Fig 1: Chronic ISO administration promoted Drp1 S616 phosphorylation and its mitochondrial translocation through CaMKII pathway.(a) Increased Drp1 phosphorylation at S616 site (Drp1S616) in the heart after ISO infusion through CaMKII-dependent pathway. N=5, 6, 4 and 4 mice in the groups of Vehicle, ISO, Prop+ISO and KN93+ISO, respectively. (b) ISO administration induced Drp1S616 phosphorylation in cultured adult rat cardiomyocytes. N=6. (c) CaMKII blocker, KN-93 (0.5 µM), prevented Drp1S616 phosphorylation by ISO (100 nM for 12 h). N=4. (d) CaMKII WT overexpression induced Drp1S616 phosphorylation in adult cardiomycytes. N=4. (e) Representative immunoblots and quantification of Drp1 proteins in mitochondrial and cytosolic fractions of the heart after ISO infusion. COX IV and ß-actin were used as mitochondrial and cytosolic markers, respectively. N=5, 6 and 4 mice in the groups of Vehicle, ISO and KN93+ISO, respectively. (f) Immunofluorescent analysis showing increased ‘punctate' Drp1 staining colocalized with mitochondria. Images are representative of 30 cells from three rats in each group. (g) Representative immunoblots and quantification of Drp1S616 or Drp1S637 phosphorylation in the mitochondrial or cytosolic fractions of the heart after 2-weeks of ISO infusion. N=4. (h) ISO treatment (1 µM or 10 µM for 24 h) induced mitochondrial fragmentation in H9C2 cardiac myoblasts. A dominant negative Drp1 mutation (Drp1 K38A) was used as positive control. Form factor (FF; the reciprocal of circularity value) and aspect ratio (AR; major axis/minor axis) were acquired by using ImageJ. Smaller values of AR and FF indicate increased mitochondrial fragmentation. N=17,714, 5,929 and 5,862 mitochondria in the groups of Vehicle, ISO 1 µM and ISO 10 µM, respectively. (i) Overexpression of CaMKII WT and CaMKII CA increased mitochondrial fragmentation in H9C2 cells. CaMKII WT potentiated the effects of ISO on mitochondrial morphological change as indicated by significant fragmentation at a low ISO dose (100 nM). N=17,714, 17,692, 9,567 and 17,035 mitochondria in the groups of Vehicle, CaMKII WT, WT+ISO and CaMKII CA, respectively. Data are mean±s.e.m. *P<0.05 versus Vehicle, #P<0.05 versus ISO. The data were analysed using One-way ANOVA followed by Turkey post test.