The Mechanics and Mechanisms of Regulation in Striated Muscle

Activation of striated muscle is a calcium-dependent process, with initial studies describing calcium binding as a switch that can turn regulated thin filaments "on" or "off". The basic regulatory apparatus is an actin-associated complex composed of tropomyosin (Tm) and troponin (Tn).

While early structural studies described regulation in terms of a three state model for the thin filament (blocked, closed, and open) this basic model lacks the ability to explain much of the behavior observed in striated muscle regulation. Developing more explicit and complex models is an area fraught with difficulty, and has been attempted by many groups. Here, we simplify the system in an elegant way to give a remarkably accurate description of a wide range of experimental behaviors using actin sliding velocities as an approximation of the level of activation observed in a system of thin filaments. Through the development of a simple steady-state model, we are able to define the level of activation of a thin filament and account for much of the behavior witnessed in regulation using only well defined parameters of actin-myosin binding kinetics and calcium regulation of striated muscle.

To measure the level of activation of a regulated thin filament, we use the in vitro motility assay, where we can measure the sliding velocities of a thin filament over a bed of myosin heads. By measuring the velocity as a function of the working calcium concentration, we can obtain the level of activation (P1) as well as the pCa50 and hill coefficient - the values which describe calcium sensitivity of muscle regulation.

The major advantage of this system is that it allows us to measure specific changes to myosin's attachment and detachment rate independently. What we have found through this is that when perturbations are made (either through mutations, chemical perturbations, or protein modification) the calcium sensitivity changes with the kinetics of actin-myosin binding. Using this information we were able to develop a minimal model that approximates the level of activation through actin sliding velocity measurements. The model states that partial activation is a function of maximal activation times the probability of actin-myosin binding, which is described by the classical approximations of actin-myosin attachment and detachment rates, as well as calcium binding to troponin.

While fascinating alone, the implications of such a model are far reaching. A multitude of diseases exist that can be directly attributed to mutations affecting the contractile apparatus such as skeletal muscle myopathies (nemaline), cardiac myopathies (familial hypertrophic (FHC) and dilated (DMC)), and distal arthrogryposis (DA). Many of these diseases exhibit behavior identical to that witnessed through our chemical perturbations of changing muscle kinetics. This model not only provides a powerful tool for analyzing these diseases and understanding the mechanics that underlie them, but also provides new perspectives and interpretations towards their treatment.

As we continue our research, we hope to further expand this model to incorporate changes in calcium sensitivity at the cellular level using intact muscle fiber mechanics, as well as correlate changes in calcium sensitivity at the cellular and ensemble molecule levels to changes in single molecule binding studies. By elucidating these relationships, we will establish a comprehensive model of thin filament regulation that can explain calcium sensitivity at all levels of muscle organization.

-Nicholas Sich, 2010