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Am J Physiol Heart Circ Physiol 289: H1399-H1407, 2005. First published May 6, 2005; doi:10.1152/ajpheart.00170.2005
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Transcriptional basis for exercise limitation in male eNOS-knockout mice with age: heart failure and the fetal phenotype

Caroline Ojaimi,1 Wei Li,1 Shintaro Kinugawa,1 Heiner Post,1 Anna Csiszar,1 Pal Pacher,2 Gabor Kaley,1 and Thomas H. Hintze1

1Department of Physiology, New York Medical College, Valhalla, New York; and 2National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland

Submitted 18 February 2005 ; accepted in final form 29 April 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Endothelium-derived nitric oxide (NO) is pivotal in regulating mitochondrial O2 consumption (O2) and glucose uptake in mice. The aim of this study was to investigate the mechanism of age- and genotype-related exercise limitation in male endothelial NO synthase (eNOS)-knockout (KO, n = 16) and wild-type (WT, n = 19) mice. Treadmill testing was performed at 12, 14, 16, 18, and 21 mo of age. O2, CO2 production, respiratory exchange ratio, and maximal running distance were determined during treadmill running. There were good linear correlations for increase of speed with increase of O2. The difference between KO and WT mice was not significant at 12 mo but was significant at 18 mo. Linear regression showed that KO mice consumed more O2 at the same absolute and relative workloads, suggesting that O2 was not inhibited by NO in KO mice. KO mice performed 30–50% less work than WT mice at each age (work = vertical distance x weight). In contrast to WT mice, the work performed by KO mice significantly decreased from 17 ± 1.4 m·kg at 12 mo to 9.4 ± 1.7 m·kg at 21 mo. Running distance was significantly decreased from 334 ± 27 m at 12 mo to 178 ± 38 m at 21 mo, and maximal O2, CO2 production, and respiratory exchange ratio per work unit were significantly higher in KO than in WT mice. Gene arrays showed evidence of a fetal phenotype in KO mice at 21 mo. In conclusion, age- and genotype-related exercise limitations in maximal work performed and maximal running distance in male eNOS-KO mice indicated that fetal phenotype and age were related to onset of heart failure.

maximal oxygen consumption; respiratory exchange ratio; work; microarray; endothelial nitric oxide synthase

NITRIC OXIDE (NO) has been well known as a potent vascular smooth muscle relaxant and regulator of cardiovascular homeostasis. NO can modulate glucose uptake (54) in mice and substrate utilization in conscious dogs with overt heart failure (44). We have reported a fall in basal cardiac production of NO during cardiac decompensation that is associated with a significant increase in myocardial glucose uptake and decrease in free fatty acid utilization (44). Our previous studies demonstrated that NO inhibits mitochondrial O2 consumption (MO2) in mouse heart (35), dog skeletal muscle (50), and humans (34). We have suggested that endogenous endothelial NO synthase (eNOS)-derived NO is a physiological regulator of myocardial O2 consumption (O2) (35).

During exercise, there is a significant increase in the release of NO from the coronary circulation in conscious dogs and greater increases in total O2 and O2 in skeletal muscle and in the heart when NO synthesis is blocked. Thus NO plays a role in matching blood flow to tissue metabolism at rest and during exercise (61). Reduced endothelium-derived NO (EDNO) synthesis or activity (e.g., heart failure) results in an inadequate exercise hyperemic response that is rate limiting for O2 transport and exercise capacity. In normal mice, L-arginine enhances exercise-induced EDNO synthesis and aerobic capacity (37). Dyke et al. (12) reported a diminution of forearm exercise hyperemia during prolonged handgrip exercise in humans with infusion of an NO synthase (NOS) inhibitor.

Laboratory mice are frequently used in aging research and exercise physiology. One study demonstrated that, in untrained C57BL/6J mice, maximal O2 (O2 max) and maximal exercise capacity declined with age (48). Cross-sectional and longitudinal studies in humans and rats demonstrated similar changes in O2 max associated with increasing age (19, 23, 39). Findings in several different mammalian species strongly support the hypothesis that O2 max and maximal exercise capacity decline as a direct result of aging (20).

Mice lacking eNOS have been generated. There is an increase in O2 and glucose uptake (54) in eNOS-knockout (KO) mouse hearts (35). Our previous data indicate that cardiac dysfunction in male eNOS-KO mice is associated with age (31). No study has been done to address the potential limitation in exercise capacity and its molecular basis in those mice with age. Therefore, the aims of this study were to investigate age- and genotype-related exercise limitation in O2 max, maximal CO2 production (CO2 max), and maximal respiratory exchange ratio (RERmax), work performed, and maximal distance run by male eNOS-KO and wild-type (WT) mice. We hypothesized that eNOS-KO mice would perform less work and run a shorter distance than WT mice with age. The potential molecular basis of these differences was determined using gene arrays.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Heterozygote eNOS-KO mice, originally developed by Shesely et al. (51), were interbred to generate eNOS+/– homozygous KO and WT mice. Sixteen male KO and 19 male WT mice were genotyped by Southern blot analysis of DNA from tail snips, as described previously (51). All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the current National Institutes of Health and American Physiological Society guidelines for the use and care of laboratory animals.

Equipment. O2 and CO2 fractions at the inlet and output ports of test chambers for calculation of O2, CO2 production (CO2), and respiratory exchange ratio (RER; i.e., ratio of CO2 to O2) were monitored with the Oxymax system and the Eco 3/6 treadmill (Columbus Instruments). Work was calculated as vertical distance * weight.

Experimental protocol. Each mouse was adapted to the testing environment by one practice trial 3 days before the experiment at 10 m/min for 10 min. The mice had free access to water but were allowed no food 3 h before the experiment. The mice were weighed and placed on the treadmill for 15 min of acclimation. O2, CO2, and RER were measured during rest, during exercise, and for ≥5 min during recovery after the animals ran. Distance run until exhaustion was measured. Mice ran at a constant 10° angle. We increased the running speed at 2-min intervals by 2 m/min until the mouse was exhausted. The mice were considered to be exhausted when they remained on the shocker plate without attempting to reengage the treadmill within 10 s. The experiments were performed when the KO and WT mice were 12.5 ± 0.2, 14.2 ± 0.3, 16.5 ± 0.1, 18.3 ± 0.2, and 21.5 ± 0.5 mo old. During this study, six KO mice died, so only the healthiest survived. There were no deaths among the WT mice.

RNA isolation. Total RNA was extracted from the left ventricle from male eNOS-KO (n = 3) and WT (n = 4) mice at 21 mo of age. RNA was extracted with a commercial RNA isolation kit using Trizol (TRI REAGENT, Sigma, St. Louis, MO) followed by the RNeasy total RNA extraction kit with modifications (for detailed protocol see http://fgc.urmc.rochester.edu/). RNA quality was assessed by electrophoresis (Agilent Bioanalyzer 2100).

Microarray labeling and hybridization. Between 1 and 10 µg of total RNA from each sample were used to generate a high-fidelity cDNA, which is modified at the 3' end to contain an initiation site for T7 RNA polymerase. On completion of cDNA synthesis, 1 µg of product was used in an in vitro transcription reaction that contains biotinylated UTP and CTP, which are utilized for detection after hybridization to the oligonucleotide microarray. Fragmentation of 20 µg of full-length cRNA, from control and enriched samples, was carried out in 200 mM Tris-acetate (pH 8.1), 500 mM KOAc, and 150 mM MgOAc at 94°C for 35 min. After fragmentation, all components generated throughout the processing procedure (cDNA, full-length cRNA, and fragmented cRNA) were analyzed by electrophoresis (Agilent Bioanalyzer 2100) to assess the appropriate size distribution before microarray hybridization (for detailed protocols for sample preparation using the Affymetrix labeling protocols see http://www.affymetrix.com).

Each cRNA was hybridized to an individual Affymetrix GeneChip Mouse Expression Array 430A, which was subsequently processed and scanned according to the manufacturer's instructions. Each array quantifies the expression level of 39,000 transcripts and variants, including >34,000 well-substantiated mouse genes (http://www.affymetrix.com). The cDNA array hybridizations and scanning were carried out by Dr. Andrew Brooks (Functional Genomics Center, University of Rochester Medical Center) through the AMDec consortium collaboration (for detailed protocols see http://fgc.urmc.rochester.edu/).

Microarray data analysis. All arrays referred to in this study were assessed for "array performance" before data analysis. This process involved the statistical analysis of control transcripts that are spiked into the samples and the hybridization cocktail for assessment of array performance. In addition, several genes have been identified on each array to help assess the overall quality of signal intensity from all arrays. The results of this analysis helped validate the reproducibility of each array at baseline, allowing us to define the lower level of sensitivity that was employed to identify small changes in biologically relevant genes.

Before analysis, data from each hybridization were processed using Microarray Suite software (version 5.0) to yield average difference values corresponding to signal intensity for each probe set. Distinct algorithms were used to determine the absolute call, which distinguishes the presence or absence of a transcript, the differential change in gene expression (increase, decrease, marginal increase, marginal decrease, and no change), and the magnitude of change, which is represented as signal logarithmic ratio (on a log base 2 scale). t-Tests were performed on the normalized signal values before further analysis.

All the hybridization data have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) with GEO accession numbers for series GSE1988. The raw pixel data were imported into GeneTraffic MULTI, and all subsequent analyses were performed on a GeneTraffic server (GeneTraffic version 3.0-27, Iobion Informatics, La Jolla, CA) at the Functional Genomics Core Facility of New York Medical College. All the microarray data were analyzed and normalized using a Robust Multi-Chip Analysis algorithm. The average of all the WT data sets was used as the baseline for the analysis.

The expression data generated from GeneTraffic were then imported into PathwayAssist software (version 2.53, Iobion Informatics). We visualized and explored biological association networks represented in the ResNet database. Microarray gene expression data were overlaid on a biological association network to show how genes are affected in eNOS-KO mice.

Statistical analysis. For the statistical analyses, the average O2 (ml·kg–1·min–1) at each speed at 12 and 18 mo for KO and WT mice was determined. Linear regression of O2 as a function of treadmill speed and linear regression of work performed and maximal running distance with age and comparison of linear regressions between KO and WT mice were conducted using Sigmastat and NCSS 2000-PASS 2000 software. The comparison of both genotypes for O2 max, CO2 max, and RERmax was performed via two-way ANOVA to determine the main effect of age and genotype on O2. Values are means ± SE. P < 0.05 was accepted as level of significance.

For the microarray analysis, statistical significance for changes in gene expression was performed in GeneTraffic using a two-class method (t-test) and with variance stabilization. Differences were considered statistically significant at a nominal significance of P < 0.05 and at least ±1.5-fold change in expression between eNOS-KO and WT mice.

Real-time quantitative RT-PCR. To independently confirm the differential expression data generated by microarray analysis, real-time quantitative RT-PCR was utilized to determine the relative expression of specific genes between male KO and WT mice. Quantitative PCR was performed to validate changes in eight selected genes using the same RNA samples analyzed with microarrays. A relative quantitation method [{Delta}{Delta}C(t)] (33) was used to evaluate the relative expression of each gene between KO and WT mice. RT-PCR of GAPDH was used as an internal control and for normalization of all data.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
For most of the mice, volitional running was easily maintained when the mice ran 10–20 m/min. Electrical shocks were necessary to keep mice running at higher speeds.

O2 and percentage of O2 max at each speed in KO and WT mice at 12 mo. In the range of treadmill speeds, O2 increased progressively as a function of running speed and could be expressed by simple linear equations, as shown previously (13). The average O2 at each speed for KO and WT mice at 12 mo is shown in Fig. 1A. In KO mice O2 increased from 33 ± 0.74 ml x kg–1·min–1 at 0 m/min to 53 ± 4.4 ml·kg–1·min–1 at 28 m/min. In WT mice, O2 increased from 42 ± 1.8 ml·kg–1·min–1 at 0 m/min to 62 ± 2.7 ml·kg–1·min–1 at 30 m/min. When we plot the linear regression, the simple linear functions are as follows: O2 = 0.46 x speed + 37.8 for KO mice and O2 = 0.45 x speed + 45.6 for WT mice. Multiple R (correlation of actual O2 with predicted value) was 0.77 for KO mice and 0.88 for WT mice. The slopes between the two regression lines were not significantly different.



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  		Fig. 1. A: average O2 consumption (O2) vs. speed in endothelial nitric oxide synthase (eNOS)-knockout (KO) and wild-type (WT) mice at 12 mo of age. Simple linear functions for KO and WT mice are shown. Slopes between the 2 regression lines were not significantly different at 21 mo. B: maximal work performed by KO and WT mice with age. Work performed by WT mice decreased (P = not significant), but work performed by KO mice significantly decreased from 17 ± 1.4 m x kg at 12 mo to 9.4 ± 1.7 m x kg at 21 mo (P < 0.001). Simple linear regressions for KO and WT mice are shown. Slopes between the 2 regression lines were significantly different (P < 0.01). Values are means ± SE.

 
Maximal work performed by KO and WT mice with age. In KO mice, a slight loss of weight from 30 ± 0.8 g at 12 mo to 29 ± 0.5 g at 18 mo was observed; in WT mice, a weight gain from 31 ± 1 g at 12 mo to 33 ± 0.9 g at 18 mo was observed. At 12 mo, KO mice weighed ~3% less than WT mice, and this difference was not significant. At 18 mo, KO mice weighed significantly less (~12%) than WT mice (P < 0.05). KO mice performed 30–50% less work than WT mice at each age. As shown in Fig. 1B, work performed by WT mice did not change with age (24 ± 1.5 and 22 ± 1.3 m x kg at 12 and 21 mo, respectively, P = not significant), but work performed by KO mice significantly decreased (from 17 ± 1.4 m x kg at 12 mo to 9.4 ± 1.7 m x kg at 21 mo). Two-way ANOVA showed that the difference was significant (P < 0.001). The changes in work performed between 12 and 21 mo for KO and WT mice can be described by simple linear functions: work = –0.79 x age + 26 and work = –0.23 x age + 27, respectively. Multiple R (correlation of actual work with predicted value) was 0.97 and 0.87 for KO and WT mice, respectively. The slopes between the two regression lines were significantly different (P < 0.01), but there was no difference between the intercepts.

Maximal distance run by KO and WT mice with age. Distance run by KO and WT mice with age is shown in Fig. 2A. Running distance was 20–60% less for KO than for WT mice at each age. Distance run by WT mice significantly decreased from 483 ± 28.6 m at 12 mo to 369 ± 25.7 m at 21 mo, and distance run by KO mice was reduced from 334 ± 27.1 m at 12 mo to 178 ± 38 m at 21 mo (P < 0.05). The changes of running distance between 12 and 21 mo for KO and WT mice can be described by simple linear functions: running distance = –15.7 x age + 515 and running distance = –9.9 x age + 568, respectively. Multiple R was 0.96 and 0.96 for KO and WT mice, respectively. The slopes and constants between the two regression lines were not significantly different.



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  		Fig. 2. A: maximal distance run by KO and WT mice vs. age. KO mice ran 20–60% less distance than WT mice at each age (P < 0.001). B: maximal O2 (O2 max) in KO and WT mice vs. age. O2 max normalized to work unit was significantly higher in KO than in WT mice from 14 to 21 mo (P < 0.05). *P < 0.05 vs. WT. Values are means ± SE.

 
O2 max in KO and WT mice with age. O2 max was defined as the plateau in O2, despite increasing work intensity. In cases in which a plateau was not reached, O2 max was approximated by the peak O2 attained by the animal before exhaustion. When a further increase in treadmill speed resulted in a decline of O2 or the mice refused to run, we chose the O2 as O2 max for that mouse. At 14 mo of age, O2 max significantly decreased in KO mice compared with their age-matched WT controls. At all the other ages, there were no significant differences between these two genotypes. When O2 max is normalized to work unit (Fig. 2B), values were significantly higher for KO mice starting from 14 mo than for WT mice (P < 0.05).

CO2 max in KO and WT mice with age. Only at 16 and 18 mo was the decrease of CO2 max in KO mice significant compared with their age-matched WT controls. When CO2 max is normalized to per work unit (Fig. 3A), values are higher from 14 to 21 mo for KO than for WT mice (P < 0.05).



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  		Fig. 3. A: maximal CO2 production (CO2 max) in KO and WT mice vs. age. CO2 max normalized to work unit was significantly higher in KO than in WT mice from 14 to 21 mo (P < 0.05). B: maximal respiratory exchange ratio (RERmax) in KO and WT mice vs. age. RERmax normalized to work unit was significantly higher in KO than in WT mice (P < 0.05). *P < 0.05 vs. WT. Values are means ± SE.

 
RERmax in KO and WT mice with age. RERmax was chosen at the point of O2 max. There was no significant difference between KO and WT mice. When RERmax is normalized to work unit (Fig. 3B), values were higher from 12 to 21 mo in KO than in WT mice (P < 0.05).

Comparison of changes in gene expression between eNOS-KO and WT mice. Comparison of RNA expression levels in KO and WT mice revealed that, of the 39,000 transcripts and variants on the microarrays, a total of 480 genes was differentially expressed at 21 mo (P < 0.05). The data showed that 293 genes were expressed at significantly greater levels in the eNOS-KO mice and 187 were significantly reduced in the KO mice (Fig. 4A).



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  		Fig. 4. A: scatter plot of 1 KO mouse vs. baseline, which is the average of all WT samples. x-Axis, basal gene expression in WT controls; y-axis, expression ratios comparing KO sample and baseline. Red and green crosses represent genes that are up- and downregulated in KO samples compared with baseline, respectively. B: percentage of known genes in each functional category that are up- and downregulated in KO mice.

 
Functional categories of differentially expressed genes in male eNOS-KO mice. All 480 genes were grouped into 7 functional categories on the basis of their Gene Ontology (GO) biological process annotations provided by PathwayAssist through the ResNet database. These categories are classified as cell signaling/communication/transport, cell structure/motility, metabolism, development, immune response/apoptosis, protein/protein expression, and unknown/other (Fig. 4B). The results showed a wide range of distribution of expression patterns within the functional groups of genes. The most striking observation was that, of the 480 genes that are differentially regulated between KO and WT mice, 178 encode proteins of unknown function (unknown/other, Fig. 4B). Of genes encoding proteins of known function, the greatest number was cell signaling/communication/transport, with 105 being differentially expressed. Interestingly, almost all the differentially expressed genes within the cell structure/motility group showed increased expression in the KO mice. Examples of these genes are {alpha}-skeletal actin, cartilage oligomeric matrix protein, and myosin heavy polypeptide 7, which were upregulated by 5.90-, 4.96-, and 3.53-fold, respectively, in KO mice. The other functional category for which a substantial number of genes are differentially regulated is metabolism: 29 are significantly upregulated (1.5- to 4.23-fold) and 41 are significantly downregulated (1.5- to 7.16-fold) in KO mice. Many genes in the metabolism category encode proteins involved in energy metabolism, and within that group a high number of lipid metabolism genes are primarily repressed. Examples of differentially regulated metabolic genes in KO mice included genes encoding latent transforming growth factor-{beta}-binding protein 2, deiodinase, aminolevulinic acid synthase 2, and phosphofructokinase, which are upregulated by 4.23-, 2.57-, 2.51-, and 2.27-fold, respectively. Downregulated genes include carboxylesterase 3, methionine adenosyltransferase II, methylmalonyl-CoA mutase, deoxyuridine triphosphatase, and cytosolic acyl-CoA thioesterase 1, which are downregulated by 7.16-, 2.30-, 2.27-, 2.25-, and 2.22-fold, respectively.

In the protein/protein expression group, a higher number of genes show increased expression in KO mice. Most of the elevated expressions are for genes encoding enzymes involved in protein modification and degradation. In the immune response/defense/apoptosis category, 36 genes are differentially expressed; of these, 22 are upregulated and 14 are downregulated in KO mice. Very few genes in the category of development are differentially expressed. (For a complete annotated list of all the genes see Supplementary Table 1 at http://ajpheart.physiology.org/cgi/content/full/00170.2005/DC1).

Heart failure is characterized at the molecular level by changes in gene expression that result in the induction of fetal genes (1). In this study, atrial natriuretic peptide (ANP) was significantly higher (2.83-fold) in KO mice. Brain natriuretic peptide showed higher expression in KO mice, but its expression was not statistically significant. In addition, {alpha}-skeletal actin was significantly upregulated by 5.90-fold in KO mice, and there was a switch from {alpha}- to {beta}-myosin heavy chain (MHC). {beta}-MHC was upregulated by 3.53-fold in KO mice.

One of the genes that was classified as unknown/other was of particular interest. This gene was designated RIKEN cDNA A830037N07 gene (Affymetrix no. 1456395_at), and it was statistically downregulated by 4.2-fold in KO mice. Recent analysis of the present data showed that this gene has been designated peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1). Another variant of this gene on the Affymetrix chips (Affymetrix no. 1460336_at) was also statistically downregulated by 1.73-fold in KO mice.

To more fully disclose the pathways relevant to the pathogenesis of heart failure, PathwayAssist was used to group the 480 differentially expressed genes according to function. Of those genes, 401 were recognized by the software and were thus subjected to subsequent analysis. The "find common targets for selected nodes" feature of PathwayAssist was used to build a network of connections starting with these 401 genes and including all available categories of interaction. Some of the observed cellular processes included apoptosis, proliferation, inflammation, motility, contraction, and regeneration. This analysis highlighted the importance of these cellular processes in the development of heart failure in KO mice.

Correlation between microarrays and real-time RT-PCR. Real-time RT-PCR was employed to validate the final microarray data. A total of eight different genes were selected for this analysis. There was a high correlation between expression values obtained by real-time PCR analysis and those measured by microarray analysis (R2 = 0.91; data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have demonstrated that the maximal work performed and distance run by KO mice were decreased significantly with age and that KO mice have greater total O2 and run at a higher relative workload at high treadmill speed. O2 max, CO2 max, and RERmax normalized to work performed was elevated in KO compared with WT mice with age. A change in genotype indicative of the development of heart failure and expression of a fetal phenotype was observed in KO mice at 21 mo.

At 12 and 18 mo, there is a good correlation for the increase of O2 with the increase of treadmill speed. The slope of linear regression was significantly higher for KO than for WT mice at 18 mo. KO mice consumed more O2 than WT mice, especially at high speed. We also plotted the linear regression curve of the relative workload at each speed; the slopes were significantly different between these two genotypes at 18 mo. The genotypic differences in the slopes of the O2 of running curves at 18 mo are likely the product of the higher relative workload in WT mice at lower treadmill speed and higher relative workload in KO mice at higher treadmill speed. For example, at a treadmill speed of 16 m/min, WT mice ran at a relative workload of 62% of O2 max and KO mice ran at 85% of O2 max. Although the WT mice reach a higher percentage of O2 max at lower treadmill speed than KO mice, the O2 required at equal relative workloads was still higher in KO mice. For instance, at a relative workload of 70% of O2 max, O2 was 55 ± 5.8 and 39 ± 3.1 ml·kg–1·min–1 for KO and WT mice, respectively (P < 0.05).

This increase in O2 in KO mice at absolute and relative work intensity may be due to the loss of shear stress-stimulated NO regulation of MO2. Our previous study showed that vascular EDNO may play an important role in the modulation of cellular respiration and O2 in parenchymal cells, such as skeletal and cardiac myocytes (50). The underlying mechanism is the ability of NO to reduce the activity of a number of enzymes in the mitochondria, including aconitase, NADH-ubiquinone oxidoreductase, and succinate-ubiquinone oxidoreductase (17, 22, 27, 46). In addition, nanomolar concentrations of NO have a significant and reversible inhibitory effect on cytochrome oxidase through a competitive binding site for O2 in the enzyme (7, 9). The feedforward mechanism is driven by the ATP metabolic products, such as ADP, H+, and Pi, to accelerate cellular respiration and O2 (57). In another study, we showed that bradykinin had no inhibitory effect on O2 in heart from eNOS-KO mice (35). Therefore, at high speed, O2 in WT mice was regulated by NO release, and this inhibition was removed in KO mice. During high-intensity exercise, factors such as increased body temperature, increased energy cost of breathing, and elevated blood catecholamine levels may augment O2 (10). Therefore, at high-intensity exercise, KO mice work inefficiently compared with WT mice. This finding is further supported by the fact that inhibition of EDNO resulted in decreased myocardial metabolic efficiency, including coupling between MO2 and cardiac performance and coupling MO2 and ATP synthesis rate (49).

One study demonstrated that, despite the increase in O2 by N-nitro-L-arginine in the isolated guinea pig heart at all levels of contractile performance, neither ATP concentration nor ATP synthesis rate changed (49). When NO synthesis was blocked systemically in the resting dogs, there was an elevation in total body O2, which was accompanied by an elevation in body temperature (4).

In contrast to 18 mo, there was no difference between the slopes of the liner regressions for the absolute and relative workload in KO and WT mice at 12 mo. There may be some compensatory mechanism at 12 mo to block the effect of gene deletion, but age uncovered the consequence of eNOS deletion. Indeed, studies have demonstrated that increased release of prostaglandins contributes to flow-induced arteriolar dilation in young eNOS-KO mice (53), and shear stress can activate neuronal NOS (nNOS) to release NO, compensating for the absence of eNOS-derived NO in coronary arteries of young KO mice (24).

Treadmill exercise has typically been the choice for exercise involving mice. Generally, treadmill running at 4 m/min with an 18° slope has been termed "submaximal" intensity (14), whereas running at 12 m/min with an 8° slope has been characterized as "moderate"-intensity exercise (5). Our mice could achieve ≥28 m/min with 10° slope until exhaustion.

KO mice performed 30–50% less work than WT mice at each age. In WT mice the work performed slightly decreased with age (by 0.8%/mo), but in KO mice the work performed decreased much faster (5%/mo). Distance run by KO mice was 20–60% shorter than distance run by WT mice at each age. In WT mice the distance run decreased 23% from 12 to 21 mo, whereas in KO mice the distance run decreased 47% (P < 0.001). The decline in maximal exercise capacity with age has been shown in several studies in aged mice (48), dogs (20), and rats (39). In addition, our previous study (31) found that KO mice developed cardiac dysfunction between 14 and 21 mo, which is responsible for the mortality we observed and may account for the marked decrease of work and running distance.

Maxwell et al. (38) showed that EDNO contributes to exercise hyperemia and is a determinant of aerobic capacity in exercising mice. They also indicated that exercise capacity depends on the integrity of the NOS pathway (37). When deletion of the eNOS gene is added to aging, exercise capacity is decreased. In KO mice, MO2 was elevated, and failure to reduce leg vascular resistance may limit the increase in leg blood flow. Consequently, perfusion in the exercising limb is unable to keep up with the rising demand for O2 transport, and the balance between supply and metabolism is severely compromised.

Several studies indicate that O2 max declines with age. Schefer and Talan (48) indicated that O2 max declined with age in mice. A decline in O2 max of ~10%/decade after 30 yr of age has been observed in studies of healthy humans (18), and Lawler et al. (28) demonstrated that the O2 cost of treadmill running is lower for 24- than for 4-mo-old rats, except at high work levels. In our study, we found only a slight decrease in O2 max from 63 ± 3.4 ml·kg–1·min–1 at 12 mo to 60 ml·kg–1·min–1 at 21 mo in WT mice. An explanation for the discrepancies may that the age range in our investigation, i.e., 9 mo, is not long enough for normal mice to present a significant aging effect.

Only at 14 mo of age for O2 max and 16 and 18 mo of age for CO2 max were decreases significant in KO mice compared with their age-matched WT controls. Because KO and WT mice had not achieved the same workload or the same cumulative distance run at the point at which we chose the values for O2 max, CO2 max, and RERmax, this way of evaluating the data may not be appropriate. We therefore normalized the data to work unit and running distance. O2 max and CO2 max are significantly higher from 14 to 21 mo in KO than in WT mice (P < 0.05).

The significant elevation in O2 at 14 mo in KO compared with WT mice correlated with our previous study, which showed significant cardiac hypertrophy in KO mice starting from 14 mo and markedly reduced cardiac function in KO mice at 21 mo (31).

CO2 also started to rise after 14 mo. Several previous studies showed that NO can regulate substrate utilization. Glucose uptake was increased in KO mice and nitro-L-arginine methyl ester-treated mice in a Langendorff heart preparation (54). Blockade of NO synthesis resulted in reductions in myocardial free fatty acid consumption for comparable levels of cardiac work (4). The acute inhibition of NOS by N-nitro-L-arginine causes a switch from fatty acids to lactate and glucose utilization by the heart that can be reversed by an NO donor (45). Basal cardiac production of NO falls below normal levels during cardiac decompensation, and substrate utilization shifts from fatty acids to glucose. It has been demonstrated in vitro that NO can stimulate ADP-ribosylation of GAPDH (60), which may inhibit enzyme activity. This augmented glucose utilization may account for the results of the present study, i.e., that CO2 max and RERmax were significantly elevated in KO compared with WT mice. ATP yield is greater for a given rate of O2 because of the higher ATP-to-O ratio from carbohydrate oxidation than from fatty acid oxidation (52).

Data from Ji et al. (26) reveal that a brief acclimation period in untrained animals does not affect the activity of working muscle metabolic enzymes. An increase in stride length and consequent reduction of stride rate could result in a lower O2 cost of running (55). O2 is total body O2, and during exercise, most of the O2 was consumed by skeletal muscle. One limitation of this study is that we could not exclude the skeletal muscle lean mass difference between these two groups. The body mass of KO mice is significantly less than that of WT mice at 18 mo, but if we assume that the lean skeletal muscle mass follows the decrease in body mass, KO mice should consume less O2; however, in our study, the O2 of KO mice is significantly higher, further supporting the change in MO2 and substrate utilization due to gene deletion.

The present study showed that the work performed by KO mice significantly decreased with age beginning at 12 mo. In addition, we previously found cardiac dysfunction in KO mice with age and 50% mortality at 21 mo. In the present study, 6 of 16 (40%) KO mice died by 21 mo. Therefore, it was of interest to investigate the gene expression profile of aged KO mice compared with WT mice to explore the genetic markers and molecular mechanisms leading to heart failure. The array data showed that the gene profile in our 21-mo-old KO mice is indicative of cardiac failure and remodeling. In KO mice, 480 genes grouped into 7 different functional categories were found to have altered expression. These categories are classified as cell signaling/communication/transport, cell structure/motility, metabolism, development, immune response/apoptosis, protein/protein expression, and unknown/other.

Previous studies have shown that heart failure is characterized at the molecular level by changes in gene expression that result in repression of adult genes, such as {alpha}-MHC and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2A), and the induction of fetal genes (1). The change in MHC expression is a logical candidate for affecting cardiac contractility, because small changes in isoform composition have been shown to affect contractility of cardiac myocytes. ANP was significantly higher (2.83-fold) in KO mice. Brain natriuretic peptide showed higher expression in KO mice, but this was not statistically significant. In addition, {alpha}-skeletal actin was significantly upregulated by 5.90-fold in KO mice, and there was a switch from {alpha}- to {beta}-MHC. {beta}-MHC was upregulated by 3.53-fold in KO mice.

Oxidative stress may play an important role in the pathophysiology of heart failure. Previous studies have shown that the overexpression of glutathione peroxidase (Gpx3) attenuates left ventricular remodeling and failure after myocardial infarction. In our study, Gpx3 was significantly upregulated by 2.33-fold in KO mice. It has been reported that the yeast prion protein Ure2 shows glutathione peroxidase activity in native and fibrillar forms (3). In our study, prion protein was also significantly upregulated by 1.85-fold in KO mice.

It has been well established that fatty acids are primary fuels for energy production in healthy hearts. However, in disease states, glucose becomes the favored energy source. Our data indicate that there might be an adaptation in expression of fatty acid metabolism genes as a means to accomplish this metabolic reprogramming. The upregulation of some of the genes that are involved in glucose metabolism (phosphofructokinase, 2,3-bisphosphoglycerate mutase, protein phosphatase 1, hexokinase 1, amylo-1,6-glucosidase, 4-{alpha}-glucanotransferase, and glucose phosphate isomerase 1) and the downregulation of several lipid metabolism genes (carboxylesterase 3, phospholipase A2, cytosolic acyl-CoA thioesterase 1, and short-chain acyl-CoA dehydrogenase) may be a reflection of altered bioenergetics in a failing heart in KO mice.

Another interesting observation is that the PGC-1 gene was statistically downregulated by 4.2-fold in KO mice. Recently, PGC-1{alpha} has been identified as an inducible upstream regulator of mitochondrial number and function (29, 59). Recent studies implicated PGC-1{alpha} in the activation of the mitochondrial biogenesis program in heart and skeletal muscle (59). Garnier et al. (15) showed that expression of the PGC-1{alpha} gene was significantly downregulated in congestive heart failure (CHF) and that PGC-1{alpha} correlated with the oxidative capacity in failing cardiac and skeletal muscles as well as in healthy muscles. Therefore, the decreased oxidative capacity in cardiac and skeletal muscles in CHF may eventually result from a decrease in mitochondrial gene expression linked to PGC-1{alpha} (15). Sano et al. (47) also showed that impaired transcription of PGC-1 and its targets likely contributes to the transition to heart failure via mitochondrial dysfunction and, at least in part, the resulting vulnerability to apoptosis.

In the heart, PGC-1{alpha} expression increases sharply at birth, coincident with a perinatal shift from glucose metabolism to fat oxidation. Consistent with its emerging role as a central regulator of energy metabolism, PGC-1{alpha} is abundantly expressed in mitochondria-rich tissues such as heart, skeletal muscle, and brown adipose tissue (42). PGC-1{alpha} expression is inducible in these tissues in response to physiological stimuli. In heart and skeletal muscle, physiological stimuli, including fasting and exercise, lead to an increase in PGC-1{alpha} gene expression (2, 16, 29); conversely, pressure overload decreases PGC-1{alpha} expression (30). Signaling pathways associated with these stimuli, including p38 MAP kinase, {beta}-adrenergic/cAMP, NO, AMP kinase, and Ca2+-calmodulin kinase, activate PGC-1{alpha} and its downstream target genes by increasing PGC-1{alpha} expression or transactivation function (6, 41, 43, 58). Nothing is known regarding the signaling pathways leading to downregulation of PGC-1 and decreased energy metabolism in CHF. Garnier et al. (15) proposed that mitogen-actived protein kinases, as well as the protein kinase Akt or protein kinase B pathway, which are not activated in hypertrophied heart, are highly activated in heart failure irrespective of the cause of the disease (21). TNF-{alpha}, angiotensin II, and endothelin-1, which are dramatically increased in heart failure, may potentially activate the Akt pathway (40). Cardiac-specific expression of a constitutively active mutant of Akt mediates a nearly threefold downregulation of PGC-1{alpha} mRNA expression (11). Overexpression of TNF-{alpha} induces heart failure, accompanied by mitochondrial abnormalities and impaired DNA repair activity (32), and TNF-{alpha} has been proposed to contribute to mitochondrial defects in CHF (36). This provides a possible link between neurohumoral activation, decreased PGC-1{alpha} expression, and altered cardiac and skeletal muscle bioenergetics in CHF.

One of the limitations of this study is that eNOS-KO mice are hypertensive and develop cardiac hypertrophy with age. For this reason, it might be difficult to differentiate the specific changes in gene expression due to the absence of NO generated by eNOS from those associated with the pressure-overload phenotype in these mice. However, in our previous study (31), we showed that female eNOS-KO mice maintain an elevated blood pressure for 21 mo, yet they do not develop cardiac dysfunction, as seen in male KO mice. Therefore, it is unlikely that changes in gene expression in hearts of male KO mice are simply due to pressure overload.

Recently, Cappola et al. (8) used Affymetrix mouse arrays (MG-U74A) from eNOS- and nNOS-KO mice to describe the cardiac expression profiles. There was a limited overlap between the genes identified in their eNOS-KO studies and those in our data set. The limited correspondence between the two data sets may be a result of substantial differences in array design and disease state. Most sequences previously represented on the GeneChip Murine Genome Array U74Av2 are represented on the GeneChip Mouse Expression Array 430A. Because of the dynamic nature of public databases, probe sets for these sequences are not identical and, in some cases, are represented by a completely new probe set. As a result, data generated with different versions of the mouse arrays may not always produce concordant results (http://www.affymetrix.com). In addition, in their study, eNOS- and nNOS-KO models showed similar hypertrophic cardiac phenotypes. In our recent study, we found that eNOS-KO mice have a reduced life expectancy because of the development of dilated cardiomyopathy (56). The eNOS-KO model used in their study was generated by Huang et al. (25); we used eNOS-KO mice generated by Shesely et al. (51).

In conclusion, there are age- and genotype-related exercise limitations in work performed and maximal distance run by KO mice. The genotype difference in the slopes of the O2 running curves at absolute and relative workload observed in this study demonstrates that KO mice work less efficiently and consume more O2 than WT mice. Starting from 14 mo, the normalized O2 max and CO2 max began to increase significantly, which correlated with the time course of cardiac dysfunction, implicating an inefficiency of O2 and an increase in skeletal muscle O2 in KO mice. This may be compounded by the switch of substrate utilization from fatty acids to glucose, which was a result of deletion of the eNOS gene and was uncovered with age.


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W. Li was supported by American Heart Association New York State Affiliate Predoctoral Fellowship 0215297. H. Post was supported by German Research Federation Grant PO 672/1-1. This work was supported by National Institutes of Health Grants PO-43023, HL-50142, and HL-61029 (to T. H. Hintze).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. H. Hintze, Dept. of Physiology, New York Medical College, Valhalla, NY 10595 (e-mail: Thomas_Hintze{at}nymc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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