Effect of a high omega-3 fatty acid diet on cardiac contractile performance in Oncorhynchus mykiss.

OBJECTIVE: Omega-3 fatty acids have been implicated in the amelioration of cardiovascular disease in humans. Since these fatty acids are found in salmonid fish and are known to be essential for all salmonids, this study was undertaken to determine the effect of a high dietary intake of omega-3 fatty acids on the function of trout myocardium. METHODS: Rainbow trout (Oncorhynchus mykiss) from a single stock population were divided into two groups and fed either a diet high in omega-3 fatty acids (i.e. 4.0%) or low in omega-3 fatty acids (i.e. 2.1%) for 3 months. Heart function was studied at the whole heart and isolated muscle level. RESULTS: In whole heart preparations, peak developed pressures in freely ejecting hearts from salmonids fed the high omega-3 fatty acid diet were significantly greater than the hearts from salmonids fed the low omega-3 fatty acid diet (21 +/- 1.5 vs. 11.5 +/- 0.9 mmHg respectively, P < 0.05). These data correlated with results from isolated muscle preparations of myocardium from fish fed high and low omega-3 fatty acid diets (4.12 +/- 0.32 vs. 3.08 +/- 0.28 mN/mm2 respectively, P < 0.05). The calcium uptake rate of heart homogenates from fish fed the high omega-3 diet was slower and sarcoplasmic reticulum Ca2+ ATPase activity was lower. The myofilament force-calcium relationship in myocardium from trout fed the low omega-3 diet was shifted leftward on the calcium axis to lower intracellular calcium concentrations (delta 0.4 pCa units) compared to mammalian myocardium. This resulted in greater activation at lower intracellular calcium concentrations. However, trouts fed diets high in omega-3 fatty acids had [Ca2+] required for half maximal activation more similar to what has been reported for mammalian myocardium (delta 0.1 pCa unit). Furthermore, the myofilaments of trout hearts appear to show less cooperativity (Hill coefficient approximately 1) than has been found in mammalian myocardium (Hill coefficient > or = 2). CONCLUSIONS: Our experimental results demonstrate for the first time that dietary omega-3 fatty acid content affects myocardial force of contraction by affecting calcium metabolism and myofilament calcium-activation.

Myosin phosphorylation augments force-displacement and force-velocity relationships of mouse fast muscle.

Two studies were conducted to examine the effect of myosin regulatory light chain (R-LC) phosphorylation on the rate and extent of shortening in submaximally activated mouse extensor digitorum longus muscles in vitro at 25 degrees C. For each study, R-LC phosphate content was increased fivefold by application of a 5-Hz, 20-s conditioning stimulus (CS) to 0.65-0.68 mol phosphate/mol R-LC; this level was sustained between 10 and 40 s after the CS. Maximum isometric twitch force and the maximum rate of force development (+dF/dtmax) were potentiated in the range 13-17% and 9-17% (P < 0.05), respectively, after the CS. In study 1, the maximal rate and extent of shortening were significantly enhanced by 10 and 21% (P < 0.001), respectively, when measured using a twitch zero-load clamp technique. In study 2, the force-velocity and force-displacement relationships were both augmented when determined with the twitch afterload technique. Displacement was enhanced between 20 and 82% for loads that ranged from 3 to 75% of active peak twitch force, whereas velocity was increased 6-8% over the same range (P < 0.05), including the predicted maximum velocity (Vmax; 5.08 vs. 4.69 muscle length/s). In both studies the increase in velocity likely represents a shift along the force-velocity relationship toward true Vmax that reflects a decrease in relative load due to force potentiation. Furthermore, with the decrease in relative load, displacement at a given load was also increased. Potentiated displacement and extent of R-LC phosphorylation also decreased in parallel when studied for 5 min after the CS. The increase in muscle shortening is a novel finding and suggests a function for R-LC phosphorylation with respect to movement because both peak work and power were also enhanced by up to 22%. These effects are consistent with an R-LC phosphorylation-induced increase in fapp, the apparent rate constant that describes the cross-bridge transition from the non-force-generating to the force-generating state.

Compensatory asymmetry in down-regulation and inhibition of the myocardial Ca2+ cycle in congestive heart failure produced in dogs by idiopathic dilated cardiomyopathy and rapid ventricular pacing.

In this study we used 2.5% myocardial homogenates to study sarcoplasmic reticulum (SR) activity of the Ca2+ pump and Ca2+ release channel (CRC) from dogs with congestive heart failure produced by either rapid ventricular pacing or idiopathic dilated cardiomyopathy. We used the fluorescent indicator dye and ratiometric spectrofluorometry to monitor Ca2+ uptake while the CRC was open and closed with ryanodine. We confirmed and extended conclusions derived from previous studies of the same dogs using isolated SR. Compared to controls, activities of dogs with either form of CHF were decreased by 36% for the Ca2+ pump (33.7 +/- 7.3 and 21.6 +/- 4.2 nM/s), 78% for the CRC (10.0 +/- 2.8 and 1.4 +/- 1.2 nM/s), 53% for total Ca2+-cycling (53.1 +/- 8.5 and 24.8 +/- 4.4 nM/s), and 17% for net Ca2+ uptake (23.7 +/- 4.0 and 19.6 +/- 4.0 nM/s). In the absence of SR and mitochondrial activity, ionized Ca2+ concentration in myocardial homogenates were 70% abnormally increased in dogs with CHF, probably due to decreased concentration of Ca2+-binding proteins. Comparison of homogenate and isolated SR activities indicated lower-than-normal membrane yields for dogs with CHF. This fractionation artefact previously resulted in up to 50% overestimation of the degree of downregulation of Ca2+-cycling activities in CHF. The CRC activity was found to be decreased due to decreased activity of the Ca2+-ATPase, decreased CRC content, and inhibition. Decreased CRC energy. Maintenance of net Ca2+-pump activity is expected to maintain the amplitude of the myocardial ionized Ca2+ transient whereas downregulation of the CRC and pump are predicted to reduce the total amount of Ca2+ cycled and slow the rise and fall of the Ca2+ transient.

Contractile properties of rat gastrocnemius muscle during staircase, fatigue and recovery.

Slowing of relaxation is one of the anticipated changes in the contraction of fatigued skeletal muscle. However, interpretation of the mechanism(s) contributing to slowed relaxation may be affected by the measurement technique employed. In this study, relaxation was measured in three ways: (i) traditional half-relaxation time; (ii) peak rate of relaxation; and (iii) late relaxation time, measured from 50 to 25% of peak developed tension. When rat gastrocnemius muscle was stimulated indirectly in situ at 10 Hz, developed tension increased in 10 s to 185%, then decreased to 39% after 1 min with little additional change over the next 4 min. After 10 s of inactivity, developed tension was 60% of the initial value, but did not recover further over the next 20 min. The half-relaxation time transiently decreased at the start of stimulation, then by 20 s was considerably prolonged. Within 10 s of recovery, half-relaxation time returned to prestimulation values but became prolonged again by 10 min of recovery. The peak rate of relaxation was proportional to the developed tension at all times except 2.5-10 s of 10 Hz stimulation, at which time acceleration of relaxation was evident, and 15-20 s of the 10 Hz stimulation when it was relatively decreased. The late relaxation time increased during the repetitive stimulation, returned to control level early in recovery, then increased again, by 5 min of recovery. The diverse responses indicated by these indices of relaxation potentially discriminate different mechanisms which contribute to slowing of relaxation in fatigue, a point which would be missed if a single method of measurement of relaxation was employed.

Role of sarcoplasmic reticulum in loss of load-sensitive relaxation in pressure overload cardiac hypertrophy.

The loss of load-sensitive relaxation observed in the pressure-overloaded heart may reflect a strategy of slowed cytosolic Ca2+ uptake to yield a prolongation of the active state of the muscle and a decrease in cellular energy expenditure. A decrease in the potential of the sarcoplasmic reticulum (SR) to resequester cytosolic Ca2+ during diastole could contribute to this attenuated load sensitivity. To test this hypothesis, both in vitro mechanical function of anterior papillary muscles and the SR Ca2+ sequestration potential of female guinea pig left ventricle were compared in cardiac hypertrophy (Hyp) and sham-operated (Sham) groups. Twenty-one days of pressure overload induced by coarctation of the suprarenal, subdiaphragmatic aorta resulted in a 36% increase in left ventricular mass in the Hyp. Peak isometric tension, the rate of isometric tension development, and the maximal rates of isometric and isotonic relaxation were significantly reduced in Hyp. Load-sensitive relaxation were significantly reduced in Hyp. Load-sensitive relaxation quantified by the ratio of a rapid loading to unloading force step in isotonically contracting papillary muscle was reduced 50% in Hyp muscles. Maximum activity of SR Ca(2+)-adenosinetriphosphatase (ATPase) measured under optimal conditions (37 degrees C; saturating Ca2+) was unaltered, but at low free Ca2+ concentrations (0.65 microM), it was decreased by 43% of the Sham response. Bivariate regression analysis revealed a significant (r = 0.84; P = 0.009) relationship between the decrease in SR Ca(2+)-ATPase activity and the loss of load-sensitive relaxation after aortic coarctation. Stimulation of the SR Ca(2+)-ATPase by the catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase resulted in a 2.6-fold increase for Sham but only a 1.6-fold increase for Hyp. Semiquantitative Western blot radioimmunoassays revealed that the changes in SR Ca(2+)-ATPase activity were not due to decreases in the content of the Ca(2+)-ATPase protein or phospholamban. Our data directly implicate a role for decreased SR function in attenuated load sensitivity. A purposeful downregulation of SR Ca2+ uptake likely results from a qualitative rather than a quantitative change in the ATPase and possibly one of its key regulators, phospholamban.

Myosin light chain phosphorylation during staircase in fatigued skeletal muscle.

It has been reported that the peak of the staircase or the enhanced tension response during low frequency stimulation is delayed in fatigued fast muscle. Our purpose was to determine if the rate and extent of regulatory myosin light chain (P-LC) phosphorylation, a molecular mechanism associated with the positive staircase, are also altered by fatigue. The staircase contractile response, muscle metabolites and phosphate incorporation by the P-LC were assessed at 0, 5, 10 or 20 s of 10-Hz stimulation, in either non-fatigued (control) or fatigued (10 Hz for 5 min, followed by 20 min of recovery) rat gastrocnemius muscle in situ. The concentration of adenosine triphosphate (ATP) in fatigued muscles, 21 +/- 0.9 mmol.kg-1 (dry weight) was significantly lower (P < 0.05) than in the control muscles, 26.1 +/- 1.5 mmol.kg-1. In both groups, ATP content was significantly lower after 20 s of 10 Hz stimulation. The P-LC phosphate content (in mol phosphate.mol-1 P-LC) was 0.10, 0.38, 0.60 and 0.72 after 0, 5, 10 or 20 s of 10 Hz stimulation in control muscles, but only 0.03, 0.08, 0.11 and 0.19 at these times in fatigued muscles. Although the absolute magnitude of tension potentiation was attenuated in proportion to the depressed twitch amplitude, these surprisingly low levels of phosphorylation were associated with 0, 48, 79 and 86% potentiation of the developed tension at these times in contrast with 0, 71, 87 and 49% potentiation in control muscles. These data demonstrate that while the rate and extent of phosphate incorporation is depressed in fatigued muscle, tension potentiation is still evident.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative mammal model of chronic rate overload: relationship of myocardial Ca-cycling to heart, metabolic and lipoperoxidation rates.

1. The cardiac cycle is generally believed to be timed by a sarcoplasmic Ca-cycle and powered by a sarcoplasmic ATP-cycle that uses fatty acids as fuel and generates toxic free radicals as a side-product. 2. This study used a comparative mammal approach to test this model and the hypothesis that these cycles were closely coupled and correlated with fatty acid oxidation and peroxidative injury. 3. Fatty acid oxidation and ATP-cycling rates correlated to log heart rate whereas Ca-cycling rate was directly coupled to heart rate in a one-to-one relationship. 4. Both Ca-pump and Ca-channel activities coordinately increased as heart rate increased across species. 5. Thus, Ca-cycling activity is constant across mammals, when normalized to heart rate. 6. Comparison of Ca-ATPase and Ca-flux rates indicated that sarcoplasmic volume was inversely correlated with log heart rate. Basal lipoperoxidation of myocardium and susceptibility of SR to lipoperoxidation correlated with metabolic rate. 7. We identified that the horse is a metabolic outlier amongst mammals, with abnormally high fatty acid oxidation and ATP-synthetase activity compared to its heart rate.

Use of a DNA-based test for the mutation associated with porcine stress syndrome (malignant hyperthermia) in 10,000 breeding swine.

To test the hypothesis that the mutation associated with porcine stress syndrome (PSS; malignant hyperthermia) was present in a large proportion of North American and English swine, a simple and rapid laboratory protocol was used for cost-effective, large-scale diagnosis of susceptibility to PSS. This PSS test was applied to 10,245 breeding swine of various breeds from 129 farms in the United States, Canada, and England. Approximately 1 of 5 swine was a heterozygous carrier of the PSS mutation, with approximately 1% being homozygotes. Prevalence of the PSS mutation was 97% for 58 Pietrain, 35% for 1,962 Landrace, 15% for 718 Duroc, 19% for 720 Large White, 14% for 496 Hampshire, 19% for 1,727 Yorkshire, and 16% for 3,446 crossbred swine. The PPS gene frequencies for these breeds were 0.72, 0.19, 0.08, 0.10, 0.07, 0.10, and 0.09, respectively. In addition to these breeds, we have identified the PSS mutation in Poland China and Berkshire breeds. These gene frequencies were 30 to 75% lower in Canadian swine than in US swine, with the exception of Yorkshires, for which the gene frequency was threefold higher in Canadian swine. English swine were similarly, or more so, affected than were US swine. Accuracy was estimated at > 99%. Cost to perform the test was < $20/animal. Depending on the perceived net balance of deleterious and beneficial effects of the mutation, the PSS test could be used to eradicate the PSS mutation from herds, or for controlled expression of the mutation.

Compensatory downregulation of myocardial Ca channel in SR from dogs with heart failure.

In this study we tested the hypothesis that the ryanodine-binding Ca-release channel activity and density of the sarcoplasmic reticulum (SR) terminal cisternae were decreased in congestive heart failure (CHF) that occurs spontaneously in doberman pinschers or experimentally with rapid ventricular pacing of mongrels. We used a novel, sensitive, and easy-to-perform microassay and demonstrated a 50% decrease in activity of the myocardial SR Ca pump and a 75% reduction in SR Ca-release channel activity in CHF. Decreases in Ca channel content were associated with increases in net Ca sequestration. 45Ca-release experiments from passively loaded SR terminal cisternae and ryanodine-binding studies confirmed a 53-68% downregulation of the Ca-release channel activity. As a consequence of release channel downregulation, there was partial restoration of net Ca sequestration activity in dogs with CHF and complete compensation in dogs with mild cardiac dysfunction. Deterioration of Ca cycling correlated with deterioration of myocardial performance, apparently due to decreased Ca-adenosinetriphosphatase (ATPase) pump and not Ca channel content. One-half the reduction in Ca-release activity could be attributed to decreased Ca sequestration and one-half to decreased Ca channel density. Downregulation of Ca channel content decreases the amplitude of the Ca cycle and maximizes the downregulation of Ca pumps that may occur. Although these adaptations may reduce cellular energy expenditure, they are likely to render the myocardium more susceptible to fatigue and failure.

Respiratory chain defect of myocardial mitochondria in idiopathic dilated cardiomyopathy of Doberman pinscher dogs.

Idiopathic dilated cardiomyopathy (IDCM) is a primary myocardial disease of unknown cause. We tested the hypothesis that IDCM was associated with a myocardial metabolic defect by determining a comprehensive biochemical profile of metabolite concentrations and enzyme activities for the major metabolic pathways of the myocardium. We used the Doberman pinscher breed as a naturally occurring canine model of IDCM and compared its myocardial profile with that of healthy adult mongrels. Compared with controls, myocardium in IDCM had markedly reduced mitochondrial electron transport activity and myoglobin concentration, in association with acidosis and energy depletion following anoxic challenge: 60% decreased NADH dehydrogenase and 50% decreased ATP synthetase activities; 90% decreased myoglobin concentration; and 30% reduced ATP and 50% increased lactate and proton concentrations. Sarcoplasmic reticulum Ca(2+)-transport ATPase was decreased by 42%. There was a 15% compensatory increase in fatty acid oxidation and Krebs cycle activity. Other biochemical changes were mild by comparison with the mitochondrial defects. We conclude that IDCM is associated with a marked impairment of mitochondrial production of ATP, arising from decreased activity of the mitochondrial electron transport system, including myoglobin. These changes may be secondary to an underlying genetic defect or may indicate a deficiency of the mitochondrial respiratory chain that predisposes this breed to heart failure.