Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function

Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-cardiac myosin contractility as a result of the R403Q mutation.

Protein kinase A does not alter unloaded velocity of sarcomere shortening in skinned rat cardiac trabeculae

Whether β-adrenergic stimulation affects the cross-bridge cycling rate independently of its effect on Ca2+ handling by the cardiac myocyte is still unknown. An increase in cross-bridge cycling rate may result in increased unloaded velocity of sarcomere shortening ( V o). To test this hypothesis directly, skinned rat cardiac trabeculae were attached between a silicon strain gauge (∼3.5 kHz resonant frequency) and a fast displacement motor. V o was measured by a modified “Edman slack test” during a single maximal activation using seven to eight sarcomere-length step releases (measured by laser diffraction) ranging between 0.12 and 0.20 μm (15.0 ± 0.1°C). β-Adrenergic stimulation was mimicked by exposing the trabeculae to the catalytic subunit of protein kinase A (PKA). Treatment with PKA (3 μg/ml; 45 min) caused a significant ( P < 0.01) increase (41 ± 13%) in the Ca2+concentration required for half-maximal steady-state tension development. Neither maximum tension nor V o was affected by treatment with PKA, suggesting that β-adrenergic stimulation does not affect the rate-limiting step of cross-bridge cycling during unloaded shortening in myocardium.

Myocardial beta-adrenergic and mechanical properties in pacing-induced heart failure in dogs

Forty-eight dogs had pacing overdrive at 250 beats/min for 4-6 wk until heart failure developed. Myocardium from pacing dogs had a decrease in tension and maximum unloaded velocity of shortening (Vmax). Pacing dogs had an increase in circulating catecholamines during exercise but a lower maximal heart rate (214 +/- 19 vs. 241 +/- 26 beats/min, P less than 0.05). A blunted chronotropic response to isoproterenol was also found. However, despite a decrease in beta-adrenergic receptor density (80 +/- 14 vs. 122 +/- 14 fmol/mg, P less than 0.001) and a decrease in beta-adrenergic signal transduction [isoproterenol-induced adenosine 3',5'-cyclic monophosphate (cAMP) production 230 +/- 45 vs. 339 +/- 64 pmol.mg-1.min-1, P less than 0.001], Vmax normalized in response to isoproterenol in pacing dogs (2.3 +/- 0.6 vs. 2.2 +/- 0.5 Lmax/s, NS, where Lmax is length at which maximum developed tension occurs). Tension did not normalize (8 +/- 2 vs. 12 +/- 2 g/mm2, P less than 0.001). Thus in this model of heart failure, despite widespread evidence of decreased beta-adrenergic signal transduction, indexes of shortening but not force generation normalize in response to isoproterenol.

Unloaded shortening velocity of skinned rat myocardium: effects of volatile anesthetics

The slack test method has been adapted for measurement of unloaded velocity of shortening in rat ventricular trabeculae that were skinned with saponin (50 micrograms/ml for 30 min). The method was sensitive enough to detect a 17% reversible change in the unloaded velocity of shortening produced by a 3 degrees C change in temperature. At pCa 5.30 (80-90% activation), halothane, enflurane, and isoflurane each slowed the shortening velocity by 25-30% at dose levels of 8 mM or greater but not at 4 mM or less. At pCa 5.48 (50-60% activation), halothane slowed the shortening velocity by 20-45% at dose levels of 4 mM or greater but not at 2 mM. The slowing effect of anesthetics on shortening velocity showed saturation at 8 mM for halothane, enflurane, and isoflurane when activation was at pCa 5.30. Saturation occurred at 4 mM for halothane when the pCa was 5.48. This result indicates that the dose-response relationship may be narrow, such that it can be demonstrated between 2 and 4 mM halothane for pCa 5.48 and between 4 and 8 mM halothane for pCa 5.30. The anesthetic dose dependence of isometric force and length axis intercept did not generally follow the same relationship as for the shortening velocity. Thus in several instances force did not significantly decrease when the velocity of shortening did. This may be interpreted as lack of simple inhibition by anesthetics on the number of interacting cross-bridges and as direct influence by anesthetics on the cross-bridge cycle.

Effect of theophylline on the velocity of diaphragmatic muscle shortening

The present study examined the effect of theophylline on the shortening velocity of submaximally activated diaphragmatic muscle (i.e., muscles were activated by the use of a level of stimulation, 50 Hz, within the range of phrenic neural firing frequencies achieved during breathing, whereas maximum activation is achieved at 300 Hz). Experiments were performed in vitro on strips of diaphragmatic muscle obtained from 21 Syrian hamsters. Muscle shortening velocity was assessed during isotonic contractions against a range of afterloads, and Hill's characteristic equation was used to calculate velocity at zero load. In addition, unloaded shortening velocity was also measured by the slack test, i.e., from the time required for muscles to take up slack after a sudden reduction in muscle length. Theophylline (160 mg/l) increased the velocity of muscle shortening against a wide range of external loads (0-14 N/cm2) and increased the extrapolated unloaded velocity of shortening from 6.4 +/- 0.9 to 7.9 +/- 1.1 (SE) lengths/s (P less than 0.01). Theophylline reduced the time required to take up slack for any given step change in muscle length, increasing the unloaded velocity of shortening assessed by the slack test from 7.6 +/- 0.9 to 9.3 +/- 1.1 lengths/s (P less than 0.002). The effect of theophylline on diaphragmatic shortening velocity was evident at concentrations as low as 40 mg/l and increased progressively as theophylline concentrations were increased to 320 mg/l. Theophylline increased the shortening velocity of fatigued as well as fresh muscles.(ABSTRACT TRUNCATED AT 250 WORDS)