Induced Pluripotent Stem Cell-derived Cardiomyocytes Research Articles

A novel ionic model for matured and paced atrial-like human iPSC-CMs integrating [formula omitted] and [formula omitted] currents

This work introduces the first atrial-specific in-silico human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) model, based on a set of phenotype-specific IKur,IKCa and IK1 membrane currents. This model is built on novel in-vitro experimental data recently published by some of the co-authors to simulate the paced action potential of matured atrial-like hiPSC-CMs. The model consists of a system of stiff ordinary differential equations depending on several parameters, which have been tuned by automatic optimization techniques to closely match selected experimental biomarkers. The new model effectively simulates the electronic in-vitro hiPSC-CMs maturation process, transitioning from an unstable depolarized membrane diastolic potential to a stable hyperpolarized resting potential, and exhibits spontaneous firing activity in unpaced conditions. Moreover, our model accurately reflects the experimental rate dependence data at different cycle length and demonstrates the expected response to a specific current blocker. This atrial-specific in-silico model provides a novel computational tool for electrophysiological studies of cardiac stem cells and their applications to drug evaluation and atrial fibrillation treatment.

Effect and mechanism of T lymphocytes on human induced pluripotent stem cell-derived cardiomyocytes via Proteomics

BackgroundAbnormalities in T cell activation play an important role in the pathogenesis of myocarditis, and persistent T cell responses can lead to autoimmunity and chronic cardiac inflammation, as well as even dilated cardiomyopathy. Although previous work has examined the role of T cells in myocarditis in animal models, the specific mechanism for human cardiomyocytes has not been investigated.MethodsIn this study, we constructed the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and established the T cell-mediated cardiac injury model by co-culturing with activated CD4 + T or CD8 + T cells that were isolated from peripheral mononuclear blood to elucidate the pathogenesis of myocardial cell injury caused by inflammation.ResultsBy combination of quantitative proteomics with tissue and cell immunofluorescence examination, we established a proteome profile of inflammatory myocardia from hiPSC-CMs with obvious cardiomyocyte injury and increased levels of lactate dehydrogenase content, creatine kinase isoenzyme MB and cardiac troponin. A series of molecular dysfunctions of hiPSC-CMs was observed and indicated that CD4 + cells could produce direct cardiomyocyte injury by activating the NOD-like receptor signals pathway.ConclusionsThe data presented in our study established a proteome map of inflammatory myocardial based on hiPSC-CMs injury model. These results can provide guidance in the discovery of improved clinical treatments for myocarditis.

P53 Activation Promotes Maturational Characteristics of Pluripotent Stem Cell-Derived Cardiomyocytes in 3-Dimensional Suspension Culture Via FOXO-FOXM1 Regulation.

Current protocols generate highly pure human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) invitro that recapitulate characteristics of mature invivo cardiomyocytes. Yet, a risk of arrhythmias exists when hiPSC-CMs are injected into large animal models. Thus, understanding hiPSC-CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC-CM maturation. Here, we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC-CM maturation in 3-dimensional suspension culture. Three-dimensional cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator, 10 μM), LOM612 (FOXO relocator, 5 μM), AS1842856 (FOXO inhibitor, 1 μM), or RCM-1 (FOXM1 inhibitor, 1 μM), starting 2 days after onset of beating, with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC-CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation, in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility, oxygen consumption, and voltage peak maximum upstroke velocity, in n=3 to 6 per group for all assays. Further, by single-cell RNA sequencing of n=2 LOM612-treated cells compared with dimethyl sulfoxide, LOM612-mediated FOXO activation promoted expression of cardiac maturational pathways. We show that p53 activation promotes FOXO and suppresses FOXM1 during 3-dimensional hiPSC-CM maturation. These results expand our understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3-dimensional culture system.

Identification of circular RNAs regulating cardiomyocyte proliferation in neonatal pig hearts.

Little is known about the expression patterns and functions of circular RNAs (circRNAs) in the heart of large mammals. In this study, we examined the expression profiles of circRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in neonatal pig hearts. Pig heart samples collected on postnatal days 1 (P1), 3 (P3), 7 (P7), and 28 (P28) were sent for total RNA sequencing. Our data revealed a total of 7,000 circRNAs in the 24 pig hearts. Pathway enrichment analysis of hallmark gene sets demonstrated that differentially expressed circRNAs were engaged in different pathways. The most significant difference was observed between P1 and the other 3 groups (P3, P7, and P28) in pathways related to cell cycle and muscle development. Out of the 10 circRNAs that were validated through real-time quantitative PCR to verify their expression, 6 exhibited significant effects on cell cycle activity in human induced pluripotent stem cell-derived cardiomyocytes following small interfering RNA-mediated knockdown. circRNA-miRNA-mRNA networks were constructed to understand the potential mechanisms of circRNAs in the heart. In conclusion, our study provided a data set for exploring the roles of circRNAs in pig hearts. In addition, we identified several circRNAs that regulate cardiomyocyte cell cycle.

The Low Tumorigenic Risk and Subtypes of Cardiomyocytes Derived from Human-induced Pluripotent Stem Cells

Background: Clinical application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a promising approach for the treatment of heart diseases. However, the tumorigenicity of hiPSC-CMs remains a concern for their clinical applications and the composition of the hiPSC-CM subtypes need to be clearly identified. Methods: In the present study, hiPSC-CMs were induced from hiPSCs via modulation of Wnt signaling followed by glucose deprivation purification. The structure, function, subpopulation composition, and tumorigenic risk of hiPSC-CMs were evaluated by single-cell RNA sequencing (scRNAseq), whole exome sequencing (WES), and integrated molecular biology, cell biology, electrophysiology, and/or animal experiments. Results: The high purity of hiPSC-CMs, determined by flow cytometry analysis, was generated. ScRNAseq analysis of differentiation day (D) 25 hiPSC-CMs did not identify the transcripts representative of undifferentiated hiPSCs. WES analysis showed a few newly acquired confidently identified mutations and no mutations in tumor susceptibility genes. Further, no tumor formation was observed after transplanting hiPSC-CMs into NOD-SCID mice for 3 months. Moreover, D25 hiPSC-CMs were composed of subtypes of ventricular-like cells (23.19%) and atrial-like cells (66.45%) in different cell cycle stages or mature levels, based on the scRNAseq analysis. Furthermore, a subpopulation of more mature ventricular cells (3.21%) was identified, which displayed significantly up-regulated signaling pathways related to myocardial contraction and action potentials. Additionally, a subpopulation of cardiomyocytes in an early differentiation stage (3.44%) experiencing nutrient stress-induced injury and heading toward apoptosis was observed. Conclusions: This study confirmed the biological safety of hiPSC-CMs and described the composition and expression profile of cardiac subtypes in hiPSC-CMs which provide standards for quality control and theoretical supports for the translational applications of hiPSC-CMs.

Loss-of-function W4645R mutation in the RyR2-caffeine binding site: implications for synchrony and arrhythmogenesis

AimsPrevious studies have identified RyR2 W4645R mutation, located in the caffeine-binding site, to associate with CPVT1 pathology. Caffeine binding to its site is thought to displace the carboxyl-terminal domain to Ca2+-binding, allowing the tryptophan residue (W4645) to regulate Ca2+ sensitivity of RyR2. To gain insights into regulation of RyR2 Ca2+-binding and its interaction with caffeine-binding site, we introduced W4645R-RyR2 point mutation via CRISPR/Cas9 gene-editing in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) and characterized their Ca2+-signaling phenotype compared to WT hiPSCCMs. Methods and ResultsW4645R-RyR2 cardiomyocytes had: (1) no significant change in ICa magnitude or voltage-dependence; (2) slightly reduced CICR; (3) altered relaxation kinetics of Ca2+-transients with no change in isoproterenol sensitivity; (4) complete loss of caffeine-triggered Ca2+ release; (5) larger SR Ca2+ leak resulting in 40 % lower SR Ca2+ content, as determined by myocytes’ response to 4-CmC; (6) lower incidence of calcium sparks and asynchronous spontaneous SR Ca2+ releases. ConclusionsW4645R-RyR2 mutation induces loss of caffeine-triggered SR Ca2+ release and enhances SR Ca2+ leak that underlie asynchronous spontaneous Ca2+ releases, triggering arrhythmia and impairing cardiac function.

Cell-specific models of hiPSC-CMs developed by the gradient-based parameter optimization method fitting two different action potential waveforms

Parameter optimization (PO) methods to determine the ionic current composition of experimental cardiac action potential (AP) waveform have been developed using a computer model of cardiac membrane excitation. However, it was suggested that fitting a single AP record in the PO method was not always successful in providing a unique answer because of a shortage of information. We found that the PO method worked perfectly if the PO method was applied to a pair of a control AP and a model output AP in which a single ionic current out of six current species, such as IKr, ICaL, INa, IKs, IKur or IbNSC was partially blocked in silico. When the target was replaced by a pair of experimental control and IKr-blocked records of APs generated spontaneously in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), the simultaneous fitting of the two waveforms by the PO method was hampered to some extent by the irregular slow fluctuations in the Vm recording and/or sporadic alteration in AP configurations in the hiPSC-CMs. This technical problem was largely removed by selecting stable segments of the records for the PO method. Moreover, the PO method was made fail-proof by running iteratively in identifying the optimized parameter set to reconstruct both the control and the IKr-blocked AP waveforms. In the lead potential analysis, the quantitative ionic mechanisms deduced from the optimized parameter set were totally consistent with the qualitative view of ionic mechanisms of AP so far described in physiological literature.

Hypoxia and re-oxygenation effects on human cardiomyocytes cultured on polycaprolactone and polyurethane nanofibrous mats

Heart diseases are caused mainly by chronic oxygen insufficiency (hypoxia), leading to damage and apoptosis of cardiomyocytes. Research into the regeneration of a damaged human heart is limited due to the lack of cellular models that mimic damaged cardiac tissue. Based on the literature, nanofibrous mats affect the cardiomyocyte morphology and stimulate the growth and differentiation of cells cultured on them; therefore, nanofibrous materials can support the production of in vitro models that faithfully mimic the 3D structure of human cardiac tissue. Nanofibrous mats were used as scaffolds for adult primary human cardiomyocytes (HCM) and immature human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). This work focuses on understanding the effects of hypoxia and re-oxygenation on human cardiac cells cultured on polymer nanofibrous mats made of poly(ε-caprolactone) (PCL) and polyurethane (PU). The expression of selected genes and proteins in cardiomyocytes during hypoxia and re-oxygenation were evaluated. In addition, the type of cell death was analyzed. To the best of our knowledge, there are no studies on the effects of hypoxia on cardiomyocyte cells cultured on nanofibrous mats. The present study aimed to use nanofiber mats as scaffolds that structurally could mimic cardiac extracellular matrix. Understanding the impact of 3D structural properties in vitro cardiac models on different human cardiomyocytes is crucial for advancing cardiac tissue engineering and regenerative medicine. Observing how 3D scaffolds affect cardiomyocyte function under hypoxic conditions is necessary to understand the functioning of the entire human heart.

Chronic Mexiletine Administration Increases Sodium Current in Non-Diseased Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

A sodium current (INa) reduction occurs in the setting of many acquired and inherited conditions and is associated with cardiac conduction slowing and increased arrhythmia risks. The sodium channel blocker mexiletine has been shown to restore the trafficking of mutant sodium channels to the membrane. However, these studies were mostly performed in heterologous expression systems using high mexiletine concentrations. Moreover, the chronic effects on INa in a non-diseased cardiomyocyte environment remain unknown. In this paper, we investigated the chronic and acute effects of a therapeutic dose of mexiletine on INa and the action potential (AP) characteristics in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) of a healthy individual. Control hiPSC-CMs were incubated for 48 h with 10 µM mexiletine or vehicle. Following the wash-out of mexiletine, patch clamp analysis and immunocytochemistry experiments were performed. The incubation of hiPSC-CMs for 48 h with mexiletine (followed by wash-out) induced a significant increase in peak INa of ~75%, without any significant change in the voltage dependence of (in)activation. This was accompanied by a significant increase in AP upstroke velocity, without changes in other AP parameters. The immunocytochemistry experiments showed a significant increase in membrane Nav1.5 fluorescence following a 48 h incubation with mexiletine. The acute re-exposure of hiPSC-CMs to 10 µM mexiletine resulted in a small but significant increase in AP duration, without changes in AP upstroke velocity, peak INa density, or the INa voltage dependence of (in)activation. Importantly, the increase in the peak INa density and resulting AP upstroke velocity induced by chronic mexiletine incubation was not counteracted by the acute re-administration of the drug. In conclusion, the chronic administration of a clinically relevant concentration of mexiletine increases INa density in non-diseased hiPSC-CMs, likely by enhancing the membrane trafficking of sodium channels. Our findings identify mexiletine as a potential therapeutic strategy to enhance and/or restore INa and cardiac conduction.

Stage specific effects of insulin and Wnt on cell fate decisions in human induced pluripotent stem cell-derived cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation Background Reactivation of the so-called fetal gene programs and pathways have been reported in failing hearts, however, it remains unclear whether this phenomenon is beneficial or detrimental to the remaining functional myocardium. During cardiac development, the orchestration of these embryonic growth pathways, facilitate temporal and stage-specific cell fate decisions such as required for specification and growth of the various myocardial compartments. Previously, we demonstrated that molecular activation of the Wnt pathway resulted in massive expansion of immature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Yet, it is unknown what exact cues guide the cell fate decisions of immature hiPSC-CMs to either proliferate or terminally differentiate into more mature cardiomyocytes. Purpose This work uncovers the embryonic growth pathways that control the cues for proliferation versus terminal differentiation in functional immature hiPSC-CMs. Methods We utilised spontaneously beating hiPSC-CMs at day 11 of differentiation and performed a combinatory screen for various dosages of CHIR99021/Wnt and Insulin/Akt pathway. Proliferation was determined by quantification of cell number and the proportion of Ki67 positive hiPSC-CMs. Terminal differentiation was evaluated by presence of more mature sarcomere markers and cell cycle exit. Results Our combinatory screen for Insulin/Akt and CHIR99021/Wnt demonstrates synergistic effects on proliferation of immature hiPSC-CMs, whereas the absence of these pathway activators leads to rapid cell cycle exit and terminal differentiation of hiPSC-CMs. Detailed characterization reveals that CHIR99021/Wnt also importantly regulates sarcomere homeostasis and architecture in hiPSC-CMs. Moreover, we identify a novel temporal interplay between CHIR99021/Wnt via TCF and Insulin/Akt via FOXO as regulators of cell-fate decision in immature hiPSC-CMs. Conclusions Molecular targeting of the embryonic growth pathways in acute and chronic forms of heart failure requires a detailed understanding of the specific effects on cardiomyocyte function and sarcomere architecture. This work provides a starting point to develop future strategies to target acute and chronic forms of heart failure via modulation of embryonic growth pathways.Graphical Summary

Impacts of DCM-linked gating pore currents on the electrophysiological characteristics of hiPSC-CM monolayers

BackgroundVariants of the SCN5A gene, which encodes the NaV1.5 cardiac sodium channel, have been linked to arrhythmic disorders associated with dilated cardiomyopathy (DCM). However, the precise pathological mechanisms remain elusive. The present study aimed to elucidate the pathophysiological consequences of the DCM-linked Nav1.5/R219H variant, which is known to generate a gating pore current, using patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in monolayers. MethodsVentricular- and atrial-like hiPSC-CM monolayers were generated from DCM patients carrying the R219H SCN5A variant as well as from healthy control individuals. CRISPR-corrected hiPSC-CMs served as isogenic controls. Simultaneous optical mapping of action potentials (APs) and calcium transients (CaTs) was employed to measure conduction velocities (CVs) and AP durations (APDs) and served as markers of electrical excitability. Calcium handling was evaluated by assessing CaT uptake (half-time to peak), recapture (tau of decay), and durations (TD50 and TD80). A multi-electrode array (MEA) analysis was conducted on hiPSC-CM monolayers to measure field potential (FP) parameters, including corrected Fridericia FP durations (FPDc). ResultsOur results revealed that CVs were significantly reduced by more than 50 % in both ventricular- and atrial-like hiPSC-CM monolayers carrying the R219H variant compared to the control group. APDs were also prolonged in the R219H group compared to the control and CRISPR-corrected groups. CaT uptake, reuptake, and duration were also markedly delayed in the R219H group compared to the control and CRISPR-corrected groups in both the ventricular- and the atrial-like hiPSC-CM monolayers. Lastly, the MEA data revealed a notably prolonged FPDc in the ventricular- and atrial-like hiPSC-CMs carrying the R219H variant compared to the control and isogenic control groups. ConclusionsThese findings highlight the impact of the gating pore current on AP propagation and calcium homeostasis within a functional syncytium environment and offer valuable insights into the potential mechanisms underlying DCM pathophysiology.

Susceptibility to innate immune activation in genetically-mediated myocarditis.

Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment for myocarditis is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis with biomarkers and pathological features indistinguishable from other forms of myocarditis. DSP-associated myocarditis can progress to dilated cardiomyopathy with heightened arrhythmia risk. To model the cardiomyocyte aspects of DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients and gene-edited healthy control hiPSC lines. Homozygous and heterozygous DSP disrupted EHTs were generated to contain 90% hiPSC-CMs and 10% healthy control human cardiac fibroblasts. We measured innate immune activation and function at baseline and in response to Toll-like receptor (TLR) stimulation in EHTs. At baseline, DSP-/- EHTs displayed a transcriptomic signature of immune activation which was mirrored by EHT cytokine release. Importantly, DSP-/- EHTs were hypersensitive to TLR stimulation demonstrating greater contractile function impairment compared to isogenic controls. Compared to homozygous DSP-/- EHTs, heterozygous DSP patient-derived EHTs had less functionally impairment but also displayed heightened sensitivity to TLR stimulation. When subjected to strain, heterozygous DSP EHTs developed greater functional deficit indicating reduced contractile reserve compared to healthy control. Colchicine or NFΚB inhibitors improved baseline force production and strain-induced force deficits in DSP EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Genetic reduction of DSP renders cardiomyocytes susceptible to innate immune activation and strain-dependent contractile deficits. EHTs replicate electrical and contractile phenotypes seen in human myocarditis implicating cytokine release as a key part of the myogenic susceptibility to inflammation. This heightened innate immune activation and sensitivity is a target for clinical intervention.

Single-cell ionic current phenotyping elucidates non-canonical features and predictive potential of cardiomyocytes during automated drug experiments.

All new drugs must go through preclinical screening tests to determine their proarrhythmic potential. While these assays effectively filter out dangerous drugs, they are too conservative, often misclassifying safe compounds as proarrhythmic. In this study, we attempt to address this shortcoming with a novel, medium-throughput drug-screening approach: we use an automated patch-clamp system to acquire optimized voltage clamp (VC) and action potential (AP) data from human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) at several drug concentrations (baseline, 3×, 10× and 20× the effective free plasma concentrations). With our novel method, we show correlations between INa block and upstroke slowing after treatment with flecainide or quinine. Additionally, after quinine treatment, we identify significant reductions in current during voltage steps designed to isolate If and IKs. However, we do not detect any IKr block by either drug, and upon further investigation, do not see any IKr present in the iPSC-CMs when prepared for automated patch experiments (i.e. in suspension) - this is in contrast to similar experiments we have conducted with these cells using the manual patch setup. In this study, we: (1) present a proof-of-concept demonstration of a single-cell medium-throughput drug study, and (2) characterize the non-canonical electrophysiology of iPSC-CMs when prepared for experiments in a medium-throughput setting. KEY POINTS: Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer potential as an in vitro model to study the proarrhythmic potential of drugs, but insights from these cells are often limited by the low throughput of manual patch-clamp. In this study, we use a medium-throughput automated patch-clamp system to acquire action potential (AP) and complex voltage clamp (VC) data from single iPSC-CMs at multiple drug concentrations. A correlation between AP upstroke and INa transients was identified and drug-induced changes in ionic currents found. We also characterize the substantially altered physiology of iPSC-CMs when patched in an automated system, suggesting the need to investigate differences between manual and automated patch experiments.

Generation of a Human Induced Pluripotent Stem Cell Line Expressing a Magnetic Resonance Imaging Reporter Gene.

The objective of the current study is to develop a new method for tracking transplanted human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) using magnetic resonance imaging (MRI). The CRISPR/dCas9 activation system is employed to overexpress ferritin heavy chain (FHC) in hiPSC-CMs. The mRNA and protein expression of FHC in hiPSC and hiPSC-CMs significantly increased after transfection. Iron chloride does not affect the cell viability in a concentration range from 0 to 2000 µm. hiPSCs overexpressing FHC (hiPSC- FHCOE) and hiPSC-CMs overexpressing FHC (hiPSC-CM-FHCOE) significantly enhanced cellular uptake of iron chloride but with no changes in electrophysiological properties compared to hiPSC-CM-Control. Furthermore, hiPSC-CM-FHCOE presented robust contrast and lower T2* values, signifying their potential as highly effective candidates for cardiac MRI. Next, hiPSC-CM-FHCOE is injected into mouse hearts and after 3 days of transplantation, MR images are obtained. hiPSC-CM-FHCOE cells exhibited clear signals in the hearts with lower T2* and rapid signal decay. Collectively, data from this proof-of-concept study demonstrated that endogenous labeling with FHC in hiPSC-CMs can be a potent strategy for enhancing the accuracy of cardiac MRI. This technology represents a significant step forward in tracking the transplanted hiPSC-CMs in the hearts of live animals.

Stress-Induced Proteasome Sub-Cellular Translocation in Cardiomyocytes Causes Altered Intracellular Calcium Handling and Arrhythmias.

The ubiquitin-proteasome system (UPS) is an essential mechanism responsible for the selective degradation of substrate proteins via their conjugation with ubiquitin. Since cardiomyocytes have very limited self-renewal capacity, as they are prone to protein damage due to constant mechanical and metabolic stress, the UPS has a key role in cardiac physiology and pathophysiology. While altered proteasomal activity contributes to a variety of cardiac pathologies, such as heart failure and ischemia/reperfusion injury (IRI), the environmental cues affecting its activity are still unknown, and they are the focus of this work. Following a recent study by Ciechanover's group showing that amino acid (AA) starvation in cultured cancer cell lines modulates proteasome intracellular localization and activity, we tested two hypotheses in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs, CMs): (i) AA starvation causes proteasome translocation in CMs, similarly to the observation in cultured cancer cell lines; (ii) manipulation of subcellular proteasomal compartmentalization is associated with electrophysiological abnormalities in the form of arrhythmias, mediated via altered intracellular Ca2+ handling. The major findings are: (i) starving CMs to AAs results in proteasome translocation from the nucleus to the cytoplasm, while supplementation with the aromatic amino acids tyrosine (Y), tryptophan (W) and phenylalanine (F) (YWF) inhibits the proteasome recruitment; (ii) AA-deficient treatments cause arrhythmias; (iii) the arrhythmias observed upon nuclear proteasome sequestration(-AA+YWF) are blocked by KB-R7943, an inhibitor of the reverse mode of the sodium-calcium exchanger NCX; (iv) the retrograde perfusion of isolated rat hearts with AA starvation media is associated with arrhythmias. Collectively, our novel findings describe a newly identified mechanism linking the UPS to arrhythmia generation in CMs and whole hearts.

Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the β-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

CD8+ T cell-mediated rejection of allogenic human-induced pluripotent stem cell-derived cardiomyocyte sheets in human PBMC-transferred NOG MHC double knockout mice

BackgroundTransplantation of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has emerged as a promising therapy to treat end-stage heart failure. However, immunogenicity of hiPS-CMs in transplanted patients has not been fully elucidated. Thus, in vivo models are required to estimate immune responses against hiPS-CMs in transplant recipients. MethodsWe transferred human peripheral blood mononuclear cells (hPBMCs) into NOD/Shi-scid IL2rgnull (NOG) MHC class I/II double knockout (NOG-ΔMHC) mice, which were reported to accept hPBMCs without xenogeneic-graft-versus-host disease (xeno-GVHD). Then, hiPS-CM sheets generated from the hiPS cell line 201B7 harboring a luciferase transgene were transplanted into the subcutaneous space of NOG-ΔMHC mice. Graft survival was monitored by bioluminescent images using a Xenogen In Vivo Imaging System. ResultsThe human immune cells were engrafted for more than three months in NOG-ΔMHC mice without lethal xeno-GVHD. The hiPS-CMs expressed a moderate level of human leukocyte antigen (HLA)-class I, but not HLA-class II, molecules even after interferon-gamma (IFN-γ) stimulation. Consistently, the allogenic IFN-γ-treated hiPS-CMs induced weak CD8+ but not CD4+, T cell responses in vitro. hiPS-CM sheets disappeared approximately 17–24 days after transplantation in hPBMC-transferred NOG-ΔMHC mice, and CD8+ T cell depletion significantly prolonged graft survival, similar to what was observed following tacrolimus treatment. ConclusionhiPS-CMs are less immunogenic in vitro but induce sufficient CD8+ T cell-mediated immune responses for graft rejection in vivo.

TAD boundary deletion causes PITX2-related cardiac electrical and structural defects

While 3D chromatin organization in topologically associating domains (TADs) and loops mediating regulatory element-promoter interactions is crucial for tissue-specific gene regulation, the extent of their involvement in human Mendelian disease is largely unknown. Here, we identify 7 families presenting a new cardiac entity associated with a heterozygous deletion of 2 CTCF binding sites on 4q25, inducing TAD fusion and chromatin conformation remodeling. The CTCF binding sites are located in a gene desert at 1 Mb from the Paired-like homeodomain transcription factor 2 gene (PITX2). By introducing the ortholog of the human deletion in the mouse genome, we recapitulate the patient phenotype and characterize an opposite dysregulation of PITX2 expression in the sinoatrial node (ectopic activation) and ventricle (reduction), respectively. Chromatin conformation assay performed in human induced pluripotent stem cell-derived cardiomyocytes harboring the minimal deletion identified in family#1 reveals a conformation remodeling and fusion of TADs. We conclude that TAD remodeling mediated by deletion of CTCF binding sites causes a new autosomal dominant Mendelian cardiac disorder.

Hydroethanolic extract of Cirsium setidens ameliorates doxorubicin-induced cardiotoxicity by AMPK-PGC-1α-SOD-mediated mitochondrial protection

BackgroundDoxorubicin (DOX) is an effective anticancer agent. However, the clinical outcomes of DOX-based therapies are severely hampered by their significant cardiotoxicity. PurposeWe investigated the beneficial effects of an ethanol extract of Cirsium setidens (CSE) on DOX-induced cardiomyotoxicity (DICT). MethodsUPLC-TQ/MS analysis was used to identify CSE metabolite profiles. H9c2 rat cardiomyocytes and MDA-MB-231 human breast cancer cells were used to evaluate the effects of CSE on DICT-induced cell death. To elucidate the mechanism underlying it, AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma co-activator l-alpha (PGC1-α), nuclear respiratory factor 1 (NRF1), NRF2, superoxide dismutase (SOD1), and SOD2 expression was detected using western blot analysis. The oxygen consumption rate (OCR), cellular ROS, and mitochondrial membrane potential were measured. Finally, we confirmed the cardioprotective effect of CSE against DICT in both C57BL/6 mice and human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) by observing various parameters, such as electrophysiological changes, cardiac fibrosis, and cardiac cell death. ResultsChlorogenic acid and nicotiflorin were the major compounds in CSE. Our data demonstrated that CSE blocked DOX-induced cell death of H9c2 cells without hindrance of its apoptotic effects on MDA-MB-231 cells. DOX-induced defects of OCR and mitochondrial membrane potential were recovered in a CSE through upregulation of the AMPK-PGC1-α-NRF1 signaling pathway. CSE accelerated NRF1 translocation to the nucleus, increased SOD activity, and consequently blocked apoptosis in H9c2 cells. In mice treated with 400 mg/kg CSE for 4 weeks, electrocardiogram data, creatine kinase and lactate dehydrogenase levels in the serum, and cardiac fibrosis, were improved. Moreover, various electrophysiological features indicative of cardiac function were significantly enhanced following the CSE treatment of hiPSCCMs. ConclusionOur findings demonstrate CSE that ameliorates DICT by protecting mitochondrial dysfunction via the AMP- PGC1α-NRF1 axis, underscoring the therapeutic potential of CSE and its underlying molecular pathways, setting the stage for future investigations into its clinical applications.