired cardiomyopathy.

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. The aim of this chapter is to describe the details of LCA surgery and provide recommendations for current and future surgical operators.

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.

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.

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 ß-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. Moreover, recent studies have demonstrated other resident noncardiomyocyte heart cells (eg, physiological fibroblasts and pathological myofibroblasts), and even experimental cardiotherapeutic cells (eg, mesenchymal stem cells) are also capable of altering cardiomyocyte ECC through paracrine mechanisms. In this review, we first focus on the paracrine-mediated effects of resident and therapeutic noncardiomyocytes on cardiomyocyte hypertrophy, electrophysiology, and calcium handling, each of which can modulate ECC, and then discuss the current knowledge about key paracrine factors and their underlying mechanisms of action. Next, we provide a case example demonstrating the promise of tissue-engineering approaches to study paracrine effects on tissue-level contractility. More specifically, we present new functional and molecular data on the effects of human adult cardiac fibroblast conditioned media on human engineered cardiac tissue contractility and ion channel gene expression that generally agrees with previous murine studies but also suggests possible species-specific differences. By contrast, paracrine secretions by human dermal fibroblasts had no discernible effect on human engineered cardiac tissue contractile function and gene expression. Finally, we discuss systems biology approaches to help identify key stem cell paracrine mediators of ECC and their associated mechanistic pathways. Such integration of tissue-engineering and systems biology methods shows promise to reveal novel insights into paracrine mediators of ECC and their underlying mechanisms of action, ultimately leading to improved cell-based therapies for patients with heart disease.

CXCR4 and CXCR7 play distinct roles in cardiac lineage specification and pharmacologic ß-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 ß-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. Gene expression and flow cytometry confirmed the pluripotent and cardiomyocyte phenotype of undifferentiated and differentiated hiPSCs, respectively. Although reduction of CXCR4 and CXCR7 expression resulted in a delayed cardiac phenotype, only knockdown of CXCR4 delayed the spontaneous beating of hiPSC-CMs. Knockdown of CXCR4 and CXCR7 differentially altered calcium transients and ß-adrenergic response in hiPSC-CMs. In engineered cardiac tissues, depletion of CXCR4 or CXCR7 had opposing effects on developed force and chronotropic response to ß-agonists. This work demonstrates distinct roles for the SDF-1/CXCR4 or CXCR7 network in hiPSC-derived ventricular cardiomyocyte specification, maturation and function.

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. METHODS AND RESULTS: Extending our previous hMSC-cardiomyocyte HC computational model, we incorporated experimentally calibrated hMSC PS effects on cardiomyocyte L-type calcium channel/sarcoendoplasmic reticulum calcium-ATPase activity and cardiac tissue fibrosis. Excitation-contraction simulations of hMSC PS-only and combined HC+PS effects on human cardiomyocytes were representative of human engineered cardiac tissue (hECT) contractile function measurements under matched experimental treatments. Model simulations and hECTs both demonstrated that hMSC-mediated effects were most pronounced under PS-only conditions, where developed force increased ≈4-fold compared with non-hMSC-supplemented controls during physiological 1-Hz pacing. Simulations predicted contractility of isolated healthy and ischemic adult human cardiomyocytes would be minimally sensitive to hMSC HC, driven primarily by PS. Dominance of hMSC PS was also revealed in simulations of fibrotic cardiac tissue, where hMSC PS protected from potential proarrhythmic effects of HC at various levels of engraftment. Finally, to study the nature of the hMSC paracrine effects on contractility, proteomic analysis of hECT/hMSC conditioned media predicted activation of PI3K/Akt signaling, a recognized target of both soluble and exosomal fractions of the hMSC secretome. Treating hECTs with exosome-enriched, but not exosome-depleted, fractions of the hMSC secretome recapitulated the effects observed with hMSC conditioned media on hECT-developed force and expression of calcium-handling genes (eg, SERCA2a, L-type calcium channel). CONCLUSIONS: Collectively, this integrated experimental and computational study helps unravel relative hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity, and provides novel insight into the role of exosomes in hMSC paracrine-mediated effects on contractility.

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. The coronary artery was casted with resin and visualized with fluorescent imaging ex vivo. LCA characteristics and MI size were analyzed individually in each animal, and MI size was correlated with left ventricular (LV) function by echocardiography. Coronary anatomy varies widely in mice, posing challenges for surgical ligation and resulting in inconsistent MI size postligation. The length of coronary arterial trunk, level of bifurcation, number of branches, and territory supplied by these branches are unique in each animal. When the main LCA trunk is ligated, this results in a large MI, but when a single branch is ligated, MI size is variable due to differing levels of LCA ligation and area supplied by the branches. During the ligation procedure, nearly 40% of LCAs are not grossly visible to the surgeon. In these situations, the surgeon blindly sutures a wider and deeper area of tissue in an attempt to catch the LCA. Paradoxically, these situations have greater odds of resulting in smaller MIs. In conclusion, variation in MI size and LV function after LCA ligation in mice is difficult to avoid. Anatomic diversity of the LCA in mice leads to inconsistency in MI size and functional parameters, and this is independent of potential technical modifications made by the operator.NEW & NOTEWORTHY In the present study, we demonstrate that left coronary artery diversity in mice is one of the primary causes of variable myocardial infarction size and cardiac functional parameters in the left coronary artery ligation model. Recognition of anatomic diversity is essential to improve reliability and reproducibility in heart failure research.

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.

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. Fractional Shortening (FS) showed a higher degree of overlap between MI and control mice even though the mean values were significantly different. Hemodynamic measurements of EF resulted in greater SD, SEM, ± 95% confidence intervals, and effect size. In comparing echocardiography at different time points, EF and FS were consistent by mean, but had apparent fluctuation in individual tracks, which were more obvious in MI than control mice. Hemodynamic measurements showed more complexity in data collection in mice in vivo. MI size showed variability that correlated with severity of cardiac function. These studies show that there is inherent variability in functional cardiac parameters after induction of heart failure by MI in mice. Analysis of these parameters by traditional statistical methods is insufficient, and we propose a more robust statistical analysis for proper data interpretation.

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.

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 ß-adrenergic pathway. This regulatory axis allows for the dynamic control of SR calcium stores and cardiac contractility. Herein we focus on hereditary mutants of phospholamban that are associated with heart failure, such as Arg(9)-Cys, Arg(9)-Leu, Arg(9)-His, and Arg(14)-deletion. Each mutant has a distinct effect on PLN function and SR calcium homeostasis. Arg(9)-Cys and Arg(9)-Leu do not inhibit SERCA, Arg(14)-deletion is a partial inhibitor, and Arg(9)-His is comparable to wild-type. While the mutants have distinct functional effects on SERCA, they have in common that they cannot be phosphorylated by protein kinase A (PKA). Arg(9) and Arg(14) are required for PKA recognition and phosphorylation of PLN. Thus, mutations at these positions eliminate ß-adrenergic control and dynamic cardiac contractility. Hydrophobic mutations of Arg(9) cause more complex changes in function, including loss of PLN function and dominant negative interaction with SERCA in heterozygous individuals. In addition, aberrant interaction with PKA may prevent phosphorylation of wild-type PLN and sequester PKA from other local subcellular targets. Herein we consider what is known about each mutant and how the synergistic changes in SR calcium homeostasis lead to impaired cardiac contractility and dilated cardiomyopathy.

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. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val(49)-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-Å decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.

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 ß-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. Deletion of Arg(14) disrupts the protein kinase A recognition motif, which abrogates phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg(9) in phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.

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. R9C is a complete loss-of-function mutant of PLN, whereas R14del is a mild loss-of-function mutant. When combined with wild-type PLN to simulate heterozygous conditions, the mutants had a dominant negative effect on SERCA function. A series of targeted mutations in this region of the PLN cytoplasmic domain ((8)TRSAIRR(14)) demonstrated the importance of hydrophobic balance in proper PLN regulation of SERCA. We found that Arg(9) → Leu and Thr(8) → Cys substitutions mimicked the behavior of the R9C mutant, and an Arg(14) → Ala substitution mimicked the behavior of the R14del mutant. The results reveal that the change in hydrophobicity resulting from the R9C and R14del mutations is sufficient to explain the loss of function and persistent interaction with SERCA. Hydrophobic imbalance in the cytoplasmic domain of PLN appears to be a predictor for the development and progression of dilated cardiomyopathy.

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. In this study, we examined the oligomeric state of phospholamban and the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the 2D crystals. On the basis of projection maps from negatively stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34Ala) was also disordered. The functional state of phospholamban was correlated with an order-to-disorder transition of the phospholamban cytoplasmic domain in the 2D co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27Ala) facilitated data collection from frozen-hydrated crystals. An improved projection map was calculated to a resolution of 8 Å, which supports the pentamer as the oligomeric state of phospholamban in the crystals. The 2D co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association.

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. Herein we propose that the role of Asn in SERCA inhibition is position-sensitive and dependent upon the distribution of hydrophobic residues. To test this hypothesis, we synthesized a series of transmembrane peptides based on a 24 amino acid polyalanine sequence having either an alternating Leu-Ala sequence (Leu 12) or Leu residues at the native positions found in PLB (Leu 9). Asn-containing Leu 9 and Leu 12 peptides were synthesized with a single Asn residue located either one amino acid (N+/-1) or one turn of the helix (N+/-4) in either direction from its native position. Co-reconstitution of these peptides with SERCA into proteoliposomes revealed effects on the apparent calcium affinity and cooperativity of SERCA that correlated with the positions of the Asn and Leu residues. The most inhibitory peptides increased the cooperativity of SERCA as indicated by the Hill coefficients, suggesting that calcium-dependent reversibility is an inherent part of the inhibitory mechanism. Kinetic simulations combined with molecular modeling of the interaction between the peptides and SERCA reveal two related mechanisms of inhibition. Peptides that resemble PLB use the same inhibitory mechanism, whereas peptides that are more divergent from PLB alter an additional step in the calcium transport cycle.