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 oftreatment (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.

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.

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.

The effect of the cardiotonic sensitizing drug EMD 57033 was studied in frog skinned skeletal muscle fibres at 12°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 µM) slightly increased the rate of relaxation (rate constant k 1 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 (k 1=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 µM) slowed the rate of relaxation (k 1=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.

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 Ca 2+ (<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, k 1 (43.4±1.8 s –1 , mean ±SEM, n =14) and k 2 (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 ( k 50 = 0.9 mM for k 1 , 1.15 mM for k 2 ). Reducing the concentration of contaminating phosphate (from 640 µM to 100 µM) led to an increase in the rate of the double-exponential phase ( k 1 =67.1±4.4 s –1 , k 2 =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 (≤1.7%). This relaxation is faster than the turnover rate of cross-bridges in steadily contracting muscle. Therefore a fall in the free calcium does not just prevent the recruitment of new cross-bridges but must also increase the rate of decay of cross-bridges which are already generating force. Increased levels of free phosphate (Pi) and free magnesium adenosine di-phosphate (MgADP) slow relaxation, indicating that during relaxation cross-bridges must undergo both a Pi and an ADP release step. In addition, we have observed that muscle relaxes in two phases. Phase 1, a linear phase, is greatly truncated in diazo-2 relaxation compared to intact fibres, supporting the theory that this phase is governed by calcium removal from the thin filament regulatory system whilst Phase 2 is a rapid exponential decline. Increased MgADP slows phase 1 consistent with a co-operative model of cross-bridge attachment where strongly-bound cross-bridges can maintain thin filament activation. The transition between phase 1 and 2 is not marked by any significant changes in sarcomere length (≤0.17%).

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 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 V 1 (adult) to V 3 (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 Ca 2+ 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 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.

For many years clinicians have had to cope with the accusation that only 10-20% of the treatments they provide have any scientific foundation. Their interventions, in other words, are seldom ''evidence based''. Is the profession guilty as charged?In April, 1995, a general medical team at a university-affiliated district hospital in Oxford, UK, studied the treatments given to all 109 patients managed during that month on whom a diagnosis had been reached. Medical sources (including databases) were then searched for randomised controlled trial (RCT) evidence that the treatments were effective. The 109 primary treatments were then classified: 82% were evidence based (ie, there was RCT support [53%] or unanimity on the team about the existence of convincing non-experimental evidence [29%]).This study, which needs to be repeated in other clinical settings and for other disciplines, suggests that earlier pessimism over the extent to which evidence-based medicine is already practised is misplaced.

For many years clinicians have had to cope with the accusation that only 10-20% of the treatments they provide have any scientific foundation. Their interventions, in other words, are seldom "evidence based". Is the profession guilty as charged? In April, 1995, a general medical team at a university-affiliated district hospital in Oxford, UK, studied the treatments given to all 109 patients managed during that month on whom a diagnosis had been reached. Medical sources (including databases) were then searched for randomised controlled trial (RCT) evidence that the treatments were effective. The 109 primary treatments were then classified: 82% were evidence based (ie, there was RCT support [53%] or unanimity on the team about the existence of convincing non-experimental evidence [29%]). This study, which needs to be repeated in other clinical settings and for other disciplines, suggests that earlier pessimism over the extent to which evidence-based medicine is already practised is misplaced.

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 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.

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)