Biomechanical evaluation of flash-frozen and cryo-sectioned papillary muscle samples by using sinusoidal analysis: cross-bridge kinetics and the effect of partial Ca²⁺ activation

We examined the integrity of flash-frozen and cryo-sectioned cardiac muscle preparations (introduced by Feng and Jin, 2020) by assessing tension transients in response to sinusoidal length changes at varying frequencies (1-100 Hz) at 25 °C. Using 70-μm-thick sections, we isolated fiber preparations to study cross-bridge (CB) kinetics: preparations were activated by saturating Ca²⁺ as well as varying concentrations of ATP and phosphate (Pi). Our results showed that, compared to ordinary skinned fibers, in-series stiffness decreased to 1/2, which resulted in a decrease of isometric tension to 62%, but CB kinetics and Ca²⁺ sensitivity were little affected. The pCa study demonstrated that the rate constant of the force generation step (2πb) is proportionate to [Ca²⁺] at < 5 μM, suggesting that the activation mechanism can be described by a simple second order reaction. We also found that tension, stiffness, and magnitude parameters are related to [Ca²⁺] by the Hill equation, with a cooperativity coefficient of 4-5, which is consistent with the fact that Ca²⁺ activation mechanisms involve cooperative multimolecular interactions. Our results support the long-held hypothesis that Process C (Phase 2) represents the CB detachment step, and Process B (Phase 3) represents the force generation step. Moreover, we discovered that constant H may represent the work-performing step in cardiac preparations. Our experiments demonstrate excellent CB kinetics with two well-defined exponentials that can be more distinguished than those found using ordinary skinned fibers. Flash-frozen and cryo-sectioned preparations are especially suitable for multi-institutional collaborations nationally and internationally because of their ease of transportation.