Cardiac Microsomal Fractions Research Articles

Alterations in cytochrome P450-derived arachidonic acid metabolism during pressure overload-induced cardiac hypertrophy

Cardiac hypertrophy is a major risk factor for many serious heart diseases. Recent data demonstrated the role of cytochrome P450 (CYP)-derived arachidonic acid (AA) metabolites in cardiovascular pathophysiology. In the current study our aim was to determine the aberrations in CYP-mediated AA metabolism in the heart during cardiac hypertrophy. Pressure overload cardiac hypertrophy was induced in Sprague Dawley rats using the descending aortic constriction procedure. Five weeks post-surgery, the cardiac levels of AA metabolites were determined in hypertrophied and normal hearts. In addition, the formation rate of AA metabolites, as well as, CYP expression in cardiac microsomal fraction was also determined. AA metabolites were measured by liquid chromatography–electrospray ionization-mass spectroscopy, whereas, the expression of CYPs was determined by Western blot analysis. Non-parametric analysis was performed to examine the association between metabolites formation and CYP expressions. Our results showed that 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), and 5-, 12-, 15-, and 20-hydroxyeicosatetraenoic acids (HETEs) levels were increased, whereas, 19-HETE formation was decreased in hypertrophied hearts. The increase in EETs was linked to CYP2B2. On the other hand, CYP1B1 and CYP2J3 were involved in mid-chain HETE metabolism, whereas, CYP4A2/3 inhibition was involved in the decrease in 19-HETE formation in hypertrophied hearts. In conclusion, CYP1B1 played cardiotoxic role, whereas, CYP2B2, CYP2J3 and CYP4A2/3 played cardioprotective roles during pressure overload-induced cardiac hypertrophy. These CYP can be valid targets for the development of drugs to treat and prevent cardiac hypertrophy and heart failure.

Tissue specificity of translation inhibition in Sprague-Dawley rats following in vivo cyclosporin A

We have shown that following the in vivo administration of cyclosporin A (CsA) to either male or female Sprague-Dawley rats, there is a time- and dose-dependent inhibition of translation elongation in renal microsomes. Experiments reported in this paper explore the tissue specificity of translation inhibition. Following 6 days of oral 50 mg/kg/day CsA or control vehicle, animals were sacrificed and renal, hepatic and cardiac microsomal and cytoplasmic fractions prepared for “run-off” translation assays. These assays demonstrated that in vivo CsA resulted in a reduction in renal microsomal 3H- L-leucine incorporation to 25% of control values, a reduction of cardiac microsomal incorporation to 60% of controls and a stimulation of hepatic microsomal incorporation to 140% of controls. Cross-over experiments involving the addition of renal cytoplasmic fractions from CsA-treated animals to renal microsomal fractions from control-vehicle-treated animals depressed 3H- L-leucine incorporation to approximately 50% of control values. When the renal cytoplasmic fraction from CsA-treated animals was added to renal, hepatic or cardiac microsomal fractions from control-vehicle-treated animals, 3H- L-leucine incorporation values were consistently reduced to approximately 50% of controls. We have reported that translation elongation is inhibited by renal cytoplasm from CsA-treated rats in the presence of control renal cytoplasm. These data suggest that inhibition arises from the presence of an inhibitor rather than from a deficiency in elongation factors. The data reported in this paper demonstrate that microsomal fractions from various tissues have equal sensitivity to CsA-mediated renal cytoplasmic translation inhibition. These observations further suggest that renal translation inhibition is a result of more inhibitor being formed in, or taken up by, renal tissues than hepatic or cardiac tissues, and not due to a tissue-specific sensitivity to inhibitor. Renal translation inhibitor was also shown to inhibit eukaryotic translation in rabbit reticulocyte translation assays and prokaryotic translation in E. coli S30 coupled transcription/translation assays. The observation that translation elongation inhibition is most prominent in renal tissues, the site of dose-limiting CsA toxicity, suggests it may contribute to CsA nephrotoxicity.

Suppressive effect of coenzyme Q 10 on phospholipase A 2 activation in cardiac cells after prolonged swimming

Phospholipase A 2 (PLA 2) activity is elevated in cardiac microsomal fractions and phospholipids (PL) are much reduced in both the cardiac mitochondria and microsomal fractions from rats subjected to prolonged swimming. Preadministration of coenzyme Q 10 (CoQ 10 i.v. 30 mg/kg) significantly suppressed these changes. Two groups of 8-week-old male Wistar rats were trained to swim, receiving 30 min of training for 4 days. On the fifth day they were given an intravenous injection of either 30 mg/kg CoQ 10 in saline or 1 ml saline. Thirty minutes later they began to swim for 3 hours carrying a weight representing 3% of body weight. On completion of the swim they were sacrified by instantaneous decapitation, and cardiac mitochondria were isolated. Mitochondria were also prepared from saline injected, unexercised control rats. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) concentrations were measured with HPLC and PLA 2 activity was assayed fluorometrically. The mitochondrial concentrations (means±SEM, n=6) of PE and PC were respectively 126 ± 22 and 140 ± 22 nmol/mg protein in the exercise-CoQ 10 group against 66 ± 4 and 50 ± 10 nmol/mg protein in the exercise-saline group. The specific PLA 2 activities (expressed as nmol degraded dipyrene phosphorylethanolamine substrate/hr/mg protein) in the microsomes was 0.20 ± 0.02 in the exercise-CoQ 10 group against 0.30 ± 0.02 in the exercise-saline group. These results suggest CoQ 10 has a protective effect against an excessive reduction in mitochondrial membrane phospholipids during prolonged exercise.

The effect of linoleic and arachidonic acid derivatives on calcium transport in vesicles from cardiac sarcoplasmic reticulum

The effect of linoleic and arachidonic acid derivatives on ATP-dependent calcium transport was studied in the isolated vesicles from cardiac sarcoplasmic reticulum of guinea-pigs. Oxidation products of linoleic and arachidonic acids, obtained either by autoxidation or incubation with soybean lipoxygenase, effectively blocked in a dose-dependent manner, the net influx of calcium in the absence or presence of 5 mM of oxalate. Unoxidized fatty acids were much weaker at lower concentrations as compared to their oxidized counterparts, except the lipoxygenase-generated product of arachidonic acid which had only a marginal effect even at high concentrations. Autoxidation products of arachidonic acid were the most potent inhibitors of calcium transport. Likewise, autoxidation products of linoleic and arachidonic acids and lipoxygenase-generated products of linoleic acid induced a dose-dependent release of calcium from vesicles previously loaded with 45Ca, and release was further enhanced in the presence of 0.5 mM of EGTA. In contrast, lipoxygenase metabolites of arachidonic acid caused a transient increase in net calcium content. The effect of the fatty acid derivatives on calcium transport did not appear to be due either to the inhibition of Ca2+-ATPase activity or to a non-specific detergent-like action. The effects of oxidized fatty acids, on ATP-dependent calcium accumulation into and release from cardiac microsomal fraction were similar but less potent than those of classical calcium ionophores, X537A or A23187.

Cardiac membrane cholesterol in dystrophic and verapamil-treated hamsters

Cardiac muscle degeneration features prominently in the pathology of the dystrophic hamster. Since cholesterol is important in maintaining membrane integrity cholesterol and cholesterol ester levels were examined in cardiac subcelullar fractions of the UMX-7.1 strain of dystrophic hamster. In 50-day-old dystrophic animals cardiac microsomal fractions contained 135 ± 18 μg cholesterol per mg protein compared to control levels of 49 ± 8 μg/mg. The cholesterol content in the mitochondrial fraction was 65 ± 5 μg/mg and 17 ± 3 μg/mg respectively. In 180-day-old animals similar differences in cholesterol content were observed. Esterified cholesterol was elevated in the microsomal and mitochondrial fractions but only at 180 days. Pretreatment of dystrophic hamsters for 20 days with verapamil, a drug which has been shown to prevent cardiac necrosis in dystrophic hamsters did not alter the cholesterol content of the subellular fractions.

Changes in cardiac norepinephrine in spontaneously hypertensive and stroke-prone rats.

The norepinephrine (NE) concentration of cardiac ventricles was determined by radioenzymatic assay in normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and stroke-prone rats (SPR) at 3-6, 14-19, and over 31 weeks of age. There was no difference between strains prior to hypertension, but a progressive decrease in cardiac NE concentration occurred in SHR and particularly in SPR relative to WKY after hypertension was established. This decrease was not due to cardiac hypertrophy. The cardiac neuronal NE storage capacity in rats over 31 weeks of age was analyzed by determining the maximum concentration of NE obtained in a cardiac microsomal fraction, after saturation in vivo with exogenous NE. The results indicated that, after a long period of hypertension, there was a reduction in cardiac NE storage capacity resulting from a loss either of sympathetic nerve endings or of storage vesicles. Moreover, in addition to this reduction in the total size of the cardiac NE store, there was an independent reduction in the degree of filling of this store in both SHR and SPR. This could reflect an increased turnover of cardiac NE in chronically hypertensive SHR and SPR.

Further observations on species-determined differences in the calcium-accumulating activity of cardiac microsomal fractions

The possible existence of a species-determined difference in the ability of cardiac microsomes to accumulate and release Ca 2+ has been further investigated, by using a dual beam spectrophotometric method to detect rapid changes in Ca 2+ flux. Guinea pig, rat and rabbit microsomal fractions were used. When incubated in an oxalate-free reaction mixture the rat, rabbit and guinea pig fractions accumulated approximately the same amount of Ca 2+ but the rat microsomes accumulated this Ca 2+ at a faster ( P < 0.001) rate than did either of the other two species. Ca 2+ was released sooner ( P < 0.001) at a faster rate ( P < 0.001) from the rat microsomes than from either of the other two species. If an oxalate-containing incubation medium was used the rat microsomes accumulated Ca 2+ at a faster ( P < 0.001) rate than did either of the other two species. Even when oxalate was present Ca 2+ was rapidly released from the rat (13.65 ± 1.40 n mol Ca 2+/mg protein/min) microsomes, reflecting the relative inability of these microsomes to retain Ca 2+. These results are discussed in terms of the hypothesis that some of the peculiar contractile behaviour which is exhibited by rat heart muscle may reflect a peculiarity in the ability of its sarcoplasmic reticulum to accumulate and release Ca 2+, rather than to the presence of a relatively active Ca 2+-stimulated myofibrillar ATPase enzyme.

Effect of drugs on the cyclic adenosine 3′ 5′ monophosphate-dependent protein kinase-induced stimulation of calcium uptake by cardiac microsomal fractions

Microsomal fractions which have been preincubated in the presence of 5 × 10 −6 m 3′ 5′ AMP and 5 μg/ml 3′ 5′ AMP-dependent protein kinase accumulate Ca 2+ more rapidly than do paired microsomal fractions which have been incubated under control conditions, in the absence of added 3′ 5′ AMP and the 3′ 5′ AMP-dependent protein kinase. Ca 2+ uptake was not significantly changed if only 3′ 5′ AMP or the protein kinase was added to the incubation medium. dl propanolol (10 −4 m) and verapamil (10 −3 m) both reduced the amount of Ca 2+ which these microsomal fractions accumulated by the uptake process. These same concentrations of propanolol and verapamil failed to cause any significant change in the 3′ 5′ AMP-dependent protein kinase-induced stimulation of Ca 2+ uptake. Probably, therefore, the drugs verapamil and propanolol do not slow the rate of Ca 2+ uptake by cardiac microsomes by interfering with the 3′ 5′ AMP-dependent protein kinase induced stimulation of Ca 2+ uptake.

Influence of hyperthyroidism on the uptake and binding of calcium by cardiac microsomal fractions and on mitochondrial structure

Microsomes prepared from heart muscle of hyperthyroid dogs accumulate and exchange calcium more readily than do microsomes isolated from euthyroid heart muscle. The activity of the calcium-activated ATPase enzyme and cardiac stores of high energy phosphates and catecholamines are not influenced by the hyperthyroid state. Electron micrographs prepared from perfusion-fixed heart muscle failed to show evidence of an altered mitochondrial morphology associated with the hyperthyroid state.

Structural and functional characterization of dog heart microsomes.

Several procedures for the preparation of microsomes from heart tissue were compared. Sucrose was found the most effective addition to preserve the activity of the sarcoplasmic reticulum vesicles. Electron microscopy of sectioned pellets shows uniform vesicles, but negative staining of microsomal suspensions allows recognition of mitochondrial fragments, which are found in cardiac microsomal fractions in greater amounts than in skeletal muscle preparations. The quantity of mitochondrial contaminations in different fractions was estimated by measuring enzymatic activities. A comparison of Ca 2+ accumulation, Ca 2+ efflux, and ATPase activity shows identical features in preparations from the heart and from skeletal muscle, although the specific activity is approximately ten times lower in those from the heart. The apparent affinity of sarcoplasmic reticulum for Ca 2+ in vitro justifies the assumption that sarcoplasmic reticulum lowers the intracellular Ca 2+ concentration in the in-vivo heart below the level required for contraction. However, in a weight unit of fresh tissue, the ratio between the maximal Ca 2+ accumulated by SR and the amount of contractile protein is much lower in the heart than in skeletal muscle. Caffeine and, more markedly, propranolol and tetracaine increase efflux of accumulated Ca 2+ and thereby reduce net uptake by fragmented sarcoplasmic reticulum. Isopropylmethoxamine is not effective at millimolar concentrations.