Cultures Of Cardiomyocytes Research Articles

Aerobic exercise training attenuates ischemia-reperfusion injury in mice by decreasing the methylation level of METTL3-associated m6A RNA in cardiomyocytes.

Ischemic heart disease (IHD) presents a significant global health challenge, with myocardial ischemia-reperfusion injury (MIRI) being a major pathophysiological contributor and lacking effective interventions. While aerobic exercise training (AET) enhances cardiovascular health, its protective mechanism in MIRI remains elusive. This study aims to elucidate the protective effect of AET in MIRI and its underlying mechanism. A mouse model of AET and MIRI was established to evaluate basic indices, cardiac ultrasound, and myocardial injury markers. Dot Blot, qRT-PCR, and Western blot were employed to assess m6A RNA methylation levels and related protein expression in myocardial tissue. In vitro, primary cardiomyocyte culture was utilized to mimic MIRI, evaluating cell viability, mitochondrial membrane potential, etc. Finally, myocardial tissues of MIRI mice were immunoprecipitated for m6A RNA methylation and sequenced to analyze related signaling pathways. AET significantly improved cardiac function and mitigated myocardial injury and fibrosis. Moreover, AET protected myocardium from MIRI by reducing m6A RNA methylation levels and modulating METTL3 expression. In vitro experiments demonstrated that the decrease in m6A RNA methylation levels and METTL3 expression conferred resistance to hypoxia/reoxygenation-induced injury. Furthermore, sequencing results indicated elevated myocardial tissue m6A RNA methylation levels during MIRI, activation of the Nrf2-related signaling pathway, and AET-mediated regulation of the Nrf2/HO-1 signaling pathway, thereby attenuating MIRI through modulation of METTL3-related m6A methylation. AET attenuates MIRI by reducing the level of METTL3-related m6A RNA methylation in cardiomyocytes and activating the Nrf2/HO-1 antioxidant signaling pathway. This finding provides a novel insight and strategy for the prevention and treatment of IHD, holding significant clinical implications.

Chromatin remodeling by the histone methyltransferase SETD2 drives cardiometabolic heart failure with preserved ejection fraction

Abstract Background Cardiometabolic heart failure with preserved ejection fraction (cHFpEF) is highly prevalent and associates with a poor outcome. Pathological gene expression in heart failure is accompanied by changes in active histone marks without major alterations in DNA methylation. Histone 3 trimethylation at lysine 36 (H3k36me3) - a chromatin signature induced by the histone methyltransferase SETD2 – has shown a strong correlation with changes in gene expression in the human failing heart; however its role in cardiac disease is poorly understood. The present study investigates the role of SETD2/ H3k36me3 in cHFpEF. Purpose To investigate the role of chromatin remodelling in obese HFpEF (obHFpEF). Methods Mice with cardiomyocyte-specific deletion of SETD2 (c-SETD2-/-) were generated and subjected to high fat diet feeding and L-NAME treatment for 15 weeks to induce cHFpEF. Cardiac function and exercise tolerance were assessed by echocardiography and Treadmill exhaustion test, respectively. ChIP-Seq datasets were employed to determine the biological pathways regulated by H3k36me3, whereas chromatin immunoprecipitation assays (ChIP) were performed to investigate SETD2/H3k36me3 enrichment on gene promoters. SETD2 gain- and loss-of-function experiments were performed in cultured cardiomyocytes (CMs) exposed to metabolic stress (palmitic acid). SETD2/H3k36me3 axis was also investigated in left ventricular (LV) myocardial specimens from patients with cHFpEF and control donors. Moreover, pharmacological inhibition of SETD2 was performed in skinned CMs from cHFpEF patients. Results Bioinformatic analysis of ChIP-Seq data in mouse CMs showed a strong involvement of SETD2/H3k36me3 in lipid-related pathways. Specifically, SETD2 and H3k36me3 were upregulated in cHFpEF vs. control mouse hearts and were highly enriched on the promoter of sterol regulatory element-binding transcription factor 1 (SREBP1) gene. SETD2 activation in cHFpEF led to SREBP1 upregulation, myocardial triglyceride accumulation and lipotoxic damage. In cHFpEF mice, cardiomyocyte-specific deletion of SETD2 prevented hypertrophic remodeling, diastolic dysfunction and lung congestion while improving exercise tolerance. Mechanistically, SETD2 deletion blunted H3K36me3 enrichment on SREBP1 promoter thus preventing SREBP1-driven lipid accumulation and leading to a significant rewiring of the cardiac lipidome. These findings were associated with restoration of autophagic flux in the cHFpEF myocardium. In CMs exposed to palmitic acid, SETD2 depletion prevented SREBP1 upregulation, whereas SETD2 overexpression recapitulated lipotoxic damage. Finally, SETD2 was upregulated in LV myocardial samples from cHFpEF patients and its pharmacological inhibition attenuated CM stiffness. Conclusions SETD2 is a new molecular target implicated in the pathophysiology of cHFpEF.

The BET inhibitor Apabetalone protects against heart failure with preserved ejection fraction by suppressing myocardial inflammation

Abstract Background Posttranslational histone modifications, play a major role in cardiac hypertrophy and dysfunction. Apabetalone (APA), a selective inhibitor of bromodomain and extraterminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating transcriptional programs in different organs. Purpose We sought to investigate whether APA could be beneficial in cardiometabolic heart failure with preserved ejection fraction (cHFpEF). Methods Mice were subjected to high fat diet feeding and L-NAME treatment for 15 weeks to induce cHFpEF. Histology, mouse echocardiography (Vevo3100) and Treadmill exhaustion test were performed. Unbiased gene expression profiling by PCR array and proteomics analysis (Olink) was employed in left ventricular (LV) myocardial specimens from HFpEF mice and control animals. Cultured cardiomyocytes (CMs) treated with palmitic acid (PA) were used as an in vitro model of metabolic stress. cHFpEF mice were chronically treated with the BET inhibitor APA (150mg/kg/day) or vehicle (DMSO). In order to translate our findings to the human setting, passive stiffness of skinned CMs collected from cHFpEF patients was assessed before and after APA treatment. Results HFpEF mice displayed LV hypertrophy, diastolic dysfunction, myocardial fibrosis, lung congestion and impaired exercise tolerance as compared to controls. Interestingly, diastolic dysfunction - assessed by E/A ratio and isovolumic relaxation time (IVRT) - and lung congestion were significantly reduced in HFpEF mice treated with APA, while exercise tolerance was improved. Transcriptomic analysis in PA-treated CMs and LV mouse specimens from cHFpEF mice showed a profound deregulation of genes controlling inflammation, namely IL-6, TNF-alpha, and IL-1beta. ChIP assays showed BRD4 occupancy on the promoter of several inflammatory genes, including IL6. Treatment with APA suppressed most of inflammatory genes, with a pronounced effect on IL6 expression. Moreover, APA reduced circulating levels of several inflammatory chemokines. The beneficial effects of APA were explained by modulation of IL-6/CaMKII/STAT3 pathway both in cHFpEF hearts and PA-treated CMs. In skinned CMs from cHFpEF patients, both APA and IL6 blockade were able to attenuate passive stiffness. Conclusions Our findings set the stage for preclinical studies and exploratory clinical trials testing APA in patients with cHFpEF.

Activation of fibroblast growth factor 21 by Canagliflozin reduces high-fat diet associated obesity-related cardiac injury: a translational study from bench to bedside

Abstract Aims/hypothesis Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are indicated for the treatment of type 2 diabetes, and evidence shows that they may confer a cardioprotective effect to reduce the incidence of cardiovascular conditions in obese type 2 diabetes patients. The mechanisms underlying this effect remain unclear, but an SGLT2i, canagliflozin (CANA), has been reported to increase levels of fibroblast growth factor-21 (FGF21) and enhance activation of its downstream target proteins. We thus aimed to study whether the effect on FGF21 by CANA can reduce obesity-related cardiac injury. Methods 1) Animal study: Sixteen-week-old normal diet and high-fat diet male mice were treated with or without 30 mg/kg CANA once a day for 24 weeks, and were subsequently examined for body weight, blood glucose levels, adipose tissue changes, lipid accumulation in the liver, and cardiac function. 2) Cell study: An in vitro study that treated H9c2 cardiomyocyte cells cultured in a simulated high-lipid environment containing palmitic acid (PA) with FGF21 recombinant protein was also conducted to examine protein expression profiles. Furthermore, the effect of CANA on FGF21 expression. 3) Human clinical trial: A randomized double-blinded control trial (NCT 05764811) was performed to test effect of CANA on MRI liver fibrosis scores of obese type 2 diabetes patients (n = 40). Results 1) High-fat diet mice treated with CANA for 24 weeks had lower average body weight than untreated controls (34 g vs. 40 g, p < 0.001). In addition, CANA treatment stimulated FGF21 protein production in liver tissue and plasma at higher levels for both normal diet and high-fat diet mice, and reduced expression levels of proteins associated with fibrosis and apoptosis. 2) H9c2 cardiomyocyte culture studies suggested that a simulated high-lipid environment with PA caused higher expression of apoptotic proteins, but the addition of FGF21 recombinant protein significantly reduced apoptosis signalling. 3) MRI before and after 4 weeks of CANA treatment in obese type 2 diabetes patients showed a significant reduction in liver fibrosis scores for those receiving CANA treatment. Conclusion CANA treatment stimulated FGF21 protein expression and reduced fibrosis and apoptosis signalling protein expression. Moreover, reductions in liver fibrosis scores were observed in obese type 2 diabetes patients treated with CANA. Taken together, this suggests that CANA-induced FGF21 expression may exert a cardioprotective effect against fibrosis and apoptosis and can potentially reduce the risk of obesity-related cardiac injury.

Pharmacological Preconditioning with Fenofibrate in Cardiomyocyte Cultures of Neonatal Rats Subjected to Hypoxia/Reoxygenation, High Glucose, and Their Combination.

Pharmacological preconditioning is an alternative to protect the heart against the consequences of damage from ischemia/reperfusion (I/R). It is based on the administration of specific drugs that imitate the effect of ischemic preconditioning (IPC). Peroxisomal proliferator-activated receptors (PPARs) can prevent apoptosis in pathologies such as I/R and heart failure. Therefore, our objective was to determine if the stimulation of PPARα with fenofibrate (feno) decreases the apoptotic process induced by hypoxia/reoxygenation (HR), high glucose (HG), and HR/HG. For that purpose, cardiomyocyte cultures were divided into the following groups: Group 1-control (Ctrl); Group 2-HR; Group 3-HR + 10 μM feno; Group 4-HG, (25 mM glucose); Group 5-HG + feno; Group 6-HR/HG, and Group 7-HR/HG + feno. Our results indicate that cell viability decreases in neonatal cardiomyocytes undergoing HR, HG, and their combination, while feno improved cell viability. Feno treatment decreased apoptosis compared with HG-, HR-, or HG/HR-vehicle-treated. Nuclear- and mitochondrial-apoptosis markers increased in neonatal cardiomyocytes from HR, HG, and HR/HG; while the cytotoxicity decreased in cells treated with feno. In addition, the expression of Bax, Bad, and caspase 9 decreased due to feno, while 14-3-3ɛ and Bcl2 were increased. Inner mitochondrial cytochrome C increased with feno in every condition, as well as mitochondrial activity. Feno treatment prevented injury in the ultrastructure and in the mitochondrial membranes. Thus, our results suggest that feno decreases apoptosis in neonatal cardiomyocytes, improving the ultrastructure of mitochondria in the pathological conditions studied.

Stochastic and alternating pacing paradigms to assess the stability of cardiac conduction

Reentry, the most common cause of severe arrhythmias, is initiated by slow conduction and conduction block. Hence, evaluating conduction velocity and conduction block is of primary importance. However, the assessment of cardiac conduction safety in experimental and clinical settings remains elusive. To identify markers of conduction instability that can be determined experimentally, we developed an approach based on new pacing paradigms. Conduction across a cardiac tissue expansion was assessed in computer simulations and in experiments using cultures of neonatal murine cardiomyocytes on microelectrode arrays. Simulated and in vitro tissues were paced at a progressively increasing rate, with stochastic or alternating variations of cycle length, until conduction block occurred. Increasing pacing rate led to conduction block near the expansion. When stochastic or alternating variations were introduced into the pacing protocol, the standard deviation and the amplitude of alternating variations of local conduction times emerged as markers of unstable conduction prone to block. In both simulations and experiments, conduction delays were prolonged at the expansion but increased only slightly during the pacing protocol. In contrast, these markers of instability increased several-fold, early before block occurrence. The first and second moments of these two metrics provided an estimation of the site of block and the accuracy of this estimation. Therefore, when beat-to-beat variations of pacing cycle length are introduced into a pacing protocol, the local variability of conduction permits to predict sites of block. Our pacing paradigms may have translational applications in clinical cardiac electrophysiology, particularly in identifying ablation targets during mapping procedures.

Simulation-Based Study of Control Strategies for Beating of Human Cardiomyocyte Cultures

Simulation-Based Study of Control Strategies for Beating of Human Cardiomyocyte Cultures

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

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

Ophthalmic acid is a bloodborne metabolite that contributes to age-induced cardiomyocyte hypertrophy.

Cardiac aging involves the development of left ventricular hypertrophy alongside a decline in functional capacity. Here, we use neutral blood exchange to demonstrate that the acute removal of age-accumulated blood factors significantly regresses cardiac hypertrophy in aged mice. The reversal of hypertrophy was not attributed to age-associated hemodynamic effects, implicating a role of blood-derived factors. In addition, the overarching paradigm of systemic aging maintains that the age-related overabundance of plasma proteins are largely responsible for causing pathological phenotypes in tissues. Our results suggest that blood metabolites, not proteins, drive cardiac hypertrophy instead. Upon analyzing serum metabolomics data sets, we identified ophthalmic acid as a circulating metabolite whose levels increase with advanced age. Treatment of adult mouse and neonatal rat cardiomyocytes in culture with ophthalmic acid increased their relative surface areas. This study uncovers a non-protein metabolite that may contribute to cardiomyocyte hypertrophy during aging. Identifying a method to counteract ophthalmic acid's hypertrophic effects may reveal novel therapeutic opportunities for cardiac rejuvenation.

Abstract Mo088: RBFOX2 haploinsufficiency impairs cardiomyocyte adhesion and contractility through faulty RNA metabolism

Background: Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease that is uniformly lethal if left untreated. Protein damaging mutations in one allele of RBFOX2, a highly conserved RNA binding protein, are enriched in HLHS patients, suggesting they are causal. However, it remains unclear how RBFOX2 haploinsufficiency contributes to disease pathogenesis as heterozygous animal models appear healthy. Methods: We generated and characterized RBFOX2 heterozygous and null human induced pluripotent stem cells (hiPSCs) and assessed their phenotypes in differentiated cardiomyocytes (iPSC-CMs) in 2D culture. To learn if altered function might be revealed in a more native microenvironment, we also generated 3D engineered heart tissues (EHTs) from RBFOX2 heterozygous hiPSC-CMs. To delineate the underlying molecular mechanisms, we performed bulk RNA-seq to reveal differential gene expression (DGE) and alternative splicing events (ASEs) between the different genetic cohorts and eCLIP-seq to detect RNAs directly bound by RBFOX2 in the CM lineage. Results: We observed dose-dependent reductions in cell adhesion, size, maturation, respiration, calcium handling, and action potential duration with no significant effect on proliferation in 2D culture. Although myofibril alignment and contractility were completely abolished in null hiPSC-CMs, these features were surprisingly preserved in heterozygous cells. However, in EHTs, we found that RBFOX2 is haploinsufficient for tissue maturation and contractility, suggesting that myocardial-intrinsic defects contribute to RBFOX2 -mediated HLHS. Integration of DGE, ASEs and eCLIP-seq datasets uncovered significant enrichment of DGE and/or ASEs of RBFOX2-bound and unbound transcripts involved in cell adhesion between the extracellular matrix (ECM) and the actin cytoskeleton/sarcomere network. Included in the direct targets with altered expression and alternative splicing was the ECM ligand FIBRONECTIN 1 ( FN1 ), which when downregulated was previously associated with HLHS. Conclusions: Our data suggest that RBFOX2 sits atop an expression and splicing hierarchy that promotes optimal CM cell growth, adhesion, and contractility that protects the heart from HLHS pathogenesis.

Abstract We023: P53 Activation Promotes Maturational Characteristics of Pluripotent Stem Cell-derived Cardiomyocytes in 3D Suspension Culture via FOXO-FOXM1 Regulation

Background: Current protocols can generate highly-pure populations of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate key characteristics of mature in vivo cardiomyocytes. Yet, there exists a risk of arrhythmias when hiPSC-CMs are injected into large animal models. This prompts further investigation into the mechanisms of hiPSC-CM maturation to facilitate clinical translation. Hypothesis: The forkhead box (FOX) family of transcription factors can regulate maturation in neonatal cardiomyocytes through a balance between FOXO and FOXM1. We also previously found that p53 activation could enhance hiPSC-CM maturation. Therefore, we hypothesized that p53 activation increases FOXO and decreases FOXM1 to promote hiPSC-CM maturation in three-dimensional (3D) suspension culture. Methodology: 3D cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator), LOM612 (FOXO activator), AS1842856 (FOXO inhibitor), or RCM-1 (FOXM1 inhibitor), starting 2 days after onset of beating. The hiPSC-CMs were assessed for maturation in metabolic, contractile, and electrophysiological properties, by Seahorse mito stress test, Multi electrode array, and VALA kinetic image cytometer respectively. Results: P53 activation promoted FOXO upregulation and FOXM1 downregulation in hiPSC-CMs, measured by RT-qPCR and immunostaining. Alongside this, p53 activation also promoted hiPSC-CM metabolic and contractile maturational characteristics, seen by increase in oxygen consumption and beat amplitude respectively. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2 and eliminated spontaneous beating. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics of hiPSC-CMs such as increase in contractility, oxygen consumption, and voltage peak maximum upstroke velocity. Further, by single-cell RNAseq, FOXO activated groups showed increase in cardiac maturational pathways compared against DMSO control groups. Conclusions: These results show that p53 activation promotes FOXO and suppresses FOXM1, which modulate hiPSC-CM maturation in 3D suspension. Our study expands current understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3D culture system.

Oxycodone attenuates lipopolysaccharide-induced myocardial injury by inhibiting inflammation, oxidation and pyroptosis via Nrf2/HO-1 signalling pathway.

Myocardial injury and cardiovascular dysfunction are the most common complications of sepsis, and effective therapeutic candidate is still lacking. This study aims to investigate the protective effect of oxycodone in myocardial injury of lipopolysaccharide-induced sepsis and its related signalling pathways. Wild-type and nuclear factor erythroid 2-related factor 2 (Nrf2)-knockout mice, as well as H9c2 cardiomyocytes cultures treated with lipopolysaccharide (LPS) were used as models of septic myocardial injury. H9c2 cardiomyocytes culture showed that oxycodone protected cells from pyroptosis induced by LPS. Mice model confirmed that oxycodone pretreatment significantly attenuated myocardial pathological damage and improved cardiac function demonstrated by increased ejection fraction (EF) and fractional shortening (FS), as well as decreased cardiac troponin I (cTnI) and creatine kinase isoenzymes MB (CK-MB). Oxycodone also reduced the levels of inflammatory factors and oxidative stress damage induced by LPS, which involves pyroptosis-related proteins including: Nod-like receptor protein 3 (NLRP3), Caspase 1, Apoptosis-associated speck-like protein contain a CARD (ASC), and Gasdermin D (GSDMD). These changes were mediated by Nrf2 and heme oxygenase-1 (HO-1) because Nrf2-knockout mice or Nrf2 knockdown in H9c2 cells significantly reversed the beneficial effect of oxycodone on oxidative stress, inflammatory responses and NLRP3-mediated pyroptosis. Our findings yielded that oxycodone therapy reduces LPS-induced myocardial injury by suppressing NLRP3-mediated pyroptosis via the Nrf2/HO-1 signalling pathway in vivo and in vitro.

Imaging of Existing and Newly Translated Proteins Elucidates Mechanisms of Sarcomere Turnover.

How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres. We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands. We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells. Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles.

Spexin inhibits excessive autophagy-induced ferroptosis to alleviate doxorubicin-induced cardiotoxicity by upregulating Beclin 1.

Doxorubicin is widely used in the treatment of malignant tumours, but doxorubicin-induced cardiotoxicity severely limits its clinical application. Spexin is a neuropeptide that acts as a novel biomarker in cardiovascular disease. However, the effects of spexin on doxorubicin-induced cardiotoxicity is unclear. We established a model of doxorubicin-induced cardiotoxicity both in vivo and in vitro. Levels of cardiac damage in mice was assessed through cardiac function assessment, determination of serum cardiac troponin T and CKMB levels and histological examination. CCK8 and PI staining were used to assess the doxorubicin-induced toxicity in cultures of cardiomyocytes in vitro. Ferroptosis was assessed using FerroOrange staining, determination of MDA and 4-HNE content and ferroptosis-associated proteins SLC7A11 and GPX4. Mitochondrial membrane potential and lipid peroxidation levels were measured using TMRE and C11-BODIPY 581/591 probes, respectively. Myocardial autophagy was assessed by expression of P62 and Beclin1. Spexin treatment improved heart function of mice with doxorubicin-induced cardiotoxicity, and attenuated doxorubicin-induced cardiotoxicity by decreasing iron accumulation, abnormal lipid metabolism and inhibiting ferroptosis. Interestingly, doxorubicin caused excessive autophagy in cardiomyocyte in culture, which could be alleviated by treatment with spexin. Knockdown of Beclin 1 eliminated the protective effects of spexin in mice with DIC. Spexin ameliorated doxorubicin-induced cardiotoxicity by inhibiting excessive autophagy-induced ferroptosis, suggesting that spexin could be a drug candidate against doxorubicin-induced cardiotoxicity. Beclin 1 might be critical in mediating the protective effect of spexin against doxorubicin-induced cardiotoxicity.

REDD1 Deletion Suppresses NF-κB Signaling in Cardiomyocytes and Prevents Deficits in Cardiac Function in Diabetic Mice.

Activation of the transcription factor NF-κB in cardiomyocytes has been implicated in the development of cardiac function deficits caused by diabetes. NF-κB controls the expression of an array of pro-inflammatory cytokines and chemokines. We recently discovered that the stress response protein regulated in development and DNA damage response 1 (REDD1) was required for increased pro-inflammatory cytokine expression in the hearts of diabetic mice. The studies herein were designed to extend the prior report by investigating the role of REDD1 in NF-κB signaling in cardiomyocytes. REDD1 genetic deletion suppressed NF-κB signaling and nuclear localization of the transcription factor in human AC16 cardiomyocyte cultures exposed to TNFα or hyperglycemic conditions. A similar suppressive effect on NF-κB activation and pro-inflammatory cytokine expression was also seen in cardiomyocytes by knocking down the expression of GSK3β. NF-κB activity was restored in REDD1-deficient cardiomyocytes exposed to hyperglycemic conditions by expression of a constitutively active GSK3β variant. In the hearts of diabetic mice, REDD1 was required for reduced inhibitory phosphorylation of GSK3β at S9 and upregulation of IL-1β and CCL2. Diabetic REDD1+/+ mice developed systolic functional deficits evidenced by reduced ejection fraction. By contrast, REDD1-/- mice did not exhibit a diabetes-induced deficit in ejection fraction and left ventricular chamber dilatation was reduced in diabetic REDD1-/- mice, as compared to diabetic REDD1+/+ mice. Overall, the results support a role for REDD1 in promoting GSK3β-dependent NF-κB signaling in cardiomyocytes and in the development of cardiac function deficits in diabetic mice.

Reduced Graphene-Oxide-Doped Elastic Biodegradable Polyurethane Fibers for Cardiomyocyte Maturation.

Conductive biomaterials offer promising solutions to enhance the maturity of cultured cardiomyocytes. While the conventional culture of cardiomyocytes on nonconductive materials leads to more immature characteristics, conductive microenvironments have the potential to support sarcomere development, gap junction formation, and beating of cardiomyocytes in vitro. In this study, we systematically investigated the behaviors of cardiomyocytes on aligned electrospun fibrous membranes composed of elastic and biodegradable polyurethane (PU) doped with varying concentrations of reduced graphene oxide (rGO). Compared to PU and PU-4%rGO membranes, the PU-10%rGO membrane exhibited the highest conductivity, approaching levels close to those of native heart tissue. The PU-rGO membranes retained anisotropic viscoelastic behavior similar to that of the porcine left ventricle and a superior tensile strength. Neonatal rat cardiomyocytes (NRCMs) and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on the PU-rGO membranes displayed enhanced maturation with cell alignment and enhanced sarcomere structure and gap junction formation with PU-10%rGO having the most improved sarcomere structure and CX-43 presence. hiPSC-CMs on the PU-rGO membranes exhibited a uniform and synchronous beating pattern compared with that on PU membranes. Overall, PU-10%rGO exhibited the best performance for cardiomyocyte maturation. The conductive PU-rGO membranes provide a promising matrix for in vitro cardiomyocyte culture with promoted cell maturation/functionality and the potential for cardiac disease treatment.

Interactions of Cardiac Proteins with Plasma-Synthesized Polypyrrole (PSPy) to Improve Adult Cardiomyocytes Culture.

Plasma-Synthesized Polypyrrole (PSPy) has been reported as a biomaterial suitable for cell growth in vitro and in vivo. An experimental duplicate was carried out that showed the growth of cardiomyocytes with PSPy, following a protocol previously reported by the working group. The cardiomyocytes cultured with the biomaterial retained their native morphological characteristics, a fundamental key to improving cardiac cell therapy procedures. Such observations motivated us to investigate the molecular characteristics of the biomaterial and the type of interactions that could be occurring (mainly electrostatic, hydrogen bonds, and non-polar). Additionally, PSPy has been studied to establish the probable mechanisms of action of the biomaterial, in particular, its action on a group of cell membrane proteins, integrins, which we know participate in the adhesion of cells to the extracellular matrix, in adhesion between cells and as bidirectional signal transducer mechanisms. In this work, we carried out studies of the interactions established between cardiac integrins α2β1 and α5β1 with different PSPy models by molecular docking studies and binding free energies (ΔGb) calculations. The models based on a previously reported PSPy molecule have three variable terminal chemical groups, with the purpose of exploring the differences in the type of interaction that will be established by modifying the position of an amino (-NH2), a hydroxyl (-OH), and a nitrile (C≡N) in (fixed) groups, as well as the length of the terminal chains (a long/short -NH2). A model with short chains for the -OH and -NH2 (lateral) group was the model with the best interactions with cardiac integrins. We experimentally verified the direct interaction of cardiomyocytes with the PSPy biomaterial observed in rat primary cultures, allowing us to validate the favorable interactions predicted by the computational analysis.

Development of a bacterial cellulose-gelatin composite as a suitable scaffold for cardiac tissue engineering.

Cardiac tissue engineering is suggested as a promising approach to overcome problems associated with impaired myocardium. This is the first study to investigate the use of BC and gelatin for cardiomyocyte adhesion and growth. Bacterial cellulose (BC) membranes were produced by Komagataeibacter xylinus and coated or mixed with gelatin to make gelatin-coated BC (BCG) or gelatin-mixed BC (mBCG) scaffolds, respectively. BC based-scaffolds were characterized via SEM, FTIR, XRD, and AFM. Neonatal rat-ventricular cardiomyocytes (nr-vCMCs) were cultured on the scaffolds to check the capability of the composites for cardiomyocyte attachment, growth and expansion. The average nanofibrils diameter in all scaffolds was suitable (~ 30-65nm) for nr-vCMCs culture. Pore diameter (≥ 10µm), surface roughness (~ 182nm), elastic modulus (0.075 ± 0.015MPa) in mBCG were in accordance with cardiomyocyte requirements, so that mBCG could better support attachment of nr-vCMCs with high concentration of gelatin, and appropriate surface roughness. Also, it could better support growth and expansion of nr-vCMCs due to submicron scale of nanofibrils and proper elasticity (~ 0.075MPa). The viability of nr-vCMCs on BC and BCG scaffolds was very low even at day 2 of culture (~ ≤ 40%), but, mBCG could promote a metabolic active state of nr-vCMCs until day 7 (~ ≥ 50%). According to our results, mBCG scaffold was the most suitable composite for cardiomyocyte culture, regarding its physicochemical and cell characteristics. It is suggested that improvement in mBCG stability and cell attachment features may provide a convenient scaffold for cardiac tissue engineering.

Levels of Small Extracellular Vesicles Containing hERG-1 and Hsp47 as Potential Biomarkers for Cardiovascular Diseases.

The diagnosis of cardiovascular disease (CVD) is still limited. Therefore, this study demonstrates the presence of human ether-a-go-go-related gene 1 (hERG1) and heat shock protein 47 (Hsp47) on the surface of small extracellular vesicles (sEVs) in human peripheral blood and their association with CVD. In this research, 20 individuals with heart failure and 26 participants subjected to cardiac stress tests were enrolled. The associations between hERG1 and/or Hsp47 in sEVs and CVD were established using Western blot, flow cytometry, electron microscopy, ELISA, and nanoparticle tracking analysis. The results show that hERG1 and Hsp47 were present in sEV membranes, extravesicularly exposing the sequences 430AFLLKETEEGPPATE445 for hERG1 and 169ALQSINEWAAQTT- DGKLPEVTKDVERTD196 for Hsp47. In addition, upon exposure to hypoxia, rat primary cardiomyocytes released sEVs into the media, and human cardiomyocytes in culture also released sEVs containing hERG1 (EV-hERG1) and/or Hsp47 (EV-Hsp47). Moreover, the levels of sEVs increased in the blood when cardiac ischemia was induced during the stress test, as well as the concentrations of EV-hERG1 and EV-Hsp47. Additionally, the plasma levels of EV-hERG1 and EV-Hsp47 decreased in patients with decompensated heart failure (DHF). Our data provide the first evidence that hERG1 and Hsp47 are present in the membranes of sEVs derived from the human cardiomyocyte cell line, and also in those isolated from human peripheral blood. Total sEVs, EV-hERG1, and EV-Hsp47 may be explored as biomarkers for heart diseases such as heart failure and cardiac ischemia.

Application of Digital Holographic Imaging to Monitor Real-Time Cardiomyocyte Hypertrophy Dynamics in Response to Norepinephrine Stimulation.

Cardiomyocyte hypertrophy, characterized by an increase in cell size, is associated with various cardiovascular diseases driven by factors including hypertension, myocardial infarction, and valve dysfunction. In vitro primary cardiomyocyte culture models have yielded numerous insights into the intrinsic and extrinsic mechanisms driving hypertrophic growth. However, due to limitations in current approaches, the dynamics of cardiomyocyte hypertrophic responses remain poorly characterized. In this study, we evaluate the application of the Holomonitor M4 digital holographic imaging microscope to track dynamic changes in cardiomyocyte surface area and volume in response to norepinephrine treatment, a model hypertrophic stimulus. The Holomonitor M4 permits non-invasive, label-free imaging of three-dimensional changes in cell morphology with minimal phototoxicity, thus enabling long-term imaging studies. Untreated and norepinephrine-stimulated primary neonatal rat cardiomyocytes were live-imaged on the Holomonitor M4, which was followed by image segmentation and single-cell tracking using the HOLOMONITOR App Suite software version 4.0.1.546. The 24 h treatment of cultured cardiomyocytes with norepinephrine increased cardiomyocyte spreading and optical volume as expected, validating the reliability of the approach. Single-cell tracking of both cardiomyocyte surface area and three-dimensional optical volume revealed dynamic increases in these parameters throughout the 24 h imaging period, demonstrating the potential of this technology to explore cardiomyocyte hypertrophic responses with greater temporal resolution; however, technological limitations were also observed and should be considered in the experimental design and interpretation of results. Overall, leveraging the unique advantages of the Holomonitor M4 digital holographic imaging system has the potential to empower future work towards understanding the molecular and cellular mechanisms underlying cardiomyocyte hypertrophy with enhanced temporal clarity.