Two cardiomyopathic mutations were expressed in human cardiac actin, using a Baculovirus/insect cell system; E99K is associated with hypertrophic cardiomyopathy whereas R312H is associated with dilated cardiomyopathy. The hypothesis that the divergent phenotypes of these two cardiomyopathies is associated with fundamental differences in the molecular mechanics and thin filament regulation of the underlying actin mutation was tested using the in vitro motility and laser trap assays. In the presence of troponin (Tn) and tropomyosin (Tm), β-cardiac myosin moved both E99K and R312H thin filaments at significantly (p<0.05) slower velocities than wild type (WT) at maximal Ca ++ . At submaximal Ca ++ , R312H thin filaments demonstrated significantly increased Ca ++ sensitivity (pCa 50 ) when compared to WT. Velocity as a function of ATP concentration revealed similar ATP binding rates but slowed ADP release rates for the 2 actin mutants compared to WT. Single molecule laser trap experiments performed using both unregulated (i.e. actin) and regulated thin filaments in the absence of Ca ++ revealed that neither actin mutation significantly affected the myosin's unitary step size (d) or duration of strong actin binding (t on ) at 20 μM ATP. However, the frequency of individual strong-binding events in the presence of Tn and Tm, was significantly lower for E99K than WT at comparable myosin surface concentrations. The cooperativity of a second myosin head binding to the thin filament was also impaired by E99K. In conclusion, E99K inhibits the activation of the thin filament by myosin strong-binding whereas R312H demonstrates enhanced calcium activation.

The modulatory role of whole cardiac myosin binding protein-C (cMyBP-C) on myosin force and motion generation was assessed in an in vitro motility assay. The presence of cMyBP-C at an approximate molar ratio of cMyBP-C to whole myosin of 1:2, resulted in a 25% reduction in thin filament velocity (P<0.002) with no effect on relative isometric force under maximally activated conditions (pCa 5). Cardiac MyBP-C was capable of inhibiting actin filament velocity in a concentration-dependent manner using either whole myosin, HMM or S1, indicating that the cMyBP-C does not have to bind to myosin LMM or S2 subdomains to exert its effect. The reduction in velocity by cMyBP-C was independent of changes in ionic strength or excess inorganic phosphate. Cosedimentation experiments demonstrated S1 binding to actin is reduced as a function of cMyBP-C concentration in the presence of ATP. In contrast, S1 avidly bound to actin in the absence of ATP and limited cMyBP-C binding, indicating that cMyBP-C and S1 compete for actin binding in an ATP-dependent fashion. However, based on the relationship between thin filament velocity and filament length, the cMyBP-C induced reduction in velocity was independent of the number of crossbridges interacting with the thin filament. In conclusion, the effects of cMyBP-C on velocity and force at both maximal and submaximal activation demonstrate that cMyBP-C does not solely act as a tether between the myosin S2 and LMM subdomains but likely affects both the kinetics and recruitment of myosin cross-bridges through its direct interaction with actin and/or myosin head.

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Intraperitoneal instillation of lidocaine was effective in reducing postoperative pain after minor gynecological laparoscopic procedures.

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