Heart Tissue Research Articles

Unexpected macrophage-mediated myocardial cGas-STING activation in a novel mouse model of stress-related sleep disturbance

Abstract Background Delayed bedtime following stress disorder is prevalent in waves of pandemics and modern life. Considered to be a specific and important contributor to cardiovascular health, stress-related sleep disturbance has an unmet need in steady preclinical models. We previously found exciting corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVH) area of mice could induce 3-hour-long wakefulness. Purpose This study aimed to induce reproducible phenotypes of stress-related sleep disturbance in mice and explore the potential impact on cardiac function. Methods The chemogenetic method of designer receptors exclusively activated by designer drugs (DREADD) system was adopted to mimic stress-related sleep disturbance. We transfected PVH CRH neurons with rAAV-hSyn-DIO-hM3Dq-mCherry and rAAV-Crh-CRE. Prolonged CRH neuron activation was induced by daily intraperitoneal injection of clozapine N-oxide (CNO, 3mg/kg) at 9 am. Bulk RNA-sequencing and bioinformatics analysis were conducted for mechanistic exploration. Results 2-week repeated chemogenetic activation of PVH CRH neurons induced a 5-fold corticosterone release, consistent with increased daily 3-hour wakefulness and corresponding decreases in both rapid eye movement (REM) as well as non-REM sleep. Over 30% of chronic CRH activation mice displayed difficulties in maintaining balance and experienced premature mortality. Mice subjected to prolonged CRH activation showed impaired left ventricular ejection fraction (67.9% versus 48.2%, p=0.0011), and a dilated appearance demonstrated by histological staining. Intriguingly, the number of circulating monocytes increased. Then, we performed bulk RNA-sequencing of heart and bone marrow from CRH-activated and control mice. Differential gene expression and gene set enrichment analysis (GSEA) indicated marked activation of interferon-beta-related pathways in both tissues. Cytosolic DNA-sensing pathway and related key effector genes (cGAS, Cxcl10, CCL5) were found up-regulated in the heart, while mitochondrial oxidative phosphorylation was suppressed. We adopted the CIBERSORT tool to estimate immune infiltration in heart tissues and characterized M1 macrophage as the main pro-inflammatory cell. Further, we screened a set of secreted factors and mediators, which might play active roles in inflamed hearts. Conclusion Taken together, we report a failing heart in a mouse model of stress-related sleep disturbance. The neuro-immune axis involvement and molecular mechanisms merit in-depth explorations.

Downregulation of CPEB3 exacerbates cardiac injury by inhibiting cardiomyocyte adaptive metabolic reprogramming after myocardial infarction

Abstract Background Adaptive metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis in hypoxia plays a protective role in cardiomyocyte survival by reducing reactive oxygen species (ROS) and increasing ATP. Cytoplasmic polyadenylation element binding protein 3 (CPEB3) influences protein translation by modulating 3' untranslated region (3'UTR) lengths through alternative polyadenylation (APA). However, the role of CPEB3 in cardiomyocyte after myocardial infarction (MI) remains unknown. Purpose This study aims to explore the function of CPEB3 in cardiomyocyte after MI. Methods Three GEO databases of mice MI (GSE110209, GSE114695 and GSE236374) were analyzed to identify CPEB3 dysregulation. Cardiomyocyte-specific CPEB3 knockout mice and CPEB3-knockdown neonatal mouse cardiomyocytes (NMCMs), RNA sequencing, flow cytometry, TUNEL staining, Seahorse, ROS and ATP measurements were used to investigate the impact of CPEB3. APA events analysis, RIP sequencing, CUT-TAG, 3’RACE, polysome profiling and dual luciferase report were performed to elucidate the mechanisms of CPEB3. Recombinant adeno-associated virus carrying cardiac troponin T promoter was used to evaluate the therapeutic efficacy of CPEB3 in mice with MI. Results CPEB3 expression was markedly reduced in the heart tissue after MI and in NMCMs after hypoxia. Cardiomyocyte-specific CPEB3 deletion aggravated cardiomyocyte apoptosis, enlarged infarct size, and exacerbated cardiac injury after MI. RNA sequencing revealed that CPEB3 deficiency predominantly inhibited glycolysis-related pathway, with a notable decrease in pyruvate dehydrogenase kinase 1 (PDK1) expression. In CPEB3-knockdown NMCMs, adaptive metabolic reprogramming from OXPHOS to glycolysis and ATP level were decreased in hypoxia, whereas ROS production was significantly enhanced, all of which resulted in apoptosis and were regulated by PDK1. Besides, PDK1 overexpression counteracted the effects of CPEB3 knockdown on metabolic pattern, ROS, ATP and apoptosis. Mechanistically, CPEB3 deficiency led to a shortened 3’UTR of the transcription factor forkhead box O3 (FOXO3), and a decline of FOXO3 protein level. CPEB3 protein was confirmed to directly bind to FOXO3 mRNA, and the translation efficiency of FOXO3 was decreased with CPEB3 knockdown. Furthermore, FOXO3 was found to activate the transcription of PDK1, and FOXO3 overexpression alleviated the decline of PDK1 and attenuated CPEB3 knockdown-induced apoptosis in hypoxia. Finally, cardiomyocyte-specific CPEB3 overexpression reduced cardiomyocyte apoptosis, suppressed myocardial fibrosis, and improved cardiac function by upregulating FOXO3 and PDK1 levels after MI. Conclusion CPEB3 could promote FOXO3 protein expression via APA of FOXO3 3’UTR, activate transcription of PDK1 and ultimately protect cardiomyocyte from apoptosis by restoring metabolic balance in hypoxia. CPEB3 may serve as a promising therapeutic target for cardiomyocyte apoptosis after MI.Schematic diagram

Investigation of the usefulness of collagen peptide in intramyocardial injection of human iPS cell-derived cardiomyocytes

Abstract Background Injecting human induced pluripotent stem (iPS) cell-derived cardiomyocytes directly into the heart muscle holds promise as a treatment for heart failure. Achieving better engraftment is crucial for maximizing therapeutic effectiveness. Collagen peptide (CP) is a form of hydrolyzed gelatin known for its resistance to solidification. By adjusting its concentration, the viscosity of CP can be easily managed, potentially influencing the retention and engraftment efficiency of transplanted cells. Purpose We investigated the impact of different CP concentrations as a solvent for intramyocardial injection of human iPS cell-derived cardiomyocytes. Methods Initially, we examined the leakage of CP solution from the injection site using chicken meat and ex vivo rat heart. Subsequently, an in vivo experiment was conducted using beating rat myocardium injected with CP solution and Indian ink to assess CP diffusion in dynamic heart tissue. Finally, we performed intramyocardial injections of human iPS cell-derived cardiomyocytes onto a myocardial infarction model in immunosuppressed rats (1.0×107 cells/rat), followed by echocardiographic evaluation of cardiac function four weeks post-transplantation (N=3-6). Results There was less leakage from the injection site at lower CP concentration (10%) compared to higher concentration (20%), particularly in chicken or ex vivo rat heart tissue under static, non-beating conditions (chicken; 10% vs. 20% CP: 3.2±0.7% vs. 11.6±0.8%, P=0.01 / rat heart; 1.5±0.9% vs. 6.8±1.1%, P=0.0072). However, when examining fluid leakage from the cross-section of ex vivo rat hearts, we observed significantly higher leakage, regardless of CP concentration (10% CP: 45.7±2.7%, 20% CP: 59.1±8.1%), suggesting that CP diffusion within myocardial tissues had a greater influence on local retention than leakage from the injection site. Subsequently, in an experiment using beating rat myocardium and Indian ink, we observed significant suppression of diffusion in tissues treated with CP solution, with the 20% concentration showing a tendency to remain at the injection site (0% CP: 24.5±6.2 mm2, 10% CP: 6.9±7.1 mm2, 20% CP: 10.6±3.3 mm2, P=0.005). This indicates that 20% CP, rather than 10% CP, may be more suitable for local retention in tissues experiencing intra-tissue pressure, such as the beating myocardium. Finally, in vivo experiment using a myocardial infarction model of rat, there was no significant difference in left ventricular ejection fraction (LVEF) when cardiomyocytes were transplanted with 10% CP (0％ CP: 56.1±8.9%, 10%CP: 61.9±5.2%, P=0.92). However, there was a trend towards improved LVEF with the 20% CP solution (0% CP: 56.1±8.9%, 20%CP: 68.8±7.9%, P=0.13), Conclusion Twenty-percent CP may be more beneficial for treating myocardial ischemia due to enhanced retention and engraftment of transplanted cardiomyocytes.

Proteomic profiling of extracellular vesicles in takotsubo syndrome

Abstract Background/Purpose Takotsubo syndrome (TS) is an acute form of heart failure characterized by transient regional wall motion abnormalities triggered by a stressful event. The mechanisms underlying its development and recovery are poorly understood, resulting in a lack of disease-specific treatments and diagnostic markers. Extracellular vesicles (EVs) play a significant role in cellular communication and have been shown to be important in various diseases. Their involvement in cardiac conditions like TS is an emerging area of research. The primary objective of this study is to isolate and characterize EVs, as well as profile their proteomic profile, from the heart tissue of rats induced with TS. Methods Rats underwent TS induction through the infusion of 1 mg/kg isoprenaline over 15 minutes (TS24h), or were given saline (control). High-resolution echocardiography verified apical ballooning at 6 hours. After 24 hours, heart tissues from apical and basal segments were collected and processed. EV isolation involved tissue slicing, enzymatic digestion, and differential centrifugation steps, including a final iodixanol cushion separation for large and small EVs (Figure 1). Tandem mass tags and mass spectrometry were utilized for global proteomics and any differentially expressed proteins (DEPs) were identified. Analytical techniques such as volcano plots, heatmaps, enrichment analysis, and protein clustering/networks were employed. The strongest clusters (lowest False Discovery Rate and highest intensity) were selected, and their Gene Ontology (GO) terms/pathways were identified. Results Pure, cup-shaped, vesicles ranging from 50-800nm were successfully isolated (figure 1). EVs from apex of control and TS24h hearts had lower protein concentrations compared to their corresponding basal segments. Global proteomic analysis revealed a distinct protein profile in EVs from the apical segment of TS hearts at 24 hours. A total of 256 (TS24h-apex vs control-apex) and 561 proteins (TS24h-apex vs TS24h-base) were differentially expressed. No DEPs were observed when comparing the basal segments of TS24h and control hearts. Functional enrichment analysis of the DEPs showed an abundant change of biological processes related to metabolic lipid processes, inflammatory response, complement activation, reorganization of extracellular matrix. A downregulation was seen for biological processes related to mitochondrial ATP synthesis coupled electron transport and muscle contraction. Conclusion This study demonstrates a distinct EV protein profile in the apical segments of TS hearts, with marked changes in proteins related to metabolic lipid processes, inflammation, and mitochondrial activity. This extensive catalogue of novel proteins sets the stage for future research aimed at identifying potential therapeutic targets and diagnostic biomarkers, and enabling proof-of-concept studies in TS.

Mid-regional pro-adrenomedullin is an independent predictor of cardiovascular outcomes in cardiac surgery, a prospective cohort study in 500 patients

Abstract Background MR-proADM (mid-regional pro-adrenomedullin) is a stable pro-peptide of Adrenomedullin, which is expressed in various cell types such as heart, lung, and renal tissue. It is a prognostic parameter for heart failure (HF) and sepsis as well as all-cause and cardiovascular (CV) mortality. Purpose Considering the characteristics of MR-proADM as a reliable biomarker for adverse events, we aimed to investigate the prognostic impact of MR-proADM in patients undergoing elective cardiac surgery. Methods We prospectively enrolled patients undergoing elective cardiac bypass and/or valve surgery at the department of cardiac surgery between May 2013 and August 2018. Blood samples were taken one day prior to surgery. MR-proADM was measured using an automated immunofluorescence assay. Patients were followed prospectively until endpoints were reached. The primary endpoint was the composite of hospitalization for heart failure (HHF) or CV mortality, the secondary endpoint was postoperative atrial fibrillation (POAF). The multivariate regression model was adjusted for age, sex, NT-proBNP, type of surgery, and CRP-level at baseline. Results A total of 500 patients (146 female [29.2%]; median age 69.8 years [IQR 60.6-75.5]) were followed over a median of 4.6 years (IQR 3.0–5.8). Valve surgery was performed in 214 (42.8%) patients, 160 (32%) underwent a bypass operation and 126 (25.2%) a combined valve and bypass surgery. The median MR-proADM value of the entire study population was 0.58 nmol/L (IQR: 0.44-0.79 nmol/L). Patients were stratified in risk categories based on their MR-proADM values (Low Risk ≤0.63nmol/L, Intermediate Risk 0.63-0.83nmol/L, High Risk >0.84nmol/L). Patients in the highest category presented with higher rates of diabetes mellitus (p <0.001), COPD (p <0.001), HF (p=0.009) and either bypass surgery (p=0.039) or the combination of bypass and valve surgery (p=0.008), compared to patients in the lowest tertile. We observed a significant increase in 5-year’s event rates for HHF/ CV Mortality in patients in higher risk groups (Low Risk 8.6% vs High Risk 37.7%, p <0.001, Figure 1). The adjusted Cox regression model found that MR-pro ADM was independently associated with a risk increase for HHF/ CV Mortality (adjusted HR of 4.45, 95% CI 2.55-8.09; p<0.001, Figure 2) comparing the High Risk to the Low Risk Group. A comparable risk increase could be observed in POAF, with an adjusted HR of 2.61 (High Risk Group vs Low Risk Group, 95% CI 1.46-4.67, p<0.001). Conclusion Within the prospective investigation, MR-pro ADM was found to be an independent predictor for HHF and CV mortality in patients undergoing cardiac bypass and/or valve surgery. Furthermore, there was a consistent predictive potential for POAF. In the era of personalized medicine, preoperative MR-pro ADM levels could help to identify patients at risk for serious adverse events, in order to apply intensified secondary prevention and reduce mortality and morbidity.

Arterial-myocardial coupling in primary AL amyloidosis. A validation study of vascular involvement

Abstract Background Primary AL amyloidosis is a rare yet lethal systemic disorder. Previously published own and others’ work indicated that vascular involvement in AL cardiac amyloidosis defined as low blood pressure or paradoxical vasodilation may independently affect survival. Mechanistic data will pave the way to validate the clinical role of vascular involvement in these patients. Purpose We sought to explore the presence of arterial-myocardial coupling in AL amyloidosis by investigating associations between markers of vascular and cardiac involvement. Methods We consecutively recruited n=152 newly diagnosed patients with AL amyloidosis that underwent thorough vascular examination including reactive vasodilation (flow mediated vasodilation, FMD and vasodilation in response to cold-pressor test) using high resolution ultrasonography. Arterial stiffness was evaluated by carotid-femoral pulse wave velocity (PWV) and aortic pressures and markers of arterial wave reflections were estimated using radial artery tonometry. In a subgroup of patients, transthoracic echocardiography (n=152) and cardiac magnetic resonance (CMR) (n=59) were performed. Results Vascular reactivity correlated with global longitudinal strain (GLS), left ventricle ejection fraction (LVEF)/GLS ratio and worse LV volumes. Vasodilation post-cold pressure test was correlated with LV mass. cSBP exhibited a negative correlation with markers of heart involvement, wall thickness markers, GLS, and CMR tissue characterization markers. A positive correlation between cSBP and LVEF, rotation and twist strain, myocardial work and contractility markers and late gadolinium enhancement (LGE). cDBP was associated with systolic function and contractility markers, with myocardial work indices and LGE. A negative relationship with GLS, LA strain, RV GLS and LA diameter and E/e’ was noted. In addition, T2 was negatively correlated with cDBP. SBP was correlated positively with LVEF, contractility markers, and LGE myocardium and negatively correlated with biomarkers of heart involvement, GLS, CMR structure and tissue characterization. DBP was positively correlated with LGE and myocardial work. A negative relation with NTproBNP, E/e’, RVSP, LA diameter and T2 mapping was noted. Time of wave reflections (Tr) was negatively correlated with rotation strain and positively correlated with circumferential strain, LA strain and LV structure. Ai@75 was positively correlated with myocardial work and contractility markers, and LGE. Ai@75 was negatively correlated with markers of heart involvement, LV structure, E/e’, worse GLS and adverse CMR tissue characterization. Conclusion Markers of peripheral vascular dysfunction were associated with cardiac structure and function acquired both with echocardiography and CMR in AL amyloidosis. This arterial-myocardial coupling supports the clinical role of peripheral vascular involvement in this disease and warrants further investigation.

Deletion of nephrocystin 4 exacerbates cardiac function and survival rate in a mouse model of cardio-renal syndrome

Abstract Background Cardiorenal syndrome (CRS), characterized by cardiac and renal dysfunction, is an increasing health problem associated with high mortality rate. Nephrocystin 4 (NPHP4) gene is the major cause of end-stage renal disease in children. We have reported NPHP4 single nucleotide polymorphism is the genetic risk for CRS and subsequent cardiovascular events in general population. However, the functional role of Nphp4 in the development of CRS has never been examined yet. Methods and Results We generated systemic Nphp4 knockout (KO) mice deleting exon 3 to 8 using CRISPR-Cas9 system. RNA sequence analysis of heart and kidney tissues demonstrated that gene-sets related to mitochondrial function were significantly downregulated in Nphp4 KO mice compared to their wild-type (WT) littermates. Nphp4 KO mice showed less cardiac hypertrophy, however, exacerbated cardiac function and survival compared to WT mice in a CRS model induced by thoracic transverse aortic constriction. Nphp4 located in endosome and cell membrane in H9C2 cardiomyocytes. Immunoprecipitation confirmed protein interaction between Nphp4 and HECT-type E3 ligase Itch. Furthermore, we found that Itch targeted Lats2 and insulin-like growth factor-1 receptor in cardiomyocytes. Knockdown of Nphp4 activated Hippo signaling and worsened mitochondrial function in H9C2 cardiomyocytes through the inhibition of Itch-dependent Lats2 degradation. Knockdown of Nphp4 inhibited insulin-like growth factor-1 signaling through the disturbance of Itch-mediated endosome function. Conclusions Deletion of Nphp4 impaired compensatory cardiac hypertrophy and exacerbated cardiac function and survival in a mouse CRS model through mitochondrial dysfunction. Nphp4 mediated Hippo signaling and insulin-like growth factor-1 signaling through the interaction with Itch. NPHP4 could be the novel therapeutic target in CRS.figure1figure2

NRF3-mediated mitochondrial superoxide promotes cardiomyocyte apoptosis and impairs cardiac functions by suppressing Pitx2

Abstract Background Myocardial infarction (MI) elicits mitochondria reactive oxygen species (ROS) production and cardiomyocyte apoptosis. Nrf3 (Nuclear factor erythroid 2-related factor 3) has an established role in regulating redox signaling and tissue homeostasis. Herein, we aimed to uncover the exact role and potential mechanism of Nrf3 in injury-induced cardiac remodeling. Methods Global (Nrf3-KO) and cardiomyocyte-specific (NRF3△CM) Nrf3 knockout mice were subjected to MI or ischemia/reperfusion (I/R) injury, followed by functional and histopathological analysis. Primary neonatal mice (NMVMs) and rat (NRVMs) ventricular myocytes as well as cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) were used to detect the possible impact of Nrf3 on cardiomyocyte apoptosis and mitochondrial ROS production. Chromatin immunoprecipitation sequencing and immunoprecipitation–mass spectrometry analysis were used to uncover potential targets of Nrf3. MitoPQ administration and cardiomyocyte-specific AAV vector were used to further confirm the in vivo relevance of the identified signal pathways. Results Increased level of Nrf3 was observed in cardiomyocytes within border zone of infarcted human heart and murine cardiac tissues post-MI. Both global and cardiomyocyte-specific Nrf3 knockout significantly decreased injury-induced mitochondrial ROS production and cardiomyocyte apoptosis, consequently improving cardiac functions. Additionally, cardiac-specific Nrf3 overexpression reversed the cardiac phenotypes observed in Nrf3-KO mice. Functional studies showed that Nrf3 promoted NMVM, NRVM and hiPSC-CM apoptosis by increasing mitochondrial ROS production. Critically, restoring mitochondrial ROS using MitoParaquat blunted the beneficial effects of Nrf3 deletion on cardiac functions and remodeling. Mechanistically, the cardiac redox regulator paired-like homeodomain transcription factor 2 (Pitx2) was identified as one of the main target genes of Nrf3. Specifically, Nrf3 binds to Pitx2 gene promoter where it increases Pitx2 gene promoter DNA methylation through recruiting heterogeneous nuclear ribonucleoprotein K and DNA-methyltransferase 1 complex, thereby inhibiting Pitx2 expression. Importantly, cardiomyocyte-specific knockdown of Pitx2 blunted the beneficial effects of Nrf3 deletion on cardiac functions and remodeling. Conclusions Nrf3 promotes injury-induced cardiomyocyte apoptosis and deteriorates cardiac functions by increasing mitochondrial ROS production via suppressing Pitx2 expression. Targeting Nrf3-Pitx2-mitochondrial ROS signal axis could represent novel therapeutics for treating MI patients.Graphic Abstract

Vaccination targeting senescence-associated transmembrane protein alleviated cardiovascular pathology in the mice

Abstract Introduction Accumulation of senescent cells is observed in various organs including vasculature, heart and white adipose tissue with age, and known to become a substrate of pathophysiology of various cardiovascular-metabolic diseases. We have previously developed a vaccine to eliminate senescent cells, so-called "senolytic vaccine" by targeting Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) as a cellular-senescence-associated cell surface protein. We have demonstrated that GPNMB senolytic vaccine could remove senescent cells and alleviate the pathology cardiovascular-metabolic diseases. Senescence-associated adhesion molecule 1 (SAAM-1) is also known as a cell-surface membrane to increase its expression during cellular senescence in endothelial cells as well as the development of cardiovascular diseases. Here, we identified SAAM-1 as another senescence antigen and created a vaccine to eliminate senescent cells by targeting SAAM-1. Purpose This study was purposed to evaluate the efficacy of SAAM-1 vaccine for the treatment of cardiovascular diseases in mice models. Methods As a pressure overload-induced heart failure (HF) model, C57BL/6N wild type (WT) mice were underwent transverse aortic constriction (TAC) or sham opretaion at 13 weeks of age. SAAM-1 or control vaccination were given at 8 and 10 weeks of age before operation. Echocardiography examination was conducted two weeks after TAC operation, followed by tissue sampling 3 weeks after the examination. As an atherosclerosis model, ApoE KO mice were subjected to a high-fat diet (HFD) starting at 4 weeks of age. Vaccination of ApoE KO mice was administered at 8 weeks of age with SAAM-1-vaccine or control-vaccine, followed by sacrifice 4 weeks later for further analysis. ApoE-KO mice were also crossed with p16-mediated Luciferase expressing mice (p16-Luc mice) and subjected to generation of atherosclerosis model. Results SAAM-1 expression was significantly increased in left ventricle (LV) in TAC mice but its increase was suppressed by SAAM-1 vaccination. Interestingly, both worsening systolic function and dilatation of LV by pressure overload were significantly alleviated by SAAM-1 vaccination. Furthermore, expression level of senescent markers including p16 and p21, as well as several inflammation markers were increased in failing heart along with development of cardiac fibrosis, but SAAM-1 vaccine could significantly attenuate this pathological change. In ApoE KO mice, SAAM-1 vaccine decreased expression level of several inflammation markers in the mice, along with a decreasing p16 signal in the aorta. Conclusions Targeting SAAM-1 for vaccination would be a novel and promising approach to alleviating age-related cardiovascular disease.

Mature cardiac tissues modeling Duchenne muscular dystrophy related cardiomyopathy

Abstract Background The major cause of mortality in patients with Duchenne muscular dystrophy (DMD) is cardiomyopathy. This disease is caused by dystrophin gene mutations that result in the loss of dystrophin protein. Gene replacement therapies have been approved for skeletal muscle symptoms of DMD. However, these therapeutic modalities are not indicated for cardiomyopathy due to their low efficiency of delivery to the heart. Despite the significant unmet needs, a limitation in this area of research is the divergence of cardiomyopathy symptoms in model animals from those in patients. Alternatively, cardiomyocytes differentiated from patient-derived iPS cells have recently been analyzed. However, existing results are based on a single time point analysis and do not model disease progression or non-cardiomyocyte involvement. Purpose Our research aims to establish an in vitro model that can reproduce the pathological progression of DMD cardiomyopathy. Methods We have generated cardiac organoids and engineered heart tissues (EHTs) from patient-derived iPS cells, consisting of cardiomyocytes and epicardial-derived non-cardiomyocytes. An iPS cell line in which the dystrophin mutation was repaired by genome editing was used as a control. The cardiac organoids or EHTs were subjected to transient activation of fatty acid metabolism to promote the maturation of iPS-derived cardiomyocytes. Pathological conditions were then induced in these 3D-models by applying loads that mimicked the DMD-disease environment. Results The mature cardiac organoids displayed adult-like gene expression profiles in the components of dystrophin-associated-protein-complex and calcium handling. After continuous mechanical loading, cell death appeared in DMD-EHTs, although there was only a slight decrease in contractility and fibrosis. Elevated mitochondrial ROS production and DNA damage were observed as a part of the cell death mechanism. Furthermore, pathological stimuli in addition to mechanical loading reproduced progressive contractile dysfunction and further increase in fibrosis in DMD-EHTs. Conclusions From these observations, we concluded that our DMD-EHT model recapitulate the pathological development of DMD-cardiomyopathy. These results suggest that the key factors involved in the progression of DMD cardiomyopathy are (i) the fragility of cardiomyocytes to stress in early stages and (ii) the involvement of non-cardiomyocytes such as fibrosis. Our model provides insight into the molecular mechanisms underlying disease progression and novel therapeutic targets.

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.

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.

Sinomenine hydrochloride alleviates autoimmune myocarditis via suppressing Th1 cell induced-M1 macrophage pyroptosis

Abstract Background Myocarditis is typical of the complex inflammatory response in the heart with high morbidity and mortality, accompanied by tissue damage and cardiac fibrosis. Previous studies have demonstrated that CD4+ T cells are crucial to the cardiac autoimmunity of myocarditis, but the role of different CD4+ T cell subsets remains unclear. Recently, sinomenine hydrochloride, a natural compound from the traditional Chinese herb Sinomenium acutum, is reported to inhibit T cell proliferation in some systemic autoimmune diseases such as rheumatoid arthritis. Given that autoimmune myocarditis is an organ-specific autoimmune disease, the effect of sinomenine hydrochloride on autoimmune myocarditis demands further study. Purpose This study aims to elucidate the role of CD4+ T-helper 1 (Th1) cells in regulating inflammatory cell death in autoimmune myocarditis and investigate the pharmacological effect of sinomenine hydrochloride on CD4+ Th1 cells and autoimmune myocarditis. Methods Male Balb/c mice were immunized with myosin heavy chain-α peptides to establish the experimental autoimmune myocarditis (EAM) model, followed by the treatments with sinomenine hydrochloride by gavage. Tohoku Hospital Pediatrics-1 (THP-1) cell line and murine T cells were used for in vitro experiments. Results In the acute phase of EAM, we observed plenty of CD4+ T cells infiltrating the heart tissue. RNA sequencing revealed that among the genes encoding typical transcription factors of CD4+ T cell subset, only Tbx21 and Stat4, both representing Th1 cells, were upregulated, which consisted with immunofluorescence staining. Further studies indicated that Th1 cells not only mediated classical activated macrophage (M1 macrophage) polarization by secreting interferon-γ (IFN-γ) but also associated with pro-inflammatory macrophage pyroptosis in the EAM, with the cleavage of gasdermin D (GSDMD), caspase-1 and IL-1β. Subsequent in vitro experiments confirmed that IFN-γ could dramatically promote macrophage pyroptosis combined with ATP, leading to the release of IL-1β and IL-18(Figure 1). To find an effective method for Th1 cell suppression and myocarditis remission, we identified that sinomenine hydrochloride impeded Th1 cell activation, differentiation and proliferation in vitro. Furthermore, sinomenine hydrochloride inhibited Th1 cell infiltration and M1 macrophage pyroptosis in the EAM heart, In the chronic phase, cardiac remodeling and fibrosis were suppressed, and cardiac systolic function was significantly improved in the treatment group (Figure 2). Conclusion Our study demonstrates that Th1 cells-induced M1 macrophage pyroptosis is an essential pathogenic mechanism of EAM. Sinomenine hydrochloride can alleviate the severity and progression of EAM, which may become a novel therapeutic strategy for treating myocarditis.Figure 1Figure 2

Cardiac secreted-HSP90alpha in cardiomyocyte hypertrophy by N-Cadherin mediated signaling under pressure overload

Abstract Background Left ventricular hypertrophy (LVH) in response to pressure overload is strongly associated with adverse cardiovascular outcomes. Heat shock protein 90 (HSP90) has been reported to be involved in cardiac remodeling under stress. Aims Here, we evaluate whether serum HSP90α (an isoform of HSP90) increases and associates with cardiac hypertrophy in patients with hypertension or severe aortic stenosis (AS), and explore the potential mechanisms in experimental pressure overload mouse model. Methods Serum HSP90α levels in patients with hypertension or aortic stenosis (AS) were determined by ELISA. Pressure overload mouse model was built by a transverse aortic constriction (TAC). Cardiac function was assessed by echocardiography. Cardiac hypertrophy was examined by histology, western blot and RNA analysis. Results Serum levels of HSP90α were higher in patients with hypertension or AS than in the control group, respectively, and the levels positively correlated with LVH. HSP90α levels also increased in heart tissues of patients with obstructive hypertrophic cardiomyopathy (HCM), and mice after TAC, in parallel with the hypertrophic markers. TAC induced the enhanced expression and secretion of HSP90α from cardiomyocytes and cardiac fibroblasts. Knockdown of HSP90α or blockade of extracellular HSP90α (eHSP90α) prevent the development of LVH under pressure overload in vivo and in vitro. By Nano-HPLC-MS/MS analysis, we identified eHSP90α interacted with N-cadherin in cardiomyocyte surface, which induced the activation of β-catenin and promoted β-catenin to bind TCF7 (a member of TCF/Lef family) in nucleus, enhanced the transcription of hypertrophic genes, contributing to cardiac hypertrophy and dysfunction under pressure overload. Conclusions Our data demonstrated that cardiac secreted-HSP90α promoted myocardial hypertrophy by N-Cadherin mediated β-catenin/TCF7 signaling under pressure overload. It indicates the therapeutic potential of targeting HSP90α-initiated signaling against cardiac hypertrophy and dysfunction under pressure overload.

The cardioprotective potential of soluble urokinase Plasminogen Activator Receptor (suPAR) in myocardial ischemia

Abstract Background and aims: Increased circulating soluble urokinase plasminogen activator receptor (suPAR) levels are associated with adverse cardiovascular outcomes, however, any cardiovascular-related functions remain unknown. This study aimed to (i) document haemodynamic profiles in isolated rat hearts under the influence of suPAR, and to (ii) explore any mechanisms triggered by suPAR following myocardial ischaemia-reperfusion. Methods We undertook a 4-pronged approach (Figure 1). A Langendorff isolated rat heart model was used to perfuse all hearts with Krebs-Henseleit solution flowing retrogradely through the aorta at 37°C. A balloon inserted into the left ventricle allowed measurements of ventricular pressure, and a pressure transducer placed above the aorta recorded perfusion pressure for coronary flow rates. The experimental protocol consisted an initial 30-minute stabilisation period, followed by 40 minutes of ischemia via buffer flow cessation. Hearts were subsequently recovered during 90 minutes of reperfusion, receiving either suPAR treatment or perfusion buffer as controls. An inhibitor arm comprising co-infusion of suPAR and a monoclonal antibody capable of binding to suPAR was also assessed. Troponin T was measured in outflow perfusates collected at specific intervals. Proteins from hearts in treatment groups were separated using gel electrophoresis, with Western blotting to visualise the phosphorylation status of kinases (Akt, STAT). High-flow respirometry and fluorometry were investigated in cardiac mitochondria to determine respiration rates and ATP production in ischaemia reperfusion under the influence of suPAR. Statistical analysis was conducted with ANOVA using SPSS. Results Hearts receiving suPAR infusion at reperfusion (n = 16) showed a significant increase in developed pressure (p = .017) and lower perfusion pressure (p = .02) compared to controls (n = 16), revealing that suPAR-treated hearts recovered with better contractility and vasodilatory properties post-ischaemia. suPAR’s haemodynamic profiles in the antibody group were similar to controls, attenuating suPAR-induced effects (n = 8). Outflow effluents in suPAR-treated hearts obtained >3 fold lower serial Troponin T values than controls (p < .001). Heart tissues receiving suPAR treatment revealed a 1.7-fold increase in Akt (p = .001) and a 3.8-fold increase in STAT3 phosphorylation compared to controls (p < .001) and the inhibitor group (p = .04). Finally, suPAR increased mitochondrial ATP production by 28% (p = .013) compared to controls after ischaemia. Conclusion This is a world-first demonstration of suPAR’s cardioprotective influences in isolated rat hearts recovering from ischaemia. suPAR’s ability to promote cardiac contractility and vasodilation was associated with lesser injury as shown by Troponin T reduction. Mechanistically, suPAR infusion upregulated two key cardioprotective pathways, which may also function to protect cardiac mitochondria.Figure 1 - Schematic of Methods

Non-cell-autonomous effects of arginase-II in macrophages on cardiomyocytes in aging

Abstract Background Aging-associated cardiac structural and functional alterations enhance cardiac susceptibility to ischemic injury. The full elucidation of the underlying molecular mechanisms remains far from complete. The mitochondrial enzyme arginase-II (Arg-II) is implicated in cardiac injury and aging. However, contradictory results have been reported and no systemic analysis of its cellular localization has been performed. The involvement of Arg-II in cardiac aging, as well as the manner in which it participates, remains uncertain. Aim and methods: This project focuses on investigation of Arg-II cellular localization and its functional role in cardiac aging. Experiments are conducted in young (3-4 months) and old (20-22 months) wt and arg-ii-/- mice and in human heart tissues. Cardiac function is assessed by Langendorff apparatus ex vivo. Cellular localization of Arg-II in cardiac tissues is investigated by confocal microscopy. Intercellular interactions are studied using ventricular cardiomyocytes, cardiac fibroblasts, endothelial cells, and macrophages isolated from wt and arg-ii-/- mice or humans. Results Cardiac Arg-II expression is enhanced with aging. Surprisingly, Arg-II is not found in cardiomyocytes from aged mice and human patients, but upregulated in non-myocytes, including macrophages, fibroblasts, endothelial cells. Arg-ii-/- mice are protected from age-associated cardiac inflammation, cardiomyocyte apoptosis, fibrosis, endothelial-mesenchymal transition (EndMT), and susceptibility to ischemic injury. Further experiments show that Arg-II mediates IL-1b release from macrophages of old mice, contributing to the above-described cardiac aging phenotype. In addition, Arg-II enhances mitochondrial reactive oxygen species (mtROS) and activates cardiac fibroblasts that is inhibited by inhibition of mtROS. Conclusion Our study demonstrates a non-cell-autonomous effect of Arg-II on cardiomyocytes, fibroblasts, and endothelial cells mediated by IL-1b from aging macrophages, which may explain the enhanced vulnerability of aging heart to ischemic injury.Role of Arg-II in cardiac aging

Predicting major adverse cardiovascular events in suspected myocarditis patients using cmr late gadolinium enhancement radiomics: a time-to-event prognostication modeling

Abstract Background Myocarditis, characterized by inflammation of the heart tissue, exhibits a wide range of clinical manifestations; however, stratifying its risk is difficult. Objective We aimed to prognosticate Major Adverse Cardiovascular Events (MACE, i.e., death, sustained ventricular arrhythmia, heart failure hospitalization, and recurrent myocarditis) in patients with suspected myocarditis using cardiac magnetic resonate (CMR) late gadolinium enhancement (LGE) radiomic features. Method We enrolled consecutive 1099 patients (age: 47.68±16.56, 39.63% female) undergoing CMR with the initial suspicion of myocarditis based on the judgment of the referring physician, recruited from two major registries. We used fully automated deep learning tools to determine the segmental extent of LGE in the left ventricle. Furthermore, we pre-processed the dataset through bias-field correction and intensity normalization to ensure the model consistency across various image acquisitions. Different radiomic features, including shape, intensity, and texture, were extracted from CMR-LGE. We employed several feature selection methods and time-to-event survival models based on radiomics features. The development process, covering pre-processing, feature selection, and hyperparameter optimization, utilized 70% of the dataset (10-fold cross-validation). The evaluation was performed on the remaining 30% of the test set. Model performance was measured using the c-index and cumulative AUC (C-AUC) on the untouched 30% test set. Results The top-performing model, employing a 90% variance threshold with the survival support gradient boosting model, achieved a C-Index of 0.76 and a cumulative C-AUC of 0.80. The Random Forest Survival model was a close second, with a C-Index of 0.75 and a C-AUC of 0.79. The conventional multivariate Cox-PH model achieved a C-Index of 0.67 and a C-AUC of 0.70. Conclusion Utilizing radiomics features from the CMR-LGE, combined with machine learning algorithms, allows accurate prediction of MACE in suspected myocarditis patients. The performance of the Radiomics model could be improved by including other modality information.

Identification of cell adhesion molecules for the maturation of engineered heart tissue

Abstract Background Engineered cardiac tissue (ECT) utilizing human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is anticipated to function as a translational in vitro model. However, hiPSC-CMs display phenotypes indicative of immaturity, and the structural organization of ECT composed of hiPSC-CMs is insuficient when compared to that of mature adult hearts. Although the mechanisms for hiPSC-CMs maturation have been reported, little is known about the mechanisms regulating the formation of in vitro tissue such as ECT. Cell adhesion molecules play important roles in heart formation in vivo through cell-cell interactions. However, specific cell adhesion molecules responsible for tissue structure formation in in vitro tissue models, such as ECTs, have not been identified. We hypothesize that cell adhesion molecules, which regulate cell-cell attachment, are crucial for in vitro tissue formation. Purpose This study aims to identify the key cell adhesion molecules involved in cell-cell interaction and assess the effects of their expression on hiPSC-CMs and EHT functions. Methods and Results We developed a cell attachment assay to evaluate the attachment ability of hiPCS-CMs and discovered that hiPSC-CMs produced via embryoid body method (EB-hiPSC-CMs) exhibited a higher attachment ability than those produced by monolayer method (Mono-hiPSC-CMs). Further analysis revealed no significant difference in anoikis sensitivity between EB-hiPSC-CMs and Mono-hiPSC-CMs, suggesting that the observed difference in attachment ability were due to differencies in cell adhesion efficiency between hiPSC-CMs generated by two methods. Moreover, RNA-seq analysis identified nine cell adhesion molecule genes that were highly expressed in EB-hiPSC-CMs. We identified the cell adhesion molecules responsible for the attachment ability of hiPSC-CMs by knockdown and over-expression experiments. Moreover, over-expression experiments using adeno-associated virus (AAV) demonstrate that hiPSC-CMs with overexpression of a cell adhesion molecule improved calcium handling and increased propagation speed, and ECTs with overexpression of a cell adhesion molecule showed enhanced sarcomere alignment and sarcomere length compared to controls. Conclusion(s) We observed differences in attachment ability between hiPSC-CMs generated by two differentiation methods. Transcriptome analysis of these hiPSC-CMs identified key cell adhesion molecules that regulate cell-cell attachment ability of hiPSC-CMs. Furthermore, increment of attachment ability of hiPSC-CMs by overexpression of a cell adhesion molecule enhanced hiPSC-CMs and ECT functions.

Complications of acute myocardial infarction in women over 50 years of age in a coronary unit

Background: Cardiovascular diseases are the main cause of mortality in women around the world. Erroneously for many years it was considered a disease of men and therefore they were the most studied in terms of risk factors, symptoms, treatments and complications. Ischemic heart disease mainly affects postmenopausal women because by decreasing the estrogen load, cardiovascular risk increases. Therefore, we will study the characteristics of acute myocardial infarction in women over 50 years of age to learn about this highly prevalent disease from another gender. Material and methods: For this study we will carry out an observational, retrospective, cross-sectional and analytical investigation. At the Instituto Cardiovascular de Buenos Aires, collecting information from the medical records of female patients over 50 years of age with acute myocardial infarction in the period May-July 2022.Results: Of 22 patients analyzed in this study, 19 of them successfully survived the cardiovascular event and only 3 had fatal consequences. These patients, who were between the ages of 53 and 94 (with almost 50% of them between the ages of 70 and 80) had comorbidities at the time of the coronary event. 72% had high blood pressure, while only 18% had diabetes. In the interview we found that 77% of them led a sedentary life, 36% were smokers, and 22% had suffered a previous myocardial infarction. When performing the admission electrocardiogram, 36% revealed ST-segment elevation while 63% presented ST-segment depression. The treatment of choice in 36% of the patients was transluminal coronary angioplasty within the first hour of admission, while 63% received the same treatment on a scheduled basis in the following days. Conclusion: Based on what was analyzed in this study, transluminal coronary angioplasty is the treatment with the highest success rate for acute-stage infarction, because it is a minimally invasive procedure and has a low mortality rate. Sometimes when performed together with the placement of a stent, the percentage of recurrence decreases since the artery is kept patent. Its main objective is to restore blood flow to the heart tissue with the intention of preventing a more complex procedure

Investigating Inherited Heart Diseases Using Human Induced Pluripotent Stem Cell-Based Models

Inherited heart diseases (IHDs) are caused by genetic mutations that disrupt the physiological structure and function of the heart. Understanding the mechanisms behind these diseases is crucial for developing personalised interventions in cardiovascular medicine. Development of induced pluripotent stem cells, which can then be differentiated to any nucleated adult cell type, has enabled the creation of personalised single-cell and multicellular models, providing unprecedented insights into the pathophysiology of IHDs. This review provides a comprehensive overview of recent advancements in human iPSC models used to dissect the molecular and genetic underpinnings of common IHDs. We examine multicellular models and tissue engineering approaches, such as cardiac organoids, engineered heart tissue, and multicellular co-culture systems, which simulate complex intercellular interactions within heart tissue. Recent advancements in stem cell models offer a more physiologically relevant platform to study disease mechanisms, enabling researchers to observe cellular interactions, study disease progression, and identify therapeutic strategies. By leveraging these innovative models, we can gain deeper insights into the molecular and cellular mechanisms underlying IHDs, ultimately paving the way for more effective diagnostic and therapeutic strategies.