Mechanical Evaluation of Frozen and Cryo-Sectioned Papillary Muscle Samples by Using Sinusoidal Analysis: Cross-bridge Kinetics and the Effect of Partial Ca 2+ activation

The use of frozen and cryo-sectioned cardiac muscle preparations, introduced recently by (Feng & Jin, 2020), offers promising advantages of easy transport and exchange of muscle samples among collaborating laboratories. In this report, we examined integrity of such preparation by studying tension transients in response to sinusoidal length changes and following concomitant amplitude and phase shift in tension time courses at varying frequencies. We used sections with 70 µm thickness, isolated fiber preparations, and studied cross-bridge (CB) kinetics: we activated the preparations with saturating Ca 2+ , and varying concentrations of ATP and phosphate (Pi). Our experiments have demonstrated that this preparation has the normal active tension and elementary steps of the CB cycle. Furthermore, we investigated the effect of Ca 2+ on the rate constants and found that the rate constant r 4 of the force generation step is proportionate to [Ca 2+ ] when it is < 5 µM. This observation suggests that the activation mechanism can be described by a simple second order reaction. As expected, we found that magnitude parameters including tension and stiffness are related to [Ca 2+ ] by the Hill equation with cooperativity of 4–5, consistent to the fact that Ca 2+ activation mechanisms involve cooperative multimolecular interactions. Our results are consistent with a long-held hypothesis that process C (phase 2 of step analysis) represents the CB detachment step, and process B (phase 3) represents the force generation step. In this report, we further found that constant H may also represent work performance step. Our experiments have demonstrated excellent CB kinetics with reduced noise and well-defined two exponentials, which are better than skinned fibers, and easier to handle and study than single myofibrils.