Myocardin Reverses Hypoxia-Inducible Factor-1α Mediated Phenotypic Modulation of Corpus Cavernosum Smooth Muscle Cells in Hypoxia Induced by Cobalt Chloride

- ¹Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China.
- ²Department of Urology, Guangzhou Eighth People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China.
- ³Department of Urology, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China.
- ⁴Department of Andrology, The Third Affiliated Hospital of Guangdong Medical University, Foshan, Guangdong, China.
Correspondence to: Anyang Wei. Department of Urology, Nanfang Hospital, Southern Medical University, North of Guangzhou Avenue 1838#, Guangzhou 510510, China. Tel: +86-020-62787210, Fax: +86-20-6161763, Email: profwei@126.com

Correspondence to: Haibo Zhang. Department of Urology, Nanfang Hospital, Southern Medical University, North of Guangzhou Avenue 1838#, Guangzhou 510510, China. Tel: +86-020-62787210, Fax: +86-20-6161763, Email: hai516@163.com

Correspondence to: Tao Wang. Department of Andrology, The Third Affiliated Hospital of Guangdong Medical University, No.39 Donghua Road, longjiang town, Shunde District, Foshan 528318, China. Tel: +86-0757-23881523, Fax: +86-757-23881857, Email: job1982@126.com

^*These authors contributed equally to this work as co-first authors.

Received November 14, 2021; Revised December 21, 2021; Accepted December 30, 2021.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

We aimed to investigate the mechanism of phenotypic transformation of corporal cavernosum smooth muscle cells (CCSMCs) under hypoxic conditions in vitro.

Materials and Methods

In this study, a hypoxia model was established using cobalt chloride (CoCl₂). CCSMCs were treated with different concentrations of CoCl₂ for varying time periods, and cell viability was assessed. Hypoxia-inducible factor-1α (HIF-1α), myocardin (Myocd) and phenotypic markers were detected in the CCSMCs. We also transfected the CCSMCs with si-HIF-1α and Ad-Myocd and evaluated the effects on phenotypic modulation of CCSMCs and the relationship between HIF-1α and Myocd was evaluated.

Results

CoCl₂ inhibited the viability of CCSMCs in a dose- and time-dependent manner, and treatment with 300 µM CoCl₂ for 48 hours were the optimal conditions for establishing the hypoxia model. The results showed increased expression levels of HIF-1α and osteopontin and decreased Myocd, alpha-smooth muscle actin, and calponin levels in CCSMCs under hypoxia. HIF-1α knockdown reversed hypoxia-induced phenotypic transformation with elevated Myocd expression. Overexpression of Myocd also reversed the effect of hypoxia on the phenotypic switch, but did not affect HIF-1α expression.

Conclusions

Our findings showed that HIF-1α was involved in the effect of hypoxia induced by CoCl₂ on CCSMC phenotypic modulation, and Myocd overexpression could inhibit this process. Thus, Myocd might be a potential therapeutic target for erectile dysfunction under hypoxia or HIF-1α activation.

Keywords

Hypoxia; Myocardin; Myocytes, smooth muscle; Phenotype

INTRODUCTION

Erectile dysfunction (ED) is the failure to attain or maintain an erection sufficient for satisfactory sexual intercourse [1]. ED can seriously affect the physical and mental health and reduce the quality of life of patients and their sexual partners. Epidemiological data indicate that ED prevalence in men varies between 2% and 74% [2].

ED is common in patients with various hypoxic diseases, especially those with obstructive sleep apnea (OSA), diabetes mellitus (DM), bilateral cavernous nerve injury (BCNI), and chronic obstructive pulmonary disease [3, 4, 5]. OSA is associated with a greater risk of ED, and ED prevalence in patients with OSA varies from 41% to 80% [6]. Moreover, the erectile function significantly improved after treatment with continuous positive airway pressure to improve hypoxia [7]. In men with chronic obstructive pulmonary diseases, the prevalence of ED ranges from 72% to 87%, with a severe disease stage related to poorer erectile function [5]. Currently, there is a global pandemic of coronavirus disease-2019, in which approximately 5% to 8% of infected patients develop hypoxia [8], and men infected with coronavirus disease-2019 can also experience sequelae of sexual dysfunction after recovery [9], demonstrating the potential correlation between hypoxia and ED. Animal studies have also shown that chronic hypoxia can cause ED [10, 11]. Nevertheless, the exact pathogenesis of ED under hypoxia remains poorly understood.

Corporal cavernosum smooth muscle cells (CCSMCs) are the primary functional cells involved in the physiological process of an erection and comprise 42% to 50% of the total cells in the corpora cavernosum [12]. Smooth muscle cells can assume two phenotypes, namely contractile and synthetic phenotype, which can be transformed into each other and exert different effects under diverse environmental stimuli [13]. The biomarkers of contractile phenotype mainly include alpha-smooth muscle actin (α-SMA), calponin, and smoothelin, while synthetic phenotypes often contain osteopontin (OPN) and collagen I [14]. Studies have shown that phenotypic transformation of CCSMCs may be an important pathological basis for ED caused by DM and BCNI in rats [14, 15]. CCSMCs also undergo phenotypic switching under hypoxic conditions [16]. Therefore, reverse phenotypic transformation may be a potentially effective treatment for ED. However, the mechanism of the phenotypic transformation of CCSMCs under hypoxia remains unclear.

Hypoxia-inducible factor-1α (HIF-1α), a key regulator of hypoxia, is vital for maintaining homeostasis and cellular function during hypoxia, and plays a significant role in the phenotypic modulation of CCSMCs [4, 16, 17]. The present study demonstrated that the phenotypic transformation of CCSMCs under the influence of HIF-1α was associated with decreased expression of myocardin (Myocd) [16]. Myocd is an essential factor in maintaining the contractional phenotype and plays a prominent role in the phenotypic transformation of smooth muscle cells [18]. Therefore, HIF-1α possibly participates in the phenotypic switch of CCSMCs under hypoxia by regulating the expression of Myocd. However, whether Myocd can reverse the phenotypic transformation of CCSMCs under hypoxic conditions remains unknown.

In this study, we constructed a hypoxia model of CCSMCs by intervention with cobalt chloride (CoCl₂) and explored the impact of HIF-1α and Myocd on the phenotypic modulation of CCSMCs simulated by CoCl₂ in vitro.

MATERIALS AND METHODS

1. Ethics statement

For animal research, Institutional animal care and use committee (IACUC) approval is required (No. NFYY-2021-0033).

2. Cell isolation and culture

Primary culture CCSMCs were obtained using the explant method as previously described [15]. Immunofluorescence staining confirmed the CCSMCs, and markers such as α-SMA and calponin were used for identification. Subsequently, the second or third passage of CCSMCs was used for further experiments.

3. Hypoxic treatment

CoCl₂ (catalog# MO63103; Sigma, St. Louis, MO, USA) was used to establish an anoxic model of CCSMCs. First, CoCl₂ was dissolved to a concentration of 100 mmol/L in phosphate-buffered saline (PBS) and then diluted to the required experimental concentration in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium.

4. Cell counting kit-8 (CCK-8) assay

The survival of CCSMCs was examined using the CCK-8 (Dojindo, Kyushu Island, Japan) assay. According to the manufacturer’s instructions, the CCSMCs were plated in 96-well plates, incubated overnight, and then treated with CoCl₂ (100, 200, 300, and 400 µM) for 24, 48, and 72 hours after the cells grew adherently. Subsequently, CCK-8 solution was added to a final concentration of 10% and incubated for 2 hours. The absorbance was measured at 450 nm.

5. Cell transfection

RNA interference was used to downregulate HIF-1α translation in this study. First, CCSMCs were cultured at 37℃ in a 5% CO₂ incubator with antibiotic-free DMEM/F12 medium. After that, the HIF-1α siRNAs (50 nmol/L; RiboBio, Guangzhou, China) were diluted in RNase-free water. Cell transfection was carried out using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions at 30% to 50% cell density. CCSMCs were transfected with a Myocd-overexpressing adenovirus (multiplicity of infection=1:50, 1×10¹⁰ pfu/mL; Hanbio Co., Shanghai, China). Knockdown and overexpression efficiencies were assessed using quantitative real-time PCR (qRT-PCR). The culture medium was replaced with normal DMEM/F12, and CCSMCs were exposed to CoCl₂ for 48 hours.

6. Immunofluorescence staining

CCSMCs were seeded and grown on slides. The CCSMCs were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.25% Triton. The slides were then incubated with antibodies against HIF-1α (1:50; Abcam, Boston, MA, USA), Myocd (1:100; Affinity, Cincinnati, Ohio, USA), α-SMA (1:100; Proteintech, Wuhan, China), and calponin (1:100; Santa Cruz, Dallas, TX, USA) at 4℃ overnight. The cells were then incubated with secondary antibodies (1:50; Proteintech) at room temperature for 1 hour. The nuclei of the cells were stained with DAPI (Solarbio, Beijing, China). Finally, the CCSMCs were analyzed using an inverted microscope (Olympus, Tokyo, Japan).

7. qRT-PCR

Total RNA was extracted from CCSMCs and used for qRT-PCR analysis. β-actin served as an internal reference, and the relative expression of target genes was calculated using the following primer sequences:β-actin: 5′-GATCAAGATCATTGCTCCTCCTG-3′ (forward), 5′-AGGGTGTAAAACGCAGCTCA-3′ (reverse); HIF-1α: 5′-GTCTCCATTACCTGCCTCTGAAACTC-3′ (forward), 5′-GTGACTCTGGGCTTGACTCTAACTTC-3′ (reverse); Myocd: 5′-CTTGCAGATGACCTCAACGA-3′ (forward), 5′-TCACGGAAGAATCCATAGGC-3′ (reverse); a-SMA: 5′-TTCAATGTCCCTGCCATGTA-3′ (forward), 5′-CATCTCCAGAGTCCAGCACA-3′ (reverse); Calponin: 5′-ATCATTGGCCTACAGATG-GGC-3′ (forward), 5′-AGCGTGTCACAGTGTTCCAT-3′ (reverse).

8. Western blotting (WB)

WB was performed as previously described [19]. The primary antibodies were as follows: HIF-1α (1:1,000, catalog#ab179483; Abcam); Myocd (1:500, catalog#AF0023; Affinity); a-SMA (1:3,000, catalog#14395-1-AP; Proteintech), calponin (1:400, catalog#Sc-58707; Santa Cruz); OPN (1:500, catalog#25715-1-AP; Proteintech), and β-tubulin (1:20,000, catalog#R66240-1-Ig; Proteintech). Secondary antibodies were purchased from Abcam and diluted to 1:15,000.

9. Statistical analysis

Statistics were performed using GraphPad Prism 8.3 (GraphPad, San Diego, CA, USA). Following the assessment of normal distribution and homogeneity of variance, parametric data sets were analyzed using Student’s t-test or one-way ANOVA. Non-parametric data were analyzed using the Kruskal–Wallis test. Statistical significance was set at p<0.05.

RESULTS

1. Isolation and identification of CCSMCs

Under an inverted phase-contrast microscope, it was observed that a few spindle cells had emerged from the edge of the tissue block during the 4th to 5th day of primary culture (Fig. 1A). On the 2nd day of cell subculture, it was observed that the cells adhered to the wall and grew well; most of them were long spindles. When the growth was dense, spindle or long spindle cells were interwoven into a network. The second-generation cells were selected for immunofluorescence identification and observed under an inverted fluorescence microscope (Fig. 1B). All nuclei in the field showed blue fluorescence, and anti-α-SMA and anti-calponin immunofluorescence were positive under a fluorescence microscope, which proved that the isolated, cultured cells were CCSMCs.

Fig. 1
Isolation and identification of primary cultured CCSMCs. (A) Cells grow out from the edge of tissue. Scale bar=200 µm. (B) Immunofluorescence identification of CCSMCs using α-SMA and calponin expression. Scale bar=100 µm. CCSMCs: corporal cavernosum smooth muscle cells, α-SMA: alpha-smooth muscle actin.

2. Establishment of chemical hypoxia model for CCSMCs under CoCl₂

1) Effects of different concentrations of CoCl₂ on CCSMCs activity

CCSMCs were treated with CoCl₂ (0, 100, 200, 300, and 400 µM) for 24, 48, and 72 hours, and the CCK-8 assay results showed that CoCl₂ inhibited cell viability in a time- and concentration-dependent manner. Cell viability did not noticeably decrease after treatment with CoCl₂ for 24 hours and when the concentration was less than 200 µM. Cell viability significantly decreased when the CoCl₂ concentration was over 300 µM and the treatment time was over 48 hours (Fig. 2A).

Fig. 2
Establishment of chemical hypoxia model for CCSMCs under CoCl₂ influence. (A) Cells reacted with different concentrations of CoCl₂ for different durations, and the OD value was measured using the CCK-8 assay. (B) qRT-PCR showed that the mRNA levels of biomarkers related to phenotypic modulation and HIF-1α at different concentrations and time periods. n=3 for each group. Comparing to control group, ^*p<0.05, ^**p<0.01, ^***p<0.001. (C) Microscopic imaging of CCSMCs under normoxia and hypoxia (treated for 48 h by 300 µM CoCl₂). Scale bar=100 µm. CCSMCs: corporal cavernosum smooth muscle cells, CoCl₂: cobalt chloride, OD: optical density, HIF-1α: hypoxia-inducible factor-1α, Myocd: myocardin, α-SMA: alpha-smooth muscle actin.

2) Effects of different concentrations of CoCl₂ on the expression of related genes

According to the cytotoxic effect of CoCl₂, 48 hours was selected as the optimal time for CoCl₂-induced hypoxia. The expression levels of related genes such as HIF-1α, α-SMA, calponin, and Myocd were detected at 0, 100, 200, and 300 µM. According to the qRT-PCR results, the expression levels of HIF-1α were significantly increased at 300 µM CoCl₂ and the expression levels of Myocd, α-SMA, and calponin were significantly decreased (Fig. 2B).

In conclusion, treatment with 300 µM CoCl₂ for 48 hours was selected as the optimal condition to induce hypoxia in CCSMCs for subsequent experiments.

3) Effect of hypoxia on morphology of CCSMCs

Most CCSMCs cultured under normoxic conditions were in the shape of a long spindle, and few cells were oval-shaped. The size of the cell population was relatively uniform, with clear contours of cell edges and clearly discernible myofilament structures, which were distributed along the longitudinal axis of the cells. After treatment with CoCl₂ for 48 hours, the morphology of the CCSMCs changed, and mainly manifested as cell hypertrophy and exhibited rounding, uneven cell population size, enlarged nucleus, loss of myofilaments, and decreased cell number (Fig. 2C).

3. Phenotypic modulation of CCSMCs under hypoxia

Immunofluorescence of CCSMCs showed that the fluorescence intensity of Myocd and α-SMA was downregulated, and the fluorescence intensity of HIF-1α protein was upregulated in hypoxic CCSMCs (Fig. 3A, 3B). WB results showed that Myocd, α-SMA, and calponin expression levels were downregulated in hypoxic CCSMCs, while the expression levels of HIF-1α and OPN were upregulated (Fig. 3C, 3D).

Fig. 3
Phenotypic modulation in CCSMCs under hypoxia. (A) Representative images of immunofluorescence detecting HIF-1α, Myocd and α-SMA. Scale bar=100 µm. (B) Quantitative results of the statistical analysis. (C) Representative western blotting results of HIF-1α, Myocd, α-SMA, calponin, and OPN. (D) Quantitative results of the statistical analysis. n=3 for each group. CCSMCs: corporal cavernosum smooth muscle cells, HIF-1α: hypoxia-inducible factor-1α, α-SMA: alpha-smooth muscle actin, Myocd: myocardin, OPN: osteopontin. ^*p<0.05, ^**p<0.01.

4. Role of HIF-1α in the phenotypic modulation of CCSMCs under hypoxia

CCSMCs were subjected to different conditions: normoxia+si-control; normoxia+si-HIF-1α; hypoxia+si-control; hypoxia+si-HIF-1α. WB results demonstrated that the protein expression of HIF-1α and OPN in the hypoxic transfection group was lower than that in the hypoxic group. In contrast, Myocd, α-SMA, and calponin expression levels in the hypoxia transfection group were higher than those in the hypoxia group. Si-HIF-1α transfection had no significant effect in the normoxic group (Fig. 4).

Fig. 4
HIF-1α knockdown reversed the phenotypic modulation of CCSMCs under hypoxia. (A) Representative western blotting results. (B-F) The relative expressions levels of various proteins in the different groups with western blotting. n=3 for each group. HIF-1α: hypoxia-inducible factor-1α, Myocd: myocardin, α-SMA: alpha-smooth muscle actin, OPN: osteopontin, CCSMCs: corporal cavernosum smooth muscle cells, ns: no significancy. ^*p<0.05, ^**p<0.01.

5. Role of Myocd in the phenotypic modulation of CCSMCs under hypoxia

CCSMCs were subjected to different conditions: normoxia+Ad-vector; normoxia+Ad-Myocd; hypoxia+Ad-vector; hypoxia+Ad-Myocd. WB results showed that OPN protein expression in the hypoxic transfection group was lower than that in the hypoxic group. On the other hand, Myocd, α-SMA, and calponin expression levels in the hypoxia transfection group were higher than those in the hypoxia group, while HIF-1α protein expression levels did not change significantly. Ad-Myocd transfection had the same effect in the normoxic group (Fig. 5).

Fig. 5
Myocd overexpression reversed the phenotypic transition of CCSMCs under hypoxia. (A) Representative western blotting results. (B-F) The relative expressions levels of various proteins in the different groups with western blotting. n=3 for each group. HIF-1α: hypoxia-inducible factor-1α, Myocd: myocardin, α-SMA: alpha-smooth muscle actin, OPN: osteopontin, CCSMCs: corporal cavernosum smooth muscle cells, ns: no significancy. ^*p<0.05, ^**p<0.01, ^***p<0.001.

DISCUSSION

Establishing a cellular hypoxia model is an important method for studying hypoxic diseases at the molecular level. For hypoxia, CCSMCs were cultured in the presence of CoCl₂, and while another way to induce hypoxia was by incubating CCSMCs in a mixture of 1.5% O₂, 5% CO₂, and balanced N₂ [20]. The use of CoCl₂ is a reliable model for the study of hypoxia; furthermore, it is a model that prevents the rapid degradation of HIF-1α by reoxygenation for several hours after removing the medium containing CoCl₂ [21]. At present, there are limited reports on using CoCl₂ to establish chemical hypoxia in CCSMCs, and the concentration adopted is directly borrowed from other cell types, ranging from 100 µM to 300 µM [22, 23]. However, the dosage and time required to establish the chemical hypoxia model differed among different cell types. In this study, using CCK-8 assay and qRT-PCR, we concluded that CCSMCs treated with 300 µM CoCl₂ for 48 hours, could maximize the effect of the drug and ensure a high cell survival rate. In the hypoxia model established under these conditions, HIF-1α protein expression was significantly upregulated, and CCSMCs showed obvious phenotypic transformation and morphological changes. Compared to the conventional hypoxia model established through physical hypoxia reported in the literature [16, 17], the results obtained herein were consistent. This also confirmed the reliability of the CoCl₂-induced chemical hypoxia model in CCSMCs.

HIF-1α is an important transcriptional regulator in hypoxia, which can regulate the expression of hundreds of downstream target genes, leading to a series of hypoxic adaptation responses [24]. However, adaptive responses induced by HIF although protective in many diseases, can be detrimental [25]. HIF-1α can induce phenotypic transformation of CCSMCs by altering the p38 mitogen-activated protein kinase or by influencing the expression of transcription factors such as ELK-1, KLF-4, and Myocd [4, 16]. In this study, we found that the expression levels of Myocd were significantly decreased under hypoxia, and HIF-1α knockdown effectively reversed the hypoxia-induced phenotype modulation of CCSMCs, accompanied by increased expression of Myocd, which is consistent with the findings of other studies [16, 26]. These results suggest that HIF-1α may mediate hypoxia-induced phenotypic transformation of CCSMCs by affecting the expression of Myocd. However, HIF-1α knockdown had little effect on the phenotypic transformation of CCSMCs under normoxic conditions, which may be related to the low expression level of HIF-1α in the normoxic state.

Myocd is a nuclear receptor transcription factor protein, which has high tissue-specific expression in the body and is restricted to the smooth muscle cells of the heart, organs, and blood vessels. Myocd plays an indispensable role in the development of visceral and vascular smooth muscle cells [27]. Binding of Myocd to the serum response factor activates the CArG box and directly promotes the transcription and translation of muscle-specific genes [28]. In this study, we showed that overexpression of Myocd reversed the phenotypic transformation of CCSMCs under hypoxia. Hypoxia plays a central role in the development of diabetes and diabetes complications [25], and the expression level of HIF-1α was significantly increased after BCNI [4]. Previous studies have shown that the phenotypic switch of CCSMCs is involved in the development of ED in rats caused by DM and BCNI, which could be improved by Myocd gene therapy [14, 29, 30]. Therefore, our findings not only further enrich the mechanism of Myocd in the treatment of ED resulting from DM and BCNI, but also extend the possibility of Myocd in the treatment of ED induced by other hypoxia diseases, such as OSA and coronavirus disease-2019. However, there was no significant change in the expression level of HIF-1α when Myocd was overexpressed. The results demonstrated that Myocd may be involved in the downstream signaling pathway of HIF-1α during the phenotypic modulation of CCSMCs promoted by hypoxia, and it is worthy of being verified by further studies.

This study has some limitations. First, the administration of CoCl₂ in vitro could not accurately reflect the complex oxygen changes that occur in vivo under hypoxia, and the cells were also affected by inflammation, osmolar pressure, pH, and other complex factors. In addition, this experiment was only validated at the cellular level, and further studies in animal models are required in the future.

CONCLUSIONS

In this study, CCSMCs were induced by CoCl₂ to establish a chemical hypoxia model. Hypoxia upregulated the expression of HIF-1α in CCSMCs, inhibited the expression of Myocd, and promoted the phenotypic transformation of CCSMCs. These results enhance our understanding of the effects of hypoxia on the phenotypic transformation of CCSMCs, showing that Myocd might be a potential therapeutic target of ED under hypoxia or HIF-1α activation.

Notes

Conflict of Interest:The authors have nothing to disclose.

Funding:This research was supported by grants from the National Natural Science Foundation of China (82171612), the Guangdong Basic and Applied Basic Research Foundation (2020A1515010114), and the Scientific Research Fund of Guangdong Medical University (GDMUZ201812).

Author Contribution:

Conceptualization: AW, HZ.
Data curation: XZ, CL, JF.
Formal analysis: XZ, CL, JF.
Funding acquisition: AW, TW.
Investigation: HZ.
Methodology: HZ, XZ.
Project administration: HZ.
Resources: AW, HZ.
Software: CL, GG.
Supervision: AW, HZ, TW.
Validation: AW, HZ.
Visualization: XZ, CL, GG.
Writing – original draft: XZ, HZ.
Writing – review & editing: all authors.

Data Sharing Statement

The data analyzed for this study have been deposited in HARVARD Dataverse and are available at https://doi.org/10.7910/DVN/GI6ELD.

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1. Zhang HB, Chen FZ, He SH, Liang YB, Wang ZQ, Wang L, et al. In vivo tracking on longer retention of transplanted myocardin gene-modified adipose-derived stem cells to improve erectile dysfunction in diabetic rats. Stem Cell Res Ther 2019;10:208
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Funding Information

National Natural Science Foundation of China
82171612
Basic and Applied Basic Research Foundation of Guangdong Province
2020A1515010114
Scientific Research Fund of Guangdong Medical University
GDMUZ201812

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