A previous study showed that high-glucose (HG) conditions induce mitochondria fragmentation through the calcium-mediated activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) in H9C2 cells. This study tested whether empagliflozin could prevent HG-induced mitochondria fragmentation through this pathway. We found that exposing H9C2 cells to an HG concentration decreased cell viability and increased cell apoptosis and caspase-3. Empagliflozin could reverse the apoptosis effect of HG stimulation on H9C2 cells. In addition, the HG condition caused mitochondria fragmentation, which was reduced by empagliflozin. The expression of mitochondria fission protein was upregulated, and fusion proteins were downregulated under HG stimulation. The expression of fission proteins was decreased under empagliflozin treatment. Increased calcium accumulation was observed under the HG condition, which was decreased by empagliflozin. The increased expression of ERK 1/2 under HG stimulation was also reversed by empagliflozin. Our study shows that empagliflozin could reverse the HG condition, causing a calcium-dependent activation of the ERK 1/2 pathway, which caused mitochondria fragmentation in H9C2 cells.

BACKGROUND: Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Ischemic heart disease is one of the most harmful conditions to cellular structure and function. After reperfusion treatment, a spectrum of adverse effects becomes evident, encompassing altered cell viability, heightened oxidative stress, activated autophagy, and increased apoptosis. Photobiomodulation (PBM) has been utilized in experimental models of cardiac hypoxia to enhance mitochondrial response and ameliorate biochemical changes in injured tissue. However, the effects of PBM on cultured cardiomyocytes subjected to hypoxia/reoxygenation are not yet well established.
METHODS: H9C2 cardiomyocytes were exposed to hypoxia with concentrations of 300 μM CoCl2 for 24 h, followed by 16 h of reoxygenation through incubation in a normoxic medium. Treatment was conducted using GaAIAs Laser (850 nm) after hypoxia at an intensity of 1 J/cm2. Cells were divided into three groups: Group CT (cells maintained under normoxic conditions), Group HR (cells maintained in hypoxia and reoxygenation conditions without treatment), Group HR + PBM (cells maintained in hypoxia and reoxygenation conditions that underwent PBM treatment). Cell viability was analyzed using MTT, and protein expression was assessed by western blot. One-way ANOVA with the Tukey post hoc test was used for data analysis. Differences were significant when p < 0.05.
RESULTS: PBM at an intensity of 1 J/cm2 mitigated the alterations in cell survival caused by hypoxia/reoxygenation. Additionally, it significantly increased the expression of proteins Nrf2, HSP70, mTOR, LC3II, LC3II/I, and Caspase-9, while reducing the expression of PGC-1α, SOD2, xanthine oxidase, Beclin-1, LC3I, and Bax.
CONCLUSIONS: PBM at intensities of 1 J/cm2 reverses the changes related to oxidative stress, mitochondrial biogenesis, autophagy, and apoptosis caused by hypoxia and reoxygenation in a culture of cardiomyocytes.

SCN5A mutations have been reported to cause various cardiomyopathies in humans. Most of the SCN5A mutations causes loss of function and thereby, alters the overall cellular function. Therefore, to understand the loss of SCN5A function in cardiomyocytes, we have knocked down the SCN5A gene (SCN5A-KD) in H9c2 cells and explored the cell phenotype and molecular behaviors in the presence and absence of isoproterenol (ISO), an adrenergic receptor agonist that induces cardiac hypertrophy. Expression of several genes related to hypertrophy, inflammation, fibrosis, and energy metabolism pathways were evaluated. It was found that the mRNA expression of hypertrophy-related gene, brain (B-type) natriuretic peptide (BNP) was significantly increased in SCN5A-KD cells as compared to \'control\' H9c2 cells. There was a further increase in the mRNA expressions of BNP and βMHC in SCN5A-KD cells after ISO treatment compared to their respective controls. Pro-inflammatory cytokine, tumor necrosis factor-alpha expression was significantly increased in \'SCN5A-KD\' H9c2 cells. Further, metabolism-related genes like glucose transporter type 4, cluster of differentiation 36, peroxisome proliferator-activated receptor alpha, and peroxisome proliferator-activated receptor-gamma were significantly elevated in the SCN5A-KD cells as compared to the control cells. Upregulation of these metabolic genes is associated with increased ATP production. The study revealed that SCN5A knock-down causes alteration of gene expression related to cardiac hypertrophy, inflammation, and energy metabolism pathways, which may promote cardiac remodelling and cardiomyopathy.

The current study aimed to assess the effectiveness of pharmacological intervention with Platycodin D (PD), a critically active compound isolated from the roots of Platycodon grandiflorum, in mitigating cardiotoxicity in a murine model of type 2 diabetes-induced cardiac injury and in H9c2 cells in vitro. Following oral administration for 4 weeks, PD (2.5 mg/kg) significantly suppressed the elevation of fasting blood glucose (FBG) levels, improved dyslipidemia, and effectively inhibited the rise of the cardiac injury markers creatine kinase isoenzyme MB (CK-MB) and cardiac troponin T (cTnT). PD treatment could ameliorate energy metabolism disorders induced by impaired glucose uptake by activating AMPK protein expression in the DCM mouse model, thereby promoting the GLUT4 transporter and further activating autophagy-related proteins. Furthermore, in vitro experiments demonstrated that PD exerted a concentration-dependent increase in cell viability while also inhibiting palmitic acid and glucose (HG-PA)-stimulated H9c2 cytotoxicity and activating AMPK protein expression. Notably, the AMPK activator AICAR (1 mM) was observed to upregulate the expression of AMPK in H9c2 cells after high-glucose and -fat exposure. Meanwhile, we used AMPK inhibitor Compound C (20 μM) to investigate the effect of PD activation of AMPK on cells. In addition, the molecular docking approach was employed to dock PD with AMPK, revealing a binding energy of -8.2 kcal/mol and indicating a tight interaction between the components and the target. PD could reduce the expression of autophagy-related protein p62, reduce the accumulation of autophagy products, promote the flow of autophagy, and improve myocardial cell injury. In conclusion, it has been demonstrated that PD effectively inhibits cardiac injury-induced type 2 diabetes in mice and enhances energy metabolism in HG-PA-stimulated H9c2 cells by activating the AMPK signaling pathway. These findings collectively unveil the potential cardioprotective effects of PD via modulation of the AMPK signaling pathway.

Background: Ischemic heart disease is a leading cause of death in patients with cardiovascular disease. Natural products containing high antioxidant activity have been used as an alternative therapy to improve the living conditions of patients. In this study, we examine the protective effect of tamarind seed (TS) on myocardial hypoxic injury. Methods: The hypoxia model was mimicked by mineral oil overlayed on H9c2 cardiomyoblasts for 4 h. TS extract was pretreated and administered during the hypoxic condition. Radical scavenging activity of TS extract was measured and exhibited very potent antioxidant activities on 2,2-diphenyl-2-picrylhydrazyl (DPPH) and 2,2\'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. Results: TS extract at a concentration of 10 µg/ml significantly reversed the effect of hypoxia-induced cell death and intracellular reactive oxygen species (ROS) production. We also observed hypoxia-induced over-expression of both inflammatory cytokine mRNA and activation of cellular apoptosis. Pretreatment of TS extract significantly reduced hypoxia-induced HIF-1a and pro-inflammatory cytokine production, IL-1b and IL-6. The Western blot analysis for apoptotic regulatory molecules, caspase 3, caspase 8 and Bax proteins, also showed hypoxia injury reversal by TS extract treatment. Conclusions: The results suggest that the anti-ischemic effect of TS extract protects against hypoxia-induced injury and has potential to be an effective alternative therapy for ischemic heart disease and oxidative-damage related disease.

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how (i) the absence or presence of different cryoprotectants, (ii) different cell-cryoprotectant incubation conditions, and (iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays.

UNASSIGNED: Luteolin is a flavone that provides defense against myocardial ischemia/reperfusion (I/R) injury. However, this compound is subjected to methylation mediated by catechol-O-methyltransferase (COMT), thus influencing its pharmacological effect. To synthesize a new flavone from luteolin that avoids COMT-catalyzed methylation and find out the protective mechanism of LUA in myocardial I/R injury.
UNASSIGNED: Luteolin and 2,2\'-azobis (2-amidinopropane) dihydrochloride (AAPH) were used to synthesize the new flavone known as LUAAPH-1 (LUA). Then, the myocardial ischemia/reperfusion injury cell model was established using H9c2 cells to detect the effect in myocardial ischemia/reperfusion regulation and to identify the underlying mechanism.
UNASSIGNED: Pretreatment with LUA (20 μmol/l) substantially increased cell viability while reducing cell apoptosis rate and caspase-3 expression induced by I/R, and the protective effect of LUA on cell viability was stronger than diosmetin, which is the major methylated metabolite of luteolin. In addition, intracellular reactive oxygen species (ROS) production and calcium accumulation were both inhibited by LUA. Furthermore, we identified that LUA markedly relieved the promotive effects of I/R stimulation upon JNK and p38 phosphorylation.
UNASSIGNED: LUT pretreatment conveys significant cardioprotective effects after myocardial I/R injury, and JNK and p38 MAPK signaling pathway may be involved.

The Sirtuins family, formally known as the Silent Information Regulator Factors, constitutes a highly conserved group of histone deacetylases. Recent studies have illuminated SIRT6\'s role in doxorubicin (DOX)-induced oxidative stress and apoptosis within myocardial cells. Nevertheless, the extent of SIRT6\'s impact on DOX-triggered myocardial cell aging and damage remains uncertain, with the associated mechanisms yet to be fully understood. In our research, we examined the influence of SIRT6 on DOX-induced cardiomyocyte senescence using β-galactosidase and γ-H2AX staining. Additionally, we gauged the mRNA expression of senescence-associated genes, namely p16, p21, and p53, through Real-time PCR. Employing ELISA assay kits, MDA, and total SOD activity assay kits, we measured inflammatory factors TNF-α, IL-6, and IL-1β, alongside oxidative stress-related indicators. The results unequivocally indicated that SIRT6 overexpression robustly inhibited DOX-induced cardiomyocyte senescence. Furthermore, we established that SIRT6 overexpression suppressed the inflammatory response and oxidative stress induced by DOX in cardiomyocytes. Conversely, silencing SIRT6 exacerbated DOX-induced cardiomyocyte injury. Our investigations further unveiled that SIRT6 upregulated the expression of genes CD36, CPT1, LCAD, MCAD associated with fatty acid oxidation through its interaction with PPARα, thereby exerting anti-aging effects. In vivo, the overexpression of SIRT6 was observed to restore DOX-induced declines in EF and FS to normal levels in mice. Echocardiography and HE staining revealed the restoration of cardiomyocyte alignment, affording protection against DOX-induced myocardial senescence and injury. The findings from this study suggest that SIRT6 holds significant promise as a therapeutic target for mitigating DOX-induced cardiomyopathy.

Increasing evidence shows that maternal hyperglycemia inhibits cardiomyocyte (CM) proliferation and promotes cell apoptosis during fetal heart development, which leads to cardiac dysplasia. Accumulating evidence suggests that the overexpression of miR-21 in CMs has a protective role in cardiac function. Therefore, we investigated whether miR-21 can rescue CM injury caused by high glucose. First, we performed biological function analysis of miR-21-5p overexpression in H9c2 cells treated with high glucose. We found that the proliferation of H9c2 cells treated with high glucose decreased significantly and was rescued after overexpression of miR-21-5p. CCK-8 and EdU incorporation assays were performed to assess cell proliferation. The cell proliferation of the miR-21-5p mimic transfection group was improved compared with that of the NC mimic group (*p < 0.05, miR-21-5p mimics vs. NC mimics) when the proliferation of H9c2 cells was reduced by high glucose (****p < 0.0001, high glucose (HG) vs. normal glucose (NG)). Then, we verified the targeted and negative regulation of miR-21-5p on Rhob using a dual-luciferase activity assay and RT-qPCR, respectively. We further demonstrated that miR-21-5p regulates Rhob to rescue the inhibition of CM proliferation induced by high glucose. The CCK-8 results showed that the cell proliferation of the siRNA-Rhob group was higher than that of the NC mimic group (***p < 0.001) and that of the cotransfection group with Up-Rhob plasmids and miR-21-5p mimics was lower than that of the miR-21-5p mimic group (*p < 0.05). Conclusion: Overexpression of miR-21-5p rescues the inhibition of high glucose-induced CM proliferation through regulation of Rhob.

Micropillars have emerged as promising tools for a wide range of biological applications, while the influence of magnetic fields on cell behavior regulation has been increasingly recognized. However, the combined effect of micropillars and magnetic fields on cell behaviors remains poorly understood. In this study, we investigated the responses of H9c2 cells to ultramicromagnetic micropillar arrays using NdFeB as the tuned magnetic particles. We conducted a comparative analysis between PDMS micropillars and NdFeB/PDMS micropillars to assess their impact on cell function. Our results revealed that H9c2 cells exhibited significantly enhanced proliferation and notable cytoskeletal rearrangements on the ultramicromagnetic micropillars, surpassing the effects observed with pure PDMS micropillars. Immunostaining further indicated that cells cultured on ultramicromagnetic micropillars displayed heightened contractility compared to those on PDMS micropillars. Remarkably, the ultramicromagnetic micropillars also demonstrated the ability to decrease reactive oxygen species (ROS) levels, thereby preventing F-actin degeneration. Consequently, this study introduces ultramicromagnetic micropillars as a novel tool for the regulation and detection of cell behaviors, thus paving the way for advanced investigations in tissue engineering, single-cell analysis, and the development of flexible sensors for cellular-level studies.