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

doi:10.21203/rs.3.rs-27647/v1

PDF

Study protocol

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

https://doi.org/10.21203/rs.3.rs-27647/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted 29 May, 2020

You are reading this latest preprint version

Background

As the number of diabetics worldwide continues to increase, diabetic cardiomyopathy has become one of the main causes of cardiovascular disease risk in diabetic patients. Currently, the pathophysiological mechanism of DCM has not been fully elucidated. 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.

Method:

Cultured rat myocardium H9C2 cells in vitro and divided into high glucose group (glucose concentration 33 mmol/L), normal group (glucose concentration 5.5 mmol/L), experimental group (5.5 mmol/L glucose and transfected with MCU siRNA) and control group (5.5 mmol/L glucose and transfected negative control siRNA). Comparative analysis of MCU expression, Ca²⁺ uptake, mitochondrial function, oxidative stress and apoptosis of two groups of cells.

Results

(1) Compared with normal group, in the high glucose group the MCU expression of myocardial cells in H9C2 rats decreased, The Ca²⁺ levels, membrane potential and mitochondrial ATP levels decreased, mitochondrial ROS levels increased, NADH⁺/NADPH ratio in cardiomyocytes increased, GSH/GSSG ratio decreased, the expression levels of cleaved caspase-3 and cleaved caspase-9 increased, bcl-2 expression decreased, the number of cardiomyocytes apoptotic cells increases. (2) Compared with the normal group and the control group, the experimental group MCU expression of myocardial cells in H9C2 rats decreased, The Ca²⁺ levels, membrane potential and mitochondrial ATP levels decreased, mitochondrial ROS levels increased, NADH⁺/NADPH ratio in cardiomyocytes increased, GSH/GSSG ratio decreased, the expression levels of cleaved caspase-3 and cleaved caspase-9 increased, bcl-2 expression decreased, the number of cardiomyocytes apoptotic cells increases.

Discussion

Cardiac & Cardiovascular Systems

Endocrinology & Metabolism

mitochondrial calcium uniporter (MCU)

mitochondrial dysfunction

oxidative stress

apoptosis

diabetic cardiomyopathy

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.

2.1 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 Scientific (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 Biotechnology Co., Ltd. (China). Skim milk powder for blocking was purchased from Solarbio Science & Technology Co., Ltd. (China). The calcium ion assay kit and MCU antibodies were provided by Abcam (USA). Goat anti-mouse and anti-rabbit polyclonal antibodies were purchased from Boster Biological Technology Co., Ltd. (China). PVDF membranes were from Millipore (USA). MCU primers were provided by TaKaRa (Japan). Reverse transcription kit and RT-PCR assay kit purchased from TIANGEN Biotech Co., Ltd. (China). TUNEL apoptosis detection kit was purchased from RiobBio Technology Co., Ltd. (China). Annexin V-FITC / PI cell apoptosis detection kit was purchased from BD Biosciences Co., Ltd. (USA). siRNA purchased from GenePharma.

2.2 Methods

2.2.1 Cell culture

H9C2 cardiomyocytes were cultured in DMEM (Hyclone) complete medium (20% fetal bovine serum, 1% streptomycin, and 100 U/L penicillin) for about 5 days (80% confluency), 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.

2.2.2 Experimental grouping

High glucose group (HG): glucose concentration is 33 mmol/L.

Normal group (NG): Glucose concentration is 5.5 mmol/L.

Experimental group (NG + MCU siRNA): 5.5 mmol/L glucose and transfected with MCU siRNA.

Control group (NG + NC siRNA): 5.5 mmol/L glucose and transfected with negative control siRNA.

2.2.3 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 Scientific) 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.

2.3 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 significant.

3.1 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 Ca²⁺ in cardiomyocytes, and fluorescence intensity is directly proportional to Ca²⁺ levels. The results showed that Ca²⁺ levels in the high glucose group were lower than normal group (Fig. 2).

In order to clarify the relationship between Ca²⁺ reduction and MCU downregulation in high glucose environment, we downregulated MCU expression in H9C2 cells and used Rhod-2 again to detect mitochondrial Ca²⁺ in myocardial cells. The results showed that the mitochondrial Ca²⁺ uptake in the experimental group was reduced compared with the normal group and the control group, but there was no difference in the mitochondrial Ca²⁺ between the control group and the normal group (Fig. 3).

3.3 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 reflect 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 fluorescence; in case of mitochondrial membrane potential, JC-1 could not aggregate in the mitochondrial matrix, and, JC-1 as a monomer could produce green fluorescence. Usually, the relative proportion of red to green fluorescence is used to measure mitochondrial depolarization. In the high glucose group, red fluorescence signals were weak, while green fluorescence signals were strong, suggesting low membrane potential; in the normal group, red fluorescence signals were strong, and green fluorescence signals were weak, suggesting high membrane potential. There were significant 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).

3.4 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 fluorescence is produced. A strong fluorescence indicates large amounts of ROS, and a weak fluorescence indicates small amounts of ROS. The results showed that the high glucose group had stronger red fluorescence 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).

3.5 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 fluorescence (DAPI dye will stain all nuclei). The results showed that the cells in the high glucose group had more red fluorescence 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 fluorescence, and PI can combine with late apoptotic cells to emit fluorescence. In the figure, 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 flow 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).

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 Ca^{2+ [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.

diabetes mellitus	DM
cardiovascular disease	CVD
heart failure	HF
Diabetic cardiomyopathy	DCM
type 2 diabetes mellitus	T2DM
mitochondrial calcium uniporter	MCU
mitochondrial membrane potential	MMP
reactive oxygen species	ROS
pyruvate dehydrogenase	PDH
α-ketoglutarate dehydrogenase	αKGDH
isocitrate dehydrogenase	IDH
electron transfer chain	ETC
glutathione reductase	GSH

Ethics approval and consent to participate

Not applicable.

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.

Funding

All sources of funding for the research reported was from the Gansu Natural Fund (Mitochondrial Calcium Uniporter Transporter Down-regulation Increases Myocardial Ischemic Vulnerability and Its Mechanism), Project code: 18JR3RA402.

Authors' contributions

Xia Chen, Wenyun Guo and Zhe Jing designed the study, analysed and interpreted the data, and drafted the paper. Yongqing Chen and Hui Chen designed and directed research. Tao Zhang, Zhaoqi Wu, Hengqiang Cui and Wenxue Zheng contributed to the experimental data collection. All authors read and approved the final manuscript.

Acknowledgements

Thanks to the members of our experimental team and instructors for their contributions in this research.

Danaei G, Finucane MM, Lu Y, et al. National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2·7 million participants[J].Lancet. 2011,378(9785):31–40. doi:10.1016/S0140-6736(11)60679-X.
Wang ZV, Hill JA. Diabetic cardiomyopathy: catabolism driving metabolism[J]. Circulation. 2015;131:771–3. doi:10.1161/CIRCULATIONAHA.115.015357.
Ryden L, Armstrong PW, Cleland JG, et al. Efficacy and safety of high-dose lisinopril in chronic heart failure patients at high cardiovascular risk, including those with diabetes mellitus: results from the ATLAS trial[J]. Eur Heart J. 2000;21:1967–78. doi:10.1053/euhj.2000.2311.
Shindler DM, Kostis JB, Yusuf S, et al. Diabetes mellitus, a predictor of morbidity and mortality in the Studies of Left Ventricular Dysfunction (SOLVD) Trials and Registry[J]. Am J Cardiol. 1996;77:1017–20. doi:10.1016/s0002-9149(97)89163-1.
Wong AK, AlZadjali MA, Choy AM, et al. Insulin resistance: a potential new target for therapy in patients with heart failure[J]. Cardiovasc Ther. 2008;26:203–13. doi:10.1111/j.1755-5922.2008.00053.
Maisch B, Alter P, Pankuweit S. Diabetic cardiomyopathy: fact or fiction[J]? Herz. 2011,36:102–115. doi: 10.1007/s00059-011-3429-4.
Jia G, DeMarco VG, Sowers JR. Insulin resistance and hyperinsulinaemia in diabetic cardiomyopathy[J]. Nat Rev Endocrinol. 2016;2:144–53. doi:10.1038/nrendo.2015.216.
Duncan JG. Mitochondrial dysfunction in diabetic cardiomyopathy[J]. Biochim Biophys Acta. 2011;1813(7):1351–9. doi:10.1016/j.bbamcr.2011.01.014.
Abel 9BuggerH. ED. Mitochondria in the diabetic heart[J]. Cardiovasc Res. 2010;88(2):229–40. doi:10.1093/cvr/cvq239. Epub 2010 Jul 16. Review.
Fan C, Fan M, Orlando BJ,et al.X-ray and cryo-EM structures of the mitochondrial calcium uniporter[J].Nature,2018,559(7715):575–579.DOI:10.1038/s41586-018-0330-9.
Baughman JM, Perocchi F, Girgis HS, et al. Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter[J]. Nature. 2011;476:341–5. doi:10.1038/nature10234.
Choi S, Quan X, Bang S, et al. Mitochondrial calcium uniporter in transfers calcium between the endoplasmic reticulum and mitochondria in oxidative stress-induced cell death[J]. J Biol Chem. 2017;292(35):14473–85. doi:10.1074/jbc.M116.765578.
Aneja A, Tang WH, Bansilal S, et al. Diabetic cardiomyopathy: insights into pathogenesis, diagnostic challenges, and therapeutic options[J]. Am J Med. 2008;121:748–57. doi:10.1016/j.amjmed.2008.03.046.
Pham T, Loiselle D, Power A, et al. Mitochondrial inefficiencies and anoxic ATP hydrolysis capacities in diabetic rat heart[J]. Am J Physiol Cell Physiol. 2014;307(6):499–507. doi: 10.1152/ajpcell.00006.2014. Epub 2014 Jun 11.
Bragg F, Holmes MV, Iona A, et al. Association between diabetes and causespecific mortality in rural and urban areas of China[J]. JAMA. 2017;317(3):280–9. doi:10.1001/jama.2016.19720.
Sorrentino A, Borghetti G, Zhou Y, et al. Hyperglycemia induces defective Ca²⁺ homeostasis in cardiomyocytes[J]. Am J Physiol Heart Circ Physiol. 2017;312(1):H150–61. doi:10.1152/ajpheart.00737.2016.
Brownlee M. Biochemistry and molecular cell biology of diabetic complications[J]. Nature. 2001;414(6865):813–20. doi:10.1038/414813a.
Kamer KJ. The molecular era of the mitochondrial calcium uniporter[J]. Nat Rev Mol Cell Biol. 2015;16(9):545–53. doi:10.1038/nrm4039.
Kwong JQ, Lu X, Correll RN, et al. The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart[J]. Cell Rep. 2015;12(1):15–22. doi: 10.1016/j.celrep.2015.06.002. Epub 2015 Jun 25.
Kwong JQ. The mitochondrial calcium uniporter in the heart: energetics and beyond[J]. J Physiol. 2017;15(12):3743–51. doi:10.1113/JP273059. Epub 2017 Feb 1. 595 ) .
Bravo-Sagua R, Parra V, López-Crisosto C, et al. Calcium Transport and Signaling in Mitochondria[J]. Comprehensive Physiology. 2017;7(2):623–34. doi:10.1002/cphy.c160013.
Plattner H. Inseparable tandem: evolution chooses ATP and Ca²⁺ to control life, death and cellular signalling[J]. Philos Trans R Soc Lond B Biol Sci. 2016,371(1700). doi:10.1098/rstb.2015.0419.
Rizzuto R, De Stefani D, Raffaello A, et al. Mitochondria as sensors and regulators of calcium signalling[J]. Nat Rev Mol Cell Biol. 2012;13(9):566–78. doi:10.1038/nrm3412.
Denton RM. Regulation of mitochondrial dehydrogenases by calcium ions[J]. Biochim Biophys Acta. 2009;1787(11):1309–16. doi:10.1016/j.bbabio.2009.01.005.
De Stefani D, Raffaello A, Teardo E, et al. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter[J]. Nature. 2011;476(7360):336–40. doi:10.1038/nature10230.
Rossi CS, Vasington FD, Carafoli E, et al. The effect of ruthenium red on the uptake and release of Ca²⁺ by mitochondria[J]. Biochem Biophys Res Commun. 1973;50(3):846–52. doi:10.1016/0006-291x(73)91322-3.
Kain V, Sawant MA, Dasgupta A. A novel SOD mimic with a redox-modulating mn (II) complex, ML1 attenuates high glucose-induced abnormalities in intracellular Ca2 + transients and prevents cardiac cell death through restoration of mitochondrial function[J]. Biochem Biophys Rep. 2016;5:296–304. doi:10.1016/j.bbrep.2016.01.003.
Liu ZW, Zhu HT, Chen KL, et al. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling pathway plays a major role in reactive oxygen species (ROS)-mediated endoplasmic reticulum stress-induced apoptosis in diabetic cardiomyopathy[J]. Cardiovasc Diabetol. 2013;112:158. doi:10.1186/1475-2840-12-158.
Evangelista I, Nuti R, Picchioni T, et al. Molecular Dysfunction and Phenotypic Derangement in Diabetic Cardiomyopathy[J]. Int J Mol Sci. 2019;20(13):1–15. doi:10.3390/ijms20133264.
Guo Y, Zhuang X, Huang Z, et al. Klotho protects the heart from hyperglycemia-induced injury by inactivating ROS and NF-κB-mediated inflammation both in vitro and in vivo[J]. Biochim Biophys Acta Mol Basis Dis. 2018;1864(1):238–51. doi:10.1016/j.bbadis.2017.09.029.
Chen Y-R, Zweier JL. Cardiac mitochondria and reactive oxygen species generation[J]. Circ Res. 2014;114(3):524–37. doi:10.1161/CIRCRESAHA.114.300559.
Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release[J]. Physiol Rev. 2014;94(3):909–50. doi:10.1152/physrev.00026.2013.
Murphy MP. Understanding and preventing mitochondrial oxidative damage[J]. Biochem Soc Trans. 2016;44(5):1219–26. doi:10.1042/BST20160108.
Nickel AG, von Hardenberg A, Hohl M, et al. Reversal of ¨ mitochondrial transhydrogenase causes oxidative stress in heart failure[J]. Cell Metab. 2015;22(3):472–84. doi:10.1016/j.cmet.2015.07.008.
Kohlhaas M, Nickel AG. Mitochondrial energetics and calcium coupling in the heart[J]. J Physiol. 2017;595(12):3753–63. doi:10.1113/JP273609.
Joseph SK, Booth DM, Young MP, et al. Redox regulation of ER and mitochondrial Ca²⁺ signaling in cell survival and death[J]. Cell Calcium. 2019;79:89–97. doi:10.1016/j.ceca.2019.02.006.
Diaz-Juarez J, Suarez J, Cividini F, et al. Expression of the mitochondrial calcium uniporter in cardiac myocytes improves impaired mitochondrial calcium handling and metabolism in simulated hyperglycemia[J]. Am J Physiol Cell Physiol. 2016;311(6):C1005–13. doi:10.1152/ajpcell.00236.2016.

PDF

Version 1

posted 29 May, 2020

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Background

Method:

Results

Discussion

Figures

1. Background

2. Methods

2.1 Materials

2.2 Methods

2.2.1 Cell culture

2.2.2 Experimental grouping

2.2.3 Research methods

2.3 Statistical treatments

3. Results

3.1 Effects of high glucose on MCU expression in H9C2 cardiomyocytes

3.3 Mitochondrial dysfunction of H9C2 cardiomyocytes caused by down-regulation of MCU in high glucose environment

3.4 Effects of MCU down-regulation in high glucose environment on oxidative stress of H9C2 cardiomyocytes

3.5 Effect of MCU down-regulation on apoptosis of H9C2 cardiomyocytes in high glucose environment

4. Discussion

List Of Abbreviations

Declarations

Ethics approval and consent to participate

Funding

Authors' contributions

Acknowledgements

References

Status:

Version 1