The loss of N-terminal PKA phosphorylation sites in cTnI-ND,Tnni3 ^−/− mouse heart does not alter PKA phosphorylation of cMBP-C and titin. (A) The SDS-PAGE gel stained for phosphoprotein using Pro-Q reagent shows the significant phosphorylation of cMBP-C and intact cTnI in WT mouse hearts treated with ISO in vivo. cTnI-ND in cTnI-ND,Tnni3^−/− mouse hearts had no detectable phosphorylation, consistent with the fact that PKA phosphorylation of cTnI is primarily at the N-terminal Ser²³/Ser²⁴. (B) Densitometry quantification showed that the loss of a major PKA substrate in cTnI-ND,Tnni3^−/− hearts did not alter the level of ISO-stimulated PKA phosphorylation of cMBP-C. (C) The Pro-Q phosphoprotein stained SDS-gel shows that the level of cMBP-C phosphorylation was increased in ex vivo working hearts upon 10 nM ISO treatment. (D) Densitometry quantification showed similar degrees of cMBP-C phosphorylation in cTnI-ND,Tnni3^−/− and WT hearts at baseline and after ISO treatment. (E and F) The agarose SDS-gels stained for total proteins and phosphoproteins and densitometry quantification showed no difference in titin splice form or in the degrees of phosphorylation in cTnI-ND,Tnni3^−/− and WT hearts. MHC, myosin heavy chain. Values are mean ± SEM. n = 3–5 mice in each group. ***P < 0.001 vs. baseline in Student’s t test.