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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: J Mol Cell Cardiol. 2012 Feb 3;52(5):1135–1144. doi: 10.1016/j.yjmcc.2012.01.021

Figure 8.

Figure 8

Oxidative activation of CaMKII by LPS or MI requires MyD88. (A) LPS treatment induces oxidation of CaMKII. Cultured neonatal cardiomyocytes were untreated (lanes 1 to 3) or treated with LPS (10 µg/ml) for 30 min (lanes 4 to 6). Cell lysates were prepared and immunoblotted under non-reducing conditions for oxidized CaMKII (Ox-CaMKII). Blots were stripped and reprobed for CaMKII (CaMKII). (B) Reduced CaMKII oxidation in post-MI MyD88−/− hearts. WT or MyD88−/− mice underwent sham or MI surgery and heart lysates were prepared 1-day post-MI. Equal amounts of protein samples were fractionated by non-reducing NuPAGE gel electrophoresis and immunoblotted for ox-CaMKII. Blots were stripped and reprobed for CaMKII. (C) Relative abundance of ox-CaMKII was quantified from scanned autoradiographs and presented as the mean of the ratios of ox-CaMKII to CaMKII. One-way ANOVA with Bonferroni’s post-test (*P ≤ 0.01). (D) Reduced phosphorylation of a target protein (phospholamban, PLN) by CaMKII in MyD88−/− hearts after MI. Lysates from 1d post-MI hearts of WT (lanes 1 and 2) and MyD88−/− (lanes 3 and 4) mice were subjected to immunoblotting using antibody to the phosphorylated Thr-17 epitope of phospholamban (PLN-T17). Blots were then stripped and reprobed with antibody specific to total phospholamban (PLN). (E) Band intensity of PLN-T17 immunoblots were normalized to PLN blots. Statistical significance was determined by unpaired t-test (***P = 0.0002). (F) Cultured neonatal cardiomyocytes were transduced with adenovirus with empty expression vector or with a Rac1 dominant negative construct (Rac1DN) and treated with LPS. Rac1DN expressing cells had significantly reduced oxCaMKII (n=3 each, ***P = 0.0005).