Expression and purification of human cardiac troponin subunits and their functional incorporation into isolated cardiac mouse myofibrils

The three subunits of the human cardiac troponin complex (hcTnC, hcTnI, hcTnT) were overexpressed in E. coli, purified and reconstituted to form the hcTn complex. This complex was then incorporated into subcellular bundles of mouse cardiac myofibrils whereby the native mcTn complex was replaced. On thus exchanged myofibrils, isometric force kinetics following sudden changes in free Ca2+ concentration were measured using atomic force cantilevers. Following the exchange, the myofibrillar force remained fully Ca2+ regulated, i.e. myofibrils were completely relaxed at pCa 7.5 and developed the same maximum Ca2+-activated isometric force upon increasing the pCa to 4.5 as unexchanged myofibrils. The replacement of endogenous mcTn by wild-type hcTn neither altered the kinetics of Ca2+-induced force development of the mouse myofibrils nor the kinetics of force relaxation induced by the sudden, complete removal of Ca2+. Preparations of functional Tn reconstituted myofibrils provide a promising model to study the role of Tn in kinetic mechanisms of cardiac myofibrillar contraction and relaxation.