In vitro Reconstitution of Skeletal Muscle Contraction using Native Thin Filaments

Abstract

Intracellular Ca2+ concentration regulate the muscle contraction by changing the conformation of Tropomyosin molecule over Actin filament. In this work we have purified native thin filaments (NTF) from Chicken Pectoralis muscle and reconstituted in in vitromotility assay (IVMA) with skeletal muscle myosin II. NTF prepared were 1.67±0.14 µm in length and showed good regulation under different Ca2+ concentrations. At pCa 4, more than 90% of NTF were continuously sliding and when solution changed to pCa 9 all the filaments were rigidly stuck to surface. Sliding velocity of NTF was VF=5.63±1.21 µm/s at 30 oC and 17% higher compared to unregulated actin filaments. Fraction of sliding filament showed a direct correlation with pCa of buffer and pCa50= 6.69 was obtained using Hills equation fit. However the sliding filament velocity was not affected under different pCa concentration. Under varying substrate [MgATP] conditions in IVMA we found k+ATP is 4.3± 0.56 x 106 M−1s−1 and k-ADP is 404.33±2.7 s−1 at 30 oC. These kinetic parameters are comparable to values obtained using unregulated actin filaments, suggesting the detachment kinetics of myosin II is not affected due to Ca2+ regulation. We studied the effect of myosin density on thin filaments activation in IVMA. Even with fully activated thin filament (pCa 4.0), decrease in myosin density leads to increase in fraction of stuck filaments. Decrease in fraction of motile filaments with myosin density in IVMA suggests there is minimum number of myosin head binding required to completely activate the NTF.Supported by Department of Science and Technology, Government of India.