Cardiomyocyte Damage Research Articles

Schisandrin C prevents Regorafenib-Induced cardiotoxicity by recovering EPHA2 expression in cardiomyocytes.

Regorafenib, an oral multikinase inhibitor of angiogenic, stromal, and oncogenic receptor tyrosine kinases, has been approved for the treatment of metastatic colorectal cancer, gastrointestinal stromal tumors and hepatocellular carcinoma by the US Food and Drug Administration and European Medicines Agency. However, regorafenib-induced cardiotoxicity increases the risk of mortality. Despite reports that regorafenib can cause mitochondrial dysfunction in cardiomyocytes, the molecular mechanism of regorafenib-induced cardiotoxicity is much less known and there is an urgent need for intervention strategies. Here, we treated mice with vehicle or 200 mg/kg regorafenib daily for 42 days by gavage or treated cardiomyocyte lines with 8, 16 or 32 μM regorafenib, and we found that regorafenib could cause apoptosis, mitochondrial injury and DNA damage in cardiomyocytes. Mechanistically, regorafenib can reduce the expression of EPHA2, which inhibits AKT signaling, leading to cardiomyocyte apoptosis and cardiotoxicity. In addition, we showed that recovering EPHA2 expression via plasmid-induced overexpression of EPHA2 or schisandrin C, a natural product, could prevent regorafenib-induced cardiotoxicity. These findings demonstrated that regorafenib causes cardiomyocyte apoptosis and cardiac injury by reducing the expression of EPHA2 and schisandrin C could prevent regorafenib-induced cardiotoxicity by recovering EPHA2 expression, which provides a potential management strategy for regorafenib-induced cardiotoxicity and will benefit the safe application of regorafenib in clinic.

Mechanistic insights into Suanzaoren Decoction's improvement of cardiac contractile function in anxiety-induced cardiac insufficiency

Ethnopharmacological relevanceAccording to traditional Chinese medicine, Anxiety-induced cardiac blood insufficiency leads to palpitations and restlessness. Suanzaoren Decoction (SD) is effective in replenishing blood and promoting blood circulation. Clinical practice has shown that it has a better therapeutic effect on cardiac insufficiency. However, its mechanism of action is still unclear. Aim of the studyThe study aims to determine the mechanism by which SD treats chronic restraint stress (CRS)-induced anxiety-induced cardiac insufficiency (ACI). Materials and methodsSD was orally administered to mice with CRS-induced ACI. Firstly, we constructed an anxiety model in mice by CRS. Subsequently, SD was investigated to assess cardiac function and pathological changes through echocardiography, H&E staining, and Masson staining. Thirdly, the function of sympathetic and parasympathetic nerves was evaluated using enzyme-linked immunosorbent assay (ELISA) and enzyme activity assays. Network pharmacology and molecular docking were employed to predict potential targets for SD treatment of cardiac insufficiency. CaMKII expression was scrutinized utilizing publicly accessible databases. CaMKII was identified as a target through immunohistochemistry and Western Blot analysis in mouse hearts. Finally, the therapeutic mechanism of SD was confirmed in injured cardiomyocytes via Western Blot and quantitative PCR. ResultsSD exerted anxiolytic effects by increasing the frequency of entries into and the duration spent in open arms while reducing the time spent in the light chamber and increasing the number of transitions between light and dark chambers. Additionally, it mitigated cardiac insufficiency, as evidenced by the enhancement of left ventricular ejection fraction (LVEF) and attenuation of cardiomyocyte damage and inflammatory infiltration.However, SD did not alleviate the elevated norepinephrine (NE) and decreased Acetylcholine (Ach) in anxiety states. To investigate the mechanism of action of SD, we constructed a Drug-Component-Target-Disease network, identifying 13 potential active compounds. Additionally, leveraging bioinformatics analysis and molecular docking targeting heart diseases characterized by clinical left ventricular ejection fraction (LVEF), we focused on the CaMKII target. The ability of SD to modulate CaMKII expression and phosphorylation in the mouse heart was investigated using immunohistochemistry and Western blotting. SD was found to alleviate NE-injured cardiomyocytes by modulating the Ca2+/CaMKII/MEF2 and GATA4 pathways. ConclusionSD is a potential formula for the treatment of chronic restraint stress (CRS)-induced ACI that ameliorates cardiomyocyte injury and improves cardiac function. Its efficacy is associated with the inhibition of the Ca2+/CaMKII/MEF2 and GATA4 signaling pathways.

NRIP1 is a downstream target of YY1 in promoting OGD/R-induced H9c2 cardiomyocyte injury and mitochondrial dysfunction.

From a clinical standpoint, myocardial ischemia/reperfusion injury (MIRI) has always been an enormous challenge for the treatment of acute myocardial infarction (AMI). Molecular targeting therapy may help overcome this challenge. The present work aimed to elucidate the possible involvement of Yin-Yang 1 (YY1)/nuclear receptor-interacting protein 1 (NRIP1) and discover the molecular mechanism of MIRI. Herein, a cardiomyocyte ischemia/reperfusion (I/R) model was established via oxygen-glucose deprivation/re-oxygenation (OGD/R) damage in H9c2 cardiomyocytes. Reverse transcription-quantitative PCR and western blotting were conducted to measure the levels of YY1 and NRIP1 at the RNA and protein levels, respectively. H9c2 cell viability and apoptosis were assayed using the Cell Counting Kit-8, flow cytometry, and western blotting. In addition, superoxide dismutase, glutathione peroxidase, and malondialdehyde levels were analyzed as markers of oxidative stress. Additionally, mitochondrial membrane potential, which was measured via JC-1 staining, ATP content, Complex I activity, mitochondrial DNA copy number, and mitochondrial permeability transition pore (mPTP) opening rate were analyzed to evaluate mitochondrial activity. Moreover, luciferase reporter and chromatin immunoprecipitation assays experimentally validated the predicted affinity of YY1 with the NRIP1 promoter according to the HumanTFDB online tool. YY1/NRIP1 were both highly expressed in OGD/R-injured H9c2 cardiomyocytes. Downregulation of NRIP1 improved cell viability, whereas it inhibited cell apoptosis and oxidative stress, and suppressed mitochondrial dysfunction in OGD/R-injured H9c2 cardiomyocytes. Importantly, it was verified that YY1 could bind to the NRIP1 promoter to positively regulate NRIP1 expression. The protective effects of NRIP1 knockdown against cardiomyocyte damage and mitochondrial dysfunction in OGD/R-injured H9c2 cardiomyocytes were partly abolished through overexpression of YY1. NRIP1 emerged as a downstream target of YY1 in promoting OGD/R-induced H9c2 cardiomyocyte injury and mitochondrial dysfunction, providing novel ideas for targeted treatments to alleviate MIRI.

Preconditioning with Ginsenoside Rg3 mitigates cardiac injury induced by high-altitude hypobaric hypoxia exposure in mice by suppressing ferroptosis through inhibition of the RhoA/ROCK signaling pathway

Ethnopharmacological relevanceGinseng has historically been utilized as a conventional herbal remedy and dietary supplement to enhance physical stamina and alleviate fatigue. The primary active component of Ginseng, Ginsenoside Rg3 (GS-Rg3), possesses diverse pharmacological properties including immune modulation and anti-inflammatory effects. Furthermore, GS-Rg3 has demonstrated efficacy in mitigating tissue and organ damage associated with metabolic disorders such as hypertension, hyperglycemia, and hyperlipidemia. Nevertheless, its potential impact on high-altitude cardiac injury (HACI) remains insufficiently explored. Aim of the studyThe aim of this study was to examine the potential cardioprotective effects of Ginsenoside Rg3, and to investigate how Ginsenoside Rg3 preconditioning can enhance high-altitude cardiac injury by inhibiting the RhoA/ROCK pathway and ferroptosis in cardiac tissue. The findings of this study may contribute to the development of novel therapeutic strategies using traditional Chinese medicine for high-altitude cardiac injury, based on experimental evidence. Materials and methodsA hypobaric hypoxia chamber was employed to simulate hypobaric hypoxia conditions equivalent to an altitude of 6000 m. Through a randomization process, groups of six male mice were assigned to receive either saline, Ginsenoside Rg3 at doses of 15 mg/kg or 30 mg/kg, or lysophosphatidic acid (LPA) at 1 mg/kg. The impact of Ginsenoside Rg3 on high altitude-induced arrhythmias was evaluated using electrocardiography. Cardiac pathology sections stained with hematoxylin and eosin were evaluated for damage, with the extent of cardiomyocyte damage observed via transmission electron microscopy. The impact of Ginsenoside Rg3 on high-altitude cardiac injury was investigated through analysis of serum biomarkers for cardiac injury (CK-MB, BNP), inflammatory cytokines (TNF, IL-6, IL-1β), reactive oxygen species (ROS) and glutathione (GSH). The expression levels of hypoxia and hypoxia-related proteins in myocardial tissues from each experimental group were assessed using Western blot analysis. Following a review of the existing literature, the traditional regulatory mechanisms of ferroptosis were examined. Immunofluorescence staining of cardiac tissues and Western blotting techniques were utilized to investigate the impact of Ginsenoside Rg3 on cardiomyocyte ferroptosis through the RhoA/ROCK signaling pathway under conditions of hypobaric hypoxia exposure. ResultsPre-treatment with Ginsenoside Rg3 improved high altitude-induced arrhythmias, reduced cardiomyocyte damage, decreased cardiac injury biomarkers and inflammatory cytokines, and lowered the expression of hypoxia-related proteins in myocardial tissues. Both Western blotting and immunofluorescence staining of cardiac tissues demonstrated that exposure to high-altitude hypobaric hypoxia results in elevated expression of ferroptosis and proteins related to the RhoA/ROCK pathway. Experimental validation corroborated that the role of the RhoA/ROCK signaling pathway in mediating ferroptosis. ConclusionsThe findings of our study suggest that preconditioning with Ginsenoside Rg3 may attenuate cardiac injury caused by high-altitude hypobaric hypoxia exposure in mice by inhibiting ferroptosis through the suppression of the RhoA/ROCK signaling pathway. These findings contribute to the current knowledge of Ginsenoside Rg3 and high-altitude cardiac injury, suggesting that Ginsenoside Rg3 shows potential as a therapeutic agent for high-altitude cardiac injury.

Exendin-4 exhibits cardioprotective effects against high glucose-induced mitochondrial abnormalities: Potential role of GLP-1 receptor and mTOR signaling

Mitochondrial dysfunction is associated with hyperglycemic conditions and insulin resistance leading to cellular damage and apoptosis of cardiomyocytes in diabetic cardiomyopathy. The dysregulation of glucagon-like peptide-1 (GLP-1) receptor and mammalian target of rapamycin (mTOR) is linked to cardiomyopathies and myocardial dysfunctions mediated by hyperglycemia. However, the involvements of mTOR for GLP-1 receptor-mediated cardioprotection against high glucose (HG)-induced mitochondrial disturbances are not clearly identified. The present study demonstrated that HG-induced cellular stress and mitochondrial damage resulted in impaired ATP production and oxidative defense markers such as catalase and SOD2, along with a reduction in survival markers such as Bcl-2 and p-Akt, while an increased expression of pro-apoptotic marker Bax was observed in H9c2 cardiomyoblasts. In addition, the autophagic marker LC3-II was considerably reduced, together with the disruption of autophagy regulators (p-mTOR and p-AMPKα) under the hyperglycemic state. Furthermore, there was a dysregulated expression of several indicators related to mitochondrial homeostasis, including MFN2, p-DRP1, FIS1, MCU, UCP3, and Parkin. Remarkably, treatment with either exendin-4 (GLP-1 receptor agonist) or rapamycin (mTOR inhibitor) significantly inhibited HG-induced mitochondrial damage while co-treatment of exendin-4 and rapamycin completely reversed all mitochondrial abnormalities. Antagonism of GLP-1 receptors using exendin-(9–39) abolished these cardioprotective effects of exendin-4 and rapamycin under HG conditions. In addition, exendin-4 attenuated HG-induced phosphorylation of mTOR, and this inhibitory effect was antagonized by exendin-(9–39), indicating the regulation of mTOR by GLP-1 receptor. Therefore, improvement of mitochondrial dysfunction by stimulating the GLP-1 receptor/AMPK/Akt pathway and inhibiting mTOR signaling could ameliorate cardiac abnormalities caused by hyperglycemic conditions.

MiR-24-3p modulates cardiac function in doxorubicin -induced heart failure via the Sp1/PI3K signaling pathway

PurposeThe goal of this research was to explore the role of miR-24-3p in heart failure (HF), with a focus on its impact on the specificity protein 1 (Sp1)/phosphoinositide 3-kinase (PI3K) pathway. MethodsHF rat and HF cell models were established using doxorubicin(Dox). Cardiac function was assessed through echocardiography, while histological changes were observed via hematoxylin-eosin (HE) staining. To further investigate the underlying mechanisms, HF cell models were treated with either an Sp1 inhibitor or a PI3K inhibitor. Additionally, models with miR-24-3p overexpression or silencing were constructed. N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were determined by ELISA. Cell apoptosis was evaluated using TUNEL staining, and lactate dehydrogenase (LDH) levels were measured by colorimetry. Reactive oxygen species (ROS) production was analyzed using flow cytometry. Related gene and protein expressions were assessed via qRT-PCR and Western blotting. Finally, the relationship between miR-24-3p and Sp1 was confirmed through dual-luciferase assays. ResultsDox treatment increased the left ventricular internal diameter (LVIDd) while decreasing ejection fraction (EF) and fractional shortening (FS), leading to disorganized cardiomyocyte arrangement, cellular edema, and necrosis in rats. In HF rats, NT-proBNP, Caspase-3, and miR-24-3p expression levels were elevated, whereas Sp1 and PI3K mRNA and protein expression levels were decreased. Similarly, Dox-induced damage in H9c2 cardiomyocytes resulted in increased NT-proBNP, apoptosis, Caspase-3, LDH, ROS, and miR-24-3p expression, along with decreased Sp1 and PI3K expression. Treatment with either Sp1 or PI3K inhibitors exacerbated the Dox-induced cardiomyocyte damage, further elevating NT-proBNP, apoptosis, Caspase-3, LDH, ROS, and miR-24-3p expression levels. Notably, Sp1 inhibition reduced PI3K expression, and PI3K inhibition, in turn, suppressed Sp1 expression. Overexpression of miR-24-3p worsened Dox-induced cardiomyocyte damage, characterized by increased NT-proBNP, apoptosis, Caspase-3, LDH, and ROS expression, alongside reduced Sp1 and PI3K expression. In contrast, silencing miR-24-3p mitigated these detrimental effects and increased Sp1 and PI3K expression. Dual-luciferase assays confirmed that miR-24-3p directly targets Sp1. ConclusionDox induces cardiomyocyte damage, impairs cardiac function, and promotes cardiomyocyte apoptosis and oxidative stress. Silencing miR-24-3p offers a protective effect by activating the Sp1/PI3K signaling pathway in heart failure.

Minocycline alleviates lipopolysaccharide-induced cardiotoxicity by suppressing the NLRP3/Caspase-1 signaling pathway.

Minocycline (Min), as an antibiotic, possesses various beneficial properties such as anti-inflammatory, antioxidant, and anti-apoptotic effects. Despite these known qualities, the precise cardioprotective effect and mechanism of Min in protecting against sepsis-induced cardiotoxicity (SIC) remain unspecified. To address this, our study sought to assess the protective effects of Min on the heart. Lipopolysaccharide (LPS) was utilized to establish a cardiotoxicity model both in vivo and in vitro. Min was pretreated in the models. In the in vivo setting, evaluation of heart tissue histopathological injury was performed using hematoxylin and eosin (H&E) staining and TUNEL. Immunohistochemistry (IHC) was employed to evaluate the expression levels of NLRP3 and Caspase-1 in the heart tissue of mice. During in vitro experiments, the viability of H9c2 cells was gauged utilizing the CCK8 assay kit. Intracellular ROS levels in H9c2 cells were quantified using a ROS assay kit. Both in vitro and in vivo settings were subjected to measurement of oxidative stress indexes, encompassing glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. Additionglly, myocardial injury markers like lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB) activity were quantified using appropriate assay kits. Western blotting (WB) analysis was conducted to detect the expression levels of NOD-like receptor protein-3 (NLRP3), caspase-1, IL-18, and IL-1β, alongside apoptosis-related proteins such as Bcl-2 and Bax, and antioxidant proteins including superoxide dismutase-1 (SOD-1) and antioxidant proteins including superoxide dismutase-1 (SOD-2), both in H9c2 cells and mouse heart tissues. In vivo, Min was effective in reducing LPS-induced inflammation in cardiac tissue, preventing cell damage and apoptosis in cardiomyocytes. The levels of LDH and CK-MB were significantly reduced with Min treatment. In vitro studies showed that Min improved the viability of H9C2 cells, reduced apoptosis, and decreased ROS levels in these cells. Further analysis indicated that Min decreased the protein levels of NLRP3, Caspase-1, IL-18, and IL-1β, while increasing the levels of SOD-1 and SOD-2 both in vivo and in vitro. Min alleviates LPS-induced SIC by suppressing the NLRP3/Caspase-1 signalling pathway in vivo and in vitro.

Exposure to Polypropylene Microplastics Causes Cardiomyocyte Apoptosis Through Oxidative Stress and Activation of the MAPK-Nrf2 Signaling Pathway.

Microplastics are a growing concern as pollutants that impact both public health and the environment. However, the toxic effects of polypropylene microplastics (PP-MPs) are not well understood. This study aimed to investigate the effects of PP-MPs on cardiotoxicity and its underlying mechanisms. The cardiotoxicity of exposure to different amounts of PP-MPs were investigated in both ICR mice and H9C2 cells. Our results demonstrated that sub-chronic exposure to 5 and 50 mg/L PP-MPs led to myocardial structural damage, apoptosis, and fibrosis in mice cardiomyocytes. Flow cytometry analysis revealed that PP-MPs could decrease mitochondrial membrane potential and induce apoptosis in H9C2 cells. Western blotting revealed decreased expression of Bcl-2, poly(ADP-ribose) polymerase (PARP) and caspase 3 and increased expression of Bax, cleaved-PARP, and cleaved-caspase 3 in PP-MPs-treated cardiac tissue and H9C2 cells. These results confirmed the apoptotic effects induced by PP-MPs. Moreover, PP-MPs treatment triggered oxidative stress, as evidenced by the increased levels of malondialdehyde; reduction in glutathione peroxidase, superoxide dismutase, and catalase activities in mice cardiac tissues; and increased reactive oxygen species levels in H9C2 cells. Finally, western blotting demonstrated that exposure to PP-MPs significantly reduced the expression levels of Nrf2 and p-ERK proteins associated with MAPK-Nrf2 pathway in both cardiac tissue and H9C2 cells. Overall, our findings indicate that PP-MPs can induce cardiomyocyte apoptosis through MAPK-Nrf2 signaling pathway, which is triggered by oxidative stress. This study provides a foundation for determining the effects of PP-MPs on cardiotoxicity and their underlying mechanisms.

N-acetylcysteine, a small molecule scavenger of reactive oxygen species, alleviates cardiomyocyte damage by regulating OPA1-mediated mitochondrial quality control and apoptosis in response to oxidative stress.

Oxidative stress-induced mitochondrial damage is the major cause of cardiomyocyte dysfunction. Therefore, the maintenance of mitochondrial function, which is regulated by mitochondrial quality control (MQC), is necessary for cardiomyocyte homeostasis. This study aimed to explore the underlying mechanisms of N-acetylcysteine (NAC) function and its relationship with MQC. A hydrogen peroxide-induced oxidative stress model was established using H9c2 cardiomyocytes treated with or without NAC prior to oxidative stress stimulation. Autophagy with light chain 3 (LC3)-green fluorescent protein (GFP) assay, reactive oxygen species (ROS) with the 2',7'-dichlorodi hydrofluorescein diacetate (DCFH-DA) fluorescent, lactate dehydrogenase (LDH) release assay, adenosine triphosphate (ATP) content assay, and a mitochondrial membrane potential detection were used to evaluate mitochondrial dynamics in H2O2-treated H9c2 cardiomyocytes, with a focus on the involvement of MQC regulated by NAC. Cell apoptosis was analyzed using caspase-3 activity assay and Annexin V-fluorescein isothiocyanate (V-FITC)/propidium iodide (PI) double staining. We observed that NAC improved cell viability, reduced ROS levels, and partially restored optic atrophy 1 (OPA1) protein expression under oxidative stress. Following transfection with a specific OPA1-small interfering RNA, the mitophagy, mitochondrial dynamics, mitochondrial functions, and cardiomyocyte apoptosis were evaluated to further explore the mechanisms of NAC. Our results demonstrated that NAC attenuated cardiomyocyte apoptosis via the ROS/OPA1 axis and protected against oxidative stress-induced mitochondrial damage via the regulation of OPA1-mediated MQC. NAC ameliorated the injury to H9c2 cardiomyocytes caused by H2O2 by promoting the expression of OPA1, consequently improving mitochondrial function and decreasing apoptosis.

KLF4-induced upregulation of SOCS1 ameliorates myocardial ischemia/reperfusion injury by attenuating AC16 cardiomyocyte damage and enhancing M2 macrophage polarization.

Reperfusion strategies, the standard therapy for acute myocardial infarction (AMI), may result in ischemia/reperfusion (I/R) damage. Suppressor of cytokine signaling1 (SOCS1) exerts a cardioprotective function in myocardial I/R damage. Here, we investigated epigenetic modulators that deregulateSOCS1 in cardiomyocytes under hypoxia/reoxygenation (H/R) conditions. Human AC16 cardiomyocytes were exposed to H/R conditions to generate a cell model of myocardial I/R damage. Expression of mRNA and protein was detected by quantitative PCR and western blot analysis, respectively. Cell migratory and invasive abilities were evaluated by transwell assay. Cell apoptosis and M2 macrophage polarization were assessed by flow cytometry. TNF-α, IL-1β, and IL-6 levels were examined by ELISA. The interaction of KLF4 with SOCS1 was verified by chromatin immunoprecipitationand luciferase assays. SOCS1 and transcription factor KLF4 protein levels were underexpressed by 75% and 57%, respectively, in H/R-exposed AC16 cardiomyocytes versus control cells. Under H/R conditions, forced SOCS1 expression (2.7 times) induced cell migration (2.2 times) and invasion (1.9 times) and hindered cell apoptosis (by 45%) of AC16 cardiomyocytes as well as enhanced M2 macrophage polarization (4.6 times). Mechanistically, KLF4 upregulation promoted SOCS1 transcription (2.6 times) and expression (2.6 times) by binding to the SOCS1 promoter. Decrease of SOCS1 (by 51%) reversed the effects of KLF4 upregulation on cardiomyocyte migration, invasion and apoptosis, and M2 macrophage polarization under H/R conditions. Additionally, SOCS1 and KLF4 were underexpressed by 56% and 63%, respectively, in AMI serum. Our study indicates that KLF4-induced upregulation of SOCS1 can attenuate H/R-triggered apoptosis of AC16 cardiomyocytes and enhance M2 macrophage polarization.

DNA damage-associated protein co-expression network in cardiomyocytes informs on tolerance to genetic variation and disease.

Cardiovascular disease (CVD) is associated with both genetic variants and environmental factors. One unifying consequence of the molecular risk factors in CVD is DNA damage, which must be repaired by DNA damage response proteins. However, the impact of DNA damage on global cardiomyocyte protein abundance, and its relationship to CVD risk remains unclear. We therefore treated induced pluripotent stem cell-derived cardiomyocytes with the DNA-damaging agent Doxorubicin (DOX) and a vehicle control, and identified 4,178 proteins that contribute to a network comprising 12 co-expressed modules and 403 hub proteins with high intramodular connectivity. Five modules correlate with DOX and represent distinct biological processes including RNA processing, chromatin regulation and metabolism. DOX-correlated hub proteins are depleted for proteins that vary in expression across individuals due to genetic variation but are enriched for proteins encoded by loss-of-function intolerant genes. While proteins associated with genetic risk for CVD, such as arrhythmia are enriched in specific DOX-correlated modules, DOX-correlated hub proteins are not enriched for known CVD risk proteins. Instead, they are enriched among proteins that physically interact with CVD risk proteins. Our data demonstrate that DNA damage in cardiomyocytes induces diverse effects on biological processes through protein co-expression modules that are relevant for CVD, and that the level of protein connectivity in DNA damage-associated modules influences the tolerance to genetic variation.

GDF15 attenuates sepsis-induced myocardial dysfunction by inhibiting cardiomyocytes ferroptosis via the SOCS1/GPX4 signaling pathway

Sepsis is a systemic inflammatory response syndrome triggered by infection, presenting with symptoms such as fever, increased heart rate, and low blood pressure. In severe cases, it can lead to multiple organ dysfunction, posing a life-threatening risk. Sepsis-induced cardiomyopathy (SIC) is a critical factor in the poor prognosis of septic patients, leading to myocardial dysfunction characterized by cell death, inflammation, and diminished cardiac function. Ferroptosis, an iron-dependent form of programmed cell death, is a key mechanism causing cardiomyocyte damage in SIC. Growth differentiation factor 15 (GDF15), a member of the TGF-β superfamily, is associated with various cardiovascular diseases and can inhibit oxidative stress, reduce reactive oxygen species (ROS), and suppress ferroptosis. Elevated serum GDF15 levels in sepsis are correlated with organ injuries, suggesting its potential as a therapeutic target. However, its role and mechanisms in SIC remain unclear. Glutathione peroxidase 4 (GPX4), the only enzyme capable of reducing lipid peroxides within cells, protects cells by reducing lipid peroxidation levels and inhibiting ferroptosis. Investigating the regulatory factors of GPX4 may provide a theoretical basis for SIC treatment. In this study, a mouse SIC model revealed that elevated GDF15 exerts a protective effect. Antagonizing GDF15 exacerbates myocardial damage. Through transcriptomic analysis and other methods, we confirmed that GDF15 inhibits the expression of SOCS1 by activating the ALK5-SMAD2/3 pathway, thereby activates the JAK2/STAT3 pathway, promotes the transcription of GPX4, inhibits ferroptosis in cardiomyocytes, and plays a myocardial protective role in SIC.

Hypoxic Cardioprotection by New Antihypertensive Compounds in High Salt-Diet Hypertensive Rats: Glucose Transport Participation and Its Possible Pathway.

Hypertension (HP) is a health condition that overloads the heart and increases the risk of heart attack and stroke. In an infarction, the lack of oxygen causes an exclusive use of glycolysis, which becomes a crucial source of ATP for the heart with a higher glucose uptake mediated by glucose transporters (GLUTs). Due to the unpleasant effects of antihypertensives, new drugs need to be researched to treat this disease. This study aimed to evaluate the cardioprotective effect of three novel antihypertensive compounds (LQMs, "Laboratorio de Química Medicinal") synthesized from Changrolin under hypoxic conditions with the participation of two primary cardiac GLUT1 and GLUT4 using a high-salt diet HP model. The model used a diet with 10% salt to increase arterial blood pressure in Wistar rats. In isolated cardiomyocytes from these rats, glucose uptake was measured during hypoxia, evaluating the participation of GLUTs with or without the animals' previous treatment with LQM312, 319, and 345 compounds. In silico calculations were performed to understand the affinity of the compounds for the trafficking of GLUTs. Results: Control cells do shift to glucose uptake exclusively in hypoxia (from 1.84 ± 0.09 µg/g/h to 2.67 ± 0.1 µg/g/h). Meanwhile, HP does not change its glucose uptake (from 2.38 ± 0.24 µg/g/h to 2.33 ± 0.26 µg/g/h), which is associated with cardiomyocyte damage. The new compounds lowered the systolic blood pressure (from 149 to 120 mmHg), but only LQM312 and LQM319 improved the metabolic state of hypoxic cardiomyocytes mediated by GLUT1 and GLUT4. In silico studies suggested that Captopril and LQM312 may mimic the interaction with the AMPK γ-subunit. Therefore, these compounds could activate AMPK, promoting the GLUT4 trafficking signaling pathway. These compounds are proposed to be cardioprotective during hypoxia under HP.

P62-autophagic pathway degrades SLC7A11 to regulate ferroptosis in doxorubicin-induced cardiotoxicity

Doxorubicin-induced cardiotoxicity (DIC) poses a significant challenge, impeding its widespread application. Emerging evidence suggests the involvement of ferroptosis in the DIC. While the downregulation of SLC7A11 expression has been linked to the promotion of ferroptosis, the precise regulatory mechanism remains unclear. Recent studies, including our own, have highlighted abnormal levels of autophagy adapter protein P62 and autophagy in DIC development. Thus, our study aimed to further investigate the role of autophagy and ferroptosis in DIC, elucidating underlying molecular mechanisms across molecular, cellular, and whole-organ levels utilizing gene knockdown, immunoprecipitation, and mass spectrometry techniques. The results of our findings unveiled cardiomyocyte damage, heightened autophagy levels, and ferroptosis in DOX-treated mouse hearts. Notably, inhibition of autophagy levels attenuated DOX-induced ferroptosis. Mechanistically, we discovered that the autophagy adaptor protein P62 mediates the entry of SLC7A11 into the autophagic pathway for degradation. Furthermore, the addition of autophagy inhibitors (CQ or BAF) could elevate SLC7A11 and GPX4 protein expression, reduce the accumulation of Fe2+ and ROS in cardiomyocytes, and thus mitigate DOX-induced ferroptosis. In summary, our findings underscore the pivotal role of the P62-autophagy pathway in SLC7A11 degradation, modulating ferroptosis to exacerbate DIC. This finding offers significant insights into the underlying molecular mechanisms of DOX-induced ferroptosis and identifies new targets for reversing DIC.

Abstract We063: CD73 EXPRESSION ON NEUTROPHILS PROTECTS AGAINST CARDIOMYOCYTE DAMAGE DURING INVASIVE STREPTOCOCCUS PNEUMONIAE INFECTION

In recent years, Streptococcus pneumoniae (pneumococcus)-induced cardiac events have emerged as one of the most serious and life-threatening infection outcomes of invasive pneumococcal disease (IPD), leading to major adverse cardiac events (MACE) with high mortality rates. S. pneumoniae has the ability to invade myocardium and damage cardiomyocytes through the release of bacterial factors and formation of bacterial-filled cardiac microlesions. We previously found that polymorphonuclear cells (PMNs) or neutrophils are crucial for host defense against S. pneumoniae lung infection and that extracellular adenosine (EAD) production, by the exonucleosidase CD73, controlled the anti-bacterial functions of these cells. The objective of this study was to explore the role of PMNs and CD73 in cardiac damage during invasive pneumococcal infection. Upon intra-peritoneal ( i.p .) injection with TIGR4, an invasive pneumococcal strain, C57BL/6 mice showed an increased influx of PMNs into the cardiac tissue as determined by flow cytometry. However, the increased PMN numbers failed to contain bacterial burden in the heart and showed a positive correlation with serum levels of the cardiac damage marker Troponin-1 as determined through ELISA. Depletion of PMNs prior infection reduced pneumococcal burden in the heart and lowered the Troponin-1 levels suggesting that PMNs induce cardiac damage during infection. While exploring the mechanisms underlying the detrimental PMN response, we found that the late stage of IPD was associated with reduced CD73 expression on PMNs. The role of CD73 in regulating cardiac damage was tested in vivo using CD73 -/- mice which had significantly higher bacterial burden and cardiac damage compared to wild-type mice despite similar PMN numbers. The role of CD73 expression on PMNs was also tested ex vivo using the HL-1 cardiomyocyte cell line which showed increased LDH release upon S. pneumoniae infection in presence of CD73 -/- PMNs, a phenotype reversed by supplementation of exogenous adenosine. Similar iincreased damage in presence of CD73 -/- PMNs was seen with C57BL/6 Mouse Primary Cardiac Microvascular Endothelial Cells which lead to increased migration of beads across endothelium. Our findings suggest that dysregulation of CD73 on PMNs during invasive pneumococcal infection leads to an inability to control bacterial numbers and increased cardiac damage, and that the EAD-pathway can be a potential pharmacological target to limit disease severity.

SARS-CoV-2 variants divergently infect and damage cardiomyocytes in vitro and in vivo

BackgroundCOVID-19 can cause cardiac complications and the latter are associated with poor prognosis and increased mortality. SARS-CoV-2 variants differ in their infectivity and pathogenicity, but how they affect cardiomyocytes (CMs) is unclear.MethodsThe effects of SARS-CoV-2 variants were investigated using human induced pluripotent stem cell-derived (hiPSC-) CMs in vitro and Golden Syrian hamsters in vivo.ResultsDifferent variants exhibited distinct tropism, mechanism of viral entry and pathology in the heart. Omicron BA.2 most efficiently infected and injured CMs in vitro and in vivo, and induced expression changes consistent with increased cardiac dysfunction, compared to other variants tested. Bioinformatics and upstream regulator analyses identified transcription factors and network predicted to control the unique transcriptome of Omicron BA.2 infected CMs. Increased infectivity of Omicron BA.2 is attributed to its ability to infect via endocytosis, independently of TMPRSS2, which is absent in CMs.ConclusionsIn this study, we reveal previously unknown differences in how different SARS-CoV-2 variants affect CMs. Omicron BA.2, which is generally thought to cause mild disease, can damage CMs in vitro and in vivo. Our study highlights the need for further investigations to define the pathogenesis of cardiac complications arising from different SARS-CoV-2 variants.

Abstract Tu130: Intracellular Galectin-3 Interacts with Cytosolically Exposed Glycans to Promote the Injury of Cardiomyocytes Under Oxidative Stress

Background: Oxidative stress is a hallmark of cardiovascular (CV) diseases. The potential involvement of Galectin-3 (Gal-3) has been proposed in CV pathological conditions, but its role in reactive oxygen species (ROS)-induced cardiomyocyte damage remains elusive. Hypothesis: Oxidative stress leads to ruptures of membrane-bound organelles including lysosome. Intracellular Gal-3 of cardiomyocytes may interact with glycans exposed by ROS-damaged organelles to modulate downstream cellular signaling. Aims: To investigate whether intracellular Gal-3-glycan interactions may occur in ROS-treated cardiomyocytes and how such interactions may regulate cell survival or death. Methods and Results: Using cardiomyoblast H9c2 cells exposed to hydrogen peroxide (H 2 O 2 ) to be the cell model, we found that oxidative stress induced significant lysosomal damage followed by cell death. The immunofluorescence imaging showed that cytosolic Gal-3 aggregated into small puncta, which were colocalized with LAMP2. To examine whether the aggregation of Gal-3 was contributed by ROS-induced damage, catalase was ectopically expressed in H 2 O 2 -treated cells and the formation of Gal-3 puncta was attenuated. These results suggest that intracellular Gal-3 interacts with glycans presented by the ruptured lysosomes in H 2 O 2 -treated H9c2 cells, leading to puncta formation. To further delineate whether these Gal-3 puncta show a functional role in cell survival under oxidative stress, we knocked endogenous Gal-3 down in H9c2 cells by RNAi and found that Gal-3 ablation protected the cells from ROS-induced injuries. Moreover, the ectopic expression of N-terminally truncated Gal-3, which loses its protein-interaction function but preserved the carbohydrate-recognition ability, also prevented H9c2 cells from oxidative stress-caused damage. Conclusions: ROS-induced lysosomal rupture leads to intracellular Gal-3-glycan interactions, further recruiting cytosolic proteins and activating downstream cell death signaling cascades. Such intracellular Gal-3-glycan interactions may contribute to the cardiomyocyte injuries under oxidative stress.

The potential treatment of N-acetylcysteine as an antioxidant in the radiation-induced heart disease.

Radiation-induced heart disease (RIHD) is a serious complication of thoracic tumor radiotherapy that substantially affects the quality of life of cancer patients. Oxidative stress plays a pivotal role in the occurrence and progression of RIHD, which prompted our investigation of an innovative approach for treating RIHD using antioxidant therapy. We used 8-week-old male Sprague-Dawley (SD) rats as experimental animals and H9C2 cells as experimental cells. N-acetylcysteine (NAC) was used as an antioxidant to treat H9C2 cells after X-ray irradiation in this study. In the present study, the extent of cardiomyocyte damage caused by X-ray exposure was determined, alterations in oxidation/antioxidation levels were assessed, and changes in the expression of genes related to mitochondria were examined. The degree of myocardial tissue and cell injury was also determined. Dihydroethidium (DHE) staining, reactive oxygen species (ROS) assays, and glutathione (GSH) and manganese superoxide dismutase (Mn-SOD) assays were used to assess cell oxidation/antioxidation. Flow cytometry was used to determine the mitochondrial membrane potential and mitochondrial permeability transition pore (mPTP) opening. High-throughput transcriptome sequencing and bioinformatics analysis were used to elucidate the expression of mitochondria-related genes in myocardial tissue induced by X-ray exposure. Polymerase chain reaction (PCR) was used to verify the expression of differentially expressed genes. X-ray irradiation damaged myocardial tissue and cells, resulting in an imbalance of oxidative and antioxidant substances and mitochondrial damage. NAC treatment increased cell counting kit-8 (CCK-8) levels (P=0.02) and decreased lactate dehydrogenase (LDH) release (P=0.02) in cardiomyocytes. It also reduced the level of ROS (P=0.002) and increased the levels of GSH (P=0.04) and Mn-SOD (P=0.01). The mitochondrial membrane potential was restored (P<0.001), and mPTP opening was inhibited (P<0.001). Transcriptome sequencing and subsequent validation analyses revealed a decrease in the expression of mitochondria-related genes in myocardial tissue induced by X-ray exposure, but antioxidant therapy did not reverse the related DNA damage. Antioxidants mitigated radiation-induced myocardial damage to a certain degree, but these agents did not reverse the associated DNA damage. These findings provide a new direction for future investigations by our research group, including exploring the treatment of RIHD-related DNA damage.

Gastrodin exerts perioperative myocardial protection by improving mitophagy through the PINK1/Parkin pathway to reduce myocardial ischemia-reperfusion injury

BackgroundAlthough blood flow is restored after treatment of myocardial infarction (MI), myocardial ischemia and reperfusion (I/R) can cause cardiac injury, which is a leading cause of heart failure. Gastrodin (GAS) exerts protective effects against brain, heart, and kidney I/R. However, its pharmacological mechanism in myocardial I/R injury (MIRI) remains unclear. PurposeGAS regulates autophagy in various diseases, such as acute hepatitis, vascular dementia, and stroke. We hypothesized that GAS could repair mitochondrial damage and regulate autophagy to protect against MIRI. Study designMale C57BL/6 mice and H9C2 cells were subjected to I/R and hypoxia-reoxygenation (H/R) injury after GAS administration, respectively, to assess the impact of GAS on cardiomyocyte phenotypes, heart, and mitochondrial structure and function. The effect of GAS on cardiac function and mitochondrial structure in patients undergoing cardiac surgery has been observed in clinical practice. MethodsThe effects of GAS on cardiac structure and function, mitochondrial structure, and expression of related molecules in an animal model of MIRI were evaluated using immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy, western blotting, and gene sequencing. Its effects on the morphological, molecular, and functional phenotypes of cardiomyocytes undergoing H/R were observed using immunohistochemical staining, real-time quantitative PCR, and western blotting. ResultsGAS significantly reduces myocardial infarct size and improves cardiac function in MIRI mice in animal models and increases cardiomyocyte viability and reduces cardiomyocyte damage in cellular models. In clinical practice, myocardial injury was alleviated with better cardiac function in patients undergoing cardiac surgery after the application of GAS; improvements in mitochondria and autophagy activation were also observed. GAS primarily exerts cardioprotective effects through activation of the PINK1/Parkin pathway, which promotes mitochondrial autophagy to clear damaged mitochondria. ConclusionGAS can promote mitophagy and preserve mitochondria through PINK1/Parkin, thus indicating its tremendous potential as an effective perioperative myocardial protective agent.

20S-O-Glc-DM treats left ventricular diastolic dysfunction by modulating cardiomyocyte mitochondrial quality and excess autophagy

BackgroundLeft ventricular diastolic dysfunction (LVDD) is a manifestation of heart failure, with both its incidence and prevalence increasing annually. Currently, no pharmacological treatments are available for LVDD, highlighting the urgent need for new therapeutic discoveries. Ginsenosides are commonly used in cardiovascular therapy. Previous research has synthesized the ginsenoside precursor molecule, 20S-O-Glc-DM (C20DM), through biosynthesis. C20DM shows greater bioavailability, eco-friendliness, and cost-effectiveness compared to traditional ginsenosides, positioning it as a promising option for treating LVDD. PurposeThis study firstly documents the therapeutic activity of C20DM against LVDD and unveils its potential mechanisms of action. It provides a pharmacological basis for C20DM as a new cardiovascular therapeutic agent. MethodsIn this study, models of LVDD in mice and ISO-induced H9C2 cell damage were developed. Cell viability, ROS and Ca2+ levels, mitochondrial membrane potential, and proteins associated with mitochondrial biogenesis and autophagy were evaluated in the in vitro experiments. Animal experiments involved administering medication for 3 weeks to validate the therapeutic effects of C20DM and its impact on mitochondria and autophagy. ResultsResearch has shown that C20DM is more effective than Metoprolol in treating LVDD, significantly lowering the E/A ratio, e'/a' ratio, and IVRT, and ameliorating myocardial inflammation and fibrosis. C20DM influences the activity of PGC-1α, downregulates PINK1 and Parkin, thereby enhancing mitochondrial quality control, and restoring mitochondrial oxidative respiration and membrane potential. Furthermore, C20DM reduces excessive autophagy in cardiomyocytes via the AMPK-mTOR-ULK1 pathway, diminishing cardiomyocyte hypertrophy and damage. ConclusionsOverall, our research indicates that C20DM has the potential to enhance LVDD through the regulation of mitochondrial quality control and cellular autophagy, making it a promising option for heart failure therapy.