Protein kinase A mediated modulation of acto-myosin kinetics.

The effects of protein kinase A (PKA) mediated phosphorylation on thin filament and cross-bridge function is not fully understood. To delineate the effects of troponin I (TnI) phosphorylation by PKA on contractile protein performance, reconstituted thin filaments were treated with PKA. With the use of the in vitro motility assay, PKA treated thin filament function was assessed relative to non-phosphorylated thin filaments in a calcium-regulated system. At maximal calcium activation, unloaded shortening velocity and force did not differ between the groups. However, at submaximal activation, an increase in calcium sensitivity of the thin filament was observed for velocity but a decrease in calcium sensitivity was observed for force. Activation of the thin filament by myosin strong-binding did not elicit a calcium-independent effect. The rightward shift in calcium sensitivity for force and the leftward shift in calcium sensitivity for velocity indicate that PKA phosphorylation of TnI directly modulates the kinetics of the myosin cross-bridge. In addition, the altered velocity dependence on thin filament length implicates reduced myosin cross-bridge binding with PKA treatment. These data highlight the importance of TnI serine 23 and 24 phosphorylation in the modulation of cardiac function.

Activation of the calcium-regulated thin filament by myosin strong binding.

The current study was undertaken to investigate the relative contribution of calcium and myosin binding to thin filament activation. Using the in vitro motility assay, myosin strong binding to the thin filament was controlled by three mechanisms: 1), varying the myosin concentration of the motility surface, and adding either 2), inorganic phosphate (Pi) or 3), adenosine diphosphate (ADP) to the motility solutions. At saturating myosin conditions, Pi had no effect on thin filament motility. However, at subsaturating myosin concentrations, velocity was reduced at maximal and submaximal calcium in the presence of Pi. Adding ADP to the motility buffers reduced thin filament sliding velocity but increased the pCa(50) of the thin filament. Thus by limiting or increasing myosin strong binding (with the addition of Pi and ADP, respectively), the calcium concentration at which half maximal activation of the thin filament is achieved can be modulated. In experiments without ADP or Pi, the myosin concentration on the motility surface required to reach maximal velocity inversely correlated with the level of calcium activation. Through this approach, we demonstrate that myosin strong binding is essential for thin filament activation at both maximal and submaximal calcium levels, with the relative contribution of myosin strong binding being greatest at submaximal calcium. Furthermore, under conditions in which myosin strong binding is not rate limiting (i.e., saturating myosin conditions), our data suggest that a modulation of myosin cross-bridge kinetics is likely responsible for the graded response to calcium observed in the in vitro motility assay.

Myosin from failing and non-failing human ventricles exhibit similar contractile properties.

In non-failing human myocardium, V1 myosin comprises a small amount (<10%) of the total myosin content, whereas end-stage failing hearts contain nearly 100% V3 myosin. It has been suggested that this shift in V1 myosin isoform content may contribute to the contractile deficit in human myocardial failure. To test this hypothesis, myosin was isolated from human failing and non-failing ventricles, and non-failing atria. Performance was assessed in in vitro motility and isometric force assays. Consistent with prior reports, a small amount of V1 myosin was present in both non-failing (6.2 +/- 1.0%) and failing (3.5 +/- 1.4%) ventricular tissues. No difference in isometric force or unloaded shortening velocity was observed for failing and non-failing ventricular myosin irrespective of myosin isoform content. Atrial tissue expressing predominantly V1 myosin (66.7 +/- 4.1%) generated half the force but greater velocity compared with ventricular tissue, expressing predominantly V3 myosin. In additional experiments, rabbit cardiac myosin was used in a calcium regulated assay system to determine if V1 and V3 isoforms differentially affect thin filament activation. Half-maximal calcium activation was similar for the two cardiac isoforms. A 1:9 mixture of V1/V3 myosin, simulating isoform composition in non-failing human myocardium, was indistinguishable from 100% V3 myosin (simulating the failing state) with regard to velocity of shortening and average force. These data suggest that the myosin isoform shift reported in human myocardial failure does not significantly contribute to the contractile deficit of this disease.

Altered myocardial thin-filament function in the failing Dahl salt-sensitive rat heart: amelioration by endothelin blockade.

BACKGROUND: Dahl salt-sensitive rats fed a high-salt diet develop compensated left ventricular hypertrophy followed by a transition to myocardial failure. We previously reported an increase in a troponin T isoform (TnT3) and a decrease in TnT phosphorylation in failing Dahl salt-sensitive rat hearts compared with low-salt controls. The present study was undertaken to determine whether the thin filament plays a role in depression of the contractile machinery in this model. METHODS AND RESULTS: Native thin filaments (NTFs) were isolated intact from rats with compensated left ventricular hypertrophy and failing hearts and compared with age-matched controls. NTF velocity was measured as a function of free calcium in the in vitro motility assay. Maximal velocity was similar in all groups. However, NTFs from failing hearts demonstrated a reduction in calcium sensitivity compared with controls, as reflected in the pCa50 (5.88+/-0.05 versus 6.22+/-0.05, respectively, P<0.001). No difference in thin-filament motility (pCa50, V(max)) was observed in rats with compensated left ventricular hypertrophy compared with controls. Protein kinase A treatment of NTFs from control and failing hearts had no effect on thin-filament calcium sensitivity. However, the endothelin receptor blocker bosentan prevented the reduction in thin-filament calcium sensitivity found in failing hearts. CONCLUSIONS: The thin filament is a key modulator of contractile performance in the transition to failure in the Dahl salt-sensitive rat model. The alteration in thin-filament function may be mediated by an endothelin-triggered pathway potentially affecting protein kinase C signaling.

Cardiac troponin T isoforms demonstrate similar effects on mechanical performance in a regulated contractile system.

Alteration of troponin T (TnT) isoform expression has been reported in human and animal models of myocardial failure. The two adult beef cardiac TnT isoforms (TnT(3) and TnT(4)) were isolated for comparative functional analysis. Thin filaments were reconstituted containing pure populations of the isoforms. The in vitro motility assay was used to directly compare the effect of the two TnT isoforms on force and unloaded shortening as a function of free calcium. We found no significant differences between the two isoforms in terms of calcium sensitivity, cooperativity, or maximal activation (velocity and force) as assessed in a fully calcium-regulated system. Activation by myosin strong binding was similar for thin filaments containing either of the two TnT isoforms. Whereas maximally activated velocity and cooperativity was depressed at pH 6.5, no difference between thin filaments containing the two isoforms was detected. From the small magnitude of the TnT isoform shifts detected in myocardial failure and the lack of significant mechanical effect detected in the motility assay, variable TnT isoform expression is unlikely to be any functional significance in heart failure.