Cardiac mitochondria in heart failure: decrease in respirasomes and oxidative phosphorylation.

Aims: 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.

Rat liver mitochondrial carnitine palmitoyltransferase-I, hepatic carnitine, and malonyl-CoA: effect of starvation.

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.

Quantification of malonyl-coenzyme A in tissue specimens by high-performance liquid chromatography/mass spectrometry.

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.

Phosphorylation of rat liver mitochondrial carnitine palmitoyltransferase-I: effect on the kinetic properties of the enzyme.

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.