Myofilament Calcium Sensitivity: Mechanistic Insight into TnI Ser-23/24 and Ser-150 Phosphorylation Integration

Figures

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  Figure 1. TnI Ser-23/24 and Ser-150 differentially alter thin filament activation through different TnI mediated mechanisms. (A) Diagrammatic representation of the biochemical states in the Siddiqui et al. model (Siddiqui et al., 2016). The rates altered to fit our previous biochemical data for thin filaments containing TnI Ser-150 (red arrow) and Ser-23/24 phosphorylation (blue arrow) are identified. Model simulation of (B) steady-state Ca2+-binding and (C) Ca2+ dissociation from TnC in human thin filaments containing WT (Tn WT, black dashed line), Ser-150 pseudo-phosphorylated (Tn S150D, red dashed line), Ser-23/24 pseudo-phosphorylated (Tn S23/24D, blue dashed line) or Ser-23/24 and Ser-150 pseudo-phosphorylated (Tn S23/24/150D, gray dashed line) TnI demonstrating a similar effect of these phosphorylations to our previously reported biochemical measurements (Nixon et al., 2014).

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  Figure 2. TnI Ser-150, but not Ser-23/24, pseudo-phosphorylation decreases TnI binding to actin. Average solid-phase TnI-actin protein binding data demonstrating that compared to WT (TnI WT: black circle, solid line), the pseudo-phosphorylation of Ser-150 (TnI S150D: black Square, dashed line) and Ser-23/24/150 (TnI S23/24/150D: gray diamond, solid line) decrease TnI binding to actin while the pseudo-phosphorylation of Ser-23/24 (TnI S23/24D: gray triangle, dashed line) do not. Protein binding assays were conducted in triplicate wells and repeated.

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  Figure 3. Tn exchange into skinned cells is similar for all investigated TnI pseudo-phosphorylations. (A) Representative TnI Western blot of myocytes following exchange demonstrates a similar presence of the faster migrating exogenous human cardiac TnI compared to the slower migrating endogenous rat cardiac TnI in all Tn's investigated. (B) Densitometry analysis of Western blots demonstrates the average percent of each exchanged exogenous Tn is not different. Control; purified recombinant rat WT cardiac TnI.

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  Figure 4. TnI Ser-150 blunts Ser-23/24 pseudo-phosphorylation in Tn exchanged single rat cardiac myocytes. Average normalized force-pCa measurements of Tn exchanged single cardiac myocytes. (A) Myocytes exchanged with WT human cardiac Tn (Tn WT: black circle, solid line; n = 8) or WT rat cardiac Tn (RcTn WT: gray triangle, dashed line; n = 8) exhibit similar Ca2+-sensitive force development. (B) The Ca2+-sensitive force development in myocytes exchanged with Tn containing Ser-150 pseudo-phosphorylated TnI (Tn S150D: gray triangle, dashed line; n = 7) is increased while Ser-23/24 pseudo-phosphorylated TnI (Tn S23/24D: gray square, dashed line; n = 7) is decreased compared to Tn containing WT TnI (Tn WT: black circle, solid line; n = 8) exchange. (C) The Ca2+-sensitive force development in myocytes exchanged with Tn containing Ser-23/24/150 pseudo-phosphorylated TnI (Tn S23/24/150D: gray triangle, solid line; n = 8) is identical to those with Tn WT exchange. (D) Comparison of pCa50. *p < 0.05 vs. WT Tn.

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  Figure 5. Single myocytes exchange with 50% pseudo-phosphorylated TnI exhibit half the phosphorylation mediated sensitivity change. Average normalized force-pCa measurements of Tn exchanged single cardiac myocytes. (A) The decrease in Ca2+-sensitive force development of myocytes exchanged with Tn containing 50% WT and 50% Ser-23/24 pseudo-phosphorylated TnI (Tn WT+S23/24D: black square, dashed line; n = 7) is half that of myocytes exchanged with Tn containing Ser-23/24 pseudo-phosphorylated TnI (Tn S23/24D: gray triangle, dashed line; n = 7) compared to WT (Tn WT: black circle, solid line; n = 8) exchange. (B) The increase in Ca2+-sensitive force development of myocytes exchanged with Tn containing 50% WT and 50% Ser-150 pseudo-phosphorylated TnI (Tn WT+S150D: black square, dashed line; n = 9) is half that of myocytes exchanged with Tn containing Ser-150 pseudo-phosphorylated TnI (Tn S150D: gray triangle, dashed line; n = 7) compared to Tn WT exchange. (C) Comparison of pCa50. *p < 0.05 vs. WT Tn.

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  Figure 6. Single myocytes exchanged with Ser-150 and Ser-23/24 pseudo-phosphorylation on different Tn exhibit Ca2+-sensitivity identical to both TnI pseudo-phosphorylations on the same Tn. (A) Average normalized force-pCa measurements of Tn exchanged single cardiac myocytes exchanged with Tn containing Ser-23/24 and Ser-150 pseudo-phosphorylation on the same TnI (Tn S23/24/150D: gray triangle, solid line; n = 8) or exchanged with 50% Ser-23/24 and 50% Ser-150 pseudo-phosphorylated TnI on different Tn (Tn S23/24D+S150D: black square, dashed line; n = 7) both exhibit identical Ca2+ dependent force development to those exchanged with WT TnI (Tn WT: black circle, solid line; n = 8). (B) Comparison of pCa50.