Efficient and reproducible generation of human iPSC-derived cardiomyocytes using a stirred bioreactor.

In the last decade human iPSC-derived cardiomyocytes (hiPSC-CMs) proved to be valuable for cardiac disease modeling and cardiac regeneration, yet challenges with scale, quality, inter-batch consistency, and cryopreservation remain, reducing experimental reproducibility and limiting clinical translation. Here, we report a robust cardiac differentiation protocol that uses Wnt modulation and a stirred suspension bioreactor to produce on average 124 million hiPSC-CMs with >90% purity using a variety of hiPSC lines (19 differentiations; 10 iPSC lines). After controlled freeze and thaw, bioreactor-derived CMs (bCMs) showed high viability (>90%), interbatch reproducibility in cellular morphology, function, drug response and ventricular identity, which was further supported by single cell transcriptomes. bCMs on microcontact printed substrates revealed a higher degree of sarcomere maturation and viability during long-term culture compared to monolayer-derived CMs (mCMs). Moreover, functional investigation of bCMs in 3D engineered heart tissues showed earlier and stronger force production during long-term culture, and robust pacing capture up to 4 Hz when compared to mCMs. bCMs derived from this differentiation protocol will expand the applications of hiPSC-CMs by providing a reproducible, scalable, and resource efficient method to generate cardiac cells with well-characterized structural and functional properties superior to standard mCMs.

Pediatric ECG-Based Deep Learning to Predict Left Ventricular Dysfunction and Remodeling.

BACKGROUND: Artificial intelligence-enhanced ECG analysis shows promise to detect ventricular dysfunction and remodeling in adult populations. However, its application to pediatric populations remains underexplored. METHODS: A convolutional neural network was trained on paired ECG-echocardiograms (≤2 days apart) from patients ≤18 years of age without major congenital heart disease to detect human expert-classified greater than mild left ventricular (LV) dysfunction, hypertrophy, and dilation (individually and as a composite outcome). Model performance was evaluated on single ECG-echocardiogram pairs per patient at Boston Children's Hospital and externally at Mount Sinai Hospital using area under the receiver operating characteristic curve (AUROC) and area under the precision-recall curve (AUPRC). RESULTS: The training cohort comprised 92 377 ECG-echocardiogram pairs (46 261 patients; median age, 8.2 years). Test groups included internal testing (12 631 patients; median age, 8.8 years; 4.6% composite outcomes), emergency department (2830 patients; median age, 7.7 years; 10.0% composite outcomes), and external validation (5088 patients; median age, 4.3 years; 6.1% composite outcomes) cohorts. Model performance was similar on internal test and emergency department cohorts, with model predictions of LV hypertrophy outperforming the pediatric cardiologist expert benchmark. Adding age and sex to the model added no benefit to model performance. When using quantitative outcome cutoffs, model performance was similar between internal testing (composite outcome: AUROC, 0.88, AUPRC, 0.43; LV dysfunction: AUROC, 0.92, AUPRC, 0.23; LV hypertrophy: AUROC, 0.88, AUPRC, 0.28; LV dilation: AUROC, 0.91, AUPRC, 0.47) and external validation (composite outcome: AUROC, 0.86, AUPRC, 0.39; LV dysfunction: AUROC, 0.94, AUPRC, 0.32; LV hypertrophy: AUROC, 0.84, AUPRC, 0.25; LV dilation: AUROC, 0.87, AUPRC, 0.33), with composite outcome negative predictive values of 99.0% and 99.2%, respectively. Saliency mapping highlighted ECG components that influenced model predictions (precordial QRS complexes for all outcomes; T waves for LV dysfunction). High-risk ECG features include lateral T-wave inversion (LV dysfunction), deep S waves in V1 and V2 and tall R waves in V6 (LV hypertrophy), and tall R waves in V4 through V6 (LV dilation). CONCLUSIONS: This externally validated algorithm shows promise to inexpensively screen for LV dysfunction and remodeling in children, which may facilitate improved access to care by democratizing the expertise of pediatric cardiologists.

Social Drivers of Health and Pediatric Extracorporeal Membrane Oxygenation Outcomes.

BACKGROUND: Relationships between social drivers of health (SDoH) and pediatric health outcomes are highly complex with substantial inconsistencies in studies examining SDoH and extracorporeal membrane oxygenation (ECMO) outcomes. To add to this literature with emerging novel SDoH measures, and to address calls for institutional accountability, we examined associations between SDoH and pediatric ECMO outcomes. METHODS: This single-center retrospective cohort study included children (<18 years) supported on ECMO (2012-2021). SDoH included Child Opportunity Index (COI), race, ethnicity, payer, interpreter requirement, urbanicity, and travel-time to hospital. COI is a multidimensional estimation of SDoH incorporating traditional (eg, income) and novel (eg, healthy food access) neighborhood attributes ([range 0-100] higher indicates healthier child development). Outcomes included in-hospital mortality, ECMO run duration, and length of stay (LOS). RESULTS: 540 children on ECMO (96%) had a calculable COI. In-hospital mortality was 44% with median run duration of 125 hours and ICU LOS 29 days. Overall, 334 (62%) had cardiac disease, 92 (17%) neonatal respiratory failure, 93 (17%) pediatric respiratory failure, and 21 (4%) sepsis. Median COI was 64 (interquartile range 32-81), 323 (60%) had public insurance, 174 (34%) were from underrepresented racial groups, 57 (11%) required interpreters, 270 (54%) had urban residence, and median travel-time was 89 minutes. SDoH including COI were not statistically associated with outcomes in univariate or multivariate analysis. CONCLUSIONS: We observed no significant difference in pediatric ECMO outcomes according to SDoH. Further research is warranted to better understand drivers of inequitable health outcomes in children, and potential protective mechanisms.

An improved reporter identifies ruxolitinib as a potent and cardioprotective CaMKII inhibitor.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) hyperactivity causes cardiac arrhythmias, a major source of morbidity and mortality worldwide. Despite proven benefits of CaMKII inhibition in numerous preclinical models of heart disease, translation of CaMKII antagonists into humans has been stymied by low potency, toxicity, and an enduring concern for adverse effects on cognition due to an established role of CaMKII in learning and memory. To address these challenges, we asked whether any clinically approved drugs, developed for other purposes, were potent CaMKII inhibitors. For this, we engineered an improved fluorescent reporter, CaMKAR (CaMKII activity reporter), which features superior sensitivity, kinetics, and tractability for high-throughput screening. Using this tool, we carried out a drug repurposing screen (4475 compounds in clinical use) in human cells expressing constitutively active CaMKII. This yielded five previously unrecognized CaMKII inhibitors with clinically relevant potency: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib. We found that ruxolitinib, an orally bioavailable and U.S. Food and Drug Administration-approved medication, inhibited CaMKII in cultured cardiomyocytes and in mice. Ruxolitinib abolished arrhythmogenesis in mouse and patient-derived models of CaMKII-driven arrhythmias. A 10-min pretreatment in vivo was sufficient to prevent catecholaminergic polymorphic ventricular tachycardia, a congenital source of pediatric cardiac arrest, and rescue atrial fibrillation, the most common clinical arrhythmia. At cardioprotective doses, ruxolitinib-treated mice did not show any adverse effects in established cognitive assays. Our results support further clinical investigation of ruxolitinib as a potential treatment for cardiac indications.

Insight into the Role of the Child Opportunity Index on Surgical Outcomes in Congenital Heart Disease.

OBJECTIVE: To use neighborhood-level Child Opportunity Index (COI) measures to investigate disparities in congenital heart surgery postoperative outcomes and identify potential targets for intervention. STUDY DESIGN: In this single-institution retrospective cohort study, children <18 years old who underwent cardiac surgery between 2010 and 2020 were included. Patient-level demographics and neighborhood-level COI were used as predictor variables. COI-a composite US census tract-based score measuring educational, health/environmental, and social/economic opportunities-was dichotomized as lower (<40th percentile) vs higher (≥40th percentile). Cumulative incidence of hospital discharge was compared between groups using death as a competing risk, adjusting for clinical characteristics associated with outcomes. Secondary outcomes included hospital readmission and death within 30 days. RESULTS: Among 6247 patients (55% male) with a median age of 0.8 years (IQR, 0.2-4.3), 26% had lower COI. Lower COI was associated with longer hospital lengths of stay (adjusted HR, 1.2; 95% CI, 1.1-1.2; P < .001) and an increased risk of death (adjusted OR, 2.0; 95% CI. 1.4-2.8; P < .001), but not hospital readmission (P = .6). At the neighborhood level, lacking health insurance coverage, food/housing insecurity, lower parental literacy and college attainment, and lower socioeconomic status were associated with longer hospital length of stay and increased risk of death. At the patient-level, public insurance (adjusted OR, 1.4; 95% CI, 1.0-2.0; P = .03) and caretaker Spanish language (adjusted OR 2.4; 95% CI, 1.2-4.3; P < .01) were associated with an increased risk of death. CONCLUSIONS: Lower COI is associated with longer length of stay and higher early postoperative mortality. Risk factors identified including Spanish language, food/housing insecurity, and parental literacy serve as potential intervention targets.

Computational design of custom therapeutic cells to correct failing human cardiomyocytes.

Background: Myocardial delivery of non-excitable cells-namely human mesenchymal stem cells (hMSCs) and c-kit+ cardiac interstitial cells (hCICs)-remains a promising approach for treating the failing heart. Recent empirical studies attempt to improve such therapies by genetically engineering cells to express specific ion channels, or by creating hybrid cells with combined channel expression. This study uses a computational modeling approach to test the hypothesis that custom hypothetical cells can be rationally designed to restore a healthy phenotype when coupled to human heart failure (HF) cardiomyocytes. Methods: Candidate custom cells were simulated with a combination of ion channels from non-excitable cells and healthy human cardiomyocytes (hCMs). Using a genetic algorithm-based optimization approach, candidate cells were accepted if a root mean square error (RMSE) of less than 50% relative to healthy hCM was achieved for both action potential and calcium transient waveforms for the cell-treated HF cardiomyocyte, normalized to the untreated HF cardiomyocyte. Results: Custom cells expressing only non-excitable ion channels were inadequate to restore a healthy cardiac phenotype when coupled to either fibrotic or non-fibrotic HF cardiomyocytes. In contrast, custom cells also expressing cardiac ion channels led to acceptable restoration of a healthy cardiomyocyte phenotype when coupled to fibrotic, but not non-fibrotic, HF cardiomyocytes. Incorporating the cardiomyocyte inward rectifier K+ channel was critical to accomplishing this phenotypic rescue while also improving single-cell action potential metrics associated with arrhythmias, namely resting membrane potential and action potential duration. The computational approach also provided insight into the rescue mechanisms, whereby heterocellular coupling enhanced cardiomyocyte L-type calcium current and promoted calcium-induced calcium release. Finally, as a therapeutically translatable strategy, we simulated delivery of hMSCs and hCICs genetically engineered to express the cardiomyocyte inward rectifier K+ channel, which decreased action potential and calcium transient RMSEs by at least 24% relative to control hMSCs and hCICs, with more favorable single-cell arrhythmia metrics. Conclusion: Computational modeling facilitates exploration of customizable engineered cell therapies. Optimized cells expressing cardiac ion channels restored healthy action potential and calcium handling phenotypes in fibrotic HF cardiomyocytes and improved single-cell arrhythmia metrics, warranting further experimental validation studies of the proposed custom therapeutic cells.

PPARdelta activation induces metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs.

In silico Cell Therapy Model Restores Failing Human Myocyte Electrophysiology and Calcium Cycling in Fibrotic Myocardium.

Myocardial delivery of human c-kit+ cardiac interstitial cells (hCICs) and human mesenchymal stem cells (hMSCs), an emerging approach for treating the failing heart, has been limited by an incomplete understanding of the effects on host myocardium. This computational study aims to model hCIC and hMSC effects on electrophysiology and calcium cycling of healthy and diseased human cardiomyocytes (hCM), and reveals a possible cardiotherapeutic benefit independent of putative regeneration processes. First, we developed an original hCIC mathematical model with an electrical profile comprised of distinct experimentally identified ion currents. Next, we verified the model by confirming it is representative of published experiments on hCIC whole-cell electrophysiology and on hCIC co-cultures with rodent cardiomyocytes. We then used our model to compare electrophysiological effects of hCICs to other non-excitable cells, as well as clinically relevant hCIC-hMSC combination therapies and fused hCIC-hMSC CardioChimeras. Simulation of direct coupling of hCICs to healthy or failing hCMs through gap junctions led to greater increases in calcium cycling with lesser reductions in action potential duration (APD) compared with hMSCs. Combined coupling of hCICs and hMSCs to healthy or diseased hCMs led to intermediate effects on electrophysiology and calcium cycling compared to individually coupled hCICs or hMSCs. Fused hCIC-hMSC CardioChimeras decreased healthy and diseased hCM APD and calcium transient amplitude compared to individual or combined cell treatments. Finally, to provide a theoretical basis for optimizing cell-based therapies, we randomized populations of 2,500 models incorporating variable hMSC and hCIC interventions and simulated their effects on restoring diseased cardiomyocyte electrophysiology and calcium handling. The permutation simulation predicted the ability to correct abnormal properties of heart failure hCMs in fibrotic, but not non-fibrotic, myocardium. This permutation experiment also predicted paracrine signaling to be a necessary and sufficient mechanism for this correction, counteracting the fibrotic effects while also restoring arrhythmia-related metrics such as upstroke velocity and resting membrane potential. Altogether, our in silico findings suggest anti-fibrotic effects of paracrine signaling are critical to abrogating pathological cardiomyocyte electrophysiology and calcium cycling in fibrotic heart failure, and support further investigation of delivering an optimized cellular secretome as a potential strategy for improving heart failure therapy.

Adult human cardiac stem cell supplementation effectively increases contractile function and maturation in human engineered cardiac tissues.

BACKGROUND: Delivery of stem cells to the failing heart is a promising therapeutic strategy. However, the improvement in cardiac function in animal studies has not fully translated to humans. To help bridge the gap between species, we investigated the effects of adult human cardiac stem cells (hCSCs) on contractile function of human engineered cardiac tissues (hECTs) as a species-specific model of the human myocardium. METHODS: Human induced pluripotent stem cell-derived cardiomyoctes (hCMs) were mixed with Collagen/Matrigel to fabricate control hECTs, with an experimental group of hCSC-supplemented hECT fabricated using a 9:1 ratio of hCM to hCSC. Functional testing was performed starting on culture day 6, under spontaneous conditions and also during electrical pacing from 0.25 to 1.0 Hz, measurements repeated at days 8 and 10. hECTs were then frozen and processed for gene analysis using a Nanostring assay with a cardiac targeted custom panel. RESULTS: The hCSC-supplemented hECTs displayed a twofold higher developed force vs. hCM-only controls by day 6, with approximately threefold higher developed stress and maximum rates of contraction and relaxation during pacing at 0.75 Hz. The spontaneous beat rate characteristics were similar between groups, and hCSC supplementation did not adversely impact beat rate variability. The increased contractility persisted through days 8 and 10, albeit with some decrease in the magnitude of the difference of the force by day 10, but with developed stress still significantly higher in hCSC-supplemented hECT; these findings were confirmed with multiple hCSC and hCM cell lines. The force-frequency relationship, while negative for both, control (- 0.687 Hz- 1; p = 0.013 vs. zero) and hCSC-supplemented (- 0.233 Hz- 1;p = 0.067 vs. zero) hECTs, showed a significant rectification in the regression slope in hCSC-supplemented hECT (p = 0.011 vs. control). Targeted gene exploration (59 genes) identified a total of 14 differentially expressed genes, with increases in the ratios of MYH7/MHY6, MYL2/MYL7, and TNNI3/TNNI1 in hCSC-supplemented hECT versus controls. CONCLUSIONS: For the first time, hCSC supplementation was shown to significantly improve human cardiac tissue contractility in vitro, without evidence of proarrhythmic effects, and was associated with increased expression of markers of cardiac maturation. These findings provide new insights about adult cardiac stem cells as contributors to functional improvement of human myocardium.

FTO-Dependent N6-Methyladenosine Regulates Cardiac Function During Remodeling and Repair.

BACKGROUND: Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. METHODS: We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. RESULTS: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. CONCLUSIONS: Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

An Introduction to Computational Modeling of Cardiac Electrophysiology and Arrhythmogenicity.

Mathematical modeling is a powerful tool to study the complex and orchestrated biological process of cardiac electrical activity. By integrating experimental data from key components of cardiac electrophysiology, systems biology simulations can complement empirical findings, provide quantitative insight into physiological and pathophysiological mechanisms of action, and guide new hypotheses to better understand this complex biological system to develop novel cardiotherapeutic approaches. In this chapter, we briefly introduce in silico methods to describe the dynamics of physiological and pathophysiological single-cell and tissue-level cardiac electrophysiology. Using a "bottom-up" approach, we first describe the basis of ion channel mathematical models. Next, we discuss how the net flux of ions through such channels leads to changes in transmembrane voltage during cardiomyocyte action potentials. By applying these fundamentals, we describe how action potentials propagate in models of cardiac tissue. In addition, we provide case studies simulating single-cell and tissue-level arrhythmogenesis, as well as promising approaches to circumvent or overcome such adverse events. Overall, basic concepts and tools are discussed in this chapter as an accessible introduction to nonmathematicians to foster an understanding of electrophysiological modeling studies and help facilitate communication with dry lab colleagues and collaborators.

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.

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.

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.

Modeling Electrophysiological Coupling and Fusion between Human Mesenchymal Stem Cells and Cardiomyocytes.

Human mesenchymal stem cell (hMSC) delivery has demonstrated promise in preclinical and clinical trials for myocardial infarction therapy; however, broad acceptance is hindered by limited understanding of hMSC-human cardiomyocyte (hCM) interactions. To better understand the electrophysiological consequences of direct heterocellular connections between hMSCs and hCMs, three original mathematical models were developed, representing an experimentally verified triad of hMSC families with distinct functional ion channel currents. The arrhythmogenic risk of such direct electrical interactions in the setting of healthy adult myocardium was predicted by coupling and fusing these hMSC models to the published ten Tusscher midcardial hCM model. Substantial variations in action potential waveform-such as decreased action potential duration (APD) and plateau height-were found when hCMs were coupled to the two hMSC models expressing functional delayed rectifier-like human ether à-go-go K+ channel 1 (hEAG1); the effects were exacerbated for fused hMSC-hCM hybrid cells. The third family of hMSCs (Type C), absent of hEAG1 activity, led to smaller single-cell action potential alterations during coupling and fusion, translating to longer tissue-level mean action potential wavelength. In a simulated 2-D monolayer of cardiac tissue, re-entry vulnerability with low (5%) hMSC insertion was approximately eight-fold lower with Type C hMSCs compared to hEAG1-functional hMSCs. A 20% decrease in APD dispersion by Type C hMSCs compared to hEAG1-active hMSCs supports the claim of reduced arrhythmogenic potential of this cell type with low hMSC insertion. However, at moderate (15%) and high (25%) hMSC insertion, the vulnerable window increased independent of hMSC type. In summary, this study provides novel electrophysiological models of hMSCs, predicts possible arrhythmogenic effects of hMSCs when directly coupled to healthy hCMs, and proposes that isolating a subset of hMSCs absent of hEAG1 activity may offer increased safety as a cell delivery cardiotherapy at low levels of hMSC-hCM coupling.