Role of SRSF1-mediated CDK1 exon skipping in promoting cardiomyocyte senescence during myocardial ischemia-reperfusion injury

Objective To investigate the role and mechanism of serine/arginine-rich splicing factor 1 (SRSF1) in promoting cardiomyocyte senescence during myocardial ischemia-reperfusion injury (MIRI). Methods ① Forty male SD rats (250-300 g) were randomly divided into a sham group and a MIRI group (n=20). The sham group underwent thoracotomy and cardiac exposure without ischemia-reperfusion, while the MIRI group underwent coronary artery ischemia-reperfusion after thoracotomy. The rats were sacrificed and myocardial tissue injury and senescence were evaluated by qRT-PCR, ELISA, Western blot, and β-galactosidase staining. ② Second-generation transcriptome sequencing was performed on the myocardial tissues from the control and MIRI groups to identify differentially expressed genes, which were then intersected with senescence-related genes. ③ SRSF1 overexpression and gene silencing lentiviruses were constructed to infect human cardiomyocytes (AC16). Cellular senescence was evaluated by qRT-PCR, Western blot, and β-galactosidase staining.④ Semi-quantitative PCR and agarose gel electrophoresis were used to detect the expression of CDK1 full-length splice variants in AC16 cells under conditions of high and low SRSF1 expression. ⑤ Forty SD rats, weighing 250-300 g, were divided into a control group and a SRSF1 overexpression group. The myocardial p16 and p21 levels with high SRSF1 expression was measured by qRT-PCR and Western blot. Results ① qRT-PCR results showed that the levels of serum cardiac enzymes CK-MB and LDH in rat myocardial tissue significantly increased after MIRI. ELISA results confirmed that the levels of creatine kinase isoenzyme (CK-MB) and serum lactate dehydrogenase (LDH) in rat blood increased after MIRI, indicating myocardial injury in rats after MIRI. Immunohistochemistry (IHC), qRT-PCR, and Western blot results confirmed that the expression of p16 and p21 in rat myocardial tissue was upregulated after MIRI (P<0.05), suggesting myocardial tissue senescence. ② Second-generation transcriptome sequencing results of myocardial tissue from the control group and the model group showed that 456 genes were up-regulated and 368 genes were down-regulated. Intersecting the up-regulated genes with senescence-associated genes revealed that SRSF1 was an up-regulated senescence-associated gene after MIRI. ③ qRT-PCR, Western blot, and β-galactosidase staining results showed that SRSF1 significantly promoted the senescence of AC16 cells. ④ Semi-quantitative PCR and agarose gel electrophoresis results confirmed that high expression of SRSF1 led to the skipping of CDK1 exon 5, inactivating CDK1, and ultimately mediating the senescence of AC16 cells. ⑤ qRT-PCR and Western blot results confirmed that high expression of SRSF1 promoted the expression of p16 and p21 in rat cardiomyocytes.Conclusions SRSF1 is upregulated after MIRI, which in turn mediates the skipping of CDK1 exon 5, induces cardiomyocyte cell cycle arrest, and ultimately leads to cardiomyocyte senescence.