A single bout of eccentric exercise results in muscle damage, but it is not known whether this is correlated with microcirculatory dysfunction. We tested the following hypotheses in the spinotrapezius muscle of rats either 1 (DH-1; n = 6) or 3 (DH-3; n = 6) days after a downhill run to exhaustion (90–120 min; −14° grade): 1) in resting muscle, capillary hemodynamics would be impaired, and 2) at the onset of subsequent acute concentric contractions, the decrease of microvascular O2 pressure (Pmvo2), which reflects the dynamic balance between O2 delivery and O2 utilization, would be accelerated compared with control (Con, n = 6) rats. In contrast to Con muscles, intravital microscopy observations revealed the presence of sarcomere disruptions in DH-1 and DH-3 and increased capillary diameter in DH-3 (Con: 5.2 ± 0.1; DH-1: 5.1 ± 0.1; DH-3: 5.6 ± 0.1 μm; both P < 0.05 vs. DH-3). At rest, there was a significant reduction in the percentage of capillaries that sustained continuous red blood cell (RBC) flux in both DH running groups (Con: 90.0 ± 2.1; DH-1: 66.4 ± 5.2; DH-3: 72.9 ± 4.1%, both P < 0.05 vs. Con). Capillary tube hematocrit was elevated in DH-1 but reduced in DH-3 (Con: 22 ± 2; DH-1: 28 ± 1; DH-3: 16 ± 1%; all P < 0.05). Although capillary RBC flux did not differ between groups (P > 0.05), RBC velocity was lower in DH-1 compared with Con (Con: 324 ± 43; DH-1: 212 ± 30; DH-3: 266 ± 45 μm/s; P < 0.05 DH-1 vs. Con). Baseline PmvO2 before contractions was not different between groups (P > 0.05), but the time constant of the exponential fall to contracting PmvO2 values was accelerated in the DH running groups (Con: 14.7 ± 1.4; DH-1: 8.9 ± 1.4; DH-3: 8.7 ± 1.4 s, both P < 0.05 vs. Con). These findings are consistent with the presence of substantial microvascular dysfunction after downhill eccentric running, which slows the exercise hyperemic response at the onset of contractions and reduces the PmvO2 available to drive blood-muscle O2 delivery.
- skeletal muscle
- downhill running
- microvascular adaptation
- capillary hemodynamics
- oxygen exchange
novel eccentric exercise that generates high intramuscular forces induces profound muscle damage and dysfunction. For example, after a single bout of eccentric exercise, serum creatine kinase activity becomes elevated (9; for review, see Ref. 48), proteolytic enzymes are increased (8, 44), and a substantial inflammatory response is manifested (15). Moreover, a select population of damaged myocytes demonstrates mononuclear cell infiltration, and there is the presence of multiple central nuclei in these fibers (23). Depending on the time elapsed after the exercise bout (i.e., 1–7 days), either an initial fiber swelling or subsequent fiber degeneration may be observed (23).
Whereas myocyte degenerative changes will impact the ability of the muscle(s) to produce high forces, the capacity for repetitive contractions such as those powering locomotory exercise will likely be affected by any impairment in the ability to deliver O2 and energetic substrates to the muscle fibers. Specifically, the probability that eccentric exercise will damage the microcirculation and therefore the ability to deliver and exchange O2 and substrates must be recognized. Such damage may be incurred directly during the eccentric exercise or secondary to myocyte swelling and/or altered intramuscular pressures. Indeed, Kano and colleagues (23) have demonstrated that eccentric exercise results in a disruption in the capillary geometry in the red and white gastrocnemius 1–7 days after electrically induced eccentric contractions. However, to date it remains unknown whether microcirculatory function and the capacity to deliver and distribute O2 within the capillary bed are impaired after eccentric exercise.
Our laboratory has demonstrated recently that downhill running represents a physiological paradigm that recruits the spinotrapezius muscle as evidenced by the presence of an exercise hyperemic response (22). This muscle stabilizes the scapula, and downhill running consequently subjects the spinotrapezius to eccentric activity. Therefore, the purpose of the present investigation was to utilize downhill running to explore the functional microvascular and O2 delivery (Q̇o2) and O2 utilization (V̇o2) [i.e., ratio between Q̇o2 and V̇o2 (Q̇o2/V̇o2)] sequelae to eccentric exercise. A combination of intravital microscopy and phosphorescence quenching techniques were utilized to address the following hypotheses. Within the spinotrapezius of naive rats, at 1 and 3 days after exhaustive downhill exercise, we hypothesized that 1) the proportion of capillaries sustaining red blood cell (RBC) flux would be decreased and capillary hemodynamics would be impaired, and 2) at the onset of acute concentric contractions initiated at 1 and 3 days after the eccentric exercise, the kinetics of the fall in microvascular oxygen pressure (PmvO2), which reflects the dynamic balance between Q̇o2 and V̇o2, would be accelerated. Specifically, if microcirculatory impairments act to reduce Q̇o2, a more rapid fall in PmvO2 would be expected at the onset of contractions. Our results substantiated these hypotheses and indicated that eccentric exercise impairs the capacity to deliver and distribute O2 within muscle 1–3 days postexercise. This scenario would be expected to impact negatively V̇o2 kinetics and therefore the oxidative contribution to muscle energetics, particularly during subsequent bouts of exercise.
Animal selection and care.
Eighteen female Sprague-Dawley rats (body mass 264 ± 4 g) were used in this study. Rats were maintained on a 12:12-h light-dark cycle and received food and water ad libitum. All experiments were conducted under the guidelines established by the National Institutes of Health and were approved by Kansas State University's Institutional Animal Care and Use Committee. Rats were randomly assigned to one of two groups: control (Con; n = 6) or downhill running (n = 12).
Rats in the downhill running group underwent a period of familiarization (1–2 wk) to running on a motor-driven treadmill that entailed exercising for 5–10 min/day at a speed of 20–30 m/min (0% grade). After acclimatization to the treadmill, each rat performed an exercise protocol designed to actively recruit the spinotrapezius muscle (22). To ensure that each rat was fatigued by the protocol, all animals ran individually on a −14° decline for 90 min (5-min bouts with 2 min of rest between successive bouts). The running speed was maintained at 40 m/min until the rat could no longer keep pace with the treadmill. At this time, the speed was decreased to 20 m/min for the subsequent exercise bouts. Each rat performed approximately nine exercise bouts at 40 m/min and nine bouts at 20 m/min. Subsequently, each rat ran continuously until it was unable to maintain pace with the treadmill despite humane encouragement. This final run constituted an average of 9 min of downhill running at 20 m/min. Thus each rat ran for a total duration of 99 min on average. Presence of fatigue was confirmed by exhibition of extreme lethargy in righting themselves when placed in the supine position. The downhill running group was further divided into two subgroups in which examination of the effects of downhill running was performed at 1 and 3 days postrunning [DH-1 (n = 6) and DH-3 (n = 6), respectively]. After the designated period of time, each rat underwent two procedures: phosphorescence quenching for determination of PmvO2 at the onset of contractions and examination of the microcirculation within the spinotrapezius muscle. Heart rate and mean arterial pressure were monitored continuously throughout both data-acquisition periods. Total duration of the experiments did not exceed 3 h. Control rats were not exercised because it has been demonstrated previously that the classical protocol of inclined running does not recruit the spinotrapezius muscle as determined by the lack of increase of blood flow measured using radiolabeled microspheres (22, 35)
Surgical preparation for phosphorescence quenching.
Before the surgical procedures, the animals were anesthetized with pentobarbital sodium (50 mg/kg ip to effect and supplemented as necessary). The rat was placed on a heating pad (38°C) to maintain body temperature. To monitor arterial blood pressure and heart rate (model 200, Digimed BPA, Louisville, KY), the left carotid artery was cannulated (polyethylene-50, Intra-Medic polyethylene tubing, Clay Adams Brands; Sparks, MD). This cannula also allowed for infusion of the phosphorescent probe [palladium meso-tetra(4-carboxyphenyl) porphyrin dendrimer (R2)] at 15 mg/kg body wt.
The spinotrapezius muscle is a postural muscle that lies in the middorsal region of the rat; it originates from the lower thoracic and upper lumbar region and inserts on the spine of the scapula. The right spinotrapezius muscle was exposed by a U-shaped skin incision to provide access for electrical stimulation and measurement of PmvO2. After the overlying skin was reflected and fascia was removed, the muscle surface was superfused with Krebs-Henseleit solution equilibrated with 5% CO2-95% N2 at 38°C and adjusted to pH 7.4. For the induction of indirect bipolar muscle contractions in the spinotrapezius, stainless steel electrodes were attached to the muscle proximal to the motor point (cathode) and across the caudal extremity (anode) close to the spinal attachment.
The phosphor R2 was infused via the arterial cannula ∼15 min before each experiment. The experiments were conducted in a darkened room to prevent contamination from ambient light. After a 10- to 15 min stabilization period after the surgery, twitch muscle contractions (1 Hz, 3–5 V, 2-ms pulse duration) were elicited for 3 min using a Grass S88 stimulator (Quincy, MA). This contraction profile provides a blood flow response consistent with moderate-intensity exercise (4). PmvO2 was determined at 2-s intervals at rest and after the rest-to-stimulation transition for 3 min. After the 3-min stimulation period, PmvO2 was measured for 3–5 min into recovery before undergoing surgical exteriorization of the contralateral (left) spinotrapezius muscle for intravital observation.
The theoretical basis for phosphorescence quenching has been detailed previously (3, 4, 16). Briefly, the Stern-Volmer relationship (43) describes quantitatively the O2 dependence of the phosphorescent probe. R2 is a nontoxic dendrimer (31) that binds completely to albumin at 38°C and pH 7.4, with a quenching constant of 409 mmHg/s and lifetime of decay in the absence of O2 of 601 μs (33, 36). In addition to binding with albumin, the net negative charge of R2 also facilitates restriction of the compound to the vascular space (38).
PmvO2 reflects the Po2 within the capillary blood, which constitutes the principal intramuscular vascular space. To determine PmvO2, a PMOD 1000 frequency domain phosphorometer (Oxygen Enterprises, Philadelphia, PA) was utilized. The common end of the bifurcated light guide was placed ∼2–3 mm above the medial region of the spinotrapezius (i.e., superficial to dorsal surface), and blood was sampled within the microvasculature up to ∼500 μm deep within a circular region ∼2 mm in diameter. The phosphorometer employs a sinusoidal modulation of the excitation light (524 nm) at frequencies between 100 Hz and 20 kHz, which allows for phosphorescence lifetime measurements from 10 μs to ∼2.5 ms. In the single-frequency mode, 10 scans (100 ms) were used to acquire the resultant lifetime of the phosphorescence (700 nm) and were repeated every 2 s (for review, see Ref. 51). To obtain the phosphorescence lifetime, the logarithm of the intensity values was taken at each time point and the linearized decay was fit to a straight line by the least squares method (7).
Modeling of PmvO2 profiles.
Curve fitting was accomplished by use of KaleidaGraph software (Synergy Software, Reading, PA) and was performed on the PmvO2 data by using a one-component exponential model by means of the following equation: where PmvO2(t) is PmvO2 at time t, ΔPmvO2 designates the decrease of PmvO2 from resting baseline to steady state during contractions, TD is the time delay, and τ is the time constant.
Goodness of model fit was determined via three criteria: 1) the coefficient of determination (i.e., r2), 2) the sum of the squared residuals term (i.e., χ2), and 3) visual inspection of the model.
Intravital microscopy studies of the microcirculation.
After the phosphorescence quenching procedures were completed, the left spinotrapezius was exteriorized as described previously (18, 25–28, 39) to examine the microcirculation. This procedure takes ∼45 min (range 40–50 min) and does not perturb the microvasculature or blood flow at rest or during 1-Hz contractions (2). Fascial removal and disturbance were minimized to avoid any associated muscle damage (34). All exposed tissue as well as the dorsal surface of the spinotrapezius was superfused with Krebs-Henseleit solution at 38°C (see Surgical preparation for phosphorescence quenching) while the muscle was sutured (6.0 silk, Ethicon, Somerville, NJ) at five equidistant points around the perimeter to a thin-wire horseshoe-shaped manifold (39). The muscle was then protected with Saran Wrap (Dow Brands, Indianapolis, IN).
The rat was placed on a circulation-heated Lucite platform, and the spinotrapezius was observed by use of an intravital microscope (Nikon, Eclipse E600-FN; ×40 objective; 0.8 numerical aperture) equipped with a noncontact, illuminated lens and a high-resolution color monitor (Sony Trinitron PVM-1954Q, Ichinonya, Japan). The spinotrapezius was maintained at physiological length (∼2.4 μm) throughout the subsequent observation period, and exposed tissue was continuously superfused with Krebs-Henseleit solution. The muscle was then transilluminated in a fashion that ensured clear resolution of the A bands of the sarcomeres within one-third to two-thirds of the muscle fibers. The final screen magnification (×1,184) was confirmed by initial calibration of the system with a stage micrometer (MA285, Meiji Techno, Saitama, Japan). This magnification is adequate for measuring all essential structural and hemodynamic variables (39).
Once the muscle was positioned on the platform, a microvascular viewing field (270 × 210 μm) containing ∼5–8 muscle fibers and 5–10 capillaries in the midcaudal (dorsal surface) region of the muscle, was selected. Approximately 8–10 fields (∼1–1.5 min each) were recorded for each rat, and images were time-referenced by frame and fields and stored on Super-VHS high-resolution videocassettes (JVC S-Master XG) by using a videocassette recorder (JVC BR-S822U, Elmwood Park, NJ) for subsequent offline analysis.
Capillary and fiber structural data analysis.
Five of the fields were chosen for each rat on the basis of clarity of sarcomeres, fibers, and capillaries. Initially, each microvascular field (i.e., capillaries, direction of RBC flow, and myocyte boundaries) was traced onto paper directly from the television monitor. Capillaries supporting RBC flow were assessed in real time, and each capillary was placed into one of two categories: 1) normal flow = 60 s of continuous flow or 2) impeded flow or stopped flow for >10 of 60 s. This was further used for determination of percentage of flowing capillaries [i.e., (no. of capillaries supporting RBC flow/total no. of visible capillaries per area) × 100]. The direction of RBC flow was used to determine the percentage of capillaries with countercurrent flow. For all capillaries in which hemodynamics were assessed and where the capillary endothelium was clearly visible on both sides of the lumen, capillary luminal diameter (dc) was measured (2–4 measurements/capillary) with calipers accurate to ± 0.25 mm (±0.17 μm at ×1,184) magnification.
Examination of the fields was conducted using a videocassette recorder (JVC BR-S822U; 30 frames/s) in real time and by frame-by-frame analysis techniques. Sarcomere length was determined from sets of 10 consecutive in-register sarcomeres (i.e., distance between 11 consecutive A bands) measured parallel to the muscle fiber longitudinal axis. The procedure was repeated three to four times where sarcomeres were visible to obtain a mean sarcomere length for each viewing field. For each muscle fiber in which both sarcolemmal boundaries were visible on the screen, the apparent fiber width perpendicular to the longitudinal muscle fiber axis was measured at three locations, and a mean fiber width was determined for each fiber. The total number of capillaries, i.e., those with and without RBC flow, were also counted, and these values were used to calculate lineal density (i.e., the number of capillaries per unit muscle width). Red blood cell velocity (VRBC) was determined in all capillaries that were continuously RBC perfused by following the RBC path length over several frames (∼5–10 capillaries/area) and for the maximum capillary length over which the RBC remained in crisp focus. Red blood cell flux (FRBC) was measured by counting the number of cells in a capillary passing an arbitrary point over not less than 5 frames per measurement. For each capillary in which hemodynamic data were gathered, capillary tube hematocrit (Hctcap) was calculated as where VolRBC is RBC volume, which was taken to be 61 μm3 (1), and capillaries were approximated as circular in cross section.
Values are expressed as means ± SE where the group mean is that of the individual muscles rather than the individual measurements across muscles. Group differences were determined by a one-way analysis of variance and a Student-Newman-Keuls post hoc test. Where a directional a priori hypothesis was tested (i.e., RBC velocity and flux and time constant of PmvO2 fall), a one-tailed test was utilized. Statistical significance was established at P < 0.05.
Body weights did not differ between Con and the downhill running groups (Con: 270 ± 8; DH-1: 261 ± 8; DH-3: 259 ± 8 g; P > 0.05), and the weight of the spinotrapezius muscle was not altered by downhill running (Con: 0.46 ± 0.01; DH-1: 0.45 ± 0.02; DH-3: 0.43 ± 0.03 g wet wt; P > 0.05). Mean arterial pressure and heart rate remained constant throughout each experimental protocol and did not differ between the protocols or between groups (Table 1; P > 0.05).
Fiber width and sarcomere length did not differ significantly between Con, DH-1, and DH-3 (P > 0.05; Table 2). However, sarcomere disruptions were observed in DH-1 and DH-3. Specifically, over large regions of the muscle fibers in DH-1 and DH-3 (but not Con), A bands were either out of register or smeared (percentage of screen area with affected sarcomeres, DH-1, 32.2 ± 5.9, DH-3, 40.7 ± 5.6, Fig. 1). Collapsed or obstructed capillaries were not apparent in either DH-1 or DH-3. However, there was a significant reduction in the percentage of capillaries that sustained continuous RBC flux in both downhill groups (Con: 90.0 ± 2.1, DH-1: 66.4 ± 5.2, DH-3: 72.9 ± 4.1%, both P < 0.01 vs. Con; Fig. 2). The percentage of capillaries with countercurrent flow was not different between groups (P > 0.05; Table 2). In DH-3, but not DH-1, mean capillary diameter was increased significantly compared with Con and DH-1 (P < 0.05; Table 2). Capillary tube hematocrit differed between groups and was highest in DH-1 and lowest in DH-3 (both P < 0.05 vs. Con; Table 2). Although capillary RBC flux did not differ between groups (P > 0.05; Table 2), RBC velocity was lower (P < 0.05) in DH-1, but not DH-3 (P > 0.05), compared with Con (Table 2). Also, DH-1 exhibited the highest frequency (%) of capillaries with the slowest velocities (i.e., 0–200 μm/s) and a lower frequency of capillaries with velocities in the 200–400 μm/s range than control muscles (P < 0.1; Fig. 3).
Microvascular PmvO2 response.
The dynamic PmvO2 profiles in response to electrical stimulation are shown in Fig. 4 for representative Con, DH-1, and DH-3 rats. No differences were found in mean values for baseline PmvO2, TD, or ΔPmvO2 values between Con, DH-1, and DH-3 (P > 0.05; Table 3). However, the time constant was faster in the DH-1 and DH-3 than Con (Con: 14.7 ± 1.4, DH-1: 8.9 ± 1.4, DH-3: 8.7 ± 1.4 s, both P < 0.05 vs. Con), but DH-1 and DH-3 were not different from one another (P > 0.05; Table 3, Fig. 4).
This investigation is the first to demonstrate that downhill running, which forces eccentric contractions within the rat spinotrapezius, impairs both muscle microcirculatory flow and also the balance between Q̇o2 and V̇o2 at the onset of contractions as evidenced by the accelerated fall of PmvO2. One consequence of this lowered O2 pressure head during the first 20–40 s of muscle contractions will be an impaired blood-myocyte O2 diffusion. These findings complement and may help explain the extensive and prolonged structural damage (23, 47) and impaired muscle function (9) that follow a single bout of eccentric exercise.
The control structural data obtained herein are in close agreement with values obtained previously for this spinotrapezius preparation in rats with body masses of 250–300 g (25–28, 39, 40). Specifically, when corrected to a sarcomere length of 2.7 μm, mean fiber diameter was 56 μm, which falls within the reported range of 50–57 μm for rats of a similar body mass. Moreover, literature values for mean capillary diameter are 5.4–6.2 μm compared with 5.2 μm herein. Also, the appearance of enlarged capillary diameters at 3 days after downhill running is consistent with several previous studies that have examined damaged muscle (23, 37, 42). The mechanism for this effect is not known. However, there are inextensible collagenous fibers that mechanically tether the capillaries to adjacent fibers, and it is possible that alterations in fiber geometry related to the 10–20% increase in fiber diameter (when corrected for sarcomere length differences among muscles) may have forced the increase observed in capillary diameter.
With respect to microcirculatory function, there is a body of previous literature that indicates that a substantial proportion of capillaries in resting skeletal muscle of healthy animals contain stationary RBCs (10, 11, 19, 29). Indeed, the notion that initially non-RBC-flowing capillaries are recruited at exercise onset has been invoked to explain the increase of muscle O2 diffusing capacity at exercise onset. In marked contrast, in vivo studies using dye injection in the rat at rest have found that essentially all muscle capillaries sustain some plasma flow irrespective of muscle fiber type (24). In healthy spinotrapezius muscles observed at a resting sarcomere length of ∼2.7 μm, our laboratory demonstrated continuous RBC flow in 80–96% of visible capillaries (25–28, 39, 40), and this range encompasses the present findings (∼90%). However, chronic disease conditions such as heart failure (25, 40) and Type 1 diabetes (28) increase the percentage of non-RBC-flowing capillaries to 30–50%. The present investigation indicates that a sizeable increase (27–34%) in non-RBC-flowing capillaries is also found 1–3 days after a single exhaustive bout of eccentric exercise.
The observation that capillary tube hematocrit was increased in DH-1 but reduced at DH-3 is intriguing. The elegant experiments of Duling and colleagues (e.g., Ref. 50) indicate that the capillary endothelial glycocalyx layer is intrinsic in setting a differential flow of plasma and RBCs in the capillary that lowers tube hematocrit below that present systemically. It is possible, therefore, that muscle damage consequent to eccentric exercise impacted the endothelial glycocalyx layer and thus its effect on capillary hemodynamics. By DH-3 any such effect was gone, and the reduction in capillary tube hematocrit below control values might be expected on the basis of the hyporemic conditions extant within these capillaries.
Within the healthy spinotrapezius muscle at the onset of 1-Hz contractions, PmvO2 does not fall immediately but rather evidences a modest delay (i.e., TD) where PmvO2 remains close to baseline before decreasing exponentially to the steady-state contracting value (4). Coupled with capillary hemodynamic measurements across the transition to contractions, it is evident that this behavior reflects an almost instantaneous increase in RBC flux (i.e., Q̇o2; Ref. 27) that is matched temporally and quantitatively with increases of V̇o2 (3). The subsequent fall in PmvO2 results from a relatively greater increase of V̇o2 than Q̇o2. The mean TD and τ for the control muscles in the present investigation (11 and 15 s, respectively) fit closely with values reported previously for the healthy spinotrapezius (i.e., TD, 11–14 s; τ, 16–19 s, Refs. 3, 5, 13).
One particularly striking finding from the present investigation was the accelerated fall (i.e., ∼40% faster) in PmvO2 in both 1- and 3-day posteccentric exercise groups (i.e., τ, < 9 s for both, Table 3). In this respect, the sequelae of downhill running resembles the PmvO2 response of rats suffering from moderate chronic heart failure (13) with both conditions being characterized by capillary hemodynamic impairments that include a significant increase in the proportion of capillaries that do not support continuous RBC flow. Therefore, it is logical to consider that the impaired microcirculatory hemodynamics might be responsible for the more rapid fall of PmvO2 found in the present investigation (see Mechanistic insights into myocyte O2 delivery below).
Mechanistic insights into myocyte O2 delivery.
The elegant modeling of Federspiel and Popel (14) and Groebe and Thews (20) suggests that the capacity of the capillary bed for blood-myocyte O2 diffusion is determined principally by the number of RBCs lying adjacent to that myocyte at any given time. Thus, within RBC-flowing capillaries, RBC flux and hematocrit, as well as the available length of capillaries, are important determinants of perfusive and diffusive O2 conductance. Capillaries that do not flow may contain stationary RBCs. However, with zero perfusive O2 flux, RBC Po2 will equilibrate fairly rapidly with intramyocyte Po2, at which time blood-myocyte O2 transport will cease and such capillaries will not supply O2. It is possible but unlikely that these nonflowing capillaries did not contain the R2 phosphorescent probe. What is more likely is that the perfusive and diffusive characteristics of the tissue were impaired, resulting in a mismatching of Q̇o2 and V̇o2 (or at least O2 demand). Thus, although there was a population of capillaries in which PmvO2 may have reached near equilibration with the low intramyocyte Po2, it is quite feasible that there may have been other capillaries either with impaired O2 diffusion characteristics or that abutted myocytes with a low O2 requirement. This latter effect may account for the absence of lowered PmvO2 at rest or during the contracting steady state in DH-1 and DH-3 rats. As mentioned above, it is pertinent that the spinotrapezius microcirculatory dysfunction found herein after eccentric exercise (i.e., reduced percentage of capillaries not supporting RBC flow, decreased RBC velocity and flux) resembles that observed in experimental heart failure of a moderate severity (25, 40). Moreover, under both of these conditions the PmvO2 profile evidences a faster fall (shorter time constant) (13). At the onset of contractions in the spinotrapezius capillaries of rats in heart failure, this PmvO2 profile was associated with an extremely sluggish increase of RBC flux and velocity and the absence of RBC flow in those vessels not flowing at rest (40). It is tempting to speculate, therefore, that in the present investigation following eccentric exercise, similar perfusion deficits to those found in heart failure may have produced the altered PmvO2 profile. The effect of this is to decrease PmvO2 transiently below values seen in control muscle, which would lower the driving pressure for O2 diffusion from capillary to myocyte across the critical transition period at the onset of contractions (i.e., 20–40 s, Fig. 4).
In healthy individuals, several lines of evidence (for review, see Ref. 17) indicate that neither pulmonary nor muscle V̇o2 kinetics at exercise onset are limited by Q̇o2 per se. Rather, some intramuscular enzymatic process(es), some of which are modulated by endogenous nitric oxide (21), are thought to set the speed of V̇o2 kinetics and therefore determine the size of the O2 deficit and the resulting intracellular perturbation (e.g., hydrogen ions and phosphocreatine among others) upon initiating muscle contractions. In contrast, within disease states such as severe chronic heart failure, muscle perfusive O2 conductance may be impaired to an extent that mandates a PmvO2 fall below levels found in healthy muscle (13). From consideration of Fick's law of diffusion, a lowered pressure driving O2 from blood into the myocyte is expected to constrain the diffusive flux of O2, thereby contributing to a slowing of V̇o2 kinetics (6, 41, 45). The present results suggest that one consequence of the microcirculatory dysfunction demonstrated after eccentric exercise may be slowed V̇o2 kinetics. In turn, these slowed V̇o2 kinetics would be associated with reduced contractile function and impaired exercise tolerance.
Downhill running has been employed effectively as a model for eccentrically induced damage in humans (46) and rats (30). We have recently demonstrated in the rat that downhill running recruits the spinotrapezius, a scapular-stabilizing muscle, as indicated from the threefold increase in blood flow above that found at rest (22). Such eccentric contractions reduce muscle force-producing capacity (9) while disrupting the extracellular matrix (47), elevating proteolytic enzyme activity (44), and causing profound ultrastructural damage to the myocytes (23). In addition, levels of heat shock proteins HSP27 and HSP70 as well as serum creatine kinase may become elevated (46, 49).
The primary focus of the present investigation was to undertake a novel evaluation of the effect of downhill running on the microcirculation and the balance of Q̇o2/V̇o2 during contractions rather than quantify myocyte damage per se. However, intravital light microscopy did reveal the presence of damaged myocytes at 1 and 3 days after downhill running (Fig. 1). Specifically, in contrast to myocytes in control (nonexercised) spinotrapezius muscles, after downhill running a population of fibers evidenced severe sarcomeric disruptions. These disruptions presented as a loss of register of the sarcomeres, and, in the extreme, sarcomeres across the myocyte thickness displayed a smeared appearance that extended for 10–40 μm or more along the length of the myocyte and averaged 32 and 41% of the fiber area at DH-1 and DH-3, respectively. Such damaged fibers were often, but not always, abutted by nonflowing capillaries (Fig. 1). An important question for future investigations is whether there is a direct relationship between nonflowing capillaries and myocyte damage and, if so, the mechanistic basis for such a relationship. With respect to supporting oxidative function during contractions and supplying nutritive flow to resting muscle, the presence of nonflowing capillaries will compromise performance and possibly recovery from injury. However, it is also possible that by reducing capillary flow adjacent to damaged myocytes, the opportunity for generation and/or delivery of reactive O2 species that may incur further damage is reduced. It will be valuable for future studies to explore the role of reactive O2 species in microvascular dysfunction after eccentric exercise.
In conclusion, the present investigation has demonstrated the presence of impaired capillary hemodynamics and a compromised Q̇o2/V̇o2 matching 1 and 3 days after novel eccentric exercise. Furthermore, these findings suggest that the spinotrapezius muscle presents a viable model for studying microvascular and myocyte injury consequent to eccentric contractions. Apart from being a highly accepted model for microvascular studies, it is pertinent that the spinotrapezius has a fiber composition (12) and oxidative capacity (32) that resemble closely that of the human quadriceps. Thus elucidation of the mechanisms responsible for eccentric muscle damage in this muscle may be applicable to prevention of postexercise damage and debilitation in humans.
This work was supported, in part, by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y. Kano) and by National Institutes of Health Grants AG-19228, HL-50306, and HL-69739.
Present address of Y. Kano: Dept. of Applied Physics and Chemistry, University of Electro-Communications, Chofugaoka, Chofu, Tokyo 182-8585, Japan.
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- Copyright © 2005 the American Physiological Society