MALAT1/miR‐185‐5p mediated high glucose‐induced oxidative stress, mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway

Abstract To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR‐185‐5p with MALAT1 and RhoA were validated by dual‐luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D‐glucose (HG) in the presence or absence of MALAT1‐shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR‐185‐5p expression were determined by real‐time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1S616, mitofusin 2 and apoptosis‐related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC‐1. MALAT1 was significantly up‐regulated while miR‐185‐5p was down‐regulated in myocardium of db/db mice and HG‐induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR‐185‐5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG‐induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR‐185‐5p and mediated HG‐induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.

in cardiomyocytes, which are considered as the critical process causing DCM. 2,3 Mitochondria play vital roles in pathophysiology of myocardium. The balance of dynamics between mitochondrial fission and fusion is very important for maintaining myocardial energy production and cardiomyocyte survival, 4,5 which are modulated by a series of dynamin-related GTP proteins, including mitofusins (Mfn1/2) and dynamin-related protein 1 (Drp1), etc.
Emerging evidence has suggested that imbalanced mitochondrial dynamics contribute to the occurrence of DCM through mitochondrial oxidative stress and mitochondrial pathway-mediated cardiomyocyte apoptosis. [6][7][8] Abnormal morphology and dysfunction of mitochondria are usually observed in heart tissues in DCM. 9 So, regaining mitochondrial homeostasis may be a potential therapeutic strategy for DCM.
Abundant evidence has demonstrated that epigenetic factors, such as non-coding RNAs (ncRNAs), occupy an important position in the pathogenesis of DCM, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). [10][11][12] MiRNAs are composed of approximately 22 nucleotide sequence, and regulate the expression of target genes at the post-transcriptional levels via binding to the untranslated regions (UTRs) of complementary message RNAs. 13 Several studies have verified that the differential expression of miR-NAs in myocardium are involved in DCM in either patients or animal models with DM. [14][15][16][17] Recently, we screened the expression profiles of miRNAs in myocardium from DCM mice, and found that miR-185-5p, one of the differentially expressed miRNAs, was markedly down-regulated in DCM mice ( Figure 2A). The miRNA candidate target mRNAs were predicted by miRDB database (http://mirdb.org/ miRDB/), and RhoA was shown to be a target gene of miR-185-5p, which were verified by NCBI (http://www.ncbi.nlm.nih.gov/). Our previous studies have shown that the RhoA/Rho kinase (ROCK) pathway in heart tissues is activated by hyperglycemia and involved in myocardial fibrosis in a rat model of Type 2 DM (T2DM), and inhibition of ROCK can improve the cardiac function of diabetic rats in vivo. 18 In vitro, the RhoA/ROCK pathway mediates HG-induced oxidative stress, cardiomyocyte apoptosis and fibroblast proliferation as well as collagen synthesis. 19,20 Plenty of evidence shows that the RhoA/ROCK pathway is closely related to mitochondrial dynamics. [21][22][23] LncRNAs are non-protein coding transcripts with more than 200 nucleotides, but they play crucial roles in multiple steps of gene regulation by serving as transcription cofactors, scaffolds for chromatin-modifying complexes, molecular guides or decoys.
LncRNAs act as competitive endogenous RNAs (ceRNAs) by sponging miRNAs, which change the expression of downstream target mRNAs. 11,24 Such, ceRNA networks consisting of lncRNA/miRNA/ mRNA interactions play an important role in the pathological process of DCM. 25,26 LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is found to participate in the pathology of cardiomyocytes in response to diverse stimuli. [27][28][29] In the present study, the interactions between MALAT1, miR-185-5p and RhoA were revealed by bioinformatics analysis. We would explore whether MALAT1, miR-185-5p and the RhoA/ROCK pathway play a role in HG-induced mitochondrial damage and cardiomyocyte apoptosis, with the aim of providing a novel therapeutic target for DCM.

| Animals
Eight-week-old male db/db mice and C57BL/6J wild-type mice were purchased from Nanjing Junke Biological Engineering Co., Ltd.
The mice were housed in cages with room temperature (21-24°C), relative humidity (50%) and 12 h light-dark cycle. All mice were fed with standard chow and distilled water. Body weight, random bold glucose and systolic arterial blood pressure (SABP) were weekly

| Echocardiographic determination
At age of 22 weeks, mice were anaesthetized using 1.5% maintenance of isoflurane under continuous heart rate monitoring. All measurements were performed with a 11-MHz linear transducer coupled to a high-resolution Ultrasound System (vivid E95, GE Healthcare, USA).
Fractional shortening (FS%) and ejection fraction (EF%) were used to assess cardiac systolic function. Peak velocity of early filling (E), atrial contraction (A) and early diastolic mitral valve flow velocity (e′) were obtained to evaluate cardiac diastolic function.

| Transmission electron microscopy
Cardiac tissues were cut into fragments of 1 mm sections, which were fist fixed in 4% glutaraldehyde at 4°C overnight before being subjected to dehydration, soaking, embedding and staining. They were eventually cut into ultrathin sections of 50-70 nm, and the ultrastructure of myocardium was observed by transmission electron microscopy (TEM) (HT7800; Hitachi).

| MiRNA sequencing
Left ventricular samples from three mice in each group were randomly selected for miRNA sequencing. The whole experiment process was performed by Kangcheng Biotechnology Co. Ltd.
Briefly, total RNA of each sample was used for preparing the miRNA sequencing library. Then, the library was denatured as singlestranded DNA, captured on Illumina flow cells, amplified in situ as clusters and subjected to 50-cycle sequencing on Illumina NextSeq 500. Differentially expressed miRNAs were defined by a threshold with fold change ≥1.5 and p ≤ 0.05.

| Culture of primary cardiomyocytesand treatment
All experimental procedures were approved by the Ethics Committee of the Second Hospital of Hebei Medical University.
Cardiac tissues were acquired from 1 to 3 day old newborn C57BL/6J mice and the primary cardiomyocytes were obtained as previously described. 30 When cardiomyocytes had been confirmed to reach 80% of confluence, they were randomized into experimental groups as follows: 5.5 mmol/L D-glucose as the normal glucose (NG) group, osmolarity control (OSM) group con-

| Western blotting and pull-down RhoA assay
Total proteins were obtained from the cell lysis buffer (Abcam) and were quantified using Bradford method. The proteins were sepa-

| Measurements of SOD activity and MDA content
The activity of superoxide dismutase (SOD) and the contents of malondialdehyde (MDA) in cellular supernatants were measured by commercial kits (Nanjing, China).

| Flow cytometry
Cell apoptosis rate was examined using the Annexin V-FITC/PI detection Kit (Neobioscience, China). Briefly, collected cardiomyocytes were resuspended in binding buffer and subsequently stained with Annexin V-FITC and propidium iodide (PI) for 15 min at room temperature in the dark. Then, cell apoptosis rate was analysed by flow cytometry.

| TdT-mediated dUTP-biotin nick end labelling (TUNEL) staining
Cell apoptosis assay (Beyotime) was performed by TUNEL staining according to the manufacturer's instructions. Briefly, cells were fixed with 4% paraformaldehyde in PBS for 30 min and permeabilized with 0.3% Triton X-100 for 5 min at room temperature. Cells were labelled with TUNEL reaction mixture and were incubated at 37°C for 1 h and sealed with anti-fluorescence quenching solution containing DAPI (Beyotime, China). TUNEL positive cells were observed with a fluorescence microscope (OLYMPUS, Japan).

| Measurement of mitochondrial membrane potential (MMP)
The alteration in MMP was determined by JC-1 assay kit (Beyotime).

| Statistical analysis
GraphPad Prism 9.0 software (CA, USA) was used for statistical analyses. The experimental data were expressed as the mean ± SEM, with three independent experiments. Data were analysed using Student's t test between two groups and one-way ANOVAs for more groups as well as followed with LSD test. A value of p<0.05 was considered statistically significant.

| Db/db mice developed DCM
Metabolic parameters and the ratio of heart weight to body weight (HW/BW) at age of 22 weeks are shown in Table 1. Compared with control mice, FBG, FINS, HbA1c, HOMA-IR and HW/BW were remarkably increased in db/db mice (p<0.001, p<0.01, respectively).
There was no significant difference in SABP between db/db and control mice. Representative pictures of echocardiography are presented in Figure 1A. Compared with control mice, the EF% and the FS% were significantly decreased while the E/A and the E/e′ were enhanced in db/db mice ( Figure 1B). The results revealed that the cardiac systolic as well as diastolic function were impaired in db/ db mice. Myocardium ultrastructure was visualized in Figure 1C.
Obvious mitochondria swelling, cristae disorder and myofibril fracture were observed in myocardium of db/db mice.

| MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG condition
Compared with the NG group, exposure of cardiomyocytes to HG significantly increased the expression of MALAT1 and decreased TA B L E 1 Metabolic parameters in db/db mice (N = 6) at the age of 22 weeks. the expression of miR-185-5p. Compared with the HG group,

MALAT1-shRNA significantly decreased the expression of MALAT1
and increased the expression of miR-185-5p in cardiomyocytes exposed to HG ( Figure 3A,B). Bioinformatic analysis predicts that there are binding sites between miR-185-5p and MALAT1 or RhoA ( Figure 2B,C). The dual-luciferase reporter assay showed that transfection with miR-185-5p mimics markedly reduced the luciferase activity of wt-MALAT1; however, mut-MALAT1 luciferase activity had no changes ( Figure 3C). Transfection with miR-185-5p mimics markedly decreased the luciferase activity of wt-RhoA, while mut-RhoA luciferase activity was not affected ( Figure 3D). Compared with the HG group, MALAT1-shRNA dramatically decreased RhoA activity in cardiomyocytes exposed to HG, which were reversed by addition of miR-185-5p inhibitor ( Figure 3E). These results show that miR- activity in cardiomyocytes exposed to HG ( Figure 3F).

| MALAT1 and the RhoA/ROCK pathway mediated oxidative stress, mitochondrial injury and cardiomyocyte apoptosis in HG condition
We investigated oxidative stress in the cellular supernatants by the content of MDA, a classic oxidative damage marker and the activity of SOD, an antioxidant marker. Compared with NG group, MDA content was significantly increased while SOD activity was significantly decreased in the HG group. Treatments of cardiomyocytes with MALAT1-shRNA and fasudil markedly decreased HG-induced increase in MDA content and enhanced SOD activity. There were no significant differences in MDA content and SOD activity between the OSM and NG groups ( Figure 4A,B).
As shown in Figure 4C, remarkably decreased MMP level was observed in cardiomyocytes exposed to HG, which were improved these changes induced by HG ( Figure 4D). There were no significant differences in the levels of MMP, Mfn2 and the phosphorylation of Drp1 S616 between the OSM and NG groups.

| MALAT1 and the RhoA/ROCK pathway mediated cardiomyocyte apoptosis in HG condition
Cardiomyocyte apoptosis is shown in Figure 5A,B, dramatically increased cell apoptosis was observed in the presence of HG.

MALAT1-shRNA and fasudil treatment significantly suppressed HG-
induced increase of cardiomyocyte apoptosis. It is well known that the occurrence of cell apoptosis is regulated by apoptosis-associated proteins. Compared with the NG group, the ratio of Bax to Bcl-2 protein (Bax/Bcl-2) and cleaved caspase-3 levels were significantly increased cardiomyocytes exposed to HG, which were counteracted by MALAT1-shRNA and fasudil ( Figure 5C,D). There were no significant differences in cell apoptosis and apoptosis-associated proteins between the OSM and NG groups, suggesting that HG-induced cardiomyocyte apoptosis was independent of high osmotic pressure.

| DISCUSS ION
Db/db mice are classically used as animal models of T2DM. In this study, db/db mice exhibited hyperglycemia and insulin resistance, Collecting evidence shows that oxidative stress plays a crucial role in development of DCM. 36,37 Glucose overload in mitochondria leads to excessive reactive oxygen species (ROS) generation and mitochondrial dysfunction, which in turn enhance oxidative stress. 38 Enhanced oxidative stress is closely associated with the induction of cell apoptosis. 39,40 Cardiomyocyte apoptosis is key step triggering the progression of DCM, which subsequently result in the remodelling and fibrosis of the myocardium, ultimately leading to impaired myocardial function. 41 In the study, we observed increased MDA and reduced SOD in the medium in HG, concurrently increased apoptosis of cardiomyocytes, Bax/Bcl-2 and cleaved caspase-3, meaning that HG induced oxidative stress and triggered mitochondrial pathway dependent cardiomyocyte apoptosis. Mitochondrial dynamics not only determine mitochondrial morphology but also regulate mitochondrial function, including energy production, oxidative stress and cell apoptosis. 9 Yanxia Chen: Validation (equal). Hong Zhou: Conceptualization (lead); project administration (lead); supervision (lead); writing -review and editing (lead).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflict of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.