Project description:Epidemiological studies reveal a strong link between low aerobic capacity and metabolic and cardiovascular diseases. Two-way artificial selection of rats based on low and high intrinsic exercise capacity has produced two strains that also differ in risk for metabolic syndrome (Koch LG, Britton SL. Artificial selection for intrinsic aerobic endurance running capacity in rats. Physiol Genomics 5:45-52, 2001). Here we investigated skeletal muscle characteristics and genotype-phenotype relationships behind high and low inherited aerobic exercise capacity and the link between oxygen metabolism and metabolic disease risk factors in rats derived from generation 18. This population (n=24) of high capacity runners (HCR) and low capacity runners (LCR) differed by 615% in maximal treadmill running capacity. LCR were significantly significantly heavier and had increased blood glucose, serum insulin and triglyceride concentration. HCR had higher resting metabolic rate than LCR. Capillaries/mm2 and capillary-to-fiber ratio were significantly greater in HCR rats in soleus and gastrocnemius and capillary-to-fiber ratio in extensor digitorum longus (EDL) muscle. Subsarcolemmal mitochondrial area was 96% (p<0.01) and intermyofibrillar area was 32% (p<0.05) larger in HCR soleus. Microarray results showed that 126 genes were significantly up-regulated and 113 genes were down-regulated in HCR (p<0.05). Functional clustering and unbiased correlation analysis of muscle microarray data revealed that genes up-regulated in HCR were related to mitochondria, carboxylic acid and lipid metabolism, and oxidoreductase activity. In conclusion, our data show that aerobic capacity is strongly linked to the architecture of energy transfer and corroborate the importance of oxygen metabolism as the determinant of metabolic health and complex metabolic diseases such as metabolic syndrome and type 2 diabetes. Total RNA obtained from gastrocnemius muscle was compared between rat strains of low and high inherited aerobic exercise capacity.

Project description:Purpose: Aerobic capacity is a strong predictor of cardiovascular mortality. To determine the relationship between inborn aerobic capacity and soleus gene expression we examined genome-wide gene expression in soleus muscle of rats artificially selected for high and low running capacity (HCR and LCR, respectively) over 16 generations. The artificial selection of LCR caused accumulation of risk factors of cardiovascular disease similar to the metabolic syndrome seen in man, whereas HCR had markedly better cardiac function. We also studied alterations in gene expression in response to exercise training in the two groups, since accumulating evidence indicates that exercise has profound beneficial effects on the metabolic syndrome. Methods:; Soleus gene expression of both sedentary and exercise trained HCR and LCR was characterized by microarray- and gene ontology analysis. Results: Although HCR and LCR had an inborn 347% difference in running capacity, only three genes were found differentially expressed in the soleus muscle between the two groups. Up-regulation of the mitochondrial enzyme leucyl-transferRNA synthetase (LARS2) was found in the sedentary LCR. Increased expression of LARS2 has been associated with a mitochondrial DNA mutation linked to maternally inherited diabetes and mitochondrial dysfunction. In line with our findings, a growing body of evidence suggests that LCR have compromised mitochondrial function. After exercise training, 58 genes were altered in the soleus muscle of HCR, in contrast to only one in the LCR group. This suggests that animals born with different levels of fitness respond different to the same type of exercise training. Adaptations to exercise in HCR seemed to be associated with increased lipid metabolism and fatty acid elongation in the mitochondria. Also, genes associated with the peroxisomes, seemed to be central in the adaptation to exercise. Conclusion: The results indicate that (i) LCR might have mitochondrial dysfunction, which may be a contributing factor of the low inborn aerobic capacity, (ii) animals born with different levels of fitness respond different to the same exercise program. Experiment Overall Design: There are 16 samples in this study.

Project description:Purpose: Aerobic capacity is a strong predictor of cardiovascular mortality. To determine the relationship between inborn aerobic capacity and soleus gene expression we examined genome-wide gene expression in soleus muscle of rats artificially selected for high and low running capacity (HCR and LCR, respectively) over 16 generations. The artificial selection of LCR caused accumulation of risk factors of cardiovascular disease similar to the metabolic syndrome seen in man, whereas HCR had markedly better cardiac function. We also studied alterations in gene expression in response to exercise training in the two groups, since accumulating evidence indicates that exercise has profound beneficial effects on the metabolic syndrome. Methods: Soleus gene expression of both sedentary and exercise trained HCR and LCR was characterized by microarray- and gene ontology analysis. Results: Although HCR and LCR had an inborn 347% difference in running capacity, only three genes were found differentially expressed in the soleus muscle between the two groups. Up-regulation of the mitochondrial enzyme leucyl-transferRNA synthetase (LARS2) was found in the sedentary LCR. Increased expression of LARS2 has been associated with a mitochondrial DNA mutation linked to maternally inherited diabetes and mitochondrial dysfunction. In line with our findings, a growing body of evidence suggests that LCR have compromised mitochondrial function. After exercise training, 58 genes were altered in the soleus muscle of HCR, in contrast to only one in the LCR group. This suggests that animals born with different levels of fitness respond different to the same type of exercise training. Adaptations to exercise in HCR seemed to be associated with increased lipid metabolism and fatty acid elongation in the mitochondria. Also, genes associated with the peroxisomes, seemed to be central in the adaptation to exercise. Conclusion: The results indicate that (i) LCR might have mitochondrial dysfunction, which may be a contributing factor of the low inborn aerobic capacity, (ii) animals born with different levels of fitness respond different to the same exercise program. Keywords: aerobic capacity, metabolic syndrome, soleus muscle, gene expression, metabolism

Project description:Left heart disease (LHD) frequently causes lung vascular remodelling and pulmonary hypertension (PH). Yet, pharmacological treatment for PH in LHD is lacking and its pathophysiological basis remains obscure. We aimed to identify candidate mechanisms of PH in LHD and to test their relevance and therapeutic potential. In rats, LHD was induced by supracoronary aortic banding. Whole genome microarray analyses were performed, candidate genes were confirmed by RT-PCR and Western blots and functional relevance was tested in vivo by genetic and pharmacological strategies. In lungs of LHD rats, mast cell activation was the most prominently upregulated gene ontology cluster. Mast cell gene upregulation was confirmed at RNA and protein levels and remodelled vessels showed perivascular mast cell accumulations. In LHD rats treated with the mast cell stabiliser ketotifen, or in mast cell deficient Ws/Ws rats, PH and vascular remodelling were largely attenuated. Both strategies also reduced PH and vascular remodelling in monocrotaline-induced pulmonary arterial hypertension, suggesting that the role of mast cells extends to noncardiogenic PH. In PH of different aetiologies, mast cells accumulate around pulmonary blood vessels and contribute to vascular remodelling and PH. Mast cells and mast cell-derived mediators may present promising targets for the treatment of PH. Whole rat genome microarray analyses were performed in lung homogenates of three rats with established PH following supracoronary aortic banding and in three sham-operated controls. Out of a total of 28,000 analysed genes, differential expression defined as 2-fold change with p<0.05 was evident for 120 genes. Of these, 76 were upregulated and 44 downregulated in aortic banding compared with control lungs gene. Enrichment analysis revealed regulation of mast cell specific genes - 13 out of 20 genes were significantly upregulated in aortic banding compared with control lungs. To test for a putative functional role of mast cells in lung vascular remodelling and PH in LHD, we applied a pharmacological approach by treatment of aortic banding rats with the mast cell stabiliser ketotifen. Microarray analysis then compared rats that were treated with untreated. In brief, congestive heart failure (CHF) was induced in juvenile rats by supracoronary aortic banding and rats were analyzed 9 weeks thereafter when they had established left heart failure with preserved ejection fraction and secondary pulmonary hypertension. Three heart failure rats were untreated, and 3 received the mast cell stabilizer ketotifen (1 mg/kg-1 bodyweight/day-1) with the drinking water. Sham rats underwent the same surgical procedure but without placement of a clip on the supracoronary aorta. Microarray analyses: Lungs from banded and control rats were excised and total RNA was extracted (Stratagene Absolutely RNA Miniprep Kit; Stratagene, La Jolla, USA). Three Âµg total RNA from three control and three banded rats each were processed according to the One-Cycle Target Labeling protocol (GeneChip Expression Analysis, Affymetrix, Santa Clara, USA). Before and after amplifications, the total RNA/complementary RNA concentrations were checked with NanoDrop ND-1000 (Thermo Scientific, Wilmington, USA) and quality was controlled (Experion electrophoresis station, BioRad; Hercules, USA). Samples were hybridized to GeneChipÂ® Rat Genome 230 2.0 arrays (Affymetrix; Santa Clara, USA) containing 31,000 probe sets covering 28,000 rat genes.

Project description:Aerobic capacity is a strong predictor of cardiovascular mortality. To determine the relationship between aerobic capacity and cardiac gene expression we examined genome-wide gene expression in hearts of rats artificially selected for high- and low running capacity (HCR and LCR, respectively) over 16 generations. HCR were born with an athletic phenotype, whereas LCR exhibited features of the metabolic syndrome. Left ventricle gene expression of both sedentary and exercise trained HCR and LCR was characterized by microarray- and gene ontology analysis. Western immunoblots and immunohistochemistry were used to validate the results at protein level. Out of 28.0000 screened genes, 1540 were differentially expressed between HCR and LCR. HCR expressed higher amounts of genes involved in lipid metabolism, whereas LCR expressed higher amounts of the genes involved in glucose metabolism and transport. By simply selecting for running capacity, we created a difference in cardiac energy substrate utilisation from normal mitochondrial fatty acid ï¢-oxidation in HCR to carbohydrate metabolism in LCR. This event often occurs in diseased hearts. Differential expression of genes involved in cardiomyocyte growth was also found. LCR rats were associated with fetal gene expression, indicating the presence of pathological remodelling signaling. In addition, different expression of genes associated with cardiac contractility and cellular stress were detected. Also, hypoxia triggered transcription seemed to be involved in several of the functional differences between HCR and LCR. In conclusion, high- versus low aerobic capacity was associated with differences in genes regulating cardiac energy substrate, growth signalling, contractility and cellular stress. Experiment Overall Design: Animals Experiment Overall Design: The experimental rats in this study are the result of artificial selection for high and low aerobic capacity, starting from the N: NIH stock obtained from the National Institutes of Health (USA). The generation of the model has been previously described. Briefly, the rats in each generation were tested for exercise capacity by treadmill running at about 11 weeks of age. The individuals with the highest and lowest aerobic capacity were selected and each group served as the mating population for the next generation of high- and low-capacity runners; HCR and LCR, respectively. Rats from generation 16 were used in this study. The study includes four groups; LCR trained (n=4), LCR sedentary (n=4), HCR trained (n=4) and HCR sedentary (n=4). Experiment Overall Design: Endurance training Experiment Overall Design: The animals in the training groups were submitted to an aerobic interval training program previously described by HÃ¸ydal et al. Briefly, after 10 minutes of warm-up, rats ran uphill (25Â°) on a treadmill for 1.5 hours, alternating between 8 minutes at an exercise intensity corresponding to 85-90% of maximal oxygen uptake (VO2max), and 2 minutes active recovery at 50 - 60%. Exercise was performed 5 days per week over 8 week; controls were age-matched rats that remained sedentary. In the exercising animals VO2max was measured every week to adjust band speed in order to maintain the intended intensity throughout the experimental period. The VO2max-measurements consisted of a 20 minutes warm-up at 50-60% of VO2max, whereupon treadmill velocity was increased by 0.03 m/s every 2 minute until VO2 plateau despite of increased workload. The apparatus and method were previously described and validated. Experiment Overall Design: Tissue collection Experiment Overall Design: At approximately 7 months of age the animals were sacrificed. A section of the left ventricle was formalin fixated for immunohistochemistry and morphological studies, whereas the rest was snap frozen in liquid nitrogen and stored at -80oC for later genetic screening and protein analysis. Experimental protocols were approved by the respective Institutional Animal Research Ethics Councils. Experiment Overall Design: Ribonuclease (RNA) isolation Experiment Overall Design: Tissue samples (20 mg) were homogenized in 100 ÂµL TRIzol (Life Technologies, Gaithersburg, MD) using a Mixer Mill MM301 at 20-25 Hz. RNA clean-up was performed using RNA Mini kit (Qiagen, Germantown, MD). Total RNA was isolated and RNA clean-up was performed according to the manufacturer's instructions. Experiment Overall Design: RNA integrity, purity and quantity were assessed by Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Nanodrop (NanoDrop Technologies, Baltimore, MD). The concentration of total RNA was measured by Nanodrop with ultraviolet spectrophotometry at 260/280 nm. RNA quality was assessed by electrophoresis on Bioanalyzer chips (Agilent Technologies). Experiment Overall Design: Experiment Overall Design: High quality RNA was classified as a 260/280 ratio above 1.8. Only samples with a 260/280 ratio between 1.8-2.2 and no signs of degradation were used for analysis. Experiment Overall Design: Processing of Affymetrix data Experiment Overall Design: Gene expression were analyzed on whole-genome RAE 230 2.0 chip from Affymetrix GeneChip (Affymetrix, Santa Clara, CA) comprised of 31,042 probe sets, analyzing over 30,000 transcripts and variants from over 28,000 substantiated rat genes. On the Affymetrix GeneChip arrays, each gene is represented by a set of 11-20 probe pairs consisting of a perfect match (PM) and a mismatch (MM) probe. The statistical analysis is based on summary expression measures for each probe set. Experiment Overall Design: Computing summary measures (RMA) Experiment Overall Design: The summary measure for each probeset is computed based on a linear statistical model for background-corrected, normalized and log-transformed PM values for each probe pair by use of the robust multiarray average (RMA) method. The PM values are normalized using the quantile normalization method, normalizing the arrays such that the empirical distribution of the expression measures is equal across arrays. Experiment Overall Design: Statistical analysis for finding differentially expressed genes Experiment Overall Design: For each gene (probeset), a linear regression model, including parameters representing the effect of aerobe capacity is specified. Based on the estimated effects, tests for significant differential expression are performed using moderated T-tests. Experiment Overall Design: To account for multiple testing, we calculate adjusted p-values controlling the False Discovery Rate (FDR), with the use of the Benjamini-Hochberg step-up procedure. Consequently, selecting differentially expressed genes based on a threshold of 0.05 on the adjusted FDR p-values means that the expected proportion of genes falsely classified as differential expressed should be below 0.05. Experiment Overall Design: All statistical analyses on the gene expression data are performed using the R language (R Development Core Team, 2004) and packages affy, affyPLM and limma from the Bioconductor project.

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Low and high aerobic capacity rats exposed to cigarette smoke, lung tissue

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