Force, sarcomere shortening velocity and ATPase activity.
We have tested the hypothesis that the transition rate (G) of the cardiac XB from the strong force generating state to the weak state is a linear function V of the sarcomere (VSL); furthermore, we tested whether the ATPase rate of the two isoforms of myosin can be held responsible for the difference between V0 of rat cardiac trabeculae containing V1 isomyosin versus those containing V3 isomyosin.


METHODS
V1 isomyosin was induced by thyroid hormone treatment of the rats for 2 weeks, V3 isomyosin by PTU treatment for 1 month. Force was measured with a strain gauge in trabeculae from the rat right ventricle in K-H solution ([Ca]o=1.5 mM, 25 degrees C). Sarcomere length (SL) was measured with laser diffraction techniques. Twitch force at constant SL, and the force response to shortening at constant VSL (0-8 microm/s; deltaSL 50-100 nm) were measured at varied time during the twitch.


RESULTS
The force response to shortening consisted of a fast initial exponential decline (tau = 2 ms) followed by a slow decrease of F. The instantaneous difference (deltaF) between isometric force (FM) and the declining force depended on shortening duration (deltat), VSL and instantaneous FM: deltaF = G1 x FM x deltat x VSL x (1-VSL/VMAX), where VMAX is the unloaded VSL and G1 was 6.15 +/- 2.12 microm(-1) (mean +/- s.d.; n=6). deltaF/FM was independent of the time onset of shortening. G1 of V1 and V3 trabeculae did not differ. V0 of V1 and V3 trabeculae differed 2-2.5 fold, as did both the ATPase rate and the velocity of actin sliding in a motility assay of the myosin purified from V1 or V3 hearts. The temperature dependence of the ATPase rate (Q10: 4.03 and 4.33, respectively; n.s.) was similar to that of V0 that has previously been reported for predominantly V1 trabeculae. Cross-linking of actin to myosin with the short chain cross linker EDC increased the ATPase rate of the two isomyosins (200-fold and 600-fold respectively) to exactly the same final level and reduced their Q10 by 50%.


CONCLUSIONS
The linear interrelation between deltaF and VSL is consistent with feedback, whereby XB kinetics depends on VSL. This feedback provides an integrated description of cardiac muscle mechanics and energetics. The results, also, suggests that it is unlikely that the hydrolytic domain of the cross bridge determines V0 and warrant ongoing experiments to investigate the role of the actin binding domain of the XB in cardiac sarcomere kinetics. In order to further investigate the role of the actin binding domain, we have expressed chimeric cardiac myosin, co-assembled with MLC, by mutual substitution of actin binding loop on alpha MHC and beta MHC.
ter Keurs
Henk E D J
HE
Department of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa, Israel.
Deis
Nathan
N
Landesberg
Amir
A
Nguyen
The-Tin T
TT
Livshitz
Leonid
L
Stuyvers
Bruno
B
Zhang
Mei Luo
ML
D000199:Actins / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D006352:Heart Ventricles / Q000473:pathology; D015151:Immunoblotting; D007313:Insecta; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009218:Myosins / Q000737:chemistry; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D011914:Rats, Inbred BN; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry; D012518:Sarcomeres / Q000737:chemistry / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D000199:Actins; D018076:DNA, Complementary; D020033:Protein Isoforms; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium