Methods for assessing cardiac myofilament calcium sensitivity

Figures

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  FIGURE 1. Illustration of reaction states for cross-bridge formation that are relevant for evaluating the constituent components of force-Ca2+ dynamics. State (A) represents the absence of Ca2+, and the myosin-binding sites of actin molecules (top panel) are sterically inhibited by tropomyosin and the troponin complex, so myosin-actin binding does not occur (lower panel). Once Ca2+ binds to TnC, a conformational change in tropomyosin and troponin removes the steric hindrance (B), which makes actin-myosin binding possible (C). The transition between (A) and (B) is described by the kinetic rates for the association (k Ca on) and dissociation (k Ca off) of Ca2+ to and from TnC, respectively (orange dashed box). The transition between States (B) and (C) is described by the kinetic rates associated with the attachment (k XB on) and detachment (k XB off) of cross-bridges (blue dashed box). Force-dependent cooperativity is indicated by the dotted black arrow, where the force by crossbridges modulates Ca2+-TnC binding affinity. This figure was created with BioRender.com.

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  FIGURE 2. (A) An increase in myofilament sensitivity is associated with a left shift of the force-Ca2+ curve (blue to orange), quantifiable as EC50. (B) A change in responsiveness alone would not change sensitivity and, hence, would not change the EC50 of steady-state force-Ca2+ curves. The Hill equation (Eq. 1) used to fit experimental data contains these parameters: F max is the maximum force, n is the Hill coefficient, which indicates the steepness, or cooperativity, of the relation, and EC50 is the concentration of Ca2+ associated with half-maximal force production (dotted line).

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  FIGURE 3. Potassium contracture techniques. Panels (A) and (B) are replicated from Varian et al. (2012). Panels (C) and (D) are reproduced from Jones et al. (2023). (A) The contracture protocol of Varian et al. exposes muscle to a high K+ superfusate solution (shaded region), with the electrical stimulation (at 1 Hz) remains on (Varian et al., 2012). Force twitches diminish upon contracture and re-develop upon washout of the high K+ superfusate. (B) Force-[Ca2+]i data were obtained from the rising (upstroke) phase of the contracture (shaded region in panel (A). The 4 Hz data were positioned left to that of the 1 Hz, which was interpreted by Varian et al. as reduced myofilament Ca2+ sensitivity (i.e., frequency-dependent myofilament desensitization). (C) The high-K+ contracture protocol of the Ward lab (Jones et al., 2023) includes high caffeine with the electrical stimulation halted. (D) Force-[Ca2+]i data were taken from the downstroke phase of the contracture (indicated by the dotted rectangle in panel (C) for curve fitting (red line) for the estimation of EC50 (dotted red line).

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  FIGURE 4. (A) Steady-state force-Ca2+ (black) and peak-force-Ca2+ relations (white) are not equivalent. (B) Isometric force-Ca2+ phase-plane loops at three different [Ca2+]o are also not equivalent to the steady-state relation (sigmoid). Steady-state force-Ca2+ relation was measured using ryanodine-induced tetani. Reproduced from Backx et al. (1995).

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  FIGURE 5. Comparison of phase-plane loop morphology under conditions where Ca2+ sensitivity is unchanged using a biophysical model (Rice et al., 2008). Temperature-dependent Ca2+ transients (25°C and 37°C) were obtained from Chung and Campbell (2013), scaled to align with the force-Ca2+ relation of the biophysical model (blue (25°C) and orange (37°C) dashed lines), and fit with a Ca2+ transient function, defined by two decaying exponentials with parameters τ1, τ2, Cadias, Caamp, and t start (see Appendix of Rice et al. (2008)). The parameter set for the “normal” Ca2+ transient at each temperature was τ1 = 34.9 ms, τ2 = 183.3 ms, Cadias = 0.078 µM, Caamp = 0.70 µM, and t start = 96.4 ms (25°C Ca2+ transient); τ1 = 54.4 ms, τ2 = 31.8 ms, Cadias = 0.21 µM, Caamp = 0.72 µM, and t start = 100 ms (37°C Ca2+ transient). The influence of Ca2+ handling on phase-plane loop morphology is illustrated in panels (A) and (B), where the amplitude of the “normal” temperature-dependent Ca2+ transient (blue) was increased (green) and decreased (yellow) by 10% by changing the Caamp parameter. These Ca2+ transients were then provided as an input to the biophysical model (upper insets; (A) and (B). The…

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  FIGURE 6. Model simulation of an increase in Ca2+ myofilament sensitivity to test identifiability. The myofilament model (Op Den Buijs et al., 2008) takes a Ca2+ transient (A) as input and produces an LV pressure transient (B) as output; a parametric plot of these two variables produces a phase-loop (C). The Ca2+ transient function is defined by two decaying exponentials with parameters τ1 = 80 ms, τ2 = 40 ms, Cadias = 0.12 µM, Caamp = 0.3 µM, and t start = 50 ms (see Appendix of Rice et al. (2008)). The myofilament model was used to simulate two sets of positive inotropic responses corresponding to an increase in the Ca2+ binding affinity by 25% (orange open circles) or 50% (green open circles) relative to control (blue line). The model parameters for the control model were taken from Table 2 of Op Den Buijs et al. (2008): k 1 = 38 s−1, k 3 = 361 s−1, α = 0.29 nM−2s−1, β = 658, k d = 212 s−1, and Cat = 117 nM. For generation of the simulation data, all parameters were kept the same except for k 1, which was increased by either 25% or 50%. The model was then fitted individually to these two data sets by constraining the Ca2+ parameters (k 1, k 3, Cat) to be unchanged from control and…