Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of rat posterior cricoarytenoid muscle from animals aged 6, 18 and 30 months

ABSTRACT: Sarcopenia is the decreased muscle mass and weakness associated with aging and a major cause of morbidity and mortality in the elderly. To what extent non-locomotive muscles are susceptible to this condition is unclear. For example, age affects laryngeal function (ventilation, airway protective reflexes, swallowing and phonation). Age-related laryngeal dysfunction may be due to effects on its intrinsic muscles that have a unique phenotype: very small, mostly fast oxidative muscle fibers. For this study, we examined how age alters the functional characteristics and gene expression profile of posterior cricoarytenoid (PCA), an intrinsic laryngeal muscle. PCA muscles from Fischer 344-Brown Norway F1 hybrid rats (6 and 30 months of age) were used for cDNA microarrays, light and electron (EM) microscopy, and in vitro contractile function. Histological analyses demonstrated a ~40% increase in mean PCA fiber size and in the number of fibers with low myosin ATPase activity. There was also evidence of ragged-red fibers, a hallmark of mitochondrial dysfunction. In turn, mitochondrial volume density, determined by EM, was significantly higher in PCA muscles at 30 months (43% vs. 21% at 6 months). In vitro function showed a decrease in velocity of unloaded shortening at 30 months. Finally, cDNA microarrays demonstrated a transcriptome shift in PCA muscle with age. Gene classes with the largest changes were: signal transduction, transcription factors, and metabolic enzymes. These data demonstrate that PCA muscles are significantly altered by age. Moreover, the observed changes in muscle fiber size, mitochondrial content and gene expression profile suggest that the PCA response to age diverges from that seen in more typical skeletal muscles. Experiment Overall Design: Total RNA was obtained with TRIzol (Invitrogen Carlsbad, CA) following the manufacturers recommended protocol. Tissues from 4 animals were combined into each RNA sample to decrease inter-subject variability. Biotinylated cRNA samples were hybridized to Affymetrix Rat Genome U34 gene chips (n=9 chips) described previously [McMullen et al. 2004]. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all three independent replicates per group were considered for further analysis. Comparisons used the 6-mo transcriptome as the baseline and the one-sided Wilcoxonâs signed rank test to estimate âincrease/no change/ decreaseâ difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 1.70 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Colleen McMullen

PROVIDER: E-GEOD-3310 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: Despite demonstrating that the overall effect of long-term rapamycin-treatment is overwhelmingly positive in aging skeletal muscle, we observed muscle-specificity in the responsiveness to rapamycin, leading us to hypothesize that the primary drivers of age-related muscle loss and therefore effective intervention strategies may differ between muscles. To address this question and dissect the key signaling nodes associated with mTORC1-driven muscle aging, we created a comprehensive multi-muscle gene expression atlas in adult, sarcopenic and rapamycin-treated mice using RNA-seq. Methods: To examine the impact of long-term rapamycin treatment, male C57BL/6 mice were fed encapsulated rapamycin incorporated into a standardized AIN93M diet at 42 ppm (i.e. mg per kg of food), corresponding to a dose of ~4 mg·kg-1·day-1, from 15- to 30-months of age. This dose of rapamycin has been shown to extend lifespan maximally in male C57BL/6 mice. We performed RNA-seq on gastrocnemius (GAS), tibialis anterior (TA), triceps brachii (TRI) and soleus (SOL) muscles from each of six mice for 10m, 30m and 30mRM groups. The four muscle types were chosen to encompass fore- and hindlimb locations (e.g. TRI and GAS); slow and fast contraction properties (e.g. SOL and GAS); anterior and posterior positioning (e.g. TA and GAS) and the extent of protection by rapamycin (TA and TRI: protected; SOL: partiallly protected; GAS: not protected). Results: Age-related gene expression changes were remarkably consistent across the four different muscles, varying only in magnitude. Despite having the smallest age-related muscle loss of the four muscles, TA had the strongest age-related gene expression response which was associated with an increased reinnervation response. Despite the strong between-muscle commonality of age-related changes, gene expression responses to prolonged rapamycin treatment differed substantially between muscles. Rapamycin partially reversed many age-related changes in mRNA expression in the TA and TRI, but not in SOL or GAS muscle. Principle component analysis (PCA) showed that rapamycin had common 'anti-aging' effects on all muscles, while also exerting muscle-specific pro-aging effects on muscles not protected by rapamycin. Conclusions: Sustained, muscle fiber-specific mTORC1 activity drives sarcopenia-like gene expression programs, and the hyperactive mTORC1 seen in sarcopenic muscle may therefore contribute to sarcopenia.

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Transcription profiling of rat posterior cricoarytenoid muscle from animals aged 6, 18 and 30 months

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