Myosin-actin crossbridge independent sarcomere length induced Ca2+ sensitivity changes in skinned myocardial fibers: Role of myosin heads

Figures

• 

  Fig. 1.. Western blot analysis of skinned myocardial fibers before (lane 1) and after (lane 2) reconstitution with the troponin complex containing wt-cTnT, cMyc-cTnI, and cTnC(13C/51C)AEDANS-DDPM. Lane 1 shows a single band at ~24 kDa of the intrinsic wt-cTnI protein extracted from the non-exchanged skinned fibers. Lane 2 shows two bands, one at ~24 kDa of the wt-cTnI and the other at ~25 kDa of the cMyc-cTnI protein extracted from the protein- exchanged skin fibers. The cMyc-tag is around 1 kDa. The protein exchange efficiency of this protocol reached 80 ± 5 % and it was analyzed by ImageLab density analysis software. Mouse monoclonal anti-cTnI antibody (Cat: ab10231, Abcam) and HRP-labeled sheep anti-mouse secondary antibody (GE Healthcare) were used in this experiment.

• 

  Fig. 2.. Tension development vs. Ca2+ titration of skinned myocardial fibers under different conditions. A: Activated tensions of the skinned myocardial fiber reconstituted with troponin containing ΔSP-cTnI reconstitution (teal) and wt-cTnI in the absence (black) and presence (pink) of 5 μM of mavacamten. B: Normalized tension development vs. Ca2+ titration at SL 1.8 (red) and 2.2 μm (black) of skinned myocardial fibers reconstituted with Group I troponin complex containing cTnC (13C/51C)AEDANS-DDPM, wt-cTnT, and cMyc-cTnI. Data (solid lines) were fit to a 4-parameter Hill equation (cyan dash lines) to derive the Ca2+ sensitivity (pCa50) and slope nH (the Hill coefficient) at different SLs, which are given in the legend. (Nfiber = 6 for B, the curves shown in both A and B represent the average curves of all measurements, and the errors calculated from STD are shown in Fig. 5 and Table 1). The maximal Ca2+ activated forces corresponding to tension-Ca2+ titrations shown in panel B were 28.9 mN/mm2 for SL of 2.2 μm and 21.3 mN/mm2 for SL of 2.8 μm, respectively.

• 

  Fig. 3.. The normalized FRET donor fluorescence intensity changes vs. Ca2+ titration at SL 1.8 (red) and 2.2 μm (black) in skinned myocardial fibers reconstituted with Group I complex containing cTnC(13C/51C)AEDANS-DDPM, wt-cTnT, and cMyc-cTnI (A) and Group II containing cTnC(13C/51C)AEDANS-DDPM, wt-cTnT, and ΔSP- cTnI (B). Data (dots) were fit to a 4 parameter Hill equation (cyan dash lines) to derive the Ca2+ sensitivity (pCa50) and nH (the Hill coefficient) at different SLs, which are listed in the legend. (Nfiber = 6 for A, and Nfiber = 6 for B, the curves shown in both A and B represent the average curves of all measurements, and the errors calculated from STD are shown in Fig. 5 and Table 1.)

• 

  Fig. 4.. The normalized FRET donor fluorescence intensity changes vs. the Ca2+ titration at SL 1.8 (red) and 2.2 μm (black) of the myocardial fibers reconstituted with Group I complex containing cTnC(13C/51C)AEDANS-DDPM, wt-cTnT, and cMyc-cTnI (A) and Group II containing cTnC(13C/51C)AEDANS-DDPM, wt-cTnT, and ΔSP-cTnI (B) in the presence of 5 μM mavacamten drug. Data (dots) were fit to a 4 parameter Hill equation (cyan dash line) to derive the Ca2+ sensitivity (pCa50) and slope nH (Hill coefficient) at different SLs, which are listed in the legend. (Nfiber = 5 for A, and Nfiber = 6 for B, the curves shown in both A and B represent the average value of all measurements, and the errors calculated from STD are shown in Fig. 5 and Table 1.)

• 

  Fig. 5.. Collective results of pCa50 (A) and ΔpCa50 (B) measured by force- and FRET-Ca2+ titrations under different cTnI reconstitution conditions in the absence (−) and presence (+) of mavacamten at short SL (filled bar) and long SL (diagonal stripes bar). Values are reported as mean ± SD. **, p < 0.01, *, P < 0.05, NS, no significant changes.

• 

  Fig. 6.. The pCa50 vs. force and FRET reconstituted with wt-cTnI (cMyc-tagged) and ΔSP-cTnI with no TEV cut, with TEV-cTnT cut, and with TEV-cTnI cut, respectively in short (filled bar) and long (diagonal stripes bar) SL (A). ΔpCa50 vs. force (filled bar) and Ca2+ sensitivity reconstituted with wt-cTnI (cMyc- tagged) (diagonal stripes bar) and ΔSP-cTnI (diamond grids bar) with no TEV cut, with TEV-cTnT cut, and with TEV-cTnI cut. Values are reported as mean ± SD. **, p < 0.01, *, P < 0.05, NS, no significant changes (B).

• 

  Graph 1.. The schematic picture of the troponin bridge mechanism underlying sarcomere length dependent activation of the D-zone of sarcomere. The picture on the left shows the relaxation sarcomere at short SL, and the one on the right shows the relaxation sarcomere at long SL which interfilament space decreased. Myosin heads in SRX and DRX, TEV cutting site for cTnI and cTnT, and potential regions for SL-induced interaction between DRX myosin heads and troponin are all shown as indicated. The shown thin filament structure composed of cTnC, cTnI, cTnT, tropomyosin and actin filament are from literature (ref??) with minor modification to show the potential region of myosin heads and troponin interactions.