Dynamic interaction between cardiac myosin isoforms modifies velocity of actomyosin sliding in vitro.

Abstract

To study the functional significance of cardiac isomyosin heterogeneity, active sliding of actin-myosin was studied using two different types of in vitro motility assay systems: (1) a sliding actin filament assay, in which fluorescently labeled actin filaments were made to slide on a myosin layer attached to a glass coverslip, and (2) a myosin-coated bead assay, in which myosin-coated latex beads were made to slide on actin cables of an alga. Two different isomyosins were obtained from 3-week-old (V,) and hypothyroid (V3) rat hearts and were mixed to form solutions with various mixing ratios [Vj1(V1+V3)]. For these myosin mixtures, both ATPase activity and sliding velocity of actin-myosin were determined. As the relative content of V, increased, both ATPase activity and velocity increased. However, in contrast to the linear relation between the mixing ratio and ATPase activity, the relation between the mixing ratio and sliding velocity was sigmoid, suggesting the existence of mechanical interaction between different isomyosins. To clarify the nature of this interaction, sliding velocity was measured for mixtures ofV1 andp-N,N'-phenylenedimaleimide-treated V1 myosin (pPDM-M). A convex relation was observed between the relative content of pPDM-M and velocity. Because pPDM-M is known to form a noncycling and weakly bound crossbridge with actin, it is expected to exert a constant internal load on V,, in contrast to the actively cycling V3. In conclusion, in actomyosin sliding, different isomyosins mechanically interact when they coexist. The interaction may be a dynamic one that cannot be explained by a simple load effect. (Circ Res. 1993;73:696-704.) KEY WoRDs * myosin * isoform * actin * heterogeneity * in vitro motility assay T hree different isoforms (V1, V2, and V3) have been identified in mammalian cardiac myosin based on their electrophoretic mobility on pyrophosphate polyacrylamide gels.' Biochemically, V, is characterized as having the highest ATPase activity, and V3, the lowest. Later studies2,3 revealed that these isoforms consist of only two heavy chains named a and 13; ie, V, and V3 are the homodimers of a and 13, respectively, and V2 is the heterodimer of a and 13. Because the relative content of these isoforms changes (myosin heterogeneity) at different stages in development,3-5 in response to the thyroid state,1'6-10 and under the influence of the hemodynamic load,79"1"2 its functional meaning has been discussed repeatedly.7'9-"1 Although these studies unanimously demonstrated a positive correlation between ATPase activity of myosin and the maximum shortening velocity of the muscle or cell, the precise role of myosin heterogeneity remains to be fully understood because of the technical problems inherent in the preparations used in these studies. First, the parts of the tissue with which ATPase assay and mechanical assay were performed were not always identical. Second, because isoform distribution was difficult to control in vivo, only rough relations between Received January 21, 1993; accepted ...

Identification and Characterization of Nonmuscle Myosin II-C, a New Member of the Myosin II Family

“…Significant alterations in cardiac function are associated with isoform shifts, evidenced by changes in the maximum unloaded shortening velocity and ATP consumption by the contractile machinery (34). This is consistent with the different mechanical and enzymatic properties of the two isoforms (1,37,38).…”

Kinetic Differences in Cardiac Myosins with Identical Loop 1 Sequences

“…29). Rat myocytes expressing ␣-MHC were used as controls and exhibited shortening velocities that were 2.2-fold faster than rat myocytes expressing ␤-MHC, which is consistent with previous results from cardiac myocytes (15) and from purified myosin in an in vitro motility assay (30). Remarkably, there was a 3-fold difference in V 0 between rat ␤-MHC and pig ␤-MHC myocytes measured under identical conditions (Table I).…”

Comparison of Unitary Displacements and Forces Between 2 Cardiac Myosin Isoforms by the Optical Trap Technique

“…In accordance with the results for V max in muscle preparations, the unloaded velocity of actin-myosin sliding in vitro has been shown to be much faster for V 1 than for V 3 isoforms. 5,6 Using the centrifuge microscope, with which constant centrifugal forces are applied as loads on in vitro actin-myosin sliding, we showed that the shape of force-velocity curves was markedly different between V 1 and V 3 isoforms, reflecting their different interaction kinetics with actin.7 Recent development of the laser optical trap technique has made it possible to study mechanical events generated by a single myosin molecule as it splits ATP and interacts with actin. 8 -10 In the present study, we used this technique to measure unitary displacements and forces generated by the 2 cardiac myosin isoforms.…”