| Background:Ischemia-reperfusion(I/R) ventricular arrhythmias were used as an indicator of recanalization after treatment with percutaneous coronary intervention or thrombolysis. However, these arrhythmias are also one of the main reasons for deadly cardiovascular events and their incidence is high. Therefore, how to decrease the occurrence of I/R-induced ventricular arrhythmias has become a major problem for clinical interventional treatment and preventing fatal cardiovascular disease. Integrin-linked kinase(ILK) is a key protein kinase expressed in many types of tissues and cells, which mediates extracellular signal molecules regulating intracellular structure and function. It has been proved that ILK was involed in regulating many cellular biological processes including cell migration, angiogenesis, cell differentiation and proliferation. ILK also has an important regulatory role in the cardiac structure, function, and cardiovascular diseases(involed in cardiomyopathy, ventricular hypertrophy, myocardial infarction, etc). However, whether ILK is involved in the occurrence of cardiac I/R ventricular arrhythmias is unclear. Numerous studies showed that connexin 43(Cx43) played an important role in arrhythmogenesis. The positive regulation of Cx43 may significantly decrease the occurrence of arrhythmias after I/R, including increasing Cx43 expression, promoting its phosphorylation or inhibiting its remodeling. It was reported that suppression of ILK could decrease the expression of Cx43, indicating that ILK may play a key role in regulating the function of Cx43. It has been reported recently that Akt could maintain Cx43 at intercalated disk by phosphorylating Cx43 at S373, thereby inhibiting the remodel of Cx43. Moreover, ILK could also phosphorylate Akt at S473 directly or indirectly. Therefore, we hypothesize that ILK ameliorates I/R ventricular arrhythmias, which might be caused by the inhibition of Cx43 remodeling via Akt activation. In this study we try to certify this hypothesis with isolated I/R rat hearts.Methods:1. The role of ILK in ventricular arrhythmias after I/R injuryWe established the model of I/R arrhythmia, followed by the pretreatment of ILK agonist LPTP and ILK inhibitor Cpd22. We devided the rats into five groups: sham group, I/R group, I/R+Cpd22 group, I/R+LPTP group and I/R+Cpd22+LPTP group. The arrhythmias incidences after I/R in each group were recorded.2. Detecting the role of Cx43 in the prevention of ILK against I/R ventricular arrhythmias.We dectected the expression and distribution of Cx43 in each group after I/R by Western-Bloting analysis or immunofluorescence staining.3. Mechanism underlying the regulaiton of ILK on Cx43(1)We examined the expression of P-Akt473 in sham group, I/R group, I/R+Cpd22 group, I/R+LPTP group, I/R+Cpd22+LPTP group by Western-Blot analysis. The change of p-Akt473 after pretreatment with the regulator of ILK activity were calculated.(2)To investigate the role of Akt on ILK regulating Cx43, we devided the rats into four groups: sham group, I/R group, I/R+LPTP group, I/R+MK2206(an Akt inhibitor) +LPTP group. The expression and distribution of Cx43 and expression of p-Akt473 in each group after I/R were detected by Western-Bloting analysis or immunofluorescence staining.ResultsPartⅠ: The effect of ILK on ventricular arrhythmia after I/R in isolated hearts1.Inhibiting ILK significantly aggravated I/R ventricular arrhythmias;2.ILK agonist significantly relieved the severity of ventricular arrhythmias induced by I/R, and this effect was impaired by ILK inhibitor.PartⅡ: The role of Cx43 in ventricular arrhythmia affected by ILK after I/R1.There were no significant differences among all groups regarding to the expression of total Cx43 after pretreatment with ILK activity regulator.2.ILK agonist could stabilize the distribution of Cx43 in intercalated disk. ILK inhibitor promoted the lateralization of Cx43, and blocked the effect of ILK agonist on inhibition of Cx43 remodeling.Part Ⅲ:The role of akt in the ILK induced inhibition of Cx43 remodelingAfter perfusion the hearts administrated of the ILK agonist, Akt inhibitor antagonized the protective effects of ILK agonist on ventricular arrhythmia induced by I/R, which simultaneously blocked the ILK agonist induced beneficial effect against I/R related distribution of Cx43.Conclusion:1. ILK could prevent I/R ventricular arrhythmias in isolated hearts;2. The mechanisms of ILK antiarrhythmic effect were related to inhibiting redistribution of Cx43;3. ILK could inhibite the redistribution of Cx43 via phosphorylating Akt473; PartⅢ: The role of Akt in the ILK induced inhibition of Cx43 remodeling... |
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