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Aging induces complex changes in myocardium bioenergetic and contractile properties. Using F344BNF1rats, we examined age-dependent changes in myocardial bioenergetic enzymes (catalytic activities and transcript levels) and mRNA levels of putative transcriptional regulators of bioenergetic genes. Very old rats (35 months) showed a 22% increase in ventricular mass with no changes in DNA or RNA per gram. Age-dependent cardiac hypertrophy was accompanied by complex changes in mitochondrial enzymes. Enzymes of the Krebs cycle and electron transport system remained within 15% of the values measured in adult heart, significant decreases occurring in citrate synthase (10%) and aconitase (15%). Transcripts for these enzymes were largely unaffected by aging, although mRNA levels of putative transcriptional regulators of the enzymes (nuclear respiratory factor (NRF) 1 and 2 α subunit) increased by about 30%–50%. In contrast, enzymes of fatty acid oxidation exhibited a more diverse pattern, with a 50% decrease in β-hydroxyacyl-CoA dehydrogenase (HOAD) and no change in long-chain acyl-CoA dehydrogenase or carnitine palmitoyltransferase. Transcript levels for fatty acid oxidizing enzymes covaried with HOAD, which declined significantly by 30%. There were no significant changes in the relative transcript levels of regulators of genes for fatty acid oxidizing enzymes: peroxisome proliferator-activated receptor-α (PPARα), PPARβ, or PPARγ coactivator-1α (PGC-1α). There were no changes in the mRNA levels of Sirt1, a histone-modifying enzyme that interacts with PGC-1α. Collectively, these data suggest that aging causes complex changes in the enzymes of myocardial energy metabolism, triggered in part by NRF-independent pathways as well as post-transcriptional regulation.Key words: PGC-1a, fatty acid oxidation, nuclear respiratory factor (NRF), PPAR, coactivator, transcriptional regulation.

Mitochondrial content of skeletal muscle varies among fiber types, and changes in complex ways during aging. We evaluated the regulatory origins of differences in mitochondrial content among muscles of varied fiber type in F344xBNF1 rats, and how these regulatory patterns are altered with aging. In adult (12 month) animals we found that units citrate synthase (CS)/g tissue, a marker for mitochondrial content, varied approximately 3-fold among 10 skeletal muscles. Stoichiometric relationships between CS and isocitrate dehydrogenase, aconitase, and cytochrome c oxidase were generally preserved across fiber types. Among the 10 muscles of adult rats, CS content correlated with nuclear content (R2= 0.36). Muscles differed widely in CS messenger RNA (mRNA)/DNA (an index of variation in transcriptional regulations) and units CS/CS mRNA (an index of variation in posttranscriptional regulations). All muscles of aged rats (35 months) showed an increase in mg DNA/g, suggestive of atrophy. Age-dependent declines in units CS/DNA were accompanied by reductions in CS mRNA/DNA and/or units CS/CS mRNA, depending on muscle fiber type. Thus, declines in units CS/DNA with age appeared to be due to transcriptional as well as translational variations. Differences in mitochondrial content among muscle fiber types and age groups may arise from variations in nuclear content and posttranscriptional processes, as well as transcriptional regulation.

Cellular aging in nucleated erythrocytes from lower vertebrates is accompanied by losses in mitochondria but it remains unclear (i) how these losses accrue (ii) if these changes alter energetics and (iii) whether such changes increase the propensity for apoptosis. We addressed these questions using trout erythrocytes that were separated into age classes using inherent differences in buoyant density. The oldest cells showed a profound decline in mtDNA transcripts, due to reductions in both transcription (90% decline in total RNA) and mtDNA copy number (35%). No alterations in the ratio of 16S rRNA to COX I mRNA were detected, nor was there an accumulation of unprocessed mtDNA transcripts. While older cells had reduced basal respiration, there were no changes in mitochondrial enzymes stoichiometries, tissue ATP levels or dinitrophenol-induced (maximal) respiration rates. Apoptosis could not be induced in either whole blood, young or old erythrocytes by pro-oxidants, mitochondrial inhibitors or staurosporine. In contrast, cyclosporin A (CsA) caused caspase 3 activation, DNA laddering and LDH leakage, but only in young cells. Both CsA and a combination of azide, oligomycin and dinitrophenol cause mitochondrial depolarization and caspase 9 activation, but only CsA induced caspase 3 and apoptosis. Caspase inhibitor studies support the conclusion that mitochondrial changes may accompany CsA-induced cell death, but are not essential in its progression. While pifithrin failed to induce cell death, it enhanced the effects of CsA, implicating a role for p53. Collectively, these studies suggest that the mitochondrial changes with aging do not compromise cellular function, although trout erythrocytes can initiate apoptosis by non-mitochondrial pathways. D 2002 Elsevier Science B.V. All rights reserved.

Chronic treatment of cultured cells with very low levels of azide (I(50)

The complete amino acid sequence of human retina and muscle aldose reductase was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes based on partial amino acid sequences of purified human psoas muscle aldose reductase. The cDNA sequence differs substantially in the noncoding and coding regions of recently published sequences of this enzyme. The mRNA for aldose reductase was abundantly expressed in HeLa cells, but only scarcely in a neuroblastoma cell line. Recombinant baculovirus containing one of the muscle cDNA clones was constructed and used to infect Spodoptera frugiperda (SF9) cells. A prominent protein with an apparent molecular size of 36 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the culture medium as well as in the homogenate of SF9 cells after 2 days of infection. Culture medium or the supernatant fraction of cell homogenates containing this protein had high aldose reductase activity which showed characteristics of the reported human enzyme. These findings indicate that the amino acid sequence reported in this paper represents human retina and muscle aldose reductase and that functional human aldose reductase can be expressed in large amounts in a baculovirus expression system. The result should facilitate refined structural analysis and the development of new specific aldose reductase inhibitors for the treatment of diabetic complications.

We used expression and reporter gene analysis to understand how changes in transcription factors impinge on mitochondrial gene expression during myogenesis of cultured murine myoblasts (C2C12 and Sol8). The mRNA levels for nuclear respiratory factor-1 (NRF-1) and NRF-2alpha increased 60% by the third day of myogenesis, whereas NRF-1 and NRF-2 reporter gene activity increased by fivefold over the same period. Although peroxisome proliferator activated receptor (PPARalpha) mRNA levels increased almost 10-fold, the activity of a PPAR reporter was unchanged during myogenesis. The PPAR coactivator PPAR-gamma coactivator-1alpha (PGC1alpha), a master controller of mitochondrial biogenesis, was not expressed at detectable levels. However, the mRNA for both PGC1alpha-related coactivator and PGC1beta was abundant, with the latter increasing by 50% over 3 days of differentiation. We also conducted promoter analysis of the gene for citrate synthase (CS), a common mitochondrial marker enzyme. The proximal promoter ( approximately 2,100 bp) of the human CS lacks binding sites for PPAR, NRF-1, or NRF-2. Deletion mutants, a targeted mutation, and an Sp1 site-containing reporter construct suggest that changes in Sp1 regulation also participate in mitochondrial biogenesis during myogenesis. Because most mitochondrial genes are regulated by PPARs, NRF-1, and/or NRF-2, we conducted inhibitor studies to further support the existence of a distinct pathway for CS gene regulation in myogenesis. Although both LY-294002 (a phosphatidylinositol 3-kinase inhibitor) and SB-203580 (a p38-MAPK inhibitor) blocked myogenesis (as indicated by creatine phosphokinase activity), only SB-203580 prevented the myogenic increase in cytochrome oxidase activity, whereas only LY-294002 blocked the increase in CS (enzyme and reporter gene activities). Collectively, these studies help delineate the roles of some transcriptional regulators involved in mitochondrial biogenesis associated with myogenesis and underscore an import role for posttranscriptional regulation of transcription factor activity.

Myogenesis induces mitochondrial proliferation, a decrease in reactive oxygen species (ROS) production, and an increased reliance upon oxidative phosphorylation. While muscles typically possess 20%–40% excess capacity of cytochrome c oxidase (COX), undifferentiated myoblasts have only 5%–20% of the mitochondrial content of myotubes and muscles. We used two muscle lines (C2C12, Sol8) and 3T3-L1 pre-adipocytes to examine if changes in COX regulation or activity with differentiation cause a shift in metabolic phenotype (i.e., more oxidative, less glycolytic, less ROS). COX activity in vivo can be suppressed by its inhibitor, nitric oxide, or sub-optimal substrate (cytochrome c) concentrations. Inhibition of nitric oxide synthase via L-NAME had no effect on the respiration of adherent undifferentiated cells, although it did stimulate respiration of myoblasts in suspension. While cytochrome c content increased during differentiation, there was no correlation with respiratory rate or reliance on oxidative metabolism. There was no correlation between COX specific activity and oxidative metabolism between cell type or in relation to differentiation. These studies show that, despite the very low activities of COX, undifferentiated myoblasts and pre-adipocytes possess a reserve of COX capacity and changes in COX with differentiation do not trigger the shift in metabolic phenotype.Key words: oxidative phosphorylation, myogenesis, nitric oxide, reactive oxygen species, cytochrome c oxidase.

Although striated muscles differ in mitochondrial content, the extent of fiber-type specific mitochondrial specializations is not well known. To address this issue, we compared mitochondrial structural and functional properties in red muscle (RM), white muscle (WM), and cardiac muscle of rainbow trout. Overall preservation of the basic relationships between oxidative phosphorylation complexes among fiber types was confirmed by kinetic analyses, immunoblotting of native holoproteins, and spectroscopic measurements of cytochrome content. Fiber-type differences in mitochondrial properties were apparent when parameters were expressed per milligram mitochondrial protein. However, the differences diminished when expressed relative to cytochrome oxidase (COX), possibly a more meaningful denominator than mitochondrial protein. Expressed relative to COX, there were no differences in oxidative phosphorylation enzyme activities, pyruvate-based respiratory rates, H2O2production, or state 4 proton leak respiration. These data suggest most mitochondrial qualitative properties are conserved across fiber types. However, there remained modest differences (∼50%) in stoichiometries of selected enzymes of the Krebs cycle, β-oxidation, and antioxidant enzymes. There were clear differences in membrane fluidity (RM > cardiac, WM) and proton conductance (H+/min/mV/U COX: WM > RM > cardiac). The pronounced differences in mitochondrial content between fiber types could be attributed to a combination of differences in myonuclear domain and modest effects on the expression of nuclear- and mitochondrially encoded respiratory genes. Collectively, these studies suggest constitutive pathways that transcend fiber types are primarily responsible for determining most quantitative and qualitative properties of mitochondria.

We used spontaneously hypertensive rats to study remodeling of cardiac bioenergetics associated with changes in blood pressure. Blood pressure was manipulated with aggressive antihypertensive treatment combining low dietary salt and the angiotensin-converting enzyme inhibitor enalapril. Successive cycles of 2 wk on, 2 wk off treatment led to rapid, reversible changes in left ventricular (LV) mass (30% change in

We have previously purified and characterized a Dictyostelium myosin II heavy chain kinase which phosphorylates threonine residues (Cote, G. P., and Bukiejko, U. (1987) J. Biol. Chem. 262, 1065-1072). The phosphorylated threonines are located within a 34-kDa fragment which can be selectively cleaved from the carboxyl terminal end of the Dictyostelium myosin II tail. Tryptic and chymotryptic digests of the 34-kDa fragment phosphorylated with the kinase have now been performed and the resulting phosphopeptides isolated and sequenced. Two phosphorylated threonine residues have been identified, corresponding to residues 1833 and 2029 in the complete amino acid sequence of the Dictyostelium myosin II heavy chain. These amino acids are 87 and 283 residues, respectively, distant from the carboxyl terminus of the Dictyostelium myosin II heavy chain and are present in sections of the tail which seem to be alpha-helical coiled coils. In contrast, the three Acanthamoeba myosin II heavy chain phosphorylation sites are located within 10 residues of each other in a small globular domain at the carboxyl terminal tip of the tail (Cote, G. P., Robinson, E. A., Appella, E., and Korn, E. D. (1984) J. Biol. Chem. 259, 12781-12787). This suggests that the mechanism by which heavy chain phosphorylation inhibits the actin-activated ATPase activity and filament-forming properties of the two myosins may be quite different.

The reaction of aldose reductase from human psoas muscle with either pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) results in a pseudo first-order 2-fold activation of the enzyme with the stoichiometric incorporation of 1 mol of either reagent per mol of enzyme. However, in addition to an increase in Vmax there was also an increase in Km for both substrate, DL-glyceraldehyde, and coenzyme, NADPH. This resulted in an overall decrease in catalytic efficiency (kcat/Km). Spectral analysis indicated that activation by both PLP and PLP-AMP was accompanied by Schiff's base formation and epsilon-pyridoxyllysine was identified in hydrolysates of the reduced enzyme PLP-complex. Digestion of either PLP-modified or PLP-AMP-modified aldose reductase with endoproteinase Lys-C followed by high performance liquid chromatography purification and amino acid sequencing of the pyridoxyllated peptide revealed that PLP and PLP-AMP had modified the same lysine residue. A 32-residue peptide containing the essential lysine was found to be highly homologous with a segment of the sequence of both human liver aldehyde reductase and rat lens aldose reductase. A tetrapeptide (Ile-Pro-Lys-Ser) containing the essential lysine was identical in all three enzymes. These results highlight the close structural similarity between members of the aldehyde reductase family.