Fluorescence lifetime of actin in the familial hypertrophic cardiomyopathy transgenic heart.
Clinical studies have revealed that the D166V mutation in the ventricular myosin regulatory light chain (RLC) can cause a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). It has been proposed that RLC induced FHC in the heart originates at the level of the myosin cross-bridge due to alterations in the rates of cross-bridge cycling. In this report, we examine whether the environment of an active cross-bridge in cardiac myofibrils from transgenic (Tg) mice is altered by the D166V mutation in RLC. The cross-bridge environment was monitored by tracking the fluorescence lifetime (tau) of Alexa488-phalloidin-labeled actin. The fluorescence lifetime is the average rate of decay of a fluorescent species from the excited state, which strongly depends on various environmental factors. We observed that the lifetime was high when cross-bridges were bound to actin and low when they were dissociated from it. The lifetime was measured every 50 ms from the center half of the I-band during 60 s of rigor, relaxation and contraction of muscle. We found no differences between lifetimes of Tg-WT and Tg-D166V muscle during rigor, relaxation and contraction. The duty ratio expressed as a fraction of time that cross-bridges spend attached to the thin filaments during isometric contraction was similar in Tg-WT and Tg-D166V muscles. Since independent measurements showed a large decrease in the cross-bridge turnover rate in Tg-D166V muscle compared to Tg-WT, the fact that the duty cycle remains constant suggests that the D166V mutation of RLC causes a decrease in the rate of cross-bridge attachment to actin.
Mettikolla
P
P
Department of Molecular Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, 3500 Camp Bowie Blvd., Fort Worth, Texas 76107, USA.
Luchowski
R
R
Gryczynski
I
I
Gryczynski
Z
Z
Szczesna-Cordary
D
D
Borejdo
J
J
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D024741:Cardiomyopathy, Hypertrophic, Familial / Q000503:physiopathology*; D005453:Fluorescence; D006321:Heart / Q000503:physiopathology*; D051379:Mice; D008822:Mice, Transgenic; D009200:Myocardial Contraction / Q000502:physiology; D009210:Myofibrils / Q000473:pathology; D010590:Phalloidine / Q000378:metabolism; D012298:Rigor Mortis / Q000503:physiopathology; D013997:Time Factors
D000199:Actins; D010590:Phalloidine