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. 2020 Aug 27;17(15):2328-2337.
doi: 10.7150/ijms.48872. eCollection 2020.

Downregulation of Cypher induces apoptosis in cardiomyocytes via Akt/p38 MAPK signaling pathway

Tianming Xuan  1 , Dongfei Wang  1 , Jialan Lv  1 , Zhicheng Pan  1 , Juan Fang  1 , Yin Xiang  1 , Hongqiang Cheng  2 , Xingxiang Wang  1 , Xiaogang Guo  1
Affiliations

Downregulation of Cypher induces apoptosis in cardiomyocytes via Akt/p38 MAPK signaling pathway

Tianming Xuan et al. Int J Med Sci. .

Abstract

Background: Dilated cardiomyopathy (DCM) is considered as the most common form of non-ischemic cardiomyopathy with a high mortality worldwide. Cytoskeleton protein Cypher plays an important role in maintaining cardiac function. Genetic studies in human and animal models revealed that Cypher is involved in the development of DCM. However, the underlying molecular mechanism is not fully understood. Accumulating evidences suggest that apoptosis in myocytes may contribute to DCM. Thus, the purpose of this study is to define whether lack of Cypher in cardiomyocytes can elevate apoptosis signaling and lead to DCM eventually. Methods and Results: Cypher-siRNA sufficiently inhibited Cypher expression in cardiomyocytes. TUNEL-positive cardiomyocytes were increased in both Cypher knockdown neonatal rat cardiomyocytes and Cypher knockout mice hearts, which were rare in the control group. Flow cytometry further confirmed that downregulation of Cypher significantly increased myocytes apoptosis in vitro. Cell counting kit-8 assay revealed that Cypher knockdown in H9c2 cells significantly reduced cell viability. Cypher knockdown was found to increase cleaved caspase-3 expression and suppress p21, ratio of bcl-2 to Bax. Cypher-deficiency induced apoptosis was linked to downregulation of Akt activation and elevated p-p38 MAPK accumulation. Pharmacological activation of Akt with SC79 attenuated apoptosis with enhanced phosphorylation of Akt and reduced p-p38 MAPK and Bax expression. Conclusions: Downregulation of Cypher participates in the promotion of cardiomyocytes apoptosis through inhibiting Akt dependent pathway and enhancing p38 MAPK phosphorylation. These findings may provide a new potential therapeutic strategy for the treatment of DCM.

Keywords: Akt; Cypher; apoptosis; dilated cardiomyopathy; p38 MAPK.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Downregulation of Cypher induced apoptosis in vitro. (A and B) Representative western blot bands and statistical results of Cypher protein expression normalized by GAPDH. *P < 0.05 versus control cells. (C) Cell viability was measured using the CCK-8 assay. *P < 0.05 Cypher-si versus control. (D) Representative images of TUNEL staining showing the apoptotic cells (stained in green) and nucleus (stained in blue with DAPI). Arrowheads in the pictures indicate apoptotic cell nuclei. (E) Annexin V-FITC/propidium iodide (PI) staining by flow cytometry was performed to detect the apoptosis of H9c2 cells. Early apoptosis (Annexin V-FITC+/PI), late apoptosis (Annexin V-FITC+/PI+), and necrosis (Annexin V-FITC/PI+). (F) The quantitative presentation of apoptotic cell population by Annexin V-FITC/PI staining. *P < 0.05 versus control. Experiments were repeated three times.
Figure 2
Figure 2
Effects of Cypher in mice hearts. (A) Representative western blot bands of Cypher protein expression was shown. (B) The effects of Cypher knockout on heart volume. (C) Representative TUNEL staining images from hearts of wild type mice (left) and Cypher knockout mice (right), scale bar: 100 µM.
Figure 3
Figure 3
Effects of Cypher knockdown on the mRNA expression of apoptosis-related proteins in H9c2 cells. The mRNA levels of Cypher, bcl-xl, bcl-2, Bax, Bim, Bak, PUMA, Fas, p53, and NF-κB were detected by real-time polymerase chain reaction procedure. The relative levels of above mentioned mRNA were calculated by the values of ΔCt by normalizing with that of GAPDH. *P < 0.05 versus control. Experiments were repeated three times.
Figure 4
Figure 4
Effects of Cypher knockdown on apoptosis regulatory protein expression in H9c2 cells. (A-L) Representative western blot bands and statistical results of protein expression. *P < 0.05 versus control cells. Experiments were repeated three times.
Figure 5
Figure 5
SC79 inhibited Cypher-siRNA-induced apoptosis of H9c2 cells by activating Akt. (A) HEK293T cells were transfected with GST-Cypher and HA-Akt plasmids. The cells were then lysed. Lysates from HEK293T co-immunoprecipitated (IP) with IgG or antibody to HA, followed by immunoblot (IB) analyses with GST antibody. (B) HEK293T cells were transfected with GST-Cypher and HA-Akt plasmids. The cells were then lysed. Lysates from HEK293T IP with IgG or antibody to GST, followed by IB analyses with HA antibody. H9c2 cells were pre-cultured in serum-free medium in the presence of Cypher-siRNA for 72 hrs, and then stimulated further with 10 µM SC79 for an additional 30 mins. The cells were then lysed, and Western blot analysis was performed. (C) Western band images are representative of three independent experiments. (D) The quantitative analysis of p-Akt-473 using total Akt as normalization, while p-p38 and Bax using GADPH as normalization. Experiments were repeated three times.
Figure 6
Figure 6
Schematic of the working signal pathway. In Cypher deficient cardiomyocytes, phosphor Akt-473 is downregulated and leads to a significant increased p-p38 MAPK. There it is assumed to activate mitochondria pathway, manifested specifically by decreasing the expression of p21 and the ratio of bcl-2 to Bax, while increasing the expression of cleaved caspase-3. These lead to an increase of apoptosis, thus ultimately leading to dilated cardiomyopathy.
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