lament data analysis was performed using the modified Hill equation 
to fit force-pCa relations. Comparisons between the cTn and cTn groups were made using an unpaired Student t-test or ANOVA, where appropriate. Values are given as means 6 S.E.M. of n myocytes. MRM data acquisition, processing and analysis were performed using Applied Biosystems/MDS Sciex Analyst software and Multiquant 1.0. Time control denotes results obtained from cardiomyocytes kept in exchange buffer without cTn complex added. Symbols used: denotes P,0.05, cTn compared to time control or cTn and { denotes P,0.05, cTn compared to time control in ANOVA followed by R-7128 Bonferroni post-hoc test. Measurements in cTn exchanged cells were repeated after treatment with PKCa. In this case denotes P,0.05, before vs. after incubation with PKCa in paired t-test. Abbreviations: Fmax, maximal force per cross-sectional area at saturating calcium concen-tration in kN/m2; Fpas, passive force per cross-sectional area in relaxing solution in kN/m2; nH, steepness of the force-pCa curves; Ktr-max, the rate of force redevelopment at maximal activating solution in s21. doi:10.1371/journal.pone.0074847.t002 4 PKCa Phosphorylation of Cardiac Troponin combined effects on Ca2+-sensitivity observed upon PKCa treatment of all contractile proteins within the myofilament lattice. PKCa-mediated phosphorylation of cTn depresses maximal force generation Exchange of endogenous cTn with cTn did not change the maximal isometric force at saturating Ca2+ concentration . However, cardiomyocytes exchanged with cTn showed a significant reduction in Fmax PKCa Phosphorylation of Cardiac Troponin 6 PKCa Phosphorylation of Cardiac Troponin and after incubation with PKCa. These results did not show a decrease in Fmax after incubation with PKCa compared to the Fmax before incubation . Myocyte measurements showed that prolongation of the incubation time to 3 hours did not have a significant effect on Fmax. This suggests that in vitro phosphorylation of the cTn complex prior to exchange is required to observe an effect of PKCa on Fmax. PKCa-mediated phosphorylation of cTn has no effect on other contractile properties Exchange 2578618 of endogenous cTn with cTn did not change passive force measured at pCa 9 compared to untreated time control cells. In addition, Fpas did not significantly differ between cTn and cTn . Recently, PKCa-mediated phosphorylation of titin has been shown to 23584186 increase passive stiffness. However, subsequent PKCa treatment of cTn exchanged cardiomyocytes did not significantly affect Fpas. In addition, no effect of cTn exchange was seen on the steepness of the force-pCa relation or the rate of force redevelopment measured at pCa 4.5 after a slack test. Furthermore, no significant differences were observed in nH and Ktr-max upon exchange with cTn compared to cTn . PKCa treatment of cTn exchanged cardiomyocytes did not alter nH or Ktr-max. This suggests that PKCa-mediated phosphorylation of cTn or of any other sarcomeric protein has no effect on cross-bridge kinetics. LC MS/MS analysis For LC MS/MS analysis, PKCa-treated recombinant cTn was digested using trypsin immediately after mixing of the solutions at time-points “0” and after 180 minutes of incubation. The amino acid sequence coverage of cTnT and cTnI was on average 47% and 40%, respectively. Both the phosphorylated and unphosphorylated cTnI peptides of NIDALsGMEGR comprising residues 193203 were observed in the PKC treated sample indicating that Ser19