Young and old male Fischer 344 pathogen-free rats (Samtako Bio, Osan, Korea) were housed in a climate-controlled room (25℃, 12-hr light-dark cycle) and allowed standard food and water ad libitum. Old rats were weight-matched and randomly assigned to a sedentary control (OC) or an exercise-training group (OT). Young rats (YC) were assigned to a control group only to determine aging effects. The number of rats in each group was at least 7. After either 12 weeks of exercise or alternatively confinement in a cage, rats (young: 6 months and old: 25 months of age) were studied. The 6-month-old rats are mature animals which had passed the rapid growth phase and 25-month-old rats represent old animals with relatively little mortality (12).

Plasma glucose levels were assessed at baseline using the glucose oxidase technique (YSI 2300 stat plus glucose/L-lactate analyzer; YSI, Yellow Springs, OH, U.S.A.). This investigation conducted in accord with the guidelines issued by the Seoul National University Hospital Institutional Animal Care and Use Committee.

Before training, all rats were familiarized with a treadmill (1004RVI Exer-4R Open Treadmill; Columbus Instruments, Columbus, OH, U.S.A.) by walking or running for 10 min/day for 10-15 days. Animals in OT commenced training at 4 m/min, for 20-30 min, 5 days/week. Rates and times were progressively increased throughout the 12-week training program. Rates at termination were 15 m/min for 45-60 min/day, 5 days/week. This speed-grade combination has been shown to be slightly less than 70-75% of maximal oxygen consumption (VO_2max) (11, 13). The YC and OC rats walked or ran on the treadmill for 10 min once a week to maintain familiarity with the treadmill for subsequent maximal exercise capacity testing. At the end of the 12-week period, maximal exercise capacities were measured twice in each rat, using a test involving walking at 10 m/min for 5 min followed by 2 m/min increases in speed every 2 min until the animal was exhausted (9).

Cardiac magnetic resonance imaging (MRI) was performed at baseline and 2 days after measuring maximal exercise capacity. Rats were anesthetized with pentobarbital sodium (Entobar; Hanlim; young rats 50 mg/kg, old rats 42.5 mg/kg) administered intraperitoneally (i.p.).

Rats were imaged with a 1.5-T clinical imaging unit (Gyroscan Intera, release 10; Philips Medical Systems, Best, Netherlands) equipped with a custom surface coil (4.7 cm in diameter). A prospectively electrocardiogram (ECG)-gated steady-state free-precession cine sequence (balanced fast field-echo 10/4.1 [repetition time msec/echo time msec], flip angle of 35°, matrix of 179×256, field of view of 80×80 mm, number of excitation:1) was applied in a true short-axis image orientation to encompass the entire heart with contiguous 2 mm sections. With ECG referencing, the temporal resolution was 10 time frames per cardiac cycle per study. A shimming procedure was performed for imaging areas to avoid magnetic field inhomogeneity. Most MR images were suitable for analysis.

MR images were analyzed by two independent readers, blinded to subjects and their results, using commercially available software on an off-line workstation (View Forum, release 3.2, Cardiac Package; Philips Medical Systems). The cine loops were viewed and end-diastolic and end-systolic frames were chosen as the images with the largest and smallest left ventricle (LV) cavities, respectively. Endocardial contours, including papillary muscles leading into the LV cavity, were traced using a semiautomated border technique. LV volumes were calculated consecutively from MR imaging data sets with software assistance.

Hemodynamic parameters were measured as previously described (14). Two days after MRI measurements, rats were anesthetized with pentobarbital sodium (young rats 50 mg/kg, old rats 42.5 mg/kg, i.p.) without endotracheal intubation while breathing room air. MPVS-300 Cardiovascular Micro-Tip multi-segment pressure-volume (P-V) transducer catheter (Millar Instruments, Huston, TX, U.S.A.) was inserted into the right carotid artery and advanced into the LV under pressure monitoring. Catheter was inserted into the right femoral artery for mean arterial pressure (MAP) measurement and into the right femoral vein for fluid administration. Heart rate (HR), maximal LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), MAP, maximal slope of systolic pressure increment (+dP/dt) and diastolic pressure decrement (-dP/dt), time constant of LV pressure decay (tau, τ), ejection fraction (EF), stroke volume (SV), LV end-diastolic volume (EDV), cardiac output (CO), and stroke work (SW) were computed using a cardiac P-V analysis program (PVAN3.3; Millar Instruments). Isovolumic relaxation was quantified by calculating -dP/dt and τ. Tau (τ) describes the rate of fall of LV pressure during isovolumic relaxation. The area contained within the left ventricular pressure-volume loop is the SW. SV and CO were calculated and corrected according to in vitro and in vivo volume calibrations using PVAN3.3. CO and SW were normalized to body weight (cardiac index [CI] and SW index [SWI]).

LV pressure-volume relations were assessed by transiently compressing the inferior vena cava. The slope of end-systolic and end-diastolic pressure-volume relation (ESPVR and EDPVR) and +dP/dt-EDV relation were calculated using PVAN 3.3. ESPVR and +dP/dt-EDV represents a parameter of chamber contractility. EDPVR slope is an index of LV chamber stiffness. The volume calibration of the conductance system was performed. At the end of each experiment, 50 µL of 15% saline was injected intravenously, and from shifts in P-V relations parallel conductance volume (V_p) was calculated using PVAN 3.3 and used to correct cardiac mass volume.

After the hemodynamic measurements, animals were sacrificed by exsanguinations (aortas were punctured to obtain blood samples). Hearts and lungs were removed, and left and right ventricles were dissected and weighed. Hearts were snapfrozen in liquid nitrogen and stored at -80℃ for hydroxyproline assay or were fixed in 10% formalin and paraffin-embedded for histological assessment.

Interstitial collagen in LV mid-free wall was measured using Picrosirius red staining with a computerizing automated image analysis system (Image-pro plus, Silver Spring, MD, U.S.A.) (5). Collagen volume fraction (CVF) was defined as the sum of all stained interstitial collagen tissue areas divided by the whole tissue area. Collagen surrounding intramyocardial coronary arteries was excluded.

Hydroxyproline (a marker of collagen content) concentrations were determined using the colorimetric assay described by Woessner (15). Collagen concentrations were calculated by assuming that collagen weighs 7.25 times more that of hydroxyproline (collagen has a molecular weight of 300,000).

Unmodified heart collagen was significantly solubilized by pepsin after 2 hr and greater than 98% of heart collagen was solubilized after 24 hr. The susceptibility of collagen to digestion by pepsin provides an index of AGEs cross-linked collagen (16, 17). Aliquots of LV were excised and mixed with 3 mL of 200 µg/mL pepsin in 0.5 M/L acetic acid at 37℃. After 2 and 24 hr of digestion, 1 mL of the supernatant was removed and hydroxyproline concentrations were measured as described above.

The results presented reflect means±SE per group. Interobserver agreement of the MRI results was determined by the Pearson correlation coefficient. An unpaired two-sided Student's t-test was used to compare old vs. young rats, and old controls vs. old trained groups. p values <0.05 were considered statistically significant, and SPSS Ver 12.0 software was used throughout (SPSS Inc., Chicago, IL, U.S.A.).

At baseline, there were no significant differences in body weight (OC 409±8 g, OT 410±9 g; p=0.877) and plasma glucose levels (OC 150±10 mg/dL, OT 157±12 mg/dL; p=0.564) in old rats. At the end of the 12-week period, LV weight and the ratio of LV to body weight were significantly higher in OC rats compared with YC rats without notable differences in body weight (aging effect). In addition, wet lung weights to body weight, an index of pulmonary congestion followed a similar pattern, indicating marked pulmonary congestion in old rats secondary to cardiac decompensation. Exercise-training significantly reduced body weight in OT rats. LV weight and the ratio of LV weight to body weight (an index of LV hypertrophy) were not different between OC and OT groups after 12 week of exercise training. Exercise training had no effect on LV weight, the ratio of LV to body weight, or the ratio of lung to body weight in old rats (Table 1).

Maximal exercise capacity was measured twice with high reproducibility (r=0.823 [p<0.001], and r²=0.677 by Pearson's rank-order correlation analysis). Twelve weeks of exercise training significantly increased maximal exercise capacity, defined as time to exhaustion using a standardized treadmill running protocol, as was expected (7.7±1.0 min in OC, 20.8±1.7 min in OT; p<0.001). Exercise training generated the OT rats with a maximal exercise capacity matched that of the YC rats (17.2±1.6 min in YC, 20.8±1.7 min in OT; p>0.05).

Typical P-V loops obtained from young and old animals shows in Fig. 1. The shift of P-V loops to the right and decreased +dP/dt in aging rats indicated a decrease in cardiac contractility. Aging was associated with significant decrease in HR, MAP, LVSP, +dP/dt, -dP/dt. In contrast, LVEDP, EDV, ESV, τ(a classic parameter of active relaxation), and EDPVR slope (a parameter of LV chamber stiffness) significantly increased in aging, indicating diastolic dysfunction (Table 2). In old rats, +dP/dt was significantly decreased but EF, SV, CI, and SWI were not different from the parameters of young rats, which resembles findings in diastolic heart failure in men. During preload manipulation, ESPVR slope was steeper in young than in old rats, suggesting decreased systolic performance with aging. In contrast, EDPVR slope was increased (with a higher EDPVR slope) in aging rats, indicating increased end-diastolic stiffness. ESPVR slope and +dP/dt were significantly increased in OT vs. OC rats. Fig. 2 shows that the slope of +dP/dt-EDV relation and SW and EDV were steeper in OT vs. OC rats, indicating increased contractility in OT rats. ESPVR was a load insensitive index of contractility but also by changes in chamber geometry and other diastolic factors. +dP/dt is a classic parameter that is sensitive to changes in contractility but dependent on changes in preload. +dP/dt-EDV represents a sensitive but less load-dependent parameter of chamber contractility. Preload recruitable SW (PRSW), the slope of the relation between SW and EDV, is sensitive to chamber contractile function, which is independent of chamber size and mass.

Exercise was also associated with significant decreases in EDV and EDPVR slope. Tau (τ) and -dP/dt were not different between OC and OT rats (Table 2). The long-term exercise training prominently improved myocardial contractility and stiffness.

At the baseline ESV and EDV measured by MRI were not different among OC and OT rats. After 12 weeks of exercise training, ESV and EDV increased in old rats, but, post-ESV and EDV had no difference in OC and OT groups. However, ΔEDV (%), the percentage of the EDV increment-to-baseline EDV in each rats was 50.5±12.7% in OC rats and 20.2±7.6% in OT rats (p=0.060) (Table 3). Interobserver measurement of MR volumes were closely correlated (R, 0.96, 95% CI, 0.55 to 1.00, p<0.05).

Total myocardial collagen amount (% dry weight) and CVF were significantly higher in OC vs. YC rats (percent collagen: 6.67±0.91% vs. 2.08±0.79%, respectively; p=0.013). Exercise training did not influence total myocardial collagen amount of LV in the old rats (OC 6.67±0.91%, OT 6.61±0.61%; p=0.962) (Fig. 3). Myocardial collagen solubility as an index of AGEs cross-linked collagen was significantly lower in old rats (YC 43.57±1.13%, OC 38.11±1.27%; p=0.006), but significantly increased in OT rats (OC 38.11±1.27%, OT 43.94±2.11%; p=0.032) (Fig. 4).