INTRODUCTION

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LONG-DISTANCE RUNNING CAPACITY is a complex trait that is determined by multiple genes as they interact with the environment in the form of a response to training. In addition, it is axiomatic that running capacity is associated with the ability to consume oxygen with either delivery or utilization operating conditionally as the limiting factor (25). Although evidence from heritability studies in both humans and animals suggests that a genetic component accounts for as much as 70 to 90% of the variation in aerobic capacity, the genes that determine the intrinsic difference between low and high capacity for aerobic exercise remain undefined (3, 10).

In 1996, we started artificial divergent selection for low and high aerobic treadmill running capacity in rats (18). The purpose was to create low-capacity runners (LCR) and high-capacity runners (HCR) that could ultimately be developed into contrasting strains for genetic studies of intrinsic (i.e., untrained) capacity. We hypothesized that our divergent models would concentrate contrasting alleles that encode for variation at essentially all levels of organization as represented in a model of running capacity developed by Joyner (17). This model is the product of three physiological variables: 1) the maximal rate at which oxygen and nutrient substrates can be used to produce energy in the form of ATP (O_2 max; ml O₂ · kg¹ · min¹), 2) the percentage of O_2 max at the threshold for lactate release (%O_2 max), and 3) the efficiency of running (km · min¹ · O₂¹). Cardiac capacity is an embedded component of the Joyner model because oxygen consumption is the product of cardiac output (CO) and peripheral oxygen extraction. Furthermore, concepts originated by A. V. Hill (14, 15) and extended by others (8, 9, 19, 26) support the contention that the ability of the heart to deliver oxygen can be a major factor that limits maximal aerobic capacity, especially in animals with high aerobic capacity (9, 25). From these ideas, we predicted that artificial selection based on capacity to perform an aerobic treadmill run would concentrate genes associated with enhanced cardiac function in the HCR.

In addition, it is well documented that aerobic exercise training induces cardiovascular changes that render animals more tolerant to myocardial ischemia (5, 6, 23). It is not known, however, if animals with high intrinsic aerobic capacity are more resistant to myocardial ischemia compared with animals with low intrinsic capacity. Thus the purpose of this work was to test the hypotheses that HCR relative to LCR have 1) greater isolated heart performance and 2) more resistance to myocardial ischemic insult. Our data support the hypothesis that the HCR have enhanced intrinsic cardiac capacity relative to the LCR. In contrast, our results provide no evidence that intrinsic aerobic capacity is differentially associated with resistance to myocardial ischemia.

	METHODS

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Animals

All rats were housed two per cage, provided food and water ad libitum, and placed on a 12:12-h light-dark cycle with the light cycle occurring during daytime. Each of these 40 rats was coded numerically, and all were studied between 11 and 30 wk of age using a single-blind design. All procedures were carried out with approval by our Institutional Animal Care and Use Committee and were conducted in accordance with the Guiding Principles in the Care and Use of Animals as approved by the Council of the American Physiological Society.

Estimation of Endurance Running Capacity

The first 2 days of introduction to treadmill running consisted of simply placing the rat on the belt that was moving at a velocity of 10 m/min (15° slope) and picking the rat up and moving it forward if it started to slide off the back of the belt. During introduction days 3-5, the belt speed was gradually increased up to 15 m/min, and failure to run caused the rats to slide off of the moving belt and onto a 15 × 15-cm electric shock grid that delivered 1.2 mA of current at 3 Hz. The rats were left on the grid for ~1.5 s and then moved forward onto the moving belt. This process was repeated until the rats learned to run to avoid the mild shock.

During the second week, each rat was evaluated for maximal endurance running capacity on 5 consecutive days. Each daily endurance trial was performed at a constant slope of 15° with the starting velocity at 10 m/min. Treadmill velocity was increased by 1 m/min every 2 min, and each rat was run until exhausted. Exhaustion was operationally defined as the third time a rat could no longer keep pace with the speed of the treadmill and remained on the shock grid for 2 s rather than run. At the moment of exhaustion, the current to the grid was stopped and the rat was removed from the treadmill and weighed.

For each of the five trials, the total distance run (m) was used as the estimate of aerobic endurance capacity. The single best daily run of five trials for each rat was considered the trial most closely associated with the heritable component of endurance running capacity. All estimates of capacity reported here are based on the single best day of running for each rat (7, 18).

Estimation of Isolated Cardiac Performance

Preparation of the heart. Hearts were removed for isolated study when the rats were between 20 and 30 wk of age. The gap between the running tests and isolated heart experiments occurred because these rats were part of an ongoing breeding paradigm. The span represents time for breeding and making certain viable offspring were produced. One hour before heart removal, 100 U/kg body wt of heparin sodium were administered via an intraperitoneal injection. The rats were then anesthetized with pentobarbital sodium (50 mg/kg body wt ip), and the hearts were removed and placed in iced Krebs-Henseleit buffer.

Perfusion of the working rat heart. Hearts were evaluated using the Langendorff-Neely isolated working heart preparation (22). The composition of the Krebs-Henseleit bicarbonate perfusion buffer was (in mM) 118 NaCl, 4.7 KCl, 1.25 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 0.32 EDTA, 25 NaHCO₃, and 11 D-glucose. Oxygenation of the perfusion buffer was achieved by aeration with 95% O₂-5% CO₂ at 37°C (pH = 7.4). To initiate isolation, the aorta was slipped over a 16-gauge bulbed needle and perfused retrograde at 80 mmHg for 15 min. During this time, the heart was instrumented for the working mode by inserting a 16-gauge cannula into the left atrium. Working mode perfusion was initiated by starting perfusion of the left atria at a constant pressure of 15 mmHg and switching the aortic flow to a cannula with an overflow set at 95.2 cm above the heart (70 mmHg). Both aortic flow (cannula overflow) and coronary artery flow (effluent from severed pulmonary artery) were measured with graduated cylinders. CO was calculated by summing aortic and coronary flow. The baseline CO, expressed per gram of heart weight, was taken as the mean of the values at 4, 8, and 12 min of starting working mode perfusion.

Ischemic period. After 12 min in the working mode, the heart was subjected to low-flow ischemia, as described previously by Waagstein et al. (24). The aortic outflow cannula was lowered in two 30-s steps to a height of 27.2 cm above the heart (providing a mean aortic pressure of 20 mmHg). Oxygenation of the buffer was ceased at this point. This low-flow ischemia was maintained for 25 min, during which time aortic flow and coronary effluent were collected and measured at 10, 15, and 25 min.

Recovery period. After 25 min of low-flow ischemia, the aortic outflow cannula was raised in two 30-s steps to its original height of 95.2 cm, restoring the mean aortic pressure to 70 mmHg, and oxygenation of the buffer was resumed. Aortic and coronary flows were measured at 4, 8, and 12 min. These values were summed to derive the CO, which was designated the recovery CO. After the last readings were taken, the heart cavities were cut open, drained, blotted dry, and weighed.

Analysis of Data

	RESULTS

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On average, LCR ran for 227 ± 7 m and the HCR ran 993 ± 11 m at exhaustion (337% difference, P < 0.001). CO averaged 33.5 ± 2.0 ml · min¹ · g¹ in LCR hearts and 49.9 ± 1.4 ml · min¹ · g¹ in HCR hearts (49% difference, P < 0.001). The CO and distances run for each of the rats are shown in Fig. 1. Although it is clear that the populations are different for both of these variables, there was no relationship between CO and distance run within either of the lines.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Isolated cardiac output and distance run to exhaustion for low-capacity runners (LCR) and high-capacity runners (HCR) of both sexes. On average, the LCR had a 49% greater isolated cardiac output and ran to exhaustion at 338% greater distance relative to the HCR. The bold crosses mark average values ± 1 SD. Distance run was evaluated at 11 wk and cardiac function at 20 to 30 wk of age.

Table 1 provides a summary of data collated by line (LCR and HCR) and sex for running capacity and heart performance before ischemic insult. For females, the HCR recorded significantly greater distances run (362%), isolated COs (38%), stroke volumes (31%), coronary flows (35%), and aortic flows (37%) compared with the LCR. The differences in CO were produced by changes in stroke volume because there were no differences in heart rate between the LCR and HCR females (240 vs. 247 beats/min). In addition, there were no differences between female LCR and HCR for body weight, heart weight, the ratio of heart weight to body weight (taken at time of heart study), or the rates of change in aortic pressure with each beat (+dP/dt and dP/dt).

For males, the HCR recorded significantly greater distances run (291%), isolated COs (62%), stroke volumes (69%), coronary flows (41%), and aortic flows (88%) compared with the LCR. Similar to the females, differences in CO were accounted for by changes in stroke volume because there were no differences in heart rate between the LCR and HCR males (253 vs. 238 beats/min). In contrast to the females, there were differences between male LCR and HCR for both body weight (464 vs. 363 g) and heart weight (1.04 vs. 0.78 g). For males, there were no differences between LCR and HCR for the ratio of heart weight to body weight or for the rates of change in aortic pressure with each beat.

Figure 2 shows summary data for the CO in the LCR and HCR before, during, and after recovery from global ischemia for females and males combined. As presented above, baseline CO averaged 33.5 ± 2.0 for the LCR and 49.9 ± 1.4 ml · min¹ · g¹ heart wt for the HCR (49% difference, P < 0.001). Twenty-five minutes of global ischemia produced by lowering aortic pressure to 20 mmHg and stopping oxygenation of the perfusate caused CO of the LCR to decrease to 11.6 ± 1.7 (188%) and that of the HCR to decrease to 17.2 ± 2.1 ml · min¹ · g¹ heart wt (190%). Return of the hearts to an oxygenated perfusate and an aortic pressure of 70 mmHg caused recovery of COs for both the LCR and HCR to values that were not different from baseline. During recovery, COs of the LCR rats averaged 30.5 ± 2.1 (91% recovery) and that of the HCR averaged 43.7 ± 2.0 ml · min¹ · g¹ heart wt (88% recovery). Because heart rates were not different between LCR and HCR during either baseline, ischemic, or recovery conditions, all of the changes in CO were accounted for by changes in stroke volume. Table 2 summarizes data collated by line (LCR and HCR) and sex for running capacity and heart performance during ischemia and recovery from ischemia. When separated by sex, only the coronary flows for the males were significantly different between the LCR and HCR. During recovery, the COs of the LCR were lower than those in the HCR for each sex but not different from baseline values.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Isolated cardiac outputs for LCR and HCR before (baseline), during, and after (recovery) 25 min of global ischemia. Outputs decreased 188% in the LCR and 190% in the HCR during ischemia. During recovery, outputs returned to within 91% of baseline for the LCR and 88% for the HCR. Baseline and recovery outputs were not significantly different within either line. Cardiac outputs were different between the LCR and HCR for baseline and recovery conditions but not during ischemia. Values are means ± 1 SE. *LCR significantly less (P < 0.001) than HCR for the same condition.

	DISCUSSION

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Aerobic capacity as measured by maximal treadmill running is a standard tool that assesses the status of a wide variety of clinically relevant organ systems (19). Indeed, the original interest in developing these selectively bred models stemmed largely, but not exclusively, from the possibility of producing low and high lines that concentrated genes for extremes of the oxygen flow pathways as exemplified by the Joyner model (17). On the other hand, other important biological factors such as motivation, educability, tractability, fear, pain, and pleasure are also likely to be segregated by the divergent selection. Amidst this complexity, it appears that differences in cardiac function will emerge as a correlated trait that can account for part of the difference in running capacity between the LCR and HCR. A correlated trait is an unselected trait that is produced by selection for another trait. The genetic component of a phenotypic correlation is produced by pleiotropy, which is the property of an allele whereby it simultaneously affects two or more traits. As such, the LCR and HCR might also serve as contrasting models to determine genes that influence intrinsic cardiac function.

One's current aerobic capacity can be considered the composite of the contribution from intrinsic aerobic genes and genes that respond to training. Although none of these genes have been unequivocally identified, current information suggests that intrinsic and exercise response genes represent different substrates. Bouchard and colleagues (4) performed a genome-wide scan to identify chromosomal regions (HERITAGE Family) linked to maximal oxygen consumption under two conditions: 1) individuals in the untrained, sedentary condition and 2) the same individuals after 20 wk of endurance training on a bicycle ergometer. For the sedentary condition, microsatellite markers on 4q, 8q, 11p, and 14q were linked with maximal oxygen consumption at P values <0.01. For the response to training, markers on 1p, 2p, 4q, 6p, and 11p were linked with maximal oxygen consumption at P values <0.01. These results suggest that the genetic factors underlying sedentary maximal oxygen consumption (i.e., intrinsic capacity) and the responses to training are not the same. The rats used in the present study presumably represent phenotypes that originated almost exclusively from intrinsic aerobic genes.

Although we selected the one best day run out of five to reduce environmental variation, this simultaneously created the possibility that a component of selection may be related to short-term adaptation (2). Indeed, for the 5 consecutive days of testing, on average, the LCR ran best on day 2.76 ± 0.40 and the HCR ran best on day 3.94 ± 0.25 (P < 0.05). Although this may represent a minor component of aerobic biochemical adaptation for the high line, other factors such as differences in behavioral responses to running may also be involved. The 8- to 18-wk gap between evaluation of running performance and evaluation of isolated cardiac performance almost assuredly precluded any influence of a greater "training" effect in the hearts from HCR. On the negative side, this time gap between evaluation of running and heart capacity may have added variation to the data.

Artificial selection at the extremes of a trait produces ideal genetic models because contrasting allelic variation can be concentrated from one generation to the next. Such contrasting models can be used to identify the allelic variants that are causative of the difference between low and high capacity for a given trait (7). Selection is possible if substantial additive genetic variance exists in a population for that trait (12). Fisher's 1930 Theorem of Natural Selection (13) predicts that traits peripherally associated with evolutionary fitness, such as morphology and complex physiology, will demonstrate more additive genetic variance because of less pressure from natural selection (21). As an extension of this logic, it appears that high intrinsic capacity for both aerobic endurance running and heart function is not tightly associated with fitness in the evolutionary sense. That is, aerobic running responded somewhat strongly to selection for high capacity (18) with high isolated heart performance as a correlated trait.

Previous work demonstrated that aerobic exercise training increases tolerance to episodic cardiac ischemia. That is, functional and metabolic recovery is greater in exercise-trained rats compared with their sedentary counterparts (5, 6, 23). Despite significant differences between LCR and HCR rats for both intrinsic aerobic running capacities (226%) and cardiac performance (49%), there were no significant differences between these lines in either the response to or recovery from 25 min of global cardiac ischemia (Fig. 2). In F344 rats, ischemic challenges similar to what we applied allowed only a 36% recovery in CO for sedentary rats (5). Intense aerobic training, however, improved the recovery in CO to 73% (5). In aggregate, these results do not allow explicit conclusions, but they suggest three working hypotheses. First, high intrinsic aerobic capacity is not associated with recovery from cardiac ischemia. Second, the protective effect of a higher aerobic capacity accrues only from that gained as an environmental response to training. Third, both intrinsic and exercise-induced increases in aerobic capacity provide for increased recovery from cardiac ischemia. Wide strain differences may exist for sensitivity to cardiac ischemia with the F344 being relatively sensitive. Indeed, it is possible that the N:NIH rat stock, from which our lines were originally derived (18), is relatively resistant to ischemia, and a larger insult would have revealed that the LCR are more sensitive to ischemia compared with the HCR.