Small G protein signaling modulator 3 (SGSM3) knockdown attenuates apoptosis and cardiogenic differentiation in rat mesenchymal stem cells exposed to hypoxia

Abstract

Connexin 43 (Cx43) may be important in cell death and survival due to cell-to-cell communication-independent mechanisms. In our previous study, we found that small G protein signaling modulator 3 (SGSM3), a partner of Cx43, contributes to myocardial infarction (MI) in rat hearts. Based on these previous results, we hypothesized that SGSM3 could also play a role in bone marrow-derived rat mesenchymal stem cells (MSCs), which differentiate into cardiomyocytes and/or cells with comparable phenotypes under low oxygen conditions. Cx43 and Cx43-related factor expression profiles were compared between normoxic and hypoxic conditions according to exposure time, and Sgsm3 gene knockdown (KD) using siRNA transfection was performed to validate the interaction between SGSM3 and Cx43 and to determine the roles of SGSM3 in rat MSCs. We identified that SGSM3 interacts with Cx43 in MSCs under different oxygen conditions and that Sgsm3 knockdown inhibits apoptosis and cardiomyocyte differentiation under hypoxic stress. SGSM3/Sgsm3 probably has an effect on MSC survival and thus therapeutic potential in diseased hearts, but SGSM3 may worsen the development of MSC-based therapeutic approaches in regenerative medicine. This study was performed to help us better understand the mechanisms involved in the therapeutic efficacy of MSCs, as well as provide data that could be used pharmacologically.

Fig 1

Time-dependent differential expression of gap junction factors (Cx43, ZO-1) and SGSM3 in normoxic and hypoxic conditions in rat MSCs as determined by qRT-PCR (A) and immunoblot analysis (B). All qRT-PCR values are shown as the normalized target gene expression level relative to the GAPDH transcript levels, and the data are representative of three independent experiments (A). Band intensity was measured as the area density and analyzed in ImageJ and relative intensity levels indicate protein levels normalized to the β-actin levels (B). Significant differences between the normoxia and hypoxia groups were determined via ANOVA, with p values indicated as *p<0.01 and **p<0.001. N, normoxia; H, hypoxia; NC, negative control cells; KD, knockdown cells.

Fig 2

Effects of Hif1a (A) and Sgsm3 (B) knockdown on Cx43 and SGSM3 expression levels in rat MSCs under normoxic and hypoxic conditions, as measured via immunoblot analysis. Band intensity was measured as the area density and analyzed in ImageJ and relative intensity levels indicate protein levels normalized to the β-actin levels. The data are representative of two independent experiments. Significant differences between groups were determined via ANOVA, with p values indicated as *p<0.05 and **p<0.01. N, normoxia; H, hypoxia; NC, negative control cells; KD, knockdown cells.

Fig 3. Effects of Sgsm3 knockdown on cell death and apoptosis under hypoxia in rat MSCs.

Changes in cell viability (A) and apoptosis marker expression (B) in MSCs with Sgsm3 knockdown under normoxic and hypoxic conditions were measured using Ez-Cytox and immunoblot analysis, respectively. Band intensity was measured as the area density and analyzed in ImageJ and relative intensity levels indicate protein levels normalized to the β-actin levels. The data are representative of two independent experiments. Significant differences between groups were determined via ANOVA, with p values indicated as *p<0.05 and **p<0.01. N, normoxia; H, hypoxia; NC, negative control cells; KD, knockdown cells.

Fig 4. Effects of Sgsm3 knockdown on cardiogenic marker expression in rat MSCs.

Changes in cardiogenic marker expression upon Sgsm3 knockdown in MSCs were measured via immunoblot analysis (A) and immunocytochemical staining (B). Band intensity was measured as the area density and analyzed in ImageJ and relative intensity levels indicate protein levels normalized to the β-actin levels (A). The nuclei were stained with DAPI (B). Scale bar = 200 μm. The data are representative of two independent experiments. Significant differences between groups were determined via ANOVA, with p values indicated as *p<0.05 and **p<0.01. N, normoxia; H, hypoxia; NC, negative control cells; KD, knockdown cells.

Fig 5. Effects of Sgsm3 knockdown on the Wnt/β-catenin pathway in rat MSCs.

Changes in Wnt/β-catenin pathway-related protein expression upon Sgsm3 knockdown in MSCs were measured via immunoblot analysis. Band intensity was measured as the area density and analyzed in ImageJ and relative intensity levels indicate protein levels normalized to the β-actin levels. The data are representative of two independent experiments. Significant differences between groups were determined via ANOVA, with p values indicated as *p<0.05 and **p<0.01. N, normoxia; H, hypoxia; NC, negative control cells; KD, knockdown cells.