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 Ca(2+) concentration were measured using atomic force cantilevers. Following the exchange, the myofibrillar force remained fully Ca(2+) regulated, i.e. myofibrils were completely relaxed at pCa 7.5 and developed the same maximum Ca(2+)-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 Ca(2+)-induced force development of the mouse myofibrils nor the kinetics of force relaxation induced by the sudden, complete removal of Ca(2+). 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.
Krüger
Martina
M
Institute of Vegetative Physiology, University of Cologne, Robert-Koch-Strasse 39, D-50931, Köln, Germany. martina.krueger@uni-koeln.de
Pfitzer
Gabriele
G
Stehle
Robert
R
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D006801:Humans; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism*; D011817:Rabbits; D014336:Troponin / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D005456:Fluorescent Dyes; D014336:Troponin