Hypertrophic cardiomyopathy (HCM) has long been viewed as the archetypal monogenic disorder caused by pathogenic variants in the genes encoding components of the sarcomere. However, the fact that only one-third of HCM cases are genotype-positive, as well as other factors such as incomplete disease penetrance and marked phenotypic heterogeneity, challenge this reductionist view. Recent advances in mass spectrometry-based proteomics have provided new opportunities to interrogate human HCM myocardium at unprecedented depth and are reshaping our understanding of HCM pathobiology. In this mini-review, we summarize insights from both top-down and bottom-up proteomics studies showing that HCM is characterized by broad molecular remodeling across multiple cellular compartments, including the sarcomere, sarcoplasmic reticulum, cytoskeleton, mitochondria, and nucleus. Together, these studies support a model in which diverse HCM genotypes converge on shared downstream proteomic phenotypes and highlight proteomics as a powerful approach for defining disease mechanisms, modifiers, and therapeutic targets.

Since the discovery of the cardiac isoform of myosin-binding protein-C (cMyBP-C), there has been continued interest in how cMyBP-C impacts cardiac function in both health and disease. cMyBP-C is a regulatory protein in the sarcomere that controls beat-to-beat changes in contractility in response to dynamic environmental demands placed upon the heart. Changes in force production during the contractile cycle are modulated through interactions of cMyBP-C with myosin and actin. Post-translational modifications (PTMs) of cMyBP-C, of which phosphorylation has received the most attention, are critical to the function of cMyBP-C in the healthy heart and is affected in many disease states. While each of the PTMs that will be discussed in this review have known and often widespread effects on important cellular processes spanning transcriptional regulation, cell signaling, and metabolism, their impact on cMyBP-C function remains poorly understood and in some cases unverified. This Review focuses on the current understanding of cMyBP-C PTMs, namely phosphorylation, S-glutathionylation, S-nitrosylation, acetylation, citrullination, carbonylation, and O-GlcNAcylation. The potential for PTMs to exert wide ranging and likely nuanced effects may influence the range of cMyBP-C's response to varied conditions and may offer opportunities to identify novel therapeutic paradigms in the setting of disease.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) has traditionally been regarded as a disease of the sarcomere; however, it is in the midst of a paradigm shift with growing recognition of contributions beyond the sarcomere to the heterogeneity of HCM phenotypes. Innovative approaches are essential to uncover novel determinants and mechanisms underlying this heterogeneity. Top-down proteomics has emerged as a powerful method for analysis of proteoforms—the myriad protein products arising from genetic variants, posttranslational modifications, and splicing isoforms from a single gene—offering a more precise lens to understand the disease heterogeneity in HCM. Yet, how proteoforms are altered on a global scale in HCM has not been investigated. METHODS: Global top-down proteomics was performed on myocardial samples from patients with advanced obstructive HCM and nonfailing controls. Specifically, serial protein extraction enabled by the photocleavable surfactant, 4-hexylphenylazosulfonate (Azo), was utilized to solubilize diverse categories of proteins from minimal tissue, including membrane proteins. Subsequently, high-sensitivity top-down mass spectrometry was used to detect and quantify proteoforms across various cellular compartments. RESULTS: Using this global top-down proteomics approach, we have detected ≈2000 proteoforms across disparate cellular compartments, including the sarcoplasmic reticulum, cytoskeleton, mitochondria, and nucleus, in advanced obstructive HCM tissues. Quantitative analysis uncovered significant alterations not only in sarcomeric but also cytoskeletal, mitochondrial, nucleosome, and sarcoplasmic reticulum proteoforms in HCM as compared with nonfailing controls. Notably, we have discovered a significant proteoform crosstalk among the sarcomere, sarcoplasmic reticulum, and cytoskeleton. Moreover, we have identified a previously unrecognized decrease in succinylated mitochondrial proteoforms as a critical feature of the advanced obstructive HCM proteoform landscape, alongside a marked reduction in acetylation of nucleosome proteins. CONCLUSIONS: This study represents the most comprehensive analysis of the proteoform landscape in HCM to date, uncovering pathways beyond the sarcomere that may contribute to HCM pathophysiology and identifying potential targets for development of therapeutic interventions.

There is a need for robust in vitro models of human atrial tissue to empower mechanistic disease research, drug discovery, toxicity screening, and precision medicine. In the present study, we used atrial-like human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-aCM) and hiPSC-cardiac fibroblasts to produce and evaluate atrial-like engineered cardiac tissue (aECT) constructs compared with adult native human atrial myocardium. Using various techniques to evaluate ultrastructure, molecular makeup, contractile function, and electrophysiology, we compare these aECT to ventricular-like engineered cardiac tissue (vECT) and native atrial myocardium. First, aECT demonstrated a higher spontaneous beating rate, lowered IRX4 mRNA expression, and an atrial-like expression of contractile mRNA and protein with higher MYL7/MLC2a and lower MYL2/MLC2v, compared with vECT, following similar patterns exhibited by native myocardium. Second, aECT exhibited ultrastructural features like native atrial myocardium, including lower cardiomyocyte circularity, higher dimensional cardiomyocyte anisotropy (i.e., rod-shaped), higher caveolae abundance, and higher sarcomere alignment. Importantly, aECT showed contractile parameters similar to those previously observed in native atrial myocardium with minimal differences between the two in twitch force, contraction and relaxation times, and contraction kinetics. Electrophysiological data also showed that aECT exhibits atrial-like action potential morphology, with shorter action potential duration, lower APD20/80 ratio, and higher repolarization fraction. Electrophysiological data were accompanied by elevated potassium channel mRNA expressions, compared with vECT. Overall, we have generated aECT with atrial-like phenotypes, compared with vECT and native atrial myocardium, that can be leveraged for drug testing and disease modeling of atrial electroanatomical remodeling and contractile dysfunction that occurs during atrial pathology.NEW & NOTEWORTHY We demonstrate an atrial-like engineered cardiac tissue that recapitulates adult human atrial contraction for both kinetics and force production. Furthermore, we include numerous previously unmeasured ultrastructure metrics such as sarcomere alignment, cardiomyocyte anisotropy, and caveolae abundance. Our constructs may provide a human cardiac tissue platform for drug testing to identify combinatorial therapies to address atrial contractile dysfunction and diseases linked to cardiomyocyte ultrastructural defects.

Background: About 45% of identified HCM-causing mutations are in cardiac myosin binding protein C (cMyBP-C). One prevalent missense mutation is R495Q, which is in a positively charged pocket and alters the predicted electrostatic properties of the C3 domain. Many known HCM-causing mutations cause hypercontractility, likely through promoting the disordered relaxed state (DRX), in which myosin heads are more likely to bind to actin to generate contractile force, relative to the super relaxed state (SRX) in which myosin heads fold back on myosin tails. Mavacamten (MAVA), an FDA-approved drug for HCM patients with left ventricular outflow obstruction, is proposed to reduce DRX:SRX toward normal by inhibiting the myosin ATPase. It is unclear if all HCM-causing mutations act via this mechanism. Question: What is the contractile phenotypic timeline of MYBPC3 R495Q in human iPSC-engineered heart tissue (EHT) and can chronic MAVA ameliorate this phenotype? Aim: Define the phenotype progression in EHTs homozygous for the R495Q mutation (QQ), compared to isogenic controls (RR). We will use this timeline, combined with pharmacologic intervention to gain mechanistic insight into the pathogenesis of the R495Q mutation. Methods: 3D EHTs were generated from CRISPR/Cas-9 edited QQ and RR human iPSC cardiomyocytes. We used capillary immunoassay to quantify cMyBP-C expression and immunocytochemistry to determine protein localization. Contractile function of EHTs was assessed weekly (weeks 1-6) with chronic treatment of MAVA (100nM) starting at day 8 of EHT culture. Results: QQ EHTs express normal cMyBP-C protein levels with appropriate incorporation into the sarcomere, yet at day 8 show increased twitch force (TF) and fast relaxation (Fig A-B). At day 36, QQs have normalized TF and slow relaxation. Importantly, MAVA normalizes relaxation time (Fig C,D). Conclusion: These data suggest that R495Q presents with an early hypercontractile phenotype. With prolonged time in culture, TF is reduced, and relaxation time slows. Chronic administration of MAVA quickened relaxation, but reduced total force compared to untreated normal EHT, suggesting that caution may be warranted to ensure that systolic function isn’t impaired.

Hypercontractility, caused by mutations that increase the number of myosin heads in the disordered relaxed (DRX) state, is a prevalent cause of hypertrophic cardiomyopathy (HCM). A subgroup of HCM mutations cause primary hypocontractility, by stabilizing super-relaxed crossbridges (SRX). Mavacamten (mava) directly targets hypercontractility by reducing the number of DRX heads and improves outcomes in ~70% patients with obstructive HCM. We here assessed the efficacy of chronic mava treatment at preventing phenotype development in human iPSC engineered heart tissue (EHT) in a cMyBP-C null model (-/-), that increases the number of DRX heads, and EHT carrying the W792R (RR) HCM mutation. While the primary contractile defect caused by the W792R mutation is unknown, we postulated that it differed from the cMyBP-C -/- model, as RR mice die prior to weaning while -/- mice do not. Twitch force (TF) measurements performed on day 7 on control (+/+), -/- and RR EHT show that -/- EHT were hypercontractile while RR EHT were hypocontractile (Fig. 1). Following measurement, EHT from each genotype were either treated with vehicle or 100nM mava and cultured for 21 days. By day 28, TF production was similar between +/+ and -/- vehicle treated EHT while RR EHT remained hypocontractile (Fig. 2). Chronic mava treatment significantly reduced TF production in +/+ EHT but not in -/- or RR EHT (Fig. 2). Consistent with diastolic dysfunction, a hallmark of HCM in humans, day 28 -/- and RR EHT displayed slow relaxation, though the effect was more pronounced in RR EHT (Fig. 3). While chronic mava treatment accelerated relaxation in -/- EHT to levels that were similar to that observed in vehicle treated +/+ EHT, it failed to do so in RR EHT (Fig. 3). cMyBP-C -/- EHTs initially present with a hypercontractile phenotype, consistent with an increase in DRX crossbridges, while W792R RR EHT present with a hypocontractile phenotype. By 4 weeks in EHT culture, both genotypes present with impaired relaxation, that is more severe in RR EHT. While chronic mava treatment prevents development of slow relaxation in -/- EHT, it failed to do so in RR EHT. These findings suggest that the primary disease mechanism of the W792R mutation is not an increase in DRX crossbridges and that mavacamten may not be an effective treatment for patients carrying this mutation, further highlighting the need for studying the pathomechansims of HCM mutations and the development of novel therapies.

Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent cell-derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the of human cardiac biology and disease. A recent study reported widely used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared with magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACS-purified hiPSC-CMs affects the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. Global proteomics revealed that lactate-purified hiPSC-CMs displayed a differential phenotype over MACS hiPSC-CMs. hiPSC-CMs were then integrated into 3D hiPSC-ECTs and cultured for 4 weeks. Structurally, there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force and Ca2+ transient measurements revealed similar functional performance between purification methods. High-resolution mass spectrometry- based quantitative proteomics showed no significant difference in protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates that lactate-and MACS purified hiPSC-CMs generate ECTs with comparable structural, functional, and proteomic features, and it suggests that lactate purification does not result in an irreversible change in a hiPSC-CM phenotype.

Missense mutations in cardiac myosin binding protein C (cMyBP-C) are known to cause hypertrophic cardiomyopathy (HCM). The W792R mutation in the C6 domain of cMyBP-C causes severe, early onset HCM in humans, yet its impact on the function of cMyBP-C and the mechanism through which it causes disease remain unknown. To fully characterize the effect of the W792R mutation on cardiac morphology and function in vivo, we generated a murine knock-in model. We crossed heterozygous W792RWR mice to produce homozygous mutant W792RRR, heterozygous W792RWR, and control W792RWW mice. W792RRR mice present with cardiac hypertrophy, myofibrillar disarray and fibrosis by postnatal day 10 (PND10), and do not survive past PND21. Full-length cMyBP-C is present at similar levels in W792RWW, W792RWR and W792RRR mice and is properly incorporated into the sarcomere. Heterozygous W792RWR mice displayed normal heart morphology and contractility. Permeabilized myocardium from PND10 W792RRR mice showed increased Ca2+ sensitivity, accelerated cross-bridge cycling kinetics, decreased cooperativity in the activation of force, and increased expression of hypertrophy-related genes. In silico modeling suggests that the W792R mutation destabilizes the fold of the C6 domain and increases torsion in the C5-C7 region, possibly impacting regulatory interactions of cMyBP-C with myosin and actin. Based on the data presented here, we propose a model in which mutant W792R cMyBP-C preferentially forms Ca2+ sensitizing interactions with actin, rather than inhibitory interactions with myosin. The W792R-cMyBP-C mouse model provides mechanistic insights into the pathology of this mutation and may provide a mechanism by which other central domain missense mutations in cMyBP-C may alter contractility, leading to HCM.

Myosin functions as the "molecular motor" of the sarcomere and generates the contractile force necessary for cardiac muscle contraction. Myosin light chains 1 and 2 (MLC-1 and -2) play important functional roles in regulating the structure of the hexameric myosin molecule. Each of these light chains has an ‘atrial' and ‘ventricular' isoform, so called because they are believed to exhibit chamber-restricted expression in the heart. However, recently the chamber-specific expression of MLC isoforms in the human heart has been questioned. Herein, we analyzed the expression of MLC-1 and -2 atrial and ventricular isoforms in each of the four cardiac chambers in adult non-failing donor hearts using top-down mass spectrometry (MS)-based proteomics. Strikingly, we detected an isoform thought to be ventricular, MLC-2v (gene: MYL2), in the atria and confirmed the protein sequence using tandem MS (MS/MS). For the first time, a putative deamidation post-translation modification (PTM) located on MLC-2v in atrial tissue was localized to amino acid N13. MLC-1v (MYL3) and MLC-2a (MYL7) were the only MLC isoforms exhibiting chamber-restricted expression patterns across all donor hearts. Importantly, our results unambiguously show that MLC-1v, not MLC-2v, is ventricle-specific in adult human hearts. Moreover, we found elevated MLC-2 phosphorylation in male hearts compared to female hearts across each cardiac chamber. Overall, top-down proteomics allowed an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs.

Many hypertrophic cardiomyopathy (HCM) causing mutations result in myocardial hypercontractility by increasing the number of myosin crossbridges in the disordered relaxed (DRX) state, or by sensitizing the sarcomere to the effects of Ca 2+ . Mavacamten (Mava) recently became the first FDA-approved treatment of obstructive HCM that directly targets hypercontractility by reducing the number of DRX cross bridges. Mava improves outcomes in patients with obstructive HCM but has not been approved in children or adults without obstructive HCM. The efficacy of chronic Mava treatment at preventing phenotype development in asymptomatic HCM mutation carriers remains largely unexplored. We recently showed that ablation of cardiac myosin binding protein-C (cMyBP-C -/-) in engineered heart tissue (EHT) causes rapid phenotype development in a dish. Initial hypercontractility progresses to hypocontractility with impaired relaxation, mediated by Ca 2+ mishandling. Here we investigate whether chronic Mava administration prevents phenotype progression. cMyBP-C +/+, +/- and -/- EHT were treated with 100nM Mava starting on day 7 of EHT culture. Serial twitch force (TF) measurements were performed weekly for 5 weeks, using the Mantarray platform. At day 7, prior to Mava treatment, +/- and -/- produced significantly more TF than +/+ EHT (392 ± 9 μN vs. 390 ± 15 μN vs. 199 ± 13 μN). At day 14, Mava treatment reduced TF by 31% (p < 0.001) in +/+ EHT, 30% (p < 0.001) in +/- EHT and 12% (p = 0.115) in -/- EHT. After an additional 4 weeks, chronic Mava treatment reduced TF by 54% (p < 0.001) in +/+ EHT, 65% (p < 0.001) in +/- EHT but increased TF by 23% (p = 0.034) in -/- EHT.These data show that chronic Mava treatment prevents/slows maladaptive remodeling in -/- EHT, but depresses contractility in +/+ and +/- ECT, suggesting that chronic Mava treatment may be detrimental to patients with mild HCM. Our ongoing studies are assessing the effect of chronic Mava treatment on other EHT models of HCM and on Ca 2+ -handling.

Background: Mutations in cardiac myosin binding protein C (cMyBP-C) are a common cause of hypertrophic cardiomyopathy (HCM) in humans. While many known cMyBP-C mutations lead to haploinsufficiency, others lead to amino acid substitutions which may impact protein function in unique ways The W792R mutation introduces a charge change and is known to be pathogenic in humans. This mutation, located remotely from both the regulatory amino terminus, and the C-terminus, has an unknown impact on the final protein structure and function. Goal: Generate a murine knock-in model to fully characterize the impact of the W792R mutation on cardiac morphology and function in vivo. Methods: We generated a W792R knock-in (KI) mouse and produced heterozygous (HT) and homozygous (KI) offspring, and wild-type (WT) littermate controls. We performed histology, RNA and protein expression analyses, and mechanics on isolated trabeculae at postnatal day 12 (KI n=5; HT n=3; and LC n=4). One-way ANOVA used to compare KI and HT to WT samples, with p-values < ; 0.05 as significant. Results: The KI mice did not live past ~20 days old with massive hypertrophy, dilated atrium, myofibrillar disarray and fibrosis. Full-length mutant protein was expressed and incorporated into the sarcomere. Survival and morphology in the HT was similar to WT. Post-natal day 10 KI mice demonstrated severely enlarged atria and ventricles, interstitial fibrosis, a trend towards increased Ca 2+ sensitivity (pCA 50 KI 5.8 vs HT 5.65 vs LC 5.67 mM; p > 0.05), a trend towards increased cross-bridge cycling kinetics (k tr KI 24.00 + 2.42 vs HT 24.33 + 1.08 vs LC 21.25 + 0.87; p > 0.05) and increased levels of β-myosin heavy chain expression compared to both LC and HT mice. Conclusions: In our mouse HCM model, homozygous expression of the W792R-cMyBP-C mutation recapitulates severe human HCM and provides mechanistic insights into the pathology of this mutation. The trend towards accelerated crossbridge kinetics suggest that the W792R mutation disrupts the inhibitory effects of cMyBP-C on myosin-actin binding implicating the potential application of current myosin ATPase inhibitors in the treatment of patients carrying this mutation. Ongoing work will expand sample sizes to evaluate significance of findings.

Truncation mutations in cardiac myosin binding protein C (cMyBP-C) are common causes of hypertrophic cardiomyopathy (HCM). Heterozygous carriers present with classical HCM, while homozygous carriers present with early onset HCM that rapidly progress to heart failure. We used CRISPR-Cas9 to introduce heterozygous (cMyBP-C+/-) and homozygous (cMyBP-C-/-) frame-shift mutations into MYBPC3 in human iPSCs. Cardiomyocytes derived from these isogenic lines were used to generate cardiac micropatterns and engineered cardiac tissue constructs (ECTs) that were characterized for contractile function, Ca2+-handling, and Ca2+-sensitivity. While heterozygous frame shifts did not alter cMyBP-C protein levels in 2-D cardiomyocytes, cMyBP-C+/- ECTs were haploinsufficient. cMyBP-C-/- cardiac micropatterns produced increased strain with normal Ca2+-handling. After 2 wk of culture in ECT, contractile function was similar between the three genotypes; however, Ca2+-release was slower in the setting of reduced or absent cMyBP-C. At 6 wk in ECT culture, the Ca2+-handling abnormalities became more pronounced in both cMyBP-C+/- and cMyBP-C-/- ECTs, and force production became severely depressed in cMyBP-C-/- ECTs. RNA-seq analysis revealed enrichment of differentially expressed hypertrophic, sarcomeric, Ca2+-handling, and metabolic genes in cMyBP-C+/- and cMyBP-C-/- ECTs. Our data suggest a progressive phenotype caused by cMyBP-C haploinsufficiency and ablation that initially is hypercontractile, but progresses to hypocontractility with impaired relaxation. The severity of the phenotype correlates with the amount of cMyBP-C present, with more severe earlier phenotypes observed in cMyBP-C-/- than cMyBP-C+/- ECTs. We propose that while the primary effect of cMyBP-C haploinsufficiency or ablation may relate to myosin crossbridge orientation, the observed contractile phenotype is Ca2+-mediated.

Cardiovascular disease is the leading cause of death in the USA and is known to be exacerbated by elevated mechanical stress from hypertension. Caveolae are plasma membrane structures that buffer mechanical stress but have been found to be reduced in pathological conditions associated with chronically stretched myocardium. To explore the physiological implications of the loss of caveolae, we used human engineered cardiac tissue (ECT) constructs, composed of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and hiPSC-derived cardiac fibroblasts, to develop a long-term cyclic stretch protocol that recapitulates the effects of hypertension on caveolae expression, membrane tension, and the β-adrenergic response. Leveraging this new stretch protocol, we identified neutral sphingomyelinases (nSMase) as mechanoregulated mediators of caveolae loss, ceramide production and the blunted β-adrenergic response in this human cardiac model. Specifically, in our ECT model, nSMase inhibition via GW4869 prevented stretch-induced loss of caveolae-like structures, mitigated nSMase-dependent ceramide production, and maintained the ECT contractile kinetic response to isoprenaline. These findings are correlated with a blood lipidomic analysis in middle-aged and older adults, which revealed an increase of the circulating levels of ceramides in adults with hypertension. Furthermore, we found that conduction slowing from increased pressure loading in mouse left ventricle was abolished in the context of nSMase inhibition. Collectively, these findings identify nSMase as a potent drug target for mitigating stretch-induced effects on cardiac function. KEY POINTS: We have developed a new stretch protocol for human engineered cardiac tissue that recapitulates changes in plasma membrane morphology observed in animal models of pressure/volume overload. Stretch of engineered cardiac tissue induces activation of neutral sphingomyelinase (nSMase), generation of ceramide, and disassembly of caveolae. Activation of nSMase blunts cardiac β-adrenergic contractile kinetics and mediates stretch-induced slowing of conduction and upstroke velocity. Circulating ceramides are increased in adults with hypertension, highlighting the clinical relevance of stretch-induced nSMase activity.

Heart disease is the largest contributor to death worldwide and research is limited by a lack of adequate human tissue in vitro models. To address this, we developed a protocol for generating 3D, atrial-like engineered cardiac tissue (ECT) comprised of cardiomyocytes (CM) and cardiac fibroblasts, derived from human-induced pluripotent stem cells (hiPSC). Control and atrial-like hiPSC-CM were produced using GiWi differentiation supplemented without and with 0.75 µM retinoic acid, respectively. ECTs were generated using hiPSC-cardiac fibroblasts and Day 30 hiPSC-CM combined in a fibrin matrix and molded via a FlexCell Tissue TrainTM system. ECTs were cultured for 30 days and evaluated for action potentials (AP) as well as mRNA and protein expression profiles. A subset of control ECTs were cyclically and incrementally stretched for 7 days up to 18% elongation. Stretch-induced changes were evaluated via transmission electron microscopy (TEM) and cell culture medium (LC-MS/MS). Atrial-like ECTs recapitulated various atrial phenotypes. They exhibit an atrial-like electrophysiology with a shorter AP duration and increased repolarization fraction (p<0.05). Atrial-like ECT show atrial-specific mRNA/protein expression, such as lower ventricular-like MYL2 and higher atrial-like MYL7 levels (p<0.05). They also demonstrate distinct contraction mechanics with faster kinetics (p<0.05) and decreased normalized contraction force, compared to control ECT. Cyclic stretch reduced cardiomyocyte membrane convolution index (estimating membrane tension) and caveolae membrane structure density (p<0.05). The latter was rescued via neutral sphingomyelinase inhibitor GW4869 (20 µM, p<0.05). LC-MS/MS of stretch-conditioned culture medium revealed decreases in relative sphingomyelins and phosphatidylcholines (p<0.05), ostensibly indicating membrane damage which was reversed by GW4869. Overall, our findings demonstrate retinoic acid-induced atrial-like phenotypes in hiPSC-CM are sustained in ECT form. We also introduce a novel ECT stretch protocol that recapitulates changes observed in animal models of heart pressure overload.

Animal atrial disease models are inadequate representations of human pathophysiology and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) in monolayer (ML) poorly model adult human myocardium, especially for long-term studies. To address this, we present a three-dimensional (3D) atrial-like engineered cardiac tissue (ECT) composed of atrial-like hiPSC-CM and hiPSC-cardiac fibroblasts co-cultured in a fibrin matrix that can be used to model chronic effects of atrial disease.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is characterized by an arrhythmogenic mechanism involving disruption of calcium handling. This genetic disease can lead to sudden death in children and young adults during physical or emotional stress. Prior CPVT studies have focused on calcium handling, but mechanical functionality has rarely been investigated in vitro. In this research we combine stem cell-derived cardiomyocytes from a CPVT patient (RyR2-H2464D mutation) and a healthy familial control with an engineered culture platform to evaluate mechanical function of cardiomyocytes. Substrates with Young's modulus ranging from 10 to 50 kPa were used in conjunction with microcontact printing of ECM proteins into defined patterns for subsequent attachment. Digital Image Correlation (DIC) was used to evaluate collections of contracting cells. The amplitude of contractile strain was utilized as a quantitative indicator of functionality and disease severity. We found statistically significant differences: the maximum contractile strain was consistently higher in patient samples compared to control samples on all substrate stiffnesses. Additionally, the patient cell line had a statistically significantly slower intrinsic contraction rate than the control, which agrees with prior literature. Differences in mechanical strain have not been previously reported, and hypercontractility is not a known characteristic of CPVT. However, functional changes can occur as the disease progresses, thus this observation may not represent behavior observed in adolescent and adult patients. These results add to the limited studies of mechanical function of CPVT CMs reported in literature and identify functional differences that should be further explored.

Branch pulmonary artery (PA) stenosis (PAS) commonly occurs in patients with congenital heart disease (CHD). Prior studies have documented technical success and clinical outcomes of PA stent interventions for PAS but the impact of PA stent interventions on ventricular function is unknown. The objective of this study was to utilize 4D flow cardiovascular magnetic resonance (CMR) to better understand the impact of PAS and PA stenting on ventricular contraction and ventricular flow in a swine model of unilateral branch PA stenosis. 18 swine (4 sham, 4 untreated left PAS, 10 PAS stent intervention) underwent right heart catheterization and CMR at 20 weeks age (55 kg). CMR included ventricular strain analysis and 4D flow CMR. 4D flow CMR measured inefficient right ventricular (RV) and left ventricular (LV) flow patterns in the PAS group (RV non-dimensional (n.d.) vorticity: sham 82 ± 47, PAS 120 ± 47; LV n.d. vorticity: sham 57 ± 5, PAS 78 ± 15 p < 0.01) despite the PAS group having normal heart rate, ejection fraction and end-diastolic volume. The intervention group demonstrated increased ejection fraction that resulted in more efficient ventricular flow compared to untreated PAS (RV n.d. vorticity: 59 ± 12 p < 0.01; LV n.d. vorticity: 41 ± 7 p < 0.001). These results describe previously unknown consequences of PAS on ventricular function in an animal model of unilateral PA stenosis and show that PA stent interventions improve ventricular flow efficiency. This study also highlights the sensitivity of 4D flow CMR biomarkers to detect earlier ventricular dysfunction assisting in identification of patients who may benefit from PAS interventions.

Mutations that cause truncation of cardiac myosin binding protein C (cMyBP-C) are common causes of hypertrophic cardiomyopathy (HCM), and cause disease through a mechanism of haploinsufficiency. Re...