Mild hypoxia-induced cardiomyocyte hypertrophy via up-regulation of HIF-1α-mediated TRPC signalling

Hypoxia-inducible factor-1 alpha (HIF-1α) is a central transcriptional regulator of hypoxic response. The present study was designed to investigate the role of HIF-1α in mild hypoxia-induced cardiomyocytes hypertrophy and its underlying mechanism. Mild hypoxia (MH, 10% O2) caused hypertrophy in cultured neonatal rat cardiac myocytes, which was accompanied with increase of HIF-1α mRNA and accumulation of HIF-1α protein in nuclei. Transient receptor potential canonical (TRPC) channels including TRPC3 and TRPC6, except for TRPC1, were increased, and Ca2+-calcineurin signals were also enhanced in a time-dependent manner under MH condition. MH-induced cardiomyocytes hypertrophy, TRPC up-regulation and enhanced Ca2+-calcineurin signals were inhibited by an HIF-1α specific blocker, SC205346 (30 μM), whereas promoted by HIF-1α overexpression. Electrophysiological voltage-clamp demonstrated that DAG analogue, OAG (30 μM), induced TRPC current by as much as 170% in neonatal rat cardiomyocytes overexpressing HIF-1α compared to negative control. These results implicate that HIF-1α plays a key role in development of cardiac hypertrophy in responses to hypoxic stress. Its mechanism is associated with up-regulating TRPC3, TRPC6 expression, activating TRPC current and subsequently leading to enhanced Ca2+-calcineurin signals.


Introduction
The lasting hypoxic exposure or vigorous exercise could lead to the adaptive cardiac hypertrophy [1,2]. Unlike the toxic effect of severe hypoxia, mild hypoxia (MH, 10% oxygen) could not induce any cytotoxicity but trigger hypertrophic responses in cultured neonatal rat cardiac myocytes [3], and the mechanisms involved remain to be elusive.
Hypoxia-inducible factor 1 alpha (HIF-1␣) is a heterodimeric subunit of the transcription factor HIF-1, which regulates the transcription of genes involved in adaptive responses to hypoxia [4]. So far, role of HIF-1␣ in the development of cardiac hypertrophy has been sparsely documented. Silter et al. [5] have shown that HIF-1␣ is critically involved in the preservation of cardiac function without affecting cardiac hypertrophy by using HIF-1␣ knockdown mice with transverse aortic constriction. Recently, Xue et al. [6] have demonstrated that cardiac-specific overexpression of HIF-1␣ could prevent deterioration of glycolytic pathway and cardiac hypertrophy in streptozotocin-induced diabetic mice. More interestingly, carvedilol, a ␤-receptor blocker, has emerged as a beneficial treatment for cardiac hypertrophy and inhibited the overexpression of HIF-1␣ in pressure-overloaded rat heart [7]. These studies regarding the role of HIF-1␣ in cardiac hypertrophy were based upon pathologic situation, and their conclusions were under the controversial arguments. The previous investigations have shown that hypoxia is a critical factor which triggers cardiac hypertrophy in vivo [2,8], therefore, the potential role of HIF-1␣ in adaptive cardiac hypertrophy, e.g. MH-induced cardiomyocytes hypertrophy, needs to be clarified.

Cell culture and MH
Cardiomyocytes from 1-or 2-day-old Wistar rats were isolated, subjected to Percoll gradient centrifugation and cultured as previously described [13]. Use of animals was in accordance with the regulations of the ethic committees of Harbin Medical University, and confirmed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The purified cardiomyocytes were plated on 35-mm dishes (1.6ϫ10 5 cells per dish) in minimum essential medium (MEM) supplemented with 5% foetus bovine serum (FBS), penicillin (100 U/ml; GIBCO, Grand Island, NY, USA), and streptomycin (100 g/ml; GIBCO). When cardiomyocytes were exposed to MH, cells were placed in a hypoxic chamber, which was kept at 37ЊC, 90% humidity. The chamber was filled with gas mixture of 10% O2/85% N2/5% CO2.

Quantitative real-time PCR
PCR primers for HIF-1␣, rTRPC1, rTRPC3, rTRPC6, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were designed based on published sequences [14][15][16][17]. For details on primer sequences and setting of the PCR reaction, see Supporting Information. 18S rRNA was used as endogenous control. PCR reactions took place in 96-well plates using SYBR Green detection. Relative gene expression was calculated to 18S rRNA expression.

Measuring intracellular calcium concentrations by flow cytometry
The intracellular calcium was measured by flow cytometry using the calcium-sensitive dyes, Fluo-3 and Fura Red (Molecular Probes, Eugene, OR, USA). Fluo-3 fluorescence at 530 nm increases with increasing Ca 2ϩ binding, whereas Fura-Red fluorescence at 670 nm decreases with increasing Ca 2ϩ binding, allowing ratiometric measurement of calcium [18]. Myocytes were resuspended in medium supplemented with 1% FBS, then stained with 4 M Fluo-3 and 10 M Fura Red for 30 min. at 37ЊC. The Fluo-3/Fura Red fluorescence ratio was read on a FACSCalibur flow cytometer (BD Biosciences).

Patch clamp
Patch clamp is done as previously described [11,19,20]. For TRPC current recording, we used the whole-cell voltage clamp technique with pipette resistances of 2-3 M⍀ when filled with internal solution. The junctional potential was corrected by zeroing the potential before the pipette tip touched the cell membrane. After the cell membrane was broken by application of additional suction, cell capacitance and series resistance were electrically compensated. After access was gained in the whole-cell voltage-clamp configuration, cardiomyocytes were allowed to equilibrate for 5 min. with the internal solution before data were collected. For details on patch clamp, see Supporting Information.

Data analysis
Data are presented as the mean Ϯ S.E.M. Differences were evaluated using the unpaired Student's t-test, and P Ͻ 0.05 was considered to be statistically significant.

MH induces hypertrophy in cultured cardiomyocytes of neonatal rats
Neonatal rat cardiomyocytes were exposed to MH for 0 (control), 1, 3, 6 or 12 hrs. Morphology changes for hypertrophy in cardiomyocytes were determined by ␣-actinin staining. In order to confirm cardiac hypertrophy, three markers, ANP, BNP and ␤-MHC, were also detected by Western blotting and real-time PCR. Figure 1A showed typical morphology of hypertrophy in cardiomyocytes exposed to MH for 6 hrs; surface area of cardiomyocytes evaluated by Imagepro-Plus software in cardiomyocytes exposed to MH for 6 and 12 hrs was increased significantly by 1.6-fold and 1.9-fold, respectively, when compared to non-hypoxia control. Moreover, Figure 1B-D showed a time-dependent increase of ANP, BNP and ␤-MHC mRNA and protein expression induced by MH in neonatal rat cardiomyocytes. These results indicate that MH is able to induce hypertrophy in cardiac myocytes.

HIF-1␣ is increased in cultured cardiomyocytes exposed to MH
To investigate whether HIF-1␣ correlates to the development of hypertrophy by MH in cultured cardiomyocytes, we detected the expression of HIF-1␣ mRNA and protein. Immunofluorescence assay showed that an elevated HIF-1␣ protein within nucleus area ( Fig. 2A) in cardiac myocytes exposed to MH for 6 hrs. HIF-1␣ mRNA level (Fig. 2B) and nucleus HIF-1␣ protein (Fig. 2C) were elevated in a time-dependent manner in cardiomyocytes exposed to MH for 1, 3, 6 or 12 hrs. In order to determine reference control proteins are not altered by MH, two reference proteins, GAPDH and ␤-actin, were used as internal control, which did not reflect significant difference under MH in cardiomyocytes; data were shown in Supporting Information (Fig. S1).

HIF-1␣ controls development of hypertrophy induced by MH in cardiomyocytes
In order to investigate whether HIF-1␣ participates in the development of hypertrophy, an HIF-1␣ specific blocker, SC205346, for the loss-of-function and HIF-1␣ transfection for the gain-offunction were used before cardiomyocytes exposed to MH. A successful transfection was demonstrated as shown in Figure 3A and B. ␣-actinin (Fig. 3C) and a statistical graph were shown from cell surface area (Fig. 3D), Figure 3C showed that SC205346 (30 M) prior to MH for 1 hr significantly inhibited cardiomyocytes hypertrophy, while HIF-1␣ overexpression promoted this process. MH-induced ␤-MHC overexpression in cardiomyocytes was abolished by SC205346, but was enhanced by HIF-1␣ transfection (Fig. 3E), and the data are consistent with ␣-actinin staining assay in Figure 3C. Real-time PCR also showed that ANP, BNP and ␤-MHC mRNA up-regulation induced by MH were blocked by SC205346, but enhanced by HIF-1␣ transfection (Fig. 3F). SC205346 as an HIF-1␣ blocker inhibited nuclear HIF-1␣ accumulation in hypoxic cardiomyocytes (Fig. S1). But SC205346 alone did not affect ANP, BNP and ␤-MHC expression when 30 M SC205346 was administered to neonatal cardiomyocytes for 6 hrs (Fig. S2). Interestingly, cardiomyocytes transfected with HIF-1␣ without hypoxia exposure showed an increased expression of ANP, BNP and ␤-MHC (Fig. S3). Taken together, HIF-1␣ controls the process of hypertrophy induced by MH in cardiac myocytes.

TRPC-mediated calcineurin signalling is involved in hypertrophic cardiomyocytes induced by MH
Previous studies have demonstrated up-regulation of TRPC1, TRPC3 and TRPC6 is involved in the development of cardiac hypertrophy in various animal models [11,12,21]; therefore, we detect their expression levels of mRNA and protein by real-time PCR and Western blotting, respectively. Figure 4A and S4A) and an increased calcineurin expression (Fig. S4B) (Fig. 4C). Calcineurin expression in hypertrophic cardiomyocytes was also inhibited by SK&F96365 (Fig. 4D). SK&F96365 also restored p-NFAT expression level, a substrate of calcineurin, induced by MH (Fig. 4E). The expression of modulatory calcineurin interacting protein 1 (MCIP1, also known as reg-ulator of calcineurin) was up-regulated in response to calcineurin activation; therefore, we further measured MCIP1 mRNA as a reflection of endogenous calcineurin activity. As shown in Figure 4F, MCIP1 mRNA expression was significantly increased in MH exposed cardiomyocytes, and the increased MCIP1 mRNA expression was inhibited by SK&F96365 treatment. These results were consistent with the previous report [22]. In addition, SK&F96365 also prevented MH-induced hypertrophic morphology in cardiomyocytes (Fig. 4G) and blocked MH-induced hypertrophy-related genes expression, for instance, ANP, BNP and ␤-MHC (Fig. 4H). Together, it suggests that TRPC-mediated Ca 2ϩ -calcineurin signalling participates in the cardiac hypertrophy induced by MH, but whether TRPC is regulated by HIF-1␣ needs to be further investigated. ␣-actinin immunostaining images by confocal microscope (original magnification, ϫ600) in cardiac myocytes exposed to MH (10% O2) for 6 hrs, and statistical graph showed calculated surface area from 40 random cardiac myocytes exposed to MH for 1, 3, 6 and 12 hrs, respectively. ANP (B), BNP (C) and ␤-MHC (D) protein and mRNA expression were also measured in cardiomyocytes exposed to MH for 1, 3, 6 or 12 hrs. *P Ͻ 0.05 versus Ctrl; n ϭ 5 independent experiments for each condition.

SC205346, as specific HIF-1␣ blocker, prevents increase of TRPC3 and TRPC6 expression and inhibits elevation of Ca 2؉ -calcineurin signals under MH condition
In order to determine whether TRPC-mediated cardiomyocyte hypertrophy is through HIF-1␣, neonatal rat cardiac myocytes were treated with 30 M SC205346 for 1 hr prior to MH stimuli. Results showed that MH-induction could increase TRPC3 and TRPC6 mRNA (Fig. 5A) and protein expression levels ( Fig. 5B and  C), which could be completely suppressed by HIF-1␣ blocker, SC205346. It also showed that the elevation of [Ca 2ϩ ]i (Fig. 5E) and enhanced expression of calcineurin (Fig. 5D) caused by MH in cardiomyocytes were also blocked by SC205346. But SC205346 alone did not affect TRPC3 and TRPC6 expression (Fig. S5A (Fig. 6A) and protein (Fig. 6B and C) 6E) and calcineurin up-regulation (Fig. 6D) by MH in cardiomyocytes. Interestingly, HIF-1␣-overexpressed cardiomyocytes under normal oxygen condition also caused an up-regulation of TRPC3 or TRPC6 protein (Fig. 7C), suggesting a tight regulatory role of HIF-1␣ in TRPC expression.

Discussion
Many efforts have been made to examine an important role of hypoxic stress in the development of cardiac hypertrophy, but the initial molecule responded to oxygen still needs to be clarified. The established studies of HIF-1␣ in oxygen-sensing responses to hypoxia set up the milestone in the field. A major advance in the understanding of oxygen-sensing processes and the mechanisms which indicates the cellular and tissular responses to hypoxia came with the discovery of HIF-1␣. Here, we demonstrate that MH can induce hypertrophy in cultured neonatal rat cardiac myocytes. MH stimulates an increase of HIF-1␣ mRNA and HIF-1␣ protein in nuclei. MH up-regulates TRPC3 and TRPC6, but not TRPC1 mRNA and protein expression, and enhances Ca 2ϩ -calcineurin signals. We further demonstrate HIF-1␣ is involved in up-regulation of TRPC3 and TRPC6 expression and elevation of Ca 2ϩ -calcineurin signals under MH condition. These results suggest HIF-1␣ plays a critical role in hypoxic adaptive cardiomyocytes hypertrophy, which gives a clue for future study in vivo.
Concerning the effect of hypoxia on HIF-1␣ expression, it varies in different cell lines and the degree of hypoxia. Hypoxia at 1% O2 induces HIF-1␣ protein accumulation after 4 hrs followed by a strong desensitization (loss of the protein) after 24 hrs to 7 days in Hela cells [23]. Belaiba et al. [24] demonstrate that HIF-1␣ mRNA level is increased in response to hypoxia (1% O2) within 0.5 hr, peaking at 1 hr and returning to basal levels after 4 hrs of hypoxic stimuli, and HIF-1␣ protein levels are rapidly increased in response to hypoxia after 0. 5 [25]. In U87 MG glioblastoma cells, MH (10% O2) causes an increase of HIF-1␣ expression at the time point of 1, 6 and 18 hrs [26]. These data are consistent with our results. In the present study, when neonatal cardiac myocytes are exposed to MH (10% O2) for 1, 3, 6 or 12 hrs, HIF-1␣ mRNA and protein levels increase in a time-dependent manner, and reach the peak at 6 hrs. In addition, hypertrophy-related proteins, ANP, BNP and ␤-MHC, are also increased when the cardiomyocytes are exposed to MH for 6 hrs. With the observation of the persistent and significant changes of HIF-1␣ and hypertrophy-related proteins at this time point, we apply 6-   The present study also shows that SC205346, a specific HIF-1␣ blocker, could abolish up-regulation of TRPC3 and TRPC6 expression, but overexpression of HIF-1␣ promotes up-regulation of TRPC3 and TRPC6 expression in cardiomyocytes exposed to MH. Most importantly, patch clamp data indicate that HIF-1␣ up-regulated a functional channel formed by TRPC3 and TRPC6 occurred only in HIF-1␣ overexpression cells. One important report has shown that HIF-1 mediates hypoxia-induced TRPC6 expression in pulmonary arterial smooth muscle cells [36], which is consistent with our data. These evidences suggest HIF-1␣ is able to regulate TRPC protein expression and function, but the detailed mechanism has not been elucidated in this study. We predict the promoter regions of rat and mouse genes encoding TRPC3 and TRPC6 may contain HIF-1␣ binding sites. It is possible that HIF-1␣ regulated the expression of TRPC3 and TRPC6 through some intermediate factors, for instance, some evidence has shown that mild hypoxia induces endothelin-1 (ET-1) expression, which is able to activate TRPC3 and TRPC6 channel current [3,37,38].

hr and remained elevated for up to 8 hrs in pulmonary artery smooth muscle cells. In Hela cells, MH (10% O2) for 30 min. is able to induce an increase of HIF-1␣ protein
In summary, when cardiomyocytes are exposed to MH, cardiac hypertrophy starts to occur, HIF-1␣ plays a critical role in the development of cardiomyocytes hypertrophy. MH