Sprague-Dawley (SD) rats (1-3-day-old, n = 3) supplied by Shanghai SLAC Laboratory Animal Co., Ltd (Certificate No. SCXK (Hu) 2017-0005, Shanghai, China) were raised under a 12-hour-light/-dark cycle with 22 $\pm$ 2 °C and 45–50% humidity. Food and water that were freely supplied. The study was approved by the Animal Experimentation Ethics Committee of Zhejiang Eyong Pharmaceutical Research and Development Center (No. SYXK (Zhe) 2021-0003, Hangzhou, China).

The rats were euthanized using excessive pentobarbital sodium, and then the ventricular tissues of rats were collected, chopped on ice and digested with type II collagenase (1148090, Merck, Darmstadt, Germany) to obtain a cell suspension. High-and low-density Percoll (P4937, Merck, Darmstadt, Germany) were prepared in advance. Low-density Percoll (0.407 g/mL) was added slowly over high-density Percoll (1.017 g/mL), and the cell suspension was added over Percoll and centrifuged at 1700 g for 30 minutes. The cells on the upper layer of the centrifuge tube were fibroblasts, and the cells on the lower layer were cardiomyocytes. The cardiomyocytes were cultured in rat cardiomyocytes complete medium (CM-R073, Procell, Wuhan, Hubei, China), and the fibroblasts were cultured in rat myocardial fibroblast complete medium (CM-R074, Procell, Wuhan, Hubei, China). All cells were maintained in a humidified atmosphere at 37 °C with 5% CO${}_2$. After the cells were cultured for 4 hours, 24 hours, 48 hours and 72 hours, the morphology and growth of the cells were observed using an optical microscope (AE2000, Motic, Shenzhen, Guangdng, China).

The second generation of cardiomyocytes and fibroblasts were inoculated into a 6-well plate with cell slides. After 48 hours of incubation, the cells were fixed with 4% paraformaldehyde (abs9179, absin, Shanghai, China) and then reacted with 0.3% Triton-X 100 (A1009, Applygen, Beijing, China) for 3 minutes. Cells were blocked with 3% BSA (abs9157, absin, Shanghai, China) and incubated with primary antibody overnight at 4 °C and with the secondary antibody at room temperature for 1 hour the following day. Cells were then sealed with antifade mounting solution with DAPI (C1211, Applygen, Beijing, China). The final results were observed under a fluorescence microscope (Ts2-FC, Nikon, Tokyo, Japan). Information on the antibodies is shown in Table 1.

Rat BMSCs were purchased from Procell Life Science&Technology Co.,Ltd (CP-R131, Wuhan, Hubei, China). Rat BMSCs were cultured in a complete medium (CM-R131, Procell, Wuhan, Hubei, China) and maintained in a humidified atmosphere at 37 °C with 5% CO${}_2$.

The gene sequence of rat PDE-5 was obtained from the NCBI database and then amplified by polymerase chain reaction (PCR). The amplified gene sequence was inserted into the pRRL WS1.6 GFP vector (K4820-01, Invitrogen, Waltham, MA, USA), named overexpression (oe)-PED-5, and the empty vector was regarded as a negative control (NC). The PDE-5 specific short hairpin RNA (shRNA) was synthesized by VectorBuilder (Guangzhou, Guangdong, China) with the target sequence (5${}^{\prime }$-GACAGCTCTAAAGACAAATTT-3${}^{\prime }$). BMSCs (5 $\times$ 10${}^5$/well) were inoculated into a six-well plate and cultured routinely for 24 hours. The vectors or shRNA were transfected into cells by lipofectamin2000 (11668-019, Invitrogen, Waltham, MA, USA). After 5 hours, the cell culture medium was replaced with the complete medium and the conventional culture was continued for 24 hours. The interference efficiency of oe-PED-5 was then detected by quantitative reverse transcription-PCR (qRT-PCR).

Cardiomyocytes or fibroblasts, were divided into 4 groups (control group, model group, PDE-5 OE group and PDE-5 shRNA group). Cardiomyocytes or fibroblasts (1 $\times$ 10${}^5$ cells/well) were inoculated in a culture dish containing normal glucose (NG, 5.5 mmol/L) or HG (30 mmol/L) for 48 hours, and then transfected BMSCs (1 $\times$ 10${}^5$ cells/well) were inoculated on a transwell insert placed above the culture dish to form a co-culture system [13]. In the control group, cardiomyocytes or fibroblasts cultured in NG were co-cultured with BMSCs transfected with NC. In the model group, cardiomyocytes or fibroblasts cultured in HG were co-cultured with BMSCs transfected with NC. In the PDE-5 OE group, cardiomyocytes or fibroblasts cultured in HG were co-cultured with BMSCs transfected with PDE-5 OE. In the PDE-5 shRNA group, cardiomyocytes or fibroblasts cultured in HG were co-cultured with BMSCs transfected with PDE-5 shRNA.

Cell counting kit 8 (CCK8, C0005, Topscience, Shanghai, China) was used to detect the cell viability. Cardiomyocytes and fibroblasts in logarithmic phase were taken and prepared into a 3 $\times$ 10${}^4$/mL cell suspension, which was then added into a 96-well plate at 100 µL per well. After 24, 48 or 72 hours of cell culture, 10 µL of CCK8 reagent was added directly to the cell culture medium and the incubation was continued for 2 hours. The absorbance at 450 nm was determined with a microplate reader (CMaxPlus, MD, San Jose, CA, USA) and cell viability was calculated according to the instructions.

Rat collagen (col)-I (ml058800, Mlbio, Shanghai, China), col-III (MM-0765R2, Meimian, Yancheng, Jiangsu, China), tissue inhibitor of metalloproteinase (TIMP)-1 (ml003040, Mlbio, Shanghai, China), desmin (MM-70789R2, Meimian, Yancheng, Jiangsu, China), Matrix metalloproteinase (MMP)-1 (ml002968, Mlbio, Shanghai, China) and troponin (Tn)-I (ml003202, Mlbio, Shanghai, China) ELISA kits were used to determine the cytokine levels in rat cardiomyocytes and fibroblasts. Briefly, the cells were centrifuged at 1000 g for 10 minutes to obtain a supernatant. The standards and cell supernatants were diluted and added to the ELISA plates for incubation for 30 minutes. The biotin-labeled antibody was used to unbind the antigen in the cell supernatant, followed by binding to biotin by affinity streptavidin–horseradish Peroxidase (HRP). After the color was developed by the substrate developing solution, the reaction was stopped. The absorbance at 450 nm was determined with a microplate reader and the cytokines levels were calculated according to the instructions.

The Annexin V-FITC/PI Apoptosis Detection Kit (556547, BD, Franklin Lakes, NJ, USA) was used to determine the apoptosis of rat cardiomyocytes and fibroblasts. The cells were collected and the cell concentration was adjusted to 1 $\times$ 10${}^6$ cells /mL. The cell suspension was mixed with binding buffer, and then stained with Annexin V-FITC and PI at room temperature in the dark. Then apoptosis was detected by flow cytometry (C6, BD, Franklin Lakes, NJ, USA).

Total protein was extracted from rat cardiomyocytes and fibroblasts by the Protein Extraction Kit (G2002, Servicebio, Wuhan, Hubei, China). Then the protein was quantified by the BCA kit (T14514, Topscience, Shanghai, China) was separated by SDS-PAGE gel and electroblotted to the PVDF membrane (WJ001, Epizyme, Cambridge, MA, USA). After blocking, the membrane was probed with the primary antibody and the secondary antibody which are shown in Table 1. Finally, the protein signals were detected by the ECL luminescence reagent (SQ202, Epizyme, Cambridge, MA, USA) using the Imaging System (610020-9Q, Qinxiang, Shanghai, China). $\beta$-actin was used as the internal control.

Total RNA was extracted from rat cardiomyocytes and fibroblasts by the Total RNA Purification Kit (EZB-RN4, HiFunBio, Shanghai, China). Then the reverse transcription and qPCR was conducted by the cDNA Synthesis Kit (EZB-RT2, HiFunBio, Shanghai, China) and SYBR Green qPCR Mix (A0001, HiFunBio, Shanghai, China) on a Real-Time PCR system (CFX Connect, Bio-rad, Hercules, CA, USA). The mRNA levels were normalized to $\beta$-actin by the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }}$^CT approach [14]. The thermal cycling conditions were as follows: 95 °C for 10 minutes followed by 40 cycles of 95 °C for 15 s, 60 °C for 60 s. The sequences of the primers are listed in Table 2.

Data are presented as mean $\pm$ SD. One-way analysis of variance (ANOVA) was used for the comparison among multiple groups, and pairwise comparison between groups was conducted by the Turkey test. Data with normal distribution and homogeneity test of variance were analyzed by the SNK test. Dunnett’s T3 test or independent sample T test was used for heterogeneity of variance. Kruskal-Wallis H test was used for the measurement data that did not conform to normal distribution. All statistical analyses were performed with SPSS 16.0 (IBM Corp., Armonk, NY, USA). A p 0.05 was considered statistically significant.

As shown in Fig. 1A, cardiomyocytes and fibroblasts began to adhere to the wall after 4 hours of culture, and their shapes gradually changed from round to spindle. After 24 hours of culture, cardiomyocytes began to extend out from pseudopodia, showing a triangular or polygonal shape. After 72 hours of culture, cardiomyocytes were interconnected and interwoven into a network, forming single-layer cardiomyocytes or cell clusters. After 24 hours and 48 hours of culture, fibroblasts also showed fusiform or polygonal shape, and scattered growth. After 72 hours of culture, cells proliferated faster, grew and gathered, and showed spiral and radial shapes. Cardiomyocytes specifically express Cardiac troponin I, Nkx2.5 and fibroblasts specifically express Vimentin, $\alpha$-SMA both of which are localized in the cytoplasm. The cells have been identified by immunofluorescence with a purity of over 90% which was listed in Table 3. We were able to successfully isolate rat cardiomyocytes and fibroblasts as shown in the results in Fig. 1B,C.

Fig. 1.

Isolation and identification of neonatal rat primary cardiomyocytes and fibroblasts. (A) The morphology of rat primary cardiomyocytes and fibroblasts cultured for 4 hours, 24 hours, 48 hours and 72 hours was observed under inverted microscope (magnification, 100 $\times$). (B) The expression of Cardiac troponin I and Nkx2.5 in rat myocardial cells (magnification, 200 $\times$). (C) The expression of Vimentin and $\alpha$-SMA in rat fibroblasts (magnification, 200 $\times$).

We determined the overexpression efficiency of PDE-5 by qRT-PCR and showed that the expression of PDE-5 significantly increased after BMSCs were transfected with PDE-5 overexpression plasmid (Fig. 2A, p 0.01). The PDE-5 gene expression levels are reduced in BMSCs after transfection with PDE-5 shRNA (Fig. 2B, p 0.01). We then determined the effects of BMSCs overexpressing or silencing PDE-5 on the biological behavior of cardiomyocytes and fibroblasts through a co-culture system. Compared with the control group, the viability of cardiomyocytes in the model group, the PDE-5 OE group, and the PDE-5 shRNA group was significantly lower at 24 hours, 48 hours and 72 hours (Fig. 2C, p 0.05 or p 0.01), while the viability of fibroblasts in the model group and the PDE-5 OE group was facilitated at 24 hours, 48 hours and 72 hours (Fig. 2D, p 0.05). However, the viability of fibroblasts in the PDE-5 shRNA group was decreased at 24 hours, 48 hours and 72 hours (p 0.05). Compared with the model group, PDE-5 silencing promoted the viability of cardiomyocytes, but inhibited the viability of fibroblasts (p 0.05 or p 0.01), while PDE-5 OE did not affect the viability of fibroblasts or cardiomyocytes (p $>$ 0.05).

Fig. 2.

The effects of PDE-5 on the viability of rat fibroblasts and cardiomyocytes. The transfection efficiency of PDE-5 overexpression plasmid (A) and the PDE-5 sh-RNA (B) in rat BMSCs (mean $\pm$ SD, n = 3). ${}^{\mathrm{▲}\mathrm{▲}}$p 0.01, ${}^{\mathrm{▲}}$indicates vs. NC group. CCK-8 was used to detect the activity of cardiomyocytes (C) and fibroblasts (D) in each group at 24 hours, 48 hours and 72 hours. ${}^{\mathrm{▲}}$p 0.05, ${}^{\mathrm{▲}\mathrm{▲}}$p 0.01; ${}^{\mathrm{★}}$p 0.05, ${}^{\mathrm{★}\mathrm{★}}$p 0.01 (${}^{\mathrm{▲}}$indicates vs. control group; ${}^{\mathrm{★}}$indicates vs. model group).

After HG culture, the contents of Col-I, Col-III, TIMP-1 and Desmin secreted by fibroblasts gradually increased, while MMP-1 secreted by fibroblasts and Tn-I secreted by cardiomyocytes decreased in a time-dependent manner (Fig. 3A–F, p 0.05 or p 0.01). Of interest, PDE-5 shRNA suppressed the secretion of Col-I, Col-III, TIMP-1 and Desmin in fibroblasts and elevated the secretion of MMP-1 in fibroblasts and Tn-I in cardiomyocytes (Fig. 3A–F, p 0.05), while PDE-5 OE did not affect the levels of these cytokines (p $\mathrm{>}$ 0.05).

Fig. 3.

The effects of PDE-5-modified MSCs on the contents of Col-Ⅰ, Col-III, Desmin, MMP-1, TIMP-1 and Tn-I. (A–E) ELISA was used to detect the contents of Col-Ⅰ, Col-III, Desmin, MMP-1, and TIMP-1 in the supernatant of rat fibroblasts. (F) ELISA was used to detect the contents of Tn-I in the supernatant of rat cardiomyocytes (mean $\pm$ SD, n = 6). ${}^{\mathrm{▲}}$p 0.05, ${}^{\mathrm{▲}\mathrm{▲}}$p 0.01; ${}^{\mathrm{★}}$p 0.05 (${}^{\mathrm{▲}}$indicates vs. control group; ${}^{\mathrm{★}}$indicates vs. model group).

HG culture increased the apoptosis of rat fibroblasts and cardiomyocytes compared to the normal group cells (Fig. 4A,B, p 0.01), while these trends were reversed by PDE-5 shRNA (Fig. 4A,B, p 0.05). However, PDE-5 OE did not affect the apoptosis of HG-induced rat fibroblasts and cardiomyocytes (p $>$ 0.05).

Fig. 4.

The effects of PDE-5-modified MSCs on apoptosis of fibroblasts and cardiomyocytes. (A,B) The apoptosis of fibroblasts and cardiomyocytes in each group was determined by flow cytometry (mean $\pm$ SD, n = 3). ${}^{\mathrm{▲}\mathrm{▲}}$p 0.01; ${}^{\mathrm{★}}$p 0.05 (${}^{\mathrm{▲}}$indicates vs. control group; ${}^{\mathrm{★}}$indicates vs. model group).

As shown in Figs. 5A–D,6A,B, we found that the protein and mRNA levels of PDE-5 were increased (p 0.05), while the protein and mRNA levels of cGMP and PKG were inhibited in untransfected cardiomyocytes and fibroblasts after HG culture (p 0.05 or p 0.01). However, PDE-5 shRNA resulted in down-regulation of PDE-5 levels and up-regulation of cGMP and PKG levels in HG-induced fibroblasts and cardiomyocytes (Figs. 5A–D,6A,B, p 0.05 or p 0.01). PDE-5 OE only elevated the PDE-5 levels in HG-induced fibroblasts and cardiomyocytes (Figs. 5A–D,6A,B, p 0.05).

Fig. 5.

The effects of PDE-5-modified MSCs on the protein levels of PDE-5, cGMP and PKG in fibroblasts and cardiomyocytes. Western blot was used to detect the protein levels of PDE-5, cGMP and PKG in rat cardiomyocytes (A,B) and fibroblasts (C,D). $\beta$-actin was used as internal control (mean $\pm$ SD, n = 3). ${}^{\mathrm{▲}}$p 0.05, ${}^{\mathrm{▲}\mathrm{▲}}$p 0.01; ${}^{\mathrm{★}}$p 0.05, ${}^{\mathrm{★}\mathrm{★}}$p 0.01 (${}^{\mathrm{▲}}$indicates vs. control group; ${}^{\mathrm{★}}$indicates vs. model group).

Fig. 6.

The effects of PDE-5-modified MSCs on the mRNA levels of PDE-5, cGMP and PKG in fibroblasts and cardiomyocytes. qRT-PCR was used to detect the mRNA levels of PDE-5, cGMP and PKG in rat cardiomyocytes (A) and fibroblasts (B). $\beta$-actin was used as internal control (mean $\pm$ SD, n = 3). ${}^{\mathrm{▲}}$p 0.05, ${}^{\mathrm{▲}\mathrm{▲}}$p 0.01; ${}^{\mathrm{★}}$p 0.05, ${}^{\mathrm{★}\mathrm{★}}$p 0.01 (${}^{\mathrm{▲}}$indicates vs. control group; ${}^{\mathrm{★}}$indicates vs. model group).