MITOCHIP assessment of differential gene expression in the skeletal muscle of Ant1 knockout mice: Coordinate regulation of OXPHOS, antioxidant, and apoptotic genes

Abstract

Genetic inactivation of the nuclear-encoded mitochondrial heart-muscle adenine nucleotide translocator-1 (ANT1), which exports mitochondrial ATP to the cytosol in both humans (ANT1-/-) and mice (Ant1-/-), results in lactic acidosis and mitochondrial cardiomyopathy and myopathy, the latter involving hyper-proliferation of mitochondria, induction of oxidative phosphorylation (OXPHOS) enzymes, increased reactive oxygen species (ROS), and excessive mtDNA damage. To understand these manifestations, we analyzed Ant1-/- mouse skeletal muscle for changes in gene expression using our custom 644 and 1087 gene MITOCHIP microarrays and for changes in the protein levels of key mitochondrial transcription factors. Thirty-four mRNAs were found to be up-regulated and 29 mRNAs were down-regulated. Up-regulated mRNAs included the mitochondrial DNA (mtDNA) polypeptide and rRNA genes, selected nuclear-encoded OXPHOS genes, and stress-response genes including Mcl-1. Down-regulated mRNAs included glycolytic genes, pro-apoptotic genes, and c-Myc. The mitochondrial regulatory proteins Pgc-1alpha, Nrf-1, Tfam, and myogenin were up-regulated and could account for the induction of the OXPHOS and antioxidant enzymes. By contrast, c-Myc levels were reduced and might account for a reduction in apoptotic potential. Therefore, the Ant1-/- mouse skeletal muscle demonstrates that energy metabolism, antioxidant defenses, and apoptosis form an integrated metabolic network.

Complete loss of expression of theANT1gene causing cardiomyopathy and myopathy

“…As observed in ANT1-KO mice, the loss of ANT1 in our patient is compatible with adult life, presumably as a result of increased mitochondrial mass sustaining significant ADP/ATP exchange mediated by ANT3, the human ubiquitous isoform of the carrier. Even if our data and others2 16 failed to demonstrate overexpression of ANT3 gene, the slow progression of the hypertrophic cardiomyopathy with no sign of heart failure argues for such a partial compensation of the ANT1 defect.…”

“…Proliferation of abnormal mitochondria in muscle, a hallmark of mitochondrial diseases, is reflected by the dramatic increase in citrate synthase activity and suggests a compensatory, but futile, response to the absence of ANT1 in our patient and in mutant mice 14 16. In addition, mitochondrial proliferation is associated with an increased level of mtDNA in skeletal muscle from our patient.…”

The pathophysiology of mitochondrial disease as modeled in the mouse

“…MITO-CHIP and differential display gene expression analysis of Ant1 À/À skeletal muscle revealed a coordinate upregulation of the mtDNA polypeptide and rRNA genes; selected nuclear-encoded OXPHOS genes; mitochondrial regulatory genes PGC-1a, Nrf-1, Tfam, and myogenin; and stress response genes including Mcl1. In contrast, glycolytic genes, proapoptotic genes, and c-myc were all down-regulated (Murdock et al 1999;Subramaniam et al 2008).…”

Severity of cardiomyopathy associated with adenine nucleotide translocator-1 deficiency correlates with mtDNA haplogroup

“…Insight into the pathophysiology can be gleaned from Ant1 null mice, which closely parallel the mitochondrial and cardiac phenotype of Mennonite patients and have similar contractile abnormalities on echocardiographic VVI (12,14). Mitochondria of Ant1 −/− mice have markedly reduced ADP-stimulated respiration rates, most apparent in the skeletal muscle mitochondria that rely almost exclusively on ANT1 as an ADP/ATP translocator and show a massive induction of mitochondrial proliferation and a down-regulation of glycolysis, presumably as a futile attempt to compensate for the diminished mitochondrial ATP export (36,37). Reduced single mitochondrion ATP flux limits sarcomere contraction and also entrains marked compensatory proliferation of cardiac mitochondria, but the adapted myocardium continues to contract inefficiently and dyssynchronously.…”