The role that remodeled myocardial extracellular matrix (ECM) plays in altered left ventricular (LV) function seen after myocardial infarction (MI) has long been recognized (2,21,29). Alterations in both the amount of collagen, the largest component of the ECM, together with changes in its tensile properties, have been seen after compensatory hypertrophy of the heart after MI. The remodeling process that takes place not only within the infarct scar itself but also within remaining viable myocardium results in region-specific increases in LV collagen concentration and mature cross-linking (20). These changes seen in myocardial ECM both proximal and distal to the infarcted (INF) region are thought to be the result of altered wall tension properties and/or hormonal changes seen consequent to MI (21,25,38).
The dynamics of this ECM remodeling process after MI have been elucidated (12,39). Immediately after MI, there is a dramatic increase in message for the principal fibrillar collagens, coincident with an up-regulation of matrix metalloprotease activity, which degrades approximately 50% of existing myocardial collagen (30). This initial degradative phase is followed by an extensive synthetic period resulting in interstitial fibrosis as well as compensatory hypertrophy of the remaining myocardium owing to alterations in ventricular geometry, loss of myocytes, and changes in myocardial hormonal profile (21,38). Myocardial collagen content increases with time in both INF and non-INF areas (20) owing to increased transcription of fibroblast procollagen Type I, III, IV, and V mRNA (5,12,39). In addition, the extent of mature collagen cross-links (hydroxylysylpyridinoline, HP), indicative of the strength and maturity of collagen, seems to increase in both scar and viable myocardium proximal to the scar by 13 wk after MI. Increased HP cross-linking has traditionally been taken to imply reduced collagen degradation because mature collagen is more heavily cross-linked and thought to be less susceptible to degradation (17). Therefore, the rapid increase in HP cross-linking simultaneous with increases in collagen biosynthesis after MI observed in these studies has yet to be fully understood.
Exercise is a commonly used therapy for patients recovering from a heart attack. Training in both patients and animal models is associated with improvements in exercise capacity as well as overall heart function after MI (11,23). These improvements in heart function include increased maximal stroke volume (22), increased maximal heart rate, and normalization of end-diastolic pressure and wall stress (23). However, it is unknown whether training-induced changes in myocardial collagen properties play a role in the improvements seen in heart function and aerobic capacity after MI.
Previous studies evaluating the effects of chronic exercise on myocardial ECM in the healthy rodent heart have found age-dependent differences in the training response for both collagen and its degree of nonreducible cross-linking. Thus, collagen concentrations in LV are either unaffected (33,34,37) or reduced with training (31), whereas a reduction in mature collagen cross-linking is a consistent finding in middle-aged and older (31,33,34), but not young, animals (33,34,37). These ECM changes in response to training thus contrast those found in the heart after MI where both collagen concentration and extent of nonreducible cross-linking are elevated not only in the infarct but also in the remaining viable regions of LV. These connective tissue increases (fibrosis) have been linked to increased chamber stiffness and dysfunction. Although HP cross-link concentrations have not been reported previously in the right ventricle (RV) after infarction of the LV, this pathologic abnormality has been shown to cause RV hypertrophy in response to elevated pressures in the pulmonary circulation. Whether or not exercise training can ameliorate aberrant collagen concentration and cross-linking profiles seen in the heart after MI is unknown. Therefore, the purpose of the present investigation was to evaluate combined effects of MI and a 10-wk progressive exercise training program on myocardial ECM properties in both RV and regionally within the LV.
METHODS
Animal selection and surgical preparation
Fifteen young adult (4–6 months old) Charles River rats were obtained from the animal facility at the University of Wyoming. All rats were familiarized with walking on a Stanhope rodent treadmill at 10–15 m·min−1 and 0% grade for a 2-wk period, before the 10-wk training period for the exercising group. Animals were then randomly divided into three groups with five rats per group: a sham–surgery sedentary control group (SHAM) and two INF groups, one sedentary (MI-SED) and the other exposed to 10 wk of progressive treadmill running after MI (MI-TR). All rats were sacrificed at 12 wk after MI.
The MI surgery was carried out as described previously (20). The surgery produced a transmural infarct scar in the LV anterior free wall by occluding the left anterior descending coronary artery. Briefly, anesthesia was induced by a ketamine/xylazine combination (50 and 3 mg·kg−1 intraperitoneally, respectively). All rats were intubated and placed on a rodent respirator (Harvard Model 683, Holliston, MA). The heart was exposed by lateral thoracotomy between the fifth and sixth ribs, and the pericardium was reflected. An attempt was made to produce smaller (nonfailing) infarcts by ligating the coronary artery at a point 4–5 mm distal to the edge of the left atrial appendage as performed previously (32). Previous experience had shown that a larger infarct is produced if the artery is occluded more proximal as opposed to more distal to the left atrium (32). Occlusion of the artery was confirmed by ECG responses (elevated ST segment, Vtach, Q-wave depression, preventricular contraction (PVC) along with blanching of the myocardium indicative of ischemia. The sham operation was performed with identical procedures except for occlusion of the coronary artery. Before and after surgery, all animals were maintained in the Animal Welfare Assurance–approved facility at the University of Wyoming on a 12-h light–12-h dark cycle and were fed ad libitum. These facilities and animal care meet the standards adhered to by the American College of Sports Medicine.
Training protocol
The training protocol consisted of a modification of a protocol used previously for training MI rats (23). The 10-wk training protocol consisted of a gradual increase in speed (13–24 m·min−1) of treadmill running at 15% grade for up to 6 × 60 min·wk−1. The MI-SED and SHAM groups were walked on the treadmill for 5 min, two sessions per week, at a speed of 10 m·min−1 and 15% grade to minimize any handling effects. After 10 wk of training, a maximal running speed test, which, in rats, has been shown to differentiate trained from sedentary groups (19), was performed with the 10-wk MI-SED and MI-TR groups. Also, citrate synthase (CS) activity in plantaris muscle was measured in all three groups according to the procedure of Shepherd and Garland (27) to evaluate any aerobic capacity differences in the skeletal muscle. All training and surgical procedures were approved by the University of Wyoming Institutional Animal Care and Use Committee.
Tissue collection
All three groups were sacrificed at a time point that represented 12 wk after MI for the MI-SED and MI-TR groups. The heart and plantaris muscles were removed immediately after euthanasia. The great vessels and atria were trimmed away before weighing the heart. Under a dissecting microscope, the RV was then trimmed away from the LV septum (LVS). In the SHAM group, the LV was carefully subdivided into free wall (LVF) and LVS using the RV attachments to delimit the LVS. In MI groups, the LV free wall scar tissue (INF) was also dissected away from the remaining surrounding viable tissue (LVF). The consolidated transmural scar was easily distinguishable from the viable myocardium by 12 wk after MI. All samples were removed from each rat, weighed, frozen in liquid nitrogen, and stored at −70°C for subsequent analysis.
Measurement of hypertrophy and infarct size
Because MI groups were somewhat lighter than SHAM controls the extent of heart, RV, and LV hypertrophy was determined by expressing the excised wet weights (mg) relative to body weight (BW) (g) (39). Because biochemical analysis was to be performed on RV, viable LVF, INF, and LVS, the size of the infarct could not be estimated by transverse measurements of INF epicardial and endocardial circumference expressed as a fraction of total LV circumference as used by us previously (32). Scar weight has been reported to be 87% of the original INF mass by 72 h after MI in the rat and 50% by 15 d after MI (9). Original infarct size was therefore computed by doubling the consolidated INF wet weight (INF scar (mg)) and expressing it relative to LV as used by us previously with this infarct model (39).
HP cross-link concentration
Quantification of HP was used to determine the degree of nonreducible cross-linking of myocardial collagen. HP was isolated from collagen by acid hydrolysis, purified by CF1 cellulose chromatography, and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) (8).
Myocardial samples (RV, LVFW, LVS, and INF) were hydrolyzed (16 h, 110°C) in 6N hydrochloric acid (volume 20 times sample dry weight) and filtered (Grade 413 Qualitative Filter Paper; VWR Scientific, Media, PA). One milliliter was extracted for later hydroxyproline (OH-Pro) analysis. The remaining 24 mL was evaporated at 50°C until dry and was loaded on to a CF1 cellulose column (12 cm × 1 cm; Whatman Inc., Piscataway, NJ). The column was washed in a mobile phase (n-butanol/acetic acid/water, 4:1:1), and the elution was performed by Millipore H2O (Millipore Corp., Billerica, MA). One hundred microliters (10.86 μg·L−1) of vitamin B6 (pyridoxamine (B6); Sigma Co, St. Louis, MO) was added as the internal standard. The mixture was dried in a centrifuge under vacuum for 12–16 h and rehydrated with 1% heptafluorobutyric acid (HFBA), filtered through a filter-tipped syringe (Acrodisc LC13 PVDF 0.45-μm syringe filter; Gelman Sciences, Ann Arbor, MI), and stored (at 4°C) after RP-HPLC analysis.
As described previously (8), RP-HPLC was used to analyze purified samples for HP concentration. A binary gradient was used with a Beckman HPLC system (110 B pumps, 421A controller; Beckman, Palo Alto, CA).
Solvent A was 5% acetonitrile, 0.001% HFBA, and 94.9% HPLC water, and solvent B was 99.998% acetonitrile and 0.002% HFBA. The naturally fluorescing B6 and HP peaks were detected (Ex295, Em380, Shimadzu RF 535 fluorescence detector; Shimadzu Scientific Instruments, Columbia, MD) and were integrated (Shimadzu 601 Integrator). A C-18 reverse-phase column (small pore, 4.6 mm × 25 cm), protected by a 25-nm guard column, was used for sample separation. The calculation of HP (mol HP·mol−1 collagen) was performed assuming that the fluorescence of 1 mol of B6 is 3.1 times that of HP and OH-Pro constitutes 14% of the collagen molecule (1/0.14 = 7.25).
Hydroxyproline concentration
Hydroxyproline analysis was carried out on whole muscle as described by Woessner (36). Hydroxyproline concentration (OH-Pro) was determined from 1 mL of hydrolyzed sample that was evaporated on a water bath at 60°C three times with Millipore H2O. This sample was brought to 25 mL in a volumetric flask with Millipore water. Two milliliters of sample was obtained for the assay. Collagen concentration (%COL) was calculated assuming that 14% of total collagen is OH-Pro and the molecular weight of collagen is 300,000.
Statistical analysis
A one-way ANOVA was performed to determine differences between the three groups in the study, namely, sham–sedentary (SHAM), INF sedentary (MI-SED), and INF-trained (MI-TR) rats that exercised for a period of 10 wk after MI. Statistical analysis was performed on the following variables separately: plantaris muscle CS activity, heart weight (HW)/BW, LV/BW, infarct size corrected to estimated original muscle mass and expressed as a percentage of LV, RV/BW, collagen concentration, and collagen cross-linking. Tukey HSD tests were used to make post hoc comparisons between specific groups when the overall F value achieved significance. A Student’s t-test was used to compare maximal running speed and infarct size between MI-SED and MI-TR groups. Significance level was set at P < 0.05.
RESULTS
Hypertrophy and infarct size
There was evidence of cardiac hypertrophy in both INF groups compared to sham controls as measured by differences in HW, LV, and RV weight relative to total BW (Table 1). The HW/BW, LV/BW, and RV/BW ratios were similarly increased (P < 0.005) relative to SHAM controls in both MI-SED and MI-TR animals, indicating that the RV contributed to the overall hypertrophy effect, with no additional hypertrophy of either ventricle induced by the training per se. Using the correction factor from Fishbein et al. (9) for tissue consolidation, initial infarct size was determined to be 19% ± 5% and 24% ± 5% of the LV in MI-SED and MI-TR groups, respectively, with no difference between the two groups (Table 1).
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TABLE 1: Effect of MI and training on HW/BW, LV/BW, INF size, and RV/BW.
Muscle aerobic capacity and running test
Both MI and treadmill running affected skeletal muscle aerobic capacity as estimated from plantaris muscle CS activity (Fig. 1). MI was associated with a 50% decrease in plantaris muscle CS activity measured 12 wk later in the MI-SED group compared to SHAM values (22.5 ± 2.2 vs 11.2 ± 1.3 μmol·min−1·g−1 muscle; P < 0.001). Training partially prevented this reduction in plantaris muscle CS activity in the MI-TR group compared to the MI-SED group (16.7 ± 0.9 μmol·min−1·g−1 muscle; P < 0.05). However, training did not return CS values to those seen in SHAM control animals (P < 0.05). Maximal running speed tests performed on the treadmill 12 wk after MI in trained and sedentary animals also demonstrated a significant (P < 0.05) training effect in the 10-wk MI-TR rats (40.5 ± 14.2 vs 25.4 ± 0.7 m·min−1).
FIGURE 1: Plantaris muscle CS activity in SHAM, MI-SED, and MI-TR groups. Values are means ± SEM for n = 5 rats in each group. MI-SED significantly reduced compared to SHAM controls, *P < 0.005; MI-TR significantly different from both SHAM and MI-SED groups, §P< 0.05.
Collagen characteristics
A comparison of %COL in different regions of the heart for the three groups is shown in Figure 2. MI resulted in a significant increase in %COL in LVF of MI-TR and MI-SED groups compared to SHAM controls (7.61 ± 0.19 and 7.14 ± 0.15 vs 3.55 ± 0.19 g collagen·100 g−1 dry weight; both P < 0.001). In LVS, a smaller, but similar increase in %COL was observed in both INF groups compared to SHAM controls (both P < 0.05). Training did not alter these infarct-induced increases in %COL in either LVF or LVS. RV %COL was unaffected by infarction alone or in combination with exercise training. However, comparing the two MI groups, training decreased %COL in the INF scar from 26.0 ± 1.3 to 21.0 ± 1.6 g collagen·100 g−1 dry weight (P < 0.05; Fig. 2).
FIGURE 2: %COL in RV, LVS, LVF, and INF regions from SHAM, MI-SED, and MI-TR groups. Values are means ± SEM for n = 5 rats ineach group. In LVS and LVF regions, %COL in MI-SED and MI-TR was significantly higher than in SHAM controls, *P < 0.05, ***P<0.001. INF scar %COL was significantly higher in both MI-SED and MI-TR compared to LVF from SHAM controls, ***P < 0.001. %COL in INF from MI-TR was significantly lower than that from MI-SED, + P < 0.05.
As shown in Figure 3, increased LVF HP cross-linking was found in MI-SED rats by 10 wk after MI compared to SHAM controls (0.43 ± 0.02 vs 0.30 ± 0.04 mol HP·mol−1 collagen; P < 0.05) that was completely abrogated in MI-TR animals (0.27 ± 0.03 mol HP·mol−1 collagen). Similarly, HP cross-linking in the RV of MI-SED rats was elevated (P < 0.001) above SHAM controls at 10 wk after MI (0.81 ± 0.08 vs 0.39 ± 0.04 mol HP·mol−1 collagen)—an increase that was prevented by training (MI-TR 0.25 ± 0.02 mol HP·mol−1 collagen). In contrast, HP cross-linking in LVS was unaltered as a result of MI or training. Both MI-SED and MI-TR groups showed similar increases in HP cross-linking in the INF scar region that were greater than those seen in viable LVF or LVS, reaching 1.01 ± 0.2 and 0.84 ± 0.14 mol HP·mol−1 collagen, respectively, approximately a threefold increase above SHAM LVF values (both P < 0.01).
FIGURE 3: HP cross-linking in RV, LVS, LVF, and INF regions from SHAM, MI-SED, and MI-TR groups. Values are means ± SEM for n=5 rats in each group. In RV and LVF, HP cross-linking significantly increased in MI-SED group compared to either SHAM or MI-TR groups, *P < 0.05, ***P < 0.001. In INF scar, HP cross-linking was significantly elevated in both MI-SED and MI-TR groups compared to HP cross-linking in LVF from SHAM controls, **P < 0.01.
DISCUSSION
The present study was designed to examine whether exercise training could favorably modify the deleterious changes in heart ECM seen after MI, particularly with respect to observed increases in concentration of both collagen and its maturity of cross-linking. This was deemed particularly relevant because exercise is a recommended interventional strategy after a heart attack for many cardiac patients, and the bulk of the literature indicates improved LV function without increased risk for subsequent arrhythmias, heart attacks, or aneurisms as a result of this recovery paradigm (13).
In the present study, documentation of a training effect was obtained by comparing maximum achievable running speeds between the two INF groups, as well as by measurement of plantaris muscle CS activity. Correlating the different maximal running speeds of MI-TR and MI-SED groups in the present study with aerobic capacity values in the study of Mazzeo et al. (19) indicated that our trained and sedentary INF animals had V˙O2max values of ∼68 and 45 mL O2·kg−1·min−1, respectively. The improvements in work capacity and oxidative enzyme activity of the skeletal muscle were similar to those reported previously for trained INF rats (23).
Heart remodeling after MI
It has been well established that structural remodeling of LV after MI includes increases in collagen deposition in all areas of the myocardium (20). In the present study, %COL in viable remaining LVF approximately doubled by 10 wk after MI compared to SHAM controls, which mirrors earlier findings (20). Observed increases in %COL are due to upregulated transcription of Type I and III procollagen mRNA in cardiac fibroblasts (5,39), affected by increases in systemic and/or myocardial renin–angiotensin–aldosterone systems or by indirectly stimulating cytokines and growth factors (7). The regional differences in %COL between LVF and LVS observed previously (20) are thought to be related to regional differences in cardiomyocyte slippage and wall thinning in the two regions in response to different wall stresses (25).
In the present study, elevations in RV HP cross-linking after MI were greater than the increases seen in LVF and were only exceeded by values observed in the INF region itself. The large increases in RV collagen cross-linking thus paralleled the degree of compensatory hypertrophy observed in this chamber compared to that seen in LV. It is recognized that the stimulus for compensatory hypertrophy differs between the two chambers with the stimulus for RV hypertrophy being pressure overload that occurs after infarction-related abnormalities in LV function and consequent elevation of pressures in the pulmonary circulation.
The mechanism(s) responsible for rapid development of mature collagen cross-links in both RV and LV from INF rats in the present study is currently unclear. HP cross-links are formed between lysyl and hydroxylysyl residues after lysyl oxidase converts them to their corresponding aldehydes. Because HP is a trivalent cross-link, which forms between homotrimers and heterotrimers of Type I and III collagens, appropriate fibril orientation is critical for residues to react and form cross-links. In addition to its contribution to cross-link formation, collagen fiber orientation plays a role in the maturation of the infarct scar and in post-MI cardiac function (35). The degree of mature covalent cross-linking is thought to be a determinant of collagen’s tensile strength (8) and, in the rat, is normally seen to gradually increase in heart over the lifespan of the animal (33,34). Thus, the elevated HP values seen in INF scar from young adult rats as early as 12 wk after MI in the current study are higher than those seen at 24 months of age in healthy senescent myocardium.
Chamber stiffness properties and LV function
There is a substantial body of evidence showing that altered ECM characteristics affect both heart stiffness and function. In one of the first studies looking at the potential relationship between ECM and LV function after MI, Litwin et al. (18) found that increased %COL in non-INF myocardium from MI rats was associated with perturbations in several measurements of systolic function, as well as increased passive stiffness characteristics, as evaluated by a rightward shift in muscle stress–strain relationships. In these early studies, analysis of HP, the major nonreducible collagen cross-link found in heart was not performed. However, the importance of cross-linked collagen to myocardial stiffness properties was highlighted by Norton et al. (24) who noted in spontaneously hypertensive rats that LV end-diastolic myocardial stiffness constant was increased as a consequence of reduced collagen solubility (which was equated with increased collagen cross-linking) rather than elevated LV collagen or Type I phenotype concentrations. Significantly, this depressed diastolic function was normalized by ACE-inhibition therapy, further implicating ECM in the observed abnormalities in LV compliance.
Training and heart matrix properties
Previous studies have shown exercise training in senescent rats to be associated with attenuation of the normal increase in HP cross-links in the heart associated with aging, and unaltered (33), or even reduced myocardial %COL (31). It has also been shown to prevent, or attenuate the decline in LV mRNA for Type I and Type III procollagens in both middle-aged and elderly animals, respectively (34). The mechanisms for training-induced reductions in HP cross-links are thought to relate to increased collagen turnover. The findings from the current study go one step further to show that even when HP cross-linking is increased in LVF after MI, exercise training prevents this increase in nonreducible collagen cross-links and may improve myocardial stiffness and contractility after MI. Although the effects of MI on HP cross-links in the RV have not been reported previously, in the current study, the training–MI combination normalized the elevated collagen cross-linking seen in this chamber in response to MI alone. This finding also points out how injury and dysfunction in LV affects ECM metabolism on the right side of the heart.
Training and heart function after MI
Most studies evaluating exercise training after MI in both humans and rodents have found either no effect or improvement in heart remodeling and LV function in conjunction with increases in aerobic or work capacity (4,22,23). Using an endocardial mapping technique to evaluate 32 separate wall segments, Cannistra et al. (4) showed that 12 wk of training improved MET capacity but did not affect percent abnormal wall motion or indices of LV sphericity in patients with predominantly small infarcts. These results confirmed earlier findings of Dubach et al. (6) and those of Giannuzzi et al. (11) in patients with larger infarcts, who also noted an improved LV ejection fraction in concert with an almost 30% improvement in work capacity after a 6-month training program. An additional important finding of the latter study was that this extended period of training prevented the increase in LV end-diastolic and end-systolic diameters seen in the sedentary-matched group.
Nonhuman studies have evaluated the effects of training after MI with a variety of infarct sizes, training modes, intensities, and durations, as well as time of intervention after MI. A swimming program initiated in rats 1 wk after MI and only lasting 2 wk did not affect systolic function as evaluated by the rate of rise of LV pressure and showed a trend toward favorable effects on infarct expansion and wall thickness indices (1). Other rodent studies in which swimming exercise was used found infarct expansion when the program was initiated 7 d (16) or 4 d (10) after MI. In the study of Gaudron et al. (10), deleterious effects were only noted in those rats with large infarcts (>35% of the LV) and were more pronounced compared to a second training group that initiated exercise 21 d after MI. In contrast, Hochman and Healy (14), who initiated a weeklong treadmill running program only 24 h after MI in rats with smaller infarcts (MI = 24% of LV), noted no deleterious effects on infarct shape or ventricular topography. When long-term (6–8 wk) training programs were used in rats with large infarcts (MI ∼ 50% of LV), diametrically opposite findings with respect to infarct thinning may have also been related to time-of-initiation of the training program (15,26). Thus, in rats that initiated a swim training program 5 wk after MI for 6 wk, no additional scar thinning was observed, whereas exercise attenuated ventricular dilation and reduced wall tension (26). Commencing treadmill running 2 wk after MI for 8 wk also attenuated dilation and improved LV-developed pressure but resulted in scar thinning, a phenomenon that was prevented in a second running group of rats that were also receiving AT1 receptor blockade (15).
Infarct scar metabolism
One of the most novel findings of the current study was that training after MI affected scar metabolism as indicated by differences in %COL between MI-TR and MI-SED groups. It is widely accepted that a phenotypic modification of myocardial fibroblasts occurs after MI resulting in the production of myofibrobasts with the capability of producing enhanced levels of contractile smooth muscle α-actin (28,29). Training has been shown to inhibit cultured cardiac fibroblast migration patterns on collagen and increase expression for α5- and β1-integrins compared to values in control hearts (3). It is currently unknown whether training alters fibroblast migration and contractility after MI and thus contributes to overall improvement in LV ejection fraction seen in some (11), but not all (4,6), human studies after MI. Putative increases in scar smooth muscle actin and integrin expression consistent with increased angiogenesis and cardiac fibroblast activity would tend to dilute scar collagen concentration resulting in the decreased values noted in the MI-TR group in the current study.
Recognized limitations to the current study are the small number of rats used in the three groups as well as the lack of directly measured cardiac function or stiffness parameters. However, in pathological models of LVH including MI and hypertension, elevations in both collagen and collagen cross-linking were implicated in the increase in stiffness observed (18,24). Furthermore, LV end-diastolic stiffness was improved by normalizing the elevated collagen cross-linking profile in hypertensive hearts as alluded to earlier (24).
In summary, findings related to ECM from both remaining viable myocardium and scar tissue in hearts after surgically induced MI indicate that 10 wk of treadmill running modulates some of the deleterious changes in connective tissue metabolism seen in response to MI alone. Our results highlight the regional differences in properties of heart ECM following MI and demonstrate that exercise can minimize the deleterious effects of fibrosis seen post-MI. Specifically, training seems to normalize the elevations in HP cross-linking seen in both LVF and RV in response to MI. The implications of these findings are a reduction in the increased stiffness characteristics and associated diastolic function abnormalities seen after a heart attack. Interestingly, a training-induced reduction in collagen cross-links was not observed in the INF scar, a reduction of which could predispose the scar to dilation and potentially deleterious effects leading to aneurism.
This study was supported by grants from the Mountain West Affiliate of the American Heart Association (9206228S and 93062665) awarded to D.P.T. and R.J.M., respectively. The authors do not have any financial disclosures to report. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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