Mouse Cardiomyocytes Research Articles

MicroRNA-146b targets HIF-1α AND attenuates cardiomyocyte apoptosis and fibrosis in doxorubicin-induced heart failure.

The global prevalence of heart failure is still growing, which imposes a heavy economic burden. The role of microRNA-146b (miR-146b) in HF remain largely unknown. This study aims to explore the role and mechanism of miR-146b in HF. Method: We applied Reverse transcription-polymerase chain reaction (RT-PCR) to search for differential microRNAs between myocardial tissues of heart failure patients and controls. We also used RT-PCR to detect the miR-146b expression in primary neonatal mouse cardiomyocytes (NMCMs) and mice models of doxorubicin induced HF. In vivo experiments, echocardiography was performed at baseline and weeks 6. After that we harvested mice' heart and evaluated the cardiomyocyte with hematoxylin and eosin (HE), Masson trichrome staining and TUNEL staining. Through bioinformatics analysis, we found HIF-1α might be the target gene of miR-146b which validated by luciferase reporter gene assay. Subsequently, mRNA and protein expression levels of HIF-1α were detected by overexpression or inhibition of miR-146b in primary NMCMs. Results: We found that miR-146b expression was decreased in myocardial tissues of HF patients compared with controls (p < 0.01). MiR-146b levels were notably down regulated in HF models. MiR-146b knockout mice showed a more pronounced decrease in cardiac function and more severe myocardial fibrosis and apoptosis than wild type. Meanwhile, over expression or repression of miR-146b in primary neonatal mouse cardiomyocytes could inhibit or upregulate HIF-1α mRNA and protein expression. Conclusion: Our study shows that miR-146b may be a protective factor for cardiomyocytes by modulating HIF-1α.

Rutin Attenuates Pirarubicin‐Induced Cardiomyocyte Injury by Knocking Down lncRNA Miat

ABSTRACTPirarubicin (THP) is a widely used chemotherapeutic agent for breast cancer, but its clinical use is limited by its cardiotoxicity. In our previous study, we found that rutin (RUT), the main active ingredient in the traditional Chinese medicine Sophora japonica, could attenuate THP‐induced cardiotoxicity. The aim of this study was to further investigate the potential role and mechanism of RUT in attenuating THP‐induced cardiotoxicity. We examined the expression of lncRNA Miat in mouse cardiomyocytes (HL‐1) under the intervention of THP/THP + RUT, respectively. Then we evaluated the protective effect of RUT on THP‐induced HL‐1 by CCK8, TUNEL, reactive oxygen species (ROS) assay and Western Blot. The mediating role of lncRNA Miat was verified by transfection of overexpression lncRNA Miat plasmid. The results showed that RUT increased cell viability, inhibited apoptosis, reduced ROS accumulation and decreased calcium overload in THP‐induced HL‐1. While overexpression of Miat significantly abrogated the therapeutic effect of rutin. In conclusion, RUT attenuates THP‐induced myocardial injury by downregulating lncRNA Miat.

MGST1 overexpression ameliorates mitochondrial dysfunction and ferroptosis during myocardial ischemia/reperfusion injury after heart transplantation.

Mitochondrial dysfunction and ferroptosis play crucial roles in myocardial ischemia/reperfusion (I/R) following heart transplantation. Microsomal glutathione s transferase 1 (MGST1) is widely distributed in mitochondria and has a protective effect against ferroptosis, and its involvement in myocardial I/R injury has not yet been elucidated. In this study, donor hearts from C57BL/6 male mice were subjected to 12h of ex-vivo cold ischemia treatment and transplanted into the abdomen of recipient mice for 24h of reperfusion. The results showed that MGST1 was significantly down-regulated in the model heart graft tissues, and overexpressing MGST1 effectively alleviated myocardial infarction, inflammation and histological damage of myocardial tissues in the model group. Subsequently, mouse cardiomyocytes HL-1 cells were subjected to oxygen-glucose deprivation/re‑oxygenation (OGD/R) condition, and MGST1 overexpression reduced cell apoptosis and inflammation in OGD/R-induced HL-1 cells. Of note, MGST1 overexpression attenuated I/R-induced mitochondrial damage and inhibited ferroptosis in vitro and in vivo. Moreover, MGST1 was found to be negatively regulated by DNA methyltransferase 1 (DNMT1)-mediated promoter methylation, and DNMT1 silence suppressed OGD/R-induced damage in HL-1 cells through restoring MGST1 expression. Altogether, targeting MGST1 hyper-methylation ameliorates mitochondrial damage and ferroptosis of cardiomyocytes, and prevents myocardial I/R injury following heart transplantation.

Trimetazidine: Activating AMPK Signal to Ameliorate Coronary Microcirculation Dysfunction after Myocardial Infarction.

Myocardial ischemia-reperfusion (I/R) injury and coronary microcirculation dysfunction (CMD) are observed in patients with myocardial infarction after vascular recanalization. The antianginal drug trimetazidine has been demonstrated to exert a protective effect in myocardial ischemia-reperfusion injury. This study aimed to investigate the role of trimetazidine in endothelial cell dysfunction caused by myocardial I/R injury and thus improve coronary microcirculation. The myocardial I/R mouse model was established, and trimetazidine was administered for 7 days before myocardial I/R model establishment. Echocardiography, 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H&E) staining, and thioflavin S staining were applied to assess myocardial injury and microvascular function. Additionally, the oxygen-glucose deprivation/reperfusion (OGD/R) model was developed in endothelial cells to simulate myocardial I/R injury in vitro. Griess reaction method, immunofluorescence, and western blotting (WB) were employed to detect the expressions of nitric oxide (NO), platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial (VE)-cadherin, zonula occludens protein 1 (ZO-1), occludin, vascular endothelial growth factor (VEGF) and adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling-related proteins in endothelial cells and mouse cardiomyocytes. AMPK pathway inhibitor compound C was used for further mechanism validation. Our research demonstrated that trimetazidine can alleviate myocardial pathological injury and cardiac function injury during myocardial I/R. Trimetazidine was observed to improve microvascular reflux phenomenon and microvascular function and barrier injury in myocardial I/R and OGD/R models. Additionally, the expressions of AMPK signal-related proteins were found to be inhibited in myocardial I/R and OGD/R models, which were then activated in mice administered trimetazidine. However, the effects of trimetazidine on endothelial cell function and barrier damage were attenuated after co-treatment with compound C and trimetazidine. Trimetazidine ameliorated myocardial I/R-induced CMD by activating AMPK signaling.

Recombinant Klotho administration after myocardial infarction reduces ischaemic injury and arrhythmias by blocking intracellular calcium mishandling and CaMKII activation.

Ischaemic heart disease (IHD) remains a major cause of death and morbidity. Klotho is a well-known anti-ageing factor with relevant cardioprotective actions, at least when renal dysfunction is present, but its actions are much less known when renal function is preserved. This study investigated Klotho as a biomarker and potential novel treatment of IHD-associated complications after myocardial infarction (MI) under preserved renal function. Association between circulating Klotho levels and cardiac injury was investigated in patients after ST-elevation MI (STEMI). Biochemical, in vivo and in vitro cardiac function and histological and molecular studies were performed to determine the effect of recombinant Klotho in the failing hearts of mice after MI. We demonstrated that STEMI patients showed lower systemic Klotho levels, with the lowest Klotho tertile in those patients with higher N-terminal pro B-type natriuretic peptide (NT-proBNP) levels. Mice also showed a decrease in systemic Klotho levels after MI induction. Furthermore, recombinant Klotho administration in mice reduced infarct area and attenuated cardiac hypertrophy and fibrosis. We also demonstrated that Klotho treatment prevented reduction in ejection fraction and MI-related ECG changes, including prolonged QRS, JT, QTc, and TpeakTend intervals and premature ventricular contractions. In adult mouse cardiomyocytes, Klotho treatment restricted systolic calcium (Ca2+) release and cell shortening disturbances after MI. Klotho prevented increased diastolic Ca2+ leak and pro-arrhythmogenic events in PMI mice by blocking activation of the Ca2+/calmodulin-dependent kinase type II (CaMKII) pathway, preventing ryanodine receptor type 2 (RyR2) hyperphosphorylation. In conclusion, Klotho supplementation protected against functional and structural cardiac remodelling and ameliorated ventricular arrhythmic events by preventing intracardiomyocyte Ca2+ mishandling in mice following MI. These data uncover a new cardioprotective role of Klotho, emerging as a biomarker of ventricular injury and potential treatment for patients after MI. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Xin-Ji-Er-Kang balances mitochondrial fusion and fission to protect cardiomyocytes in mice with heart failure by regulating the ERα/SIRT3 pathway

Xin-Ji-Er-Kang balances mitochondrial fusion and fission to protect cardiomyocytes in mice with heart failure by regulating the ERα/SIRT3 pathway

TLR4 Inhibition Attenuated LPS-Induced Proinflammatory Signaling and Cytokine Release in Mouse Hearts and Cardiomyocytes.

Sepsis is associated with myocardial injury and early mortality. The innate immune receptor Toll-like receptor 4 (TLR4) can recognize pathogen-associated-molecular-patterns (PAMPs) and damage-associated molecular patterns (DAMPs); the latter are released during tissue injury. We hypothesized that TLR4 inhibition reduces proinflammatory signaling and cytokine release in: (1) LPS or Escherichia coli-treated isolated mouse heart; (2) LPS-treated mouse primary adult cardiomyocytes; and (3) the isolated heart during ischemia-reperfusion. Isolated C57BL/6N male mouse hearts were perfused for 120 min, with either LPS, E. coli, with and without CLI-095 (TLR4 inhibitor). Primary adult mouse cardiomyocytes were treated with LPS or LPS + CLI-095. Isolated hearts, exposed to 35 min of global ischemia, were treated with either vehicle or CLI-095 during reperfusion. Infarct size was quantified by triphenyltetrazolium staining. Cytokine expression was analyzed with ELISA, western blot analysis, and qPCR. In isolated hearts, E. coli increased the expression of proinflammatory cytokines (IL-6 and CXCL2), which was not attenuated with TLR4 inhibition. TLR4 inhibition reduced expression (p = 0.004) and release of IL-6 (p < 0.0001) in LPS-exposed isolated hearts. LPS activated the nuclear-factor κ-light-chain-enhancer of activated B cells signaling pathway (NF-κB) in primary adult cardiomyocytes. Moreover, TLR4 inhibition reduced LPS-induced mRNA expression and release of IL-6 in primary adult cardiomyocytes. Isolated hearts treated with CLI-095 during reperfusion after ischemia (induced DAMPs release) showed reduced infarct size (39 ± 17% to 26 ± 8%, p = 0.034) and decreased IL-6 release (p = 0.006). Inhibition of TLR4 reduced proinflammatory signaling and cytokine release in LPS-treated and ischemia-reperfused isolated mouse hearts and in primary adult murine cardiomyocytes.

Prx5 overexpression protect against doxorubicin-induced cardiotoxicity by inhibiting oxidative stress and inflammation via the TLR4/NF-κB pathway.

The clinical application of Doxorubicin (DOX) is constrained due to its cardiotoxic side effects. Oxidative stress and inflammation are crucial mechanisms driving doxorubicin-induced cardiotoxicity (DIC). Peroxiredoxin 5 (Prx5) is central to these inflammatory responses. However, the specific role of Prx5 in DIC remains unclear. This study aims to investigate the impacts of Prx5 on DIC and the underlying mechanisms. A cardiac-specific Prx5-overexpressing mice was used to establish a doxorubicin (DOX)-induced cardiotoxicity (DIC) model. Neonatal mouse cardiomyocytes (NMCMs) were cultured and stimulated with DOX. Prx5 overexpression or knockdown in cardiomyocytes was achieved using a Prx5-overexpressing adenovirus or small interfering RNA (siRNA), respectively. Echocardiography, histopathological assessments, and molecular techniques were employed to examine the effects and mechanisms of Prx5 on DIC. Prx5 expression is upregulated in cardiac tissues following DOX administration. In DOX-exposed mice, overexpression of Prx5 significantly improved cardiac function and reduced myocardial injury. It inhibited myocardial hypertrophy and fibrosis, and diminished oxidative stress and inflammatory responses. Conversely, Prx5 knockdown in vitro aggravated DOX-induced cardiomyocyte inflammation and oxidative stress. Mechanistically, overexpression of Prx5 also resulted in the downregulation of Toll-like receptor 4 (TLR4) and phosphorylated P65 expression. Moreover, the protective effects of Prx5 were significantly abrogated by a TLR4 agonist. Prx5 overexpression could protect against DOX-induced cardiac oxidative stress and inflammation. Mechanistically, Prx5 overexpression potentially inhibits the TLR4/NF-κB signaling pathway to improve DOX-induced myocardial injury. These findings strongly suggest that Prx5 could be a potential candidate target for the treatment of DOX-induced myocardial injury.

A New target of ischemic ventricular arrhythmias-ITFG2.

ITFG2 is an intracellular protein known to modulate the immune response of T-cells. Our previous investigation revealed that ITFG2 specifically targets ATP5b to regulate ATP energy metabolism and maintain mitochondrial function, thereby protecting the heart from ischemic injury. However, the role of ITFG2 in ischemic ventricular arrhythmias and its underlying mechanisms have not been previously reported. In this study, we found ITFG2 overexpression, induced by an adeno-associated virus serotype 9 vector, partially reduced the incidence of ischemic ventricular arrhythmias and shortened the duration of ventricular arrhythmias in mice after myocardial infarction. Conversely, shRNA-mediated knockdown of endogenous ITFG2 aggravated ischemic ventricular arrhythmias. ITFG2 overexpression also shortened the prolonged QRS complex and increased the epicardial conduction velocity in MI mice. Additionally, the hearts from ITFG2 overexpression mice exhibited a higher maximal upstroke velocity at phase 0 of transmembrane action potential compared to MI mice. Patch-clamp analyses demonstrated a 50% increase in the peak current of voltage-dependent Na+ channel by ITFG2 overexpression in isolated ventricular cardiomyocytes post MI. In cultured neonatal mouse cardiomyocytes under hypoxic conditions, ITFG2 up-regulated Nav1.5 protein expression by inhibiting its ubiquitination. Co-immunoprecipitation experiments showed that ITFG2 reduces the binding affinity between NEDD4-2 and Nav1.5, thereby inhibiting Nav1.5 ubiquitination. Taken together, our data highlight the critical role of ITFG2 in reducing susceptibility to ischemic ventricular arrhythmias by down-regulating Nav1.5 ubiquitination. These findings suggest that ITFG2 may serve as a novel target for treating ischemic ventricular arrhythmias.

Simple Method to Isolate Individual Mouse Cardiomyocytes Using Enzymatic Digestion via Retrograde Perfusion.

Isolated single cardiomyocytes are commonly used to understand fundamental cardiac cell physiology and cell signaling at the molecular level. The protocol to isolate cardiomyocytes is under continual optimization between laboratories, despite being conducted for many years. The delicate nature of these cells coinciding with laboratory equipment variations can make the consistency of isolation challenging. The protocol described here presents a simple method to routinely isolate high-quality cardiomyocytes using Langendorff perfusion. This method provides cells well suited for analysis of physiological cellular calcium handling and spontaneous calcium release.

Cardiac Applications of CRISPR/AAV-Mediated Precise Genome Editing.

The ability to efficiently make precise genome edits in somatic tissues will have profound implications for gene therapy and basic science. CRISPR/Cas9 mediated homology-directed repair (HDR) is one approach that is commonly used to achieve precise and efficient editing in cultured cells. Previously, we developed a platform capable of delivering CRISPR/Cas9 gRNAs and donor templates via adeno-associated virus to induce HDR (CASAAV-HDR). We demonstrated that CASAAV-HDR is capable of creating precise genome edits in vivo within mouse cardiomyocytes at the neonatal and adult stages. Here, we report several applications of CASAAV-HDR in cardiomyocytes. First, we show the utility of CASAAV-HDR for disease modeling applications by using CASAAV-HDR to create and precisely tag two pathological variants of the titin gene observed in cardiomyopathy patients. We used this approach to monitor the cellular localization of the variants, resulting in mechanistic insights into their pathological functions. Next, we utilized CASAAV-HDR to create another mutation associated with human cardiomyopathy, arginine 14 deletion (R14Del) within the N-terminus of Phospholamban (PLN). We assessed the localization of PLN-R14Del and quantified cardiomyocyte phenotypes associated with cardiomyopathy, including cell morphology, activation of PLN via phosphorylation, and calcium handling. After demonstrating CASAAV-HDR utility for disease modeling we next tested its utility for functional genomics, by targeted genomic insertion of a library of enhancers for a massively parallel reporter assay (MPRA). We show that MPRAs with genomically integrated enhancers are feasible, and can yield superior assay sensitivity compared to tests of the same enhancers in an AAV/episomal context. Collectively, our study showcases multiple applications for in vivo precise editing of cardiomyocyte genomes via CASAAV-HDR.

Nicotinamide mononucleotide protects septic hearts in mice via preventing cyclophilin F modification and lysosomal dysfunction.

Myocardial dysfunction is a decisive factor of death in septic patients. Cyclophilin F (PPIF) is a major component of the mitochondrial permeability transition pore (mPTP) and acts as a critical mPTP sensitizer triggering mPTP opening. In sepsis, decreased NAD+ impairs Sirtuin 3 function, which may prevent PPIF de-acetylation. Repletion of NAD+ with nicotinamide mononucleotide (NMN) reduces myocardial dysfunction in septic mice. In addition, administration of the mPTP inhibitor cyclosporine-A attenuated sepsis-induced myocardial dysfunction, and deletion of PPIF reduced lung and liver injuries in sepsis, leading to increased survival. It is plausible that NAD+ repletion with NMN may prevent mPTP opening in protecting septic hearts through PPIF de-acetylation and/or inhibition of mitochondrial ROS-mediated PPIF oxidation. In this study we investigated how NMN alleviated myocardial dysfunction in septic mice. Sepsis was induced in mice by injection of LPS (4 mg/kg, i.p.). Then mice received NMN (500 mg/kg, i.p.) or mito-TEMPO (0.7 mg/kg, i.p.) right after LPS injection, and subjected to echocardiography for assessing myocardial function. At the end of experiment, the heart tissues and sera were collected for analyses. In vitro experiments were conducted in neonatal mouse cardiomyocytes treated with LPS (1 µg/mL) in the presence of NMN (500 µmol/L) or mito-TEMPO (25 nmol/L). We showed that LPS treatment markedly increased mitochondrial ROS production and induced lysosomal dysfunction and aberrant autophagy in cardiomyocytes and mouse hearts, leading to inflammatory responses and myocardial injury and dysfunction in septic mice. NMN administration attenuated LPS-induced deteriorative effects. Selective inhibition of mitochondrial superoxide production with mito-TEMPO attenuated lysosomal dysfunction and aberrant autophagy in septic mouse hearts. Notably, LPS treatment significantly increased acetylation and oxidation of PPIF, which was prevented by NMN in mouse hearts. Knockdown of PPIF replicated the beneficial effects of NMN or mito-TEMPO on ROS production, lysosomal dysfunction, aberrant autophagy, and myocardial injury/dysfunction in sepsis. In addition, administration of NMN abrogated LPS-induced ATP5A1 acetylation and increased ATP5A1 protein levels and ATP production in septic mouse hearts. This study demonstrates that NMN modulates the interplay of mitochondrial ROS and PPIF in maintaining normal lysosomal function and autophagy and protecting ATP5A1 and ATP production during sepsis.

Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function.

Protein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiac myocytes, and mutations in PQC pathway proteins, such as CRYAB (arginine to glycine at position 120, R120G) and BAG3 (proline to lysine at position 209, P209L) induce protein aggregate pathology with cardiomyopathy in humans. Novel observations in yeast and mammalian cells demonstrate mitochondrial uptake of cytosolic protein aggregates. We hypothesized that mitochondrial uptake of cytosolic protein aggregates and their removal by mitophagy, a lysosomal degradative pathway essential for myocardial homeostasis, facilitates cytosolic protein quality control in cardiac myocytes. Mice with inducible cardiac myocyte specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assessed for protein aggregates with biochemical fractionation and super-resolution imaging in comparison to floxed controls. Induced pluripotent stem cell (iPSC)-derived cardiac myocytes with R120G knock-in to the CRYAB locus were assessed for localization of the CRYAB protein. Transgenic mice expressing R120G CRYAB protein (R120G-TG) were subjected to both TRAF2 gain-of-function (with AAV9-cardiac Troponin T promoter-driven TRAF2 transduction) and TRAF2 loss-of-function (with tamoxifen-inducible ablation of one Traf2 allele) in cardiac myocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology. Cardiomyocyte-specific ablation of TRAF2 results accumulation of mitochondrial and cytosolic protein aggregates and DESMIN mis-localization to protein aggregates. Isolated mitochondria take up cardiomyopathy-associated aggregate-prone cytosolic chaperone proteins, namely arginine to glycine (R120G) CRYAB mutant and proline to lysine (P209L) BAG3 mutant. R120G-CRYAB mutant protein increasingly localizes to mitochondria in human and mouse cardiomyocytes. R120G-TG mice demonstrate upregulation of TRAF2 in the mitochondrial fraction with increased mitophagy as compared with wild type. Adult-onset inducible haplo-insufficiency of TRAF2 resulted in accelerated mortality, impaired left ventricular systolic function and increased protein aggregates in R120G-TG mice as compared with controls. Conversely, AAV9-mediated TRAF2 transduction in R120G-TG mice reduced mortality and attenuated left ventricular systolic dysfunction, with reduced protein aggregates and restoration of normal localization of DESMIN, a cytosolic scaffolding protein chaperoned by CRYAB, as compared with control AAV9-GFP group. TRAF2-mediated mitophagy in cardiac myocytes facilitates removal of cytosolic protein aggregates and can be stimulated to ameliorate proteotoxic cardiomyopathy.

Cardiomyocyte-derived small extracellular vesicle-transported let-7b-5p modulates cardiac remodeling viaTLR7 signaling pathway.

Cardiac remodeling is the major pathological change of heart failure. And let-7 family has been implicated in the development and pathogenesis of cardiovascular diseases. However, the mechanisms underlying let-7b-5p-mediated cellular pathogenesis of cardiac remodeling are not well understood. The present study aimed to explore the effects of let-7b-5p on cardiac remodeling and the corresponding regulatory mechanism. Invivo results indicated that cardiac let-7b-5p was upregulated in the mouse model of Angiotensin II (Ang II)-induced cardiac remodeling. Additionally, let-7b-5p knockdown ameliorated the effects of Ang II-induced cardiac remodeling, whereas let-7b-5p overexpression facilitated cardiac remodeling. Invitro, let-7b-5p mimics induced cardiomyocyte hypertrophy, fibroblast transdifferentiation, and the expression of inflammatory factors in neonatal mouse cardiomyocytes (NMCMs), neonatal mouse cardiac fibroblasts (NMCFs), and bone marrow-derived macrophages (BMDMs), respectively. Furthermore, let-7b-5p exerted its cardiac pro-remodeling effects at least partially through a small extracellular vesicle (SEV)-based delivery strategy. We found that let-7b-5p was enriched in SEVs derived from Ang II-treated NMCMs (NMCM-SEV) but not from Ang II-treated NMCFs (NMCF-SEV). Mechanistic analyses revealed that NMCM-SEV promoted TLR7 and MyD88 expression, which increased NF-κB phosphorylation levels. Knockdown of let-7b-5p, TLR7 or MyD88 in NMCMs, NMCFs, and BMDMs abolished the cardiac pro-remodeling effects of NMCM-SEV. These results uncover that let-7b-5p-containing NMCM-SEVs promote cardiac remodeling via the TLR7/MyD88/NF-κB pathway, implicating let-7b-5p as a potential therapeutic target for heart failure.

An optimized Langendorff-free method for isolation and characterization of primary adult cardiomyocytes

Isolation of adult mouse cardiomyocytes is an essential technique for advancing our understanding of cardiac physiology and pathology, and for developing therapeutic strategies to improve cardiac health. Traditionally, cardiomyocytes are isolated from adult mouse hearts using the Langendorff perfusion method in which the heart is excised, cannulated, and retrogradely perfused through the aorta. While this method is highly effective for isolating cardiomyocytes, it requires specialized equipment and technical expertise. To address the challenges of the Langendorff perfusion method, researchers have developed a Langendorff-free technique for isolating cardiomyocytes. This Langendorff-free technique involves anterograde perfusion through the coronary vasculature by clamping the aorta and intraventricular injection. This method simplifies the experimental setup by decreasing the need for specialized equipment and eliminating the need for cannulation of the heart. Here, we introduce an updated Langendorff-free method for isolating adult mice cardiomyocytes that builds on the Langendorff-free protocols developed previously. In this method, the aorta is clamped in situ, and the heart is perfused using a peristaltic pump, water bath, and an injection needle. This simplicity makes cardiomyocyte isolation more accessible for researchers who are new to cardiomyocyte isolation or are working with limited resources. In this report, we provide a step-by-step description of our optimized protocol. In addition, we present example studies of analyzing mitochondrial structural and functional characteristics in untreated isolated cardiomyocytes and cardiomyocytes treated with the acute inflammatory stimulus lipopolysaccharide (LPS).

Correlation between indole-3-acetic acid and left ventricular hypertrophy in hemodialysis patients.

Among hemodialysis patients, left ventricular hypertrophy (LVH) is a prevalent cardiac abnormality. The uremic toxin indole-3-acetic acid (IAA) is elevated in uremia patients, but the connection between IAA and LVH in individuals undergoing hemodialysis remains uncertain. Hence, the objective of this research was to examine the correlation between blood IAA levels and LVH in individuals undergoing hemodialysis. In total, 205 individuals undergoing hemodialysis were chosen and categorized into two groups, with (143 patients) and without LVH (62 patients). Patient clinical data were collected, and serum creatinine, calcium, phosphorus, hemoglobin, and IAA levels were measured. Compared to the non-LVH group, the LVH group had higher IAA and serum phosphorus but lower hemoglobin. The serum IAA concentration was positively correlated with both left ventricular mass (LVM) and left ventricular mass index (LVMI) but negatively correlated with both left ventricular ejection fraction (LVEF) and the ratio of left ventricular transmitral early peak flow velocity to left ventricular transmitral late peak flow velocity (E/A). Logistic regression analysis indicated that increased IAA levels are a risk factor for LVH and are not influenced by other factors. In addition, we exposed primary neonatal cultured mouse cardiomyocytes to varying concentrations of IAA in a controlled environment. Cardiomyocyte hypertrophy was induced by IAA in a concentration-dependent manner. Serum IAA is correlated with alterations in both the function and structure of the left ventricle. The serum IAA concentration is an independent risk factor for LVH. IAA may be a novel biomarker of LVH in hemodialysis patients.

Abstract 4122257: Inhibition of Neutrophil Extracellular Traps Prevents Heart Failure by Preserving Mitochondrial Function of Cardiomyocytes in Mice

Background: We have previously demonstrated that neutrophil extracellular traps (NETs) in myocardial tissue are associated with cardiac dysfunction and adverse outcomes in patients with heart failure with reduced ejection fraction. Here, we aimed to elucidate the underlying mechanisms and to clarify whether inhibiting NETs could rescue the cardiac phenotype using ex vivo and in vivo models. Methods and Results: Given that peptidyl arginine deiminase 4 (PAD4) is essential for the formation of NETs, PAD4 knockout mice were used for this study. First, we assessed the mitochondrial function of cardiomyocytes by conducting real-time measurements of the oxygen consumption rate in ex vivo . Extracellular flux analysis revealed that NETs-containing conditioned medium from wild-type neutrophils or purified NET components led to impaired mitochondrial oxygen consumption of adult mouse cardiomyocytes, while these effects were abolished when PAD4 in neutrophils was genetically ablated (Figure 1). In a murine model of pressure overload-induced heart failure, transverse aortic constriction (TAC) operation was performed on wild-type and PAD4 knockout mice. Immunohistological analysis demonstrated that TAC induced the formation of NETs in the myocardium, with the greatest number of NETs occurring at 1 day, persisting into the failing stage at 4 weeks in wild-type mice, while PAD4-deficient hearts displayed no presence of NETs in the heart tissue (Figure 2). Four weeks after TAC, left ventricular ejection fraction was reduced in wild-type mice, whereas PAD4 knockout mice displayed preserved left ventricular ejection fraction (Figure 2). Finally, we investigated the bioenergetics of cardiomyocytes isolated from PAD4 knockout mice after TAC. Maximal mitochondrial oxygen consumption rate in cardiomyocytes isolated from TAC-operated PAD4 knockout mice was significantly higher than in those from TAC-operated wild-type mice (Figure 3). Conclusion: Our data suggest that NETs directly derived impairment of mitochondrial function in cardiomyocytes. Inhibiting NETs represents a potential therapeutic approach for heart failure, preventing mitochondrial dysfunction of cardiomyocytes.

Abstract 4118993: β1 Adrenergic Receptor Autoantibodies Promote Heart Failure Though Activation of Prostaglandin E2 Receptor EP1/Phosphodiesterase 4B Pathway

Background: β1 adrenergic receptor (β1-AR) autoantibodies (β1-AA) can cause heart failure (HF) through activating of β1-AR; However, β-AR antagonists cannot completely reverse the cardiac injury induced by β1-AA and improve the survival rate of patients with β1-AA-positive HF. However, the β1-AR-independent mechanisms of heart failure induced by β1-AA are unclear. Methods: Proteomics of mice hearts was used to identify the specific changed proteins induced by β1-AA. The expression of phosphodiesterase 4B (PDE4B) was detected by western blot in neonatal mice mouse cardiomyocytes (NMCMs) and heart tissues. The binding proteins with β1-AA were enriched by immunoprecipitation (IP) and identified by mass spectrum. The interaction between β1-AA and prostaglandin E2 Receptor EP1 was detected by co-IP and surface plasmon resonance. The cardiac function and cardiac remodeling of PDE4B transgenic (TG) mice and EP1 knockout (KO) mice. Results: Firstly, PDE4B was changed the most proteins induced by β1-AA compared to β1 adrenergic receptor dobutamine. Moreover, the decrease of PDE4B induced by β1-AA cannot be reversed by metoprolol in vivo and in vitro. Secondly, the cAMP/PKA pathway and cardiac injury/dysfunction induced by β1-AA were inhibited after activation of PDE4B or PDE4B-TG in vivo and in vitro. Thirdly, β1-AA can bind with EP1 and activate Gq/IP3 pathway. The activation effect was abolished by EP1 antagonist. Lastly, blocked EP1 or EP1-KO will reverse the decreased PDE4B and cardiac injury/dysfunction induced by β1-AA. Conclusions: Our work reveals that β1-AA can activate of EP1/PDE4B pathway to result in heart failure.

Abstract 4134837: Checkpoint Kinase 1 Attenuates Myocardial Ischemia-Reperfusion Injury Through Maintaining SIRT1-Dependent Mitochondrial Homeostasis

Background: Mitochondrial dysfunction is linked to myocardial ischemia-reperfusion (I/R) injury. Checkpoint kinase 1 (CHK1) could facilitate cardiomyocyte proliferation and cardiac repair post myocardial infarction, however, its role on mitochondrial function in I/R injury remains unknown. Research Questions: The purpose of this study is to explore the potential effects and mechanisms of CHK1 on mitochondrial homeostasis during myocardial I/R injury. Methods: To investigate the role of CHK1 on mitochondrial function following I/R injury, cardiomyocyte-specific knockout/knock-in mouse models were generated. Mass spectrometry-proteomics analysis and protein co-immunoprecipitation assays were conducted to dissect the molecular mechanism of CHK1. The interaction between CHK1 and SIRT1 was explored using truncated plasmids. Results: CHK1 was downregulated in myocardium post I/R and neonatal mouse cardiomyocytes (NMCMs) post oxygen-glucose deprivation/re-oxygenation (OGD/R). In vivo , CHK1 overexpression protected against myocardial I/R injury, while heterogenous CHK1 knockout exacerbated cardiomyopathy. In vitro , CHK1 inhibited OGD/R-induced cardiomyocyte apoptosis and bolstered cardiomyocyte survival. Mechanistically, CHK1 attenuated oxidative stress and preserved mitochondrial metabolism in cardiomyocytes under I/R. Moreover, disrupted mitochondrial homeostasis in I/R myocardium was restored by CHK1 through the promotion of mitochondrial biogenesis and mitophagy. Through mass spectrometry analysis following co-immunoprecipitation, SIRT1 was identified as a direct target of CHK1. The 266-390 domain of CHK1 interacted with the 160-583 domain of SIRT1. Importantly, CHK1 phosphorylated SIRT1 at Thr530 residue, thereby inhibiting SMURF2-mediated degradation of SIRT1. CHK1 Δ390 amino acids (aa) mutant functioned similarly to full-length CHK1 in scavenging ROS and maintaining mitochondrial dynamics. Consistently, cardiac-specific SIRT1 knockdown partially attenuated the protective role of CHK1 in I/R injury. Conclusions: Our findings revealed that CHK1 mitigates I/R injury and restores mitochondrial dynamics in cardiomyocytes through a SIRT1-dependent mechanism.

Effects of intermittent restriction diet and moderate-intensity exercise on the expression of cardiomyocytes in mice with high-glucose loads

Effects of intermittent restriction diet and moderate-intensity exercise on the expression of cardiomyocytes in mice with high-glucose loads