Rho-kinase signaling pathway promotes the expression of PARP to accelerate cardiomyocyte apoptosis in ischemia/reperfusion

Figures

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  Figure 1.. Inhibition of the Rho-kinase signal pathway diminishes PARP expression in I/R-inducing injury. (A) Diagram of the experimental protocol in the current study. (B) Western blot analysis of the protein levels of p-MYPT-1 and PARP in different groups of H9C2 cells (Sham, DMSO + I/R, Y-27632 + I/R and 3-AB + I/R). (C) Relative quantitative analysis of p-MYPT-1 and PARP expression. **P<0.01 vs. Sham; #P<0.05 vs. DMSO + I/R; NS vs. DMSO + I/R. Similar observations were obtained in three independent experiments. PARP, poly adenosine diphosphate-ribose polymerase; I/R, ischemia/reperfusion; p-, phosphorylated; MYPT-1, myosin phosphatase target subunit 1; DMSO, dimethyl sulfoxide; 3-AB, 3-aminobenzamide; NS, nonsignificant.

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  Figure 2.. ROCK regulating PARP expression promotes myocardial cell apoptosis in I/R-inducing injury. (A) Hoechst staining was completed (magnification, ×200; red arrows, chromatin condensation, DNA fragmentation and chromatin margination). (B) The histogram present the rates of Hoechst-positive cells. (C) The expression levels of Bcl-2, Bax and caspase-3 were measured in different groups of H9C2 cells by western blotting. (D) Relative quantitative analysis of Bcl-2, Bax and caspase-3 expression. ***P<0.001 vs. Sham; #P<0.05, ##P<0.01 and ###P<0.001 vs. DMSO + I/R; $P<0.05, vs. Y-27632 + I/R. Similar observations were obtained in three independent experiments. ROCK, Rho-kinase; PARP, poly adenosine diphosphate-ribose polymerase; I/R, ischemia/reperfusion; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; DMSO, dimethyl sulfoxide.

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  Figure 3.. ROCK and PARP inhibition during I/R attenuated myocardial cell apoptosis and infarct size in vivo. Changes in (A) myocardial AAR and (B) infarct size of different groups (Sham, DMSO + I/R, Y-27632 + I/R, 3-AB + I/R) in the rat hearts. The content of (C) CK-MB, (D) LDH release and (E) NO levels were detected in the serum of rats. (G) Representative photomicrographs of ventricular tissue stained for TUNEL for DNA breaks in different groups. (F) The histogram shows the percentage of TUNEL positive cells. n=15; ***P<0.001 vs. Sham; #P<0.05, ##P<0.01 and ###P<0.001 vs. DMSO + I/R; $P<0.05, vs. Y-27632 + I/R. ROCK, Rho-kinase; PARP, poly adenosine diphosphate-ribose polymerase; I/R, ischemia/reperfusion; DMSO, dimethyl sulfoxide; 3-AB, 3-aminobenzamide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; AAR, area at risk; CK-MB, creatine kinase-MB; LDH, lactate dehydrogenase; NO, nitric oxide.

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  Figure 4.. ERK signal pathway is an important in I/R-inducing injury. (A) NAD+ level in the left ventricle were measured. (B) TNF-α and (C) IL-6 in the serum of rats were determined by ELISA. (D) Western blot analysis of protein level of p-ERK in different groups. (E) Relative quantitative analysis of p-ERK expression. **P<0.01 and ***P<0.001 vs. Sham; #P<0.05 and ##P<0.01 vs. DMSO + I/R; $P<0.05 vs. Y-27632 + I/R. Similar observations were obtained in three independent experiments. ERK, extracellular signal-related kinase; I/R, ischemia/reperfusion; NAD, nicotinamide adenine dinucleotide; TNF-α, tumor necrosis factor α; IL-6, interleukin 6; p-, phosphorylated; DMSO, dimethyl sulfoxide; 3-AB, 3-aminobenzamide.