The R21C Mutation in Cardiac Troponin I Imposes Differences in Contractile Force Generation between the Left and Right Ventricles of Knock-In Mice

Figure 5

Assessment of protein phosphorylation in LV and RV of R21C^+/+ mice compared with LV and RV of WT mice. (a) Representative ProQ/Coomassie gels of myofibrillar preparations from LV and RV of R21C^+/+ mice compared with LV and RV of WT mice. MyBP-C, myosin binding protein C; F/A, F-actin; TnT, troponin T; Tm, tropomyosin; TnI, troponin I, ELC, myosin essential light chain; RLC, myosin regulatory light chain; +P hcTnI std., phosphorylated human cardiac TnI standard; and +P RLC std., phosphorylated human cardiac RLC standard. (b) Representative Western blot of myofibrillar preparations from LV and RV of R21C^+/+ mice compared with LV and RV of WT mice. The level of RLC phosphorylation was determined with phosphospecific RLC antibodies (+P-RLC ab) and compared to the total RLC content assessed with a rabbit polyclonal RLC antibody (CT-1 ab) recognizing total RLC protein. −P RLC std., nonphosphorylated RLC and +P RLC std., phosphorylated RLC standard proteins. (c) Quantification of phosphorylated cTnI was assessed by independent SDS-PAGE (ProQ/Coomassie gels) analyses of two to three preparations from LV and RV ventricles per group. (d) Quantification of RLC phosphorylation was assessed by SDS-PAGE analyses (Western blots and ProQ/Coomassie gels) of two to three LV and RV preparations from WT and R21C^+/+ mice. Note no cTnI phosphorylation in R21C^+/+-KI mouse model. There were no differences in cTnI phosphorylation between the LV and RV of WT mice. Note significantly enhanced phosphorylation in the RV-R21C^+/+ compared to LV-R21C^+/+ () and to LV-WT (). No statistically significant differences in myosin RLC phosphorylation were observed between the LV and RV of WT mice. Errors bars are SEM.

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