Mitofusin-2 Enhances Mitochondrial Contact With the Endoplasmic Reticulum and Promotes Diabetic Cardiomyopathy.

Diabetic cardiomyopathy has been associated with mitochondrial damage. Mitochondria-endoplasmic reticulum (ER) contact is an important determinant of mitochondrial function and ER homeostasis. We therefore investigated whether hyperglycemia can damage the mitochondria by increasing their contact with the ER in cardiomyocytes. We found that hyperglycemia induced mitochondria-ER contact in cardiomyocytes, as evidenced by the increased MMM1, MDM34, and BAP31 expressions. Interestingly, the silencing of Mfn2 reduced the cooperation between the mitochondria and the ER in cardiomyocytes. Mfn2 silencing improved cardiomyocyte viability and function under hyperglycemic conditions. Additionally, the silencing of Mfn2 markedly attenuated the release of calcium from the ER to the mitochondria, thereby preserving mitochondrial metabolism in cardiomyocytes under hyperglycemic conditions. Mfn2 silencing reduced mitochondrial reactive oxygen species production, which reduced mitochondria-dependent apoptosis in hyperglycemia-treated cardiomyocytes. Finally, Mfn2 silencing attenuated ER stress in cardiomyocytes subjected to high-glucose stress. These results demonstrate that Mfn2 promotes mitochondria-ER contact in hyperglycemia-treated cardiomyocytes. The silencing of Mfn2 sustained mitochondrial function, suppressed mitochondrial calcium overload, prevented mitochondrial apoptosis, and reduced ER stress, thereby enhancing cardiomyocyte survival under hyperglycemic conditions.

Introduction

Diabetic cardiomyopathy is marked by hyperglycemia-induced cardiomyocyte apoptosis and cardiac fibroblast proliferation, resulting in myocardial fibrosis and cardiac dysfunction (Jiang et al., 2020). Under physiological conditions, the primary energetic substrates within cardiomyocytes are fatty acids; however, under hyperglycemic conditions, cardiomyocytes preferentially employ glucose to generate adenosine triphosphate (ATP) due to excessive glucose uptake (Abbas et al., 2020; Adapala et al., 2020). Fatty acids and glucose are metabolized through the tricarboxylic acid cycle in the mitochondria, and a dysregulated cardiomyocyte metabolism induces mitochondrial oxidative stress (Zhou et al., 2018b; Capasso et al., 2020). Specifically, increased glucose metabolism and reduced fatty acid oxidation stimulate reactive oxygen species (ROS) production, thus activating mitochondrial fission, reducing the mitochondrial membrane potential, impairing mitochondrial metabolism, and inducing mitochondrial apoptosis (Klinge, 2020; Lubos et al., 2020; Wang et al., 2020b). However, the upstream regulatory signals of hyperglycemia-induced mitochondrial damage are not clear, so targeted therapies are not available in clinical practice to prevent cardiomyocyte damage in hyperglycemic patients.

Contact with the endoplasmic reticulum (ER) can modify the function and structure of the mitochondria (Zhou et al., 2018a). Mitochondrial glucose oxidation and fatty acid metabolism are induced upon the uptake of calcium from the ER (the primary calcium factory in cardiomyocytes), and this transfer strongly depends on mitochondria–ER contact (Zhang et al., 2016; Zhou et al., 2018a). ROS are mainly generated by mitochondrial respiratory complexes I and III, and higher calcium concentrations are associated with greater ROS production in the mitochondria (Lindner et al., 2020; Zhang J. et al., 2020). The mitochondrial morphology is also altered by mitochondria–ER contact, with increased contact promoting mitochondrial fission and reduced contact enhancing mitochondrial fusion (Li et al., 2020; Wang et al., 2020e; Zhu and Zhou, 2021). These observations led us to wonder whether hyperglycemia-induced cardiomyocyte apoptosis could be due to abnormal mitochondria–ER contact.

Mitofusin-2 (Mfn2) is expressed on the surface of both the mitochondria and the ER (Hughes et al., 2020) and has been reported to promote their contact, thus increasing mitochondrial calcium levels and ROS production. Mfn2 is known to be involved in cardiovascular disorders; for example, large tumor suppressor kinase 2 was found to activate oxidative stress-induced apoptosis by suppressing the peroxiredoxin 3–Mfn2 signaling pathway in cardiomyocytes (Tian et al., 2019; Wang and Zhou, 2020; Wang et al., 2020a,b). Mfn2-induced mitophagy and mitochondrial fusion were shown to attenuate hypertension-associated cardiomyocyte injury by inhibiting cardiomyocyte apoptosis (Xiong et al., 2019; Tan et al., 2020). On the other hand, in a mouse model of cardiac ischemia–reperfusion injury, the downregulation of Mfn2 significantly reduced myocardial damage (Qin et al., 2018). However, the function of Mfn2 in diabetic cardiomyocytes is not fully understood.

In the present study, we assessed whether hyperglycemia-induced cardiomyocyte damage was associated with mitochondrial dysfunction and whether such dysfunction was due to greater mitochondria–ER contact. In addition, we investigated whether mitochondria–ER contact in cardiomyocytes depended on Mfn2 under hyperglycemic conditions.

Materials and Methods

Cell Culture and High-Glucose Model

Cardiomyocytes were purchased from the American Type Culture Collection (Manassas, VA, United States) and cultured in RPMI 1640 medium (Corning Inc., Corning, NY, United States) supplemented with 20% fetal bovine serum (Corning Inc.), 100 U/ml penicillin G, and 100 mg/ml streptomycin (Hausenloy et al., 2020). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. For the induction of the high-glucose model, cardiomyocytes were treated for 24 h with medium containing 25 mM glucose, as previously described (Detter et al., 2020).

Measurement of Mitochondrial ROS Production

Cells were grown in clear-bottom 96-well black plates (Corning Inc.) and then incubated in the presence or absence of Mfn2 small interfering RNA (siRNA) for 48 h at 37°C. Then, the cells were washed with phosphate-buffered saline (pH 7.4) and further incubated with MitoSOX red mitochondrial superoxide indicator (Molecular Probes, Eugene, OR, United States) for 18 h at 37°C (Bakhta et al., 2020). Subsequently, the cells were washed twice with phosphate-buffered saline (pH 7.4) and the fluorescence of MitoSOX was read at excitation/emission wavelengths of 485/530 nm (Islam, 2020; Mills et al., 2020).

TUNEL Staining

Cardiomyocyte apoptosis was determined using a one-step terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions (Dieterich et al., 2020; Jusic and Devaux, 2020). A mouse anti-troponin T (TnT) antibody (1:100; Millipore Corporation, Billerica, MA, United States) was used to label the cardiomyocytes and 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) used to counterstain the nuclei. Images were captured with a fluorescence microscope (Olympus, Tokyo, Japan), and apoptosis was assessed based on the overlap between TnT and TUNEL staining (Domingues et al., 2020; Heimerl et al., 2020).

Assessment of Cardiomyocyte Viability

After being cultured for 48 h, the cells were digested and seeded into 96-well plates at a density of 3,000 cells/well. Five replicate wells were used for each group, and 20 μl of 5 g/L 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution was added to each well. The cells were placed in an incubator for 4 h (Fukada and Kajiya, 2020; Lubos et al., 2020), after which the supernatants were completely removed, 150 μl of dimethyl sulfoxide was added to each well, and the plates were shaken for 10 min. After the purple crystals had fully dissolved, the absorbance was measured at a wavelength of 570 nm on a microplate reader and the cell proliferation rate calculated (Wang et al., 2020c,d).

Mfn2 siRNA Transfection Under High-Glucose Conditions

Cardiomyocytes under normal or high-glucose conditions were transfected with siRNA against Mfn2 using the Lipofectamine 2000 transfection reagent. Cardiomyocytes in the logarithmic growth phase were collected, adjusted to a concentration of 3 × 106 cells/ml, plated in a six-well plate, and placed in a 5% CO2 incubator at 37°C for 12 h (Jiang and Li, 2020; Zhang Y. J. et al., 2020). When cell density fusion reached 70–80%, 5 ml of Lipofectamine 2000 was added to 200 μl of serum-free medium, incubated for 15 min, combined with another 200 ml of the serum-free medium, and then incubated for another 15 min at room temperature. The Lipofectamine 2000 mixture was then combined with the siRNA against Mfn2 and incubated for 30 min at room temperature. Subsequently, the cell serum in the six-well plate was removed and the cells were gently rinsed with phosphate-buffered saline (Zhang L. et al., 2020; Zhao et al., 2020). Then, 1.6 ml of the serum-free medium was added, followed by the siRNA/Lipofectamine mixture, and the cells were returned to the incubator. The culture solution was changed after 6 h to continue the culture.

Western Blotting

Total proteins were extracted from cells using a radioimmunoprecipitation assay lysis buffer (Boster, Wuhan, China) according to the manufacturer’s instructions. Then, the proteins were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Lahiri et al., 2020) and transferred to polyvinylidene difluoride membranes (Millipore Corporation) (Mossoba et al., 2020; Yang et al., 2020). After blocking with 5% skimmed milk in Tris-buffered saline/Tween-20 for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: Bcl-2 (1:1,000; #3498, Cell Signaling Technology, Danvers, MA, United States), Bax (1:1,000; #2772, Cell Signaling Technology), caspase-9 (1:1,000; #9504, Cell Signaling Technology), c-IAP (1:1,000; #4952, Cell Signaling Technology), survivin (1:1,000; #2808, Cell Signaling Technology), and Bad (1:1,000; #ab90435, Abcam, Cambridge, United Kingdom). The membranes were washed three times with Tris-buffered saline/Tween-20 and then incubated with a horseradish peroxidase-conjugated secondary antibody (1:3,000; Abcam) for 1 h at room temperature. Images were visualized using a chemiluminescent substrate (Boster) and analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, United States) (García-Gómez and Valiente, 2020).

qRT-PCR

Total RNA was extracted using an RNAiso Plus kit (TaKaRa, Dalian, China) and reverse-transcribed using a Prime Script RT reagent kit with gDNA Eraser (TaKaRa) according to the manufacturer’s instructions on a CFX96 detection system (Bio-Rad) (Fournier et al., 2020). Then, quantitative real-time PCR (qRT-PCR) was performed using SYBR Premix Ex TaqII (TaKaRa) with the following primers: CHOP: forward 5′-CATGGCAGTGTCTTAGCTGGTT-3′, reverse 5′-CAGTGCAGGGTCCGAGGTAT-3′; PERK: forward 5′-CTCGCTTCGGCAGCACA-3′, reverse 5′-AACGCTTCACGAATTTGCGT-3′. GAPDH was used to standardize the transcription of the target RNAs (Ko et al., 2020; Mossoba et al., 2020).

Mitochondrial Calcium Level and Mitochondrial Membrane Potential

The mitochondrial calcium level was determined using Rhod-2 (Molecular Probes) according to the manufacturer’s instructions (Sanchez et al., 2020). The mitochondrial membrane potential was measured using a mitochondrial membrane potential assay kit with JC-1 (cat. no. C2006, Beyotime) according to the manufacturer’s instructions (Zhou et al., 2020).

Statistical Analysis

GraphPad Prism 5.03 (GraphPad Software, Inc., La Jolla, CA, United States) was used for all statistical analyses. One-way analysis of variance was used for multiple comparisons. Statistical significance was established at p < 0.05.

Results

Mfn2 Silencing Protected Cardiac Function Under Hyperglycemic Conditions

In this study, we first evaluated Mfn2 expression in cardiomyocytes under hyperglycemic conditions. As shown in Figures 1A,B, the Mfn2 protein levels increased significantly after the cardiomyocytes were exposed to hyperglycemia, suggesting that Mfn2 is activated by high glucose.

FIGURE 1

Mfn2 silencing protects cardiac function under hyperglycemic conditions. (A,B) Western blotting was used to analyze Mfn2 expression in cardiomyocytes under hyperglycemic conditions. (C–H) The contractile properties of cardiomyocytes were determined using Fura-2AM. + dL/dt, maximal velocity of shortening; -dL/dt, maximal velocity of re-lengthening; TPS, time to peak shortening; TR90, time to 90% re-lengthening. ∗p < 0.05.

To assess the involvement of Mfn2 in hyperglycemia-induced cardiomyocyte injury, we transfected the cardiomyocytes with siRNA against Mfn2. Then, we measured the contraction and relaxation functions of single cardiomyocytes using Fura-2-acetoxymethyl ester (Fura-2AM). Neither hyperglycemia nor Mfn2 siRNA transfection impaired the lengths of the cardiomyocytes (Figures 1C–H). However, hyperglycemia impaired the peak shortening, while Mfn2 siRNA treatment improved it, suggesting that Mfn2 silencing could normalize cardiomyocyte contractility under hyperglycemic conditions. Hyperglycemia also reduced the maximal velocity of shortening in cardiomyocytes, while Mfn2 siRNA transfection reversed this effect (Figures 1C–H). Hyperglycemia impaired not only the contraction abilities but also the relaxation properties of cardiomyocytes, including the maximal velocity of re-lengthening, the time to peak shortening, and the time to 90% re-lengthening (Figures 1C–H). Mfn2 siRNA transfection improved these relaxation features. Thus, Mfn2 silencing attenuated hyperglycemia-induced cardiomyocyte dysfunction.

Hyperglycemia Promoted Mitochondrial Apoptosis in Cardiomyocytes Through Mfn2

The primary pathogenesis of diabetic cardiomyocyte damage is apoptosis; therefore, we assessed whether hyperglycemia-induced cardiomyocyte apoptosis depended on Mfn2. Firstly, we used a Cell Counting Kit 8 (CCK-8) assay to measure cell viability. Hyperglycemia-exposed cardiomyocytes exhibited a significantly lower viability than that of control cells, but Mfn2 siRNA transfection markedly inhibited hyperglycemia-induced cardiomyocyte apoptosis (Figure 2A). We also performed TUNEL staining to determine the extent of cardiomyocyte apoptosis in response to hyperglycemia and Mfn2 silencing. As shown in Figures 2B,C, hyperglycemia increased the proportion of TUNEL-positive (apoptotic) cardiomyocytes, whereas Mfn2 silencing reversed this trend.

FIGURE 2

Hyperglycemia activates mitochondrial apoptosis in cardiomyocytes through Mfn2. (A) A CCK-8 assay was used to observe the changes in cardiomyocyte viability in response to Mfn2 silencing under hyperglycemic conditions. (B,C) TUNEL staining was used to determine the number of apoptotic cardiomyocytes in response to Mfn2 silencing under hyperglycemic conditions. (D–I) Proteins were isolated from hyperglycemia-treated cardiomyocytes, and Western blotting was used to analyze the expressions of mitochondrial apoptotic proteins. ∗p < 0.05.

To further evaluate cardiomyocyte apoptosis, we analyzed the expressions of pro-apoptotic proteins. Bax, Bad, and caspase-9 expressions in cardiomyocytes rapidly increased in response to hyperglycemia treatment (Figures 2D–I). Loss of Mfn2 significantly reduced the expressions of these pro-apoptotic proteins. In addition, Bcl-2 and cellular inhibitor of apoptosis protein 1 (c-IAP1) were downregulated upon hyperglycemia treatment, whereas Mfn2 silencing restored the expressions of these proteins in cardiomyocytes (Figures 2D–I). Thus, Mfn2 activation induced cardiomyocyte apoptosis during hyperglycemic stress.

Mfn2 Promoted Mitochondria–ER Contact and Induced Calcium Release From the ER to the Mitochondria in Cardiomyocytes

To determine the molecular mechanism through which Mfn2 induced cardiomyocyte damage, we assessed the interaction between the ER and the mitochondria. Firstly, we used Western blotting to measure the markers of mitochondria–ER contact. As shown in Figures 3A–C, maintenance of mitochondrial morphology protein 1 (Mmm1), mitochondrial distribution and morphology protein 34 (Mdm34), and B cell receptor-associated protein 31 (BAP31) were significantly upregulated in hyperglycemia-treated cardiomyocytes, suggesting that hyperglycemic stress increases mitochondria–ER contact. However, the silencing of Mfn2 suppressed the upregulation of these proteins, implying that Mfn2 promotes mitochondria–ER contact (Figures 3A–C).

FIGURE 3

Mfn2 promotes mitochondria–endoplasmic reticulum (ER) contact and induces calcium release from the ER to the mitochondria in cardiomyocytes. (A–C) Western blot was used to analyze the expressions of mitochondria–ER contact markers, including Mmm1, Mdm34, and BAP31. (D–G) Immunofluorescence was used to detect the calcium levels in the ER and the mitochondria. ∗p < 0.05.

Increased mitochondria–ER contact promotes calcium release from the ER to the mitochondria. Thus, we used immunofluorescence to observe the concentrations of calcium in the mitochondria and the ER in cardiomyocytes. Calcium levels in the ER were significantly elevated in hyperglycemia-treated cells, and mitochondrial calcium levels accordingly increased. Interestingly, loss of Mfn2 significantly reduced the calcium concentrations in both the ER and the mitochondria (Figures 3D–G). These results demonstrated that Mfn2 promotes calcium release from the ER to the mitochondria.

Mfn2 Silencing Sustained Mitochondrial Function in Cardiomyocytes Under Hyperglycemic Conditions

To determine whether the enhanced mitochondria–ER contact under hyperglycemic conditions influenced mitochondrial function in cardiomyocytes, we detected the mitochondrial membrane potential. The mitochondrial membrane potential was significantly lower in hyperglycemia-treated cells than that in control cells (Figures 4A,B). Hyperglycemia treatment also increased mitochondrial ROS production in cardiomyocytes (Figures 4C,D). Loss of Mfn2 sustained the mitochondrial membrane potential and suppressed mitochondrial ROS production in hyperglycemia-treated cardiomyocytes (Figures 4C,D).

FIGURE 4

Mfn2 silencing sustains mitochondrial function in cardiomyocytes under hyperglycemic conditions. (A,B) A JC-1 probe was used to detect changes in the mitochondrial membrane potential. (C,D) Immunofluorescence was used to observe mitochondrial ROS levels in hyperglycemia-treated cardiomyocytes. (E) ATP production was determined through an enzyme-linked immunosorbent assay (ELISA). (F,G) An ELISA was used to analyze mitochondrial respiratory complex I and III activity. ∗p < 0.05.

Cardiomyocyte metabolism greatly depends on mitochondrial ATP production. Interestingly, ATP generation was blunted in hyperglycemia-treated cardiomyocytes, but Mfn2 silencing prevented this alteration (Figure 4E), indicating that Mfn2-dependent mitochondria–ER contact impairs mitochondrial ATP production. At the molecular level, mitochondrial ATP production primarily relies on mitochondrial respiratory complexes I and III. We found that hyperglycemia rapidly inhibited mitochondrial respiratory complexes I and III, while Mfn2 silencing reversed this effect (Figures 4F,G). Thus, Mfn2 silencing maintained mitochondrial function in cardiomyocytes under hyperglycemic conditions.

Mfn2 Silencing Reduced ER Stress in Cardiomyocytes Under Hyperglycemic Conditions

The above data suggested that an enhanced mitochondria–ER contact is associated with mitochondrial dysfunction during the development of diabetic cardiomyopathy. To determine whether Mfn2-dependent mitochondria–ER contact also disturbed ER homeostasis in hyperglycemia-treated cardiomyocytes, we first used qRT-PCR to measure the markers of ER stress. As shown in Figures 5A–C, the protein kinase R-like endoplasmic reticulum kinase (PERK), C/EBP-homologous protein (CHOP), and activating transcription factor 6 (ATF-6) messenger RNA (mRNA) levels in cardiomyocytes were significantly upregulated in response to hyperglycemia treatment. Mfn2 silencing markedly repressed the activation of PERK, CHOP, and ATF-6, suggesting that mitochondria–ER contact contributes to ER stress.

FIGURE 5

Mfn2 silencing reduces endoplasmic reticulum (ER) stress in cardiomyocytes under hyperglycemic conditions. (A–C) qRT-PCR was used to determine the mRNA levels of PERK, CHOP, and ATF-6. (D) ELISA was used to detect caspase-12 activity. (E,F) Western blotting was used to analyze caspase-12 expression in hyperglycemia-treated cardiomyocytes. ∗p < 0.05.

Uncontrolled ER stress can promote cardiomyocyte apoptosis through a mechanism involving caspase-12 activation. Therefore, we used enzyme-linked immunosorbent assay (ELISA) and Western blotting to analyze caspase-12 activity and expression in cardiomyocytes under hyperglycemic conditions. Caspase-12 activity (Figure 5D) and protein levels (Figures 5E,F) were both greater in hyperglycemia-treated cardiomyocytes than in control cells. Mfn2 silencing significantly prevented caspase-12 activation in both of these assays (Figures 5D–F). Thus, Mfn2 silencing attenuated ER stress during diabetic cardiomyopathy.

Discussion

Diabetes mellitus, a metabolic disease characterized by chronic hyperglycemia resulting from insulin deficiency, is currently one of the most prevalent chronic medical conditions. Diabetes mellitus has been recognized as a major cardiovascular risk factor as it increases both cardiovascular morbidity and mortality (Dia et al., 2020; Li et al., 2021). According to the American Diabetes Association, chronic hyperglycemia can cause long-term damage, dysfunction, and failure in various organs, including the eyes, kidneys, nerves, heart, and blood vessels.

When blood cholesterol and glucose are high, the mitochondria are one of the most important targets impacted (Pflüger-Müller et al., 2020; Qiao et al., 2020). Mitochondria are remarkably dynamic organelles that perform diverse yet interconnected functions by producing ATP and multiple biosynthetic intermediates that participate in cellular stress responses, autophagy, and apoptosis (Ma et al., 2020; Pflüger-Müller et al., 2020; Sawashita et al., 2020). The mitochondrial membrane potential is a key indicator of mitochondrial activity as it reflects the processes of electron transport and oxidative phosphorylation, which enable ATP production (Schinner et al., 2020; Sørensen et al., 2020). High-glucose conditions have been shown to disrupt the mitochondrial membrane potential and increase the ATP levels (Nesti et al., 2020). High glucose can also promote the reduction of glutathione (a major non-protein thiol, cellular antioxidant, and redox regulator) in the mitochondria. Moreover, high glucose has been reported to increase the levels of malondialdehyde, an oxidative stress marker that induces lipid peroxidation, mitochondrial membrane depolarization, and mitochondrial dysfunction (Jaque-Fernandez et al., 2020; Ollauri-Ibáñez et al., 2020). A previous study has indicated that mitochondrial dysfunction contributed significantly to the development and progression of diabetic cardiomyopathy (Ahmad and Hoda, 2020). In the present study, we found that a high-glucose treatment induced mitochondrial dysfunction in cardiomyocytes by reducing the mitochondrial membrane potential and increasing the mitochondrial ROS production.

Although the mitochondria have been identified as potential targets of hyperglycemia-induced cardiomyocyte damage, the upstream trigger of such mitochondrial damage has not been clear (Jin et al., 2018; Zhu et al., 2018). In the present study, we found that mitochondria–ER contact may induce mitochondrial damage in cardiomyocytes (Moon et al., 2020; Nawaz et al., 2020). Greater mitochondria–ER contact during hyperglycemia promoted the release of calcium from the ER to the mitochondria, resulting in mitochondrial calcium overload, an early event in mitochondrial dysfunction. Excessive mitochondrial calcium disrupted mitochondrial metabolism and inhibited mitochondrial ATP production by interrupting the citric acid cycle (Chang et al., 2021; Zhu et al., 2021). Enhanced mitochondria–ER contact was followed by an increased mitochondrial ROS production, an indicator of mitochondrial oxidative stress. In fact, some mitochondrial ROS are derived from xanthine oxidase, which is localized on the surface of the ER (Selvaraju et al., 2020; Tian et al., 2021); thus, the closer the mitochondria are to the ER, the more ROS production can occur. Due to the increased oxidative stress and abnormal calcium accumulation in the mitochondria, mitochondrial function in cardiomyocytes was impaired, as evidenced by the reduced mitochondrial metabolism and activated apoptosis. Thus, mitochondria–ER contact may be an upstream inhibitor of mitochondrial function in hyperglycemic cardiomyocytes.

Mfn2 is a mitochondrial fusion-related protein. It has been demonstrated to regulate cardiomyocyte viability and function; for example, Mfn2 overexpression was associated with cardiomyocyte hypertrophy (Wang et al., 2019). Mfn2-induced mitochondrial fusion was found to promote cardiomyocyte differentiation through the Notch signaling pathway (Kasahara et al., 2013). Cardiomyocyte senescence was also shown to depend on Mfn2-induced changes in mitochondrial dynamics (Song et al., 2017). Importantly, Mfn2 inhibition was recently reported to prevent diabetic cardiomyopathy (Hu et al., 2019), although this effect was primarily attributed to the suppression of Mfn2-induced mitochondrial fusion. In the present study, we found that Mfn2 is also important for mitochondria–ER contact. Hyperglycemic stress induced Mfn2 expression, while silencing Mfn2 attenuated mitochondria–ER contact, thus improving mitochondrial function and preserving ER homeostasis in cardiomyocytes. These data have revealed further mechanisms whereby Mfn2 impairs the viability of diabetic cardiomyocytes.

Overall, our results demonstrated that hyperglycemia-induced cardiomyocyte damage is associated with increased mitochondria–ER contact. The upregulation of Mfn2 promotes mitochondria–ER contact, thus stimulating the release of calcium from the ER to the mitochondria, inducing mitochondrial dysfunction and causing ER stress in cardiomyocytes. Based on these findings, novel therapies should be designed to inhibit mitochondria–ER contact via Mfn2 for the treatment of diabetic cardiomyopathy.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Author Contributions

JZ, FZ, and YW designed and performed the research. JZ analyzed the data. FZ and YW wrote the manuscript. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.