Cardiomyocyte Model Research Articles

The Role of Human-Induced Pluripotent Stem Cells in Studying Cardiac Channelopathies

Cardiac channelopathies are inherited diseases that increase the risk of sudden cardiac death. While different genes have been associated with inherited channelopathies, there are still subtypes, e.g., catecholaminergic polymorphic ventricular tachycardia and Brugada syndrome, where the genetic cause remains unknown. Various models, including animal models, heterologous expression systems, and the human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSCs-CMs) model, have been used to study the pathophysiological mechanisms of channelopathies. Recently, researchers have focused on using hiPSCs-CMs to understand the genotype–phenotype correlation and screen drugs. By combining innovative techniques such as Clustered Regularly Interspaced Short Palindromic Repeats/Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9)-mediated genome editing, and three-dimensional (3D) engineered heart tissues, we can gain new insights into the pathophysiological mechanisms of channelopathies. This approach holds promise for improving personalized drug treatment. This review highlights the role of hiPSCs-CMs in understanding the pathomechanism of Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia and how these models can be utilized for drug screening.

Cellular viability in an in vitro model of human ventricular cardiomyocytes (RL-14) exposed to gold nanoparticles biosynthesized using silk fibroin from silk fibrous waste

In nanotechnology, tissue engineering proposes obtaining nanomaterials of natural or synthetic origin, looking to incorporate components that exhibit a defined shape, diameter, colloidal stability, and biological identity to promote and regulate the events that occur in a cardiac cell microenvironment. This research aimed to evaluate cellular viability in an in vitro model of human fetal ventricular cardiomyocytes on interaction with gold nanoparticles biosynthesized using silk fibroin from silk fibrous waste. The Physicochemical properties were characterized by UV-visible spectroscopy, Fourier-transform infrared spectroscopy, electrokinetic potential, and scanning transmission electron microscopy. Moreover, the MTT assay was used to determine the cell viability of cardiomyocytes exposed to gold nanoparticles. The results showed that the variation of the pH of the reaction allows the synthesis of different geometries of nanoparticles with diameters between 6 and 334 nm. Furthermore, it was found that the nanoparticles with a tendency to sphericity favor the cell viability of cardiomyocytes.

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.

MiR-421-mediated suppression of FGF13 as a novel mechanism ameliorates cardiac hypertrophy by inhibiting endoplasmic reticulum stress

Pathological cardiac hypertrophy is an independent risk factor for heart failure. Currently, clinical treatments offer limited effectiveness, and both mortality and morbidity from cardiac hypertrophy and heart failure continue to be significant. Therefore, it is extremely urgent to find new intervention targets to prevent and alleviate pathological cardiac hypertrophy. In this study, we explored FGF13 expression and its upstream regulators in hypertrophic hearts. Firstly, we observed an increase in FGF13 expression levels in human hypertrophic myocardium tissues, as well as in mouse models of TAC-induced hypertrophy and in neonatal rat cardiomyocyte (NRCM) models induced by isoproterenol (ISO). Moreover, these elevated levels of FGF13 were shown to positively correlate with hypertrophic markers, including ANP and BNP. By using both gain-of-function and loss-of-function approaches in an in vitro hypertrophy model, we demonstrated that FGF13 knockdown could inhibit endoplasmic reticulum stress (ERS), thereby ameliorating cardiomyocyte hypertrophy. Meanwhile, we investigated the upstream regulators of FGF13 in hypertrophic hearts, and a dual-luciferase reporter assay confirmed that FGF13 is a direct target of miR-421. Overexpression of miR-421 decreased the protein level of FGF13 and ameliorated ISO-induced cardiomyocyte hypertrophy via modulating ER stress. In contrast, overexpression of FGF13 attenuated the ameliorative effect of miR-421 on ISO-induced cardiomyocyte hypertrophy. Taken together, the present results suggested that miR-421 ameliorated ISO-induced cardiomyocyte hypertrophy by negatively regulating FGF13 expression. This finding may offer a novel approach for the treatment of cardiac hypertrophy.

Association of genetically predicted human metabolomic gene expression with incident heart failure

Abstract Background Heart failure (HF) represents a significant challenge both for patients and healthcare systems worldwide. Despite ongoing efforts, therapeutic options are limited. The underlying pathophysiology involves intricate changes in cardiac metabolism that strongly influence the clinical phenotype, thus potentially offering novel options for therapeutic targeting. Systematic prioritisation of metabolic genes regarding their phenotypic impact on HF is outstanding. Purpose We investigate the association of genetically predicted metabolic gene expression with the onset of HF within UK Biobank (UKB). We followed up on identified candidate genes using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) models of cardiomyocyte hypertrophy. Methods Utilising genomic data of 484,372 UKB participants and left ventricle-specific expression quantitative trait loci (eQTL) information from Genotype-Tissue Expression (GTEx) project, we predicted genetically regulated gene expression using established methods (PrediXcan) for all metabolic genes. Subsequently, we employed per-gene Cox proportional hazards (COX-PH) models to assess the association of genetically predicted metabolic gene expression with incident HF. We adjusted models for clinical risk using established risk models (Pooled Cohort Equations to Prevent HF (PCP-HF)). To validate our findings in vitro, we subjected hiPSC-CMs to a sympathomimetic stimulus via treatment with endothelin-1 (ET-1; 10 nM). We evaluate alterations in expression via real-time quantitative polymerase chain reaction (RT-qPCR) and establish methods for assessing hypertrophic remodelling. Results COX-PH models revealed the predicted expression of 104/1515 metabolic genes to be independently associated with the onset of HF. Subsequent refinement with a meta-analysis of commonly dysregulated genes in end-stage HF narrowed our findings to 46 candidate genes, of which several were known for their role in HF pathophysiology. We further traced nine genes for validation in hiPSC-CM models and showed significant dysregulation of several genes following the treatment with ET-1. Flow cytometry measured hiPSC-CM hypertrophy following sympathomimetic stimulation (p=0.036). Extracellular flux analysis revealed canonical changes following treatment with ET-1. Conclusions The study revealed several metabolic genes associated with incident HF, thus confirming previous findings and highlighting novel, putative therapeutic target genes. We confirmed transcriptional changes in an in vitro model. Ongoing work includes further validation via silencing candidate genes in hiPSC-CMs and subjection to the established phenotypic in vitro models.

Modulating TTN gene expression in human iPSC-CMs to investigate mechanisms and therapies in TTN-associated dilated cardiomyopathy

Abstract Background Familial dilated cardiomyopathy (DCM) is a common and harmful condition, and no available therapies target the underlying genetic mechanisms. Truncating variants in TTN are the most commonly identified genetic cause of DCM. However, it remains unclear how TTN variants drive disease. Contemporary thinking identifies haploinsufficiency, dominant negative effects, or a combination of both as likely driving mechanisms. There is competing evidence as to whether I-band and A-band mutations differ in severity or in the relative roles of haploinsufficiency and dominant-negative effects. Clarifying disease mechanisms will help pave the way to targeted genetic therapies. Purpose To modulate TTN gene expression in human-induced stem cell-derived cardiomyocyte (hiPSC-CM) models of TTN-associated DCM carrying I-band or A-band variants to provide insight into the comparative roles of haploinsufficiency and dominant negative mechanisms in DCM, and to identify novel therapeutic strategies. Methods We used CRISPR-Cas9 gene-editing to generate hiPSC lines with known disease-causing heterozygous truncating mutations in the I-band or A-band of TTN. These were screened for off-target mutations and pluripotency. hiPSCs were differentiated into hiPSC-CMs for phenotyping. We assayed TTN mRNA and titin protein levels. We trialled approaches for modulating TTN expression including CRISPR activation. Results Two hiPSC lines with heterozygous mutations in the I-band and A-band of TTN were generated using CRISPR/Cas-9 gene-editing. These hiPSC lines were successfully differentiated into hiPSC-CMs. Both cell lines demonstrated unchanged TTN mRNA expression compared to wildtype controls, but had reduced expression of full-length titin protein and identifiable truncated protein products. TTN mRNA expression was subsequently increased in these models using modulators of gene expression. Conclusions hiPSC-CM models of DCM with truncating mutations in the I-band or A-band of TTN were generated and phenotyped. These models were then used to trial gene expression modulation methods. This provides a human cellular system that can be used to better understand disease mechanisms of TTN-associated DCM, with the ultimate aim of establishing gene therapies for TTN-associated DCM.

Effect and mechanism of Adipokine Omentin-1 on cardiac hypertrophy in heart failure with preserved ejection fraction

Abstract Background Omentin-1 secreted by epicardial adipose tissue can inhibit ventricular remodeling and improve early cardiac function after myocardial infarction. Our previous studies have identified the omentin-1 receptor as integrin receptors αvβ3 and αvβ5. However, the effect of omentin-1 on heart failure with preserved ejection fraction (HFpEF) remains poorly understood. Purpose The present study aims to study the role and mechanism of omentin-1 in cardiac function and myocardial cell structure in HFpEF. Methods and Results The influences of omentin-1 were investigated in HFpEF mice (HFD (60% calories from lard) and L-NAME (0.5 g/L in drinking water)), which were treated in vivo. Echocardiography showed better diastolic function in HFpEF mice treated with omentin-1 (5 ug/kg/h) subcutaneously for 4 weeks, and tissue staining showed alleviated myocardial fibrosis and myocardial hypertrophy. The RNA sequencing analysis revealed variations in the gene expression related to ketone body metabolism in HFpEF mice after omentin-1 treatment (600 ng/ml). The supernatant was obtained from mouse bone marrow macrophages treated with ultrapure endotoxin and ATP and was utilized as the macrophage-conditioned medium for culturing primary cardiomyocytes, which were stimulated with isoproterenol for 48 hours to construct the HFpEF cardiomyocyte model. In the heart tissue of HFpEF mice and HFpEF cardiomyocytes treated with omentin-1, there was a notable increase in the expression of a pivotal enzyme of ketone body metabolism- succinyl-CoA transferase (SCOT), and a decrease in the expression of hypertrophy signal. The myocardial hypertrophy model was established by stimulating the myocardial cell line H9C2 with isoproterenol. Following the inhibition of the integrin receptor and PI3K-AKT pathway, the impacts of omentin-1 on SCOT and myocardial hypertrophy signal expression in hypertrophic cardiomyocytes were eliminated. Conclusion This study provides evidence for the effects of omentin-1 on cardiac function and myocardial structure in HFpEF and proposes that omentin-1 may stimulate the downstream PI3K/AKT pathway via integrin receptors on cardiomyocytes, leading to increased expression of SCOT and improvement of cardiomyocyte hypertrophy.

Using a Failing Human Ventricular Cardiomyocyte Model to Re-Evaluate Ca2+ Cycling, Voltage Dependence, and Spark Characteristics

Previous studies have observed alterations in excitation–contraction (EC) coupling during end-stage heart failure that include action potential and calcium (Ca2+) transient prolongation and a reduction of the Ca2+ transient amplitude. Underlying these phenomena are the downregulation of potassium (K+) currents, downregulation of the sarcoplasmic reticulum Ca2+ ATPase (SERCA), increase Ca2+ sensitivity of the ryanodine receptor, and the upregulation of the sodium–calcium (Na=-Ca2+) exchanger. However, in human heart failure (HF), debate continues about the relative contributions of the changes in calcium handling vs. the changes in the membrane currents. To understand the consequences of the above changes, they are incorporated into a computational human ventricular myocyte HF model that can explore the contributions of the spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). The reduction of transient outward K+ current (Ito) is the main membrane current contributor to the decrease in RyR2 open probability and L-type calcium channel (LCC) density which emphasizes its importance to phase 1 of the action potential (AP) shape and duration (APD). During current-clamp conditions, RyR2 hyperphosphorylation exhibits the least amount of Ca2+ release from the SR into the cytosol and SR Ca2+ fractional release during a dynamic slow–rapid–slow (0.5–2.5–0.5 Hz) pacing, but it displays the most abundant and more lasting Ca2+ sparks two-fold longer than a normal cell. On the other hand, under voltage-clamp conditions, HF by decreased SERCA and upregulated INCX show the least SR Ca2+ uptake and EC coupling gain, as compared to HF by hyperphosphorylated RyR2s. Overall, this study demonstrates that the (a) combined effect of SERCA and NCX, and the (b) RyR2 dysfunction, along with the downregulation of the cardiomyocyte’s potassium currents, could substantially contribute to Ca2+ mishandling at the spark level that leads to heart failure.

Deciphering heart failure: an integrated proteomic and transcriptomic approach with experimental validation.

This study analyzed transcriptomic and proteomic data to identify molecular changes during heart failure (HF). Additionally,we embarked on an exploration of the prospect of therapeutic intervention through the manipulation of proteins implicated in ferroptosis. Three publicly available microarray datasets (GSE135055, GSE147236, GSE161472) profiling left ventricular samples from HF patients and healthy controls were obtained. Differentially expressed genes were identified in each dataset and cross-analyzed to determine shared gene signatures. Enrichment analysis of Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and gene set enrichment analysis were performed. Differentially expressed proteins were obtained from published proteomic studies and integrated with the transcriptomic results. To validate findings, a HF mouse model was generated and ferroptosis-related proteins were evaluated. Additionally, the effect of suppression of ferroptosis on hypoxia-induced ischemia model in HL-1 cardiomyocytes was assessed by knocking down Acyl-CoA synthetase long-chain family member 4 (ACSL4) using small interfering RNA (siRNA).Cross-analysis of differentially expressed genes (DEGs) in the GSE135055, GSE147236 and GSE161472 datasets revealed 224 up-regulated and 187 down-regulated potential genes which showed high enrichment in immune, inflammatory and metabolic pathways. Notably, four proteins, among them ACSL4, displayed consistent alterations at both the transcriptional and protein levels. In the HF mouse model, ACSL4 exhibited an elevation, whereas negative regulators of ferroptosis witnessed a decrement. Subsequently, knockdown of ACSL4 in a hypoxia-induced ischemic HL-1 cardiomyocyte cell model upregulated the expression of ferroptosis inhibitory protein and decreased the levels of reactive oxygen species (ROS), malondialdehyde (MDA)., and free iron and increased cell viability. Comprehensive multi-omics analysis revealed that the expression of the molecular target ACSL4 was increased in HF. Targeting ACSL4 to inhibit ferroptosis may represent a novel therapeutic strategy for HF treatment.

The Role of BAG3 Protein Interactions in Cardiomyopathies.

Bcl-2-associated athanogene 3 (BAG3) plays an important function in cellular protein quality control (PQC) maintaining proteome stability. Mutations in the BAG3 gene result in cardiomyopathies. Due to its roles in cardiomyopathies and the complexity of BAG3-protein interactions, it is important to understand these protein interactions given the importance of the multifunctional cochaperone BAG3 in cardiomyocytes, using an in vitro cardiomyocyte model. The experimental assay was conducted using high pressure liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the human AC16 cardiomyocyte cell line with BioID technology. Proteins with BAG3-interaction were identified in all the 28 hallmark gene sets enriched in idiopathic cardiomyopathies and/or ischemic disease. Among the 24 hallmark gene sets enriched in both idiopathic cardiomyopathies and ischemic disease, 15 gene sets had at least 3 proteins with BAG3-interaction. This study highlights BAG3 protein interactions, unveiling the key gene sets affected in cardiomyopathies, which help to explain the molecular mechanisms of the cardioprotective effects of BAG3. In addition, this study also highlighted the complexity of proteins with BAG3 interactions, implying unwanted effects of BAG3.

Ling gui shen fu decoction ameliorates cardiac injury in ischemic heart disease rats by regulating ANP32A/HIF2α/HMGB1 signal route

Purpose: To investigate the effect of ling gui shen fu decoction (LGSFD) on cardiac injury and cardiomyocyte apoptosis in rats with ischemic heart disease (IHD), and to elucidate the underlying molecular mechanism. Methods: An IHD rat model was established by performing coronary artery occlusion surgery on the left anterior descending (LAD) of rats. The rats were assigned equally to 5 groups: sham-operated group (sham group), IHD group, IHD + perindopril group, IHD + low- dose LGSFD group (IHD + LGSFD-L group), and IHD + high-dose LGSFD group (IHD+LGSFD-H group). The LGSFD was given via gavage. A hypoxia-induced cardiomyocyte model was established by subjecting H9C2 cells to hypoxia. Then, LGSFD-containing serum or blank serum was used to treat the hypoxic rat cardiomyocytes (H9C2). The severity of cardiac injury and extent of apoptotic changes in cardiomyocytes was determined using hematoxylin-eosin (H&E) staining and TUNEL staining, while immunoblotting was used to measure the protein expressions of ANP32A, p-AKT, AKT, HIF-2α, p-mTOR, mTOR and HMGB1. Results: The cardiac injury of rats in the LGSFD groups was significantly reversed, relative to the IHD group (p < 0.05). Moreover, LGSFD reduced the level of apoptosis in hypoxia- induced H9C2 cells and upregulated the concentrations of ANP32A, p-AKT, HIF-2α and p-mTOR. However, the expression levels of HMGB1 were decreased in the heart tissues of IHD rats and hypoxic H9C2 cells. Silencing of ANP32A with ANP32A siRNA (siANP32A) down- regulated ANP32A, p-AKT, HIF-2α and p-mTOR, but up-regulated apoptosis and expression levels of HMGB1 in hypoxic H9C2 cells. Conclusion: LGSFD ameliorates cardiac injury in IHD rats by inhibiting cardiomyocyte apoptosis via the ANP32A/HIF-2α/HMGB1 axis. Further investigations are recommended into the specific mechanism of LGSFD effect using clinical samples, in order to facilitate its clinical development.

Defining Cardiomyocyte Repolarization Response to Pharmacotherapy in Long-QT Syndrome Type 3.

Long-QT syndrome is a primary cardiac ion channelopathy predisposing a patient to ventricular arrhythmia through delayed repolarization on the resting ECG. We aimed to establish a patient-specific, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes model of long-QT syndrome type 3 (LQT3) using clustered regularly interspaced palindromic repeats (CRISPR/Cas9), for disease modeling and drug challenge. HiPSCswere generated from a patient with LQT3 harboring an SCN5A pathogenic variant (c.1231G>A; p.Val411Met), and an unrelated healthy control. The same SCN5A pathogenic variant was engineered into the background healthy control hiPSCs via CRISPR/Cas9gene editing to generate a second disease model of LQT3 for comparison with an isogenic control. All 3 hiPSC lines were differentiated into cardiomyocytes. Both the patient-derived LQT3 (SCN5A+/-) and genetically engineered LQT3 (SCN5A+/-) hiPSC-derived cardiomyocytesshowed significantly prolonged cardiomyocyte repolarization compared with the healthy control. Mexiletine, a cardiac voltage-gated sodium channel (NaV1.5) blocker, shortened repolarization in both patient-derived LQT3 and geneticallyengineered LQT3 hiPSC-derived cardiomyocytes, but had no effect in the control. Notably, calcium channel blockers nifedipine and verapamil showed a dose-dependent shortening of repolarization, rescuing the phenotype. Additionally, therapeutic drugs known to prolong the corrected QT in humans (ondansetron, clarithromycin, and sotalol) demonstrated this effect invitro, but the LQT3 clones were not more disproportionately affected compared with the control. We demonstrated that patient-derived and genetically engineered LQT3 hiPSC-derived cardiomyocytesfaithfully recapitulate pathologic characteristics of LQT3. The clinical significance of such an invitro model is in the exploration of novel therapeutic strategies, stratifying drug adverse reaction risk and potentially facilitating a more targeted, patient-specific approach in high-risk patients with LQT3.

LncRNA ZFAS1/miR-186-5p axis is involved in oxidative stress inhibition of myocardial ischemia-reperfusion injury by targeting BTG2

ABSTRACT Objective To probe the involvement of long noncoding RNA zinc finger antisense 1 (ZFAS1)/microRNA (miR)-186-5p axis in inhibiting oxidative stress in myocardial ischemia-reperfusion injury (MIRI) by targeting B-cell translocation gene 2 (BTG2). Methods The MIRI mice model was established by ligating the left anterior descending branch of the left coronary artery in C57BL/6 mice. The in vitro MIRI model was constructed by hypoxia and reoxygenation of HL-1 cardiomyocytes. Cardiomyocyte apoptosis and the extent of myocardial injury in mice were detected. The apoptosis rates, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in HL-1 cells were assessed. The relationship among ZFAS1, miR-186-5p, and BTG2 was verified. Results High ZFAS1 and BTG2 levels and low miR-186-5p levels were demonstrated in I/R-injured myocardial tissues and in H/R-treated cardiomyocytes. Interference with ZFAS1 or elevation of miR-186-5p inhibited apoptosis and oxidative stress in H/R model cardiomyocytes and I/R-injured myocardial tissues. Overexpressing BTG2 impaired the ameliorative effects of miR-186-5p on MIRI. ZFAS1 negatively regulated miR-186-5p expression by acting as a molecular sponge. miR-186-5p targeted to regulate BTG2 negatively. Conclusion Interfering with ZFAS1 can upregulate miR-186-5p and thus inhibit BTG2 expression, thereby ameliorating MIRI.

Huangqi-Danshen decoction improves heart failure by regulating pericardial adipose tissue derived extracellular vesicular miR-27a-3p to activate AMPKα2 mediated mitophagy

BackgroundHuangqi-Danshen decoction (HDD) is a classic traditional Chinese medicine for treating heart failure. Pericardial adipose tissue (PAT) has recently gained increasing attention in cardiovascular diseases. PurposeThis study aimed to investigate the effect of pericardial adipose tissue-derived extracellular vesicles on heart failure, the protective effect of HDD on myocardial remodel in heart failure rats, and identify the potential molecular mechanisms involved. MethodsUPLC-MS/MS identified active components of HDD. Extracellular vesicles (EVs) from pericardial adipose tissue of sham-operated and HF rats were identified through transmission electron microscopy, nanoparticle tracking analysis and western blot. EVs were co-cultured with H9c2 cardiomyocytes in order to examine their uptake and effects. MicroRNA sequencing, dual-luciferase reporter assay and PCR were conducted for exploring specific mechanisms of EVs on hypertrophic cardiomyocytes. In vivo, heart failure was modeled in rats via transverse aortic constriction (TAC). In vitro, the hypertrophic cardiomyocyte model were established using Ang II-induced H9c2 cardiomyocytes. ResultsUPLC-MS/MS identified 11 active components in serum of HDD administrated rats. Echocardiography showed HDD improved cardiac function in TAC model rats. HE and Masson staining indicated HDD ameliorated myocardial hypertrophy and fibrosis. MicroRNA sequencing found that HDD treatment resulted in 37 differentially expressed miRNAs (DMEs) (P < 0.05 and |log2FC| ≥ 1). KEGG analysis revealed that DEMs were enriched in the AMPK signaling pathway. PCR identified miR-27a-3p with the greatest difference in AMPK-related DMEs. Dual-luciferase reporter assay and Targetscan website were utilized to identify the target relationship between miR-27a-3p and PRKAA2 (AMPKα2). The miR-27a-3p negatively regulated AMPKα2 to inhibit mitophagy mediated by PINK1/Parkin pathway. HDD inhibited miR-27a-3p secretion from failing heart pericardial adipose tissue-derived extracellular vesicles, thereby improving inflammation, cardiac function, and myocardial remodeling through above pathways. ConclusionHDD inhibited the PAT-derived extracellular vesicular miR-27a-3p in failing hearts to activate AMPK/PINK1/Parkin signaling-mediated mitophagy, which improved cardiomyocyte energy metabolism, myocardial remodeling and heart failure.

Transient Notch Activation Converts Pluripotent Stem Cell-Derived Cardiomyocytes Towards a Purkinje Fiber Fate.

Cardiac Purkinje fibers form the most distal part of the ventricular conduction system. They coordinate contraction and play a key role in ventricular arrhythmias. While many cardiac cell types can be generated from human pluripotent stem cells, methods to generate Purkinje fiber cells remain limited, hampering our understanding of Purkinje fiber biology and conduction system defects. To identify signaling pathways involved in Purkinje fiber formation, we analyzed single cell data from murine embryonic hearts and compared Purkinje fiber cells to trabecular cardiomyocytes. This identified several genes, processes, and signaling pathways putatively involved in cardiac conduction, including Notch signaling. We next tested whether Notch activation could convert human pluripotent stem cell-derived cardiomyocytes to Purkinje fiber cells. Following Notch activation, cardiomyocytes adopted an elongated morphology and displayed altered electrophysiological properties including increases in conduction velocity, spike slope, and action potential duration, all characteristic features of Purkinje fiber cells. RNA-sequencing demonstrated that Notch-activated cardiomyocytes undergo a sequential transcriptome shift, which included upregulation of key Purkinje fiber marker genes involved in fast conduction such as SCN5A, HCN4 and ID2, and downregulation of genes involved in contractile maturation. Correspondingly, we demonstrate that Notch-induced cardiomyocytes have decreased contractile force in bioengineered tissues compared to control cardiomyocytes. We next modified existing in silico models of human pluripotent stem cell-derived cardiomyocytes using our transcriptomic data and modeled the effect of several anti-arrhythmogenic drugs on action potential and calcium transient waveforms. Our models predicted that Purkinje fiber cells respond more strongly to dofetilide and amiodarone, while cardiomyocytes are more sensitive to treatment with nifedipine. We validated these findings in vitro , demonstrating that our new cell-specific in vitro model can be utilized to better understand human Purkinje fiber physiology and its relevance to disease.

Creating cell-specific computational models of stem cell-derived cardiomyocytes using optical experiments.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have gained traction as a powerful model in cardiac disease and therapeutics research, since iPSCs are self-renewing and can be derived from healthy and diseased patients without invasive surgery. However, current iPSC-CM differentiation methods produce cardiomyocytes with immature, fetal-like electrophysiological phenotypes, and the variety of maturation protocols in the literature results in phenotypic differences between labs. Heterogeneity of iPSC donor genetic backgrounds contributes to additional phenotypic variability. Several mathematical models of iPSC-CM electrophysiology have been developed to help to predict cell responses, but these models individually do not capture the phenotypic variability observed in iPSC-CMs. Here, we tackle these limitations by developing a computational pipeline to calibrate cell preparation-specific iPSC-CM electrophysiological parameters. We used the genetic algorithm (GA), a heuristic parameter calibration method, to tune ion channel parameters in a mathematical model of iPSC-CM physiology. To systematically optimize an experimental protocol that generates sufficient data for parameter calibration, we created in silico datasets by simulating various protocols applied to a population of models with known conductance variations, and then fitted parameters to those datasets. We found that calibrating to voltage and calcium transient data under 3 varied experimental conditions, including electrical pacing combined with ion channel blockade and changing buffer ion concentrations, improved model parameter estimates and model predictions of unseen channel block responses. This observation also held when the fitted data were normalized, suggesting that normalized fluorescence recordings, which are more accessible and higher throughput than patch clamp recordings, could sufficiently inform conductance parameters. Therefore, this computational pipeline can be applied to different iPSC-CM preparations to determine cell line-specific ion channel properties and understand the mechanisms behind variability in perturbation responses.

Ginsenoside Rg1 treats chronic heart failure by downregulating ERK1/2 protein phosphorylation.

In this study, we investigated the potential therapeutic mechanism of ginsenoside Rg1 (GRg1) in chronic heart failure (CHF), focusing on its regulation of ERK1/2 protein phosphorylation. H9c2 cardiomyocytes and SD rats were divided into the control group, CHF (ADR) group, and CHF+ginsenoside Rg1 group using an isolated cardiomyocyte model and an in vivo CHF rat model induced by adriamycin (ADR). Cell viability, proliferation, apoptosis, and the expression of relevant proteins were measured to assess the effects of GRg1. The results showed that treatment with GRg1 increased cell activity and proliferation, while significantly reducing levels of inflammatory and apoptotic factors compared to the CHF (ADR) group. Moreover, the CHF+ginsenoside Rg1 group exhibited higher levels of Bcl-2 mRNA and protein expression, as well as lower levels of Caspase3 and Bax mRNA and protein expression, compared to the CHF (ADR) group. Notably, the CHF+ginsenoside Rg1 group displayed decreased serum NT-proBNP levels and heart weight/body weight (HW/BW) index. Furthermore, the electrocardiogram of rats in the CHF+ginsenoside Rg1 group resembled that of rats in the control group. Overall, our findings suggested that GRg1 alleviated CHF by inhibiting ERK1/2 protein phosphorylation, thereby inhibiting apoptosis, enhancing cell activity and proliferation, and reducing cardiac inflammatory responses.

Enhanced Ca2+-Driven Arrhythmogenic Events in Female Patients With AtrialFibrillation: Insights From Computational Modeling.

Substantial sex-based differences have been reported in atrial fibrillation (AF), but the underlying mechanisms are poorly understood. This study sought to gain a mechanistic understanding of Ca2+-handling disturbances and Ca2+-driven arrhythmogenic events in male vs female atrial cardiomyocytes and establish their responses to Ca2+-targeted interventions. We integrated reported sex differences and AF-associated changes (ie, expression and phosphorylation of Ca2+-handling proteins, cardiomyocyte ultrastructural characteristics, and dimensions) into our human atrial cardiomyocyte model that couples electrophysiology with spatially detailed Ca2+-handling processes. Sex-specific responses of atrial cardiomyocytes to arrhythmia-provoking protocols and Ca2+-targeted interventions were evaluated. Simulated quiescent cardiomyocytes showed increased incidence of Ca2+ sparks in female vs male myocytes inAF, in agreement with previous experimental reports. Additionally, our female model exhibited elevated propensity to develop pacing-induced spontaneous Ca2+ releases (SCRs) and augmented beat-to-beat variability in action potential (AP)-elicited Ca2+ transients compared with the male model. Sensitivity analysis uncovered distinct arrhythmogenic contributions of each component involved in sex and/or AF alterations. Specifically, increased ryanodine receptor phosphorylation emerged as the major SCR contributor in female AF cardiomyocytes, whereas reduced L-type Ca2+ current was protective against SCRs for male AF cardiomyocytes. Furthermore, simulated Ca2+-targeted interventions identified potential strategies (eg, t-tubule restoration, and inhibition of ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca2⁺-ATPase) to attenuate Ca2+-driven arrhythmogenic events in women, and revealed enhanced efficacy when applied in combination. Sex-specific modeling uncovers increased Ca2+-driven arrhythmogenic events in female vs male atria inAF, and suggests combined Ca2+-targeted interventions are promising therapeutic approaches in women.

Characterisation of infantile cardiomyopathy in Alström syndrome using ALMS1 knockout induced pluripotent stem cell derived cardiomyocyte model

Alström syndrome (AS) is an inherited rare ciliopathy characterised by multi-organ dysfunction and premature cardiovascular disease. This may manifest as an infantile-onset dilated cardiomyopathy with significant associated mortality. An adult-onset restrictive cardiomyopathy may also feature later in life. Loss of function pathogenic variants in ALMS1 have been identified in AS patients, leading to a lack of ALMS1 protein. The biological role of ALMS1 is unknown, particularly in a cardiovascular context. To understand the role of ALMS1 in infantile cardiomyopathy, the reduction of ALMS1 protein seen in AS patients was modelled using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), in which ALMS1 was knocked out. MuscleMotion analysis and calcium optical mapping experiments suggest that ALMS1 knockout (KO) cells have increased contractility, with altered calcium extrusion and impaired calcium handling dynamics compared to wildtype (WT) counterparts. Seahorse metabolic assays showed ALMS1 knockout iPSC-CMs had increased glycolytic and mitochondrial respiration rates, with ALMS1 knockout cells portraying increased energetic demand and respiratory capacity than WT counterparts. Using senescence associated β-galactosidase (SA-β gal) staining assay, we identified increased senescence of ALMS1 knockout iPSC-CMs. Overall, this study provides insights into the molecular mechanisms in AS, particularly the role of ALMS1 in infantile cardiomyopathy in AS, using iPSC-CMs as a ‘disease in a dish’ model to provide insights into multiple aspects of this complex disease.

Antisense Oligonucleotide Therapy for Calmodulinopathy.

Calmodulinopathies are rare inherited arrhythmia syndromes caused by dominant heterozygous variants in CALM1, CALM2, or CALM3, which each encode the identical CaM (calmodulin) protein. We hypothesized that antisense oligonucleotide (ASO)-mediated depletion of an affected calmodulin gene would ameliorate disease manifestations, whereas the other 2 calmodulin genes would preserve CaM level and function. We tested this hypothesis using human induced pluripotent stem cell-derived cardiomyocyte and mouse models of CALM1 pathogenic variants. Human CALM1F142L/+ induced pluripotent stem cell-derived cardiomyocytes exhibited prolonged action potentials, modeling congenital long QT syndrome. CALM1 knockout or CALM1-depleting ASOs did not alter CaM protein level and normalized repolarization duration of CALM1F142L/+ induced pluripotent stem cell-derived cardiomyocytes. Similarly, an ASO targeting murine Calm1 depleted Calm1 transcript without affecting CaM protein level. This ASO alleviated drug-induced bidirectional ventricular tachycardia in CalmN98S/+ mice without a deleterious effect on cardiac electrical or contractile function. These results provide proof of concept that ASOs targeting individual calmodulin genes are potentially effective and safe therapies for calmodulinopathies.