MiR‐103 inhibiting cardiac hypertrophy through inactivation of myocardial cell autophagy via targeting TRPV3 channel in rat hearts

Abstract Cardiac hypertrophy is a common pathological change frequently accompanied by chronic hypertension and myocardial infarction. Nevertheless, the pathophysiological mechanisms of cardiac hypertrophy have never been elucidated. Recent studies indicated that miR‐103 expression was significantly decreased in heart failure patients. However, less is known about the role of miR‐103 in cardiac hypertrophy. The present study was designed to investigate the relationship between miR‐103 and the mechanism of pressure overload‐induced cardiac hypertrophy. TRPV3 protein, cardiac hypertrophy marker proteins (BNP and β‐MHC) and autophagy associated proteins (Beclin‐1 and LC3‐II) were up‐regulated, as well as, miR‐103 expression and autophagy associated proteins (p62) were down‐regulated in cardiac hypertrophy models in vivo and in vitro respectively. Further results indicated that silencing TRPV3 or forcing overexpression of miR‐103 could dramatically inhibit cell surface area, relative fluorescence intensity of Ca2+ signal and the expressions of BNP, β‐MHC, Beclin‐1 and LC3‐II, but promote p62 expression. Moreover, TRPV3 protein was decreased in neonatal rat ventricular myocyte transfected with miR‐103, but increased by AMO‐103. Co‐transfection of the miR‐103 with the luciferase reporter vector into HEK293 cells caused a sharp decrease in luciferase activity compared with transfection of the luciferase vector alone. The miR‐103‐induced depression of luciferase activity was rescued by an AMO‐103. These findings suggested that TRPV3 was a direct target of miR‐103. In conclusion, miR‐103 could attenuate cardiomyocyte hypertrophy partly by reducing cardiac autophagy activity through the targeted inhibition of TRPV3 signalling in the pressure‐overloaded rat hearts.


| INTRODUCTION
Cardiac hypertrophy is an adaptive response to the increased blood pressure and afterload, and is also a common pathophysiological process in many cardiovascular diseases such as hypertension, myocardial infarction, valvular heart disease, cardiomyopathy and so on. 1,2 Despite, the hypertrophic response is a compensatory mechanism that augments cardiac output, sustained hypertrophy can lead to dilated cardiomyopathy, heart failure and sudden cardiac death. In the western country, approximately half a million individuals are diagnosed with heart failure each year, with a mortality rate approaching 50%. There have been major advances in the identification of genes and signalling pathways involved in this disease process, but the overall complexity of hypertrophic remodelling suggests that additional regulatory mechanisms remain to be identified. Therefore, identifying new molecular mechanisms mediating cardiac hypertrophy and providing new targets for clinical treatment need to be addressed.
MiR-103 is a member of the miR-103/107 family located on human chromosome 5. 10 It is widely distributed in 13 kinds of human normal tissues (liver, placenta, brain, heart, stomach, lung, bladder, prostate, colon, thymus, ovaries, fat and uterus), and five kinds of carcinoma. 11 Recent studies have also shown that miR-103 expression was significantly lowered in heart failure patients than healthy volunteers. 12 However, the role of miR-103 in protecting against cardiac hypertrophy has never been elucidated.
We used targetscan software to predict the downstream of miR-103, and found that TRPV3 was the underlying target of miR-103.
TRPV3, a non-selective cation channel, belongs to the Ca 2+ -permeant TRP channel family, and which functions in the formation of the skin barrier, hair growth, wound healing and temperature sensation. [13][14][15][16] TRPV3 can be activated by warm temperatures above 33°C and natural herbs such as carvacrol and inhibited by ruthenium red. In recent years, many studies have expanded the functions of TRP channels, and confirmed that they participated in the regulation of various diseases such as anxiety, asthma, obesity and metabolic disorders. 17 In particular, TRP channels are also involved in the regulation of cardiovascular disease. TRPV4 is an important regulator of intracellular Ca 2+ concentration in cardiac fibroblasts. 18 It has also been reported that TRPV1 was up-regulated in pathological myocardial hypertrophy. 19 According to our previous study, TRPV3 activation exacerbated cardiac fibrosis by promoting cardiac fibroblast proliferation through TGF-β 1 /CDK2/cyclin E pathway in the pressureoverloaded rat hearts. 20 Interestingly, the overall sequence of TRPV3 has 40% homology to TRPV1. 21 Therefore, we speculate whether TRPV3 is also involved in the occurrence of cardiac hypertrophy.
Autophagy is a conserved, tightly regulated intracellular catabolic process, in which mammalian cells degrade and recycle the damaged and dysfunctional macromolecules and organelles. Recent findings suggest that autophagy exploits a variety of physiological functions to maintain the balance of cardiac homeostasis. 22 For example, a certain extent of autophagy is crucial for proper heart function, whereas exaggerated autophagic activity may foster many cardiovascular diseases such as cardiac hypertrophy, cardiac ischaemia/reperfusion injury, heart failure and so on. [23][24][25] In tumour cells, intracellular Ca 2+ , an important regulatory factor for autophagy, induced autophagy by death-associated protein kinase (DAPK). 26  were induced to myocardial hypertrophy via abdominal aorta coarctation. All animal experiments were conducted in accordance with the protocols approved by the Experimental Animal Ethics Committee of Harbin Medical University. In short, the male Wistar rats were anaesthetized with sodium pentobarbital (40 mg/kg, i.p.), and the abdomen was opened on aseptic conditions. Under sterile conditions, the abdominal aorta was separated for 1 cm segment above the double renal artery, and a diameter of 0.7 mm silver wire was placed on the surface of the abdominal aorta. Then, the abdominal aorta and wire were tightly tied together by surgical silks, and a constriction of the abdominal aorta was provided. The wire was withdrawn from the ligature, so that remaining orifice of the abdominal aorta could close to that of the wire. The rats were divided into three groups randomly: control group, model group and sham group.

| Haematoxylin-Eosin staining
The left ventricle of male Wistar rat was placed in 4% paraformaldehyde solution at 4°C overnight, then, added 30% sucrose solution.
The left ventricle tissue was sunk to the bottom of 30% sucrose solution, embedded in OCT-freeze medium and frozen at −20°C.
Sections of several micron thickness were knifed and transferred to 3-aminopropyl triethoxysilane-coated slides. These sections were stained with haematoxylin-eosin (HE), and the histopathological abnormalities were investigated under the light microscope. The photographs were captured with an Olympus BX60 microscope (Olympus Optical Co. Ltd., Tokyo, Japan).

| Cell culture and microRNA transfection
Myocardial cells were isolated and cultured as described. 27 In brief, the hearts removed from neonatal rats (1to3-day-old) were finely Ang II for 48 hours and treated with miR-103 transfection for 24 hours; (4) AMO-103 group: cells were exposed to 100 nmol L −1 Ang II for 48 hours and treated with miR-103 and AMO-103 transfection for 24 hours; (5) N.C group: cells were exposed to 100 nmol L −1 Ang II for 48 hours and treated with N.C transfection for 24 hours; (6) AMO-N.C group: cells were exposed to 100 nmol L −1 Ang II for 48 hours and treated with AMO-N.C transfection for 24 hours; (7) control+miR-103 group: cells were treated with miR-103 transfection for 24 hours.
Cells were divided into five groups: (1) control group; (2) model group: cells were treated with 100 nmol L −1 Ang II for 48 hours; (3) siTRPV3 group: cells were pre-treated with TRPV3-siRNA transfection for 24 hours and finally exposed to 100 nmol L −1 Ang II for 48 hours; (4) N.C group: cells were transfected with TRPV3-sc for 24 hours followed by treatment with 100 nmol L −1 Ang II for 48 hours; (5) control+siTRPV3 group: cells were treated with TRPV3-siRNA transfection for 24 hours.

| Western blot
Protein samples were extracted from tissues and cells with the same procedures described previously. Briefly, the heart tissues and myocardial cells were lysed in RIPA buffer and then centrifuged at 4°C

| Immunofluorescence staining
Immunofluorescence staining was performed to detect the expression of α-SMA in myocardial cells. In short, the cells were cultured on coverslips and received the desired treatment. At the end of the treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde and permeabilized using 0.2% Triton X−100 . Cells were incubated with anti-α-SMA at 4°C overnight. Then, the second antibody was incubated in the dark. In the end, DAPI (Beyotime Biotechnology, Shanghai, China) was counterstained for the identification of nucleus. Photographs were acquired using fluorescence microscope (Leica, Heidelberg, Germany).

| Tandem mRFP-GFP fluorescence microscopy
Cells transiently expressing mRFP-GFP-LC3 lentivirus were treated as designated and observed by laser microscopy. The number of GFP and mRFP puncta per cell were quantified manually.

| Statistical analysis
All data were expressed as mean ± SEM and analysed by using SPSS 19.0 software. Statistical analysis was performed with one-way ANOVA of variance. If the ANOVA was significant, SNK-q was used to evaluate the statistical significance of differences between two groups. P < 0.05 was considered to be a statistically significant difference.

| Successfully established cardiac hypertrophy models in vivo and in vitro
To ensure the accuracy of the experiment, firstly we evaluated whether the cardiac hypertrophy models were successfully established  Meanwhile, p62 expression was dramatically decreased ( Figure 2E and F). Above results suggested that autophagy was activated in hypertrophic hearts and cardiomyocytes.

| Effects of TRPV3 activation on cardiac hypertrophy
To better understand TRPV3-offered detrimental effects on cardiac hypertrophy, cardiac hypertrophy related mark proteins and cell sur-

| Effects of TRPV3 activation on the activity of cardiac autophagy
To further explore the mechanism underneath by which TRPV3 aggravated cardiac hypertrophy, cardiac autophagy was detected.
As displayed in Figure 4, autophagy related proteins Beclin-1 and LC3-II ( Figure 4A and B) were significantly increased, as well as p62 protein ( Figure 4C) was decreased in model group, whereas silencing TRPV3 inhibited the expressions of Beclin-1 and LC3-II, and enhancing p62 expression. These data indicated that TRPV3 activation might promote cardiac autophagy, finally resulting in cardiac hypertrophy.

| Effects of miR-103 on cardiac hypertrophy
To determine the effect of miR-103 on cardiac hypertrophy, we used real-time PCR to measure the expression of miR-103 in rat myocardial tissues and neonatal cardiomyocytes. The results were shown in Figure 5A  These results implied that miR-103 suppressed cardiac hypertrophy partly via reducing the activity of cardiac autophagy.

| TRPV3 gene is a target of miR-103
Subsequently, we need to examine whether miR-103 directly targeted TRPV3. We used targetscan software to predict, and found a conserved binding site for the miR-103 in the 3′ UTR of the TRPV3 gene. To further test this binding profile, miR-103 was transfected into cultured neonatal rat ventricular myocytes, and the protein level of TRPV3 induced by Ang II was remarkably reduced. Conversely, TRPV3 was significantly up-regulated when AMO-103 was transfected into neonatal rat ventricular myocytes, which indicates that Interest in autophagy has increased in recent years as this highly conserved cellular process had been proved to implicate in a growing number of diseases including infection, cancer, neurodegeneration and ageing. 28 At the same time, it has also been reported that autophagy can promote the activation of inflammatory factors, 29 and plays a role in neurodegenerative diseases such as Parkinson's disease. 30 In recent years, studies have shown that autophagy is involved in the development of a variety of cardiovascular diseases. Huang reported that berberine alleviated cardiac ischaemia/reperfusion injury by inhibiting excessive autophagy in cardiomyocytes. 31 In addition, it has been reported that excessive cardiac autophagy can lead to cardiac hypertrophy. 32 Consistent with previous reports, our results indicated that excessive cardiac autophagy exacerbated cardiac hypertrophy in vivo and in vitro models.
The activation of TRPV3 channel is involved in the development of hyperphagia and obesity in obesity-prone rats by reducing food intake, 33 and also plays an important role in scars with post-burn pruritus by thymic stromal lymphopoietin (TSLP). 34 In addition to the above-mentioned effects, the TRPV3 channel is also involved in the  3′-untranslated region from rat containing the binding sites of miR-103. Data were represented by mean ± SEM (n = 3-5). *P < 0.05 vs control group; # P < 0.05 vs model group; @ P < 0.05 vs miR-103 group of autophagy after TRPV3 knockdown. As we all know, intracellular calcium is an important regulator of autophagy. Therefore, we speculate that the activation of TRPV3 channels can increase the intracellular calcium concentration, and thus promote myocardial cell autophagy, ultimately leading to cardiac hypertrophy.
MicroRNAs are endogenous regulators of gene expression. Considering that cardiomyocyte hypertrophy, a key cellular event in hypertrophic heart, depends on gene expression. And it is reasonable to suggest that miRNAs may be involved in pressure overloadinduced cardiac hypertrophy. It should be noted that several studies have demonstrated that miRNAs protect against cardiac hypertrophy. MiR-133a was down-regulated in thyroid hormone-regulated cardiac hypertrophy, 37 and miR-23a acted downstream of NFATc3 to regulate cardiac hypertrophy, 38 and overexpression of miR-27b could induce cardiac hypertrophy. 39 Wang reported that miR-103/ 107 could regulate programmed necrosis and myocardial ischaemia/ reperfusion injury through the FADD pathway. 40 However, whether miR-103 plays a role in cardiac hypertrophy has not been addressed.
The results of this experiment showed that miR-103 expression was decreased when cardiac hypertrophy occurred. This finding sug- These findings prompted that miR-103 reduced intracellular calcium, thereby suppressed cardiac autophagy, finally inhibited cardiac hypertrophy. Furthermore, by using targetscan software, the current study predicted a putative miR-103 binding sequence in the TRPV3 mRNA. To validate that TRPV3 was a target of miR-103, we transfected miR-103 or inhibitors into neonatal rat ventricular myocytes, and found that miR-103 could affect TRPV3 protein level. Subsequently, it was verified that TRPV3 was a direct miR-103 target using luciferase reporter assays in HEK293 cells. These results indicated that miR-103 directly modulated TRPV3 expression by binding to the 3′ UTR of TRPV3 and, thus, confirmed that TRPV3 was a novel target of miR-103.
In summary, miR-103 could suppress the expression of TRPV3 by binding to the 3′ UTR, thereby reducing relative fluorescence intensity of Ca 2+ signal, leading to the decrease in the activation of cardiac autophagy and ultimately inhibiting the development of cardiac hypertrophy (schematized in Figure 8). Our studies revealed a novel hypertrophic signalling pathway (miR-103/TRPV3/autophagy), and manipulation of this signalling axis may provide innovative approaches to prevent cardiac hypertrophy.

CONFLI CT OF INTEREST
The authors declare that they have no conflict of interest.