1、Supplementary information Inhibition of late sodium current suppresses calcium-related ventricular arrhythmias by reducing the phosphorylation of CaMK- and sodium channel expressions Xiao-Hong Wei1,2, Shan-Dong Yu1,2, Lu Ren1,2, Si-Hui Huang1,2, Qiao-Mei Yang1,2, Ping Wang1, Yan-Peng Chu1,2, Wei Yan
2、g1,2, Yan-Sheng Ding1,2, Yong Huo1,2,*, and Lin Wu1,2,* 1Department of Cardiology, Peking University First Hospital, Beijing, 100034, China 2Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, 100191, China. *Corresponding authors: Lin Wu, ; and Yong Huo, . 2 Methods
3、 Myocardial ischemia/reperfusion (I/R) model establishment and myocardial infarct size assessment in rats. To determine the effect of late sodium current inhibitors on a disease model (myocardial ischemia/reperfusion injury) associated with late sodium current, male Sprague-Dawley rats weighing 240-
4、260 g were subjected to left anterior descending coronary artery (LADCA) occlusion by ligation for 30 min followed by reperfusion for 90 min. Animals were divided into three groups: Sham, I/R model, and ranolazine + I/R. Ranolazine (10 M) was administrated by intravenous for 30 min before ischemia,
5、and was continuously administrated till the end of reperfusion at a speed of 1 ml/h. At the end of reperfusion, the LADCA was re-ligated, and 2 ml of 4% Evans blue (Sigma Aldrich, Missouri, USA) was injected into the right femoral vein via intravenous catheter to delineate the area at risk (AAR). Th
6、en the heart was excised at a 5 mm level above the apex cordis and sliced transversely into five sections (1 mm thick). The slices were incubated in 0.375% 2,3,5-triphenyltetrazolium chloride (TTC) (AMRESCO, Ohio, USA) at 37 C for 15 min to identify the infarct size. AAR, infarct size and left ventr
7、icle (LV) were determined using Image-Pro Plus 5.0 software (Media Cybernetic, Maryland, USA). Results Eleclazine decreased the reverse use dependence (RUD) effect of Bay K 8644 on left ventricular epi-MAPD90. As shown in Supplementary Fig. S1, eleclazine exhibited similar effect to TTX on Bay K 864
8、4-induced RUD phenomenon of MAPD90. Bay K 8644 (200 nM) prolonged epi-MAPD90 at all stimulation rates (n=5, P0.05), and the prolongation was greater at longer cycle length (CL)s (e.g., 75.8 4.4 ms at a CL of 2000 ms) than at shorter CLs (19.6 3 5.2 ms at a CL of 400 ms, P0.05). In the continued pres
9、ence of Bay K 8644 in same group of hearts, eleclazine significantly attenuated the RUD of epi-MAPD90, especially at longer CLs (n=5, P0.05). Supplementary Figure S1. Effects of eleclazine on the RUD of MAPD90 caused by Bay K 8644. Values of MAPD90 were measured in the absence (Ctrl) and presence of
10、 Bay K 8644 (200 nM), Bay K 8644 (200 nM) plus eleclazine (n=5). *P 0.05 vs. cycle length (CL) of 400 ms; #P 0.05 vs. Ctrl at the same CL; P 0.05 vs. Bay K 8644 alone at the same CL. Inhibition of late INa by ranolazine (10 M) diminished the infarct size after myocardial I/R. As shown in Supplementa
11、ry Fig. S2, ranolazine caused a reduced ratio of infarct size/AAR in hearts treated compared to that from I/R model (Supplementary Fig. S2, (a) and (c). However, the ratios of AAR/LV were similar in all three groups, indicating similar tension and placement of the ligature across the three groups (S
12、upplementary Fig. S2(b). 4 Supplementary Figure S2. Ranolazine (10 M) reduced the infarct size in hearts with ischemia/reperfusion (I/R) injury. (a), representative images of Evans blue-TTX staining in Sham, I/R 90 min, and ranolazine plus I/R 90 min. (b) and (c), the statistic results of AAR/LV (b) and Infarct size/AAR (c) among the three groups. AAR: area at risk; LV: left ventricle. n=3, *P 0.05 vs. Sham; #P 0.05 vs. I/R 90 min.
