Quercetin inhibits necroptosis in cardiomyocytes after ischemia-reperfusion via DNA-PKcs-SIRT5-orchestrated mitochondrial quality control.

We investigated the mechanism by which quercetin preserves mitochondrial quality control (MQC) in cardiomyocytes subjected to ischemia-reperfusion stress. An enzyme-linked immunosorbent assay was employed in the in vivo experiments to assess myocardial injury markers, measure the transcript levels of SIRT5/DNAPK-cs/MLKL during various time intervals of ischemia-reperfusion, and observe structural changes in cardiomyocytes using transmission electron microscopy. In in vitro investigations, adenovirus transfection was employed to establish a gene-modified model of DNA-PKcs, and primary cardiomyocytes were obtained from a mouse model with modified SIRT5 gene. Reverse transcription polymerase chain reaction, laser confocal microscopy, immunofluorescence localization, JC-1 fluorescence assay, Seahorse energy analysis, and various other assays were applied to corroborate the regulatory influence of quercetin on the MQC network in cardiomyocytes after ischemia-reperfusion. In vitro experiments demonstrated that ischemia-reperfusion injury caused changes in the structure of the myocardium. It was seen that quercetin had a beneficial effect on the myocardial tissue, providing protection. As the ischemia-reperfusion process continued, the levels of DNA-PKcs/SIRT5/MLKL transcripts were also found to change. In vitro investigations revealed that quercetin mitigated cardiomyocyte injury caused by mitochondrial oxidative stress through DNA-PKcs, and regulated mitophagy and mitochondrial kinetics to sustain optimal mitochondrial energy metabolism levels. Quercetin, through SIRT5 desuccinylation, modulated the stability of DNA-PKcs, and together they regulated the "mitophagy-unfolded protein response." This preserved the integrity of mitochondrial membrane and genome, mitochondrial dynamics, and mitochondrial energy metabolism. Quercetin may operate synergistically to oversee the regulation of mitophagy and the unfolded protein response through DNA-PKcs-SIRT5 interaction.

Mitochondrial disorder and treatment of ischemic cardiomyopathy: Potential and advantages of Chinese herbal medicine.

Mitochondrial dysfunction is the main cause of damage to the pathological mechanism of ischemic cardiomyopathy. In addition, mitochondrial dysfunction can also affect the homeostasis of cardiomyocytes or endothelial cell dysfunction, leading to a vicious cycle of mitochondrial oxidative stress. And mitochondrial dysfunction is also an important pathological basis for ischemic cardiomyopathy and reperfusion injury after myocardial infarction or end-stage coronary heart disease. Therefore, mitochondria can be used as therapeutic targets against myocardial ischemia injury, and the regulation of mitochondrial morphology, function and structure is a key and important way of targeting mitochondrial quality control therapeutic mechanisms. Mitochondrial quality control includes mechanisms such as mitophagy, mitochondrial dynamics (mitochondrial fusion/fission), mitochondrial biosynthesis, and mitochondrial unfolded protein responses. Among them, the increase of mitochondrial fragmentation caused by mitochondrial pathological fission is the initial factor. The protective mitochondrial fusion can strengthen the interaction and synthesis of paired mitochondria and promote mitochondrial biosynthesis. In ischemia or hypoxia, pathological mitochondrial fission can promote the formation of mitochondrial fragments, fragmented mitochondria can lead to damaged mitochondrial DNA production, which can lead to mitochondrial biosynthesis dysfunction, insufficient mitochondrial ATP production, and mitochondrial ROS. Burst growth or loss of mitochondrial membrane potential. This eventually leads to the accumulation of damaged mitochondria. Then, under the leadership of mitophagy, damaged mitochondria can complete the mitochondrial degradation process through mitophagy, and transport the morphologically and structurally damaged mitochondria to lysosomes for degradation. But once the pathological mitochondrial fission increases, the damaged mitochondria increases, which may activate the pathway of cardiomyocyte death. Although laboratory studies have found that a variety of mitochondrial-targeted drugs can reduce myocardial ischemia and protect cardiomyocytes, there are still few drugs that have successfully passed clinical trials. In this review, we describe the role of MQS in ischemia/hypoxia-induced cardiomyocyte physiopathology and elucidate the relevant mechanisms of mitochondrial dysfunction in ischemic cardiomyopathy. In addition, we also further explained the advantages of natural products in improving mitochondrial dysfunction and protecting myocardial cells from the perspective of pharmacological mechanism, and explained its related mechanisms. Potential targeted therapies that can be used to improve MQS under ischemia/hypoxia are discussed, aiming to accelerate the development of cardioprotective drugs targeting mitochondrial dysfunction.

ß-tubulin contributes to Tongyang Huoxue decoction-induced protection against hypoxia/reoxygenation-induced injury of sinoatrial node cells through SIRT1-mediated regulation of mitochondrial quality surveillance.

BACKGROUND: TYHX-Tongyang Huoxue decoction has been used clinically for nearly 40 years. The ingredients of TYHX are Radix Astragali (Huangqi), Red Ginseng (Hongshen), Rehmannia Glutinosa (Dihuang), Common Yam Rhizome (Shanyao) and Cassia-bark-tree Bark (Rougui). Our previous experiments confirmed that TYHX can protect sinoatrial node cells. However, its mechanism of action is not completely understood yet. PURPOSE: The present study aimed to determine the protective effects of TYHX against Sinus node cell injury under hypoxic stress and elucidate the underlying mechanisms of protection. METHODS: Through RNA sequencing analysis and network pharmacology analysis, we found significant differences in mitochondrial-related genes before and after hypoxia-mimicking SNC, resolved the main regulatory mechanism of TYHX. Through the intervention of TYHX on SNC, a series of detection methods such as laser confocal, fluorescence co-localization, mitochondrial membrane potential and RT-PCR. The regulatory effect of TYHX on ß-tubulin in sinoatrial node cells was verified by in vitro experiments. The mechanism of action of TYHX and its active ingredient quercetin to maintain mitochondrial homeostasis and protect sinoatrial node cells through mitophagy, mitochondrial fusion/fission and mitochondrial biosynthesis was confirmed. RESULTS: Through RNA sequencing analysis, we found that there were significant differences in mitochondrial related genes before and after SNC was modeled by hypoxia. Through pharmacological experiments, we showed that TYHX could inhibit the migration of Drp1 to mitochondria, inhibit excessive mitochondrial fission, activate mitophagy and increase the mitochondrial membrane potential. These protective effects were mainly mediated by ß-tubulin. Furthermore, the active component quercetin in TYHX could inhibit excessive mitochondrial fission through SIRT1, maintain mitochondrial energy metabolism and protect SNCs. Our results showed that protection of mitochondrial function through the maintenance of ß-tubulin and activation of SIRT1 is the main mechanism by which TYHX alleviates hypoxic stress injury in SNCs. The regulatory effects of TYHX and quercetin on mitochondrial quality surveillance are also necessary. Our findings provide empirical evidence supporting the use of TYHX as a targeted treatment for sick sinus syndrome. CONCLUSION: Our data indicate that TYHX exerts protective effects against sinus node cell injury under hypoxic stress, which may be associated with the regulation of mitochondrial quality surveillance (MQS) and inhibition of mitochondrial homeostasis-mediated apoptosis.

Role of Chinese Herbal Medicines in Regulation of Energy Metabolism in Treating Cardiovascular Diseases.

Recently, studying myocardial energy metabolism pathways or improving myocardial metabolism through drugs is another effective strategy for treating ischemic heart disease. Many active components of Chinese herbal medicines (CHMs) have been found to modulate energy metabolism in myocardial cells, cerebral vascular cells, endothelial cells and tumour cells. This paper reviews the advances in studies on the active components of CHMs that modulating energy metabolism in treating cardiovascular diseases over the past five years.

The Effect of Astragaloside on Pacemaker Current and the Cytoskeleton in Rabbit Sinoatrial Node Cells Under the Ischemia and Reperfusion Condition.

Objective: We investigated the role of astragaloside in the treatment of sick sinus syndrome (SSS). Methods: Neonatal New Zealand rabbits were selected for the study. Rabbit sinoatrial node (SAN) cells were isolated by the method of dual enzymatic digestion and differential adherence. The injury model was prepared through simulated ischemia and reperfusion (I/R), and changes in the pacemaker current (If) were recorded using the whole-cell patch-clamp technique. The proteins F-actin and vinculin were examined between various groups of SAN cells using a microplate reader and laser scanning confocal microscopy. The mRNA level and protein expression of hyperpolarization-activated cyclic nucleotide gated potassium channel 4 (HCN4) were assessed by q-PCR and western blot method. Results: The peak current density of If was decreased to -19.64 ± 2.14 pA/pF in SAN cells after simulated I/R, and the difference was highly significant (P < 0.01). Following simulated I/R, 100, 200, or 300 µmol L-1 astragaloside was added to the extracellular solution of SAN cells; the peak current density of the If increased to -30.43 ± 1.98, -34.83 ± 1.6, and -52.72 ± 1.7 pA/pF, respectively (P < 0.01). Adding 100 µmol L-1 astragaloside to normal SAN cells also led to an enhanced peak current density of the If (P < 0.05). In a concentration-dependent manner, especially at 300 µmol/L, astragaloside was capable of increasing the expression of HCN4 and protecting the structural stability of F-actin and vinculin in the damaged SAN cells. Conclusion: We estimated that astragaloside could shorten the action potential duration 20 (APD20) and APD50 in damaged SAN cells of neonatal rabbits, thereby increasing the expression of HCN4 and the If current density in damaged SAN cells of neonatal rabbits in a voltage-dependent manner, accelerating the steady-state activation of the If channels, and protecting damaged cytoskeleton.

[Effect of Zishen Huoxue Recipe on Pathomorphology in Coronary Heart Disease Rats with Shen Deficiency Blood Stasis Syndrome].

OBJECTIVE: To observe the effect of Zishen Huoxue Recipe (ZHR) on pathomorphology in coronary heart disease (CHD) rats with Shen deficiency blood stasis syndrome (SDBSS). METHODS: Totally 60 healthy Wistar rats were divided into the blank control group, the model group, high, middle, and low dose ZHR groups according to random digit table, 12 in each group. Myocardial ischemia SDBSS rat model was prepared by ligating the left anterior descending coronary artery and injecting hydrocortisone. ZHR physic liquor was administered to rats in high, middle, and low dose ZHR groups at the daily dose of 21.6, 10.8, 5.4 g/kg by gastrogavage for 7 successive days, equal volume of pure water was administered to rats in the blank control group and the model group by gastrogavage for 7 successive days. Rat heart was collected for pathomorphological observation under light microscope. RESULTS: In the model group the heart muscle fiber was swollen and deformed with widened space, loose and dropsy tissues. Blood vessels in myocardial mesenchymal were dilated, infiltrated with more inflammatory cells. Myocardial cells were markedly swollen, degenerated, or necrotic, with caryolysis or disappearance of partial nuclear. A large amount of collagen fibrous tissue became hyperplasia. Endocardial blood vessels were swollen and degenerated with infiltration of few inflammatory cells. Epicardium tissue and structure were destroyed and got hyperplasia. Swollen, degenerated, or necrotic vessels could be seen, with infiltration of more inflammatory cells and collagen deposition. Pathomorphological injuries were alleviated in each ZHR group. The higher ZHR concentration, the milder the injury degree of myocardial tissue, the more limited range of damage. CONCLUSION: ZHR could attenuate pathomorphological injuries of myocardial ischemia rats with SDBSS and regulate myocardial function, thus improving myocardial ischemia in CHD rats with SDBSS.