Heart Muscle Homogenates Research Articles

Messenger RNA rescues medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts from patients and a murine model.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid β-oxidation (FAO) in humans. Patients exhibit clinical episodes often associated with fasting. Symptoms include hypoketotic hypoglycemia and Reye-like episodes. With limited treatment options, we explored the use of human MCAD (hMCAD) mRNA in fibroblasts from patients with MCAD deficiency to provide functional MCAD protein and reverse the metabolic block. Transfection of hMCAD mRNA into MCAD- deficient patient cells resulted in an increased MCAD protein that localized to mitochondria, concomitant with increased enzyme activity in cell extracts. The therapeutic hMCAD mRNA-lipid nanoparticle (LNP) formulation was also tested in vivo in Acadm-/- mice. Administration of multiple intravenous doses of the hMCAD mRNA-LNP complex (LNP-MCAD) into Acadm-/- mice produced a significant level of MCAD protein with increased enzyme activity in liver, heart and skeletal muscle homogenates. Treated Acadm-/- mice were more resistant to cold stress and had decreased plasma levels of medium-chain acylcarnitines compared to untreated animals. Furthermore, hepatic steatosis in the liver from treated Acadm-/- mice was reduced compared to untreated ones. Results from this study support the potential therapeutic value of hMCAD mRNA-LNP complex treatment for MCAD deficiency.

High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle.

Impaired myocardial bioenergetics is a hallmark of many cardiac diseases. There is a need of a simple and reproducible method of assessment of mitochondrial function from small human myocardial tissue samples. In this study we adopted high-resolution respirometry to homogenates of fresh human cardiac muscle and compare it with isolated mitochondria. We used atria resected during cardiac surgery (n = 18) and atria and left ventricles from brain-dead organ donors (n = 12). The protocol we developed consisting of two-step homogenization and exposure of 2.5% homogenate in a respirometer to sequential addition of 2.5 mM malate, 15 mM glutamate, 2.5 mM ADP, 10 μM cytochrome c, 10 mM succinate, 2.5 μM oligomycin, 1.5 μM FCCP, 3.5 μM rotenone, 4 μM antimycin and 1 mM KCN or 100 mM Sodium Azide. We found a linear dependency of oxygen consumption on oxygen concentration. This technique requires < 20 mg of myocardium and the preparation of the sample takes <20 min. Mitochondria in the homogenate, as compared to subsarcolemmal and interfibrillar isolated mitochondria, have comparable or better preserved integrity of outer mitochondrial membrane (increase of respiration after addition of cytochrome c is up to 11.7±1.8% vs. 15.7±3.1%, p˂0.05 and 11.7±3.5%, p = 0.99, resp.) and better efficiency of oxidative phosphorylation (Respiratory Control Ratio = 3.65±0.5 vs. 3.04±0.27, p˂0.01 and 2.65±0.17, p˂0.0001, resp.). Results are reproducible with coefficient of variation between two duplicate measurements ≤8% for all indices. We found that whereas atrial myocardium contains less mitochondria than the ventricle, atrial bioenergetic profiles are comparable to left ventricle. In conclusion, high resolution respirometry has been adapted to homogenates of human cardiac muscle and shown to be reliable and reproducible.

Kinetic characteristics of propofol-induced inhibition of electron-transfer chain and fatty acid oxidation in human and rodent skeletal and cardiac muscles.

IntroductionPropofol causes a profound inhibition of fatty acid oxidation and reduces spare electron transfer chain capacity in a range of human and rodent cells and tissues–a feature that might be related to the pathogenesis of Propofol Infusion Syndrome. We aimed to explore the mechanism of propofol-induced alteration of bioenergetic pathways by describing its kinetic characteristics.MethodsWe obtained samples of skeletal and cardiac muscle from Wistar rat (n = 3) and human subjects: vastus lateralis from hip surgery patients (n = 11) and myocardium from brain-dead organ donors (n = 10). We assessed mitochondrial functional indices using standard SUIT protocol and high resolution respirometry in fresh tissue homogenates with or without short-term exposure to a range of propofol concentration (2.5–100 μg/ml). After finding concentrations of propofol causing partial inhibition of a particular pathways, we used that concentration to construct kinetic curves by plotting oxygen flux against substrate concentration during its stepwise titration in the presence or absence of propofol. By spectrophotometry we also measured the influence of the same propofol concentrations on the activity of isolated respiratory complexes.ResultsWe found that human muscle and cardiac tissues are more sensitive to propofol-mediated inhibition of bioenergetic pathways than rat’s tissue. In human homogenates, palmitoyl carnitine-driven respiration was inhibited at much lower concentrations of propofol than that required for a reduction of electron transfer chain capacity, suggesting FAO inhibition mechanism different from downstream limitation or carnitine-palmitoyl transferase-1 inhibition. Inhibition of Complex I was characterised by more marked reduction of Vmax, in keeping with non-competitive nature of the inhibition and the pattern was similar to the inhibition of Complex II or electron transfer chain capacity. There was neither inhibition of Complex IV nor increased leak through inner mitochondrial membrane with up to 100 μg/ml of propofol. If measured in isolation by spectrophotometry, propofol 10 μg/ml did not affect the activity of any respiratory complexes.ConclusionIn human skeletal and heart muscle homogenates, propofol in concentrations that are achieved in propofol-anaesthetized patients, causes a direct inhibition of fatty acid oxidation, in addition to inhibiting flux of electrons through inner mitochondrial membrane. The inhibition is more marked in human as compared to rodent tissues.

PM378 Connexin45 Over-Expression Causes Arrhythmia in Rat Hearts

Introduction: Cancer-cachexia state promotes intense involuntary weight loss, wasting skeletal muscle protein due to higher proteolysis and/or decreased protein synthesis. This process can be improved by branched-chain amino acids, as leucine, which provides energy source by skeletal muscle and acts as a cell signaling. Objectives: This work aim to evaluate whether the leucine-rich diet (3%>leu) can modulate theelectrocardiogram (ECG) and cardiac biomarkers in the cachectic tumorbearing rats. Methods: Adult rats were distributed into 4 groups: C–control rats; WWalker-256 tumor tumor-bearing rats; L–leucine supplemented diet (leu-3%) and LW-leucine supplemented diet tumor-bearing rats (5 animals per group). The electrocardiogram was evaluated after anesthesia (ketamine 100mg/Kg. plus xelazine 7mg/Kg intramuscular) during pre-agonic state assayed by LabChart software. Cardiac biomarkers (MPO, TIMP, PAI-total) were evaluated in heart muscle homogenate assayed by multiplex assay (Millipore-Luminex). Difference among groups used one-way ANOVA analyses followed by Bonferroni’s test (P<0.05). Results: ECG parameters: the heart rate reduced in both tumor-bearing groups; QT-c tended to increase in W group, but was recovered in LW rats; T wave amplitude tended to increase in W group and was similar in LW group when compared to control rats. Cardiac biomarkers: MPO, TIMP and PAI increased only in W group, despite having tumor the LW group had similar values as the control rats.

Luminescence Amplification by Enzymatic Eu2+ Oxidation to Eu3+ for Time-Resolved Peroxidase Activity Measurement

A novel peroxidase activity assay was developed for horseradish peroxidase (HRP) and myeloperoxidase (MPO), in which substrate Eu(2+) was catalytically oxidized to Eu(3+), and the Eu(3+) luminescence was enhanced by the addition of sensitizer 4,4'-bis(1″,1″,1″,2″,2″,3″,3″-hepatafluoro-4″,6″-hexanedione-6″-yl)chlorosulfo-o-terphenyl (BHHCT) for time-resolved measurement of the BHHCT-Eu(3+) complex. Since BHHCT-Eu(3+) has a long lifetime (more than 500 μs), typical of Eu(3+) oxidation state, and the emission wavelength (615 nm) is totally different from those of Eu(2+) complexes, time-resolved luminescence measurement of the Eu(3+) complex enabled suppressed background and high signal/background ratio. The present method was successfully applied to monitor the oxidative stress level, which is closely associated with peroxidase activity level, in rat heart muscle homogenates. Notable parallel temporal change was observed for peroxidase activity and 4-hydroxynonenal (HNE) concentration after lipopolysaccharide (LPS) injection for induction of oxidative stress in rats. Such a relation does not contradict the oxidative stress mechanism that HNE is produced via lipid peroxidation, which is caused by the (•)OH radical generated by peroxidase activity.

Upregulation of Fatty Acyl-CoA Thioesterases in the Heart and Skeletal Muscle of Rats Fed a High-Fat Diet

In rodent models of diet-induced obesity, prolonged high-fat feeding increases the cellular uptake of fatty acids and causes lipotoxicity in the heart and skeletal muscle, where substrate overload to beta-oxidation generates mitochondrial stress. We examined the hypothesis that, because of its catalytic properties, acyl-CoA thioesterase (ACOT) would counteract these detrimental situations by modulating intracellular acyl-CoA levels. Rats were fed a low- or high-fat diet for up to 20 weeks, and the expressions of ACOT isoforms and fatty acid beta-oxidation enzymes were analyzed by western blotting. The expressions of ACOT1, ACOT2 and ACOT7 proteins in the heart and soleus muscle were significantly increased, by 2.0-7.6-fold, in rats fed the high-fat diet as compared with the low-fat diet group. These effects were accompanied by increases in carnitine palmitoyltransferase and acyl-CoA oxidase expression. However, ACOT was not induced in the extensor digitorum longus muscle or the liver. Subcellular fractionation of heart and soleus muscle homogenates confirmed expression of both the cytosolic and mitochondrial ACOT isoforms. These results underscore the functional relationship between ACOT and fatty acid oxidation, and suggest adaptive upregulation of ACOT to protect against fatty acid oversupply in the heart and skeletal muscle.

Investigation of mitochondrial protein expression and oxidation in heart muscle in low and high feed efficient male broilers in a single genetic line

The objective of this study was to assess the expression of mitochondrial proteins and oxidized proteins in heart muscle homogenate obtained from male broilers exhibiting either high or low feed efficiency (G:F) phenotypes. Tissue homogenate was prepared from ventricular tissue obtained from broilers with high (0.80+/-0.01, n=8) and low (0.62+/-0.02, n=8) FE. The levels of specific immunoreactive proteins and oxidized proteins (carbonyls) were determined using Western blot analysis. The expression of 6 electron transport chain proteins [complex II, 70S subunit (CII 70S); iron-sulfur-containing protein (ISP), cytochrome b (Cyt b), cytochrome (Cyt c1) (of complex III); and cytochrome oxidase subunit II (COX II) (of complex IV)] and adenine nucleotide translocator 1 (ANT1) were higher in low feed efficiency heart mitochondria, but 1 protein [NAD subunit 6c (NAD6c) (complex I)] was higher in high feed efficiency birds. Protein carbonyl levels, indicative of oxidized proteins, were higher in heart tissue of low compared with high feed efficiency broilers.

Modulation of antioxidant enzymes and lipid peroxidation in salivary gland and other tissues in mice by moderate treadmill exercise.

The current experiments were designed to study the effect of moderate treadmill training exercise on lipid peroxides and antioxidant enzyme activity in various tissues of ICR mice. Three-month-old female mice were trained on a treadmill to run daily from 45 to 50 minutes, at 1 km per hour, 6 days a week for a total of 8 weeks. At the end of the 8-week endurance-training period, both sedentary control (SC) and exercise-trained (ET) mice were sacrificed, and various tissues were collected to measure antioxidant enzyme activity. The results showed weight gain and serum lipid peroxides significantly decreased in ET mice compared to SC mice. Also, although lipid peroxide levels in kidney and salivary glands were found to be significantly decreased in ET mice, these mice showed higher lipid peroxide levels in the liver compared to SC mice. No change was observed in heart and calf muscle tissue of the ET mice. Exercise was also noted to increase superoxide dismutase (SOD) activity in kidney, heart, and calf muscle homogenates. Increases in catalase activity were present in liver, heart, calf muscle, and salivary gland homogenates of ET mice compared to their SC counterparts. Exercise was also shown to increase glutathione peroxidase activity in liver, kidney, and heart homogenates, as well as glutathione transferase activity in liver and salivary gland homogenates. In addition, exercise training was found to increase reduced and total glutathione levels in heart, calf muscle, and salivary gland. These results indicate that moderate exercise is beneficial to the lowering of lipid peroxides and the increasing of antioxidant enzyme activity specifically in the salivary gland, and also in various organs. However, its beneficial effect on elevation of antioxidant enzymes and suppression of lipid peroxide, varies from organ to organ.

Pathogenesis of Heart Myofibril Lesion in Experimental Vitamin D-Induced Cardionecrosis.

Multifocal cardionecrosis has been produced in rats by treatment with 3 × 100,000 in vitamin D 3 (calciol). The effects of hypervitaminosis D on rat heart myofibril structure and total protease activity was investigated. Protcolytic enzyme activity of heart muscle homogenate was determined in two independent ways, and was approximately two times higher in the necrotic heart homogenate than in the control rat heart. Electron microscopic examination showed structural derangements. Myofibrils isolated from necrotic heart exhibited significant changes of ultrastructure in the region of Z-line and I-brand. Myofibril enzyme activity (Mg 2+-ATPase) measurements demonstrated functional deficits as well. Under different conditions of myofibril isolation, it was shown that both ultrastructural and enzymatic lesions appear to be mediated by calcium-activated proteolytic enzymes operating in situ. Our results indicate that the increased proteolytic activity caused by vitamin D treatment leads to the in situ damages of proteins of the heart contractile systems.

The effect of di(ethylhexyl)phthalate on fatty acid oxidation and carnitine palmitoyltransferase in various rat tissues

Male rats were fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. Total and peroxisomal oxidation rates of palmitic and arachidonic acid were increased in homogenates of liver and kidney after DEHP administration. The relative peroxisomal contribution to the total oxidation was only higher in liver. The activities of acyl-CoA oxidase and carnitine palmitoyltransferase were also higher in both tissues. Immunoblots showed that the increase of fatty acid oxidation was associated with a higher concentration of enzymes of peroxisomal and mitochondrial β-oxidation. DEHP did not change total and peroxisomal fatty acid oxidation and activity of carnitine palmitoyltransferase of homogenates of heart and skeletal muscle. The cause for the tissue-specific response is discussed.

CGMP-stimulated protein kinase phosphorylates pyruvate kinase in an anoxia-tolerant marine mollusc

A cystosolic protein kinase that phosphorylates pyruvate kinase (PK) in vitro has been identified in crude homogenates of heart, radular retractor, and foot muscle from the anoxia-tolerant marine whelk Busycon canaliculatum. Protein kinase action was measured by following changes in PK kinetic parameters: phosphorylated PK has a higher K m value for phosphoenolpyruvate and a lower I50 value for l-alanine. The crude protein kinase readily phosphorylated PK in a Mg2+-and ATP-dependent manner in the absence of any added effector. This activity was not affected by the addition of either cAMP (a stimulator of protein kinase A) or Ca2+ plus phorbol 12-myristate 13-acetate (stimulators of protein kinase C) to the incubation medium. Addition of cGMP to the homogenate, however, increased the rate of PK phosphorylation giving a 3–4-fold increase in the rate of change in PK kinetic parameters that was readily apparent after 5h. Complete time-courses of changes in PK kinetic parameters in the presence and absence of cGMP showed that cGMP increased the rate, but not the final extent, of PK phosphorylation. These results indicate that PK inactivation by enzyme phosphorylation in response to anoxia in whelk tissues may be mediated by a cyclic GMP stimulated protein kinase in response to changing levels of cGMP. This conclusion was further supported by data indicating that the total activity of protein kinase was the same in both anoxic and aerobic animals, and that the total PK phosphatase activity was also constant. Changes in PK phosphorylation during anoxia are not, therefore, the result of changes in the total amount of protein kinase or phosphatase.

Acyl-CoA oxidase activity and peroxisomal fatty acid oxidation in rat tissues

Acyl-CoA oxidase, the first enzyme of the peroxisomal β-oxidation, was proved to be rate-limiting for this process in homogenates of rat liver, kidney, adrenal gland, heart and skeletal muscle. Acyl-CoA oxidase activity, based on H 2O 2-dependent leuko-dichlorofluorescein oxidation in tissue extract, was compared with radiochemically assayed peroxisomal β-oxidation rates. Dichlorofluorescein production was a valid measure of peroxisomal fatty acid oxidation only in liver and kidney, but not in adrenal gland, heart or skeletal muscle. Production of 14C-labeled acid-soluble products from 1- 14C-labeled fatty acids in the presence of antimycin-rotenone appears to be a more accurate and sensitive estimate of peroxisomal β-oxidation than the acyl-CoA oxidase activity on base of H 2O 2 production. Chain-length specificity of acyl-CoA oxidase changed with the acyl-CoA concentrations used. Below 80 μM, palmitoyl-CoA showed the highest activity of the measured substrates in rat liver extract. No indications were obtained for the presence in rat liver of more forms of acyl-CoA oxidase with different chain-length specificity.

Theory of relaxation of mobile water protons induced by protein NH moieties, with application to rat heart muscle and calf lens homogenates

Kimmich and co-workers (cf., Winter, F., and R. Kimmich. 1982. Biochim. Biophys. Acta. 719:292-298) discovered peaks in the magnetic field-dependent longitudinal relaxation rate (1/T1) of water protons of muscle tissue, cells, and dehydrated protein in the field range 0.5-5 MHz (proton Larmor frequency), and argued that the peaks resulted from cross relaxation associated with quadrupolar splittings of the 14N nuclei of protein NH groups. More recently, analogous peaks were found in homogenates of calf eye lens (Beaulieu, C.F., J.I. Clark, R.D. Brown III, M. Spiller, and S. H. Koenig, 1987. Abstr. Soc. Magn. Res. Med., 6th, New York. 598-599), which are essentially concentrated protein solutions, and were measured with sufficient precision to allow resolution of the relaxation spectra into several peaks and the intrinsic linewidths to be determined. Here, we analyze these relaxation spectra, as well as earlier data on rat heart (Koenig, S. H., R. D. Brown III, D. Adams, D. Emerson, and C. G. Harrison. 1984. Invest. Radiol. 19:76-81) in some detail, and suggest a specific pathway for the cross relaxation to which we apply the theory of relaxation quantitatively. The view that emerges is that, at fields such that the proton Zeeman energy of the NH protons matches an 14N quadrupolar splitting, relaxation of these protons is by cross relaxation to the 14N nuclei which in turn transfer excess energy to the protein. The correlation time for the NH proton interaction is the T2 of the 14N nuclei, approximately 10(-6) s, whereas T1 of the NH protons is approximately 1.25 ms. At these energy level crossings, the NH protons become relaxation sinks for protons of rapidly exchanging (-3 x 109 s-1) water molecules hydrogen bonded to the same backbone carbonyl oxygens as the NH protons. The lifetime of this hydrogen bond (-3 x 10-10 s) then becomes the correlation time for the water proton-NH proton interaction which, though short, is much longer than the analogous correlation time (-5 x 10-12 s) in pure water; the enhanced interaction results in peaks in the field-dependent 1/ T, of the solvent protons. There are few data on the lifetime of such bonds, but the results here conform with the recent considerations of Bennett, H. F., R. D. Brown III, S. H. Koenig, and H. M. Swartz. 1987. Magn. Reson. Med. 4:93-111, regarding hydrogen bond lifetimes for water molecules bound to macromolecules. The recent precise field-dependent relaxation data, here combined with both a quantitative theory and the fact that the magnitude of the 14N peaks is very concentration sensitive, allow, at least for lens proteins, a study of protein-protein interactions difficult to investigate by other methods.

Antioxidants as stabilizers of the Ca++ transport enzyme system in sarcoplasmic reticulum membranesin vivo

It has been demonstrated that natural and synthetic antioxidants of different chemical structures (alpha-tocopherol, butylated hydroxytoluene, 2-ethyl-6-methyl-3-hydroxypyridine) were capable of stabilizing enzymatic Ca2+ transport in sarcoplasmic membranes of the heart and skeletal muscles in vivo. Chronic administration of water-soluble antioxidant 2-ethyl-6-methyl-3-hydroxypyridine to young and old rats resulted in the increased rate of Ca2+ transport into sarcoplasmic reticulum vesicles of the heart and skeletal muscle homogenates. Keeping rats on vitamin E-rich diets supplemented with synthetic antioxidant butylated hydroxytoluene led to stabilization of Ca2+-ATPase against thermal denaturation in sarcoplasmic reticular membranes.

Effect of dietary fat on total and peroxisomal fatty acid oxidation in rat tissues

In this study the effect of dietary trans fatty acids on the peroxisomal and mitochondrial β-oxidation is compared with that of saturated or cis-monounsaturated fatty acids. Oxidation of [1- 14C]- and [16- 14C] palmitate was assayed in the absence as well as in the presence of antimycin plus rotenone in homogenates of liver, heart and skeletal muscle of four groups of rats fed diets containing 40 energy% fat of different fatty acid composition. Three groups were given fat blends rich in C 16,C 18 saturated (cocoa butter), cis-monounsaturated (low-linoleic-acid olive oil) or trans fatty acids (partially hydrogenated soybean oil), respectively. The fourth group received a mixture of these fats with half the amount of trans fatty acids of the third group. Total oxidation rates of [1- 14C]- and [16- 14C]palmitate in the absence of antimycin were not significantly influenced by the type of dietary fat in the investigated tissues. The antimycin-insensitive [1- 14C]palmitate oxidation rate and the proportion of peroxisomal oxidation of the total oxidation were lower in all tissues of those animals fed the mixed dietary fat than in those fed the other diets; both parameters were higher in the liver of cocoa butter-fed rats than in those of the other groups. Comparison of the results with literature data and with previous results obtained with a low-fat diet (Veerkamp and Van Moerkerk (1986) Biochim. Biophys. Acta 875, 301–310) indicates that high-fat diets only induce peroxisomal β-oxidation activity if they also contain C 20,C 22 fatty acids. High dietary concentrations of trans C 18 is fatty acids do not result in a higher peroxisomal activity than that observed for other fatty acids with the same chain length.

Ouabain binding and inotropy in acute potassium depletion in guinea pigs.

In hypokalaemia the incidence of cardiac toxicity with digitalis is increased, possibly through changes in the affinity or capacity of the digitalis receptor in the heart. Previous studies have reported an increased ouabain binding capacity or Na+-K+ ATPase activity after hypokalaemia in human erythrocytes and rabbit and guinea pig hearts and no change or decreases in rabbit or rat skeletal muscles with no changes in ouabain affinity. The present study determined (a) the effect of potassium on 3H-ouabain binding to normokalaemic guinea pig cardiac cell membranes, (b) the effect of acute hypokalaemia induced by a potassium deficient diet for 14-18 days in guinea pigs on 3H-ouabain binding to erythrocytes and cardiac and skeletal muscle homogenates, and (c) ouabain induced inotropy in isolated contracting guinea pig left atria, right ventricular papillary muscles, and soleus muscle strips from normokalaemic and hypokalaemic guinea pigs. 3H-ouabain binds to cardiac cell membranes in the presence of magnesium and inorganic phosphate with an affinity (KD value) of 1.13 X 10(-7) mol X litre-1. Potassium decreased this affinity without changing the binding capacity. Erythrocytes and heart muscle homogenates showed the same affinity as cardiac cell membranes, whereas soleus muscle homogenates had a higher affinity for ouabain (KD 5.1 X 10(-8) mol X litre-1). After hypokalaemia, the ouabain affinity did not change in any tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms

1. 1. After incubation of muscle preparations with [U- 14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. 2. 2. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. 3. 3. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. 4. 4. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. 5. 5. With liver homogenates the degradation of both 2-oxo acids was more complete. 6. 6. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. 7. 7. The degradation of valine was markedly limited by the release of these metabolites. 8. 8.Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO 2 and glutamine and glutamate. 9. 9. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. 10. 10. Radioactivity was never found in lactate, pyruvate or alanine. 11. 11. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.

Inhibition of long-chain fatty acid oxidation by methylglyoxal bis(guanylhydrazone)

Methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of spermidine and spermine biosynthesis and clinically used anti-cancer drug, powerfully inhibited carnitine-dependent fatty acid oxidation in heart muscle homogenates. Equipotent inhibition was also produced by spermine whereas spermidine and putrescine were less effective. MGBG appeared to act as a competitive inhibitor in respect to carnitine. Even though MGBG and spermine equally effectively depressed palmitate oxidation in muscle homogenates in vitro, a striking difference existed between the compounds as regards their effects on fatty acid oxidation in cultured tumor cells. Micromolar concentrations of MGBG distinctly impaired palmitate utilization also in cultured L 1210 leukemia cells, whereas similar concentrations of spermine markedly enhanced the oxidation of the fatty acid. The inhibitory effect of MGBG in cultured tumor cells was, at least partly, reversed upon addition of exogenous carnitine. The finding indicating that MGBG impairs fatty acid utilization may be an explanation for the known hypoglycemic effect produced by the drug in most animal species as well as for some of the side-effects associated with its clinical use, most notably severe myalgia.

Lipoprotein Lipase Activities in Heart Muscle of Streptozotocin-Induced Diabetic Rats

Triglyceride lipase (TGL) activities in the homogenates of the rat heart muscle were studied. TGL activity per mg protein of heart muscle was the highest in heart muscle homogenate utilizing 2.1 M glycine buffer, pH 8.3 among the assays investigated. The effects of NaCl, serum and heparin on TGL activities in heart muscle homogenates indicated the characteristics of lipoprotein lipase (LPL). Twelve-hour fasting increased heart muscle LPL activity, while enzyme activities in 48 hour- and 72 hour-fasted rats were lower than those in fed rats. LPL activities in heart muscle homogenates in streptozotocin (STZ)-induced diabetic rats either 3 days or 4 weeks after STZ injection, were decreased significantly as compared with those of control rats.

An analysis of contractile proteins in developing chick heart by SDS polyacrylamide gel electrophoresis and electron microscopy

Chick heart development was studied using transmission electron microscopy and SDS-polyacrylamide gel electrophoresis in combination with densitometry. Myosin heavy chain, alpha-actinin, actin and tropomyosin accumulations were analysed in developing hearts from preheartbeat stage 9 (Hamburger-Hamilton staging series) through 2 days after hatching. At the preheartbeat stage, electron microscopy revealed a significant number of thin filaments scattered throughout the cytoplasm of the myoblasts; however, very few thick filaments were seen. There was no obvious association between the two filament types. SDS-polyacrylamide tube gels of heart muscle homogenates demonstrated the presence of all five proteins in hearts at the preheartbeat stage. Further analyses of the proteins by gel densitometry indicated that both actin and myosin accumulated rapidly during heart development while alpha-actinin and tropomyosin levels remained relatively static. Our results show that detectable quantities of myosin heavy chain, alpha-actinin, actin and tropomyosin accumulate in myocardial cells prior to the appearance of myofibrils and initiation of the contractile function.