Cardiac Cell Apoptosis Research Articles

Silenced long non-coding RNA RMST ameliorates cardiac dysfunction and inflammatory response in doxorubicin-induced heart failure in C57BL/6 mice via the modulation of the microRNA-10b-5p/TRAF6 axis.

Long non-coding RNA rhabdomyosarcoma 2-associated transcript (RMST) has been found to exert effects on cardiovascular diseases. However, the research for probing its role in heart failure (HF) is limited. Our study intends to unravel the regulatory effects of RMST on HF via the microRNA (miR)-10b-5p/tumor necrosis factor receptor-associated factor 6 (TRAF6) axis. The mouse model of HF was induced by doxorubicin. The expression levels of RMST, miR-10b-5p and TRAF6 were detected. The virus carrying RMST, miR-10b-5p or TRAF6 vectors were injected into doxorubicin-induced HF mice to examine the cardiac function, inflammatory response, pathological changes and cell apoptosis in doxorubicin-induced HF mice. The target relationships among RMST, miR-10b-5p and TRAF6 were confirmed. RMST and TRAF6 were elevated and miR-10b-5p was reduced in doxorubicin-induced HF mice. RMST or TRAF6 silencing or miR-10b-5p overexpression could improve doxorubicin-induced cardiac dysfunction, and inflammatory response, and reduce cardiomyocyte apoptosis. Down-regulation of miR-10b-5p or overexpression of TRAF6 were both able to inverse the therapeutic effect of silencing RMST on doxorubicin-induced HF mice. RMST bound to miR-10b-5p that targeted TRAF6. RMST silencing could attenuate inflammatory response and cardiomyocyte apoptosis and upregulate cardiac function in mice with doxorubicin-induced HF by modulating the miR-10b-5p/TRAF6 axis. The study provides novel therapeutic targets for HF treatment.

Cardioprotective effect of tofisopam against isoprenaline-induced myocardial infarction in rats via modulation of NLRP3\\IL-1β\\caspase-1 pathway

Purpose Cardiovascular diseases (CVDs) are a leading cause of morbidity and mortality worldwide. Ischemic heart diseases, particularly acute myocardial infarction (MI), represent the most common cause of death. MI is influenced by multiple factors, including the release of inflammatory mediators. A significant percentage of individuals with CVD experience psychological effects, such as anxiety and depression, which are linked to an increased risk of coronary heart disease. Certain anti-anxiety medications have demonstrated immunomodulatory and anti-inflammatory effects. Tofisopam, a 2,3-benzodiazepine with anxiolytic properties, has been shown to exert in vitro anti-inflammatory and immunomodulatory effects. The present study investigates the potential of tofisopam as a protective adjuvant against isoprenaline-induced MI in rats and explores the possible underlying mechanisms. Methods The study included four groups: a control group, a group pretreated with tofisopam, an isoprenaline toxic group, and an isoprenaline toxic group pretreated with tofisopam. Results The findings demonstrated that isoprenaline significantly increased cardiac enzyme levels, as well as elevated oxidative and inflammatory stress parameters, along with evident apoptosis in cardiac cells. In contrast, the tofisopam-pretreated group showed a significant reversal of the cardiac damage induced by isoprenaline. Conclusions Tofisopam protects against isoprenaline-induced MI through its antioxidant, anti-inflammatory, and anti-apoptotic properties.

Protective effects of mesenchymal stem cells-derived extracellular vesicles against ischemia-reperfusion injury of hearts donated after circulatory death: Preliminary study in a pig model

IntroductionInsufficient supply of cardiac grafts represents a severe obstacle in heart transplantation. Donation after Circulatory Death (DCD), in addition to conventional donation after brain death, is one promising option to overcome the organ shortage. However, DCD organs undergo an inevitable more extended period of warm unprotected ischemia between circulatory arrest and graft procurement. Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have shown remarkable protective effects against ischemia-reperfusion injury. Thus, we aimed to enhance grafts preservation from DCD donors, through treatment with MSC-EVs. MethodsFemale pigs were euthanized by barbiturate overdose and after 20 min of a flat EKG, the chest was opened, the heart harvested and subsequently connected to an extracorporeal perfusion machine. MSC-EVs, isolated by ion exchange chromatography, were added to the perfusion solution (1×1011 particles) and the heart was perfused for 2 h. Then, heart tissue biopsies were taken to assess histological changes, mitochondrial morphology, antioxidant enzyme activity and inflammation mediators’ expression. Biochemical parameters of myocardial viability were assessed in the perfusate. ResultsThe treatment with MSC-EVs significantly prevented mitochondria swelling, mitochondrial cristae loss and oxidative stress in cardiac tissue. The protective effect of MSC-EVs was confirmed by the delayed increase of the cardiac-specific enzymes CK and TnC in the perfusate and the reduction of caspase-3+ cells in tissue sections. ConclusionMSC-EVs improve graft quality by preserving the mitochondrial ultrastructure protecting the myocardium against oxidative stress, reducing apoptosis of cardiac cells and preventing the increase of pro-inflammatory cytokines.

Abstract Mo001: Inhibition of TAOK1-mediated Cardiomyocyte Death Attenuates Doxorubicin-Induced Cardiotoxicity

Background and Objective: Doxorubicin (Dox)-induced cardiotoxicity (DIC) is one of the most serious side effects of cancer treatments. However, no effective treatment is currently available. In this study, we aimed to identify novel therapeutic targets using CRISPR/Cas9-based screening of human cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). Methods and Results: We performed a kinase-focused CRISPR gene knockout screen in hPSC-CMs and identified thousand and one amino acid protein kinase 1 (TAOK1 ) as a potential regulator of DOX-induced cardiomyocyte death. We generated TAOK1-knockout (KO) hPSC-CMs using CRISPR/Cas9 and found that TAOK1-KO hPSC-CMs showed higher cell viability than control KO cells after 1μM DOX treatment (49.3% vs. 30.5%, P = 0.03). TAOK1-KO decreased DOX-induced ROS production (-58.8%, P = 0.01) and DNA damage (-28.8%, P < 0.01) in hPSC-CMs. Transcriptome analysis using RNA-seq of TAOK1-KO hPSC-CMs identified 483 differentially expressed genes, and Hallmark pathway analysis revealed upregulation of the oxidative phosphorylation pathway (FDR < 0.01) in TAOK1-KO hPSC-CMs compared to the control. Extracellular flux analysis demonstrated higher basal and maximal respiratory capacity in TAOK1-KO hPSC-CMs compared to control after DOX treatment (1.2 vs. 0.6, and 3.8 vs. 2.0 pmol/min/μg, respectively, P < 0.01 for all). Next, we overexpressed TAOK1 in hPSC-CMs using adenovirus vector and found that TAOK1 overexpression (OE) augmented DOX-induced cell viability reduction in hPSC-CMs compared to control after 0.5μM DOX treatment (-63.4 % vs. -19.2%, P < 0.01). Western blot analysis showed that DOX-induced p38 phosphorylation was suppressed by TAOK1-KO (P < 0.01) and enhanced (P = 0.01) by TAOK1-OE in hPSC-CMs. Augmented DOX-induced cardiomyocyte death in TAOK1-OE hPSC-CMs was partially rescued by inhibitors of p38 MAPK or apoptosis. Lastly, we demonstrated that TAOK1 suppression using AAV-mediated gene silencing mitigated the decline in the left ventricular ejection fraction in a mouse model of DIC (-30.0% vs. -17.7%, P < 0.01). Histological analysis of heart tissue revealed that TAOK1 suppression reduced myocardial fibrosis and apoptosis of cardiac cells in a mouse model of DIC (6.6% vs. 4.3%, P = 0.02, and 0.31% vs. 0.18%, P< 0.01, respectively). Conclusion: Our results suggest that TAOK1 regulates DOX-induced cardiomyocyte death; thus, its inhibition could be a potential therapeutic approach for DIC.

Abstract Tu140: Evaluation of the effects of mitoquinone on doxorubicin-induced acute cardiac damage

Background: Doxorubicin (DOX)-induced cardiotoxicity remains a high priority to overcome due to irreversibility and lack of abundant preventions. DOX damages the heart tissue principally by overproduction of mitochondrial superoxide and intercalation with the topoisomerase II in the nucleus. Ultimately, cardiac cells become apoptotic, and heart function declines. Our lab has shown that Mitoquinone (MQ), a mitochondria-targeted antioxidant, improves H9c2 cell viability in the presence of DOX. We hypothesize that MQ would counteract DOX to preserve cardiac cells and function in isolated rat hearts, possibly by reducing mitochondrial superoxide production and intracellular accumulation. Methods: H9c2 cells (passages < 18) were incubated with DOX (40 µM) with or without MQ (0.005 μM - 5 μM) for 24 hours before measuring mitochondrial superoxide anion levels and DOX intracellular accumulation. Furthermore, heart rate (HR) and left ventricular end-systolic pressure (LVESP) were recorded before and after a 60-minute infusion of DOX or DOX with MQ in isolated rat hearts. TUNEL staining was performed on heart tissue slides to assess apoptosis. All the data was expressed as a ratio between treatment and DOX alone or between final to initial values. Statistical significance was evaluated using ANOVA with Student Newman Keuls post-hoc analysis. Results: Hearts perfused with DOX (25 µM, n = 5) displayed lower final LVESP with 39 ± 5% of the initial baseline. By contrast, hearts treated with 0.25 µM MQ (n = 4) or 1 µM MQ (n = 5) manifested exhibited a higher final LVESP to 44 ± 8% and 90 ± 6% of initial baselines, respectively. Additionally, MQ administration significantly reduced cardiac cell apoptosis to 8 ± 1% (0.25 µM, n = 2) and 6 ± 1% (1 µM, n = 5, p < 0.05) when compared to DOX (88 ± 2%, n = 4). In contrast to DOX, MQ (1 µM, n = 4) showed 27 ± 9% (n = 4) reduction in mitochondrial superoxide levels and 54 ± 6% (n = 6, p < 0.05) less intracellular DOX deposition in H9c2 cells. Conclusion: This study suggests acute DOX administration to the heart severely compromises cardiac systolic function with higher induction of cell apoptosis. MQ exerts cardio-protection with higher systolic function and cell viability by attenuating mitochondrial superoxide levels and intracellular DOX accumulation.

Paeonol attenuated high glucose-induced apoptosis via up-regulating miR-223-3p in mouse cardiac microvascular endothelial cells

To investigate the role of miR-223-3p in the modulatory effect of paeonol (Pae) on high glucose (HG)-induced endothelial cell apoptosis. HG (25 mmol/L) was used to induce cellular damage and apoptosis in the mouse cardiac microvascular endothelial cells (MCMECs). Various concentration of Pae was tested and 60 μmol/L Pae was selected for the subsequent studies. MCMECs were transfected with exogenous miR-223-3p mimics or anti-miR-223-3p inhibitors. Cell viability was assessed by MTT assay and apoptosis was quantified by flow cytometry. The expression of miR-223-3p and NLRP3 mRNA was measured using real-time quantitative RT-PCR, and protein level of NLRP3 and apoptosis-related proteins was detected by immunoblotting. Pae significantly attenuated HG-induced apoptosis of MCMECs in a concentration-dependent manner. In addition, Pae (60 µmol/L) significantly reversed HG-induced down-regulation of miR-223-3p and up-regulation of NLRP3. Pae (60 µmol/L) also significantly blocked HG-induced up-regulation of Bax and Caspase-3 as well as down-regulation of Bcl-2. Moreover, exogenous miR-223-3p mimics not only significantly attenuated HG-induced apoptosis, but also significantly suppressed NRLP-3 and pro-apoptotic proteins in the MCMECs. In contrast, transfection of exogenous miR-223-3p inhibitors into the MCMECs resulted in not only significantly increased apoptosis of the cells, but also significant suppression of NLRP3 and pro-apoptotic proteins in the cells. Pae attenuated HG-induced apoptosis of MCMECs in a concentration-dependent manner. MiR-223-3p may mediate the modulatory effects of Pae on MCMEC survival or apoptosis through targeting NLRP3 and regulating apoptosis-associated proteins.

The ischemia-enhanced myocardial infarction protection-related lncRNA protects against acute myocardial infarction.

Long non-coding RNA RP11-64B16.4 (myocardial infarction protection-related lncRNA [MIPRL]) is among the most abundant and the most upregulated lncRNAs in ischemic human hearts. However, its role in ischemic heart disease is unknown. We found MIPRL was conserved between human and mouse and its expression was increased in mouse hearts after acute myocardial infarction (AMI) and in cultured human and mouse cardiomyocytes after hypoxia. The infarcted size, cardiac cell apoptosis, cardiac dysfunction, and cardiac fibrosis were aggravated in MIPRL knockout mice after AMI. The above adverse results could be reversed by re-expression of MIPRL via adenovirus expressing MIPRL. Both in vitro and in vivo, we identified that heat shock protein beta-8 (HSPB8) was a target gene of MIPRL, which was involved in MIPRL-mediated anti-apoptotic effects on cardiomyocytes. We further discovered that MIPRL could combine with the messenger RNA (mRNA) of HSPB8 and increase its expression in cardiomyocytes by enhancing the stability of HSPB8 mRNA. In summary, we have found for the first time that the ischemia-enhanced lncRNA MIPRL protects against AMI via its target gene HSPB8. MIPRL might be a novel promising therapeutic target for ischemic heart diseases such as AMI.

Novel Antiarrhythmic and Cardioprotective Effects of Brilliant Blue G

In this study, we investigated the effects of the purinergic P2X7 receptor antagonist brilliant blue G (BBG) on cardiac arrhythmia and myocardial injury induced by intravenously (i.v.) administered epinephrine in anesthetized rats. We also examined the possible involvement of beta-adrenergic receptors or cholinergic mechanisms in the effects of BBG. Sprague-Dawley rats were treated with epinephrine (10 μg/kg, i.v.). Brilliant blue G (100 mg/kg) was intraperitoneally (i.p.) administered thirty minutes prior to i.v. epinephrine. The effects of pretreatment with propranolol (2 mg/kg, i.p.) or atropine (2 mg/kg, i.v.) given prior to BBG and epinephrine were examined. The control group received saline. Moreover, the effects of only BBG on electrocardiogram (ECG) parameters were investigated. Results showed that compared with the saline control, BBG caused significant bradycardia (from 405.8 ± 1.18 to 239.4 ± 6.69 beats/min), increased RR interval (from 0.149 ± 0.002 to 0.254± 0.007 sec) and PR interval (from 0.051 ± 0.0008 to 0.059 ± 0.0004 sec), increased R wave amplitude (from 0.238 ± 0.019 to 0.548 ± 0.009 mv), and shortened QTc interval (from 0.169 ± 0.006 to 0.141 ± 0.003 sec) over 15 minutes after of BBG administration. BBG did not cause cardiac arrhythmia. Meanwhile, epinephrine produced significant bradycardia (209.8 ± 28.78 vs. 405.8 ± 1.18 beats/min), increased PR interval, prolonged the QRS complex, shortened QTc interval, decreased R wave amplitude and induced ventricular tachycardia. Brilliant blue G given prior to epinephrine increased heart rate and completely suppressed the epinephrine-induced ventricular arrhythmia. The inhibitory effect of BBG on the arrhythmia caused by epinephrine was prevented by atropine. In contrast the epinephrine induced arrhythmia was completely suppressed with propranolol and BBG. The histopathological study showed that epinephrine caused necrosis and apoptosis of cardiac muscle cells, degeneration of cardiac muscle fibers, and interstitial haemorrhages. These changes were markedly prevented by BBG alone, propranolol/BBG and to a less extent by atropine/BBG pretreatment. The study provided the first evidence for a cardioprotective and anti-arrhythmogenic actions for BBG against epinephrine-induced arrhythmia and myocardial damage, and suggested that cholinergic mechanisms are involved in its anti-arrhythmogenic action.

Bone marrow mesenchymal stem cells-derived exosomal lncRNA GAS5 mitigates heart failure by inhibiting UL3/Hippo pathway-mediated ferroptosis

BackgroundExosomes (Exos) are involved in the therapeutic effects of bone marrow mesenchymal stem cells (BMSCs) on heart failure (HF). We investigated the molecular mechanisms underlying the involvement of BMSC-Exos in ferroptosis on HF.MethodsA rat model of HF and cellular model of hypoxia were established. BMSC-Exos were injected into model rats or co-cultured with model cells. In model rats, the cardiac function (echocardiography), oxidative stress (commercial kits), pathological damage (HE staining), fibrosis (MASSON staining), iron deposition (Prussian blue staining), and cell apoptosis (TUNEL staining) were examined. Viability (cell counting kit-8; CCK-8), cell cycle (flow cytometry), oxidative stress, and Fe2+ levels were detected in the model cells. GAS5, UL3, YAP, and TAZ expression were detected using qRT-PCR, western blotting, and immunohistochemistry analyses.ResultsBMSC-Exos restored cardiac function and inhibited oxidative stress, apoptosis, pathological damage, fibrosis, and iron deposition in myocardial tissues of HF rats. In hypoxic cells, BMSC-Exos increased cell viability, decreased the number of G1 phase cells, decreased Fe2+ levels, and inhibited oxidative stress. Ferrostatin-1 (a ferroptosis inhibitor) exhibited a synergistic effect with BMSC-Exos. Additionally, GAS5 was upregulated in BMSC-Exos, further upregulating its target UL3 and Hippo pathway effectors (YAP and TAZ). The relieving effects of BMSC-Exos on HF or hypoxia-induced injury were enhanced by GAS5 overexpression, but weakened by UL3 silencing or verteporfin (a YAP inhibitor).ConclusionsGAS5-harbouring BMSC-Exos inhibited ferroptosis by regulating the UL3/Hippo pathway, contributing to HF remission in vivo and in vitro.

Effect of miR-98/IL-6/STAT3 on Autophagy and Apoptosis of Cardiac Stem Cells Under Hypoxic Conditions In vitro.

The heavy burden of cardiovascular diseases demands innovative therapeutic strategies dealing with cardiomyocyte loss. Cardiac Stem Cells (CSCs) are renewable cells in the myocardium with differentiation and endocrine functions. However, their functions are significantly inhibited in conditions of severe hypoxia or inflammation. The mechanism of hypoxia affecting CSCs is not clear. Interleukin-6 (IL-6) appears active in both hypoxic and inflammatory microenvironments. The aim of this study was to explore whether IL-6 is related to CSC apoptosis and autophagy under severe hypoxia. In this study, rat CSCs were extracted by alternate digestion. The interaction of miR-98 and IL-6 mRNA was detected by the dual luciferase method, and qPCR was applied to confirm the effect of miR-98 on IL-6 expression. The effect of IL-6 on CSC apoptosis was measured by flow cytometry and the effect of IL-6 on CSC autophagy by transmission electron microscopy. The western blot method was applied to detect the effect of IL-6 on the expressions of proteins related to apoptosis and autophagy. ANOVA and Dunnett T3's test were employed in the statistical analysis. When p < 0.05, the difference was significant. Under severe hypoxia conditions, IL-6 increased CSC apoptosis and decreased p-STAT3 expression significantly. CSC apoptosis increased significantly after inhibition of the STAT3 signaling pathway under severe hypoxia. IL-6 could also significantly inhibit CSCs' autophagy and block their autophagy flow under severe hypoxic conditions. Meanwhile, it was confirmed that miR-98 had a binding site on IL-6 mRNA and miR-98 significantly inhibited IL-6 mRNA expression in CSCs under severe hypoxic conditions. miR-98/IL-6/STAT3 has been found to be involved in the regulation of CSCs' apoptosis and autophagy under severe hypoxic conditions and there might be a mutual linkage between CSCs' apoptosis and their autophagy.

Platelets Induce Cell Apoptosis of Cardiac Cells via FasL after Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the leading causes of death worldwide. Cell apoptosis in the myocardium plays an important role in ischemia and reperfusion (I/R) injury, leading to cardiac damage and dysfunction. Platelets are major players in hemostasis and play a crucial role in vessel occlusion, inflammation, and cardiac remodeling after I/R. Here, we studied the impact of platelets on cell apoptosis in the myocardium using a close-chest mouse model of AMI. We found caspase-3-positive resident cardiac cells, while leukocytes were negative for caspase-3. Using two different mouse models of thrombocytopenia, we detected a significant reduction in caspase-3 positive cells in the infarct border zone after I/R injury. Further, we identified platelet FasL to induce cell apoptosis via the extrinsic pathway of Fas receptor activation of target cells. Mechanistically, hypoxia triggers platelet adhesion to FasR, suggesting that platelet-induced apoptosis is elevated after I/R. Platelet-specific FasL knock-out mice showed reduced Bax and Bcl2 expression, suggesting that platelets modulate the intrinsic and extrinsic pathways of apoptosis, leading to reduced infarct size after myocardial I/R injury. Thus, a new mechanism for how platelets contribute to tissue homeostasis after AMI was identified that should be validated in patients soon.

Clinical value of BRE-AS1 in myocardial infarction and its role in myocardial infarction-induced cardiac muscle cell apoptosis

Objectives. The aim of this study was to investigate the expression of long non-coding RNA (lncRNA) brain and reproductive organ-expressed protein (BRE) antisense RNA 1 (BRE-AS1) in patients with acute myocardial infarction (AMI) and its effect on ischemia/reperfusion (I/R)-induced oxidative stress and apoptosis of cardiomyocytes. Methods. Serum BRE-AS1 levels in patients with AMI was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic and prognostic values of BRE-AS1 were evaluated. H9c2 cells were treated with hypoxia/reoxygenation to establish an in vitro myocardial infarction cell model. The levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). Levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were determined by commercial kits. Cell counting kit-8 (CCK-8) and flow cytometry were used to evaluate the cell viability and cell apoptosis. Results. The expression of BRE-AS1 in serum of patients with AMI is upregulated, which shows the clinical diagnostic value for AMI. In the I/R injury cell model, the knockout of BRE-AS1 can significantly alleviate the increase in TNF-α, IL-1β, and IL-6 levels, inhibit the production of LDH and MDA, increase the activities of SOD and GSH-Px, promote the cell viability and suppress cell apoptosis. Conclusions. Abnormally elevated BRE-AS1 has a high diagnostic value for AMI as well as a prognostic value for major adverse cardiovascular events (MACEs). The elevation of BRE-AS1 promoted oxidative stress injury and cell apoptosis in vitro.

USP7 cardiomyocyte specific knockout causes disordered mitochondrial biogenesis and dynamics and early neonatal lethality in mice

BackgroundUbiquitination is an enzymatic modification involving ubiquitin chains, that can be reversed by deubiquitination (DUB) enzymes. Ubiquitin-specific protease 7 (USP7), which is also known as herpes virus-associated ubiquitin-specific protease (HAUSP), has been shown to play a vital role in cardiovascular diseases. However, the underlying molecular mechanism by which USP7 regulates cardiomyocyte function has not been reported. MethodsTo understand the physiological function of USP7 in the heart, we constructed cardiomyocyte-specific USP7 conditional knockout mice. ResultsWe found that homozygous knockout mice died approximately three weeks after birth, while heterozygous knockout mice grew normally into adulthood. Severe cardiac dysfunction, hypertrophy, fibrosis, and cell apoptosis were observed in cardiomyocyte-specific USP7 knockout mice, and these effects were accompanied by disordered mitochondrial dynamics and cardiometabolic-related proteins. ConclusionsIn summary, we investigated changes in the growth status and cardiac function of cardiomyocyte-specific USP7 knockout mice, and preliminarily explored the underlying mechanism.

Atractylenolide-I Alleviates Hyperglycemia-Induced Heart Developmental Malformations through Direct and Indirect Modulation of the STAT3 Pathway

BackgroundGestational diabetes could elevate the risk of congenital heart defects (CHD) in infants, and effective preventive and therapeutic medications are currently lacking. Atractylenolide-I (AT-I) is the active ingredient of Atractylodes Macrocephala Koidz (known as Baizhu in China), which is a traditional pregnancy-supporting Chinese herb. PurposeIn this study, we investigated the protective effect of AT-I on the development of CHD in embryos exposed to high glucose (HG). Study design and MethodsFirst, systematic review search results revealed associations between gestational diabetes mellitus (GDM) and cardiovascular malformations. Subsequently, a second systematic review indicated that heart malformations were consistently associated with oxidative stress and cell apoptosis. We assessed the cytotoxic impacts of Atractylenolide compounds (AT-I, AT-II, and AT-III) on H9c2 cells and chick embryos, determining an optimal concentration of AT-I for further investigation. Second, immunofluorescence, western blot, Polymerase Chain Reaction (PCR), and flow cytometry were utilized to delve into the mechanisms through which AT-I mitigates oxidative stress and apoptosis in cardiac cells. Molecular docking was employed to investigate whether AT-I exerts cardioprotective effects via the STAT3 pathway. Then, we developed a streptozotocin-induced diabetes mellitus (PGDM) mouse model to evaluate AT-I's protective efficacy in mammals. Finally, we explored how AT-I protects hyperglycemia-induced abnormal fetal heart development through microbiota analysis and untargeted metabolomics analysis. ResultsThe study showed the protective effect of AT-I on embryonic development using a chick embryo model which rescued the increase in the reactive oxygen species (ROS) and decrease in cell survival induced by HG. We also provided evidence suggesting that AT-I might directly interact with STAT3, inhibiting its phosphorylation. Further, in the PGDM mouse model, we observed that AT-I not only partially alleviated PGDM-related blood glucose issues and complications but also mitigated hyperglycemia-induced abnormal fetal heart development in pregnant mice. This effect is hypothesized to be mediated through alterations in gut microbiota composition. We proposed that dysregulation in microbiota metabolism could influence the downstream STAT3 signaling pathway via EGFR, consequently impacting cardiac development and formation. ConclusionsThis study marks the first documented instance of AT-I's effectiveness in reducing the risk of early cardiac developmental anomalies in fetuses affected by gestational diabetes. AT-I achieves this by inhibiting the STAT3 pathway activated by ROS during gestational diabetes, significantly reducing the risk of fetal cardiac abnormalities. Notably, AT-I also indirectly safeguards normal fetal cardiac development by influencing the maternal gut microbiota and suppressing the EGFR/STAT3 pathway.

Pretreatment with interleukin-15 attenuates inflammation and apoptosis by inhibiting NF-κB signaling in sepsis-induced myocardial dysfunction.

Sepsis-induced myocardial dysfunction (SIMD) is associated with poor prognosis and increased mortality in patients with sepsis. Cytokines are important regulators of both the initiation and progression of sepsis. Interleukin-15 (IL-15), a pro-inflammatory cytokine, has been linked to protective effects against myocardial infarction and myocarditis. However, the role of IL-15 in SIMD remains unclear. We established a mouse model of SIMD via cecal ligation puncture (CLP) surgery and a cell model of myocardial injury via lipopolysaccharide (LPS) stimulation. IL-15 expression was prominently upregulated in septic hearts as well as cardiomyocytes challenged with LPS. IL-15 pretreatment attenuated cardiac inflammation and cell apoptosis and improved cardiac function in the CLP model. Similar cardioprotective effects of IL-15 pretreatment were observed in vitro. As expected, IL-15 knockdown had the opposite effect on LPS-stimulated cardiomyocytes. Mechanistically, we found that IL-15 pretreatment reduced the expression of the pro-apoptotic proteins cleaved caspase-3 and Bax and upregulated the anti-apoptotic protein Bcl-2. RNA sequencing and Western blotting further confirmed that IL-15 pretreatment suppressed the activation of nuclear factor kappa B (NF-κB) signaling in mice with sepsis. Besides, the addition of NF-κB inhibitor can significantly attenuate cardiomyocyte apoptosis compared to the control findings. Our results suggest that IL-15 pretreatment attenuated the cardiac inflammatory responses and reduced cardiomyocyte apoptosis by partially inhibiting NF-κB signaling in vivo and in vitro, thereby improving cardiac function in mice with sepsis. These findings highlight a promising therapeutic strategy for SIMD.

Diosgenin improves post-myocardial infarction cardiac function via HAND2-induced angiogenesis

While diosgenin has been demonstrated effective in various cardiovascular diseases, its specific impact on treating heart attacks remains unclear. Our research revealed that diosgenin significantly improved cardiac function in a myocardial infarction (MI) mouse model, reducing cardiac fibrosis and cell apoptosis while promoting angiogenesis. Mechanistically, diosgenin upregulated the Hand2 expression, promoting the proliferation and migration of endothelial cells under hypoxic conditions. Acting as a transcription factor, HAND2 activated the angiogenesis-related gene Aggf1. Conversely, silencing Hand2 inhibited the diosgenin-induced migration of hypoxic endothelial cells and angiogenesis. In summary, these findings provide new insights into the protective role of diosgenin in MI, validating its effect on angiogenic activity and providing a theoretical basis for clinical treatment strategies.

Lysophosphatidic Acid-Mediated Inflammation at the Heart of Heart Failure.

The primary aim of this review is to provide an in-depth examination of the role bioactive lipids-namely lysophosphatidic acid (LPA) and ceramides-play in inflammation-mediated cardiac remodeling during heart failure. With the global prevalence of heart failure on the rise, it is critical to understand the underlying molecular mechanisms contributing to its pathogenesis. Traditional studies have emphasized factors such as oxidative stress and neurohormonal activation, but emerging research has shed light on bioactive lipids as central mediators in heart failure pathology. By elucidating these intricacies, this review aims to: Bridge the gap between basic research and clinical practice by highlighting clinically relevant pathways contributing to the pathogenesis and prognosis of heart failure. Provide a foundation for the development of targeted therapies that could mitigate the effects of LPA and ceramides on heart failure. Serve as a comprehensive resource for clinicians and researchers interested in the molecular biology of heart failure, aiding in better diagnostic and therapeutic decisions. Recent findings have shed light on the central role of bioactive lipids, specifically lysophosphatidic acid (LPA) and ceramides, in heart failure pathology. Traditional studies have emphasized factors such as hypoxia-mediated cardiomyocyte loss and neurohormonal activation in the development of heart failure. Emerging research has elucidated the intricacies of bioactive lipid-mediated inflammation in cardiac remodeling and the development of heart failure. Studies have shown that LPA and ceramides contribute to the pathogenesis of heart failure by promoting inflammation, fibrosis, and apoptosis in cardiac cells. Additionally, recent studies have identified potential targeted therapies that could mitigate the effects of bioactive lipids on heart failure, including LPA receptor antagonists and ceramide synthase inhibitors. These recent findings provide a promising avenue for the development of targeted therapies that could improve the diagnosis and treatment of heart failure. In this review, we highlight the pivotal role of inflammation induced by bioactive lipid signaling and its influence on the pathogenesis of heart failure. By critically assessing the existing literature, we provide a comprehensive resource for clinicians and researchers interested in the molecular mechanisms of heart failure. Our review aims to bridge the gap between basic research and clinical practice by providing actionable insights and a foundation for the development of targeted therapies that could mitigate the effects of bioactive lipids on heart failure. We hope that this review will aid in better diagnostic and therapeutic decisions, further advancing our collective understanding and management of heart failure.

Diallyl trisulfide (DATS) protects cardiac cells against advanced glycation end-product-induced apoptosis by enhancing FoxO3A-dependent upregulation of miRNA-210

Diabetic cardiomyopathy is a common complication of diabetes, resulting in cardiac hypertrophy and heart failure associated with excessive reactive oxygen species and mitochondria-mediated apoptosis generation. Mitogen-activated protein kinase-c-Jun N-terminal kinase (MAPK-JNK), regulated by microRNA (miR)-210, affects mitochondrial function and is activated by advanced glycation end-products (AGE) in cardiac cells. Diallyl trisulfide (DATS), an antioxidant in garlic oil, inhibits stress-induced cardiac apoptosis. This study examined whether DATS enhances miR-210 expression to attenuate cardiac apoptosis. We investigated the DATS-mediated attenuation mechanism of AGE-enhanced cardiac apoptosis by modulating miR-210 and its upstream transcriptional regulator, FoxO3a. We found FoxO3a binding sites in the miR-210 promoter region. Our results indicated that DATS treatment inhibited AGE-induced JNK activation, phosphoprotein c-Jun nuclear transactivation, and cardiac apoptosis and reversed the AGE-induced reduction in cardiac miR-210 levels. The luciferase activity after DATS treatment was significantly lower than that of the control and was reversed following AGE treatment. We also showed that FoxO3a, upregulated by DATS treatment, may bind to the miR-210 promoter to enhance its expression and downregulates JNK expression to attenuate AGE-induced cardiac apoptosis. Oral administration of DATS enhanced FoxO3a expression in the heart and reduced diabetes-induced heart apoptosis. Our findings indicate that DATS mediates AGE-induced cardiac cell apoptosis attenuation by promoting FoxO3a nuclear transactivation to enhance miR-210 expression and regulate JNK activation. Our results suggest that DATS can be used as a cardioprotective agent, and miR-210 is a critical regulator in inhibiting diabetic cardiomyopathy.

Pinacidil ameliorates cardiac microvascular ischemia-reperfusion injury by inhibiting chaperone-mediated autophagy of calreticulin.

Calcium overload is the key trigger in cardiac microvascular ischemia-reperfusion (I/R) injury, and calreticulin (CRT) is a calcium buffering protein located in the endoplasmic reticulum (ER). Additionally, the role of pinacidil, an antihypertensive drug, in protecting cardiac microcirculation against I/R injury has not been investigated. Hence, this study aimed to explore the benefits of pinacidil on cardiac microvascular I/R injury with a focus on endothelial calcium homeostasis and CRT signaling. Cardiac vascular perfusion and no-reflow area were assessed using FITC-lectin perfusion assay and Thioflavin-S staining. Endothelial calcium homeostasis, CRT-IP3Rs-MCU signaling expression, and apoptosis were assessed by real-time calcium signal reporter GCaMP8, western blotting, and fluorescence staining. Drug affinity-responsive target stability (DARTS) assay was adopted to detect proteins that directly bind to pinacidil. The present study found pinacidil treatment improved capillary density and perfusion, reduced no-reflow and infraction areas, and improved cardiac function and hemodynamics after I/R injury. These benefits were attributed to the ability of pinacidil to alleviate calcium overload and mitochondria-dependent apoptosis in cardiac microvascular endothelial cells (CMECs). Moreover, the DARTS assay showed that pinacidil directly binds to HSP90, through which it inhibits chaperone-mediated autophagy (CMA) degradation of CRT. CRT overexpression inhibited IP3Rs and MCU expression, reduced mitochondrial calcium inflow and mitochondrial injury, and suppressed endothelial apoptosis. Importantly, endothelial-specific overexpression of CRT shared similar benefits with pinacidil on cardiovascular protection against I/R injury. In conclusion, our data indicate that pinacidil attenuated microvascular I/R injury potentially through improving CRT degradation and endothelial calcium overload.

Empagliflozin improves mitochondrial dysfunction in diabetic cardiomyopathy by modulating ketone body metabolism and oxidative stress

Ketone bodies are considered as an alternative energy source for diabetic cardiomyopathy (DCM) and can improve the energy supply of the heart muscle, suggesting that it may be an important area of research and development as a therapeutic target for DCM. Cumulative cardiovascular trials have shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events in diabetic populations. Whether SGLT2 inhibitors improve DCM by enhancing ketone body metabolism remains and whether they help prevent oxidative damage remains to be clarified. Here, we present the combined results of nine GSE datasets for diabetic cardiomyopathy (GSE215979, GSE161931, GSE145294, GSE161052, GSE173384, GSE123975, GSE161827, GSE210612, and GSE5606). We found significant up-regulated gene 3-hydroxymethylglutaryl CoA synthetase 2 (HMGCS2) and down-regulated gene 3-hydroxybutyrate dehydrogenase (BDH1) and 3-oxoacid CoA-transferase1 (OXCT1), respectively. Based on the analysis of the constructed protein interaction network, it was found that HMGCS2 was in the core position of the interaction network. In addition, Gene ontology (GO) enrichment analysis mainly focused on redox process, acyl-CoA metabolic process, catalytic activity, redox enzyme activity and mitochondria. The activity of HMGCS2 in DCM heart was increased, while the expression of ketolysis enzymes BDH1 and OXCT1 was inhibited. In vivo, Empagliflozin (Emp) treated DCM group significantly decreased ventricular weight, myocardial cell cross-sectional area, and myocardial fibrosis. In addition, Emp further promoted the activity of BDH1 and OXCT1, increased the utilization of ketone bodies, further promoted the activity of HMGCS2 in DCM, and increased the synthesis of ketone bodies, prevented mitochondrial breakage and dysfunction, increased myocardial ATP to provide sufficient energy, inhibited oxidative stress and apoptosis of cardiac cells ex vivo, and improved the myocardial dysfunction of DCM. Emp can improve mitochondrial dysfunction in diabetic cardiomyopathy by regulating ketone body metabolism and oxidative stress. These findings provide a theoretical basis for evaluating Emp as a treatment for DCM.