Effects of the mutation R145G in human cardiac troponin I on the kinetics of the contraction-relaxation cycle in isolated cardiac myofibrils.
Familial hypertrophic cardiomyopathy (FHC) has been linked to mutations in sarcomeric proteins such as human cardiac troponin I (hcTnI). To elucidate the functional consequences of the mutation hcTnI(R145G) on crossbridge kinetics, force kinetics were analysed in murine cardiac myofibrils carrying either the mutant or the wild-type protein. The mutation was introduced into the myofibrils in two different ways: in the first approach, the endogenous Tn was replaced by incubation of the myofibrils with an excess of reconstituted recombinant hcTn containing either hcTnI(WT) or hcTnI(R145G). Alternatively, myofibrils were isolated either from non-transgenic or transgenic mice expressing the corresponding mcTnI(R146G) mutation. In myofibrils from both models, the mutation leads to a significant upward shift of the passive force-sarcomere length relation determined at pCa 7.5. Addition of 5 mm BDM (2,3-butandione-2-monoxime), an inhibitor of actomyosin ATPase partially reverses this shift, suggesting that the mutation impairs the normal function of cTnI to fully inhibit formation of force-generating crossbridges in the absence of Ca(2)(+). Maximum force development (F(max)) is significantly decreased by the mutation only in myofibrils exchanged with hcTnI(R145G) in vitro. Ca(2)(+) sensitivity of force development was reduced by the mutation in myofibrils from transgenic mice but not in exchanged myofibrils. In both models the rate constant of force development k(ACT) is reduced at maximal [Ca(2)(+)] but not at low [Ca(2)(+)] where it is rather increased. Force relaxation is significantly prolonged due to a reduction of the relaxation rate constant k(REL). We therefore assume that the impairment in the regulatory function of TnI by the mutation leads to modulations in crossbridge kinetics that significantly alter the dynamics of myofibrillar contraction and relaxation.
Kruger
M
M
Department of Vegetative Physiology, University of Cologne, Koeln, Germany. makruger@uni-muenster.de
Zittrich
S
S
Redwood
C
C
Blaudeck
N
N
James
J
J
Robbins
J
J
Pfitzer
G
G
Stehle
R
R
D000818:Animals; D001120:Arginine / Q000235:genetics*; D005998:Glycine / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D009119:Muscle Contraction / Q000235:genetics* / Q000502:physiology; D009126:Muscle Relaxation / Q000235:genetics / Q000502:physiology; D009154:Mutation; D009206:Myocardium; D009210:Myofibrils / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D019210:Troponin I / Q000235:genetics* / Q000502:physiology
D019210:Troponin I; D001120:Arginine; D005998:Glycine
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