Induced Pluripotent Stem Cell-derived Cardiomyocytes Research Articles

Minimally Invasive Transthoracic Intramyocardial Cellular Transplantation Under Echocardiographic Guidance for Myocardial Impairment

Abstract Objective: To explore the approach of minimally invasive transthoracic intramyocardial cellular transplantation under echocardiographic guidance to promote ischemic myocardial repair in a preclinical big-animal study. Methods: Female Guangxi Bama miniature pigs (weight: 25–30 kg) were randomly allocated into the sham group, untreated myocardial infarction (MI) group (MI group), the MI and surgical intramyocardial injection (SIM) group (MI-SIM group), and the MI and transthoracic echocardiography-guided percutaneous intramyocardial injection (TTEPIM) group (MI-TTEPIM group) (n = 4 each) using a lottery method. A swine MI model was established in the 3 groups excluding the sham group, and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) labeled with the herpes simplex virus type-1 thymidine kinase reporter gene (hiPS-CMTK+) were transplanted by SIM in MI-SIM group and TTEPIM in MI-TTEPIM group. The operation time, postoperative recovery time of animals and volume of blood loss were collected for comparison between MI-SIM group and MI-TTEPIM group. 9-(4-[18F] fluoro-3-(hydroxymethyl) butyl) guanine positron emission tomography/computed tomography imaging was performed to track the hiPS-CMTK+ in vivo. Cardiac function and morphology were evaluated by echocardiography. Results: The operation time and postoperative recovery time of MI-TTEPIM group were significantly shorter than those of MI-SIM group ((28.3 ± 3.6) min vs. (97.0 ± 6.7) min, P < 0.001; (1.3 ± 0.3) d vs. (7.5 ± 0.9) d, P < 0.001). MI-TTEPIM also showed significantly lesser volume of blood loss during cell transplantation than MI-SIM group ((4.3 ± 0.8) mL vs. (47.0 ± 4.1) mL, P < 0.001). The transplanted cells could be traced more accurately in vivo in MI-TTEPIM than in MI-SIM. The circumferential strain of intervention region in the MI-TTEPIM group (–25.07% ± 0.27%) was significantly higher than that of the MI-SIM (–20.39% ± 0.67%) and MI groups (–19.68% ± 0.67%), respectively (P < 0.01). Conclusion: A minimally invasive TTEPIM protocol with stem cells for treating the ischemic myocardium was established in this study. Transplantation of hiPS-CMTK+ with this method could promote the recovery of the circumferential strain of the ischemic myocardium. The findings of this study lay a foundation for the clinical transformation of this auxiliary means of treatment in the future.

Abstract 4137339: Application of PLGA-PEG-PLGA Thermosensitive Hydrogel Loaded with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Ginsenoside Rb3 in the Treatment of Acute Myocardial Infarction

Background: Ginsenoside Rb3 (GSRb3) is a traditional Chinese medicine monomer. We aim to investigate the effects of GSRb3 on the proliferation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and the reparative effects of PLGA-PEG-PLGA thermosensitive hydrogel loaded with hiPSC-CMs and GSRb3 (NP GSRb3 @Gel+iCM) on myocardial infarction (MI). Methods: 1. Western Blot, RT-qPCR, cell immunofluorescence staining, and CCK-8 assays were employed to assess the proliferative effects of different concentrations of GSRb3 on neonatal mouse primary cardiomyocytes (NCMs) and hiPSC-CMs. 2. Transcriptomic gene sequencing technology was utilized to investigate the mRNA expression changes of hiPSC-CMs induced by GSRb3; The NP GSRb3 @Gel+iCM was injected into the left ventricular myocardium of the MI mice; the same volume injections of hiPSC-CMs (iCM group) and PLGA-PEG-PLGA thermosensitive hydrogel loaded with GSRb3 (NP GSRb3 @Gel group) served as control; Additionally, MI model group without additional treatment and sham group were established. 3. At 4 weeks post-MI, echocardiography, Sirius Red/Fast Green staining and immunofluorescence staining were utilized to evaluate the heart function, infarction size and survival rate of transplanted hiPSC-CMs. Results: GSRb3 inhibit the apoptosis of NCMs while promote proliferation of NCMs and hiPSC-CMs significantly. After 4 weeks of treatment, the engraftment rate of hiPSC-CMs in NP GSRb3 @Gel+iCM group (36.7%) was 4.8 times that of iCM group (7.7%); Compared to the MI group, NP GSRb3 @Gel group and iCM group, NP GSRb3 @Gel+iCM group showed significant improvement in cardiac function; The myocardial repair effect in NP GSRb3 @Gel+iCM group was significantly superior to NP GSRb3 @Gel group and iCM group, with a 47% reduction in infarct size compared to MI group, and further reduction in left ventricular anterior wall thickness compared to NP GSRb3 @Gel group and iCM group. Conclusion: GSRb3 promotes the proliferation of hiPSC-CMs both in vitro and in vivo. NP GSRb3 @Gel+iCM significantly increase the engraftment of hiPSC-CMs, promote angiogenesis, reduce infarction size, inhibit left ventricular remodeling and improve cardiac function.

Abstract 4136605: Effect Of A Bone Morphogenetic Protein Receptor Type 2 Mutation On The Cardiomyocyte Response To Pressure Overload

Introduction: A mutation in the Bone Morphogenetic Protein Receptor Type 2 ( BMPR2 ) gene is in 70% of hereditary pulmonary arterial hypertension (hPAH) patients the causative mutation of the disease. Previous work demonstrated that right ventricular function is more impaired in hPAH patients with a BMPR2 mutation. However, the underlying mechanism remains elusive. Moreover, natriuretic peptides, such as N-terminal Brain Natriuretic Peptides (NTproBNP) and Mid Regional proAtrial Natriuretic Peptides (MRproANP), are secreted from cardiomyocytes upon stretch as adaptation to increased pressure overload. We hypothesize that the BMPR2 mutation impairs the response to increased pressure overload. For this purpose, we make use of ventricular induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of hPAH patients carrying a BMPR2 mutation and their isogenic controls in which the mutation was corrected. Aim: To study the effect of a BMPR2 mutation on the response to pressure overload in vitro . Methods: iPSCs from 2 hPAH patients and its isogenic controls were used. iPSCs were differentiated into ventricular iPSC-CMs and stretched 10% equiaxially for 24h at 1Hz on the Flexcell FX-6000 system. Natriuretic peptides gene expressions were quantified via RT-PCR. NTproBNP and MRproANP release were measured on cell supernatants throughout specific immunoassays. Normality of the data was checked, and statistics were performed accordingly. Results: No differences were observed on natriuretic peptides gene expression on static and stretched iPSC-CMs. There is a clear correlation between NTproBNP and MRproANP release (R 2 =0.83, p<0.0001, Figure 1A). Interestingly, hPAH iPSC-CMs showed higher baseline values for NTproBNP release compared to isogenic iPSC-CMs (p<0.01, Figure 1B). Furthermore, stretching resulted in increased NTproBNP and MRproANP secretion in both hPAH and isogenic iPSC-CMs (Figure 1B-C). Conclusions: Secretion of NTproBNP and MRproANP was closely correlated in iPSC-CMs. iPSC-CMs of hPAH patients with a BMPR2 mutation showed higher baseline values of NTproBNP. Increased secretion of NTproBNP and MRproANP upon mechanical stimulation was similar between hPAH and isogenic iPSC-CMs. Experiments in atrial iPSC-CMs are currently ongoing to test potential differences in natriuretic peptide secretion between ventricular and atrial cardiomyocytes as the right atrium is also under stretch in PAH.

Abstract 4138380: Cardiac protection of hiPSC-CMs loaded chitosan cardiac patch in myocardial infarcted swine

Background: The low grafting rate of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) following direct injection into the myocardium limits the efficacy of cardiac repair after myocardial infarction (MI). Chitosan-based scaffolds have emerged as functional biomaterials for designing delivery systems in cardiac therapies. This study aimed to enhance the potency of cardiac repair using a hiPSC-CMs loaded chitosan cardiac muscle patch (hCCMP). Methods: The patches were fabricated using chitosan. Characterization of the chitosan patches involved assessing their swelling rate and porosity. The biocompatibility of the hCCMP was analyzed through scanning electron microscopy (SEM) and the CCK8 assay. 16 Yorkshire swine (15-20 kg, 45-50 days of age) were randomly divided into 4 groups (n=4 per group): Sham, MI without further intervention, MI with an empty patch (without hiPSC-CMs), and MI with hCCMP implantation. One and four weeks post-MI, heart function, Infarction size, myocardial perfusion and metabolism were evaluated via cardiac PET-MRI. Engraftment was assessed through human cardiac troponin T staining. Histological analysis including left ventricular anterior wall thickness, cardiac remodeling, angiogenesis, arterialization, and apoptosis in the infarct border area were analyzed using immunofluorescence. Results: SEM showed that the chitosan patch had regular pore size and good 3D structure; porosity was about (96.7±0.37)%. On day3, the patch swelling rate was about (715±64.55)%. SEM, CCK8 assay showed a normal cells growth in the chitosan patch. The hiPSC-CMs grafted well with chitosan patch delivery systems and was associated with significantly improvement in LV function, infarction size, angiogenesis and cardiomyocyte apoptosis in pigs 4 weeks after MI. Conclusions: The 3D chitosan cardiac patch with hiPSC-CMs loaded can enhance the potency of cardiac repair in pigs after MI and present a novel and promising therapic strategy.

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.

Hypoxia and TNF-alpha modulate extracellular vesicle release from human induced pluripotent stem cell-derived cardiomyocytes.

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in the heart under homeostatic and pathological conditions, such as myocardial infarction (MI). However, the basic mechanisms driving cardiomyocyte-derived EV (CM-EV) production following stress are poorly understood. In this study, we generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that express NanoLuc-tetraspanin reporters. These modified hiPSC-CMs allow for quantification of tetraspanin-positive CM-EV secretion from small numbers of cells without the need for time-consuming EV isolation techniques. We subjected these cells to a panel of small molecules to study their effect on CM-EV biogenesis and secretion under basal and stress-associated conditions. We observed that EV biogenesis is context-dependent in hiPSC-CMs. Nutrient starvation decreases CM-EV secretion while hypoxia increases the production of CM-EVs in a nSmase2-dependent manner. Moreover, the inflammatory cytokine TNF-α increased CM-EV secretion through a process involving NLRP3 inflammasome activation and mTOR signalling. Here, we detailed for the first time the regulatory mechanisms of EV biogenesis in hiPSC-CMs upon MI-associated stressors.

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.