Caveolin 1 Reduces High Glucose-Induced Hypertrophy and Inammatory Fibrosis of H9C2 Cardiomyocytes By Inhibiting NF-κB Signaling

Background: Cardiac hypertrophy and inammatory brosis are the basic pathological changes of the prevalent microvascular diabetes complication known as diabetic cardiomyopathy (DCM). Previous studies of the Caveolin 1 protein and the lipid raft structures known as caveolae—small invaginations of the plasma membranes of certain cell types (including cardiomyocytes)—have linked this protein to fat metabolism disorders, inammation, diabetes, and cardiovascular disease. However, there are no reports linking Caveolin 1 to DCM. Here, we show that Caveolin 1 plays a vital role in the hypertrophy and inammatory brosis of H9C2 cardiomyocytes induced by high glucose (HG) via inhibiting the NF-κB signaling. Methods: The H9C2 cardiomyocytes induced by HG were used as the experimental model in this study to explicit the effect of Caveolin 1 on DCM and the underlying mechanisms involved. Unpaired Student’s t test was used for comparison between two groups. For multiple comparisons, data were analyzed by one-way ANOVA with Bonferroni post hoc test multiple comparisons. Results: (1) Exposure of H9C2 cardiomyocytes to HG activated NF-κB signaling and induced the expression of downstream NF-κB target genes including both hypertrophic factors and inammatory brosis factors; (2) Intriguingly, exposure of cardiomyocytes to HG led to decreased Caveolin 1 levels, and this decrease occurred in an HG-exposure-time-dependent manner; (3) Overexpression of Caveolin 1 reduces phosphorylation of p65 and blocks the nuclear accumulation of this NF-κB component in HG-exposed cardiomyocytes, and inhibits transcriptional activation of NF-κB target genes; (4) Knocking down Caveolin 1 exacerbates the HG-induced promotion of the aforementioned NF-κB hypertrophic factors and inammatory brosis factors, all of which have been linked to DCM pathogenesis. Conclusions: Our study supports that Caveolin 1 can protect against hypertrophy and inammatory brosis induced by HG, specically by suppressing NF-κB In the present study, our results showed that both the phosphorylation and nuclear translocation of the NF-κB signaling component p65 increased signicantly in HG-stimulated H9C2 cardiomyocytes, whereas the total NF-κB p65 levels remain unchanged. Moreover, treatment with HG for 24 h distinctly increased the accumulation of hypertrophic proteins (ANF and BNP) and inammatory brosis factors (ICAM-1, TNF-α, IL-6, MCP-1, FN, and TGF-β1), collectively demonstrating that treatment with HG for 24 h induced inammatory brosis and hypertrophy in H9C2 cardiomyocytes and establishing that NF-κB signaling activation functionally contributes to this pathogenic process. IL-6, MCP-1 and hypertrophic genes ANF and BNP in H9C2 cardiomyocytes. These HG-induced the effects on inammatory brosis and hypertrophy in H9C2 cardiomyocytes were reversed upon overexpression of Caveolin 1 but were exacerbated by Caveolin 1 knockdown in these HG-exposed cardiomyocytes. Considering that Caveolin 1 confers resistance against both inammatory brosis and hypertrophy of H9C2 cardiomyocytes, our work supports the idea that Caveolin 1 may represent a potential therapeutic target for developing treatments for DCM.


Background
Cardiovascular disease (CVD) is the leading cause of death in people with type 2 diabetes mellitus (T2DM) [1]. Diabetic cardiomyopathy (DCM) is one of the major microvascular complications of diabetes that affects approximately 12% of diabetic patients, leading to overt heart failure and death [2]. The main pathologic changes of DCM include cardiac hypertrophy, myocardial brosis and ventricular dysfunction [3][4][5], which induce cardiac remodeling, cardiac dilatation, cardiac dysfunction and congestive heart failure, and eventually develop into heart failure, arrhythmia and cardiogenic shock [6][7][8][9].
In ammation is an important feature of DCM [10][11][12][13][14]. Activation of the nuclear factor NF-κB lead to excessive expression of its down-stream target genes, such as in ammatory brosis components including bronectin (FN), intercellular adhesionmolecule-1 (ICAM-1), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1), and hypertrophic genes including atrial natriuretic facor (ANF) and brain natriuretic peptide (BNP), which promote the pathological process of cardiac hypertrophy and interstitial brosis of DCM [15,16]. Therefore, inhibition of the expression of FN, ICAM-1, IL-6, MCP-1, TNF-α, TGF-β1, ANF and BNP induced by high glucose (HG) through NF-κB signaling is bene cial to protect against DCM, and it is of great signi cance to explore the potential targets of anti-DCM which can inhibit the activation of NF-κB in ammatory signaling. Caveolin 1 is a major integral membrane protein in the cave-like invaginations on the cell surface (Caveolae). It participates in the regulation of a variety of cellular behaviors, including the formation and localization of Caveolae, maintenance of cellular cholesterol homeostasis and internalization of pathogens, and mediates maintaining the integrity of Caveolae and signal transduction [17][18][19][20]. Caveolin 1 knockout mice showed strong in ammatory responses and increased activation of NF-κB and STAT3 pathways in the lungs, liver and kidneys [21], indicating Caveolin 1 regulates in ammatory response.
Recently, Caveolin 1 was found to play vital roles in diabetes and cardiovascular disease. In skeletal muscle biopsies of patients with type 2 diabetes, the expression of Caveolin 1 and eNOS were 50 percent lower than in normal tissue [22]. The Caveolin 1 levels decreased in mesenteric vascular smooth muscle cell from type 2 diabetic Goto-Kakizaki rats [23]. Besides, Caveolin 1 was also mediates β-cell apoptosis and proliferation [24], implying that Caveolin 1 may inhibit the development of diabetes. In the heart tissue of rat models of abdominal aortic constriction constructed left ventricular hypertrophy, the levels of Caveolin 1 decreased along with the increase of apelin receptor (APJ). Moreover, Caveolin 1 overexpression reduced the diameter and volume of H9C2 cells induced by Apelin-13 [25], demonstrating that Caveolin 1 alleviates the cardiac hypertrophy. All of these provided theoretical basis for Caveolin 1 as a potential target for the treatment of diabetes and cardiac hypertrophy.
Given the essential roles of Caveolin 1 in regulating the in ammation, diabetes and cardiac hypertrophy, our study aimed to elucidate the effects of Caveolin 1 on the HG-induced hypertrophy and in ammatory brosis of H9C2 cardiomyocytes, with special focus on the NF-κB signaling pathway, which will contribute to serve Caveolin 1 as a new target for the study of DCM.

Cell culture
Rat H9C2 cardiomyocytes were purchased from GuangZhou Jennio Biotech Co., Ltd (Jennio, Guangzhou, China). They were maintained in low glucose' DMEM in the presence of 10% fetal bovine serum (CellMax, Beijing, China) at 37 °C. Cells culture at 80% con uence were rendered quiescent by incubation for 12 h in serum-free medium before treating with HG' DMEM.

Western blot assays
Western blotting was carried out as previously described [26] to detect the protein levels of Caveolin 1, FN, ICAM-1, IL-6, MCP-1, TGF-β1, TNF-α, ANF, BNP phosphorylation and total NF-κB p65. The signals were visualized with a GE ImageQuant 350 instrument, and were quanti ed by densitometry using a Gel Doc XR System (Bio-Rad, USA); the signals were analyzed using Quantity One Protein Analysis Software (Bio-Rad).
Confocal laser scanning uorescence microscopy Immuno uorescence assay was carried out as previously described [27]. The images were collected using a DMi8 laser confocal uorescence microscope (Nikon, Tokyo, Japan).

Nuclear protein extraction
The nuclear proteins were extracted with a Nuclear Extract Kit (BestBio, Shanghai, China). Cells were collected after centrifuging at 500 g for 3 minutes at 4 °C. PBS was used to wash the cells two times and the 200 μL cold Buffer A was added with 2μL protease inhibitor cocktail. Subsequently, the cells were vortexes with maximal velocity for 15 s and placed in the ice for 10 min. After being vortexes with maximal velocity for 5 s, the cells were centrifuged at 16,000g for 5 min at 4 °C and the supernatants (cytoplasm protein) were collected, besides, the sediments were added with 200 μL cold Buffer B with 2μL protease inhibitor cocktail. Afterwards, the sediments were vortexed with maximal velocity for 15 s and placed in the ice for 40 min. The supernatants (nuclear protein) were collected after centrifuged at 16,000g for 10 min at 4 °C.
Electrophoretic mobility shift assay (EMSA) The EMSA assay was carried out as previously described [28]. Activation of NF-κB signaling was determined based on EMSA using nuclear extracts, the sequence of the biotin-labeled oligonucleotide probes of nf-κb was as follows: 5'-AGTTGAGGGGACTTTCCCAGGC-3', 3'-TCAACTCCCCTGAAAGGGTCCG -5' containing the acknowledged NF-κB binding site. The procedures were performed following the instructions of the manufacturer (Light Shift Chemiluminescent EMSA Kit, Pierce, USA). The images were captured and quanti ed using Image Quant 350 (GE Healthcare, USA).

Dual luciferase reporter assay
The dual luciferase reporter assays were carried out as previously described [29]. H9C2 cardiomyocytes were seeded in 96-well culture plates and cotransfected with 0.1 μg of pNF-κB -luciferase (Beyotime, Haimen, China) and 0.02 μg of pRL-TK (Beyotime, Haimen, China) in the presence or absence of 0.05 μg of Caveolin 1 plasmid or Caveolin 1 si-RNA. After various treatments, cells were lysed and the luciferase activity was determined using the Dual-Luciferase reporter assay system kit (Solarbio, Beijing, China) to analyze the effect of Caveolin 1 on the activation of NF-κB pathway. Luciferase activity was normalized to the renilla luciferase activity.

Statistical analysis
Values were expressed as mean ± SEM. All data were assessed by the Graphpad Prism 5.0 software. Unpaired Student's t test was used for comparison between two groups. For multiple comparisons, data were analyzed by one-way ANOVA with Bonferroni post hoc test multiple comparisons. Independent experiments were performed at least thrice with similar results. P < 0.05 was considered statistically signi cant.

Results
HG induction causes hypertrophy and in ammatory brosis of H9C2 cardiomyocytes by activating NF-κB signaling NF-κB is a vital transcription factor that mediates in ammatory and its activation leads to cardiomyocyte injury in diabetes [30]. It consists of ve members, including p65 (RelA), RelB, c-Rel, NF-κB1, and NF-κB2 [31]. HG is known to activate the NF-κB signaling in the H9C2 cardiomyocytes [32]. It was found that phosphorylation of the NF-κB p65 subunit and nuclear translocation of NF-κB p65 lead to the selective transcription of downstream proin ammatory genes [33,34]. To verify the pathogenic impacts of HG on H9C2 cardiomyocytes, we detect the NF-κB signaling activation and accumulation of its down-stream target genes hypertrophic proteins (ANF and BNP) as well as in ammatory brosis factors (FN, ICAM-1, TGF-β1, IL-6, MCP-1 and TNF-α) levels.
Western blotting showed that the phosphorylation and nuclear accumulation of NF-κB p65 rstly upregulated and then down-regulated with HG treatment compared to cells cultured in low glucose' DMEM, with a paramount up-regulation at 30 min, whereas total NF-κB p65 was not apparently changed ( Fig. 1A-B). Meanwhile, the DNA-binding activity of NF-κB was measured by EMSA. The band intensity of the NF-κB-DNA oligonucleotide complex, which suggested the DNA-binding of NF-κB to its consensus DNA sequences was rstly increased and then decreased, with the maximal effect occurred after 30 min of HG treatment (Fig. 1C). Immuno uorescence results also showed that the cytoplasmic uorescence of p65 dramatically attenuated, but the nuclear p65 signi cantly aggregated (Fig. 1D) after about 30 min incubation with HG. Besides, the ANF, BNP, FN, ICAM-1, TGF-β1, IL-6, MCP-1 and TNF-α levels signi cantly increased in the HG media experimental group compared to cells cultured in low glucose' DMEM, and these increases were in an exposure-time-dependent manner (Fig. 1E-G), indicating that our experimental cell model works and treatment with HG induces the hypertrophy and in ammatory brosis of H9C2 cardiomyocytes.

HG reduces Caveolin 1 levels in H9C2 cardiomyocytes
Previous study demonstrated that the Caveolin 1 expression decreased in skeletal muscle biopsies of patients with type 2 diabetes and the mesenteric vascular smooth muscle cells from type 2 diabetic rats [22,23]. To investigate the effects of Caveolin 1 in HG-induction H9C2 cardiomyocytes, we performed western blotting to test whether HG affects the accumulation of Caveolin 1 rstly. Compared to cells cultured in low glucose media, the Caveolin 1 levels decreased dramatically in the HG media experimental group, with the extent of reduction increasing in an exposure-time-dependent manner ( Fig. 2A), suggesting HG is a stimulator of Caveolin 1.

Caveolin 1 decreased the HG-induced accumulation of hypertrophic proteins and in ammatory brosis factors in H9C2 cardiomyocytes
We transfected with negative control (NC) and three pairs of siRNA oligonucleotides targeting Caveolin 1 in H9C2 cardiomyocytes cultured with low glucose' DMEM. Preliminary testing showed that strongest interference effect was obtained for oligonucleotide pair 223 (Fig. 3A). We subsequently tested siRNAmediated knockdown of Caveolin 1 in HG-induction H9C2 cardiomyocytes and found that cells transfected with the Caveolin 1 siRNA expressed lower overall Caveolin 1 levels than scramble control cells cultured in HG media (Fig. 3B), implying we successfully perform Caveolin 1 knockdown. Furthermore, we also transfected the hCaveolin 1 plasmids in HG-induction H9C2 cardiomyocytes to over expression Caveolin 1 (Fig. 3C). Fig.1 demonstrated that HG induces the hypertrophy and in ammatory brosis of H9C2 cardiomyocytes. Next, we knocked down Caveolin 1 and transfected the hCaveolin 1 plasmids to detect the crucial role of Caveolin 1 in the hypertrophy and in ammatory brosis of H9C2 cardiomyocytes. Western blotting results showed that Caveolin 1 knockdown led to increased accumulation of ANF, BNP, FN, ICAM-1, TGF-β1, IL-6, MCP-1 and TNF-α compared to scramble siRNA control HG culture cells (Fig. 4A-C). Moreover, overexpression of Caveolin 1 effectively inhibited HG induced up-regulation of ANF, BNP, FN, ICAM-1, TGF-β1, IL-6, MCP-1 and TNF-α compared to cells cultured in low glucose' DMEM ( Fig. 4D-F).

Results in
Caveolin 1 knockdown activates NF-κB signaling and enhances the DNA-binding and transcriptional activities of NF-κB Results above showed that the NF-κB signaling was activated induced by HG. However, whether the role of Caveolin 1 in resisting the hypertrophy and in ammatory brosis of H9C2 cardiomyocytes related with the activation of NF-κB signaling is unknown. To investigate the relationship between Caveolin 1 and the activation of NF-κB signaling, we knocked down Caveolin 1 levels in HG-induction H9C2 cardiomyocytes and the activation of the NF-κB signaling was detected.
It was shown that compared to cells cultured with low glucose' DMEM, Caveolin 1 knockdown dramatically increased the HG-induced phosphorylation and nuclear distribution of NF-κB p65, but decreased the cytoplasm distribution of p65 at 0.5 h (Fig. 5A-C). Meanwhile, the total expression of NF-κB p65 had no signi cant change (Fig. 5A). Immuno uorescence analyses showed that after Caveolin 1 was knocked down, the HG-induced subcellular distribution of p65 in the nucleus was dramatically increased (Fig. 5D).
Moreover, EMSA and luciferase reporter assays results demonstrated that Caveolin 1 knockdown further enhanced the HG-induced DNA-binding and transcriptional activities of NF-κB ( Fig. 5E-F).

Caveolin 1 overexpression inhibits NF-κB signaling activation, and the DNA-binding and transcriptional activities of NF-κB
To recon rm the effects of Caveolin 1 on the activation of NF-κB signaling, we transfected Caveolin 1 plasmids in H9C2 cells cultured with HG DMEM to detect the NF-κB signaling activation.
Present results showed that compared with cells treated with low glucose' DMEM, overexpression of Caveolin 1 effectively suppressed the HG-induced phosphorylation and nuclear accumulation of NF-κB p65 and enhanced p65 levels in the cytoplasm, whereas the total NF-κB p65 levels were unchanged (Fig.  6A-C). Immuno uorescence analyses also revealed that the HG-induced subcellular distribution of p65 in the nucleus were obviously decreased by transfection with hCaveolin 1 plasmids (Fig. 6D), which was consistent with the western blot data.
Besides, to verify the effects of Caveolin 1 on the inhibition of HG-induced hypertrophy and in ammatory brosis of H9C2 cardiomyocytes were related with suppression the activation of NF-κB signaling pathway, we conducted the EMSA and luciferase reporter assay to detect the roles of Caveolin 1 on the DNA-binding and transcriptional activities of NF-κB. Results showed that overexpression of Caveolin 1 suppressed the HG-induced increase of the DNA-binding and transcriptional activities of NF-κB compared to cells treated with low glucose' DMEM ( Fig. 6E-F).

Discussion
Cardiovascular complications, including DCM, account for signi cant morbidity and mortality in the diabetic population, and there is presently a lack of effective means to cure DCM [35]. Exploring new anti-DCM targets and therapeutic methods is therefore an urgent pursuit in DCM research. Multiple studies have used H9C2 cardiomyocytes exposed to HG for 24 h to induce an in vitro DCM model. HG treatment activates NF-κB signaling [36,37]. NF-κB signaling activation triggers excessive and persistent expression of proin ammatory cytokines, including (among others) TNF-α, IL-6, and ICAM-1 [31,[38][39][40][41][42], as well as hypertrophic genes including ANF and BNP [43,44]. These collectively exert numerous autocrine, pleiotropic effects in heart cells when exposed to various conditions, and can lead to ischemic myocardial injury, dilated cardiomyopathy, and HF, among other pathogenic phenotypes [45]. Moreover, it is known that activated NF-κB signaling in the nucleus strongly induces TGF-β1 expression, which in turn up-regulates FN expression, resulting in excessive ECM in cardiac brosis [46].
In the present study, our results showed that both the phosphorylation and nuclear translocation of the NF-κB signaling component p65 increased signi cantly in HG-stimulated H9C2 cardiomyocytes, whereas the total NF-κB p65 levels remain unchanged. Moreover, treatment with HG for 24 h distinctly increased the accumulation of hypertrophic proteins (ANF and BNP) and in ammatory brosis factors (ICAM-1, TNF-α, IL-6, MCP-1, FN, and TGF-β1), collectively demonstrating that treatment with HG for 24 h induced in ammatory brosis and hypertrophy in H9C2 cardiomyocytes and establishing that NF-κB signaling activation functionally contributes to this pathogenic process.
Caveolin 1, one of three known caveolin proteins (Caveolin 1, 2, and 3), occupies ask-shaped plasma membrane invaginations (also called caveolae) and serve as the structural components of caveolae [47]. Caveolin 1 is expressed ubiquitously in mammals as is caveolin 2, whereas Caveolin 3 is mostly found in skeletal muscle [47][48][49]. Previous studies have focused on correlations between Caveolin 3 and DCM [50,51]. Induction of the caveolin-3/eNOS complex by nitroxyl (HNO) has been shown to ameliorate development of DCM [51]. However, it is still unknown whether Caveolin 1 or Caveolin 2 participates in mediating the pathological process of DCM. Notably, Caveolin 2 was shown to be coexpressed and to colocalize with Caveolin 1, and Caveolin 2 is known to require Caveolin 1 for proper membrane targeting [52]. Moreover, Caveolin 3 is more similar to Caveolin 1 than to Caveolin 2 at the protein sequence level [53].
These previous observations motivated our interested in assessing the potential effects of Caveolin 1 in DCM and in exploring any underlying mechanisms. Brie y, our results showed that HG-decreased Caveolin 1 expression in H9C2 cardiomyocytes (in an exposure-time-dependent manner), indicating a potential link between down-regulation of Caveolin1 and the observed increases in in ammatory brosis factors like ICAM-1, TNF-α, IL-6, MCP-1, FN, and TGF-β1 as well as hypertrophic proteins like ANF and BNP. Furthermore, our study showed that overexpression of Caveolin 1 had an inhibitory effect on both HG-induced nuclear translocation of p65 and transcriptional activation of additional NF-κB signaling components, while knocking down Caveolin 1 exacerbated these HG-induced, DCM-linked pathogenic effects on H9C2 cardiomyocytes.
Accordingly, we infer that Caveolin 1 can somehow counteract the pathogenic impacts of HG on H9C2 cardiomyocytes through modulation of NF-κB signaling activation. Elucidating the mechanisms through which Caveolin 1 regulates NF-κB signaling will require further experimental work; at present, we hypothesize that Caveolin 1 may suppress the degradation of IκBα, thereby blocking the release of NF-κB dimers for translocation into the nucleus [54]. Given the known impact of the NEMO-containing IKK complex in promoting degradation of classical IκBs (IκBα, IκBβ, or IκBε) [55], it may be informative to explore potential interactions and impacts of Caveolin 1 with the kinase and/or regulatory subunits of the IKK complex.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
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Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests
The authors declare that they have no competing interests.  min. (C) EMSA was performed to investigate the DNA-binding activity of NF-κB. (D) Immuno uorescence staining for the subcellular distribution of p65 at the indicated time points in H9C2 cardiomyocytes exposed to HG. Original magni cation, ×630. The accumulation of ANF and BNP (E), FN, ICAM-1 and TGF-β1 (F), IL-6, MCP-1 and TNF-α (G), measured by western blotting of HG-exposed H9C2 cardiomyocytes at the indicated time points. *P 0.05, **P 0.01. All experiments were conducted at least three times and similar results were obtained each time.

Figure 2
HG reduces Caveolin 1 levels in H9C2 cardiomyocytes. The Caveolin 1 levels (A) were detected by western blotting of HG-exposed H9C2 cardiomyocytes at the indicated time points. **P 0.01, ***P 0.001 vs 0 h. Caveolin 1 decreased the HG-induced accumulation of hypertrophic proteins and in ammatory brosis factors in H9C2 cardiomyocytes. ANF and BNP (A and D), FN, ICAM-1 and TGF-β1 (B and E), IL-6, MCP-1 and TNF-α levels (C and F), measured by western blotting of HG-exposed Caveolin 1 knockdown or overexpression H9C2 cardiomyocytes. *P < 0.05 vs NG, #P < 0.05 vs HG. This experiment was performed at least three times, with similar results obtain each time. Caveolin 1 knockdown activates NF-κB signaling and enhances the DNA-binding and transcriptional activities of NF-κB. Western blotting of HG-exposed, Caveolin 1 knockdown H9C2 cardiomyocytes to examine: phosphorylation of NF-κB p65 and total NF-κB p65 (A); nuclear p65 (B), and cytoplasmic p65 (C). *P < 0.05, **P 0.01 vs NG, #P < 0.05 vs HG. (D) Immuno uorescence staining for the subcellular distribution of p65. Original magni cation, ×630. The DNA-binding (E) and transcriptional activity (F) of NF-κB were assessed using EMSA and luciferase reporter assay. *P < 0.05, **P < 0.01 vs NG, #P < 0.05 vs HG. All experiments were conducted at least three times and similar results were obtained each time.