Chronic stimulation of rat skeletal muscle induces coordinate increases in mitochondrial and nuclear mRNAs of cytochrome‐c‐oxidase subunits

Abstract

Fast-twitch tibialis anterior muscle of the rat was subjected to chronic low-frequency (10 Hz, 10 h daily) nerve stimulation in order to investigate the time course of changes in cytochrome-c-oxidase activity, as well as in tissue levels of specific mitochondrially and nuclear-encoded, cytochrome-c-oxidase-subunit mRNAs. Chronic stimulation induced a progressive increase in cytochrome-c-oxidase activity which was threefold elevated after 35 days. A similar increase was recorded for citrate-synthase activity. Glyceraldehyde-3-phosphate dehydrogenase, which was studied as a glycolytic reference enzyme, moderately decreased, as did the tissue level of its corresponding mRNA. There was a parallel increase in the tissue levels of the two cytochrome-c-oxidase-subunit mRNAs over the entire stimulation time course. The extent of increase (stimulated/control) was 2.4 0.3 and 1.8 f 0.2 (means SEM) for the mitochondrial and nuclear subunit mRNAs, respectively. This parallel increase suggested a coordinate regulation of the two subunits. The increase in cytochrome-c-oxidase activity initially corresponded to the changes at the mRNA level. However, with longer stimulation times (beyond 14 days), the increase in cytochrome-c-oxidase activity clearly exceeded that of the two mRNAs. This divergence was progressive and was interpreted to indicate that the increase in cytochrome-c-oxidase content was brought about not only by changes in the levels of the specific mRNAs, but also by alterations at the level of translation.It is well established that the tissue levels of enzymes involved in energy metabolism are adjusted, in mammalian skeletal muscle, to the functional demands imposed. Thus, increased contractile activity induces elevations in the mitochondrial content and, consequently, in the tissue content of enzymes functioning in aerobic-substrate and end-oxidation [I]. Cytochrome-c oxidase, the terminal enzyme of substrate end-oxidation, is composed of subunits which are derived from both the nuclear and the mitochondrial genomes [2, 31. This raises the question as to the degree to which these two systems are regulated under conditions of increased mitochondrial biogenesis, such as sustained contractile activity. In order to address this question, we have subjected f a ttwitch muscle of the rat to chronic low-frequency stimulation. This protocol has been extensively shown to result in a large and time-dependent increase in mitochondria and mitochondrial enzyme activities [4 -lo]. In order to investigate whether the two genomic systems are coordinately regulated, we have followed the time courses of changes in tissue levels of mRNAs encoding subunits I11 and VIc of cytochrome-c oxidase. Subunit I11 represents a mitochondrially encoded peptide, whereas subunit Vlc is coded for by the nuclear genome [2, 31.The changes in the levels of these two mRNAs were related to alterations in the tissue level of cytochrome-c-oxidase activity which was used as a measure of the holoenzyme's tissue concentration. In addition to addressing the...

Large Functional Range of Steady-State Levels of Nuclear and Mitochondrial Transcripts Coding for the Subunits of the Human Mitochondrial OXPHOS System

“…Support for a transcriptional coordination between nuclear and mitochondrial genes has been provided by numerous reports (Hood et al 1989;Gagnon et al 1991;Van den Bogert et al 1993;Connor et al 1996;Heddi et al 1999). However, data from analysis of changes in mitochondrial transcription in response to thyroid hormones (Enriquez et al 1999) as well as data from reporter gene activity (Sewards et al 1994) have indicated that mitochondrial and nuclear transcription systems operate independently.…”

Measurement of mitochondrial DNA synthesis in vivo using a stable isotope-mass spectrometric technique

“…We report here that mtDNA does turn over independent of somatic growth in hindlimb and cardiac muscle, that human platelets represent a highly accessible tissue, and that the method is well tolerated in people as well as rodents. Many possible research questions can be addressed by this approach, including inborn errors of mtDNA or mitochondrial biogenesis, assessment of aerobic fitness or aerobic demand on tissues, and toxicities of antiretroviral drugs (9,11,17,20,21). Future applications include isolation of mtDNA from other tissues, such as adipose or human skeletal muscle.…”

Tissue-specific regulation of cytochromecoxidase subunit expression by thyroid hormone

“…RNA (10 -30 g) was then electrophoresed on 1% agarose gels containing 0.02% formaldehyde, transferred, and fixed to nylon membranes (Hybond N; Amersham Pharmacia Biotech, Mississauga, ON, Canada). The membranes were then prehybridized and hybridized overnight at 42°C with a 32 P-labeled cDNA probe encoding either COX III (20) or COX Vb. The cDNA probes were radiolabeled with [␣-32 P]dCTP and a random primer labeling kit (New England Biolabs, Beverly, MA).…”

“…COX regulation is therefore a useful indicator of mitochondrial biogenesis, since a functional holoenzyme requires the coordination of both of these genomes. Although the coordinate induction of COX subunit mRNA derived from the nuclear and mitochondrial genomes is well established with contractile activity (20,33), this does not appear to be the case in response to altered T 3 status in which nuclear-encoded mRNAs appear to remain unchanged (40) or change with different kinetics (44) from mtDNA-encoded transcripts. Therefore, the purposes of our study were 1) to determine the extent of coordination between the nuclear and mitochondrial genomes at the protein level of expression, 2) to compare this to functional measures of holoenzyme activity, 3) to examine the role of transcriptional activation in the T 3 -induced response, and 4) to compare our measure of transcriptional activation with the expression of TR isoforms in tissues that are known to be responsive to T 3 .…”