DCM is a disorder of the cardiac muscle among DM patients in the absence of hypertension and structural heart diseases, such as valvular heart disease and CAD [13]. Its main clinical feature is abnormal systolic and diastolic function of the heart [14]. The high incidence of heart failure in DM patients has an important connection with DCM, and DCM has become one of the main causes of death in DM patients [15]. Currently, the pathophysiological mechanism of DCM has not been fully elucidated, and the existing treatment methods are limited, and the patient has a poor prognosis. In the multifactorial pathogenesis of DCM, the reduction of mitochondrial Ca2+ uptake, Mitochondrial calcium homeostasis leads to abnormal mitochondrial electron transport chain and mitochondrial membrane potential and Oxidative stress enhanced caused by imbalanced mitochondrial calcium homeostasis in the high glucose environment is considered one of the important pathological mechanisms of DCM [16, 17]. MCU is the main channel for mitochondrial Ca2+ uptake, playing an essential role in the regulation of mitochondrial calcium homeostasis [18]. In the present study, relevant pathological changes of cardiomyocytes in the high glucose environment were simulated by in vitro culture of rat H9C2 cardiomyocytes, to explore the mechanism by which MCU induces apoptosis in cardiomyocytes.
MCU is the main channel for mitochondrial Ca2+ uptake. As a mitochondrial calcium uniporter, its downregulation directly leads to abnormal mitochondrial calcium uptake, resulting in imbalanced mitochondrial calcium homeostasis [19, 20]. We tested the expression level of MCU and mitochondrial Ca2+ in rat H9C2 cardiomyocytes cultured in simulated high glucose environment and normal glucose culture. The results showed that MCU mRNA expression and mitochondrial calcium uptake in the high glucose group was lower than that of the normal control group. However, is the decrease in mitochondrial calcium uptake in cardiomyocytes caused by a decrease in MCU expression? We culture H9C2 cardiomyocytes in a normal environment in vitro to down-regulate the expression of MCU, the results showed that the decreased MCU expression in H9C2 cardiomyocytes caused a decrease in mitochondrial calcium uptake. Therefore, it can be seen that the loss of MCU expression in cardiomyocytes in a high glucose environment significantly affects the mitochondrial calcium uptake of cardiomyocytes, which may be one of the important causes of myocardial damage caused by the imbalance of mitochondrial calcium homeostasis in DM patients.
As the second messenger factor, Ca2+ is an important signal in the transmission mechanism of mitochondrial energy activity which regulates multiple mitochondrial functions from metabolism to apoptosis [21]. In the complexes Ⅰ, Ⅲ, Ⅳ and Ⅴ in the oxidative respiratory chain in mitochondria, Ca2+ can enhance the activity of oxidative phosphorylation, thereby increasing the production of ATP [22]. And when Ca2+ enters the mitochondrial matrix, it can activate three important Ca2+-dependent dehydrogenases in the rate-limiting enzymes in the tricarboxylic acid cycle, i.e. pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (αKGDH) and isocitrate dehydrogenase (IDH), furthermore, it strengthens the mitochondrial tricarboxylic acid cycle reaction and ATP production [23, 24]. At the same time, MCU is the most highly selective channel for mitochondrial Ca2+ uptake, and its transport to Ca2+ depends on the electrochemical gradient of mitochondrial membrane potential [25]. Previous studies have shown that after the application of MCU inhibitors, mitochondrial calcium homeostasis has been disturbed and further led to mitochondrial oxidative respiratory dysfunction and membrane potential loss [26]. Mitochondrial Ca2+ uptake depends on the mitochondrial membrane potential, and the myocardial cell mitochondrial membrane potential decreases in a high-glucose environment, thus forming a vicious cycle, which ultimately leads to cell apoptosis [27]. The present experimental results showed that the calcium homeostasis high glucose group H9C2 cardiomyocytes had reduced ATP production, decreased membrane potential, and increased apoptosis. We also cultured H9C2 cardiomyocytes in a normal environment in vitro to down-regulate the expression of MCU, which further proves that the increase in apoptosis caused by mitochondrial dysfunction of cardiomyocytes in high glucose environment is caused by the decrease in MCU expression.
Oxidative stress is considered to be one of the important factors that trigger the occurrence of DCM. Persistent hyperglycemia and fluctuations in blood glucose in patients with DM can cause acute oxidative stress, resulting in increased production of ROS, cell dysfunction, and death [28, 29]. Overproduction of ROS has been identified as one of the initial pathogenic factors of DCM. Related studies have shown that enhancing the endogenous antioxidant capacity of the myocardium can effectively reduce the myocardial damage in patients with DM [30]. In the electron transfer chain (ETC), superoxide anion radicals (O2−) are produced at the regions I and III of the complex, and disproportionate to hydrogen peroxide (H2O2) by Mn2+-dependent superoxide dismutase [31–33]. Then, H2O2 is detoxified by glutathione reductase (GSH), and thioredoxin and peroxiredoxin systems; all these reactions require NADPH, which is produced by the tricarboxylic acid cycle [34]. Therefore, Ca2+-regulated tricarboxylic acid cycle not only provides energy, but also maintains the body's redox equilibrium. Once The imbalance of mitochondrial calcium homeostasis,it will leads to altered redox equilibrium in cardiomyocytes, enhanced oxidative stress and increased production of ROS, eventually it will lead to cardiomyocyte apoptosis [35, 36]. Through experiments, we have found that under high glucose environment, the mitochondrial Ca2+ uptake of H9C2 cardiomyocytes is reduced, and the production of cardiac NADPH and GSH is reduced, which leads to weakened antioxidant capacity, increased ROS production and myocardial apoptosis. Simultaneously, an appeal phenomenon was also found in H9C2 cardiomyocytes that regulated the expression of MCU under normal circumstances. Therefore, we speculate that the apoptosis caused by the increased oxidative stress of cardiomyocytes in high glucose environment is caused by the down-regulation of MCU. This may be an important cause of myocardial damage caused by DCM.
This study suggested that MCU expression in rat H9C2 cardiomyocytes was decreased in the high glucose environment, causing abnormal mitochondrial calcium uptake and imbalanced calcium homeostasis, which may further contribute to mitochondrial dysfunction (decreased mitochondrial membrane potential and reduced ATP production) and enhanced oxidative stress (increased ROS production, increased ratio of NADP+/NADPH and reduced GSH/GSSG ratio) in cardiomyocytes. Mitochondrial dysfunction and enhanced oxidative stress ultimately led to apoptosis in cardiomyocytes. In conclusion, MCU may be vital in the development and progression of DCM. Previous studies have also demonstrated that recovery of MCU level can restore high glucose-induced metabolic changes and normalize Ca2+ [37]. Thus, MCU, as the main channel involved in mitochondrial Ca2+ uptake, could be considered a novel potential target for the treatment of DCM, and is worthy of further research.