Gene expression profile was obtained with high-density oligonucleotide microarrays. Of 9,977 genes represented on the microarray, 249 transcripts in the young mice, 298 transcripts in the middle-aged mice, and 256 transcripts in the old mice displayed a significant change in mRNA levels (ANOVA, P < 0.01).
Among these, a total of 55 transcripts were determined to be paraquat responsive for all age groups. Genes commonly induced in all age groups include those associated with stress, inflammatory, immune, and growth factor responses. Interestingly, only young mice displayed a significant increase in expression of all three isoforms of GADD45, a DNA damage-responsive gene.
Additionally, the number of immediate early response genes (IEGs) found to be induced by paraquat was considerably higher in the younger animals. These results demonstrate that, at the transcriptional level, there is an age-related impairment of specific inducible pathways in the response to oxidative stress in the mouse heart.
Although the increased susceptibility of older animals to various forms of stress has been well documented, little is known about the genetic basis underlying this change (31, 37). There is also evidence to suggest that longevity and the ability to resist oxidative and metabolic stress are related processes. Several long-lived mutants have been identified in Saccharomyces cerevisiae (13), Drosophila (27), and Caenorhabditis elegans (35) that exhibit increased resistance to a wide range of physiological and pharmacological insults.
The basic molecular defense systems employed by these organisms are very similar to those utilized in mammals (40, 52), suggesting that stress responses may also play a role in aging of longer lived species. In mammals, the only intervention that is proven to extend lifespan is caloric restriction (CR) (53), and CR rodents display increased resistance to heat shock (18, 21) and oxidative damage (45).
The endogenous production of reactive oxygen species (ROS), a by-product of cellular respiration, may contribute to the aging phenotype (19). The heart is an organ that is likely to be particularly vulnerable to increases in oxidative stress, since cardiomyocytes depend heavily on mitochondrial function. The ability to cope with cardiovascular injury has been shown to decline with age (23, 29, 30), and many heart-related stresses, such as myocardial ischemia and reperfusion, generate ROS that may contribute to pathology (11). Possibly, the age-associated increase in the production of ROS contributes to the observed decline in the ability to recover from cardiac-related trauma in the aged animal (23, 29, 30).
Previous studies using high-density oligonucleotide arrays have characterized the basal transcriptional response to the aging process in skeletal muscle (25), brain (26), and heart (24) of mice. However, this technology has not been used to characterize the transcriptional response to acute oxidative stress as a function of age.
Accordingly, we investigated the transcriptional response to oxidative damage in the heart by challenging 5-mo-old (young), 15-mo-old (middle-aged), and 25-mo-old mice with paraquat. Paraquat is a toxin that reacts with molecular oxygen in vivo to generate ROS in several tissues and has been used previously to elicit oxidative stress in rodents (2, 10, 50). Paraquat ingestion in rats and humans leads to severe heart damage (36, 39) and an increase in the levels of 8-hydroxydeoxyguanosine in the heart, a marker of oxidative DNA damage (48).
Messenger RNA (mRNA) is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression. Following transcription of primary transcript mRNA (known as pre-mRNA) by RNA polymerase, processed, mature mRNA is translated into a polymer of amino acids: a protein, as summarized in the central dogma of molecular biology.
As in DNA, mRNA genetic information is in the sequence of nucleotides, which are arranged into codons consisting of three base pairs each. Each codon encodes for a specific amino acid, except the stop codons, which terminate protein synthesis. This process of translation of codons into amino acids requires two other types of RNA: Transfer RNA (tRNA), that mediates recognition of the codon and provides the corresponding amino acid, and ribosomal RNA (rRNA), that is the central component of the ribosome’s protein-manufacturing machinery.