I. P. Mulligan

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

Abstract In vivo, two effects of β-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°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.

The effect of EMD 57033, a novel cardiotonic agent, on the relaxation of skinned cardiac and skeletal muscle produced by photolysis of diazo-2, a caged calcium chelator

SummaryEMD 57033 is thought to produce its potentiating effect by increasing the apparent calcium sensitivity of myofibrils. We have investigated the effect of 10μM EMD 57033 on relaxation speed, induced by flash photolysis of 2mM diazo-2 (a caged Ca2+ chelator), in skinned semitendinosus frog muscle fibres and guineapig trabeculae. 10μM EMD 57033 has no effect on the relaxation speed of semitendinosus fibres. In trabeculae, EMD 57033 slightly increases the relaxation speed slightly, in contrast to ADP which produces a slowing. 1mM ADP combined with 10μM EMD 57033 slows relaxation but not to the degree seen with ADP alone. Like ADP, EMD 57033 increases the number of cross-bridges in the force producing state, but unlike ADP does not affect the transition rates involved in relaxation.

The effect of phosphate on the relaxation of frog skeletal muscle

Abstract The effect of phosphate on the relaxation of isometrically contracting single skinned fibres from the semitendinosus muscle of the frog Rana temporaria has been investigated using laser pulse photolysis of the photolabile caged calcium-chelator diazo-2 to rapidly reduce the Ca2+ (<2 ms) within the fibre and produce >90% relaxation of force. Relaxation occurred in two phases – an initial linear shoulder which lasted approximately 20 ms followed by a double-exponential phase which gave two rate constants, k1 (43.4±1.8 s–1, mean ±SEM, n=14) and k2 (15.6±0.3 s–1, mean ±SEM, n=14) at 12°C. Increased phosphate concentrations did not affect the linear phase, but slowed the double-exponential phase following photolysis of diazo-2 in a dose-dependent fashion (k50= 0.9 mM for k1, 1.15 mM for k2). Reducing the concentration of contaminating phosphate (from 640 µM to 100 µM) led to an increase in the rate of the double-exponential phase (k1=67.1±4.4 s–1, k2=19.7±0.6 s–1, mean ±SEM, n=12). Time-resolved measurements of sarcomere length during relaxation, both in control fibres and in the presence of a raised phosphate concentration, reveal a <2% change throughout the whole relaxation transient, and less than 0.1% at the end of the linear phase. This finding implies that gross changes in sarcomere length do not contribute to the decay of the relaxation transient seen upon diazo-2 photolysis. Our results suggest that cross-bridges in states prior to phosphate release are already committed to force generation and must relax by releasing phosphate, rather than by a reversal of the force-generating step to a weakly bound, low-force phosphate-bound state. These findings also indicate that an increase in the phosphate concentration within muscle fibres plays an important part in the slowing of relaxation observed in skeletal muscle fatigue and that the relaxation transients observed upon diazo-2 photolysis represent a disengagement of the cross-bridges.

The thiadiazinone EMD 57033 speeds the activation of skinned cardiac muscle produced by the photolysis of nitr-5.

EMD 57033 is thought to produce its potentiating effect by increasing the apparent Ca2+ sensitivity of the myofilaments, possibly by altering the kinetics of actomyosin interaction. We have investigated the effect of 10μM EMD 57033 upon activation speed, induced by flash photolysis of 2mM nitr-5 (caged Ca2+), in chemically skinned trabeculae from the guinea-pig at 12°C. EMD 57033 increases the half time of activation from 238±18.5 msec (n=6) to 132.1± 34.0 msec (n=8)(mean ± s.e.m.) and suggests that this Ca2+ sensitiser has an important effect upon fapp, that is the transition from the non-force to force generating cross-bridge states.

Skeletal muscle relaxation with diazo-2 : the effect of altered pH

In a fatigued muscle fibre, the concentrations of ADP, Pi and H+ are all increased and relaxation is slowed. We have used the technique of laser flash photolysis of the caged calcium-chelator, diazo-2, to investigate the direct effect of changes in pH (pH 6.5, 7.0, 7.5) upon tension during relaxation of single chemically skinned fibres, when the effects of the sarcoplasmic reticulum are absent. The relaxation transients were closely fitted with 2 exponentials, a fast (42.3 +/- 1.4; pH 7.0) and a slow process (12.0 +/- 0.7; pH 7.0). The fast phase of relaxation was pH sensitive; lowering pH leading to a slowing of the rate of force decline and raising pH leading to an increase of the rate. The rate of the slow phase was unaltered by changing pH over the range investigated. Thus the slowing of relaxation in fatigued muscle may be due, in part, to the direct action of protons on the myofilaments independent of any effects upon the sarcoplasmic reticulum.

Striated scallop muscle relaxation: Fast force transients produced by photolysis of Diazo-2

Relaxation of the myosin regulated striated adductor muscles of Pecten maximus was initiated by the photolysis of the caged Ca2+ chelator, Diazo-2. The fibres relaxed to approximately 30% of the maximum tension with a mean half-time of 17.9 +/- 1.6 ms (n = 7, temp 12 degrees C), much faster than the rates observed in intact muscle at the same temperature. This indicates that in the intact adductor muscle the slower relaxation rate is determined by the speed of Ca2+ removal from the sarcoplasm. The faster rate of relaxation of scallop muscle in vitro, compared with frog skeletal muscle may reflect different mechanisms of regulation of the crossbridge cycle.

Effects of troponin C isoform on the action of the cardiotonic agent EMD 57033

The effects of the cardiotonic potentiator EMD 57033 on different TnC (troponin C) isoforms were investigated. Endogenous skeletal TnC was extracted from glycerinated, permeabilized rabbit psoas fibres and replaced with either purified native rabbit psoas TnC (fast TnC) or human recombinant cTnC (cardiac TnC) (3 mg/ml in relaxing solution for 30 min). In both conditions, 10 microM EMD 57033 increased maximal calcium-activated force (Pmax) and gave a leftward shift in the pCa-tension curve. With cTnC, the increase in Pmax was much greater (228%) compared with the effect seen for fast TnC (137%), which was the same as that in unextracted control fibres. When the whole troponin was replaced rather than just TnC, the effects of EMD 57033 on fibres replaced with cTn were the same as with the cTnC subunit alone, except that the force at low Ca2+ concentrations was not increased as much. If TnC was only partially extracted, it was found that the degree of extraction did not influence the effect of EMD 57033, except when force was decreased to below 10% of the pre-extraction Pmax. Dynamic stiffness was not altered by EMD 57033 in any of the preparations. The rate of tension recovery following a release-restretch method (ktr) was decreased by EMD 57033. We conclude that EMD 57033 acts by a rate-modulating effect, and that the quantitative response of this effect is dependent on the TnC isoform present.

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

Abstract. 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.