Investigating the relaxation rate, following diazo-2 photolysis, of a skinned trabecular preparation from guinea-pig hypertrophied left ventricle.

Cardiac hypertrophy in the guinea-pig is not accompanied by a large shift in the expression of the predominant isoform of myosin in the left ventricle; however, in this species, thin filament proteins do change. We examined the relaxation, following laser flash photolysis of the photolabile caged Ca2+ chelator diazo-2, of a skinned trabecular preparation from the left ventricle of guinea-pigs that had undergone abdominal aortic banding. Sham-operated animals were used as controls; no guinea-pigs showed any signs of heart failure. We report that mild cardiac hypertrophy does not affect the relaxation rate of Triton-skinned trabeculae from the guinea-pig. However, there was a 35% reduction in the maximum force generated by trabeculae from the left ventricle of the abdominal aortic-banded animals. Additionally, alterations in key troponin subunits occur in the left ventricle of guinea-pigs with mild hypertrophy. We conclude that the thin filament protein changes do not influence trabecular relaxation rates, even though they probably affect maximal force generation. The cellular membrane systems of the intact guinea-pig heart, which were not a factor in this present study, appear to have an important role in the altered cardiac relaxation rates seen in hypertrophy.

Effect of pH, phosphate, and ADP on relaxation of myocardium after photolysis of diazo 2.

The aim of this study was to examine the effect of the metabolites H+, ADP, and Pi on the rate of cardiac relaxation. We used guinea pig right ventricular trabeculae that had been chemically skinned, allowing the myofilaments to be studied in isolation. Laser-flash photolysis of the caged Ca2+ chelator diazo 2, causing a rapid fall in intracellular Ca2+, enabled investigation of relaxation independently of the rate of Ca2+ diffusion. On the photolysis of diazo 2, the trabeculae relaxed biphasically with exponential rate constants (k1 and k2) of 10.07 and 4.23 s-1, respectively, at 12 degrees C and 18.35 and 2.52 s-1, respectively, at a nominal 20 degrees C. Increasing the concentration of both protons (pH 7.2-6.8) and MgADP (0.5-3.4 mM) slowed the two phases of the relaxation transients. Raising the concentration of Pi from the control level of 1.36 mM to 15.2 mM increased the rate of both phases, with relaxation becoming monoexponential at 19.4 mM Pi (with a k of 20.31 s-1 at 12 degrees C). Cardiac muscle was compared with skeletal muscle under identical conditions; in cardiac muscle 19.4 mM Pi increased the rate of relaxation, whereas in skeletal muscle this concentration of Pi slowed relaxation. We conclude that the mechanism of relaxation differs between cardiac and skeletal muscle. This study is a direct demonstration of the effects of ATP metabolites on cardiac myofilament processes during relaxation.

Investigating the relaxation, following diazo-2 laser flash photolysis, of a skinned trabecular preparation from SHR hypertrophied left ventricle.

Rat models of cardiac hypertrophy are characterised by a shift in left ventricular myosin isoform from V1 (adult) to V3 (foetal), the latter being associated with a slowing of the acto-myosin ATPase rate. The aim of this study was to examine hypertrophy effects on relaxation by investigating a chemically skinned cardiac preparation from the SHR, where all the cellular membranes are rendered non-functional allowing the myofibrils to be studied in isolation. On comparison, following photolysis of the photolabile caged Ca2+ chelator diazo-2, it can be seen that the SHR fibres relax at a slower rate than their age-matched WKY counterparts. We suggest that, since the thin filament regulatory proteins seem not to be affected by cardiac hypertrophy in the rat, this result can be attributed to the shift in left ventricular myosin isoforms. The reduced relaxation rate in the SHR could be the result of a slowing of the dissociation of actin and myosin during the cross-bridge cycle. These results have previously been published in abstract form [1].

Troponin I phosphorylation does not increase the rate of relaxation following laser flash photolysis of diazo-2 in guinea-pig skinned trabeculae.

In vivo, two effects of beta-adrenergic stimulation in cardiac muscle are phosphorylation of troponin I and an increase in relaxation rate. In vitro, cardiac TnI can be phosphorylated by protein kinase A (PKA). We have used the technique of laser flash photolysis of the calcium chelator diazo-2 to investigate the effect of phosphorylation of TnI on the relaxation rate of skinned trabeculae from the guinea-pig at 12 degrees C. The fibres were phosphorylated by PKA, and double exponential curve fits of the average relaxation transients showed no significant difference between the rate constants of the phosphorylated and control cases. We conclude that TnI phosphorylation has no effect on the rate of relaxation in skinned trabeculae from the guinea-pig following diazo-2 photolysis.