Distinct Mechanisms for Increased Cardiac Contraction Through Selective Alteration of Either Myosin or Troponin Activity

Figures

• 

  Figure 1. Myofibril Steady-State ATPase Activity (A) The steady-state ATPase activity in whole heart myofibrils isolated from tWt (black), AAA (red), and DDD (blue) transgenic mice. AAA myofibrils exhibit significantly lower steady-state ATPase activity when compared with tWt or DDD myofibrils (1-way analysis of variance, Kruskal-Wallis’ multiple comparison test [∗∗ P < 0.01, ∗∗∗ P < 0.001]). (B to D) Both the M (filled circles) and T (half circles) increased steady-state ATPase activity in myofibrils isolated from all 3 transgenic lines: tWt (B), AAA (C), and DDD (D) (2-way analysis of variance, Sidak’s multiple comparison test [†, ‡P < 0.05, ††,‡‡ P < 0.01, †††,‡‡‡ P < 0.001 for M and T, respectively]). M exhibited a modest but significant increase steady-state ATPase activity when compared with T (2-way analysis of variance, Sidak’s multiple comparison test [∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001]). Data are represented as the group mean ± SEM. The curves were fitted to the equation of log(agonist) vs normalized response -variable slope 4 parameters variable slope model (GraphPad Prism 9.3). Four independent experiments were performed for myofibrils isolated from each transgenic line. AAA…

• 

  Figure 2. M and T Increased Cardiomyocyte Contraction Using Distinct Mechanisms Bar graph comparison of the % change over baseline in response to treatment for (A) cell shortening, (B) contraction time, (C) maximal contraction velocity and (D) maximal relaxation velocity in single cardiomyocytes isolated from Twt (black), AAA (red), and DDD (blue) mice, in presence of 1× (open circles) and 3× (half-filled circles) dose of M (left) and T (right). Statistically significant values were calculated by performing a 1-way analysis of variance, Tukey’s multiple comparison test (†,‡,∗P < 0.05; ††, ‡‡, ∗∗P < 0.01; and †††, ‡‡‡, ∗∗∗P < 0.001 for 1× vs 3× M, 1× Vs 3× T, and M Vs T, respectively). Data represented as the group mean ± SEM. N = 11-15 cardiomyocytes per group. Cells were isolated from 2 to 4 animals for each group. Abbreviations as in Figure 1.

• 

  Figure 3. Force Generation in Skinned Cardiac Papillary Fibers (A and B) Maximal force vs the calcium concentration in presence of M or T produced in skinned papillary fibers isolated from tWt (black), AAA (red), and DDD (blue) at 1× dose (empty circle, dashed line) and 3× dose (half-filled circle, dotted line). (C and D) Bar graph comparison of maximal force produced at pCa 4.5 (C) and calcium sensitivity (D) in skinned cardiac papillary fibers isolated from tWt, AAA, and DDD transgenic mice in the presence of 1× and 3× dose of M and T. Statistically significant values were calculated by performing a 2-way analysis of variance, Tukey’s multiple comparison test (†, ‡, ∗P < 0.05; ††, ‡‡, ∗∗P < 0.01; and †††, ‡‡‡, ∗∗∗P < 0.001 for 1× vs 3× M, 1× vs 3× T, and M vs T, respectively; §P < 0.5, §§P < 0.01, §§§P < 0.001 untreated tWt vs AAA; ¶P < 0.05, ¶¶P < 0.01, ¶¶¶P < 0.001 untreated AAA vs DDD for the equivalent dose). n = 5 fibers per compound treatment per dosage made across fibers isolated from each transgenic line. Data represented as the group mean ± SEM. The equation used to fit the force curves was log(agonist) vs normalized response-variable slope (GraphPad Prism 9.3). Abbreviations as…

• 

  Figure 4. T Increased the Tension Redevelopment Rate (Ktr) (A) One-phase association fit curves for rate of force redevelopment in skinned papillary fibers isolated from transgenic tWt (black), AAA (red), and DDD (blue) at 1× (dashed) and 3× (dotted) concentrations of M (left) and T (right). (B) Bar graph comparison for ktr values across tWt, AAA, and DDD muscle fibers in presence of M (left) and T (right) at 1× (open circles) and 3× (half-filled circles) concentrations. (C) Bar graph comparison of the drop in maximal force across tWt, AAA, and DDD transgenic muscle fibers in presence of M (left graph) and T (right graph) at 1× (open circles) and 3× (half-filled circles). Statistically significant values were calculated by performing a 2-way analysis of variance, Tukey’s multiple comparison test (†, ‡, ∗P < 0.05; ††, ‡‡, ∗∗P < 0.01; and †††, ‡‡‡, ∗∗∗P < 0.001 for 1× vs 3× M, 1× vs 3× T, and M vs T, respectively; ǁP < 0.5, ǁǁP < 0.01, ǁǁǁP < 0.001 untreated tWt vs DDD; ¶P < 0.05, ¶¶P < 0.01, ¶¶¶P < 0.001 untreated AAA vs DDD for the equivalent dose). n = 5 fibers per compound treatment in fibers isolated from each transgenic line. Data represented as the group mean ± SEM. The curves were…

• 

  Figure 5. M Decreased the Mant-ATP Exchange Rate in Skinned Left Ventricle Muscle Fibers Raw traces of fluorescent decay for Mant-ATP exchange with ATP in Twt (black), AAA (red), and DDD (blue) in the presence of (A) M and (B) T at 1× (open circles) and 3× (half-filled circles) concentrations. (C) Bar graphs comparing the slow phase fluorescent decay across Twt, AAA, and DDD muscle fibers in the presence of M (Left) and T (Right) at 1× (open circles) and 3× (half-filled circles) concentrations. Statistically significant values were calculated by performing a 1-way analysis of variance, Tukey’s multiple comparison test (†, ‡ P < 0.05; ††, ‡‡ P < 0.01; and †††, ‡‡‡ P < 0.001 for 1× vs 3× M And 1× vs 3× T, respectively; ǁP < 0.5, ǁǁ P < 0.01, ǁǁǁ P < 0.001 untreated Twt vs DDD; ¶ P < 0.05, ¶¶ P < 0.01, ¶¶¶ P < 0.001 untreated AAA vs DDD for the equivalent dose). N = 9-11 fibers per compound treatment in fibers isolated from each transgenic line. Data represented as the group mean ± SEM. The curves were fitted to custom 2-phase exponential decay model.