Rat Cardiomyocytes Research Articles

Dynamic map illuminates Hippo-cMyc module crosstalk driving cardiomyocyte proliferation.

Numerous regulators of cardiomyocyte (CM) proliferation have been identified, yet how they coordinate during cardiac development or regeneration is poorly understood. Here, we developed a computational model of the CM proliferation regulatory network to obtain key regulators and systems-level understanding. The model defines five modules (DNA replication, mitosis, cytokinesis, growth factor, Hippo pathway) and integrates them into a network of 72 nodes and 88 reactions that correctly predicts 73 of 78 (93.6%) independent experiments from the literature. The model predicts that in response to YAP activation, the Hippo module crosstalks to the growth factor module via PI3K and cMyc to drive cell cycle activity. This predicted YAP-cMyc axis is validated experimentally in rat cardiomyocytes and further supported by YAP-stimulated cMyc open chromatin and mRNA in mouse hearts. This validated computational model predicts how individual regulators and modules coordinate to control CM proliferation.

N-acetylneuraminic acid promotes ferroptosis of H9C2 cardiomyocytes with hypoxia/reoxygenation injury by inhibiting the Nrf2 axis.

To investigate the mechanism through which N-acetylneuraminic acid (Neu5Ac) exacerbates hypoxia/reoxygenation (H/R) injury in rat cardiomyocytes (H9C2 cells). H9C2 cells were cultured in hypoxia and glucose deprivation for 8 h followed by reoxygenation for different durations to determine the optimal reoxygenation time. Under the optimal H/R protocol, the cells were treated with 0, 5, 10, 20, 30, 40, 50, and 60 mmol/L Neu5Ac during reoxygenation to explore the optimal drug concentration. The cells were then subjected to H/R injury followed by treatment with Neu5Ac, Fer-1 (a ferroptosis inhibitor), or both. The changes in SOD activity, intracellular Fe2+ and lipid ROS levels in the cells were evaluated, and the cellular expressions of Nrf2, GPX4, HO-1, FSP1, and xCT proteins were detected using Western blotting. Following hypoxia and glucose deprivation for 8 h, the cells with reoxygenation for 6 h, as compared with other time lengths of reoxygenation except for 9 h, showed the lowest expression levels of Nrf2, GPX4, HO-1, and FSP1 proteins (P<0.001). Neu5Ac treatment of dose-dependently decreased the viability of the cells with H/R injury with an IC50 of 30.07 mmol/L. Reoxygenation for 3 h with normal glucose supplementation and a Neu5Ac concentration of 30 mmol/L were selected as the optimal conditions in the subsequent experiments. The results showed that Neu5Ac could significantly increase SOD activity, Fe2+ and lipid ROS levels and reduce Nrf2, GPX4, HO-1, and FSP1 protein expressions in H9C2 cells with H/R injury, but its effects were significantly attenuated by treatment with Fer-1. Neu5Ac exacerbates ferroptosis of myocardial cells with H/R injury by inhibiting the Nrf2 axis to promote the production of ROS and lipid ROS.

Total Glycosides of Paeony Activates PI3K/Akt Pathway to Alleviate Cardiomyocyte Hypertrophy Induced by AngII.

Total glucosides of paeony (TGP) have been investigated for their effects on cardiomyocyte hypertrophy induced by angiotensin II (Ang II). In this study, rat cardiomyocyte H9c2 cells were treated with various doses of TGP (0, 12.5, 25, 50, 100, 200, and 400 μmol/L), and cell viability was assessed using the MTT method to determine an optimal dose. To establish the cardiomyocyte hypertrophy model, Ang II (1 μmol/L) was used. The experimental groups included the control (Ctrl) group, the hypertrophy group (Ang II), the TGP treatment group (TGP+Ang II), and a combined treatment group (TGP+Ang II+LY), where LY294002, a PI3K/Akt inhibitor, was used. The surface area of H9c2 cells was analyzed using image analysis software, and apoptosis was assessed via flow cytometry. Western blotting was employed to evaluate markers related to cell proliferation, cardiac hypertrophy, apoptosis, and autophagy, as well as the phosphorylation of the PI3K/Akt pathway. The results revealed that Ang II inhibited cell viability and increased cell surface area, apoptosis, and autophagy, all of which were significantly reversed by TGP treatment. Moreover, the addition of LY294002 partially attenuated the effects of TGP, reducing cell viability and promoting hypertrophy, apoptosis, and autophagy. Additionally, Ang II reduced PI3K/Akt signaling activity, while TGP restored it. LY treatment reversed the effects of TGP and suppressed the PI3K/Akt pathway. In conclusion, TGP improves cardiomyocyte hypertrophy induced by Ang II by activating the PI3K/Akt signaling pathway.

Engineering Highly Aligned and Densely Populated Cardiac Muscle Bundles via Fibrin Remodeling in 3D-Printed Anisotropic Microfibrous Lattices.

Replicating the structural and functional features of native myocardium, particularly its high-density cellular alignment and efficient electrical connectivity, is essential for engineering functional cardiac tissues. Here, novel electrohydrodynamically printed InterPore microfibrous lattices with anisotropic architectures are introduced to promote high-density cellular alignment and enhanced tissue interconnectivity. The interconnected pores in the microfibrous lattice enable dynamic, cell-mediated remodeling of fibrous hydrogels, resulting in continuous, mechanically stable tissue bundles. Compared to lattices with isolated pores, the engineered aligned cardiac tissues from neonatal rat cardiomyocytes exhibit improved electrophysiological properties and synchronous contractions. Using a multiseeding strategy, an equivalent cell seeding density of 8 × 107 cellsmL-1, facilitating the formation of multicellular, vascularized cardiac structures with maintained tissue viability and integrity, is achieved. As a demonstration, human-induced pluripotent stem cell-derived cardiac tissues are engineered with progressive maturation and functional integration over time. These findings underscore the potential of InterPore microfibrous lattices for applications in cardiac tissue engineering, drug discovery, and therapeutic development.

High fructose levels inhibit the proliferation of cardiomyocytes via the Notch1 signaling pathway.

Fructose, as a natural and simple sugar, is not significantly harmful to the human body when consumed in moderation and can provide energy for the body. High-fructose diets have been linked to an increased risk of a range of metabolic disorders, including hypertriglyceridemia, hypertension, and diabetes mellitus. These conditions are known to be associated with an elevated risk of developing cardiometabolic diseases. Cardiomyocytes in mammals possess the capacity to proliferate from the moment of their birth. However, this capacity diminishes over time, and cardiac growth is ultimately achieved through cardiomyocyte (CM) hypertrophy. Prior studies have demonstrated that fructose metabolism is enhanced in the heart during pathological hypertrophy [1]. The consumption of foods containing high levels of fructose has been linked to an increase in the size of cardiomyocytes, which can lead to damage to the heart. The impact of high fructose on cardiomyocytes at the point of their initial capacity for proliferation has not been previously documented. In this experiment, our purpose was to explore the impact of high fructose in cardiomyocyte proliferation. To establish an apical resection model in neonatal mice, neonatal ICR mice were randomly divided into a sham-operated group (Sham + PBS), a sham-operated combined high-fructose group (Sham + fructose), an apical resection alone group (AR+PBS) and an apical resection combined with a high-fructose group (AR+fructose). Next, echocardiography was employed to assess the cardiac function of all mice. Masson staining was carried out to analyze cardiac fibrosis. Immunostaining was performed by extracting primary rat cardiomyocytes after the high-fructose intervention to see if proliferation-related markers (Ki67, PH3, Aurora-B) changed, qRT-PCR and immunofluorescence were used to determine changes in the expression profile of Notch1 in the neonatal heart. The results suggest that high fructose could inhibit cardiomyocyte proliferation in vivo and in vitro, The possible mechanism is that high fructose levels inhibit cardiomyocyte proliferation through suppression of Notch1 signaling pathway. In conclusion, high fructose levels inhibit the proliferation of cardiomyocytes via the Notch1 signaling pathway.

Low-density lipoprotein receptor-related protein 6 ameliorates cardiac hypertrophy by regulating CTSD/HSP90α signaling during pressure overload.

Pressure overload induces pathological cardiac remodeling, including cardiac hypertrophy and fibrosis, resulting in cardiac dysfunction or heart failure. Recently, we observed that the low-density lipoprotein receptor-related protein 6 (LRP6), has shown potential in enhancing cardiac function by mitigating cardiac fibrosis in a mouse model subjected to pressure overload. In this study, we investigated the role of LRP6 as a potential modulator of pressure overload-induced cardiac hypertrophy and elucidated the underlying molecular mechanisms. We performed transverse aortic constriction (TAC) to induce pressure overload in cardiomyocyte-specific LRP6 overexpression mice (LRP6-overmice) and in control mice (α-myosin heavy chain (α-MHC) Mer-Cre-Mer Tg mice or namedMCM mice). Cardiac function and hypertrophy were assessed using echocardiography. LRP6-over mice showed improved cardiac function and reduced hypertrophy after TAC, compared with MCM mice. We also applied mechanical stretch to cultured neonatal rat cardiomyocytes to model pressure overload in vitro. Mass spectrometry analysis showed that LRP6 interacts with HSP90α and cathepsin D (CTSD) in cardiomyocytes under mechanical stress. Further analysis demonstrated that LRP6 facilitates CTSD-mediated degradation of HSP90α, consequently inhibiting β-cateninactivation and reducing cardiac hypertrophy post-TAC. Treatment with recombinantHSP90α protein or the CTSD inhibitor, pepstatin A, partly abolished the protective effect of LRP6 overexpression on myocardial hypertrophy and cardiac function after TAC in mice. Collectively, our data suggest that LRP6 protects against pressure overload-induced myocardial remodeling and that the CTSD/HSP90α/β-catenin axis may be a potential therapeutic target.

Novel Selective Cardiac Myosin-Targeted Inhibitors Alleviate Myocardial Ischaemia–Reperfusion Injury

Abstract Purpose Reperfusion of the ischaemic heart is essential to limit myocardial infarction. However, reperfusion can cause cardiomyocyte hypercontracture. Recently, cardiac myosin-targeted inhibitors (CMIs), such as Mavacamten (MYK-461) and Aficamten (CK-274), have been developed to treat patients with cardiac hypercontractility. These CMIs are well tolerated and safe in clinical trials. We hypothesised that, by limiting hypercontraction, CMIs may reduce hypercontracture and protect hearts in the setting of ischaemia and reperfusion (IR). Methods We investigated the ability of MYK-461 and CK-274 to inhibit hypercontracture of adult rat cardiomyocytes (ARVC) in vitro following ATP depletion. A suitable dose of CMIs for subsequent in vivo IR studies was identified using cardiac echocardiography of healthy male Sprague Dawley rats. Rats were anaesthetized and subject to coronary artery ligation for 30 min followed by 2 h of reperfusion. Prior to reperfusion, CMI or vehicle was administered intraperitoneally. Ischaemic preconditioning (IPC) was used as a positive control group. Infarct size was assessed by tetrazolium chloride staining and extent of hypercontracture was assessed by histological staining. Results Treatment with CMIs inhibited ARVC hypercontracture in vitro. MYK-461 (2 mg/kg) and CK-274 (0.5 mg/kg to 2 mg/kg) significantly reduced infarct size vs. vehicle. IR caused extensive contraction band necrosis, which was reduced significantly by IPC but not by CMIs, likely due to assay limitations. GDC-0326, an inhibitor of PI3Kα, abrogated CK-274-mediated protection following IR injury. GDC-0326 reduced phosphorylation of AKT when administered together with CK-274. Conclusion This study identifies CMIs as novel cardioprotective agents in the setting of IR injury.

Deubiquitinase USP47 Ameliorates Cardiac Hypertrophy Through Reducing Protein O-GlcNAcylation.

Cardiac hypertrophy is a crucial risk factor for heart failure when the heart is confronted with physiologic or pathologic stimuli. The ubiquitin-proteasome system plays a critical role in the pathogenesis of cardiac hypertrophy. However, as a key component of the ubiquitin-proteasome system, the role of deubiquitinating enzymes in cardiac hypertrophy is not well understood. In this study, we observed that the expression level of deubiquitinase USP47 was increased in hypertrophic hearts and angiotensin II (Ang II)-stimulated neonatal rat cardiomyocytes. Adenovirus-mediated gain- and loss-of-function approaches indicated that USP47 overexpression significantly attenuated Ang II-induced cardiac hypertrophy in vitro and in vivo, whereas endogenous USP47 deficiency promoted the prohypertrophic effect of Ang II. Further investigation demonstrated that USP47 inhibited O-GlcNAcylation in cardiomyocytes by controlling the expression of O-GlcNAcase. Mechanistically, USP47 bound, deubiquitinated, and stabilized protein arginine methyltransferase 5 (PRMT5), thus upregulating O-GlcNAcase expression. We found that the restoration of PRMT5 abolished the prohypertrophic effects of USP47 silence in vitro. Therefore, our results provide the first evidence of the involvement of USP47 in cardiac hypertrophy and identify USP47 as a potential target for hypertrophic therapy.

Astragali Radix-Notoginseng Radix et Rhizoma medicine pair prevents cardiac remodeling by improving mitochondrial dynamic balance.

Astragali Radix (AR) and Notoginseng Radix et Rhizoma (NR) are frequently employed in cardiovascular disease treatment. However, the efficacy of the AR-NR medicine pair (AN) in improving cardiac remodeling and its underlying mechanism remains unclear. This study aimed to evaluate AN's cardioprotective effect and potential mechanism on cardiac remodeling using transverse aortic constriction (TAC) in mice and angiotensin II (Ang II)-induced neonatal rat cardiomyocytes (NRCMs) and fibroblasts in vitro. High-performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (HPLC-Q-TOF-MS/MS) characterized 23 main components of AN. AN significantly improved cardiac function in the TAC-induced mice. Furthermore, AN considerably reduced the serum levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP), cardiac troponin T (CTn-T), and interleukin-6 (IL-6) and mitigated inflammatory cell infiltration. Post-AN treatment, TAC-induced heart size approached normal. AN decreased cardiomyocyte cross-sectional area and attenuated the upregulation of cardiac hypertrophy marker genes (ANP, BNP, and MYH7) in vivo and in vitro. Concurrently, AN alleviated collagen deposition in TAC-induced mice. AN also reduced the expression of fibrosis-related indicators (COL1A1 and COL3A1) and inhibited the activation of the transforming growth factor-β1 (TGF-β1)/mothers against decapentaplegic homolog 3 (Smad3) pathway. Thus, AN improved TAC-induced cardiac remodeling. Moreover, AN downregulated p-dynamin-related protein (Drp1) (Ser616) expression and upregulated mitogen 2 (MFN-2) and optic atrophy 1 (OPA1) expression in vivo and in vitro, thereby restoring mitochondrial fusion and fission balance. In conclusion, AN improves cardiac remodeling by regulating mitochondrial dynamic balance, providing experimental data for the rational application of Chinese medicine prescriptions with AN as the main component in clinical practice.

Suppression of Dad1 induces cardiomyocyte death by weakening cell adhesion.

As cardiomyocyte loss causes heart failure, inhibition of cardiomyocyte death may be a therapeutic strategy against heart failure. In this study, we have identified defender against cell death 1 (Dad1) as a candidate regulator of cardiomyocyte death, using complementary DNA microarray and siRNA knockdown screening. Dad1 is a subunit of oligosaccharyltransferase (OST) complex that is responsible for protein N-glycosylation; however, its function in cardiomyocytes remains unknown. Importantly, the knockdown of Dad1 using siRNA reduced the viability of neonatal rat cardiomyocytes (NRCMs), accompanied by cleaved caspase3 expression, independent of endoplasmic reticulum stress. Dad1 knockdown impaired cell spreading and reduced myofibrillogenesis in NRCMs, suggesting that Dad1 knockdown induced anoikis, apoptosis by disrupting cell-matrix interactions. Consistently, knockdown of Dad1 impaired N-glycosylation of integrins α5 and β1, accompanied by inactivation of focal adhesion kinase. When cell adhesion was enhanced using adhesamine, fibronectin, or collagen type IV, cardiomyocyte death induced by Dad1 knockdown was reduced. Dad1 knockdown decreased the expression of staurosporine and temperature-sensitive 3 A (Stt3A), a catalytic subunit of OST complex. Interestingly, Stt3A knockdown using Stt3A siRNA reduced the expression of Dad1, indicating that both Dad1 and Stt3A were required for OST stabilization. In conclusion, Dad1 plays an important role in maintaining the expression of mature N-glycosylated integrins and their downstream signaling molecules to suppress cardiomyocyte anoikis.NEW & NOTEWORTHY This study found for the first time that the knockdown of Dad1 induced cardiomyocyte death, accompanied by impairment of myofibrillogenesis and cell spreading. Dad1 regulates the N-glycosylation of integrins in cooperation with Stt3A and preserves cell adhesion activity, promoting cardiomyocyte survival. This is the first demonstration that Dad1 contributes to the maintenance of cardiac homeostasis through the posttranslational modification of integrins, providing a novel insight into the biological significance of OST complex in cardiomyocytes.

VPO1 Promotes Programmed Necrosis of Cardiomyocytes in Rats with Chronic Heart Failure by Upregulating CYLD.

Chronic heart failure (CHF) is a serious cardiovascular condition. Vascular peroxidase 1 (VPO1) is associated with various cardiovascular diseases, yet its role in CHF remains unclear. This research aims to explore the involvement of VPO1 in CHF. CHF was induced in rats using adriamycin, and the expression levels of VPO1 and cylindromatosis (CYLD) were assessed. In parallel, the effects of VPO1 on programmed necrosis in H9c2 cells were evaluated through cell viability assays, lactate dehydrogenase (LDH) level measurements, and analysis of receptor-interacting protein kinase 1/receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein (RIPK1/RIPK3/MLKL) pathway-related proteins. The impact of CYLD on RIPK1 protein stability and ubiquitination was also investigated, along with the interaction between VPO1 and CYLD. Additionally, cardiac structure and function were assessed using echocardiography, Hematoxylin-eosin (HE) staining, Masson staining, and measurements of myocardial injury-related factors, including N-terminal prohormone of brain natriuretic peptide (NT-proBNP), Aspartate aminotransferase (AST), LDH, and creatine kinase-myocardial band (CK-MB). VPO1 expression was upregulated in CHF rats and in H9c2 cells treated with adriamycin. In cellular experiments, VPO1 knockdown improved cell viability, inhibited necrosis and the expression of proteins associated with the RIPK1/RIPK3/MLKL pathway. Mechanistically, VPO1 promoted cardiomyocyte programmed necrosis by interacting with the deubiquitinating enzyme CYLD, which enhanced RIPK1 ubiquitination and degradation, leading to activation of the RIPK1/RIPK3/MLKL signaling pathway. At animal level, overexpression of CYLD counteracted the cardiac failure, cardiac hypertrophy, myocardial injury, myocardial fibrosis, and tissue necrosis caused by VPO1 knockdown. VPO1 exacerbates cardiomyocyte programmed necrosis in CHF rats by upregulating CYLD, which activates the RIPK1/RIPK3/MLKL signaling pathway. Thus, VPO1 may represent a potential therapeutic target for CHF.

High Glucose Sensitizes Male and Female Rat Cardiomyocytes to Wnt/β-Catenin Signaling.

Wnt/β-catenin signaling has been shown to regulate gene expressions in cardiomyocytes. However, it is not known if this effect is dependent on the sex of cells or the glucose level in the culture medium. In the present study, ventricular myocytes were prepared from male and female neonatal rats and maintained in either a glucose-rich (25 mM) medium or a low-glucose (3 mM), lipid-rich medium. Real-time quantitative PCR was used to measure changes in target genes (Axin2, Scn5a, and Tbx3) after treatment with 1, 3, or 10 µM of CHIR-99021, an activator of Wnt/β-catenin signaling. CHIR induced similar changes in Axin2, Tbx3, and Scn5a transcripts in male and female NRVMs in both media, suggesting the absence of sex difference. However, cells in a high-glucose medium showed greater increases in Axin2 and Tbx3 transcripts than cells in a low-glucose medium. In addition, a low concentration of CHIR (1 µM) reduced the Scn5a transcript in cells in a high-glucose medium but not in a low-glucose medium, suggesting an increased sensitivity to Wnt signaling by high glucose. A non-linear relationship was identified between Axin2 transcript upregulation and Scn5a transcript downregulation in CIHR-treated NRVMs. These data suggest that high glucose sensitizes both male and female cardiomyocytes to Wnt/β-catenin signaling.

Ilexgenin A Alleviates Myocardial Ferroptosis in Response to Ischemia Reperfusion Injury via the SIRT1 Pathway.

Myocardial ischemia-reperfusion (I/R) injury has emerged as an increasingly serious cardiovascular health concern worldwide, with ferroptosis playing a pivotal role as the underlying pathogenic process. This study aimed to investigate the pharmacological effect and mechanism of Ilexgenin A on cardiomyocyte ferroptosis induced by myocardial I/R injury. Invivo, we established a murine anterior descending artery ligation/recanalization model to evaluate the cardioprotective effect of Ilexgenin A. Bioinformatics analysis, molecular docking, and Surface Plasmon Resonance imaging were conducted to predict the pharmacological targets of Ilexgenin A. Invitro experiments, the neonatal rat cardiomyocytes (NRCMs) were utilized to further explore the mechanism of Ilexgenin A in inhibiting ferroptosis using chemiluminescence and immunofluorescence staining, electron microscopy, biochemical assay, RT-qPCR, western blotting, and so on. The results showed that Ilexgenin A protected against cardiac dysfunction, ameliorated myocardial ferroptosis and mitochondrial damage induced by murine myocardial I/R injury via the silence information regulator 1 (SIRT1) pathway, the trend was consistently observed in NRCMs. Additionally, the SIRT1 knockdown by siRNA delivery partially abrogated the beneficial effects of Ilexgenin A on ameliorating mitochondrial damage, and then aggravated erastin-induced ferroptosis in NRCMs. Overall, Our research demonstrated that the inhibition of ferroptosis via the SIRT1 pathway was one of the mechanisms by which Ilexgenin A exerted cardioprotective effect.

Exosomes derived from cardiac fibroblasts with Ang-II stimulation provoke myocardial hypertrophy via miR-15b-5p/PTEN-L axis.

This study aimed to examine the impact of exosomes derived from Ang II-stimulated cardiac fibroblasts (CFs) on myocardial hypertrophy. Neonatal rat CFs were isolated and identified using Vimentin immunofluorescence. Following Ang II stimulation, exosomes were collected, characterized, and subjected to miRNA sequencing. Myocardial hypertrophy models were induced both in vitro and in vivo using Ang II. CFs were transfected with miR-15b-5p mimics or inhibitors, and their exosomes were co-cultured with rat cardiomyocytes (H9C2). Changes in cell viability, myocardial hypertrophy, and the expression levels of PTEN-L, PINK1, and Parkin proteins were assessed using the CCK-8 assay, cell surface area evaluation, and Western blot analysis. Cardiac tissue pathology and myocardial hypertrophy were evaluated through HE and WAG staining, respectively, while PTEN-L expression was detected by immunohistochemistry. The results demonstrated successful isolation of CFs and their exosomes, with miR-15b-5p significantly enriched in the exosomes derived from Ang II-stimulated CFs (Ang II-CFs-Exos). Ang II-CFs-Exos inhibited cell viability, exacerbated myocardial hypertrophy, and activated mitophagy via miR-15b-5p in the in vitro myocardial hypertrophy model. PTEN-L was identified as a downstream target of miR-15b-5p, with its overexpression reversed the effects of miR-15b-5p mimic on myocardial hypertrophy and mitophagy. Additionally, mitochondrial inhibitors also countered the effects of the miR-15b-5p mimic on myocardial hypertrophy. Furthermore, Ang II-CFs-Exos exacerbated myocardial hypertrophy in rats, while knockout of miR-15b-5p in Ang II-CFs-Exos mitigated this effect. To sum up, Ang II-CFs-Exos promote myocardial hypertrophy by modulating PINK1/Parkin signaling -mediated mitophagy through the miR-15b-5p/PTEN-L axis.

Sodium, Potassium-Adenosine Triphosphatase as a Potential Target of the Anti-Tuberculosis Agents, Clofazimine and Bedaquiline.

Multidrug-resistant tuberculosis (MDR-TB) patients are treated with a standardised, short World Health Organization (WHO) regimen which includes clofazimine (CFZ) and bedaquiline (BDQ) antibiotics. These two antibiotics lead to the development of QT prolongation in patients, inhibiting potassium (K+) uptake by targeting the voltage-gated K+ (Kv)11.1 (hERG) channel of the cardiomyocytes (CMs). However, the involvement of these antibiotics to regulate other K+ transporters of the CMs, as potential mechanisms of QT prolongation, has not been explored. This study determined the effects of CFZ and BDQ on sodium, potassium-adenosine triphosphatase (Na+,K+-ATPase) activity of CMs using rat cardiomyocytes (RCMs). These cells were treated with varying concentrations of CFZ and BDQ individually and in combination (1.25-5 mg/L). Thereafter, Na+,K+-ATPase activity was determined, followed by intracellular adenosine triphosphate (ATP) quantification and cellular viability determination. Furthermore, molecular docking of antibiotics with Na+,K+-ATPase was determined. Both antibiotics demonstrated dose-response inhibition of Na+,K+-ATPase activity of the RCMs. The greatest inhibition was demonstrated by combinations of CFZ and BDQ, followed by BDQ alone and, lastly, CFZ. Neither antibiotic, either individually or in combination, demonstrated cytotoxicity. Molecular docking revealed an interaction of both antibiotics with Na+,K+-ATPase, with BDQ showing higher protein-binding affinity than CFZ. The inhibitory effects of CFZ and BDQ, individually and in combination, on the activity of Na+,K+-ATPase pump of the RCMs highlight the existence of additional mechanisms of QT prolongation by these antibiotics.

Cardioprotective Role of Tinospora cordifolia against Trimethylamine-N-Oxide and Glucose Induced Stress in Rat Cardiomyocytes.

Type 2 diabetes has become a concern issue that affects the quality of life and can increase the risk of cardiac insufficiency elevating the threat to the life safety of patients. A recognized cause of cardiac insufficiency is diabetic cardiomyopathy, chronic hyperglycemia, and myocardial lipotoxicity which can reduce the myocardial contractile performance, and enhance the cardiomyocyte hypertrophy and interstitial fibrosis. The cause of diabetic cardiomyopathy is multi-factorial which includes oxidative stress, insulin resistance, inflammation, apoptosis, and autophagy. Recent clinical studies have suggested the dysbiosis of gut microbiota, secretion of metabolites, and their diffusion in to the host as to have direct detrimental effects on the cardiac contractility. In the present paper, we have done in silico studies including molecular interaction of phytoconstituents of Tinospora cordifolia against reactive oxygen species producing proteins. Whereas, in vitro studies were conducted on H9C2 cardiac cells including cell morphological examination, level of reactive oxygen species, cell count-viability, apoptotic status, in the presence of high glucose, trimethylamine-n-oxide, and plant extracts which were determined through cell analyzer and microscopic assays. The treatment of high glucose and trimethylamine-n-oxide was found to be increase the cardiac stress approximately two fold by attenuating hypertrophic conditions, oxidative stress, and apoptosis in rat cardiomyocytes, and Tinospora cordifolia was found to be a cardioprotective agent. Conclusively, our study has reported that the Indian medicinal plant Tinospora cordifolia has the ability to treat diabetic cardiomyopathy. Our study can open up a new herbal therapeutic strategy against diabetic cardiomyopathy.

Lipotoxicity-induced upregulation of FIS1 exacerbates mitochondrial fragmentation and promotes NLRP3-dependent pyroptosis in diabetic cardiomyopathy.

Lipotoxicity is a significant factor in the pathogenesis of diabetic cardiomyopathy (DbCM), a condition characterized by mitochondrial fragmentation and pyroptosis. Mitochondrial fission protein 1 (FIS1) plays a role in mitochondrial fission by anchoring dynamin-related protein 1 (DRP1). However, the specific contribution of FIS1 to DbCM remains unclear. This study aims to clarify how lipotoxicity-induced upregulation of FIS1 affects mitochondrial fragmentation and the mechanisms linking this fragmentation to NLRP3-dependent pyroptosis in DbCM. To model lipotoxicity in DbCM, we used db/db mice and primary neonatal rat cardiomyocytes (NRCMs) treated with palmitic acid (PA) and conducted a series of in vivo and in vitro experiments. Gain- and loss-of-function studies on NRCMs were performed using pharmacological inhibitors and small interfering RNA (siRNA) transfection, and we assessed the expression and function of FIS1, mitochondrial dynamics, mitochondrial reactive oxygen species (mitoROS) production, NLRP3-dependent pyroptosis, and their interrelationships. Our results show that in the myocardium of db/db mice, NLRP3-dependent pyroptosis is associated with upregulation of FIS1, mitochondrial fragmentation, and increased oxidative stress. In NRCMs subjected to PA, the application of VX-765 and MCC950 to inhibit caspase-1 and NLRP3, respectively, significantly reduced pyroptosis. Additionally, pretreatment with Mito-TEMPO (MT) demonstrated that mitoROS are critical initiators for NLRP3 inflammasome activation and subsequent pyroptosis. Furthermore, PA-induced upregulation of FIS1 exacerbates mitochondrial fragmentation. Downregulation of FIS1 or inhibition of FIS1/DRP1 interaction reversed mitochondrial fragmentation, reduced mitoROS levels, and mitigated pyroptosis. Lipotoxicity-induced FIS1 upregulation exacerbates mitochondrial fragmentation through its interaction with DRP1, leading to increased mitoROS production and the initiation of NLRP3-dependent pyroptosis in DbCM. Therefore, targeting FIS1 emerges as a potential therapeutic approach for managing DbCM.

IGFBP5 affects cardiomyocyte survival and functional recovery in mice following myocardial ischemia

Insulin-like growth factor-binding protein 5 (IGFBP5) has been shown to be useful for the diagnosis and treatment of multiple tumors and cerebrovascular diseases. However, it is unknown whether IGFBP5 is involved in myocardial repair following myocardial infarction (MI). Here we show high expression of IGFBP5 in multiple models of ischemic and hypoxic injury. IGFBP5 affected the proliferation of neonatal rat cardiomyocytes (NRCMs) and the cardiomyocyte apoptosis induced by oxygen-glucose deprivation (OGD). Subsequently, heart-specific IGFBP5 knockdown inhibited myocardial apoptosis and increased cardiomyocyte proliferation in mice with MI. During the chronic remodeling stage, heart-specific regulation of IGFBP5 ameliorated pathological cardiac remodeling and dysfunction. Mechanistically, IGFBP5 regulated cardiomyocyte survival through the insulin-like growth factor 1 (IGF1) receptor (IGF1R)/protein kinase B (PKB/AKT) pathway. In summary, our results provide mechanistic insights into the effect of IGFBP5 on cardiomyocyte during cardiac repair. IGFBP5 may represent a therapeutic target for myocardial ischemic injury.

Non-thermal atmospheric pressure plasma-irradiated cysteine protects cardiac ischemia/reperfusion injury by preserving supersulfides

Ischemic heart disease is the main global cause of death in the world. Abnormal sulfide catabolism, especially hydrogen sulfide accumulation, impedes mitochondrial respiration and worsens the prognosis after ischemic insults, but the substantial therapeutic strategy has not been established. Non-thermal atmospheric pressure plasma irradiation therapy is attracted attention as it exerts beneficial effects by producing various reactive molecular species. Growing evidence has suggested that supersulfides, formed by catenation of sulfur atoms, contribute to various biological processes involving electron transfer in cells. Here, we report that non-thermal plasma-irradiated cysteine (Cys∗) protects mouse hearts against ischemia/reperfusion (I/R) injury by preventing supersulfide catabolism. Cys∗ has a weak but long-lasting supersulfide activity, and the treatment of rat cardiomyocytes with Cys∗ prevents mitochondrial dysfunction after hypoxic stress. Cys∗ increases sulfide-quinone oxidoreductase (SQOR), and silencing SQOR abolishes Cys∗-induced supersulfide formation and cytoprotection. Local administration of mouse hearts with Cys∗ significantly reduces infarct size with preserving supersulfide levels after I/R. These results suggest that maintaining supersulfide formation through SQOR underlies cardioprotection by Cys∗ against I/R injury.

Empagliflozin improves pressure-overload-induced cardiac hypertrophy by inhibiting the canonical Wnt/β-catenin signaling pathway.

Empagliflozin (EMPA) is an SGLT-2 inhibitor that can control hyperglycemia. Clinical trials have indicated its cardio-protective effects against cardiac remodeling in diabetes or non-diabetes patients. However, the underlying molecular mechanisms of EMPA's cardio-protective effects remain elusive. We evaluated whether the EMPA attenuated the pressure-overload-induced cardiac hypertrophy by inhibiting the Wnt/β-catenin pathway. Furthermore, the effects of the EMPA on a mouse model of transverse aortic constriction (TAC) induced cardiac hypertrophy was also evaluated. Mice were administrated with 0.5% CMC-Na as a vehicle or EMPA (10mg/kg/day, daily, throughout the study) by intragastric gavage. The in vivo echocardiography and histologic morphological analyses revealed that EMPA attenuated TAC-induced cardiac hypertrophy. Moreover, it also ameliorated TAC-induced cardiac fibrosis and decreased the cell size of the cardiomyocytes in isolated adult cardiomyocytes. Molecular mechanism analysis revealed that the EMPA reduced the TAC-induced enhanced expression of the Wnt/β-catenin pathway in vivo. For in vitro assessments, isolated neonatal rat cardiomyocytes (NRCMs) were treated with Angiotensin II (AngII) and EMPA; the results showed that in the absence of EMPA, the expression of the Wnt/β-catenin pathway was enhanced. In the trans-genetic heterozygous β-catenin deletion mice, EMPA attenuated TAC-induced cardiac remodeling by reducing the Wnt/β-catenin pathway. In addition, molecular docking analysis indicated that EMPA interacts with FZD4 to inhibit the TAC and AngII induced Wnt/β-catenin pathway in cardiomyocytes. Our study illustrated that EMPA might directly interact with FZD4 to inhibit the TAC and AngII-induced activation of the Wnt/β-catenin pathway to attenuate the adverse cardiac remodeling.