The mitochondrial calcium uniporter induces apoptosis in cardiomyocytes cultured with high-glucose medium by affecting mitochondrial function

the the main of cardiovascular disease in the pathophysiological mechanism of DCM has not been fully 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.


Background
Over the past two decades, diabetes mellitus (DM) prevalence has been increasing worldwide. Meanwhile, the risk of severe cardiovascular disease (CVD) is 2-4 fold higher in DM patients compared with individuals suffering from hypertension, coronary artery disease and heart failure (HF), and the overall mortality rate is 3 fold higher than that of cases without DM [1] . Diabetic cardiomyopathy (DCM) has become an increasingly serious public health issue [2] . Various clinical trials have shown that HF prevalence in DM patients is as high as 19-26% [3,4] . It has been reported in the Framingham Heart Study that 19% of HF patients have type 2 diabetes mellitus (T2DM), which increases the risk of HF by 2-8 fold [5,6] . It was also shown that DM may be an independent risk factor for HF [7] . Mitochondrial dysfunction constitutes an important pathological mechanism of DCM development. Recent studies have demonstrated that unbalanced calcium homeostasis caused by abnormal mitochondrial calcium transport is an important cause of mitochondrial dysfunction [8,9] . Meanwhile, mitochondrial calcium homeostasis is crucial in regulating cytosolic calcium homeostasis, mitochondrial energy metabolism, ATP production, intracellular calcium signaling, oxidative stress, autophagy and apoptosis [8] . The mitochondrial calcium uniporter (MCU) is an important component of the mitochondrial calcium uniport complex [10] , which is the main channel by which calcium ions enter the mitochondrial matrix from the intermembrane space and plays a key role in regulating mitochondrial calcium homeostasis [11,12] . This study aimed to elucidate the mechanism by which MCU affects mitochondrial function in DCM, providing new strategies and targets for the clinical diagnosis and treatment of DCM.

Materials
H9C2 rat cardiomyocytes were provided by the Cell Bank of Chinese Academy of Sciences. DMEM, fetal bovine serum, streptomycin was purchased from Hyclone (USA) and Teizol from Thermo Fisher Scienti c (USA). The mitochondrial membrane potential assay kit (JC-1), ATP assay kit, NADP + /NADPH detection kit (WST-8 method), GSH and GSSG detection kit and RIPA lysis buffer were manufactured by Beyotime streptomycin, and 100 U/L penicillin) for about 5 days (80% con uency), and used for experiments after 2-3 passages. The cultured H9C2 cardiomyocytes were assigned to the experimental and control groups.
The experimental group was cultured with high glucose (33 mmol/L), and control cells were cultured with glucose (25 mmol/L). Experiments were conducted after culturing for 24 h.

Experimental grouping
High glucose group (HG): glucose concentration is 33 mmol/L. Control group (NG + NC siRNA): 5.5 mmol/L glucose and transfected with negative control siRNA.

Research methods
Real-time quantitative polymerase chain reaction and FastKing One Step RT-qPCR Kit (TIANGEN) was used to detect MCU mRNA expression, and Western blot was used to detect MCU protein expression. JC-1 Mitochondrial membrane potential detection kit (Beyotime) detects mitochondrial membrane potential, ATP detection kit (Beyotime) detects cardiomyocyte mitochondrial ATP activity, mitochondrial calcium dye Rhod-2 (Abcam) was used to detect myocardial cell mitochondrial calcium uptake capacity, and Mito-SOX reagent (Thermo Fisher Scienti c) was used to detect Changes of reactive oxygen species (ROS) in cardiac mitochondria. NADP + /NADPH detection kit (Beyotime) and GSH/GSSG detection kit (Beyotime) were used to detect myocardial cells NADP + /NADPH, GSH/GSSG. Western blot was used to detect the expression of caspase-3 (Abcam), cleared caspase-9 (Abcam) and Bcl-2 (Abcam). Flow cytometry combined with Annexin V-FITC/PI staining (BD) and TUNEL apoptosis detection kit (RIBO) was used to detect cardiomyocyte apoptosis.

Statistical treatments
Statistical analyses were performed with the SPSS 22.0 software. Measurement data conforming to normal distribution were presented as Mean ± SD and compared by the t test. Count data were presented as absolute number or percentage. P < 0.05 was considered statistically signi cant.

Effects of high glucose on MCU expression in H9C2 cardiomyocytes
MCU mRNA expression was detected by real-time polymerase chain reaction. The results showed that MCU mRNA expression in the high glucose group was lower than that of the normal group (Fig. 1a). The protein expression of MCU was detected by Western blot. The results also showed that MCU protein amounts in the high glucose group were lower than normal group values (Fig. 1b).
3.2 Down-regulation of MCU in a high glucose environment causes a decrease in mitochondrial Ca 2+ uptake in H9C2 cardiomyocytes Rhod-2 was used to detect mitochondrial Ca 2+ in cardiomyocytes, and uorescence intensity is directly proportional to Ca 2+ levels. The results showed that Ca 2+ levels in the high glucose group were lower than normal group (Fig. 2).
In order to clarify the relationship between Ca 2+ reduction and MCU downregulation in high glucose environment, we downregulated MCU expression in H9C2 cells and used Rhod-2 again to detect mitochondrial Ca 2+ in myocardial cells. The results showed that the mitochondrial Ca 2+ uptake in the experimental group was reduced compared with the normal group and the control group, but there was no difference in the mitochondrial Ca 2+ between the control group and the normal group (Fig. 3).

Mitochondrial dysfunction of H9C2 cardiomyocytes caused by down-regulation of MCU in high glucose environment
Mitochondrial membrane potential (MMP) is one of the important parameters that re ect the functional status of mitochondria in cells. When mitochondrial function is abnormal, mitochondrial membrane potential decreases. JC-1 was used to detect mitochondrial membrane potential in H9C2 cardiomyocytes. A high mitochondrial membrane potential resulted in JC-1 aggregation in the mitochondrial matrix to form a polymer, which could produce red uorescence; in case of mitochondrial membrane potential, JC-1 could not aggregate in the mitochondrial matrix, and, JC-1 as a monomer could produce green uorescence. Usually, the relative proportion of red to green uorescence is used to measure mitochondrial depolarization. In the high glucose group, red uorescence signals were weak, while green uorescence signals were strong, suggesting low membrane potential; in the normal group, red uorescence signals were strong, and green uorescence signals were weak, suggesting high membrane potential. There were signi cant differences between the two groups ( Fig. 4a).
Mitochondria are the main sites for ATP production, and generally a decrease in ATP levels indicates impaired or decreased mitochondrial function. An ATP assay kit was used to detect mitochondrial ATP activity in cardiomyocytes. The results showed that mitochondrial ATP levels in H9C2 cardiomyocytes of the high glucose group were lower than normal group (Fig. 4b).
In order to clarify whether the decrease of mitochondrial membrane potential and the decrease of ATP level in high glucose environment were caused by the down-regulation of MCU, we down-regulated the expression of MCU in H9C2 cells and used JC-1 again to detect the mitochondrial membrane potential of myocardial cells. The potential level is lower than that in the normal group and the control group, and there is no difference in the mitochondrial membrane potential level between the control group and the normal group, (Fig. 5a). Using the ATP detection kit to detect ATP activity of mitochondria in myocardial cells, the results showed that the ATP concentration in the experimental group was lower than that in the control group and the normal group, and there was no difference between the control group and the normal group (Fig. 5b).

Effects of MCU down-regulation in high glucose environment on oxidative stress of H9C2 cardiomyocytes
Mito SOX™-Red reagent penetrates into living cells and selectively targets the mitochondria. It could be rapidly oxidized by superoxide ROS, and after binding of the oxidation product to nucleic acids, red uorescence is produced. A strong uorescence indicates large amounts of ROS, and a weak uorescence indicates small amounts of ROS. The results showed that the high glucose group had stronger red uorescence signals (elevated ROS amounts) compared with the normal group (Fig. 6a).
NADPH and GSH are both important components of the oxidized respiratory chain. NADPH is also used to maintain the reduced state of GSH. When the cell is under oxidative stress, both NADPH and GSH decrease, so the NADP + /NADPH ratio increases and the GSH/GSSG ratio decreases. The NADP + /NADPH assay kit was used to detect NADP + /NADPH in H9C2 cardiomyocytes. The results suggested that the NADP + /NADPH ratio in the high glucose group was higher than that of the control group (Fig. 6b). GSH and GSSG assay kits were used to detect GSH and GSSG (glutathione and oxidized glutathione), respectively, in H9C2 cardiomyocytes; the results demonstrated that the GSH/GSSG ratio in the high glucose group was lower than that of the control group (Fig. 6b).
To clarify the relationship between H9C2 cardiomyocyte oxidative stress enhancement and MCU, we down-regulated the expression of MCU in H9C2 cells and used Mito SOX™-Red reagent to detect the amount of ROS. The results showed that the experimental group had more ROS than the control group and the normal group, and there was no difference between the control group and the normal group ( Fig. 7a). Using NADP + /NADPH detection kit to detect H9C2 cardiomyocytes NADP + / NADPH, the results showed that the experimental group NADP + /NADPH was higher than the control group and the normal group, the control group and the normal group were no different (Fig. 7b). The GSH and GSSG detection kits detect HSH2C cardiomyocytes GSH/GSSG, the results show that the experimental group GSH/GSSG is less than the control group and the normal group, there is no difference between the control group and the normal group (Fig. 7b).

Effect of MCU down-regulation on apoptosis of H9C2 cardiomyocytes in high glucose environment
Western blot to detect the expression of apoptosis-related proteins caspase-3, caspase-9 and apoptosis antagonist protein Bcl-2. The results revealed that these apoptosis-related proteins were altered in the high glucose group compared with the normal group (Fig. 8a).
Use the TUNEL apoptosis detection kit to detect the apoptosis of two groups of cells. Apoptotic cells will be incorporated into TAM-dUTP at the 3 -OH terminals of DNA by the catalysis of TdT enzyme, producing red uorescence (DAPI dye will stain all nuclei). The results showed that the cells in the high glucose group had more red uorescence than the normal group, and the cells in the high glucose group had more apoptosis than the normal group (Fig. 8b).
Flow cytometry combined with Annexin-V-FITC/PI staining was used to detect the apoptosis of H9C2 cardiomyocytes in the two groups. Annexin-V-FITC can combine with early apoptotic cells to emit uorescence, and PI can combine with late apoptotic cells to emit uorescence. In the gure, the LL quadrant represents living cells, the UL quadrant represents cell debris, the LR quadrant represents early apoptotic cells, and the UR quadrant represents late apoptotic cells. Adding the data of the LR and UR quadrants gives the number of apoptotic cells. The results still showed that the apoptosis of cardiomyocytes in high glucose culture was more than that in normal glucose group (Fig. 8c).
In order to clarify the relationship between increased apoptosis of H9C2 cardiomyocytes and MCU, we downregulated the expression of MCU in H9C2 cells, and used Western Blot again to detect the expression of caspase-3, caspase-9 and Bcl-2. The results showed that the expression of caspase-3 and caspase-9 in the experimental group was higher than that in the normal group and the control group, the expression of Bcl-2 was lower than that in the normal group and the control group. There was no statistical difference in protein expression between the control group and the normal group (Fig. 9a).
Using TUNEL apoptosis kit and ow cytometry combined with Annexin-V-FITC/PI staining to detect H9C2 cardiomyocyte apoptosis. The results showed that the apoptosis of myocardial cells in the experimental group was more than that in the normal group and the control group. There was no statistical difference between the control group and the normal group ( Fig. 9b and Fig. 9c).

Discussion
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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 signi cantly 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, Ca 2+ 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 , , and in the oxidative respiratory chain in mitochondria, Ca 2+ can enhance the activity of oxidative phosphorylation, thereby increasing the production of ATP [22] . And when Ca 2+ enters the mitochondrial matrix, it can activate three important Ca 2+ -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 Ca 2+ uptake, and its transport to Ca 2+ 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 Ca 2+ 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 uctuations 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 identi ed 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 (O 2− ) are produced at the regions I and III of the complex, and disproportionate to hydrogen peroxide (H 2 O 2 ) by Mn 2+ -dependent superoxide dismutase [31][32][33] . Then, H 2 O 2 is detoxi ed by glutathione reductase (GSH), and thioredoxin and peroxiredoxin systems; all these reactions require NADPH, which is produced by the tricarboxylic acid cycle [34] . Therefore, Ca 2+ -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 Ca 2+ 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 downregulation 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 Ca 2+ [37] . Thus, MCU, as the main channel involved in mitochondrial Ca 2+ uptake, could be considered a novel potential target for the treatment of DCM, and is worthy of further research.

Consent for publication
All the authors agree.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   Cell mitochondrial Ca2+ assessment by Rhod-2. Ca2+ levels in the high glucose group were lower than those of the normal group (***P 0.001).

Figure 2
Cell mitochondrial Ca2+ assessment by Rhod-2. Ca2+ levels in the high glucose group were lower than those of the normal group (***P 0.001).

Figure 4
Detection of mitochondrial function of H9C2 cardiomyocytes in high glucose environment. a: JC-1 mitochondrial membrane potential reagent was used to detect the mitochondrial membrane potential of H9C2 cardiomyocytes (compared with NG group, ****P<0.0001). b: ATP reagent was used to detect the ATP production concentration of H9C2 cardiomyocytes (compared with NG group, ***P<0.001).

Figure 4
Detection of mitochondrial function of H9C2 cardiomyocytes in high glucose environment. a: JC-1 mitochondrial membrane potential reagent was used to detect the mitochondrial membrane potential of H9C2 cardiomyocytes (compared with NG group, ****P<0.0001). b: ATP reagent was used to detect the ATP production concentration of H9C2 cardiomyocytes (compared with NG group, ***P<0.001).

Figure 6
Detection of oxidative stress in H9C2 cardiomyocytes under high glucose conditions. a: Mito-SOX ™ Red reagent detects the level of ROS (compared with NG group, ***P<0.001). b: NADP+/NADPH detection kit detects the value of NADP+/NADPH in H9C2 cardiomyocytes (compared to NG group, **P<0.01) The GSH/GSSG detection kit was used to detect the GSH / GSSG value of H9C2 cardiomyocytes (compared to the NG group, *P<0.05).

Figure 6
Detection of oxidative stress in H9C2 cardiomyocytes under high glucose conditions. a: Mito-SOX ™ Red reagent detects the level of ROS (compared with NG group, ***P<0.001). b: NADP+/NADPH detection kit detects the value of NADP+/NADPH in H9C2 cardiomyocytes (compared to NG group, **P<0.01) The GSH/GSSG detection kit was used to detect the GSH / GSSG value of H9C2 cardiomyocytes (compared to the NG group, *P<0.05).