Upregulation of Reg IV and Hgf mRNAs by Intermittent Hypoxia via Downregulation of microRNA-499 in Cardiomyocytes

Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), and is a risk factor for cardiovascular disease (CVD) and insulin resistance/Type 2 diabetes. However, the mechanisms linking IH stress and CVD remain elusive. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes to experimental IH or normoxia for 24 h to analyze the mRNA expression of several cardiomyokines. We found that the mRNA levels of regenerating gene IV (Reg IV) and hepatocyte growth factor (Hgf) in H9c2 and P19.CL6 cardiomyocytes were significantly increased by IH, whereas the promoter activities of the genes were not increased. A target mRNA search of microRNA (miR)s revealed that rat and mouse mRNAs have a potential target sequence for miR-499. The miR-499 level of IH-treated cells was significantly decreased compared to normoxia-treated cells. MiR-499 mimic and non-specific control RNA (miR-499 mimic NC) were introduced into P19.CL6 cells, and the IH-induced upregulation of the genes was abolished by introduction of the miR-499 mimic, but not by the miR-499 mimic NC. These results indicate that IH stress downregulates the miR-499 in cardiomyocytes, resulting in increased levels of Reg IV and Hgf mRNAs, leading to the protection of cardiomyocytes in SAS patients.


Introduction
Sleep apnea syndrome (SAS) is a common disorder characterized by repetitive episodes of oxygen desaturation during sleep, the development of daytime sleepiness, and the deterioration of the patient's quality of life [1,2]. SAS leads to intermittent hypoxia (IH) [3,4], hypercapnia, and subsequent reoxygenation, as well as disruption of sleep architecture such as sleep fragmentation. SAS has been reported to affect middle-aged and older individuals, with the prevalence estimated to be around 22% in men and 17% in women [5]. SAS is associated with many systemic complications, such as obesity; type 2 diabetes [6,7]; dyslipidemia [8]; cardiovascular diseases, including hypertension, coronary disease, heart failure, and stroke [9][10][11]; pulmonary hypertension [12]; neurocognitive deficits [13,14]; depression [15]; and impaired memory [16].
Observational studies have indicated that SAS is associated with a high risk of serious cardiovascular disease (CVD), including sudden death, atrial fibrillation, stroke, and coronary artery disease, leading to heart failure. It has been reported that SAS is a major independent risk factor for CVD, such as systemic and pulmonary hypertension, congestive heart failure, and stroke [17], as well as myocardial infarction, cerebrovascular dysfunction, and idiopathic sudden death [9]. IH-induced cardiomyocyte damage occurs with the increases of intracellular reactive oxygen species during reoxygenation following hypoxia [18][19][20]. Moreover, IH may cause lipid peroxidation [21], protein oxidation, DNA damage [22], and attenuation of antioxidant enzyme capacity, thus reducing cardiomyocyte numbers by cell death [23]. The prevalence of SAS in patients with heart failure ranges from 15% to 59%, and the mortality rate of patients with severe SAS is significantly high [24][25][26][27]. In addition, cardiac function is impaired with left ventricular hypertrophy in obese patients with severe SAS [28]. Hypertension, cardiac remodeling, and other complications of SAS have been studied using rodent models of IH [29].
In this study, we investigated the direct effect of IH, a hallmark of SAS, using rat and mouse cardiomyocytes and an in vitro IH system. For in vitro IH, nitrogen and oxygen are delivered by a controlled system that regulates the flow of gases. We investigated the direct effect of IH on the gene expression of cytokines and cardiac protective/regenerative factors, such as regenerating gene (Reg) family genes and hepatocyte growth factor (Hgf ). Significant increases in the mRNA levels of Reg IV and Hgf, which both generate growth factors with proliferative and anti-apoptotic effects, were detected in rat and mouse cardiomyocytes in response to IH treatment via the downregulation of microRNA (miR)-499.

Gene Expressions of Reg IV and Hgf Were Increased by IH in Cardiomyocytes
We exposed rat H9c2 cardiomyocytes and cardiomyocytic differentiated mouse P19.CL6 cells to normoxia, IH, or sustained hypoxia (SH) for 24 h. After the treatment, we measured the mRNA levels of cardiomyocytic inflammation related interleukin genes, chemokine genes, cytokine genes), genes of cardiomyocytic growth/regeneration factors and receptors, and genes of cardiomyocyte functioning: interleukin (Il)-6, Il-17A, Il-18, Il-33, transforming growth factor (Tgf)β1, C-C motif chemokine ligand 2 (Ccl2), C-X-C motif chemokine ligand 12 (Cxcl12), tumor necrosis factor-α (Tnfα), vascular endothelial growth factor A (Vegf-A), Fms-like tyrosine kinase 1 (Flt-1: Vegf receptor [Vegfr] 1), fetal liver kinase receptor 1 (Flk-1), cluster of differentiation 38 (Cd38: encoding ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase), Reg I, pancreatitis associated protein (PAP) I, PAP II, PAP III, Reg IV, Exostosin-like 3 (Extl 3)/Reg receptor, Hgf, and tyrosine-protein kinase Met (c-Met: encoding Hgf receptor) in rat H9c2 cells. We measured mRNA levels of Il-6, Il-8, Il-17A, Il-18, Tgf-β1, Ccl2, Cxcl12, Tnfα, Vegf-A, Flt-1, Flk-1, Cd38, Reg I, Reg II, Reg IIIα, Reg IIIβ, Reg IIIγ, Reg IIIδ, Reg IV, Extl3, Hgf, and c-Met in mouse P19.CL6 cells by using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). As shown in Figure 1, significant increases in Tgfβ1, Ccl2, Tnfα, Flt-1, Reg IV, and Hgf were detected in IH-treated rat H9c2 cells. However, Tgf-β1, Ccl2, Tnfα, and Flt-1 were not specifically increased by IH in mouse P19.CL6 cardiomyocytes. In contrast, the mRNAs of Reg IV and Hgf were significantly and specifically increased by IH in mouse P19.CL6 cells ( Figure 2).  I, PAP I, PAP II, PAP III, Reg IV, Extl3, Hgf, and c-Met in rat H9c2 cardiomyocytes. Rat H9c2 cells were treated with normoxia or IH for 24 h. The mRNA levels were measured by real-time RT-PCR and normalized by rat insulinoma gene (Rig)/ribosomal protein S15 (RpS15) as an internal standard. The mRNA levels exposed to normoxia were set to 1.0. Open and closed circles indicate values of relative mRNA expression of cells exposed to normoxia and IH, respectively. Data are expressed as the mean ± SD of the samples. Statistical analyses were performed using Student's t-test. IH significantly increased the mRNA levels of Tgfβ1, Tnfα, Ccl2, Flt-1, Hgf, and Reg IV in rat H9c2 cells. IH significantly decreased the mRNA levels of Il-6, Il-33, Il-18, and Cxcl12 in rat H9v2 cells. The other gene expressions (Il-17A, Cd38, Reg I, PAP I, PAP II, PAP III, Extl3, c-Met, Vegf-A, and Flk-1) did not show significant changes.  I, PAP I, PAP II, PAP III, Reg IV, Extl3, Hgf, and c-Met in rat H9c2 cardiomyocytes. Rat H9c2 cells were treated with normoxia or IH for 24 h. The mRNA levels were measured by real-time RT-PCR and normalized by rat insulinoma gene (Rig)/ribosomal protein S15 (RpS15) as an internal standard. The mRNA levels exposed to normoxia were set to 1.0. Open and closed circles indicate values of relative mRNA expression of cells exposed to normoxia and IH, respectively. Data are expressed as the mean ± SD of the samples. Statistical analyses were performed using Student's t-test. IH significantly increased the mRNA levels of Tgfβ1, Tnfα, Ccl2, Flt-1, Hgf, and Reg IV in rat H9c2 cells. IH significantly decreased the mRNA levels of Il-6, Il-33, Il-18, and Cxcl12 in rat H9v2 cells. The other gene expressions (Il-17A, Cd38, Reg I, PAP I, PAP II, PAP III, Extl3, c-Met, Vegf-A, and Flk-1) did not show significant changes.   The mRNA levels of mouse Il-6, Il-8, Il-17A, Il-18, Tgfβ1, Ccl2, Cxcl12, Tnfα, Vegf-A, Flt-1, Flk-1, Cd38, Reg I, Reg II, Reg IIIα, Reg IIIβ, Reg IIIγ, Reg IIIδ, Reg IV, Extl3, Hgf, and c-Met in mouse P19.CL6 cardiomyocytes. Mouse P19.CL6 cells were treated with normoxia, IH, or SH for 24 h. The mRNA levels were measured by real-time RT-PCR and normalized by Rig/RpS15 as an internal standard. The mRNA levels exposed to normoxia were set to 1.0. Open and closed circled circles indicate values of relative mRNA expression of cells exposed to normoxia and IH, respectively. Data are expressed as the mean ± SD of the samples. Statistical analyses were performed using Student's t-test. IH significantly increased the mRNA levels of Reg IV and Hgf in mouse P19.CL6 cells. SH significantly increased the mRNA levels of Il-6, Il-8, Ccl2, Cxcl12, Tnfα, Reg II, Reg IIIβ, Reg IIIγ, and Reg IIIδ in P19.CL6 cells. IH and/or SH decreased mRNA levels of Cd38, Extl3, Vegf-A, Flt-1, and Flk-1. The other gene expressions (Il-17A, Il-18, Tgfβ1, Reg I, Reg IIIα, and c-Met) did not show significant changes. We further measured Reg IV and Hgf proteins in the culture medium of differentiated P19.CL6 cells by the enzyme-linked immunosorbent assay (ELISA). We found that the levels of Reg IV and Hgf were significantly increased by IH (Reg IV [  .CL6 cardiomyocytes were treated by normoxia or IH condition for 24 h. The concentrations of Reg IV and Hgf were measured by ELISA. Open and closed circles indicate values of culture medium of cells exposed to normoxia and IH, respectively. Data are expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.

Reg IV and Hgf Act as Autocrine/Paracrine Growth and Anti-Apoptotic Factors in SH/IH Condition(s) for Cardiomyocytes
To evaluate the direct effects of Reg IV and Hgf on cardiomyocyte proliferation, differentiated P19.CL6 cells were incubated with Reg IV and Hgf for 24 h. Following the SH treatment, cell viability was determined by using a WST-8 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium monosodium salt) assay. P19.CL6 cell proliferation was significantly increased by 0.1 ng/mL Reg IV ( Figure 4A) and 10-100 ng/mL Hgf ( Figure 4B), and the 0.1 ng/mL Reg IV-induced proliferation was further enhanced by the combined addition of Hgf ( Figure 4B). P19.CL6 cell numbers were significantly increased by IH and dramatically reduced by SH ( Figure 4C).  P19.CL6 cardiomyocytes were treated by normoxia or IH condition for 24 h. The concentrations of Reg IV and Hgf were measured by ELISA. Open and closed circles indicate values of culture medium of cells exposed to normoxia and IH, respectively. Data are expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.

Reg IV and Hgf Act as Autocrine/Paracrine Growth and Anti-Apoptotic Factors in SH/IH Condition(s) for Cardiomyocytes
To evaluate the direct effects of Reg IV and Hgf on cardiomyocyte proliferation, differentiated P19.CL6 cells were incubated with Reg IV and Hgf for 24 h. Following the SH treatment, cell viability was determined by using a WST-8 (2-[2-methoxy-4nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium monosodium salt) assay. P19.CL6 cell proliferation was significantly increased by 0.1 ng/mL Reg IV ( Figure 4A) and 10-100 ng/mL Hgf ( Figure 4B), and the 0.1 ng/mL Reg IV-induced proliferation was further enhanced by the combined addition of Hgf ( Figure 4B). P19.CL6 cell numbers were significantly increased by IH and dramatically reduced by SH ( Figure 4C). We further measured Reg IV and Hgf proteins in the culture medium of differentiated P19.CL6 cells by the enzyme-linked immunosorbent assay (ELISA). We found that the levels of Reg IV and Hgf were significantly increased by IH (Reg IV [30.16 pg/mL vs. 91.83 pg/mL, p = 0.0025], and Hgf [101.9 pg/mL vs. 106.3 pg/mL, p = 0.0046]) ( Figure 3).

Figure 3.
Concentrations of (A) Reg IV and (B) Hgf in a mouse P19.CL6 cardiomyocyte culture medium. P19.CL6 cardiomyocytes were treated by normoxia or IH condition for 24 h. The concentrations of Reg IV and Hgf were measured by ELISA. Open and closed circles indicate values of culture medium of cells exposed to normoxia and IH, respectively. Data are expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.

Reg IV and Hgf Act as Autocrine/Paracrine Growth and Anti-Apoptotic Factors in SH/IH Condition(s) for Cardiomyocytes
To evaluate the direct effects of Reg IV and Hgf on cardiomyocyte proliferation, differentiated P19.CL6 cells were incubated with Reg IV and Hgf for 24 h. Following the SH treatment, cell viability was determined by using a WST- cell proliferation was significantly increased by 0.1 ng/mL Reg IV ( Figure 4A) and 10-100 ng/mL Hgf ( Figure 4B), and the 0.1 ng/mL Reg IV-induced proliferation was further enhanced by the combined addition of Hgf ( Figure 4B). P19.CL6 cell numbers were significantly increased by IH and dramatically reduced by SH ( Figure 4C).  To see why the cell numbers were reduced by SH, we also measured apoptosis of IH/SH-stimulated P19.CL6 cells using the TUNEL (TdT-mediated dUTP nick end labeling) method. We found that SH stimulation significantly increased cell apoptosis compared to normoxia/IH ( Figure 5A) and that the addition of Reg IV and Hgf in the cultured medium in SH significantly reduced the apoptosis ( Figure 5B). were measured by a WST-8 assay. Data are expressed as mean ± SD for each group. The statistica analyses were performed using Student's t-test.
To see why the cell numbers were reduced by SH, we also measured apoptosis o IH/SH-stimulated P19.CL6 cells using the TUNEL (TdT-mediated dUTP nick end label ing) method. We found that SH stimulation significantly increased cell apoptosis com pared to normoxia/IH ( Figure 5A) and that the addition of Reg IV and Hgf in the cultured medium in SH significantly reduced the apoptosis ( Figure 5B).

Figure 5. (A)
Anti-apoptotic effects of Reg IV and Hgf in differentiated P19.CL6 cardiomyocytes i normoxia, IH, or SH. IH did not increase apoptosis and SH increased apoptosis (p < 0.0001 vs normoxia; p < 0.0001 vs. IH). (B) Anti-apoptotic effects of Reg IV and/or Hgf in SH. Apoptosis wa quantified using the TUNEL method. Mouse recombinant Reg IV (0.1 ng/mL) and Hgf (0.1 ng/mL were added to differentiated mouse P19.CL6 cell culture medium and incubated in normoxia, IH or SH for 24 h. Data are expressed as mean ± SD for each group. The statistical analyses were per formed using Student's t-test. We then measured the replicative DNA synthesis of SH-treated P19.CL6 cells by 5 iodo-2′-deoxyuridine (IdU: pyrimidine analog) incorporation. As shown in Figure 6, rep licative DNA synthesis was significantly increased by the addition of Reg IV and/or Hg in SH-treated cardiomyocytes. Figure 6. Replicative DNA synthesis of mouse P19.CL6 cardiomyocytes incubated in SH in the pres ence/absence of Reg IV (0.1 ng/mL) and/or Hgf (0.1 ng/mL). P19.CL6 cells were exposed to SH (1% O2) for 24 h, and replicated DNA synthesis was measured by IdU incorporation. Data were ex pressed as mean ± SD for each group. The statistical analyses were performed using Student's t-tes Figure 5. (A) Anti-apoptotic effects of Reg IV and Hgf in differentiated P19.CL6 cardiomyocytes in normoxia, IH, or SH. IH did not increase apoptosis and SH increased apoptosis (p < 0.0001 vs. normoxia; p < 0.0001 vs. IH). (B) Anti-apoptotic effects of Reg IV and/or Hgf in SH. Apoptosis was quantified using the TUNEL method. Mouse recombinant Reg IV (0.1 ng/mL) and Hgf (0.1 ng/mL) were added to differentiated mouse P19.CL6 cell culture medium and incubated in normoxia, IH, or SH for 24 h. Data are expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.
We then measured the replicative DNA synthesis of SH-treated P19.CL6 cells by 5-iodo-2 -deoxyuridine (IdU: pyrimidine analog) incorporation. As shown in Figure 6, replicative DNA synthesis was significantly increased by the addition of Reg IV and/or Hgf in SH-treated cardiomyocytes. were measured by a WST-8 assay. Data are expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.
To see why the cell numbers were reduced by SH, we also measured apoptosis of IH/SH-stimulated P19.CL6 cells using the TUNEL (TdT-mediated dUTP nick end labeling) method. We found that SH stimulation significantly increased cell apoptosis compared to normoxia/IH ( Figure 5A) and that the addition of Reg IV and Hgf in the cultured medium in SH significantly reduced the apoptosis ( Figure 5B).

Figure 5. (A)
Anti-apoptotic effects of Reg IV and Hgf in differentiated P19.CL6 cardiomyocytes in normoxia, IH, or SH. IH did not increase apoptosis and SH increased apoptosis (p < 0.0001 vs. normoxia; p < 0.0001 vs. IH). (B) Anti-apoptotic effects of Reg IV and/or Hgf in SH. Apoptosis was quantified using the TUNEL method. Mouse recombinant Reg IV (0.1 ng/mL) and Hgf (0.1 ng/mL) were added to differentiated mouse P19.CL6 cell culture medium and incubated in normoxia, IH, or SH for 24 h. Data are expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.
We then measured the replicative DNA synthesis of SH-treated P19.CL6 cells by 5iodo-2′-deoxyuridine (IdU: pyrimidine analog) incorporation. As shown in Figure 6, replicative DNA synthesis was significantly increased by the addition of Reg IV and/or Hgf in SH-treated cardiomyocytes. Figure 6. Replicative DNA synthesis of mouse P19.CL6 cardiomyocytes incubated in SH in the presence/absence of Reg IV (0.1 ng/mL) and/or Hgf (0.1 ng/mL). P19.CL6 cells were exposed to SH (1% O2) for 24 h, and replicated DNA synthesis was measured by IdU incorporation. Data were expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test. Figure 6. Replicative DNA synthesis of mouse P19.CL6 cardiomyocytes incubated in SH in the presence/absence of Reg IV (0.1 ng/mL) and/or Hgf (0.1 ng/mL). P19.CL6 cells were exposed to SH (1% O 2 ) for 24 h, and replicated DNA synthesis was measured by IdU incorporation. Data were expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.
The results fitted well with those of previous papers which reported that Reg protein and Hgf functioned as anti-apoptotic and growth/differentiation factors for cardiomy-ocytes [30][31][32][33][34][35], and that Hgf acts as an anti-apoptotic factor against high concentration Reg-induced apoptosis [36].

The Promoter Activities of Reg IV and Hgf Were Not Increased by IH
To determine whether the IH-induced increases in Reg IV and Hgf mRNAs were caused by the activation of transcription, a 2037 bp fragment containing 2008 bp of the mouse Reg IV promoter was fused to the luciferase gene of pGL4.17. The mouse Reg IV promoter construct and the rat Hgf promoter construct, which had a 1395 bp fragment containing 1336 bp of the rat Hgf promoter inserted into a pGL3-Basic vector [36], were transfected into differentiated P19.CL6 cells. After IH stimulation, the promoter activities of Reg IV and Hgf were measured. We found that Reg IV and Hgf promoter activities were not activated by IH in the differentiated P19.CL6 cells (Figure 7: p = 0.6289 and p = 0.3407, respectively). These results suggested that the gene expression of Reg IV and Hgf in response to IH was not regulated by transcription. The results fitted well with those of previous papers which reported that Reg protein and Hgf functioned as anti-apoptotic and growth/differentiation factors for cardiomyocytes [30][31][32][33][34][35], and that Hgf acts as an anti-apoptotic factor against high concentration Reginduced apoptosis [36].

The Promoter Activities of Reg IV and Hgf Were Not Increased by IH
To determine whether the IH-induced increases in Reg IV and Hgf mRNAs were caused by the activation of transcription, a 2037 bp fragment containing 2008 bp of the mouse Reg IV promoter was fused to the luciferase gene of pGL4.17. The mouse Reg IV promoter construct and the rat Hgf promoter construct, which had a 1395 bp fragment containing 1336 bp of the rat Hgf promoter inserted into a pGL3-Basic vector [36], were transfected into differentiated P19.CL6 cells. After IH stimulation, the promoter activities of Reg IV and Hgf were measured. We found that Reg IV and Hgf promoter activities were not activated by IH in the differentiated P19.CL6 cells (Figure 7: p = 0.6289 and p = 0.3407, respectively). These results suggested that the gene expression of Reg IV and Hgf in response to IH was not regulated by transcription.  [36], were transfected into P19.CL6 cells. After the cells were exposed to either IH or normoxia for 24 h, the cells were lysed, and the promoter activities of Reg IV and Hgf were measured. The promoter activity was normalized for variations in transfection efficiency using β-galactosidase activity as an internal standard. The promoter activities exposed to normoxia were set to 1.0. All data are represented as the mean ± SD of the samples. The statistical analyses were performed using Student's t-test.

The miR-499 Level Was Significantly Decreased by IH
We considered the possible explanation that IH-induced up-regulation of Reg IV and Hgf was controlled post-transcriptionally. Therefore, we searched the targeted miRNA using the MicroRNA.org program (http://www.microrna.org/microrna/home.do, accessed on 29 October 2021), which revealed that Reg IV and Hgf mRNAs have a potential target sequence for miR-499. There were no other miRNA candidates targeting both genes. We measured the miR-499 levels of IH-treated cells by RT-PCR and found that the level was significantly lower than that of normoxia-treated cells (0.3229 folds vs. normoxia, p = 0.0029). The possible reasons as to why the level of miR-499 was decreased by IH include the following: mRNA levels of some enzymes involved in miRNA biosynthesis are influenced by IH, and the level of miR-499 was specifically decreased by IH either via decreased biosynthesis or enhanced degradation. We measured the mRNA levels of ribonuclease type III (Drosha) and endoribonuclease Dicer (Dicer), which are involved in the biosynthesis of miRNAs [37,38] and found that their expression was unchanged by IH (Figure 8: p = 0.2200 and p = 0.1299, respectively).  [36], were transfected into P19.CL6 cells. After the cells were exposed to either IH or normoxia for 24 h, the cells were lysed, and the promoter activities of Reg IV and Hgf were measured. The promoter activity was normalized for variations in transfection efficiency using β-galactosidase activity as an internal standard. The promoter activities exposed to normoxia were set to 1.0. All data are represented as the mean ± SD of the samples. The statistical analyses were performed using Student's t-test.

The miR-499 Level Was Significantly Decreased by IH
We considered the possible explanation that IH-induced up-regulation of Reg IV and Hgf was controlled post-transcriptionally. Therefore, we searched the targeted miRNA using the MicroRNA.org program (http://www.microrna.org/microrna/home.do, accessed on 29 October 2021), which revealed that Reg IV and Hgf mRNAs have a potential target sequence for miR-499. There were no other miRNA candidates targeting both genes. We measured the miR-499 levels of IH-treated cells by RT-PCR and found that the level was significantly lower than that of normoxia-treated cells (0.3229 folds vs. normoxia, p = 0.0029). The possible reasons as to why the level of miR-499 was decreased by IH include the following: mRNA levels of some enzymes involved in miRNA biosynthesis are influenced by IH, and the level of miR-499 was specifically decreased by IH either via decreased biosynthesis or enhanced degradation. We measured the mRNA levels of ribonuclease type III (Drosha) and endoribonuclease Dicer (Dicer), which are involved in the biosynthesis of miRNAs [37,38] and found that their expression was unchanged by IH ( Figure 8: p = 0.2200 and p = 0.1299, respectively).
These results suggest that miR-499 plays a key role in the post-transcriptional regulation of mRNA levels of Reg IV and Hgf. To investigate whether Reg IV and Hgf expression in IH is regulated by miR-499, miR-499 mimic and non-specific control RNA (miR-499 mimic NC) were introduced into differentiated P19.CL6 cells with IH/normoxia exposure, and the mRNA levels of Reg IV and Hgf were measured by real-time RT-PCR. As shown in Figures 9  and 10, we found that IH-induced increases in Reg IV and Hgf mRNAs, and IH-induced increases in Reg IV and Hgf in the culture medium, were abolished by the introduction of the miR-499 mimic, but not by the miR-499 mimic NC. These findings indicate that IH stress downregulated the miR-499 level in cardiomyocytes (Figure 8), and that the levels of Reg IV and Hgf mRNAs were increased via a miR-499 mediated mechanism. The miR-499/mRNA levels exposed to normoxia were set to 1.0. Data are expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.
These results suggest that miR-499 plays a key role in the post-transcriptional regulation of mRNA levels of Reg IV and Hgf. To investigate whether Reg IV and Hgf expression in IH is regulated by miR-499, miR-499 mimic and non-specific control RNA (miR-499 mimic NC) were introduced into differentiated P19.CL6 cells with IH/normoxia exposure, and the mRNA levels of Reg IV and Hgf were measured by real-time RT-PCR. As shown in Figures 9 and 10, we found that IH-induced increases in Reg IV and Hgf mRNAs, and IH-induced increases in Reg IV and Hgf in the culture medium, were abolished by the introduction of the miR-499 mimic, but not by the miR-499 mimic NC. These findings indicate that IH stress downregulated the miR-499 level in cardiomyocytes (Figure 8), and that the levels of Reg IV and Hgf mRNAs were increased via a miR-499 mediated mechanism. The miR-499/mRNA levels exposed to normoxia were set to 1.0. Data are expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.  U6 (for miR-499) and Rig/RpS15 (for Dicer and Drosha) as endogenous controls. The miR-499/mRNA levels exposed to normoxia were set to 1.0. Data are expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.
These results suggest that miR-499 plays a key role in the post-transcriptional regulation of mRNA levels of Reg IV and Hgf. To investigate whether Reg IV and Hgf expression in IH is regulated by miR-499, miR-499 mimic and non-specific control RNA (miR-499 mimic NC) were introduced into differentiated P19.CL6 cells with IH/normoxia exposure, and the mRNA levels of Reg IV and Hgf were measured by real-time RT-PCR. As shown in Figures 9 and 10, we found that IH-induced increases in Reg IV and Hgf mRNAs, and IH-induced increases in Reg IV and Hgf in the culture medium, were abolished by the introduction of the miR-499 mimic, but not by the miR-499 mimic NC. These findings indicate that IH stress downregulated the miR-499 level in cardiomyocytes (Figure 8), and that the levels of Reg IV and Hgf mRNAs were increased via a miR-499 mediated mechanism. PCR, as described in the Materials and Methods section. The expression of Reg IV and Hgf mRNA were measured by real-time RT-PCR, using Rig/RpS15 as an endogenous control. The mRNA levels exposed to normoxia were set to 1.0. The figure represents (A) Reg IV mRNA expression in miR-499 mimic NC-introduced cells, (B) Reg IV mRNA expression in miR-499 mimic-introduced cells, (C) Hgf mRNA expression in miR-499 mimic NC-introduced cells, and (D) Hgf mRNA expression in miR-499 mimic-introduced cells. Data are expressed as mean ± SD for each group. The statistical analyses were performed using Student's t-test.

Discussion
In this study, we demonstrated that IH exposure induced increases in Reg IV and Hgf mRNA levels, and that Reg IV and Hgf functioned as anti-apoptotic factor(s) in hypoxia (SH/IH)-exposed cardiomyocytes. We further studied the mechanisms by which IH upregulates the mRNA levels of Reg IV and Hgf and found the possibility of post-transcriptional miRNA-regulated mechanisms in which miR-499 is involved.
HGF is well known as a mesenchyme-derived multifunctional protein that plays a critical role in cell survival, proliferation, migration, and differentiation [89]. Earlier

Discussion
In this study, we demonstrated that IH exposure induced increases in Reg IV and Hgf mRNA levels, and that Reg IV and Hgf functioned as anti-apoptotic factor(s) in hypoxia (SH/IH)-exposed cardiomyocytes. We further studied the mechanisms by which IH upregulates the mRNA levels of Reg IV and Hgf and found the possibility of posttranscriptional miRNA-regulated mechanisms in which miR-499 is involved.
HGF is well known as a mesenchyme-derived multifunctional protein that plays a critical role in cell survival, proliferation, migration, and differentiation [89]. Earlier studies demonstrated that the HGF receptor, a receptor tyrosine kinase, encoded by the c-met proto-oncogene, was expressed in various cells of epithelial origin, including the cardiomyocytes [90]. HGF is also shown to promote cardiomyocyte differentiation, proliferation, and regeneration [91], and to protect from myocardial infarction [92] and/or ischemia/reperfusion injury [93]. Post-infarction treatment with HGF improves left ventricular remodeling and heart function [94]. In addition, HGF also improves heart functionality and promotes the proliferation of myocardial progenitor cells in doxorubicin-induced cardiomyopathy [31].
Until now, only a few studies have reported on miR-499 in cardiomyocytes. The miR-499 is reported to be expressed specifically in the heart and skeletal muscles of humans and mice [95][96][97], contributing to the cardiac differentiation of mesenchymal stem cells [98], late-stage cardiomyocyte differentiation [97], and the expression of the voltage-dependent calcium channel β-2 subunit [99]. A number of studies have indicated that miRNAs play a role in the regulation of many biological processes in the cardiomyocytes (migration, cell proliferation, apoptosis, differentiation, etc.).
Reg IV and Hgf were revealed in this study to function as anti-apoptotic/growthpromoting factors in cardiomyocytes, and both Reg IV and Hgf were up-regulated in cardiomyocytes in the IH condition, but not in the SH. This suggests that both Reg IV and Hgf protect cardiomyocytes from cell death/stress due to decreased oxygen concentrations in IH, but not in SH. The possible protection of cardiomyocytes from decreased oxygen concentrations may be achieved by the expression of Reg IV/Hgf or by the inhibition of miR-499.
In conclusion, this study revealed that the gene expressions of Reg IV and Hgf were increased via the downregulation of the miR-499 level in IH-treated cardiomyocytes and that both Reg IV and Hgf acted as anti-apoptotic factors in the cardiomyocytes. It is suggested that, in SAS patients, the upregulation of REG IV and HGF may function against the apoptosis of cardiomyocytes, leading to the maintenance of cardiac functions, and that miR-499 could play a crucial role in the regulation of these gene expressions.

Measurement of Mouse Reg IV and Hgf in Culture Medium by ELISA
Differentiated P19.CL6 cardiomyocytes were exposed to either normoxia or IH for 24 h. The culture medium was collected, and the concentrations of mouse Reg IV and Hgf were measured using the ELISA Kit for mouse Reg IV (Cloud-Clone Corp., Katy, TX, USA) [75] and for mouse Hgf (R&D Systems, Inc., Minneapolis, MN, USA), respectively.

Measurement of Viable Cell Numbers by Tetrazolium Salt Cleavage
P19.CL6 cells differentiated to cardiomyocytes (7.4 × 10 4 cells/100 µL in 96-well plate) were incubated at 37 • C overnight, and the medium was replaced with fresh MEMα + 10% FCS just before the addition of recombinant mouse Reg IV protein (R&D Systems) or mouse Hgf (R&D Systems). After a 24-h treatment with Reg IV or Hgf, the viable cell numbers were determined by a Cell Counting kit-8 (Dojindo Laboratories, Mashiki-machi, Japan), according to the manufacturer's instructions. Briefly, WST-8 solution was added to cells in 96-well plates, and the cells were incubated at 37 • C for 30 min. The optical density of each well was read at 450 nm (reference wave length at 650 nm) using a Sunrise TM microplate reader (Tecan, Männedorf, Switzerland), as described [36,58,75,79,85,108].

Measurement of Apoptosis
P19.CL6 cells (2.5 × 10 4 cells/100 µL in 96-well plate) were incubated and differentiated into cardiomyocytes by incubating with 1% DMSO for 10 days [100]. After the cells were differentiated into cardiomyocytes, they were exposed to normoxia, IH, or SH with/without 0.1 ng/mL recombinant Reg IV (R&D Systems) and 0.1 ng/mL recombinant mouse Hgf (R&D Systems) for 24 h, and apoptosis was detected by the TUNEL method using an apoptosis screening kit (FUJIFILM Wako). The optical density of each well was read at 490 nm (reference wave length at 650 nm) using a Sunrise TM microplate reader (Tecan), as described [36,58,108,110].

Measurement of Replicative DNA Synthesis
IdU solution was added to the culture medium of differentiated P19.CL6 cells (2.0 × 10 4 cells/100 µL in 96-well plate). After 1 h incubation in the presence of recombinant mouse Reg IV (0.1 ng/mL) and/or recombinant mouse Hgf (0.1 ng/mL), IdU incorporation was measured using a DNA-IdU Labeling and Detection kit (Takara Bio) as described [53,108,110]. The optical density of each well was read at 490 nm (reference wave length at 650 nm) using a Sunrise TM microplate reader (Tecan).

Data Analysis
Results are expressed as mean ± SE. Statistical significance was determined by Student's t-test using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data are available on request from the authors.

Conflicts of Interest:
All authors state that they have no conflict of interest.