, Eq. (1)

where F_pas corresponds to passive stress under relaxed conditions; F_act corresponds to maximal Ca²⁺-activated stress; pCa₅₀represents the free Ca²⁺ concentration required to develop half the maximum Ca²⁺-activated stress, and n_H is the Hill coefficient.

Sinusoidal length-perturbations of 0.125% myocardial strip length (clip-to-clip) were applied at 41 discreet frequencies from 0.125-100 Hz to measure the complex modulus as a function of angular frequency (Kawai and Brandt, 1980; Mulieri et al., 2002; Palmer et al., 2007). The complex modulus represents viscoelastic myocardial stiffness, which arises from the change in stress divided by the change in muscle length that is in-phase (elastic modulus) and out-of-phase (viscous modulus) with the oscillatory length change at each frequency.

Characteristics of the elastic and viscous moduli responses over the measured frequency range provide a signature of cross-bridge binding and cycling kinetics. Shifts in the elastic modulus are useful for assessing changes in the number of bound cross-bridges between experimental conditions. Shifts in the viscous modulus are useful for assessing changes in the work-producing and work-absorbing characteristics of the myocardium that arise from force-generating cross-bridges. Frequencies producing negative viscous moduli represent regions of work-producing muscle function. The “dip frequency” or frequency of the minimum viscous modulus describes force-generating events and cross-bridge recruitment rate (Mulieri et al., 2002; Campbell et al., 2004). Frequencies producing positive viscous moduli represent regions of work-absorbing muscle function. The “peak frequency” or frequency of the maximum viscous modulus describes cross-bridge distortion events and cross-bridge detachment rate (Campbell et al., 2004; Palmer et al., 2007, 2011). These characteristic regions of minima and maxima in the viscous modulus vs. frequency relationship were used to assess effects of mavacamten on cross-bridge kinetics under maximal Ca²⁺-activated conditions. Given that viscous moduli were only measured at discrete frequencies, these regions of minima viscous modulus (using 0.125-4 Hz data) and maxima viscous modulus (using 3-40 Hz data) were fit to a polynomial using MATLAB to create fitted curves at 0.05 Hz resolution. From these interpolated curves we extracted the frequency of minimum viscous modulus and frequency of maximum viscous modulus.