PNPO of rat single permeabilized slow-twitch skeletal muscle fibers before and after exchange of human cardiac Tn complex. (A) Top left: Slow-twitch fiber attachment photomicrograph and illustration of Tn exchange protocol. Top right: Coomassie-stained gel of recombinant human cardiac Tn complex containing cTnT, cTnI, and cTnC and a Western blot to assess the relative amount of endogenous ssTn compared to exogenous cTn complex after Tn exchange. Lanes 1 and 3: Rat permeabilized slow-twitch fibers that did not undergo Tn exchange. Lanes 2 and 4: Rat permeabilized slow-twitch fibers that underwent Tn exchange. Bottom: Representative length and force traces during the load clamps to measure loaded shortening velocities. (B–E) Representative force–velocity and power–load curves before and after exchange of endogenous ssTn (which lacks the N-terminal TnI phosphorylation site) with recombinant human cardiac Tn complex which contained N-terminal cTnI phosphorylation sites. The WT cTnI has serines at sites 23 and 24, but those sites are not phosphorylated. The Ser23/24Asp (S2323DD) cTnI is PKA pseudo-phosphorylated, while the Tyr26Glu (Y26E) cTnI is non-PKA pseudo-phosphorylated. Source data are available for this figure: SourceData F2.