Induced Pluripotent Stem Cell-derived Cardiomyocytes Research Articles

Cellular calcium handling and electrophysiology are modulated by chronic physiological pacing in human induced pluripotent stem cell-derived cardiomyocytes.

Electric pacing of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) has been increasingly used to simulate cardiac arrhythmias in vitro and to enhance cardiomyocyte maturity. However, the impact of electric pacing on cellular electrophysiology and Ca2+ handling in differentiated hiPSC-CM is less characterized. Here we studied the effects of electric pacing for 24 h or 7 days at a physiological rate of 60 beats/min on cellular electrophysiology and Ca2+ cycling in late-stage, differentiated hiPSC-CM (>90% troponin+, >60 days postdifferentiation). Electric culture pacing for 7 days did not influence cardiomyocyte cell size, apoptosis, or generation of reactive oxygen species in differentiated hiPSC-CM compared with 24-h pacing. However, epifluorescence measurements revealed that electric pacing for 7 days improved systolic Ca2+ transient amplitude and Ca2+ transient upstroke, which could be explained by elevated sarcoplasmic reticulum Ca2+ load and SERCA activity. Diastolic Ca2+ leak was not changed in line-scanning confocal microscopy, suggesting that the improvement in systolic Ca2+ release was not associated with a higher open probability of ryanodine receptor (RyR)2 during diastole. Whereas bulk cytosolic Na+ concentration and Na+/Ca2+ exchanger (NCX) activity were not changed, patch-clamp studies revealed that chronic pacing caused a slight abbreviation of the action potential duration (APD) in hiPSC-CM. We found in whole cell voltage-clamp measurements that chronic pacing for 7 days led to a decrease in late Na+ current, which might explain the changes in APD. In conclusion, our results show that chronic pacing improves systolic Ca2+ handling and modulates the electrophysiology of late-stage, differentiated hiPSC-CM. This study might help to understand the effects of electric pacing and its numerous applications in stem cell research including arrhythmia simulation.NEW & NOTEWORTHY Electric pacing is increasingly used in research with human induced pluripotent stem cell cardiomyocytes (hiPSC-CM), for example to simulate arrhythmias but also to enhance maturity. Therefore, it is mandatory to understand the effects of pacing itself on cellular electrophysiology in late-stage, matured hiPSC-CM. This study provides an electrophysiological characterization of the effects of chronic electric pacing at a physiological rate on differentiated hiPSC-CM.

Flavin-containing monooxygenase 2 confers cardioprotection in ischemia models through its disulfide-bond catalytic activity.

Myocardial infarction (MI) is characterized by massive cardiomyocytes death and cardiac dysfunction, and effective therapies to achieve cardioprotection are sorely needed. Here we reported that flavin containing monooxygenase 2 (FMO2) level was markedly increased in cardiomyocytes both in ex vivo and in vivo models of ischemia injury. Genetic deletion of FMO2 resulted in reduced cardiomyocyte survival and enhanced cardiac dysfunction, whereas cardiomyocyte-specific FMO2 overexpression exerted a protective effect in infarcted rat hearts. Mechanistically, FMO2 inhibited the activation of endoplasmic reticulum (ER) stress-induced apoptotic proteins, including caspase 12 and C/EBP homologous protein (CHOP), by down-regulating unfolded protein response (UPR) pathway. Furthermore, we identified FMO2 as a chaperone that catalyzed disulfide-bond formation in unfolded/misfolded proteins through its GVSG motif. GVSG-mutated FMO2 failed to catalyze disulfide-bond formation and lost its protection against ER stress and cardiomyocyte death. Finally, we demonstrated the protective effect of FMO2 in human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. Collectively, this study highlights FMO2 as a key modulator of oxidative protein folding in cardiomyocytes and underscores its therapeutic potential for treating ischemic heart disease.

Cellular Alignment and Matrix Stiffening Induced Changes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes.

Biological processes are inherently dynamic, necessitating biomaterial platforms capable of spatiotemporal control over cellular organization and matrix stiffness for accurate study of tissue development, wound healing, and disease. However, most in vitro platforms remain static. In this study, a dynamic biomaterial platform comprising a stiffening hydrogel is introduced and achieved through a stepwise approach of addition followed by light-mediated crosslinking, integrated with an elastomeric substrate featuring strain-responsive lamellar surface patterns. Employing this platform, the response of human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) is investigated to dynamic stiffening from healthy to fibrotic tissue stiffness. The results demonstrate that culturing hIPSC-CMs on physiologically relevant healthy stiffness significantly enhances their function, as evidenced by increased sarcomere fraction, wider sarcomere width, significantly higher connexin-43 content, and elevated cell beating frequency compared to cells cultured on fibrotic matrix. Conversely, dynamic matrix stiffening negatively impacts hIPSC-CM function, with earlier stiffening events exerting a more pronounced hindering effect. These findings provide valuable insights into material-based approaches for addressing existing challenges in hIPSC-CM maturation and have broader implications across various tissue models, including muscle, tendon, nerve, and cornea, where both cellular alignment and matrix stiffening play pivotal roles in tissue development and regeneration.

Characterisation of the novel SCN5A-P1891A variant, implicated in left ventricular noncompaction cardiomyopathy in a Finnish family, using human induced pluripotent stem cell-derived cardiomyocytes

Abstract Introduction Left ventricular noncompaction (LVNC) is a heterogenous cardiomyopathy characterised by an extensively trabeculated left ventricle (LV). Several genetic variants that perturb trabeculae compaction, and thus LV maturation, have been reported [1]. However, the precise mechanisms underpinning this have proven contentious with, for example, differentially aberrant cardiomyocyte proliferation implicated in the development of LVNC [2,3]. Purpose In this study, we investigate a novel SCN5A variant, P1891A, which is associated with LVNC in a Finnish family. We ascertain the effects of this variant through the characterisation of variant-carrying human induced pluripotent stem cell-derived cardiomyocytes (P1891A-hiPSC-CMs). Methods We isolated dermal fibroblasts from a symptomatic member of a Finnish family carrying the SCN5A-P1891A variant; derived P1891A-hiPSCs via nucleofection with reprogramming plasmids [4]; and differentiated these into hiPSC-CMs alongside healthy control-hiPSCs. We determined the action potential (AP) and sodium- (INa) and calcium-current (ICa) densities of hiPSC-CMs via patch-clamp. We investigated gene expression in hiPSC-CMs through qPCR and employed Multiple Approaches Combined (MCA) proteomics [5] in SCN5A-transfected HEK293-like cells to elucidate protein-protein interactions (PPI). We subjected hiPSC-CMs to mechanical stretch [6] to examine their stress response and assessed their proliferation capacity via high-content analysis for the percentage of BrdU positive hiPSC-CMs following mitogenic stimulation (5 µM CHIR99021 and 1 µM SB203580). Finally, we ascertained contractile kinetics and apparent contractile force using fibrin hydrogel-based engineered heart tissues (EHTs) [7]. Results The P1891A-hiPSC-CMs demonstrated elevated INa density alongside enhanced arrhythmogenicity relative to healthy control hiPSC-CMs although, AP kinetics and ICa density were unperturbed (Fig. 1). Under baseline conditions, P1891A-hiPSC-CMs exhibited higher SCN5A gene expression and proteomics revealed a reduction in PPI. Baseline proliferation was unchanged; however, aged P1891A-hiPSC-CMs had enhanced proliferative capacity following mitogenic stimulation. Further, the P1891A-hiPSC-CMs displayed impaired tolerance to mechanical stretch as evidenced by upregulated NPPB and ACTA1 expression. In the context of the EHT, P1891A-hiPSC-CMs yielded disparate phenotypes. Whilst a subset compacted fully and yielded weaker contractile parameters relative to healthy control EHTs, the majority of P1891A-hiPSC-CM EHTs failed to fully compact thereby recapitulating the LVNC phenotype (Fig. 2). Conclusions This study presents a unique aetiology of LVNC and links, for the first time, SCN5A mutations with enhanced human cardiomyocyte proliferation. The ability to recapture the noncompaction phenotype in EHTs presents a powerful model for future mechanistic research and drug development.

Novel mitochondria-targeted therapy AP39 limits mitochondrial dysregulation and diastolic dysfunction in 2D & 3D human models of diabetic cardiomyopathy

Abstract Introduction Mitochondrial dysregulation has been implicated in many complications of diabetes. Mitochondrial-targeted therapies AP39 and SS31 are renoprotective, but their cardioprotective potential in diabetes has not been fully resolved. Purpose We investigated AP39 and SS31 in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-hCM) and endothelial cells (iPSC-hEC) independently in 2D culture, as well as collectively in a 3D cardiac organoid (iPSC-hCO) model also comprising fibroblasts (iPSC-hFB), under type 2 diabetes (T2D)-like conditions. Methods Cells (iPSC-hCM, iPSC-hEC, iPSC-hFB; foreskin-2 cell line) were exposed to either 5.55mM glucose (control) or T2D conditions (20mM glucose plus fatty acids, endothelin-1 and cortisol) ± AP39 (50nM) or SS31 (1µM) for 48h. Cardiac organoids were subjected to 5.55mM glucose (control) or T2D for 4 days, with AP39 (50nM) or SS31 (1µM) added on day 2. Data are expressed as mean±SEM (fold) with n=4-6 from 2-3 independent experiments. One-way ANOVA with Dunnett`s post hoc analysis was performed with P<0.05 considered significant. Results 2D human cells: After 48h, T2D increased mitochondrial superoxide in iPSC-hCM (2.4±0.1 fold vs control) and iPSC-hEC (3.2±0.29 fold vs control), both P<0.05 (measured by MitoSOX). This was blunted by both AP39 (to 1.1±0.17) and SS31 (to 1.5±0.17) in T2D iPSC-hCM (P<0.05), with similar impact in iPSC-hEC, to 2.9±0.26 and 1.3±0.16 fold, both P<0.05. T2D increased cell death in both iPSC-hCM (1.6±0.09 fold vs control) and iPSC-hEC (1.6±0.12 fold, both P<0.05 measured by propidium iodide staining. Interestingly, AP39 selectively blunted cell death in T2D iPSC-hCM (to 1.1±0.06, P<0.05), mimicked by SS31 in T2D iPSC-hEC only (to 1.0±0.10, P<0.05). Further, T2D-induced mitochondrial hyperpolarisation in iPSC-hCM (1.5±0.09 fold vs control, assessed using TMRM), was only protected by AP39 (1.2±0.06 fold, P<0.05), but not SS31. 3D human cardiac organoids: After 4 days, T2D increased total contraction duration (1.6±0.10 fold control), time to peak (1.7±0.11) and relaxation duration (1.5±0.13, indicative of diastolic dysfunction), assessed by Olympus IX71 with a DP72 camera. All 3 contractile parameters were attenuated with AP39 (to 1.5±0.06, 1.3±0.09 and 1.0±0.04 fold, respectively) or SS31 (to 1.1±0.05, 1.2±0.15 and 1.2±0.12 fold, respectively), all P<0.05. T2D increased mitochondrial superoxide (2.7±0.16 fold control), blunted by AP39 (to 1.8±0.16) and SS31 (to 1.9±0.17), both P<0.05. Cellular injury measured by LDH was evident in T2D iPSC-hCO (2.1±0.23 fold control) was limited by AP39 (to 1.2±0.09) and SS31 (to 1.1±0.07), both P<0.05. Conclusion Our results highlight the cardioprotective potential of AP39 and SS31 to limit mitochondrial dysregulation, cell death and contractile dysfunction in human in vitro models of T2D. These findings may facilitate their clinical progression as therapeutic options for diabetic cardiomyopathy.

Burst-like transcription of MYH7, allelic and contractile imbalance already in early stage hiPSC-cardiomyocytes indicate these as pathogenic factors in Hypertrophic Cardiomyopathy

Abstract Background Hypertrophic Cardiomyopathy (HCM) is caused by heterozygous mutations in sarcomeric proteins, which can cause hyper- or hypocontractiliy of cardiomyocytes. Previously we also observed highly variable force generation (contractile imbalance) among individual cardiomyocytes of HCM-patients with myosin (MYH7) or myosin-binding-protein-C mutations (MYBPC3), ranging from normal to highly altered values. Contractile imbalance is presumably caused by stochastic, burst-like transcription of mutant and wildtype allele, which induces unequal ratios of mutant vs. wildtype mRNA among individual cardiomyocytes. Variable force generation most likely is the result of also unequal fractions of mutated protein from cell to cell. Aim With this study we aimed to examine whether in addition to the effects of the mutation on sarcomere function, also allelic and contractile imbalance already exist at the beginning of disease development or whether both result from disease-related alterations in symptomatic HCM-patients. Methods and Results We studied this in patient-derived human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with MYH7-mutation R723G as model for an early disease stage. These early, fetal-like cardiomyocytes developed typical HCM-related features like hypertrophy and increased myofibrillar disarray compared to WT-hiPSC-CMs. Calcium sensitivity was reduced, twitch kinetics were slowed down. To test whether MYH7 in these early-stage hiPSC-CMs also is transcribed in bursts or continuously, we performed RNA-fluorescence in situ hybridization (RNA-FISH). Nuclei without, with one and with two or more active transcription sites were observed which indicates stochastic, burst-like and independent transcription of the two MYH7-alleles. Ratios of mRNA from both alleles were quantified in individual cardiomyocytes by allele-specific single cell RT-PCR. We found highly variable ratios of mutated vs. wildtype mRNA among individual cardiomyocytes, similar to HCM-patient’s cardiac tissue, indicating unequal expression of mutant and wildtype mRNA from cell-to-cell. Furthermore, functional measurements on myofibrils from R723G-hiPSC-CMs revealed substantial variability in force generation at physiological calcium, compared to much smaller heterogeneity among WT-hiPSC-CMs. In addition, variability of twitch kinetics of intact hiPSC-CMs was also substantially increased among R723G- compared to WT-hiPSC-CMs. Altogether, this suggests that functional heterogeneity results from burst-like transcription in heterozygous HCM cardiomyocytes. Conclusions Our results in HCM-patient derived hiPSC-cardiomyocytes with a fetal-like developmental stage suggest that allelic and contractile imbalance among individual cardiomyocytes together with the mutation are present at the beginning of the disease. Thus, they most likely are exacerbating factors that contribute development of HCM hallmarks.

Indoxyl sulfate deteriorates cardiac electrophysiological function and increases the likelihood of arrhythmias in human ventricular myocardial cells

Abstract Background Approximately 10% of the world population is suffering from chronic kidney disease (CKD). In these patients, an increased mortality and morbidity, predominantly derived from cardiovascular diseases, can be observed. Purpose The impact of CKD on myocardial cell mechanisms is only partially understood. This study aims to investigate the impact of the uremic toxin indoxyl sulfate (IS) on human ventricular myocardial cells. Methods Human left ventricular myocardium was obtained from patients with severe aortic stenosis who underwent surgical valve replacement. In addition, human ventricular induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) obtained from donors without any known cardiovascular disease were used. Before measurements were conducted, isolated single primary human cardiomyocytes were incubated for 15 minutes with an IS concentration of 120 mg/l (highest measured IS-level in dialysis patients). Further, iPSC-CM were incubated for one week with IS 45 mg/l and IS 120 mg/l, with IS 45 mg/l representing the average dialysis patients IS-level. Epifluorescence and confocal microscopy were performed to investigate Ca2+hemostasis and patch clamp measurements assessed cardiac action potentials and the occurrence of arrythmias. Results Experiments were conducted using human ventricular myocardium from 10 patients with a mean left ventricular ejection fraction of 51.5±12% and a mean glomerular filtration rate of 66.3±22.7 ml/min. Compared to the control group, higher systolic Ca2+ transient amplitudes were observed after acute incubation with IS 120 mg/l in single primary human cardiomyocytes. Measurements after 7 days of IS incubation (IS 45 mg/l and 120 mg/l) in iPSC-CM confirmed these previous results by showing higher Ca2+ transient amplitudes (Figure 1). Additionally, cells showed a diminished diastolic Ca2+ level compared to controls. An increased Ca2+ spark frequency in IS-treated iPSC-CM versus the control group was unveiled by confocal microscopy measurements. While an increased number of delayed afterdepolarizations could be observed, IS seemed to have no effect on cardiac action potential duration in iPSC-CM. Conclusion For the first time, we could reveal the impact of IS on cellular electrophysiology of primary human myocardium cells and iPSC-CM and identify IS as a proarrhythmogenic trigger. Therefore, IS may be of relevance in explaining the high cardiovascular mortality in end-stage CKD patients. To improve the outcome of these patients, new therapeutic approaches will be necessary since IS, a protein-bound uremic toxin, is only insufficiently removed by dialysis.

Novel role of human epicardial adipocyte-derived SPARCL1 in suppression of postoperative atrial fibrillation in patients undergoing cardiovascular surgery

Abstract Background Postoperative atrial fibrillation (POAF) occurs in 20-50% after cardiovascular surgery and is associated with the short and long-term morbidity/mortality. The epicardial adipose tissue (EAT) may have a potential role to regulate the incidence of POAF. Purpose We explored the possible role of human epicardial adipocyte-derived adipokines in the regulation of the myocardial redox state and POAF. Methods We prospectively evaluated 149 patients without known AF who underwent scheduled open-heart cardiovascular surgery between May 2020 and November 2022. They were divided into POAF or Non-POAF group and followed up after discharge (476.6 ± 290.0 days). POAF was defined as AF lasting more than 30 seconds after the surgery. Human EAT samples were obtained from these patients during surgery and the preadipocytes were isolated. Preadipocytes were terminally differentiated to mature adipocytes and mRNAs were extracted. Genome-wide expression profiling of 6 vs. 6 matched samples of the POAF and Non-POAF groups was performed using Microarray analysis, and the results were validated with qPCR in all the cohort samples. The effects of secreted adipokines on the cardiomyocytes were assessed in terms of oxidative stress. Superoxide and whole reactive oxygen species (ROS) were evaluated with luminometry and live-cell fluorescent probes respectively. As the oxidative stress associated signalling, the phosphorylation of ERK and p38-MAPK were evaluated with western blotting. Angiotensin II (Ang-II) was used to induce oxidative stress and phosphorylation of MAPK. Results POAF was observed in 53 patients (36%). Microarray analysis (n = 6) revealed that there were 132 down- or up-regulated cording genes in epicardial adipocytes of the POAF group compared to the Non-POAF group. Of these genes, 11 genes cording secretable molecules were validated by qPCR (n = 69), showing that only SPARCL1 had significantly downregulated in the POAF group compared to the Non-POAF group (P = 0.005). Superoxide and ROS were induced by Ang II (P < 0.01) and attenuated by SPARCL1 dose-dependent manner (P < 0.01) in neonatal rat cardiomyocytes. The phosphorylation of ERK and p38-MAPK were induced by Ang II (P < 0.01) and attenuated by SPARCL1 (P < 0.01) dose-dependently. Co-culture of human induced pluripotent stem cell-derived atrial cardiomyocytes (iPS-ACM) and human epicardial adipocytes revealed that the adipocytes derived from Non-POAF group suppressed the superoxide (P = 0.002) and ROS (P = 0.02) in iPS-ACM compared to POAF group. Kaplan-Meier survival analysis revealed that the POAF group (P = 0.01) and low SPARCL1 expression in epicardial adipocytes (P = 0.018) were associated with a higher incidence of long-term adverse cardiovascular events after surgery. Conclusion: SPARCL1 derived from epicardial adipocytes may play an important role in the suppression of POAF and long-term cardiovascular events via improving the myocardial redox state.

Serum exosomal long non-coding RNA H19 as a theragnostic target for atrial fibrillation

Abstract Background/Introduction Exosomes are membrane vesicles of 30- to 150-nm secreted by most cell types that can mediate intercellular communication by transmitting various forms of RNAs. Long non-coding RNAs (lncRNAs, ≥ 200 nucleotides length) have been reported to play important roles in the pathophysiological processes that promote the development and progression of many diseases. Interestingly, recent studies have shown that exosomal lncRNAs exhibit different expression profiles in various diseases and are being extensively studied in the medical field as an ideal target for the diagnosis and treatment. However, there is still little known about their role in atrial fibrillation (AF). Purpose The aim of this study was to identify a novel theragnostic target exosomal lncRNA for AF. Methods First, serum exosomes from controls and patients with persistent AF were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blot. Next, serum exosomal lncRNA expression profiles were explored using RNA-sequencing analysis. In addition, the expression levels of candidate exosomal lncRNAs were confirmed using qRT-PCR (n = 50 per group). Finally, AF-related pathophysiological mechanisms of exosomal lncRNA were investigated in angiotensin II (Ang II)-treated human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-atrial CMs). Results After RNA-sequencing analysis, we identified 27 differentially expressed lncRNAs (i.e., 4 upregulated and 23 downregulated lncRNAs with a |fold change| ≥ 2 and p < 0.05) in serum exosomes from patients with persistent AF compared with the controls. In fact, qRT-PCR reveled that exosomal lncRNA H19 was consistently downregulated in the serum of patients with persistent AF compared with the controls (p < 0.01). In addition, exosomal lncRNA H19 exhibited significant diagnostic validity for AF. Notably, we confirmed that exosomal lncRNA H19 was involved in AF-related pathophysiological mechanisms. In Ang II-treated iPSC-atrial CMs, inhibition of lncRNA H19 significantly aggravated Ang II-induced increases in levels of hypertrophic markers (ANP, BNP and β-MHC), cell surface area and inflammation (p < 0.05). Mechanistically, we found that loss of lncRNA H19 weakens the inhibition of its direct target microRNAs, miR-141-3p and miR-200a-3p, leading to downregulation of their target PTEN, thereby promoting the atrial remodeling and the consequent AF. Conclusion In conclusion, serum-derived exosomal lncRNA H19 could serve as a potential target for the diagnosis and treatment of AF.

Robotic manipulation of cardiomyocytes to identify gap junction modifiers for arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a leading cause of sudden cardiac death among young adults. Aberrant gap junction remodeling has been linked to disease-causative mutations in plakophilin-2 (PKP2). Although gap junctions are a key therapeutic target, measurement of gap junction function in preclinical disease models is technically challenging. To quantify gap junction function with high precision and high consistency, we developed a robotic cell manipulation system with visual feedback from digital holographic microscopy for three-dimensional and label-free imaging of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The robotic system can accurately determine the dynamic height changes in the cells' contraction and resting phases, microinject drug-treated healthy and diseased iPSC-CMs in their resting phase with constant injection depth across all cells, and deposit a membrane-impermeable dye that solely diffuses between cells through gap junctions for measuring the gap junction diffusion function. The robotic system was applied toward a targeted drug screen to identify gap junction modulators and potential therapeutics for ACM. Five compounds were found to dose-dependently enhance gap junction permeability in cardiomyocytes with PKP2 knockdown. In addition, PCO 400 (pinacidil) reduced beating irregularity in a mouse model of ACM expressing mutant PKP2 (R735X). These results highlight the utility of the robotic cell manipulation system to efficiently assess gap junction function in a relevant preclinical disease model, thus providing a technique to advance drug discovery for ACM and other gap junction-mediated diseases.

An endogenous cholinergic system controls electrical conduction in the heart.

The cholinergic system is distributed in the nervous system, mediating electrical conduction through acetylcholine (ACh). This study aims to identify whether the heart possesses an intact endogenous cholinergic system and to explore its electrophysiological functions and relationship with arrhythmias in both humans and animals. The components of the heart's endogenous cholinergic system were identified by a combination of multiple molecular cell biology techniques. The relationship of this system with cardiac electrical conduction and arrhythmias was analysed through electrophysiological techniques. An intact cholinergic system including ACh, ACh transmitter vesicles, ACh transporters, ACh metabolic enzymes, and ACh receptors was identified in both human and mouse ventricular cardiomyocytes (VCs). The key components of the system significantly regulated the conductivity of electrical excitation among VCs. The influence of this system on electrical excitation conduction was further confirmed both in the mice with α4 or α7 nicotinic ACh receptors (nAChRs) knockouts and in the monolayers of human induced pluripotent stem cell-derived cardiomyocytes. Mechanistically, ACh induced an inward current through nAChRs to reduce the minimum threshold current required to generate an action potential in VCs, thereby enhancing the excitability that acts as a prerequisite for electrical conduction. Importantly, defects in this system were associated with fatal ventricular arrhythmias in both patients and mice. This study identifies an integrated cholinergic system inherent to the heart, rather than external nerves that can effectively control cardiac electrical conduction. The discovery reveals arrhythmia mechanisms beyond classical theories and opens new directions for arrhythmia research.

Hypertrophic cardiomyopathy-associated mutations drive stromal activation via EGFR-mediated paracrine signaling.

Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the left ventricular wall, diastolic dysfunction, and fibrosis, and is associated with mutations in genes encoding sarcomere proteins. While in vitro studies have used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study HCM, these models have not examined the multicellular interactions involved in fibrosis. Using engineered cardiac microtissues (CMTs) composed of HCM-causing MYH7-variant hiPSC-CMs and wild-type fibroblasts, we observed cell-cell cross-talk leading to increased collagen deposition, tissue stiffening, and decreased contractility dependent on fibroblast proliferation. hiPSC-CM conditioned media and single-nucleus RNA sequencing data suggested that fibroblast proliferation is mediated by paracrine signals from MYH7-variant cardiomyocytes. Furthermore, inhibiting epidermal growth factor receptor tyrosine kinase with erlotinib hydrochloride attenuated stromal activation. Last, HCM-causing MYBPC3-variant CMTs also demonstrated increased stromal activation and reduced contractility, but with distinct characteristics. Together, these findings establish a paracrine-mediated cross-talk potentially responsible for fibrotic changes observed in HCM.

Extracellular vesicles from human cardiac stromal cells up-regulate cardiomyocyte protective responses to hypoxia

BackgroundCell therapy can protect cardiomyocytes from hypoxia, primarily via paracrine secretions, including extracellular vesicles (EVs). Since EVs fulfil specific biological functions based on their cellular origin, we hypothesised that EVs from human cardiac stromal cells (CMSCLCs) obtained from coronary artery bypass surgery may have cardioprotective properties.ObjectivesThis study characterises CMSCLC EVs (C_EVs), miRNA cargo, cardioprotective efficacy and transcriptomic modulation of hypoxic human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). C_EVs are compared to bone marrow mesenchymal stromal cell EVs (B_EVs) which are a known therapeutic EV type.MethodsCells were characterised for surface markers, gene expression and differentiation potential. EVs were compared for yield, phenotype, and ability to protect hiPSC-CMs from hypoxia/reoxygenation injury. EV dose was normalised by both protein concentration and particle count, allowing direct comparison. C_EV and B_EV miRNA cargo was profiled and RNA-seq was performed on EV-treated hypoxic hiPSC-CMs, then data were integrated by multi-omics. Confirmatory experiments were carried out using miRNA mimics.ResultsAt the same dose, C_EVs were more effective than B_EVs at protecting CM integrity, reducing apoptotic markers, and cell death during hypoxia. While C_EVs and B_EVs shared 70–77% similarity in miRNA content, C_EVs contained unique miRNAs, including miR-202-5p, miR-451a and miR-142-3p. Delivering miRNA mimics confirmed that miR-1260a and miR-202/451a/142 were cardioprotective, and the latter upregulated protective pathways similar to whole C_EVs.ConclusionsThis study demonstrates the potential of cardiac tissues, routinely discarded following surgery, as a valuable source of EVs for myocardial infarction therapy. We also identify miR-1260a as protective of CM hypoxia.

Chronic Activation of Tubulin Tyrosination Improves Heart Function.

Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and associated with left ventricular hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with the findings that microtubule detyrosination (dTyr-MT) is markedly elevated in heart failure. Acute reduction of dTyr-MT by inhibition of the detyrosinase (VASH [vasohibin]/SVBP [small VASH-binding protein] complex) or activation of the tyrosinase (TTL [tubulin tyrosine ligase]) markedly improved contractility and reduced stiffness in human failing cardiomyocytes and thus posed a new perspective for HCM treatment. In this study, we tested the impact of chronic tubulin tyrosination in an HCM mouse model (Mybpc3 knock-in), in human HCM cardiomyocytes, and in SVBP-deficient human engineered heart tissues (EHTs). Adeno-associated virus serotype 9-mediated TTL transfer was applied in neonatal wild-type rodents, in 3-week-old knock-in mice, and in HCM human induced pluripotent stem cell-derived cardiomyocytes. We show (1) TTL for 6 weeks dose dependently reduced dTyr-MT and improved contractility without affecting cytosolic calcium transients in wild-type cardiomyocytes; (2) TTL for 12 weeks reduced the abundance of dTyr-MT in the myocardium, improved diastolic filling, compliance, cardiac output, and stroke volume in knock-in mice; (3) TTL for 10 days normalized cell area in HCM human induced pluripotent stem cell-derived cardiomyocytes; (4) TTL overexpression activated transcription of tubulins and other cytoskeleton components but did not significantly impact the proteome in knock-in mice; (5) SVBP-deficient EHTs exhibited reduced dTyr-MT levels, higher force, and faster relaxation than TTL-deficient and wild-type EHTs. RNA sequencing and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-deficient versus TTL-deficient EHTs. This study provides the first proof of concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the nonsarcomeric cytoskeleton in heart disease.

Senescent CD8+ T cells: a novel risk factor in atrial fibrillation.

Immune cell alterations may play a role in the development of atrial fibrillation (AF). Our objective was to comprehensively characterize immune cells in AF, and investigate the potential mechanisms. Single-cell RNA sequencing and multicolor flow cytometry revealed that T cells constituted the most significant subset alterations in AF, and senescent CD8+ T cells were AF-associated subset. Senescent CD8+ T cells increased in both peripheral veins (p < 0.0001) and the left atria (p < 0.05) in patients with AF compared to non-AF control. Senescent CD8+ T cells were independently associated with AF prevalence (odds ratio = 2.876, p < 0.05) and postprocedural recurrence (hazard ratio = 22.955, p < 0.0001) using a cross-sectional study and a subsequent prospective cohort study. Senescent CD8+ T cells secreted an increased amount of interferon (IFN)-γ, which induces Ca2+ handling abnormalities in human induced pluripotent stem cell-derived atrial cardiomyocytes, and translated into an increased susceptibility to AF assessed by heart optical mapping. An increased amount of senescent CD8+ T cells may be a hallmark of the immune senescence phenotype in AF and potentially serve as a valid biomarker for assessing prevalence and postprocedural recurrence of AF. By connecting immune senescence with electrophysiological disturbances in AF, this research provides a potential mechanism for the involvement of senescent CD8+ T cells in proarrhythmic calcium disorders and suggests novel avenues for developing new immune-modulatory and senolytic therapies for AF.

Metabolic changes of human induced pluripotent stem cell-derived cardiomyocytes and teratomas after transplantation

Metabolic changes of human induced pluripotent stem cell-derived cardiomyocytes and teratomas after transplantation

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.

Blunted Cardiac Mitophagy in Response to Metabolic Stress Contributes to HFpEF.

Metabolic remodeling and mitochondrial dysfunction are hallmarks of heart failure with reduced ejection fraction. However, their role in the pathogenesis of HF with preserved ejection fraction (HFpEF) is poorly understood. In a mouse model of HFpEF, induced by high-fat diet and Nω-nitrol-arginine methyl ester, cardiac energetics was measured by 31P NMR spectroscopy and substrate oxidation profile was assessed by 13C-isotopmer analysis. Mitochondrial functions were assessed in the heart tissue and human induced pluripotent stem cell-derived cardiomyocytes. HFpEF hearts presented a lower phosphocreatine content and a reduced phosphocreatine/ATP ratio, similar to that in heart failure with reduced ejection fraction. Decreased respiratory function and increased reactive oxygen species production were observed in mitochondria isolated from HFpEF hearts suggesting mitochondrial dysfunction. Cardiac substrate oxidation profile showed a high dependency on fatty acid oxidation in HFpEF hearts, which is the opposite of heart failure with reduced ejection fraction but similar to that in high-fat diet hearts. However, phosphocreatine/ATP ratio and mitochondrial function were sustained in the high-fat diet hearts. We found that mitophagy was activated in the high-fat diet heart but not in HFpEF hearts despite similar extent of obesity suggesting that mitochondrial quality control response was impaired in HFpEF hearts. Using a human induced pluripotent stem cell-derived cardiomyocyte mitophagy reporter, we found that fatty acid loading stimulated mitophagy, which was obliterated by inhibiting fatty acid oxidation. Enhancing fatty acid oxidation by deleting ACC2 (acetyl-CoA carboxylase 2) in the heart stimulated mitophagy and improved HFpEF phenotypes. Maladaptation to metabolic stress in HFpEF hearts impairs mitochondrial quality control and contributed to the pathogenesis, which can be improved by stimulating fatty acid oxidation.

Human induced pluripotent stem cell-derived cardiomyocytes to study inflammation-induced aberrant calcium transient.

Heart failure with preserved ejection fraction (HFpEF) is commonly found in persons living with HIV (PLWH) even when antiretroviral therapy suppresses HIV viremia. However, studying this condition has been challenging because an appropriate animal model is not available. In this article, we studied calcium transient in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in culture to simulate the cardiomyocyte relaxation defect noted in PLWH and HFpEF and assess whether various drugs have an effect. We show that treatment of hiPSC-CMs with inflammatory cytokines (such as interferon-γ or TNF-α) impairs their Ca2+ uptake into sarcoplasmic reticulum and that SGLT2 inhibitors, clinically proven as effective for HFpEF, reverse this effect. Additionally, treatment with mitochondrial antioxidants (like mito-Tempo) and certain antiretrovirals resulted in the reversal of the effects of these cytokines on calcium transient. Finally, incubation of hiPSC-CMs with serum from HIV patients with and without diastolic dysfunction did not alter their Ca2+-decay time, indicating that the exposure to the serum of these patients is not sufficient to induce the decrease in Ca2+ uptake in vitro. Together, our results indicate that hiPSC-CMs can be used as a model to study molecular mechanisms of inflammation-mediated abnormal cardiomyocyte relaxation and screen for potential new interventions.

Circulating Autoantibodies Targeting TREK-1 in Patients With Short-Coupled Ventricular Fibrillation.

Short-coupled ventricular fibrillation (SCVF) is increasingly being recognized as a distinct primary electrical disorder and cause of otherwise unexplained cardiac arrest. However, the pathophysiology of SCVF remains largely elusive. Despite extensive genetic screening, there is no convincing evidence of a robust monogenic disease gene, thus raising the speculations for alternative pathogeneses. The role of autoimmune mechanisms in SCVF has not been investigated so far. The objective of this study was to screen for circulating autoantibodies in patients with SCVF and assess their role in arrhythmogenesis. This is a prospective, single-center, case-control study enrolling cardiac arrest survivors diagnosed with SCVF or idiopathic ventricular fibrillation (IVF) between 2019 and 2023 at the Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval Inherited Arrhythmia Clinic in Canada. Plasma samples were screened for autoantibodies targeting cardiac ion channels using peptide microarray technology. Identified target autoantibodies were then purified from pooled plasma samples for subsequent cellular electrophysiological studies. Fourteen patients with SCVF (n=4 [29%] female patients; median age, 45 years [36, 58]; n=14 [100%] non-Hispanic White) and 19 patients with idiopathic ventricular fibrillation (n=8 [42%] female patients; median age, 49 years [38, 57]; n=19 [100%] non-Hispanic White) were enrolled in the study and compared with 38 (n=20 [53%] female subjects; median age, 45 years [29, 66]; n=36 [95%] non-Hispanic White) sex-, age- and ethnicity-matched healthy controls. During the study period, 11 (79%) SCVF probands experienced ventricular fibrillation recurrence after a median of 4.3 months (interquartile range, 0.3-20.7). Autoantibodies targeting cardiac TREK-1 (TWIK [tandem of pore-domains in a weakly inward rectifying potassium channel]-related potassium channel 1 were identified in 7 (50%) patients with SCVF (P=0.049). Patch clamp experiments demonstrated channel-activating properties of anti-TREK-1 autoantibodies that are antagonized by quinidine in both HEK293 cells and human induced pluripotent stem cell-derived cardiomyocytes. Patients with SCVF harbor circulating autoantibodies against the cardiac TREK-1 channel. Anti-TREK-1 autoantibodies not only present the first reported biomarker for SCVF, but our functional studies also suggest a direct implication in the arrhythmogenesis of SCVF.