1 Introduction
Cardiac hypertrophy is an adaptive compensatory change in the heart that is activated in response to various stimuli, such as hemodynamic overload; ischemia; or defects in genes encoding cardiac structural proteins [
1]. Sustained pressure overload gradually leads to pathological hypertrophy, which is characterized by cardiomyocyte enlargement and cardiac dysfunction. Various signaling pathways, such as those involved in sarcomere structure alteration, metabolic reprogramming, and mitochondrial dysfunction, are triggered in cardiac hypertrophy [
2]. These pathological changes cause the disorganization of the cytoskeletal structure or deficiencies in energy production, promote maladaptive cardiac remodeling and dysfunction, and eventually progress to heart failure. Pathological cardiac hypertrophy is usually accompanied by marked alterations in energy metabolism and mitochondrial function [
3,
4].
Mitofusin 2 (Mfn2) is a transmembrane GTPase located on the mitochondrial outer membrane that contributes to mitochondrial fusion [
5]. Apart from mitochondrial fusion, Mfn2 plays an important role in the endoplasmic reticulum (ER)–mitochondria tether and cellular apoptosis and proliferation [
6,
7]. Mfn2 dysfunctions have been found to contribute to cardiovascular diseases, such as ischemia–reperfusion injury, cardiac hypertrophy, and heart failure [
8,
9]. The cardiac knockout of Mfn2 manifests a unique pathological form of cardiac hypertrophy by provoking massive, progressive mitochondrial accumulation that severely distorts cardiomyocyte sarcomere architecture [
10]. In cardiac hypertrophy, Mfn2 expression could be regulated at the post-transcriptional level by microRNAs (miRNAs) [
11,
12]. However, the role of lncRNAs in regulating Mfn2 expression during cardiac hypertrophy remains unknown.
Long noncoding RNAs (lncRNAs) are a class of RNAs more than 200 nucleotides in length and lack protein-coding capacity [
13]. In cardiovascular diseases, lncRNAs regulate various physiological and pathological processes through multiple molecular mechanisms [
14–
16]. For example, lncRNA H19 reverses pressure overload–induced cardiac hypertrophy and heart failure through epigenetic regulation [
17,
18]. lncRNA ChAST (cardiac hypertrophy-associated transcript) promotes cardiac hypertrophy by disrupting beneficial autophagic processes by downregulating the expression of Pleckstrin homology domain–containing family M member 1 expression by trans regulation[
19]. We previously reported that lncRNA ZNF593 antisense (ZNF593-AS) remarkably decreases in the decompensated phase of heart failure and contributes to the improvement in cardiac Ca
2+ handling and contractile function [
20]. In cardiac hypertrophy, an early phase of heart failure, ZNF593-AS is repressed and mitochondrial function is impaired. In the present study, the function of ZNF593-AS in the pathogenesis of cardiac hypertrophy was further explored.
2 Materials and methods
2.1 Materials
Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO (Life Technologies Corporation, Carlsbad, CA, USA). Lipofectamine 2000 (Lipo2000) (Cat No.: 11668019) reagent and phenylephrine (PE) were obtained from Invitrogen (Carlsbad, CA, USA) and MedChemExpress (New Jersey, USA), respectively. Chemically synthesized GapmeR-control and GapmeR-ZNF593-AS were obtained from Exiqon (Copenhagen, Denmark). Antibodies against Mfn2 (Cat No.: A19678), ATP5A1 (Cat No.: A11217), UQCRCC2 (Cat No.: A4181), SDHB (Cat No.: A1809), MT-CO2 (Cat No.: A3843), NDUFB8 (Cat No.: A19732), p62 (Cat No.: A19700), LC3B (Cat No.: A19665), GAPDH (Cat No.: AC033), and tubulin (Cat No.: AC008) were procured from ABclonal (Wuhan, China). Polyvinylidene difluoride (PVDF) membranes were purchased from Millipore (Merck KGaA, Darmstadt, Germany).
2.2 Animal study
All animal experimental protocols complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health as well as the guidelines of Animal Research: Reporting of
In Vivo Experiments. This study was approved by the Institutional Animal Research Committee of Tongji Medical College. Mice (male, 20–25 g) were housed at the animal care facility of Tongji Medical College at 25 °C with 12/12 h light/dark cycles and allowed free access to normal mouse chow and water throughout the study period. Pressure overload was induced in ZNF593-AS transgenic or knockout and wild-type (WT) littermate mice at the age of 8 weeks through transverse aortic constriction (TAC). At 2 weeks after the operation, all the animals underwent cardiac structure and function detection and sacrificed. Their organs were collected, frozen in liquid nitrogen, and stored at −80 °C. Meanwhile, portions of the organs were fixed with neutralizing formalin for histological analysis as previously described [
21].
2.3 Generation of ZNF593-AS knockout mice and cardiac-specific transgenic mice
Global ZNF593-AS knockout (KO) or cardiac-specific transgenic mice (MHC-Tg) were generated by GemPharmatech Co., Ltd. (Nanjing, China) as described previously to explore the function of ZNF593-AS
in vivo [
22]. Briefly, zygotes were injected with Cas9 mRNA and gRNA targeting ZNF593-AS generated by using the CRISPR-Cas9 system and transplanted into C57BL/6 female mice to generate ZNF593-AS KO mice. The genotypes of KO mice were confirmed by PCR assays with the forward primer 5ʹ-CCTTTGTCACCCTCAAGCCC-3ʹ and reverse primer 5ʹ-CGTGCCCACAGAAGTGTCCA-3ʹ. ZNF593-AS cardiomyocyte–specific transgenic mice were generated under the control of the α-myosin heavy chain promoter (α-MHC promoter). The genotypes of MHC-Tg mice were confirmed by PCR assays by using the forward primer 5ʹ-CTACATTGTTGGCTGTCCTCG-3ʹ and reverse primer 5ʹ-CAGGAGGTACTTCATCGATTCAG-3ʹ. The mice were housed at the animal care facility of Tongji Medical College at 25 °C with 12/12 h light/dark cycles and allowed free access to normal mouse chow and water throughout the study period.
2.4 TAC
Pressure overload–induced cardiac hypertrophy was induced by TAC as indicated previously [
23]. In brief, a 7-0 polypropylene suture was looped around the aortic arch, and a stenosis was placed by using a 27G needle as a space holder. The sham mice underwent the same operation without aortic constriction. Two weeks after the operation, all animals were subjected to cardiac structure and function detection. Subsequently, the mice were anesthetized with intraperitoneal injections of a xylazine (5 mg/kg) and ketamine (80 mg/kg) mixture and sacrificed. Their organs were collected, frozen in liquid nitrogen, and stored at −80 °C.
2.5 Echocardiography
Echocardiography was performed by using VisualSonics Vevo 1100 (VisualSonics, Toronto, ON, Canada) to assess cardiac structure and function. Mice were sedated with 3% isoflurane (Baxter International, Deerfield, Illinois, USA). Isoflurane concentration was reduced to a minimum (1%–2%) to achieve constant heart rates during examination. The maximum dimension of the left ventricle (LV) in the parasternal long axis view was acquired in the form of M-mode images. Scanhead was taken to visualize the LV in its maximum dimension from the apex to the base while recording B-mode images in the parasternal long axis view for 2DE analyses. Image analysis was performed by a single observer by using the dedicated software package VevoLAB Version 3.0. Cardiac parameters, such as LV wall thickness, ejection fraction (EF), and fractional shortening (FS), were evaluated on the basis of the acquired M-mode images as previously described [
21].
2.6 RNA extraction and qPCR
RNA expression levels were detected through real-time quantitative PCR. Total RNA was extracted from heart tissue or cell lysate by using Trizol reagent (Invitrogen, Carlsbad, CA) and transcribed into cDNA with Hifair® II 1st Strand cDNA Synthesis SuperMix (Yeasen Biotechnology, Shanghai, China) in accordance with the manufacturer’s instructions. mRNA and lncRNA expression levels were quantified by qPCR by using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China) on a 7900HT FAST real-time PCR system (Life Technologies, Carlsbad, CA, USA). The results were analyzed through the 2
−ΔΔCt method as previously described [
21].
2.7 RNA sequencing
Sequencing was performed by Epibiotek Co. (Guangzhou, China) on a Hiseq X ten platform/NovaSeq 6000 (Illumina, San Diego, CA, USA) as previously described [
24]. A reference genome index was built with Bowtie2 (v2.4.1), and high-quality sequences were mapped to the reference genome by using HISAT2 (v2.1.0). The resulting
P values were adjusted through the Benjamini and Hochberg approach for controlling false discovery rates. Genes with adjusted
P < 0.05 and absolute log
2FC > 1 were considered as differentially expressed.
2.8 Histological analysis
Heart samples were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 μm slices. Morphology was detected by hematoxylin/eosin and wheat germ agglutinin (WGA) staining and quantified by using Image-Pro Plus Version 6.0 software (Media Cybermetics, Washington, USA) as described previously [
10].
2.9 Cells culture and treatments
AC16 cells (human myocytes) were cultured in DMEM supplemented with 10% FBS in a humidified atmosphere containing 95% air and 5% CO
2 at 37 °C as previously described to explore the
in vitro effects of ZNF593-AS on myocyte size [
20]. HL-1 (mouse myocytes) cells were cultured in Claycomb medium, which was purchased from Sigma-Aldrich and supplemented with 10% FBS, 4 mmol/L L-glutamine, and 100 μmol/L norepinephrine. Myocytes were separately transfected with GapmeR-ZNF593-AS or pcDNA3.1-ZNF593-AS by using Lipofectamine 2000 in accordance with the manufacturer’s protocol. Cells were treated with 1 mmol/L PE for another 12 h at 24 h after transfection. Myocyte size was detected through FITC–phalloidin staining, and the expression of ZNF593-AS was detected through qPCR assays.
2.10 FITC–phalloidin staining
Cell morphology was detected through FITC–phalloidin staining to evaluate myocyte size. Cells were fixed in 4% paraformaldehyde at room temperature for 30 min and incubated in 0.1% Triton X-100 for 10 min. Cells were washed with PBS, incubated in FITC–phalloidin at room temperature for 90 min, and subsequently visualized under fluorescence microscopy as previously described [
25].
2.11 Western blot analysis
The levels of proteins involved in mitochondrial complexes in ZNF593-AS KO or MHC-Tg mice were detected by Western blot analysis. Proteins from frozen animal tissues were extracted and homogenized in ice-cold lysis buffer. Lysates (20 mg protein/lane) were subjected to 10% SDS-PAGE gel for separation and transferred onto PVDF membranes. After nonspecific sites were blocked with 5% BSA for 2 h at room temperature, the membranes were incubated with primary antibodies (1:2000 dilution) overnight at 4 °C. After being washed with Tris-buffered saline Tween, the membranes were incubated with a peroxidase-conjugated secondary antibody for 2 h at room temperature. Bands were visualized with enhanced chemiluminescence reagents in accordance with the manufacturer’s recommendations.
2.12 Statistical analysis
Data are shown as means ± SEM. Statistical tests were performed by using GraphPad Prism (v8.0) (GraphPad Software, San Diego, CA, USA). A value of P < 0.05 was statistically significant. Statistical differences in multiple comparisons were analyzed by using one-way ANOVA, and Student’s t-test was used to compare the statistical significance between two groups.
3 Results
3.1 ZNF593-AS is downregulated in cardiac hypertrophy
The decreased transcriptional activity of ZNF593-AS has been identified not only in heart failure but also in the early stage of cardiac hypertrophy. For the further confirmation of the above phenomena, C57BL/6 mice were subjected to TAC to induce cardiac hypertrophy and heart failure. The time course of ZNF593-AS expression in the heart was detected at weeks 1, 2, 4, or 6 after TAC surgery. Consistent with a previous study, this work found that at week 1 after TAC surgery, ZNF593-AS expression showed low activation, whereas cardiac function was unimpaired (Fig.1), suggesting that the downregulation of ZNF593-AS is a direct cause of cardiac hypertrophy and heart failure. Primary cardiomyocytes and noncardiomyocytes were isolated from hypertrophic hearts, and ZNF593-AS was found to have mainly localized and decreased in primary cardiomyocytes (Fig.1). RNA-FISH also showed that ZNF593-AS expression decreased in cardiomyocytes in heart tissue (Fig.1). Thus, we hypothesized that lncRNA ZNF593-AS may contribute to the pathogenesis of cardiac hypertrophy.
We analyzed the area of cardiomyocytes in our previous animal models to investigate the function of ZNF593-AS in cardiac hypertrophy. Interestingly, the TAC-induced increase in cardiomyocyte size was exacerbated by ZNF593-AS inhibition (Fig.1) but reversed by ZNF593-AS overexpression (Fig.1). The role of ZNF593-AS in cardiac hypertrophy was further investigated on the basis of these findings.
3.2 ZNF593-AS knockout aggravates TAC-induced cardiac hypertrophy
ZNF593-AS KO mice were generated by using the CRISPR/Cas9 system to further confirm the effect of ZNF593-AS on cardiac hypertrophy. PCR revealed that cardiac ZNF593-AS expression was significantly knocked out (Fig. S1A and S1B), and the knockout of ZNF593-AS had no effect on the mRNA expression of the antisense transcripts of ZNF593 (Fig. S1C).
ZNF593-AS KO or WT mice underwent TAC surgery for 2 weeks and subjected to cardiac structure and function detection. As illustrated in Fig.2, ZNF593-AS KO mice that underwent TAC surgery showed exacerbated TAC-induced increases in heart weight/body weight (HW/BW) ratio compared with WT mice. FITC-conjugated WGA staining also revealed that the TAC-induced hypertrophic growth of cardiomyocytes was aggravated in TAC-treated ZNF593-AS-KO mice (Fig.2). Heart structure and function were further assessed in detail through echocardiography. The TAC-induced increases in the thicknesses of the left ventricular anterior wall and posterior wall at the systolic and diastolic phases were worse in ZNF593-AS-KO mice than in WT mice (Fig.2 and 2D). EF and FS showed no changes in ZNF593-AS-KO and WT mice subjected to TAC or sham surgery for 2 weeks (Fig.2).
These findings indicate that the downregulation of ZNF593-AS aggravates pressure overload–induced cardiac hypertrophy.
3.3 ZNF593-AS overexpression protects against TAC-induced cardiac hypertrophy
Next, gain-of-function experiments were performed by using transgenic mice with the cardiomyocyte-specific α-MHC-driven expression of ZNF593-AS (Fig. S1D). The cardiac expression of ZNF593-AS was approximately 6-fold greater in ZNF593-AS-Tg mice than in WT mice (Fig. S1E).
We compared cardiac morphology and heart weight in WT and ZNF593-AS Tg mice that underwent sham or TAC surgery for 2 weeks. Heart size and heart weight/bodyweight ratio significantly increased in WT mice following TAC surgery; however, these changes reversed in ZNF593-AS-Tg mice (Fig.3). Similar findings were also observed in cardiomyocyte size through FITC-conjugated WGA staining (Fig.3). Moreover, echocardiography revealed that the thicknesses of the left ventricular anterior wall and posterior wall at the systolic and diastolic phases in TAC-treated mice had markedly increased compared with those in sham mice, whereas these changes were attenuated in ZNF593-AS-Tg mice (Fig.3 and 3D). We observed no differences in EF or FS between ZNF593-AS-Tg mice and WT mice subjected to sham or TAC surgery for 2 weeks (Fig.3).
These results suggest that ZNF593-AS overexpression alleviates pressure overload–induced cardiac hypertrophy.
3.4 Antihypertrophic role of ZNF593-AS is conserved between humans and mice
Given that lncRNAs show low conservation across species, the function of ZNF593-AS in humans and mice was studied. First, ZNF593-AS expression in human myocytes (AC16 cells) was knocked down by using antisense LNA GapmeRs. QPCR assays showed that GapmeR-ZNF593-AS significantly downregulated ZNF593-AS (Fig. S1F). FITC staining revealed that the knockdown of human ZNF593-AS aggravated PE-induced AC16 cell hypertrophy (Fig.4). Furthermore, we downregulated mouse-derived ZNF593-AS expression in mouse cardiomyocytes (HL-1 cells). HL-1 cells treated with GapmeR-ZNF593-AS showed similar phenotypes (Fig.4).
Moreover, we overexpressed ZNF593-AS in human cardiomyocytes (AC16) and mouse cardiomyocytes (HL-1). As shown in Fig.4, ZNF593-AS overexpression in human cardiomyocytes alleviated the PE-induced hypertrophy detected by FITC staining. Consistent with the effects on human cardiomyocytes, ZNF593-AS overexpression in mouse cardiomyocytes alleviated PE-induced hypertrophy in vitro (Fig.4). Primary cardiomyocytes were isolated from neonatal mice and treated with GapmeR-ZNF593-AS or ZNF593-AS to further confirm the above result. The results showed that PE-induced hypertrophy was aggravated by ZNF593-AS knockdown but was alleviated by ZNF593-AS overexpression (Fig. S2A and S2B).
In general, these results indicate that the antihypertrophic role of ZNF593-AS is conserved between humans and mice.
3.5 ZNF593-AS regulates oxidative phosphorylation in cardiac hypertrophy
RNA-seq and gene enrichment analysis were performed on heart tissues from ZNF593-AS KO and MHC-Tg mice to demonstrate the mechanism through which ZNF593-AS regulates cardiac hypertrophy (Fig.5). Gene enrichment analysis indicated that several pathways, among which the oxidative phosphorylation (OXPHOS) pathway significantly changed, were dysregulated in ZNF593-AS KO and MHC-Tg mice (Fig.5 and 5C).
Proteins involved in OXPHOS, such as NDUFB8, SDHB, UQCRC2, CO2, and ATP5A, were detected to further confirm the above result. In mouse hearts, the expression levels of SDHB, UQCRC2, and ATP5A reduced under pressure overload, whereas ZNF593-AS overexpression significantly alleviated these changes (Fig.5). Moreover, the TAC-induced reduction in cardiac ATP levels was reversed by ZNF593-AS (Fig.5). Furthermore, the pressure overload–induced reduction in SDHB, UQCRC2, and ATP5A aggravated after ZNF593-AS knockout (Fig.5). In vitro, the knockdown of ZNF593-AS in HL-1 cells also significantly reduced OXPHOS levels, as reflected by OCR levels (Fig.5). In addition, mitochondrial ROS production was elevated after ZNF593-AS knockdown (Fig.5).
These results suggest that ZNF593-AS controls cardiac hypertrophy by regulating OXPHOS.
3.6 ZNF593-AS regulates mitochondrial function by targeting Mfn2
We sought to identify ZNF593-AS-governed genes during OXPHOS in cardiac hypertrophy. Differentially expressed genes were screened from ZNF593-AS KO and MHC-Tg mice. As shown in Fig.6, 29 genes that were upregulated in MHC-Tg mice reduced in KO mice, suggesting that these genes are regulated by ZNF593-AS. Our previous study showed that RYR2 and IRF3 are the target genes of ZNF593-AS in heart failure and diabetic cardiomyopathy, respectively. However, IRF3 expression remained unchanged during cardiac hypertrophy (Fig. S3A). Although RYR2 was regulated by ZNF593-AS in cardiac hypertrophy (Fig. S3B and S3C), Mfn2 displayed a higher expression level in heart tissue than RYR2 and was selected for further study (Fig.6). The knockout of ZNF593-AS significantly reduced Mfn2 expression, whereas the overexpression of ZNF593-AS increased Mfn2 expression (Fig.6 and 6D). In addition, the knockdown of Mfn2 had no effect on RYR2 expression or vice versa (Fig. S3D and S3E), suggesting that the function of ZNF593-AS/Mfn2 axis is independent of RYR2. On thebasis of our data, we considered the possibility that ZNF593-AS plays a positive regulatory role in Mfn2 expression. Our previous study showed that ZNF593-AS primarily localizes in the cytoplasm of human AC16 cardiomyocytes and isolated murine primary cardiomyocytes. Interestingly, ZNF593-AS not only localized in the cytoplasm of cardiomyocytes, it also localized in the nuclei (20%–30%) of myocytes to a lesser extent. Our findings showed that ZNF593-AS directly interacted with the RNA binding protein HNRNPC in the nuclei and cytoplasm of myocytes. CO-IP assays on hypertrophic heart tissues also showed that ZNF593-AS directly bound with HNRNPC (Fig.6). The binding site of ZNF593-AS on the Mfn2 promoter was predicted by using the ENCORI database (Fig. S4A). The relative luciferase activity of the Mfn2 promoter reporter significantly increased under ZNF593-AS cotransfection into 293T cells relative to that under vector-con transfection (Fig.6), suggesting that ZNF593-AS promotes Mfn2 expression by directly binding to its promoter. The ChIP-qPCR analysis of HL-1 cells showed that HNRNPC has a binding site in the Mfn2 promoter (Fig.6). These data suggest that ZNF593-AS interacts with hnRNPC to regulate Mfn2 expression.
Mfn2 plays important roles in controlling mitochondrial autophagy. TAC-induced autophagy was aggravated by ZNF593-AS knockout as reflected by decreases in p62 expression and increases in LC3B II/LC3B I level, whereas this phenotype was reversed by ZNF593-AS overexpression (Fig.6 and 6I). These results were further confirmed in vitro. The knockdown of ZNF593-AS or Mfn2 reduced mitochondrial fusion (Fig. S4B).
Collectively, our findings show that ZNF593-AS regulates Mfn2 expression by binding with HNRNPC to control mitochondrial autophagy and OXPHOS in cardiac hypertrophy (Fig.6).
4 Discussion
In this study, we found that ZNF593-AS is downregulated under the stimulation of hypertrophy. This effect leads to the decrease in the expression of Mfn2 and then further causes OXPHOS disorders and cardiac hypertrophy. The recovery of ZNF593-AS expression attenuates TAC-induced cardiac hypertrophy and cardiac dysfunction (such as OXPHOS disorder and ATP deficiency) by binding to the promoter region of Mfn2 and subsequently activating Mfn2 transcription. Our study reveals a previously uncharacterized lncRNA in pressure overload–induced pathological cardiac hypertrophy. Our observations provide a theoretical basis for developing lncRNA-based therapeutics against cardiac hypertrophy and dysfunction.
Mammalian cardiomyocytes are terminally differentiated in adults and do not proliferate under physiological or pathological conditions. The law of Laplace states that the growth or shrinkage of cardiomyocytes is an adaptive response to stimuli but becomes maladaptive under sustained stress [
26]. Cardiac hypertrophy is classified as physiological with normal cardiac function or as pathological accompanied by cardiac dysfunction. Pathological hypertrophy is associated with the production of neurohumoral mediators, hemodynamic overload, and metabolism of cardiomyocytes [
1,
27]. Changes in cardiac metabolism, such as a shift from fatty acid oxidation to glucose metabolism and overall reductions in oxidative metabolism, occur early during the development of cardiac hypertrophy [
28]. The heart is a high energy-demanding tissue, and almost 70% of the energy used by the heart originates from OXPHOS in the mitochondria [
29]. Mitochondrial dysfunction has two adverse consequences: the accumulation of cytotoxic oxygen radicals and decline in ATP production. Mitochondrial fission or fusion and mitochondrial biogenesis and mitophagy are the most important steps necessary to maintain mitochondrial quality and normal OXPHOS [
30–
32]. The impairment of mitophagy induces mitochondrial dysfunction, thereby exacerbating cardiac hypertrophy and heart failure [
33,
34]. Mitochondria are in a constant state of fission and fusion balance, which is essential to maintain their quality and quantity. Dynamin-related protein 1 (Drp1) is a major mitochondrial fission-associated protein whose activity is tightly regulated to clear damaged mitochondria via mitophagy [
35]. Drp1 insufficiency abolishes mitochondrial autophagy and exacerbates the development of mitochondrial dysfunction and cardiac hypertrophy [
36].
Mitochondrial membrane fusion is mediated by Mfn1/2. Mfn1 and Mfn2 form homodimers or heterodimers on mitochondria to complete the fusion of the outer mitochondrial membrane [
37]. Studies have reported that Mfn2 participates in autophagy in cardiomyocytes, and autophagosomes accumulate in Mfn2-knockout cardiomyocytes; this phenomenon appears to be caused by the impaired fusion of autophagosomes with lysosomes in the absence of Mfn2 [
38,
39]. Impairing mitochondrial fusion by knocking out Mfn2 is sufficient to decrease OXPHOS levels and cell proliferation [
40]. These findings indicate that Mfn2 is a vital regulator in OXPHOS and mitochondrial function. Mfn2 expression could be regulated by various signaling pathways. Peroxisome proliferator—activated receptor gamma coactivator-1 alpha stimulates the transcriptional activity of a 2 kb fragment of the Mfn2 promoter after transfection into several different cell types [
41]. Ubiquitylation and phosphorylation have been suggested to control the activation and degradation of Mfn2 [
42,
43]. Mfn2 expression could also be regulated by several miRNAs, such as miR-93, miR-195-5p, and miR-214 [
12,
44,
45]. In this study, we found that lncRNA ZNF593-AS directly regulates Mfn2 expression to control mitophagy. Given that ZNF593-AS may regulate Mfn2 expression at transcriptional or post-transcriptional levels, normal ZNF593-AS levels might be sufficient to maintain Mfn2 expression. In our study, we found that under normal conditions, ZNF593-AS KO could lead to the downregulation of Mfn2, whereas ZNF593-AS overexpression could not lead to the upregulation of Mfn2. This condition may be similar to the barrel effect. Moreover, the maintenance of the normal expression of Mfn2 requires the participation of multiple factors. The underlying mechanism through which ZNF593-AS regulates Mfn2 expression needs to be studied further.
The functions of lncRNAs in cardiac hypertrophy have recently begun to be uncovered. Some lncRNAs with critical roles in cardiac hypertrophy have been identified. For example, lncRNA cardiac physiological hypertrophy-associated regulator (CPhar) increases in heart tissue with exercise training and is necessary for exercise-induced physiologic cardiac growth by binding with DEAD-Box Helicase 17 to regulate activating transcription factor 7 [
46,
47]. Exercise-regulated cardiac lncRNAs, namely, lncExACTs, are evolutionarily conserved and decrease in exercised hearts but increase in heart failure. The inhibition of lncExACT1 induces physiological hypertrophy, whereas the cardiac overexpression of lncExACT1 causes pathological hypertrophy and heart failure [
47]. lncRNA CHAST has recently been shown to be crucial for the pathogenesis of pressure overload–induced cardiac remodeling. Chast aggravates TAC-induced cardiac hypertrophy and dysfunction by interacting with the enhancer of zeste homolog 2 subunits of PRC2 [
48]. In this study, we discovered that lncRNA ZNF593-AS attenuates cardiac hypertrophy through the regulation of cardiac mitochondrial OXPHOS by targeting Mfn2.
In this work, we found that ZNF593-AS is downregulated prior to cardiac hypertrophy, indicating that ZNF593-AS reduction may be a trigger of cardiac hypertrophy and subsequent heart failure. Cardiac hypertrophy occurs as an adaptive response to increased workload to maintain cardiac function [
49]. However, prolonged cardiac hypertrophy causes heart failure [
50,
51]. The low levels of some molecules and pathways could aggravate deteriorative phenotypes over time. However, the expression of ZNF593-AS remains stable rather than gradually decreasing during the progress from adaptive hypertrophy to heart failure. We questioned how the same reduction in ZNF593-AS triggered cardiac hypertrophy and subsequent heart failure. We reasoned that this phenomenon could be explained as follows. (1) In this study, the ZNF593-AS inhibition–induced downregulation of Mfn2 significantly reduced mitochondrial function and ATP production. Enlarged cardiomyocytes require mitochondrial biogenesis to produce additional ATP, which is necessary to enhance protein synthesis to accommodate cell size expansion and increase contractility to overcome stress overload. The vicious cycle of energy supply and demand imbalance leads to the deterioration of heart function. (2) In addition to Mfn2, RYR2 serves as a target of ZNF593-AS. RYR2 plays a key role in Ca
2+ homeostasis and cardiomyocyte contractility by mediating sarcoplasmic reticulum Ca
2+ release. Intracellular Ca
2+ homeostasis is a key determinant of cardiac contractile function. ZNF593-AS inhibition resulted in a decrease in RYR2 expression and activity, which further led to a deterioration in cardiac function. These results suggest that similarly low levels of ZNF593-AS may cause cardiac hypertrophy and heart failure. (3) The mechanism involved in regulating ZNF593-AS expression during cardiac hypertrophy and heart failure is unknown. However, the pressure overload–induced dysregulation or activity of transcription factors and epigenetic modification might participate in these processes, which need further exploration.
In summary, we demonstrated that ZNF593-AS is essential for the inhibition of pathological cardiac hypertrophy by upregulating Mfn2 expression and improving mitochondrial function. These observations provide a theoretical basis for developing lncRNA-based therapeutics against cardiac hypertrophy.
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