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To date, no studies have described the import of proteins in mitochondria obtained from skeletal muscle. In this tissue, mitochondria consist of the functionally and biochemically distinct intermyofibrillar (IMF) and subsarcolemmal (SS) subfractions, which are localized in specialized cellular compartments. This mitochondrial heterogeneity in muscle could be due, in part, to differential rates of protein import. To evaluate this possibility, the import of precursor malate dehydrogenase and ornithine carbamyltransferase proteins was investigated in isolated IMF and SS mitochondria in vitro. Import of these was 3-4-fold greater in IMF compared with SS mitochondria as a function of time. This could account for the higher malate dehydrogenase enzyme activity in IMF mitochondria. Divergent import rates in IMF and SS mitochondria likely result from a differential reliance on various components of the import pathway. SS mitochondria possess a greater content of the molecular chaperones hsp60 and Grp75, yet import is lower than in IMF mitochondria. On the other hand, adriamycin inhibition studies illustrated a greater reliance on acidic phospholipids (i.e. cardiolipin) for the import process in SS mitochondria. Matrix ATP levels were 3-fold higher in IMF mitochondria, but experiments in which ATP depletion was performed with atractyloside and oligomycin illustrated a dissociation between import rates and levels of ATP. In contrast, a close relationship was found between the rate of ATP production (i.e. mitochondrial respiration) and protein import. When respiratory rates in IMF and SS mitochondria were equalized, import rates in both subfractions were similar. These data indicate that 1) import rates are more closely related to the rate of ATP production than the steady state ATP level, 2) import into IMF and SS mitochondrial subfractions is regulated differently, and 3) mitochondrial heterogeneity within a cell type can be due to differences in the rates of protein import, suggesting that this step is a potentially regulatable event in determining the final mitochondrial phenotype.

We previously demonstrated that subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subfractions import proteins at different rates. This study was undertaken to investigate 1) whether protein import is altered by chronic contractile activity, which induces mitochondrial biogenesis, and 2) whether these two subfractions adapt similarly. Using electrical stimulation (10 Hz, 3 h/day for 7 and 14 days) to induce contractile activity, we observed that malate dehydrogenase import into the matrix of the SS and IMF mitochondia isolated from stimulated muscle was significantly increased by 1.4- to 1.7-fold, although the pattern of increase differed for each subfraction. This acceleration of import may be mitochondrial compartment specific, since the import of Bcl-2 into the outer membrane was not affected. Contractile activity also modified the mitochondrial content of proteins comprising the import machinery, as evident from increases in the levels of the intramitochondrial chaperone mtHSP70 as well as the outer membrane import receptor Tom20 in SS and IMF mitochondria. Addition of cytosol isolated from stimulated or control muscles to the import reaction resulted in similar twofold increases in the ability of mitochondria to import malate dehydrogenase, despite elevations in the concentration of mitochondrial import-stimulating factor within the cytosol of chronically stimulated muscle. These results suggest that chronic contractile activity modifies the extra- and intramitochondrial environments in a fashion that favors the acceleration of precursor protein import into the matrix of the organelle. This increase in protein import is likely an important adaptation in the overall process of mitochondrial biogenesis.

An effective tool for the global analysis of both DNA methylation status and protein–chromatin interactions is a microarray constructed with sequences containing regulatory elements. One type of array suited for this purpose takes advantage of the strong association between CpG Islands (CGIs) and gene regulatory regions. We have obtained 20 736 clones from a CGI Library and used these to construct CGI arrays. The utility of this library requires proper annotation and assessment of the clones, including CpG content, genomic origin and proximity to neighboring genes. Alignment of clone sequences to the human genome (UCSC hg17) identified 9595 distinct genomic loci; 64% were defined by a single clone while the remaining 36% were represented by multiple, redundant clones. Approximately 68% of the loci were located near a transcription start site. The distribution of these loci covered all 23 chromosomes, with 63% overlapping a bioinformatically identified CGI. The high representation of genomic CGI in this rich collection of clones supports the utilization of microarrays produced with this library for the study of global epigenetic mechanisms and protein–chromatin interactions. A browsable database is available on-line to facilitate exploration of the CGIs in this library and their association with annotated genes or promoter elements.

Chronic low-frequency (10-Hz) electrical stimulation was used to investigate mitochondrial biogenesis in rat tibialis anterior muscle. Succinate dehydrogenase and citrate synthase were used as mitochondrial enzymes, and cardiolipin (CL) was used as a phospholipid index of the inner membrane. Stimulation was via the peroneal nerve (24 h/day) for 1, 2, 3, 5, 7, 10, 14, 21, and 28 days (n = 3-9 rats/day). After each period, endurance performance was evaluated in situ. The contralateral side (CON) served as control nonstimulated muscle. Endurance performance gradually improved after 5 days of stimulation to approximately twofold higher than CON muscle beyond 10 days. Succinate dehydrogenase activity rose to 2.4-fold above CON muscle (4.8 +/- 0.2 U/g; n = 54) by 10 days (half time = 6.1 days) and then remained constant. Citrate synthase demonstrated a similar change. The improved performance with stimulation was correlated (r = 0.61, P < 0.05) to these increases in enzyme activities. CL concentration increased from CON (0.35 +/- 0.02 mumol/g; n = 30) to 3.6- and 3.8-fold above CON at 10 and 14 days (half time = 4.2 days). This increase in CL was greater (P < 0.05) than for either enzyme during the same period. These data are consistent with a model of mitochondrial membrane biogenesis in which enzyme proteins are inserted into a presynthesized lipid bilayer.

Endstage OA fat pads demonstrated a significant upregulation of genes for fat metabolism and energy homeostasis and a mixed result for inflammatory cytokines.

The relationship between adipokines, such as leptin and adiponectin, and cartilage degeneration is being increasingly recognized. We asked what the relationship is between these hormones and patient-reported knee osteoarthritis (OA) pain. We collected demographic data, Short Form McGill Pain scores, Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain scores, and synovial fluid (SF) samples from 60 consecutive patients with severe knee OA at the time of joint replacement surgery. SF samples were analyzed for leptin and adiponectin using specific ELISA. Non-parametric correlations and linear regression modeling were used to identify the relationship between the adipokines and pain levels. The correlations between the individual adipokines and the pain scales were low to moderate and consistently less than that for the corresponding adiponectin/leptin (A/L) ratio. Linear regression modeling showed that the A/L ratio was a significant predictor of a greater level of pain on the MPQ-SF (p=0.03) but not the WOMAC pain scale (p=0.77). A greater A/L ratio was associated with less pain with severe knee OA and this metabolic pathway may represent a target for novel therapeutics.

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