[Show abstract][Hide abstract] ABSTRACT: Mitochondrial dysfunction is a major factor in heart failure (HF). A pronounced variability of mitochondrial electron transport chain (ETC) defects is reported to occur in severe acquired cardiomyopathies without a consistent trend for depressed activity or expression. The aim of this study was to define the defect in the integrative function of cardiac mitochondria in coronary microembolization-induced HF.
Studies were performed in the canine coronary microembolization-induced HF model of moderate severity. Oxidative phosphorylation was assessed as the integrative function of mitochondria, using a comprehensive variety of substrates in order to investigate mitochondrial membrane transport, dehydrogenase activity and electron-transport coupled to ATP synthesis. The supramolecular organization of the mitochondrial ETC also was investigated by native gel electrophoresis. We found a dramatic decrease in ADP-stimulated respiration that was not relieved by an uncoupler. Moreover, the ADP/O ratio was normal, indicating no defect in the phosphorylation apparatus. The data point to a defect in oxidative phosphorylation within the ETC. However, the individual activities of ETC complexes were normal. The amount of the supercomplex consisting of complex I/complex III dimer/complex IV, the major form of respirasome considered essential for oxidative phosphorylation, was decreased.
We propose that the mitochondrial defect lies in the supermolecular assembly rather than in the individual components of the ETC.
Cardiovascular Research 09/2008; 80(1):30-9. · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatic mitochondrial fatty acid oxidation and ketogenesis increase during starvation. Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-controlling step in the overall pathway and retains its control over beta-oxidation under fed, starved and diabetic conditions. To determine the factors contributing to the reported several-fold increase in fatty acid oxidation in perfused livers, we measured the V(max) and K(m) values for palmitoyl-CoA and carnitine, the K(i) (and IC(50)) values for malonyl-CoA in isolated liver mitochondria as well as the hepatic malonyl-CoA and carnitine contents in control and 48 h starved rats. Since CPT-I is localized in the mitochondrial outer membrane and in contact sites, the kinetic properties of CPT-I also was determined in these submitochondrial structures. After 48 h starvation, there is: (a) a significant increase in K(i) and decrease in hepatic malonyl-CoA content; (b) a decreased K(m) for palmitoyl-CoA; and (c) increased catalytic activity (V(max)) and CPT-I protein abundance that is significantly greater in contact sites compared with outer membranes. Based on these changes the estimated increase in mitochondrial fatty acid oxidation is significantly less than that observed in perfused liver. This suggests that CPT-I is regulated in vivo by additional mechanism(s) lost during mitochondrial isolation or/and that mitochondrial oxidation of peroxisomal beta-oxidation products contribute to the increased ketogenesis by bypassing CPT-I. Furthermore, the greater increase in CPT-I protein in contact sites as compared to outer membranes emphasizes the significance of contact sites in hepatic fatty acid oxidation.
Archives of Physiology and Biochemistry 07/2008; 114(3):161-70.
[Show abstract][Hide abstract] ABSTRACT: We present a validated high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the quantification of malonyl-coenzyme A (CoA) in tissues. The assay consists of extraction of malonyl-CoA from tissue using 10% trichloroacetic acid, isolation using a reversed-phase solid-phase extraction column, HPLC separation, and detection using electrospray MS. Quantification was performed using an internal standard ([(13)C(3)]malonyl-CoA) and multiple-point standard curves from 50 to 1000pmol. The procedure was validated by performing recovery, accuracy, and precision studies. Recoveries of malonyl-CoA were determined to be 28.8+/-0.9, 48.5+/-1.8, and 44.7+/-4.4% (averages+/-SD, n=5) for liver, heart, and skeletal muscle, respectively. Accuracy was demonstrated by the addition of known amounts of malonyl-CoA to tissue samples. The malonyl-CoA detected was compared with the malonyl-CoA added, and the resulting relationships were linear with slopes and regression coefficients equal to 1. Precision was demonstrated by repetitive analysis of identical samples. These showed a within-run variation between 5 and 11%, and the interbatch repeatability was essentially the same. This procedure was then applied to rat liver, heart, and skeletal muscle, where the malonyl-CoA contents were found to be 1.9+/-0.6, 1.3+/-0.4, and 0.7+/-0.2nmol/g wet weight, respectively, for these tissues. This analytical approach can be extended to the quantification of other acyl-CoA species with no significant modification.
[Show abstract][Hide abstract] ABSTRACT: Hepatic carnitine palmitoyltransferase-I (CPT-IL) isolated from mitochondrial outer membranes obtained in the presence of protein phosphatase inhibitors is readily recognized by phosphoamino acid antibodies. Mass spectrometric analysis of CPT-IL tryptic digests revealed the presence of three phosphopeptides including one with a protein kinase CKII (CKII) consensus site. Incubation of dephosphorylated outer membranes with protein kinases and [gamma-32P]ATP resulted in radiolabeling of CPT-I only by CKII. Using mass spectrometry, only one region of phosphorylation was detected in CPT-I isolated from CKII-treated mitochondria. The sequence of the peptide and position of phosphorylated amino acids have been determined unequivocally as FpSSPETDpSHRFGK (residues 740-752). Furthermore, incubation of dephosphorylated outer membranes with CKII and unlabeled ATP led to increased catalytic activity and rendered malonyl-CoA inhibition of CPT-I from competitive to uncompetitive. These observations identify a new mechanism for regulation of hepatic CPT-I by phosphorylation.
Journal of Biological Chemistry 10/2004; 279(39):41104-13. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Frequencies of external and grossly visible liver tumors were high in brown bullheads Ictalurus nebulosus from Black River, an industrialized Lake Erie tributary. Skin and liver tumors were lacking in brown bullheads from Buckeye Lake, a reference site, and the incidence of lip tumors was low (
Transactions of the American Fisheries Society 01/1987; 116(1):79-86. · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Within the last decade unusually high frequencies of neoplasms have been reported in feral fish populations from a variety of locations. At many of these locations organic carcinogens have been noted as a potential cause. We sought to identify toxic effects including neoplasia in fish exposed to an organic carcinogen, and to quantify these effects through time. We exposed guppies (Poecilia reticulata) to multiple doses of DEN, an organic carcinogen. Fish were then subsampled and examined for liver histopathology at 2-month intervals over 12 months. Necrotic zones, macrophage centers, bile duct proliferations, enlarged lipid deposits, neoplastic foci, cholangiocarcinomas and hepatoblastomas were quantified by frequency of occurrence and the percentage of liver area involved. DEN toxicity resulted in necrotic zones that peaked in frequency at the first sample period (2 months). Lipid deposits increased, then plateaued in guppies, indicating a more chronic toxic effect. Similarly, macrophage centers increased through the sampling period. Bile duct proliferation appeared to be of two types: a reversible toxic response which peaked at 4 months and then declined and a less frequent irreversible proliferation which continued to develop into cholangiocarcinoma. Neoplastic foci of mixed hepatocytes and cholangiocytes increased in livers of exposed guppies from the second month, developing into hepatoblastomas, which occurred in almost 100% of exposed guppies by the twelfth month. The irreversible bile duct proliferations and the neoplastic foci had cellular densities different from corresponding control tissue and similar to cellular densities of cholangiocarcinomas and hepatoblastomas, respectively.
Journal of Applied Toxicology 09/1985; 5(4):265-72. · 3.17 Impact Factor