After chronic occlusion, collateral-dependent coronary arteries exhibit alterations in both vasomotor reactivity and associated myoplasmic free Ca2+ levels that are prevented by chronic exercise training. We tested the hypotheses that coronary occlusion diminishes Ca2+ uptake by the sarcoplasmic reticulum (SR) and that exercise training would prevent impaired SR Ca2+ uptake. Ameroid constrictors were surgically placed around the proximal left circumflex (LCx) artery of female swine 8 wk before initiating 16-wk sedentary (pen confined) or exercise-training (treadmill run) protocols. Twenty-four weeks after Ameroid placement, smooth muscles cells were enzymatically dissociated from both the LCx and nonoccluded left anterior descending (LAD) arteries of sedentary and exercise-trained pigs, and myoplasmic free Ca2+ was studied using fura 2 microfluorometry. After the SR Ca2+ store was partially depleted with caffeine (5 mM), KCl-induced membrane depolarization produced a significant decrease in the time to half-maximal (t ½) myoplasmic free Ca2+ accumulation in LCx versus LAD cells of sedentary pigs. Furthermore, inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA; 10 μM cyclopiazonic acid) significantly reduced t ½ in cells isolated from the LAD but not from the LCx. Exercise training did not prevent the differences in t ½ myoplasmic free Ca2+ accumulation observed between LCx and LAD cells. Occlusion or exercise training did not alter SERCA protein levels. These results support our hypothesis of impaired SR Ca2+ uptake in coronary smooth muscle cells isolated distal to chronic occlusion. Impaired SR Ca2+ uptake was independent of SERCA protein levels and was not prevented by exercise training.
- vascular smooth muscle
- exercise training
results from different laboratories (7, 11, 17, 20, 24-28) have indicated that collateral-dependent coronary arteries exhibit altered vasomotor reactivity compared with nonoccluded control arteries. Furthermore, experiments evaluating simultaneous changes in contractile tension and myoplasmic free Ca2+ have revealed enhanced constriction and impaired relaxation of coronary arterial rings from the collateral-dependent vasculature associated with increased myoplasmic free Ca2+ concentrations (11,20). Recent studies also indicate that impaired adenosine-induced relaxation and the associated increase in myoplasmic free Ca2+ levels in collateral-dependent coronary arteries are reversed with exercise training (11). The mechanisms responsible for altered Ca2+ regulation in the collateral-dependent coronary vasculature and its reversal with exercise training remain undefined.
Our preliminary experiments demonstrated that after the sarcoplasmic reticulum (SR) had been partially depleted of Ca2+ by exposure to caffeine, KCl-induced membrane depolarization resulted in a greater rate of myoplasmic free Ca2+ accumulation in smooth muscle cells isolated from collateral-dependent left circumflex (LCx) versus nonoccluded left anterior descending (LAD) coronary arteries. In contrast, in the absence of prior SR depletion, the rate of myoplasmic free Ca2+ accumulation was not different between smooth muscle cells of collateral-dependent LCx and nonoccluded LAD arteries. On the basis of these findings, we hypothesized that chronic coronary occlusion impairs SR Ca2+ uptake, leading in turn to altered myoplasmic free Ca2+ regulation in the smooth muscle of collateral-dependent arteries. We also hypothesized that exercise training would correct the impaired SR Ca2+ uptake proposed in collateral-dependent vasculature. The present studies were designed to evaluate potential Ca2+ regulatory mechanisms that contribute to altered vasomotor reactivity in coronary arteries distal to chronic occlusion and the effects of exercise training on these mechanisms.
MATERIALS AND METHODS
Animals and surgical procedures.
All animal protocols were in accordance with the “Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training” and approved by the University of Missouri Animal Care and Use Committee. Thirty-two adult female Yucatan miniature swine (Charles River; Wilmington, MA) were surgically instrumented with Ameroid constrictors around the proximal LCx artery as described previously (7, 11). Animals were preanesthetized with glycopyrrolate (0.004 mg/kg im) and midazolam (0.5 mg/kg im). Anesthesia was induced with ketamine (20 mg/kg im) and maintained with 3% isoflurane-97% O2 throughout the aseptic surgery. Animals recovered from the surgery for 8 wk before the experimental protocols were initiated. Twelve additional control animals did not undergo surgery.
Ameroid-occluded animals were randomly assigned to either a sedentary or an exercise-training group. All control (nonoccluded) pigs used for this study remained sedentary. Exercise-trained pigs underwent a progressive treadmill exercise-training program (5 days/wk for 16 wk) as described previously (7, 11). Effectiveness of the exercise-training program was determined by comparing the heart weight-to-body weight ratio and skeletal muscle citrate synthase activity as previously described (7, 11).
Preparation of coronary arteries.
After the 16-wk exercise-training protocol or sedentary confinement was completed, animals were anesthetized with ketamine (35 mg/kg im), rompun (2.25 mg/kg im), and pentothal sodium (10 mg/kg iv), followed by administration of heparin (1,000 U/kg iv). Animals were euthanized by removal of the heart, which was immediately placed in cold Krebs bicarbonate buffer (4°C) and weighed. With the aid of a dissection microscope, segments of the LCx and LAD arteries from both occluded and control pigs were trimmed of fat and connective tissue, and vessel diameter was measured with a calibrated Filar micrometer eyepiece (Hitschfel Instruments; St. Louis, MO). Visual inspection of the Ameroid occluder during dissection of the LCx artery indicated 100% occlusion in all animals that had undergone surgery for this study; the LCx was thus termed a “collateral-dependent” coronary artery.
Myoplasmic free Ca2+ measurement.
Smooth muscle cells from segments of the LCx and LAD arteries were enzymatically dissociated and loaded with the fluorescent Ca2+ indicator fura 2-AM (2.5 μM) as previously described (29). Fura 2-loaded cells were placed in a superfusion chamber and observed using an epifluorescence microscopy system (Nikon; Garden City, NY). Excitation light from a 300-W xenon arc lamp, passed via a liquid light guide, was directed through alternating 340- and 380-nm band-pass filters. Fluorescence emission (510 nm) from user-selected smooth muscle cells was synchronized with the appropriate excitation wavelength and reflected to an integrating charge-coupled device monochrome video camera (Cohu; San Diego, CA) with a dichroic mirror. Fluorescent images were acquired using InCa dual-wavelength calcium imaging software 2.1 (Intracellular Imaging; Cincinnati, OH). This microfluorometry system permits simultaneous evaluation of fura 2 fluorescence from multiple smooth muscle cells throughout an experimental protocol. Background fluorescence was determined before the experiment start for on-line subtraction during data collection. After the background fluorescence was subtracted, images obtained at 340 and 380 nm were ratioed on a pixel-by-pixel basis. Cells were continually superfused (∼2.7 ml/min) under gravity flow. All experiments were conducted at room temperature (22–25°C), and fluorescence data were sampled every 2 s.
Unless otherwise specified, cells were superfused with physiological saline solution (PSS) containing (in mM) 2 CaCl2, 143 NaCl, 1 MgCl2, 5 KCl, 10 HEPES, and 10 glucose; pH 7.4. Cells were depolarized with PSS in which 80 mM KCl replaced equimolar amounts of NaCl. For Ba2+ protocols, Ca2+ was replaced with equimolar amounts of Ba2+, and low Na+ (5 mM Na+) was used to inhibit Ba2+ extrusion via Na/Ca exchange. Caffeine (5 mM), cyclopiazonic acid (CPA; 10 μM), and thapsigargin (1 μM) additions were made directly to PSS.
Sarco(endo)plasmic reticulum Ca2+-ATPase immunoblots.
After the LCx and LAD coronary arteries were isolated from occluded hearts, arterial rings (inner diameter 0.8–1.2 mm; length ∼0.5 mm) were cut, quick-frozen, and stored at −80°C for later immunoblot analysis of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2b. Identification of SERCA2b was performed using a site-directed anti-peptide monoclonal antibody (Affinity BioReagents; Golden, CO) specifically against the SERCA2b isoform of the protein. Previous studies have documented that the SERCA of smooth muscle is transcribed exclusively from the SERCA2 isoform (8, 15), with SERCA2b accounting for ∼80% of the total SERCA2 mRNA and the remainder encoding SERCA2a (5). Individual arterial rings were homogenized in 60 μl of SDS-polyacrylamide gel loading buffer containing 62.5 mM Tris · HCl (pH 6.8), 6 M urea, 2% SDS, 150 mM dithiothreitol, and 0.001% bromophenol blue. Samples were centrifuged at 14,000 g for 10 min at room temperature to remove insoluble material. Total protein content in the extract was assessed using the Bradford assay (Bio-Rad; Hercules, CA). Equal protein amounts (15 μg) were loaded onto each lane for SDS-PAGE (4–21% polyacrylamide precast gels; Bio-Rad). Direct electrophoretic transfer of proteins from the gel to a polyvinylidene difluoride membrane (Amersham; Arlington Heights, IL) was completed using a transfer buffer containing 0.7 M glycine and 25 mM Tris base. The membrane was blocked for 1 h in Tris-buffered saline (TBS; 50 mM Tris · HCl and 0.2 M NaCl; pH 7.4), 0.1% Tween, and 5% nonfat milk to reduce nonspecific binding and then incubated overnight in blocking buffer plus the SERCA2b antibody. After this incubation, the membrane was washed (TBS and 0.1% Tween) and incubated with a secondary anti-mouse antibody conjugated to horseradish peroxidase (Sigma; St. Louis, MO) for 1 h. The membrane was washed again, and the signal was developed using enhanced chemiluminescence (ECL; Amersham). Scanning densitometry (NIH Image 1.55) was used to quantify signal density from luminograms.
Heart weight-to-body weight ratio and citrate synthase activity in sedentary and exercise-trained animals were evaluated using unpaired Student's t-tests. Data for all fura 2 experiments were analyzed as a randomized complete block design in which the treatments were arranged in a 2 × 2 factorial. Mean differences were ascertained using Fisher's least significant difference test. Comparisons of immunoblot band density were accomplished using paired Student's t-tests in which responses from sedentary and exercise-trained LAD coronary arteries served as controls for the respective LCx arteries. Analyses for all fura 2 experiments and immunoblot band density were performed on a per vessel basis, where each animal was represented by a single LAD and LCx arterial segment. For all analyses, a P value ≤0.05 was considered significant. Data are presented as means ± SE, and then values presented reflect the number of animals and the number of cells.
The effectiveness of the 16-wk exercise-training program was demonstrated by significant increases in skeletal muscle oxidative enzyme activity and increased heart weight-to-body weight ratios in exercise-trained pigs (Table 1).
Baseline myoplasmic free Ca2+concentrations.
Baseline fura 2 ratios, indicative of myoplasmic free Ca2+concentrations, were not significantly different between smooth muscle cells isolated from the occluded LCx and nonoccluded LAD arteries of sedentary and exercise-trained pigs (sedentary LAD 0.92 ± 0.07,n = 14 vessels; sedentary LCx 0.91 ± 0.06,n = 16 vessels; exercise LAD 0.89 ± 0.07,n = 15 vessels; exercise LCx 0.84 ± 0.07,n = 15 vessels).
Fura 2 measurements of myoplasmic free Ca2+ accumulation.
Figure 1 A illustrates our protocol for evaluation of myoplasmic free Ca2+accumulation in response to membrane depolarization with 80 mM KCl and a representative recording from a single sedentary control (LAD) cell. As shown in Fig. 1 B, during membrane depolarization, the time to half-maximal (t ½) myoplasmic free Ca2+ accumulation was significantly prolonged in the presence (80K2) versus the absence (80K1) of partially depleted caffeine-sensitive SR Ca2+ stores. Furthermore, the initial maximal rate (slope, 10 s) of free Ca2+accumulation in response to membrane depolarization was significantly greater during 80K1 versus 80K2 for both the LAD and LCx. These findings indicate that partial depletion of the SR Ca2+store with caffeine increases the buffering capacity of the SR adequately to significantly decrease the rate of myoplasmic free Ca2+ accumulation in response to membrane depolarization (29).
The t ½ myoplasmic free Ca2+accumulation during 80K1 was similar in smooth muscle cells isolated from LCx and LAD arteries (Fig. 1 B). In contrast,t ½ myoplasmic free Ca2+ accumulation in response to membrane depolarization during 80K2 was reduced 25% in smooth muscle cells isolated from the collateral-dependent LCx versus nonoccluded LAD artery (59 ± 6 vs. 79 ± 5 s; Fig.1 B), with no significant difference in maximum myoplasmic free Ca2+ levels (80K2; Fig. 1 A) in cells from LCx versus LAD (Δfura 2 ratio = 0.51 ± 0.07 vs. 0.42 ± 0.06, respectively). The reduced t ½myoplasmic free Ca2+ accumulation in LCx versus LAD smooth muscle cells only after partial depletion of caffeine-sensitive SR Ca2+ stores suggests impaired SR Ca2+ uptake in smooth muscle cells from collateral-dependent vasculature of sedentary animals. However, after partial depletion of caffeine-sensitive SR Ca2+ stores (CAF1), subsequent membrane depolarization (80K2) replenished SR Ca2+ stores similarly (CAF2) in LCx and LAD smooth muscle cells (CAF2/CAF1 = 95 ± 4 and 93 ± 4%), suggesting long-term (7 min) membrane depolarization allowed for complete refilling of SR Ca2+ stores. Importantly, caffeine-sensitive Ca2+ stores (CAF1; Fig. 1 A) were similar between cells from the LCx and LAD (Δfura 2 ratio = 0.96 ± 0.14 vs. 1.00 ± 0.20, respectively).
Inhibition of SR Ca2+ sequestration.
In additional experiments, we used the SERCA inhibitor CPA (10 μM) to further investigate SR Ca2+ uptake in smooth muscle cells from the collateral-dependent vasculature of sedentary animals. The protocol for these experiments and a representative recording from a single sedentary control (LAD) cell are illustrated in Fig.2 A. As presented in Fig.2 B, in the absence of CPA, t ½myoplasmic free Ca2+ accumulation in response to high KCl-induced membrane depolarization (80K2) was significantly reduced in smooth muscle cells from the collateral-dependent LCx when compared with the nonoccluded LAD artery. The addition of CPA significantly reduced t ½ myoplasmic free Ca2+ accumulation in the nonoccluded LAD but had little effect on t ½ myoplasmic free Ca2+ accumulation in the collateral-dependent LCx (59 ± 7 vs. 54 ± 6 s). Thapsigargin (1 μM) also decreased t ½ myoplasmic free Ca2+ from control values in nonoccluded LAD but not collateral-dependent LCx smooth muscle cells (58 ± 3 vs. 62 ± 4 s, respectively). Thus both SERCA inhibitors had minimal effect on the rate of myoplasmic free Ca2+ accumulation in LCx smooth muscle cells, indicating reduced SR Ca2+ uptake in smooth muscle distal to chronic occlusion. Additional experiments in control nonoccluded animals conducted in our laboratory (Fig.2 C) indicate that normal pigs do not demonstrate regional differences (LCx vs. LAD) in t ½free Ca2+ accumulation in response to high KCl-induced membrane depolarization in the absence or presence of SERCA inhibition.
Exercise training and SR Ca2+sequestration.
We also evaluated SR Ca2+ uptake in smooth muscle cells isolated from collateral-dependent LCx and nonoccluded LAD coronary arteries of exercise-trained animals. Similar to our findings in sedentary animals, in the presence of partially depleted caffeine-sensitive SR stores, t ½ myoplasmic free Ca2+ accumulation in response to high KCl-induced membrane depolarization was significantly reduced in smooth muscle cells from the collateral-dependent LCx compared with the nonoccluded LAD artery (Fig. 2 D). Furthermore, the addition of CPA significantly reduced t ½ myoplasmic free Ca2+ accumulation in the nonoccluded LAD but had little effect on t ½ myoplasmic free Ca2+accumulation in the collateral-dependent LCx (Fig. 2 D). These data are contrary to our hypothesis and suggest exercise training does not correct impaired SR Ca2+ uptake in collateral-dependent coronary smooth muscle cells.
Fura 2 measurements of Ba2+ influx.
In additional experiments, we assessed divalent cation influx through voltage-gated Ca2+ channels using 2 mM Ba2+ in the presence of low extracellular Na+ (5 mM) to determine whether the enhanced rate of myoplasmic free Ca2+accumulation in smooth muscle cells from the LCx artery was partially dependent on enhanced Ca2+ influx at the plasma membrane. Our protocol for these experiments and a representative recording from a single sedentary control (LAD) cell are presented in Fig.3 A. As shown in Fig.3 B, neither maximal rate nor net Ba2+accumulation was significantly enhanced in smooth muscle cells isolated from the collateral-dependent LCx of sedentary animals. These findings suggest that alterations in Ca2+ influx do not contribute to the reduced t ½ myoplasmic free Ca2+ accumulation observed in response to membrane depolarization in smooth muscle cells isolated from the LCx artery.
We also evaluated Ba2+ influx through voltage-gated Ca2+ channels in collateral-dependent LCx and nonoccluded LAD coronary arteries of exercise-trained animals (Fig. 3 C). Similar to our observations in cells from sedentary pigs (Fig.3 B), neither maximal rate nor net Ba2+accumulation was different between smooth muscle cells isolated from the LCx and LAD of exercise-trained animals (Fig. 3 C). These findings indicate that the reduction in t ½ free Ca2+ accumulation in cells from the occluded LCx artery, which persists after exercise training, is not attributable to enhanced Ca2+ influx through voltage-gated Ca2+ channels of the LCx artery.
Myoplasmic free Ca2+ removal during repolarization.
Myoplasmic free Ca2+ removal rate during recovery from 80 mM KCl-induced membrane depolarization was assessed to evaluate the function of Ca2+-removal mechanisms in smooth muscle cells isolated from collateral-dependent vasculature of sedentary and exercise-trained pigs. We further evaluated free Ca2+ removal both in the absence (Fig. 1 A;minutes 18–20) and presence of CPA (Fig. 2 A;minutes 18–20) to determine the specific contribution of SR sequestration in Ca2+ removal after depolarization.t ½ myoplasmic free Ca2+ removal during repolarization was not significantly different in the absence or presence of CPA in both sedentary and exercise-trained animals (Fig.4, A and B, respectively). These data suggest that impaired SR Ca2+uptake in LCx smooth muscle cells, evident during depolarization, was not detected during repolarization most likely because depolarization-induced refilling of the SR Ca2+ store limited subsequent Ca2+ sequestration during repolarization. Furthermore, t ½ myoplasmic free Ca2+ removal during repolarization was not significantly different between smooth muscle cells isolated from the collateral-dependent LCx and nonoccluded LAD of both sedentary and exercise-trained pigs (Fig. 4, A and B, respectively). Taken together, these findings indicate that the remaining Ca2+-removal mechanisms, primarily sarcolemmal Ca2+-ATPase and Na/Ca exchange, function similarly in smooth muscle cells isolated from the collateral-dependent LCx and nonoccluded LAD of both sedentary and exercise-trained animals.
We also assessed SERCA2b protein content in arterial rings from collateral-dependent LCx and nonoccluded LAD coronary arteries of sedentary (n = 7) and exercise-trained (n = 7) animals to determine whether decreases in protein content are responsible for impaired Ca2+ uptake by the SR. Luminograms of SERCA2b protein content (Fig.5) were quantified using densitometric analysis; band density of the LCx artery was expressed relative to the respective control LAD from the same heart. Our experiments indicated that the LCx artery demonstrated 99 ± 9 and 99 ± 8% of LAD SERCA2b protein content in sedentary and exercise-trained animals, respectively, suggesting decreased protein content did not contribute to impaired SR Ca2+ uptake.
The current study demonstrates that SR Ca2+ uptake is impaired in intact smooth muscle cells isolated from coronary arteries distal to chronic occlusion. These data also suggest that exercise training does not correct the impaired SR Ca2+sequestration in these smooth muscle cells. Furthermore, alterations in SERCA2b protein content were not responsible for impaired SR Ca2+ sequestration in smooth muscle cells isolated from the LCx artery of either sedentary or exercise-trained animals.
Collateral-dependent coronary arteries exhibit altered vasomotor reactivity in vitro that can be partially attributed to alterations in myoplasmic Ca2+ handling (11, 20). Impaired adenosine-induced relaxation in these collateral-dependent arteries is associated with an impaired adenosine-stimulated reduction in myoplasmic free Ca2+, which is corrected with exercise training (11). Increases in cAMP-dependent protein kinase, a product of adenosine stimulation, have been reported to affect multiple Ca2+ regulatory mechanisms to reduce intracellular Ca2+ levels, including SR Ca2+ uptake from the bulk myoplasm (19). We postulated that the impaired adenosine-mediated reduction in myoplasmic Ca2+ in the collateral-dependent artery (11) might be attributable to impaired SR Ca2+ sequestration. Although our present data suggest that SR Ca2+ uptake is impaired in the smooth muscle distal to chronic occlusion, the impaired SR Ca2+sequestration was not corrected with exercise training. These results indicate that exercise training corrects impaired adenosine-stimulated reductions in myoplasmic free Ca2+ in collateral-dependent arteries (11) by other mechanisms. Figures 3 Cand 4 B present results demonstrating that Ca2+entry via voltage-gated Ca2+ channels and Ca2+removal via Ca2+-ATPase and Na/Ca exchange are also not altered in cells from collateral-dependent arteries of exercise-trained pigs. Thus the available evidence indicates that the previously reported (11) training-induced correction of adenosine-mediated reduction in intracellular Ca2+ levels results from adaptation of other cellular mechanisms involved in Ca2+ regulation that are specific to adenosine-mediated decreases in Ca2+. For example, adenosine-induced relaxation is partly mediated by K+ channel activation (16), and increased K+ channel dependence of arterial tone has been demonstrated in coronary arteries isolated from exercise-trained versus sedentary pigs not subjected to coronary occlusion (2). Therefore, adenosine-mediated decreases in intracellular Ca2+ levels, reported in collateral-dependent arteries from trained pigs, may be the result of increased K+ channel activation.
The finding that the t ½ myoplasmic free Ca2+ accumulation was more rapid in the absence (80K1) versus the presence (80K2) of depleted caffeine-sensitive SR Ca2+ stores (Fig. 1 B) confirms previous results in normal coronary arteries (29). Caffeine depletes the SR adequately to potentiate SERCA activity (5, 29) and, therefore, examine the role of Ca2+ uptake by SERCA in coronary smooth muscle cells. Furthermore, numerous studies have reported that the superficial SR sequesters a portion of the Ca2+ that enters through the plasmalemma of smooth muscle cells during high KCl-induced depolarization (14, 29, 31,32). Accumulation of incoming Ca2+ by the SR near the site of Ca2+ entry functions to limit Ca2+entry into the bulk myoplasm and subsequent activation of smooth muscle contractile proteins (14, 31, 32). Our data demonstrate that only after SR depletion (80K2), t ½myoplasmic free Ca2+ accumulation in response to membrane depolarization was significantly reduced in smooth muscle cells isolated from the collateral-dependent LCx versus nonoccluded LAD (Fig.1 B). These findings suggest either impaired SERCA function or disruption of the structural/functional relationship between voltage-gated Ca2+ channels and SERCA in smooth muscle cells isolated from collateral-dependent vasculature. Furthermore, the prolonged t ½ myoplasmic free Ca2+accumulation (Fig. 2, C vs. B) in the LAD of control nonoccluded (∼110 s) versus occluded (∼80 s) animals suggests that the presence of chronic occlusion may produce global alterations in Ca2+ handling in both collateral-dependent and nonoccluded vasculature of chronically occluded hearts.
Hypothetically, the enhanced rate of depolarization-induced free Ca2+ accumulation observed in the presence of SR depletion may also reflect changes in Ca2+ influx at the sarcolemma or alterations in Ca2+ extrusion via sarcolemmal Ca2+-ATPase or Na/Ca exchange. Evaluation of Ba2+ accumulation in the presence of low extracellular Na+ has been used previously as a measure of unidirectional divalent cation influx in smooth muscle cells (13). Ba2+ is transported via voltage-gated Ca2+channels (1) and Na/Ca exchanger (3) but is a poor substrate for ATP-dependent Ca2+ pumps (23). Ba2+ produces a shift in the fura 2 excitation wavelength spectrum, with increasing concentrations similar to that observed with Ca2+, although fura 2 has a higher affinity for Ca2+ versus Ba2+ (12,23). However, Ba2+ is not sequestered by intracellular organelles (3, 23) and, therefore, allows us to evaluate influx at the plasma membrane under conditions in which corresponding Ca2+ fluxes would be difficult to interpret due to multiple pathways for Ca2+ removal and sequestration. Depolarization-induced Ba2+ influx in the presence of low extracellular Na+ revealed that divalent cation influx at the sarcolemma is not different between smooth muscle cells isolated from nonoccluded LAD and collateral-dependent LCx arteries of both sedentary and exercise-trained pigs (Fig. 3,B and C, respectively).
Furthermore, one might speculate that partial depletion of the SR with caffeine may stimulate Ca2+ entry through store-operated channels, refilling the SR and thereby reducingt ½ myoplasmic free Ca2+accumulation. However, previous experiments indicate that SR store depletion does not activate Ca2+ influx in our porcine coronary smooth muscle cell preparation (4). Furthermore, data from the present study demonstrate that myoplasmic free Ca2+ concentration returned to baseline levels (Fig.1 A; minute 11) after partial depletion of the SR in cells isolated from all groups (sedentary LAD 0.82 ± 0.10,n = 14 vessels; sedentary LCx 0.84 ± 0.08,n = 16 vessels; exercise LAD 0.91 ± 0.08,n = 15 vessels; exercise LCx 0.88 ± 0.08,n = 15 vessels), indicating neither exercise training nor coronary occlusion enhances Ca2+ influx through store-operated channels in these smooth muscle cells.
The rate of reduction in myoplasmic free Ca2+ during repolarization, measured after KCl-induced membrane depolarization, was not different between LAD and LCx smooth muscle cells of sedentary or exercise-trained pigs. These observations indicate that the Ca2+ extrusion mechanisms, sarcolemmal Ca2+-ATPase and Na/Ca exchange, were not discernibly different between cells of these arteries. These findings further support our hypothesis that impaired SR Ca2+ uptake contributes to altered myoplasmic free Ca2+ regulation in smooth muscle cells from collateral-dependent vasculature and potentially subsequent alterations in vasomotor reactivity distal to chronic occlusion.
Our findings that t ½ Ca2+ removal (time for half-recovery of free Ca2+ to minimum levels) and net Ca2+ removal after CAF1 exposure (minutes 9–11) were not significantly different between smooth muscle cells of the LAD and LCx do not appear to support the hypothesis of reduced SR Ca2+ uptake. Generally, after washout of caffeine, recovery of myoplasmic Ca2+ is largely attributed to Ca2+ uptake by the SR (6). However, one might speculate that SR sequestration of Ca2+ entering the cell (e.g., membrane depolarization) may be a different cellular process than SR sequestration of myoplasmic free Ca2+elevated by intracellular release (e.g., caffeine). Alternatively, disruption of the structural/functional relationship between voltage-gated Ca2+ channels and SERCA in smooth muscle cells isolated from collateral-dependent vasculature may explain the discrepancy between SR sequestration of Ca2+ entering the cell versus myoplasmic free Ca2+ elevated by intracellular release. We also report that after partial depletion of caffeine-sensitive SR Ca2+ stores (CAF1), subsequent membrane depolarization (80K2) replenished SR Ca2+ stores (CAF2) similarly in LCx and LAD smooth muscle cells. Although these data do not support our hypothesis of impaired SR Ca2+uptake, the long duration of membrane depolarization (7 min) may have been adequate for complete refilling of the SR stores even in the presence of a reduced rate of Ca2+ sequestration by the SR of LCx smooth muscle cells.
Thus several sets of data from the present study indicate decreased SR Ca2+ uptake in vascular smooth muscle of collateral-dependent coronary arteries. Our results indicate that this decreased sequestration is present with normal amounts of coronary smooth muscle SERCA protein. Our results do not reveal the cause of decreased SR Ca2+ uptake; however, there is evidence in the literature that hearts subjected to global ischemia demonstrate impaired SR Ca2+ uptake and reduced SERCA mRNA and protein levels in cardiac muscle cells (30), changes that may be associated with ischemia-mediated production of oxygen-derived free radicals (18, 33). In turn, the production of reactive oxygen species has been documented to inactivate SERCA and increase SR membrane permeability to Ca2+ in porcine coronary smooth muscle (9, 10). These studies (9,10) demonstrate that even low concentrations of reactive oxygen species (e.g., 3 × 10−6 M hydrogen peroxide) can reduce SR Ca2+ uptake by ∼20%, a reduction similar to that observed in our study. Furthermore, studies (21, 22) using a porcine model of chronic occlusion have demonstrated that exercise-induced myocardial ischemia persists in these animals long after coronary collateral development is adequate to restore blood flow and regional myocardial function to normal resting levels in the collateral-dependent region. Thus available evidence supports the notion that SERCA activity in smooth muscle cells isolated from collateral-dependent vasculature in our model of chronic coronary occlusion may be altered by reactive oxygen species generated by ischemic conditions. Reactive oxygen species-induced injury to SERCA of the collateral-dependent LCx artery may impair the activity of this Ca2+-transport mechanism.
We conclude that these data provide strong evidence for impairment of SR Ca2+ uptake at the level of the single smooth muscle cell in collateral-dependent coronary arteries. Impaired SERCA activity or disruption of the structural/functional relationship between voltage-gated Ca2+ channels and SERCA in coronary smooth muscle of collateral-dependent vasculature could explain a reduced ability of the SR to effectively sequester incoming Ca2+, thus leading to increased availability of Ca2+ to contractile proteins in the bulk myoplasm. The resulting enhanced contractile responsiveness of vasculature distal to chronic occlusion would further compromise blood flow to collateral-dependent myocardium.
The authors gratefully acknowledge the technical and surgical expertise of Millie Mattox. The computer programming expertise of Dr. Nancy Dietz contributed considerably to data analyses for these experiments and is greatly appreciated.
This study was supported by National Heart, Lung, and Blood Institute Research Grants P01-HL-52490 and R01-HL-64931. C. L. Heaps was supported by a predoctoral fellowship from the American Heart Association, Missouri Affiliate.
Address for reprint requests and other correspondence: C. L. Heaps, E102 Veterinary Biomedical Sciences, Univ. of Missouri, Columbia, MO 65211 (E-mail:).
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- Copyright © 2001 the American Physiological Society