MgADP Promotes Myosin Head Movement toward Actin at Low [Ca2+] to Increase Force Production and Ca2+-Sensitivity of Contraction in Permeabilized Porcine Myocardial Strips

Figures

• 

  Figure 1. Representative X-ray diffraction patterns from permeabilized porcine myocardium at different concentrations of ADP. Characteristic relaxed patterns with intense layer line reflections in the absence of ADP (top panel). The myosin-based layer lines (e.g., MLL1) become weaker at 1 mM ADP (bottom left panel), and disappeared completely at 5 mM ADP (bottom right panel). The sixth order actin-based reflection (ALL6) remained relatively unchanged with changes in [ADP].

• 

  Figure 2. Equatorial reflection changes with increasing [ADP] under relaxing conditions (pCa 8.0). (A) Changes in the equatorial intensity ratio (ΔI1,1/I1,0) and (B) lattice spacing (d1,0) were plotted against [ADP], in the presence and absence of creatine phosphate (CP) and creatine phosphokinase (CPK). Two-way linear mixed models were utilized to characterize the main effects of ADP and CP/CPK and their interaction. Panel A depicts ΔI1,1/I1,0 relative to 0 mM [ADP] for each condition with a significant main effect for ADP (p < 0.001). In Panel B there were significant main effects of ADP (p < 0.001), CP/CPK (p = 0.004), and an ADP*CP/CPK interaction (p = 0.02), with * indicating a Fisher’s LSD post hoc difference between −CP/CPK and +CP/CPK at a single [ADP] at p < 0.05. 9 fibers were used for each different CP/CPK group.

• 

  Figure 3. Myosin heads became more disordered as [ADP] increased under relaxing conditions. Intensity of the (A) first order myosin-based layer line (IMLL1), (B) third order myosin-based meridional reflections (IM3), (C) sixth order of myosin-based meridional reflection (IM6), and (D) spacing of the sixth order of myosin-based meridional reflection (SM6) were plotted against [ADP] in the presence and absence of CP and CPK. Intensity values were normalized to the 0 mM [ADP] value within each fiber for data shown in panels (A–C). Two-way linear mixed model analysis showed significant (p < 0.001) main effects of ADP for data in all panels. In Panel (B), there was also a main effect of CP/CPK (p = 0.03), with * indicating a Fisher’s LSD post hoc difference between −CP/CPK and +CP/CPK at a single [ADP] at p < 0.05. The number of fibers was 9 for each group.

• 

  Figure 4. Tension increased with increased [ADP] under low [Ca2+] (pCa 8.0) relaxing conditions. Steady-state isometric stress (= force per cross-sectional area) was plotted against [ADP] in the presence and absence of CP and CPK. Nested two-way linear mixed models analysis showed main effects of ADP (p < 0.001), CP/CPK (p = 0.03), and an ADP*CP/CPK interaction (p < 0.001), with * indicating a Fisher’s LSD difference between −CP/CPK and +CP/CPK at a single [ADP] at p < 0.05. n fibers was 10 for each group, from two hearts.

• 

  Figure 5. Viscoelastic myocardial stiffness under low [Ca2+] relaxing conditions. (A) Elastic and (B) viscous modulus values were plotted against frequency for 0 and 5 mM [ADP] in the presence and absence of CP and CPK. Nested two-way linear mixed models were utilized to characterize main effects frequency and treatment group ([ADP], with or without CP/CPK). There were significant main effects of frequency (p < 0.001) and treatment group (5 mM [ADP] without CP/CPK, p = 0.004) in Panel (A). A significant main effect of frequency (p < 0.001) and the frequency *CP/CPK interaction (p < 0.001) occurred for Panel (B). * indicates a Fisher’s LSD post hoc difference at a single frequency between the treatment group at 5 mM [ADP] without CP/CPK and all other treatment groups (p < 0.05). n fibers was 10 for each group, from 2 hearts.

• 

  Figure 6. Ca2+-activated tension-pCa relationships. Steady-state isometric stress plotted against pCa, showing the increase in force from relaxing conditions at pCa 8.0 as strips were titrated to pCa 4.8. (A) Effects of [ADP] on Ca2+-activated force in the absence of CP and CPK, compared to control strips (=0 mM [ADP], +CP/CPK). Data were fit to a 3-parameter Hill equation, with parameters shown for (B) maximal Ca2+-activated stress (Maxfit), (C) Hill coefficient (nH), and (D) calcium sensitivity(pCa50). Nested two-way linear mixed models analyses were used to characterize main effects of pCa and treatment group ([ADP] −/+ CPK). In Panel A, there was a significant main effect of pCa (p < 0.001), treatment group (p < 0.001), and a pCa* treatment group interaction (p < 0.001) were observed, with * indicating a Fisher’s LSD post hoc difference between all treatment groups at a single pCa (p < 0.05). † indicates a Fisher’s LSD post hoc difference between the treatment group of 5 mM ADP without CP/CPK and all other treatment groups at a single pCa (p < 0.05). Nested one-way linear mixed models were utilized to characterize the main effect of treatment group ([ADP] −/+ CPK) in Panels B, C, and…

• 

  Figure 7. Viscoelastic myocardial stiffness at maximal Ca2+-activation. (A) Elastic and (B) viscous modulus values were plotted against frequency for 0 and 5 mM [ADP] in the presence and absence of CP and CPK at pCa 4.8. Nested two-way linear mixed models analysis was used to assess main effects of frequency, treatment group ([ADP] ± CP/CPK) and the frequency * treatment group interaction. In both panels there was a main effect of frequency (p < 0.001), treatment (p < 0.001), and their interaction (p < 0.001). Within both panels Fisher’s LSD post hoc analysis indicated all treatment groups were significantly different from each other at a single frequency (p < 0.05, where obviously different) except at intercept points where moduli values cross over each other. n fibers were 13 for 0 mM ADP (+CP/CPK), 12 for 0 mM ADP (+CP/CPK), and 11 for 5 mM ADP (+CP/CPK), from 2 hearts.