Delaine K. Ceholski

Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy

A number of genetic mutations is associated with cardiomyopathies. A mutation in the coding region of the phospholamban (PLN) gene (R14del) is identified in families with hereditary heart failure. Heterozygous patients exhibit left ventricular dilation and ventricular arrhythmias. Here we generate induced pluripotent stem cells (iPSCs) from a patient harbouring the PLN R14del mutation and differentiate them into cardiomyocytes (iPSC-CMs). We find that the PLN R14del mutation induces Ca2+ handling abnormalities, electrical instability, abnormal cytoplasmic distribution of PLN protein and increases expression of molecular markers of cardiac hypertrophy in iPSC-CMs. Gene correction using transcription activator-like effector nucleases (TALENs) ameliorates the R14del-associated disease phenotypes in iPSC-CMs. In addition, we show that knocking down the endogenous PLN and simultaneously expressing a codon-optimized PLN gene reverses the disease phenotype in vitro. Our findings offer novel strategies for targeting the pathogenic mutations associated with cardiomyopathies.

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.

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.

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 &bgr;-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 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. 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 β-adrenergic response in hiPSC-CMs. In engineered cardiac tissues, depletion of CXCR4 or CXCR7 had opposing effects on developed force and chronotropic response to β-agonists. This work demonstrates distinct roles for the SDF-1/CXCR4 or CXCR7 network in hiPSC-derived ventricular cardiomyocyte specification, maturation and function.

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.

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.

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.