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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.

The effect of the cardiotonic sensitizing drug EMD 57033 was studied in frog skinned skeletal muscle fibres at 12 degrees C to provide a baseline for skeletal muscle studies and for comparison with cardiac fibres. The activation and relaxation of fibres were induced by laser flash photolysis of the caged calcium NP-EGTA, and caged calcium chelator diazo-2 respectively. EMD 57033 (10 microM) slightly increased the rate of relaxation (rate constant k1 changing from 24.0 +/- 2.9 s-1 in control to 28.1 +/- 3.2 s-1) but had no significant effect on the rate of activation (k1 = 9.6 +/- 0.9 s-1 in control conditions, 9.7 +/- 1.6 s-1 with EMD 57033). The effect of the optical isomer of EMD 57033, EMD 57439, was examined on steady-state force and relaxation rate. EMD57439 (10 microM) slowed the rate of relaxation (k1 = 20.5 +/- 2.4 s-1) but had no effect on the maximal calcium-activated force whereas EMD 57033 increased it by 16.5 +/- 5.7%. These results are compared to earlier results from this laboratory in guinea-pig skinned trabeculae, and a possible model for the action of EMD 57033 whereby the drug enhances force per cross-bridge is discussed.

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.

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 degreesC. 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 microM to 100 microM) 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.

When free calcium is rapidly removed from skinned fibres using the photolabile Ca2+ chelator diazo-2, they relax without an appreciable change in sarcomere length (

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.

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].

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.

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 10 microM 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 degrees 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.

Objective: To study QT dispersion in left ventricular hypertrophy and chronic heart failure and to determine the relation to ventricular arrhythmias., Setting: Investigational laboratory of a tertiary referral centre., Study Design: Patients with left ventricular hypertrophy and normal systolic function (n = 14) and patients with chronic heart failure (n = 18) were matched with controls (n = 17). The QT dispersion was examined in relation to abnormalities in resting mechanical and autonomic function and to the findings of 24 hour Holter monitoring., Main Outcome Measures: QT dispersion is the difference between the maximum and the minimum QT values from the 12 lead electrocardiogram. Mean(SD) QT dispersion from the 10 lead electrocardiogram was also examined once the 12 lead minimum and maximum values had been removed. The QT distribution is the curve describing the distance from the mean for all QT intervals (ms)., Results: All measures of QT dispersion were increased significantly in left ventricular hypertrophy and tended to increase in those with heart failure. The QT distribution was abnormal in both heart failure and left ventricular hypertrophy. There was no relation between the degree of change in QT dispersion and the incidence of ventricular arrhythmia on 24 hour Holter monitoring. Also there was no relation between QT dispersion and autonomic or mechanical abnormalities. The QT dispersion was related to QRS duration., Conclusion: Though QT dispersion and distribution are abnormal in left ventricular hypertrophy these findings do not support the hypothesis that QT dispersion reflects arrhythmic risk in either hypertrophy or heart failure.

EMD 57033 is thought to produce its potentiating effect by increasing the apparent calcium sensitivity of myofibrils. We have investigated the effect of 10 microM EMD 57033 on relaxation speed, induced by flash photolysis of 2mM diazo-2 (a caged Ca2+ chelator), in skinned semitendinosus frog muscle fibres and guinea-pig trabeculae. 10 microM 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 10 microM 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.

In this review, aspects of the ways in which Ca2+ is transported and regulated within muscle cells have been considered, with particular reference to crustacean muscle fibres. The large size of these fibres permits easy access to the internal environment of the cell, allowing it to be altered by microinjection or microperfusion. At rest, Ca2+ is not in equilibrium across the cell membrane, it enters the cell down a steep electrochemical gradient. The free [Ca2+] at rest is maintained at a value close to 200 nM by a combination of internal buffering systems, mainly the SR, mitochondria, and the fixed and diffusible Ca(2+)-binding proteins, as well as by an energy-dependent extrusion system operating across the external cell membrane. This system relies upon the inward movement of Na+ down its own electrochemical gradient to provide the energy for the extrusion of Ca2+ ions. As a result of electrical excitation, voltage-sensitive channels for Ca2+ are activated and permit Ca2+ to enter the cell more rapidly than at rest. It has been possible to determine both the amount of Ca2+ entering by this step, and what part this externally derived Ca2+ plays in the development of force as well as in the free Ca2+ change. The latter can be determined directly by Ca(2+)-sensitive indicators introduced into the cell sarcoplasm. A combination of techniques, allowing both the total and free Ca2+ changes to be assessed during electrical excitation, has provided valuable information as to how muscle cells buffer their Ca2+ in order to regulate the extent of the change in the free Ca2+ concentration. The data indicate that the entering Ca2+ can only make a small direct contribution to the force developed by the cell. The implication here is that the major source of Ca2+ for contraction must be derived from the internal Ca2+ storage sites within the SR system, a view reinforced by caged Ca2+ methods. The ability to measure the free Ca2+ concentration changes within a single cell during activation has also provided the opportunity to analyse, in detail, the likely relations between free Ca2+ and the process of force development in muscle. The fact that the free Ca2+ change precedes the development of force implies that there are delays in the mechanism, either at the site of Ca2+ attachment on the myofibril, or at some later stage in the process of force development that were not previously anticipated.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of Ca2+ on the time course of force generation in frog skinned muscle fibres has been investigated using laser flash photolysis of the caged-calcium, either nitr-5 or DM-Nitrophen. Gradations in the rate and extent of contraction could be achieved by changing the energy of the laser pulse, which varied the amount of caged Ca2+ photolysed and hence the amount of calcium released. The half-time for force development at 12 degrees C was noticeably calcium-sensitive when small amounts of calcium were released (low energy pulses) but did not change appreciably for calcium releases which produced a final tension of more than 50% of the maximal tension at pCa 4.5. This result is unlikely to be due to calcium binding to the regulatory sites of troponin C when on the thin filament, as this process is considered rapid (kon 10(8) M-1 s-1, koff 100 s-1). Our experimental results show that force develops relatively rapidly at intermediate Ca2+ which produce only partial activation (i.e. 50% Pmax or greater). This would not be the case if the affinity of the regulatory sites changes slowly with crossbridge attachment. The kinetics of calcium exchange with the regulatory sites may be much more rapid than crossbridge cycling, so that if calcium binding to a particular functional unit induces crossbridge attachment and force production, the force producing state may be maintained long after calcium has dissociated from that particular functional unit. The relaxation of skinned muscle fibres has also been successfully studied following the rapid uptake of Ca2+ by a photolabile chelator Diazo-2, a photolabile derivative of BAPTA, which is rapidly (> 2000 s-1) converted from a chelator of low Ca2+ affinity (Kd 2.2 microM) to a high affinity chelator (Kd 0.073 microM). We have used single skinned muscle fibres from both frog (actin regulated) and scallop striated muscle (myosin regulated), to study the time course of muscle relaxation. This procedure has enabled us to examine the effects of the intracellular metabolites, ADP, Pi and H+ upon the rate of relaxation.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

The ability of Ca2+ or inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to release Ca2+ from cardiac sarcoplasmic reticulum (SR) was investigated using saponin-skinned ventricular trabeculae from rats. To overcome diffusion delays, rapid increases in the concentrations of Ca2+ and Ins(1,4,5)P3 were produced by laser photolysis of "caged Ca2+" (Nitr-5) and "caged Ins(1,4,5)P3". Photolysis of Nitr-5 to produce a small jump in [Ca2+] from pCa 6.8 to 6.4 induced a large and rapid force response (t1/2 = 0.89 s at 12 degrees C); the source of the Ca2+ that activated the myofibrils was judged to be the SR, since it was blocked by 0.1 mM ryanodine or 5 mM caffeine. A smaller, slower, and less consistent release of SR Ca2+ was produced by photorelease of Ins(1,4,5)P3. The results demonstrate that these caged compounds can be used to study excitation-contraction coupling in skinned multicellular preparations of cardiac muscle. The data are consistent with a major role for Ca2(+)-induced Ca2+ release in cardiac activation, whereas the role for Ins(1,4,5)P3 may be to modulate, rather than directly stimulate, SR Ca2+ release.

The effects of the angiotensin converting enzyme inhibitor enalapril on myocardial sympathetic tone, as represented by noradrenaline overflow, were studied in 14 men with congestive heart failure (mean ejection fraction 20%) in a double blind crossover comparison with placebo. Arterial and coronary sinus catecholamine concentrations and oxygen content, and coronary sinus blood flow, were measured at rest and during peak symptom limited upright exercise on a bicycle ergometer. There were no significant changes four hours after the first dose of enalapril, but after six weeks of treatment (10-20 mg/day) enalapril reduced myocardial overflow of noradrenaline at peak exercise. The external workload (exercise duration) increased from baseline values after both placebo and enalapril, and there was no difference between placebo and enalapril at six weeks. Heart work, however, was lower after enalapril: stroke work index was reduced at rest and the double product was lower at peak exercise. The reduction in maximal myocardial oxygen consumption after enalapril did not reach statistical significance. Coronary sinus adrenaline concentrations after enalapril and after placebo were not significantly different. The long term reduction of myocardial sympathetic activity on exercise may represent a significant benefit from angiotensin converting enzyme inhibition in heart failure and may reflect a reduced cardiac workload.

Diazo-2 is a calcium chelator based on BAPTA [(1989) J. Biol. Chem., in press], whose electron withdrawing diazoacetyl group may be rapidly (2000 s-1) converted photochemically to an electron donating carboxymethyl group by exposure to near ultraviolet light, producing an increase in its calcium affinity (Kd changes from 2.2 microM to 0.073 microM) without steric modification of the metal binding site. Photolysis of a 2 mM solution of this compound with a brief flash of light from a frequency-doubled ruby laser (347 nm) caused single skinned muscle fibres from the semitendinosus muscle of the frog Rana temporaria to relax with a mean half-time of 60.4 +/- 5 ms (range 30-100 ms, n = 15) at 12 degrees C, which is faster than the relaxation observed in intact muscles (half-time 133 ms at 14 degrees C [(1986) J. Mol. Biol. 188, 325-342]) and similar to the rate of the fast phase of tension decay in intact single fibres (20 s-1 at 10 degrees C [(1982) J. Physiol. 329, 1-20]).

Because acute myocardial infarction may affect plasma lipid concentrations it is commonly recommended that assessment of these concentrations should be delayed until about three months after the acute event. A study was therefore conducted of fasting plasma lipid concentrations in 58 patients with acute myocardial infarction. Measurements were made during their stay in hospital (days 1, 2, and 9) and three months later. Triglyceride concentrations remained unchanged throughout. Values of total cholesterol, low density lipoprotein, and high density lipoprotein all fell significantly between the first two days and day 9. Total cholesterol and low density lipoprotein also showed significant falls between days 1 and 2. Nevertheless, fasting plasma lipid concentrations showed no significant difference at any time during the first 48 hours from values measured three months later. After the infarction 26 patients changed to eating less fat or less energy, or both. More patients had hypercholesterolaemia in the first 48 hours than at three months. These results suggest that lipid state may be assessed as accurately, and possibly more accurately, during the first 48 hours after acute myocardial infarction than at three months.

We performed a randomized double-blind placebo controlled cross-over study of enalapril in 16 patients with chronic congestive heart failure, to assess haemodynamic and hormonal effects at rest and on exercise. Acute effects were measured 4 h after enalapril 10 mg, and chronic effects after 6 weeks treatment with enalapril 10-20 mg per day. Exercise tolerance, assessed by the duration of a maximal bicycle ergometer test, was not altered by enalapril. Mean blood pressure was reduced after enalapril, at rest and on exercise, acutely by 7% and 8% respectively, and chronically by 14% and 16%. Systemic vascular resistance was reduced by 16% at rest both acutely (NS) and chronically (p less than 0.05). The resting pulmonary capillary wedge pressure was reduced by 28% with chronic treatment. In the acute study, total body oxygen consumption on exercise was 26% higher after enalapril. Chronically, resting oxygen consumption was reduced by 13% after enalapril, with mixed venous oxygen saturation increasing by 16%. In the acute study enalapril increased plasma renin activity at rest and on exercise by 181% and by 189%, and reduced aldosterone by 49% (NS) and 39% (p less than 0.05), and these effects were sustained after 6 weeks. Enalapril increased antidiuretic hormone concentrations at rest acutely by 73% (NS) and chronically by 34% (p less than 0.05) but not on exercise; the increase in the acute study correlated with plasma enalaprilat levels (r = 0.66, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)