Gender influences coronary L-type Ca2+ current and adaptation to exercise training in miniature swine
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《应用生理学杂志》
Department of Veterinary Biomedical Sciences, College of Veterinary Medicine and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211
ABSTRACT
Endurance exercise training increases smooth muscle L-type Ca2+ current density in both resistance and proximal coronary arteries of female miniature swine. The purpose of the present study was to determine 1) whether gender differences exist in coronary smooth muscle (CSM) L-type Ca2+ current density and 2) whether endurance training in males would demonstrate a similar adaptive response as females. Proximal, conduit (~1.0 mm), and resistance [~200 μm (internal diameter)] coronary arteries were obtained from sedentary and treadmill-trained swine of both sexes. CSM were isolated by enzymatic digestion (collagenase plus elastase), and voltage-gated Ca2+-channel current (ICa) was determined by using whole cell voltage clamp during superfusion with 75 mM tetraethylammonium chloride and 10 mM BaCl2. Current-voltage relationships were obtained at test potentials from 60 to 70 mV from a holding potential of 80 mV, and ICa was normalized to cell capacitance (pA/pF). Endurance treadmill training resulted in similar increases in heart weight-to-body weight ratio, endurance time, and skeletal muscle citrate synthase activity in male and female swine. ICa density was significantly greater in males compared with females in both conduit (7.57 ± 0.58 vs. 4.14 ± 0.47 pA/pF) and resistance arteries (11.25 ± 0.74 vs. 6.49 ± 0.87 pA/pF, respectively). In addition, voltage-dependent activation of ICa in resistance arteries was shifted to more negative membrane potentials in males. Exercise training significantly increased ICa density in both conduit and resistance arteries in females (7.01 ± 0.47 and 9.73 ± 1.13 pA/pF, respectively) but had no effect in males (8.61 ± 0.50 and 12.04 ± 1.07 pA/pF, respectively). Thus gender plays a significant role in determining both the magnitude and voltage dependence of ICa in CSM and the adaptive response of ICa to endurance training.
keywords:electrophysiology; vascular smooth muscle; microcirculation; voltage-gated calcium channels
INTRODUCTION
GENDER EXERTS SIGNIFICANT INFLUENCE on coronary vascular physiology and pathophysiology. For example, whereas coronary heart disease (CHD) is a leading cause of mortality in both men and women, men show a greater prevalence of CHD compared with premenopausal women (1). Similarly, the incidence of hypertension is greater in men and postmenopausal women compared with premenopausal women (20). Mechanisms underlying gender differences in the development of complex cardiovascular disease are likely multifaceted. However, gender differences in several candidate mechanisms impacting cardiovascular disease have been reported, including endothelial nitric oxide synthase (NOS) content, NO production (25), smooth muscle proliferation (11), and smooth muscle responsiveness to agonists (30), e.g., endothelin (29). Recently, functional studies have provided indirect evidence that gender differences in dihydropyridine-sensitive, voltage-gated Ca2+ channels (VGCC) may also contribute to differences in vascular function between males and females (12). Crews et al. (12) found that depolarization-induced contraction and Ca2+ influx is greater in thoracic aorta of male rats compared with female rats, suggesting a gender difference in plasmalemmal VGCC activity. VGCC activity plays a central role in regulation of vascular resistance and, therefore, total blood flow and distribution (32). In addition, VGCCs have been proposed to contribute to the incidence and severity of both acute and long-term cardiovascular events, such as vasospasm and CHD (18, 28). If present, gender differences in VGCC activity in coronary smooth muscle (CSM) could play a significant role in determining gender-related differences in vascular function in both health and disease.
Endurance exercise training also influences ion-channel activity and Ca2+ regulation in CSM (6, 8, 9, 14, 17). In female miniature swine, L-type Ca2+-channel current (ICa) was increased in both conduit and resistance arterial smooth muscle after treadmill training (8, 14). Given the potential influence of gender on vascular smooth muscle Ca2+ regulation, the purpose of the present study was to determine 1) whether gender differences exist in CSM L-type Ca2+-current density and 2) whether males demonstrate a similar adaptive response to exercise training as females.
MATERIALS AND METHODS
Animals. Sexually mature, adult male and female miniature swine weighing 25-40 kg were obtained from the breeder (Charles River) and housed in pens at the College of Veterinary Medicine. All pigs included in this study were familiarized with treadmill exercise over a 1- to 2-wk period. Treadmill performance tests were administered to each animal. Pigs of each sex were then randomly divided into two groups. One group (Ex; n = 16, 6 females, 10 males) underwent a progressive treadmill training program used previously in our laboratory (7, 8, 14). The second group of pigs was restricted to their pens (6 × 12 ft) for 16-20 wk and served as sedentary controls (Sed; n = 15, 7 females, 8 males). Animal protocols were approved by the University of Missouri Animal Care and Use Committee in accordance with the "Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training."
Training procedures and treadmill performance tests. Ex pigs underwent a 16- to 20-wk treadmill endurance program followed by treadmill performance tests as described previously (8, 9, 14, 24). During the final 4-8 wk of training, a typical training session consisted of the following 85-min workout: 1) 5-min warm-up run at 2.5 miles/h (mph), 2) 15-min sprint at speeds of 5-8 mph, 3) 60-min endurance run at 4-5 mph, and 4) 5-min cool-down run at 2 mph. Treadmill performance tests were administered before and at the completion of the training (Ex) or pen confinement (Sed).
Skeletal muscle oxidative enzyme activity. At the time of death, muscle samples were taken from the medial and lateral heads of the triceps brachii and deltoid, frozen in liquid nitrogen, and stored until processed. Citrate synthase activity was measured spectrophotometrically from whole muscle homogenates (34).
Preparation of coronary arteries. Pigs 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). Hearts were removed and placed in iced (4°C) Krebs bicarbonate solution during vessel isolation. Main right conduit arteries (1.0-mm ID) were dissected free from the heart beginning ~2 cm distal to the ostia and proceeding to the origin of the posterior descending artery and cleaned of connective and adipose tissue at 4°C in physiological saline solution (PSS) containing (in mM) 2 CaCl2, 138 NaCl, 1 MgCl2, 5 KCl, 10 HEPES, and 10 glucose, pH 7.4. Similarly, epicardial resistance vessels 175-225 μm ID were obtained from the apex region of the anterior left ventricular free wall at 4°C in PSS.
Cell dispersion. All experiments were performed on freshly dispersed cells by using methods modified from those described previously (35-37, 39). Arteries were incubated in enzyme solution consisting of low Ca2+ (0.5 mM) physiological solution plus 294 U/ml collagenase (CLS II, Worthington Biochemical, Lakewood, NJ), 6.5 U/ml elastase (Worthington), 2 mg/ml bovine serum albumin (fraction V, Sigma Chemical, St. Louis, MO), 1 mg/ml soybean trypsin inhibitor (type I-S, Sigma Chemical), and 0.4 mg/ml DNase I (type IV, Sigma Chemical) for 45-60 min at 37°C. The enzyme solution was then immediately replaced with enzyme-free low-Ca2+ solution, and isolated single cells were obtained with gentle trituration. Cell suspensions were stored in low-Ca2+ (0.5 mM) buffer at 4°C until use (0-6 h).
Whole cell voltage clamp. Whole cell currents were determined by using standard voltage-clamp techniques as used routinely (36-38). Cells were initially superfused with PSS during gigaohm seal formation. After whole cell configuration was obtained, the superfusate was switched to PSS with the following substitutions: tetraethylammonium chloride (TEACl; 75 mM) substituted isomolar for NaCl and 10 mM Ba2+ for Ca2+ as the charge carrier. The pipette solution contained (in mM) 120 CsCl, 10 TEACl, 1 MgCl2, 20 HEPES, 5 Na2ATP, 0.5 Tris-GTP, and 10 EGTA, pH 7.1. TEACl was included in both the superfusate and pipette solution to eliminate competing outward K+ current. Ionic currents were amplified by an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). Whole cell currents were low-pass filtered with a cutoff frequency of 1,000 Hz, digitized at 2.5 kHz, and stored on computer. Current densities (pA/pF) were obtained for each cell by normalization of whole cell current to cell capacitance to account for differences in cell membrane surface area. Capacity currents were measured for each cell during 10-ms pulses from a holding potential of 80 mV to a test potential of 75 mV. Capacity currents were filtered at a low-pass cutoff frequency of 5 kHz. Leak subtraction was not performed. Data acquisition and analysis were accomplished by using pCLAMP 7.0 software (Axon Instruments). Current-voltage (I-V) relationships were determined by measuring peak inward current during a 500-ms step depolarization to membrane potentials from 60 to 70 mV in 10-mV increments (holding potential = 80 mV). Voltage-dependent activation was determined as g/gmax = peak IBa/[gBa (V Erev)], where gBa is the maximal conductance obtained from the linear regression of the positive limb of the I-V relationship through the apparent reversal potential (Erev), V is the step potential, and IBa is the peak inward current at the corresponding step potential. Activation data were fit to a conventional Boltzmann distribution equation, 1/{1+exp[(V0.5 V)/k]}, where V0.5 is the membrane potential producing half-maximal activation and k is the slope factor. For voltage dependent inactivation, peak inward current during a 500-ms step depolarization to 20 mV was determined after a 4-s prepulse at membrane potentials from 80 to +50 mV in 10-mV increments. Relative peak current (I/Imax) data were fit to a conventional Boltzmann distribution equation, 1/{1 + exp[(V0.5 V)/k]}. All experiments were conducted at room temperature (22-25°C). Cells were continually superfused under gravity flow.
Statistics. Data are expressed as means ± SE with each cell (voltage-clamp) or animal (training efficacy variables) counted as an observation (n). For voltage-clamp experiments, three to five cells were examined for each vessel from each animal (6-10 animals per group). Repeated-measures ANOVA was used for comparisons of I-V relationships, utilizing a Greenhouse-Geisser adjustment for within-subjects factors and Sidak adjustment for comparison of between-subjects effects (27). Multivariate ANOVA was used for comparison of single variables among groups. A P value <0.05 was set as the criterion for significance in all comparisons.
RESULTS
Efficacy of exercise training. Male swine tended to have greater body and heart weights compared with females; however, heart weight-to-body weight ratio, treadmill endurance time, and skeletal muscle citrate synthase activity levels were similar in sedentary male and female pigs (Table 1). Consistent with previous reports using the treadmill-trained miniature swine model (24), Ex animals of both sexes demonstrated overall similar adaptations to endurance training, including an increase in citrate synthase activity in the medial and lateral heads of the triceps brachii, increased treadmill endurance time, and increased heart weight-to-body weight ratio (Table 1).
Gender and cell size. In both conduit and resistance arteries, CSM cells from male animals were significantly larger than cells from female animals, as indicated by a greater cell membrane surface area (i.e., cell capacitance, pF; Table 2). Cell size was unaffected by exercise training with the exception of a smaller mean cell size in resistance arterioles of Ex compared with Sed males. Whether the smaller cell size in resistance arteries of Ex males is an effect of training or due to unintentional bias is unclear; however, the latter is unlikely as the experimenter was blinded to the treatment of the animal. Furthermore, a similar trend was noted for Ex females in this and a previous study (8). Therefore, it is likely that this is a true response to exercise training and may indicate vascular remodeling. Due to variations among groups in cell membrane surface area, all ion currents were normalized to cell capacitance and presented as current density (pA/pF). Series resistance during voltage clamp was minimal (<10 M) and similar in all groups (data not shown).
ICa in CSM. Figure 1, A and B, shows representative peak inward currents during step depolarizations from a holding potential of 80 mV to 40, 0, and 20 mV in CSM from conduit (A) and resistance (B) arteries of a sedentary male swine. Depolarization steps produced characteristic voltage- and time-dependent inward currents with slow inactivation. Only traces from sedentary male swine CSM, presented as currents, were similar in Ex males. Similar data have been published previously for Sed and Ex female swine (8). Vascular smooth muscle from other arterial beds has been reported to contain two distinct types of voltage-gated Ca2+ currents, L- and T-type, with the relative contribution of each type dependent on both vascular bed and species (3, 5, 38). The channel subtype responsible for whole cell currents can be distinguished by several characteristics, including dihydropyridine sensitivity and time- and depolarization-dependent inactivation characteristics. L-type channels are highly sensitive to inhibition by dihydropyridines (3, 32), whereas T-type channels are insensitive to this class of drugs (32). Furthermore, T-type channels are activated by small depolarizations and inactivate quickly, whereas L-type channels require greater depolarization for activation and inactivate slowly (5). As shown previously for female porcine CSM (7, 8), inward currents in CSM in the present study were completely inhibited by nifedipine (Fig. 1, C and D) and showed characteristic L-type voltage dependence of activation and slow inactivation (3, 32, 38). Together, these data indicate that L-type Ca2+ channels are the predominant channel type in CSM from both male and female porcine coronary conduit and resistance arteries. Furthermore, in agreement with data shown previously for females (8), endurance training in males does not alter this L-type Ca2+-current predominance.
Gender, exercise training, and ICa I-V relationship. The effect of gender and exercise training on Ca2+ I-V relationships for conduit and resistance arteries are shown in Fig. 2. Exercise training, gender, and arterial size all exert significant effects on the I-V relationship. With regard to gender, ICa density was greater in males compared with females in CSM from both conduit and resistance arteries. As described previously (7), we observed a greater ICa density in CSM of resistance arteries compared with conduit arteries, confirming the significant effect of vessel size on CSM ICa density. Exercise training significantly increased ICa density in both conduit and resistance arteries of females; however, this effect was gender specific, as exercise training had no effect on ICa in either artery from male swine. Figure 3 shows the maximum peak ICa obtained, irrespective of membrane potential. Similar to effects on the I-V relationship, peak ICa density in males was greater than in females in both conduit and resistance coronary arteries. Exercise training significantly increased peak ICa density in both conduit and resistance arteries from females, with no effect in males. In addition, peak ICa density was greater in resistance vessels compared with conduit arteries in both sedentary and exercise trained groups of both sexes.
Voltage dependence of ICa. VGCCs switch between conducting (open) and nonconducting (closed) states primarily in response to changes in membrane potential (32). Whereas the open probability (Po) of VGCCs is increased by membrane depolarization, steady-state depolarization also results in channel inactivation. The relative number of open vs. inactivated channels determines steady-state Ca2+ conductance at a given membrane potential. Thus changes in the voltage dependence of channel activation or inactivation can significantly impact the steady-state Ca2+ influx in the cell. Figure 4 depicts the effect of gender, exercise, and arterial size on voltage-dependent activation of ICa in CSM. Exercise training had no effect on voltage-dependent activation of ICa in arteries of either female or male swine. However, both gender and arterial size influenced voltage-dependent activation. Half-maximal activation voltages (V0.5) derived from the Boltzmann equation fits to activation curves are shown in Table 3. In addition to a higher ICa density in males, voltage-dependent activation was shifted approximately 2-3 mV negative compared with females. Steady-state voltage-dependent inactivation is shown in Fig. 5, with corresponding V0.5 values provided in Table 3. Exercise training had no effect on voltage-dependent inactivation. However, gender influenced channel inactivation, as indicated by an approximate 5-mV negative shift in V0.5 for both resistance and conduit arteries in males compared with females. Neither exercise training, gender, nor arterial size had any effect on the slope of either voltage-dependent activation or inactivation (data not shown).
DISCUSSION
The present study provides the first direct evidence that gender plays a significant role in determining both basal ICa in CSM and the adaptive response of this current to endurance exercise training. Voltage-clamp data demonstrate that CSM from male swine exhibits both an increased ICa density and a negative shift in voltage-dependent activation and inactivation compared with female swine. A second novel and important finding of the present study is the influence of gender on the adaptive response of ICa to exercise training. Exercise training increased current density in CSM from both conduit and resistance arteries in a gender-specific manner [i.e., an increase in females as previously demonstrated (8, 14) with no effect in males]. This gender influence on ICa density may play an important role in determining previously reported gender-related differences in vascular reactivity and, perhaps, the incidence and severity of cardiovascular disease.
Gender and ICa. On the basis of existing epidemiological and experimental evidence, the initial hypothesis was that CSM from males would exhibit increases in ICa density and/or a negative shift in voltage-dependent activation compared with females. This original hypothesis was based on epidemiological studies that demonstrated a greater incidence of hypertension and CHD in men and postmenopausal women compared with premenopausal women (1, 20) and the proposed role of Ca2+ influx via VGCCs in the etiology of both diseases (18). Furthermore, in vitro studies have demonstrated increased contractile responses and 45Ca2+ influx in aortic smooth muscle from males compared with females (12), providing indirect evidence for an increased VGCC activity in aortic smooth muscle of males compared with females. The present findings in porcine coronary artery provide the first direct evidence to support this hypothesis. Smooth muscle ICa from both conduit and resistance coronary arteries was significantly greater in Sed male swine compared with Sed female swine. In addition, voltage-dependent activation and inactivation of ICa in CSM from male swine demonstrated a negative shift compared with female swine. This can be interpreted as a negative shift in the "window current" of ICa (i.e., the range of membrane potential over which Ca2+ channels are active) in males compared with females. Knot and Nelson (21) have demonstrated a dihydropyridine-sensitive, steep relationship between membrane potential, intracellular Ca2+, and diameter in cerebral microvessels, indicating that small changes in membrane potential can have significant effects on arteriolar diameter. Thus, although small, negative shifts in voltage-dependent activation in males may have a substantial impact on Ca2+ influx and vasomotor tone. The Boltzmann distribution fits derived in this study predict that a 3-mV negative shift in V0.5 would increase relative VGCC conductance (i.e., Po) at a given membrane potential by ~50%. All other factors being equal (e.g., Ca2+ buffering, extrusion), both the increased ICa density and negative shift in voltage-dependent activation in arteries of males would increase Ca2+ influx via VGCCs in response to vasoactive agonists, which directly or indirectly activate VGCCs, resulting in an increased contractile response. In addition, long-term increases in Ca2+ influx in CSM of males may contribute to increases in the incidence and severity of cardiovascular disease such as atherosclerosis and/or hypertension (18, 28).
Gender and exercise training. Previously, exercise training has been shown to increase L-type ICa in conduit, small artery, and arteriolar CSM of female miniature swine (8, 14). A primary purpose of the present study was to determine whether similar training adaptations occur in males. In contrast to females, exercise training did not affect ICa in conduit or resistance arteries from males, indicating that the adaptation of CSM ICa to endurance training is gender specific. As noted previously (8), the increased ICa density in females appears paradoxical within the context of VGCC-dependent Ca2+ influx as a mediator of increased vasomotor activity and cardiovascular disease (as discussed above for gender differences in sedentary individuals). This "ICa paradox" is perhaps resolved by the finding of Heaps et al. (14) that increased L-type Ca2+ influx in CSM of Ex female swine is compensated such that cytosolic Ca2+ responses are unchanged. Thus the increased ICa density that occurs with training may be a single component in a coordinated adaptation in CSM Ca2+ regulation involving sarcoplasmic reticulum Ca2+ unloading, coupled K+-channel activation, and depressed contractile responses to vasoactive agonists (6, 9, 10, 23). Similar potential adaptations in cellular Ca2+ regulation following exercise training may occur in males to reduce sensitivity of coronary arteries to vasoactive agents separate from changes in ICa (17). In pathological conditions, such as hypertension, increases in ICa may develop separately from the associated compensating mechanisms that occur with exercise training, resulting in increased cytosolic Ca2+ and contractile responses (12, 30) and, in the long term, contribute to increased vascular disease.
Potential mechanisms. As whole cell ICa is the product of the number and Po of active channels, differences in ICa density due to gender, training, and arterial size must result from differences in synthesis and membrane targeting of functional channels and/or modulation of channel Po. It is reasonable to speculate, especially for gender differences, that hormones may be responsible for ICa differences. Testosterone, estrogen, glucocorticoids, catecholamines, and aldosterone have been shown to influence L-type Ca2+-channel synthesis and activity in cultured vascular smooth muscle and other cell types (4, 13, 33). Recent sequencing of the Cav1.2 gene promoter region has provided evidence for a hormone response element strongly activated by testosterone (26). Conversely, estrogen inhibits L-type Ca2+ channels (31), whereas L-type Ca2+ channel expression in cardiac muscle is increased in estrogen-receptor knockout mice (16). If estrogen acts in vivo to decrease ICa, this would provide a logical candidate mechanism for the lower ICa in females compared with males. However, it is unlikely that training-induced reductions in circulating estrogens is a mechanism for increased ICa in exercise-trained female swine, as this training model does not alter estrous cycle length or estrogen or progesterone levels (24). Another consideration regarding this model is that male swine have higher circulating levels of 17-estradiol than females (2, 24). Whether the effect of higher estrogen levels in males is overridden by a dominant effect of testosterone, as has been proposed for sex hormone effects on endothelin receptors in porcine CSM (2), is unknown. Clarification of the role of either androgens or estrogens in determining gender-related difference in ICa will require additional studies (e.g., hormone replacement in gonadectomized animals of both sexes).
Apart from increased channel synthesis, changes in channel Po may account, in part or whole, for the ICa differences observed. As phosphorylation plays a dominant role in regulating Po of VGCCs, kinase/phosphatase regulation may be central to the observed gender and exercise-training differences in ICa. Accordingly, an intriguing candidate is protein kinase C (PKC). Recently, Kanashiro and Khalil (19) reported increased PKC-, -, and - levels in aorta of male, compared with female, rats and PKC activation increased L-type ICa in porcine CSM (15). Furthermore, exercise training has been reported to increase PKC- levels in porcine coronary arterioles (22). Whether PKC-dependent increases in VGCC activity contribute to gender and training-induced differences in coronary ICa remains to be determined.
In conclusion, the present study provides the first direct evidence that gender significantly influences CSM L-type Ca2+-channel activity and the subsequent adaptive response to endurance exercise training. Future studies directed at understanding the underlying mechanisms and functional consequences of these differences may aid in determining the basis for gender-related differences in cardiovascular disease and the cardioprotective effect of endurance training.
ACKNOWLEDGEMENTS
The authors thank Cathy Galle for invaluable technical assistance in this project and Dr. Cris Heaps and Joyce Warwick for critical reading.
FOOTNOTES
This work was supported by National Heart, Lung, and Blood Institute Grant HL-52490.
Original submission in response to a special call for papers on "Genome and Hormones: Gender Differences in Physiology."
Address for reprint requests and other correspondence: D. K. Bowles, E102 Veterinary Medicine, Univ. of Missouri, Columbia, MO 65211 (E-mail: bowlesd@missouri.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 June 2001; accepted in final form 14 August 2001.
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ABSTRACT
Endurance exercise training increases smooth muscle L-type Ca2+ current density in both resistance and proximal coronary arteries of female miniature swine. The purpose of the present study was to determine 1) whether gender differences exist in coronary smooth muscle (CSM) L-type Ca2+ current density and 2) whether endurance training in males would demonstrate a similar adaptive response as females. Proximal, conduit (~1.0 mm), and resistance [~200 μm (internal diameter)] coronary arteries were obtained from sedentary and treadmill-trained swine of both sexes. CSM were isolated by enzymatic digestion (collagenase plus elastase), and voltage-gated Ca2+-channel current (ICa) was determined by using whole cell voltage clamp during superfusion with 75 mM tetraethylammonium chloride and 10 mM BaCl2. Current-voltage relationships were obtained at test potentials from 60 to 70 mV from a holding potential of 80 mV, and ICa was normalized to cell capacitance (pA/pF). Endurance treadmill training resulted in similar increases in heart weight-to-body weight ratio, endurance time, and skeletal muscle citrate synthase activity in male and female swine. ICa density was significantly greater in males compared with females in both conduit (7.57 ± 0.58 vs. 4.14 ± 0.47 pA/pF) and resistance arteries (11.25 ± 0.74 vs. 6.49 ± 0.87 pA/pF, respectively). In addition, voltage-dependent activation of ICa in resistance arteries was shifted to more negative membrane potentials in males. Exercise training significantly increased ICa density in both conduit and resistance arteries in females (7.01 ± 0.47 and 9.73 ± 1.13 pA/pF, respectively) but had no effect in males (8.61 ± 0.50 and 12.04 ± 1.07 pA/pF, respectively). Thus gender plays a significant role in determining both the magnitude and voltage dependence of ICa in CSM and the adaptive response of ICa to endurance training.
keywords:electrophysiology; vascular smooth muscle; microcirculation; voltage-gated calcium channels
INTRODUCTION
GENDER EXERTS SIGNIFICANT INFLUENCE on coronary vascular physiology and pathophysiology. For example, whereas coronary heart disease (CHD) is a leading cause of mortality in both men and women, men show a greater prevalence of CHD compared with premenopausal women (1). Similarly, the incidence of hypertension is greater in men and postmenopausal women compared with premenopausal women (20). Mechanisms underlying gender differences in the development of complex cardiovascular disease are likely multifaceted. However, gender differences in several candidate mechanisms impacting cardiovascular disease have been reported, including endothelial nitric oxide synthase (NOS) content, NO production (25), smooth muscle proliferation (11), and smooth muscle responsiveness to agonists (30), e.g., endothelin (29). Recently, functional studies have provided indirect evidence that gender differences in dihydropyridine-sensitive, voltage-gated Ca2+ channels (VGCC) may also contribute to differences in vascular function between males and females (12). Crews et al. (12) found that depolarization-induced contraction and Ca2+ influx is greater in thoracic aorta of male rats compared with female rats, suggesting a gender difference in plasmalemmal VGCC activity. VGCC activity plays a central role in regulation of vascular resistance and, therefore, total blood flow and distribution (32). In addition, VGCCs have been proposed to contribute to the incidence and severity of both acute and long-term cardiovascular events, such as vasospasm and CHD (18, 28). If present, gender differences in VGCC activity in coronary smooth muscle (CSM) could play a significant role in determining gender-related differences in vascular function in both health and disease.
Endurance exercise training also influences ion-channel activity and Ca2+ regulation in CSM (6, 8, 9, 14, 17). In female miniature swine, L-type Ca2+-channel current (ICa) was increased in both conduit and resistance arterial smooth muscle after treadmill training (8, 14). Given the potential influence of gender on vascular smooth muscle Ca2+ regulation, the purpose of the present study was to determine 1) whether gender differences exist in CSM L-type Ca2+-current density and 2) whether males demonstrate a similar adaptive response to exercise training as females.
MATERIALS AND METHODS
Animals. Sexually mature, adult male and female miniature swine weighing 25-40 kg were obtained from the breeder (Charles River) and housed in pens at the College of Veterinary Medicine. All pigs included in this study were familiarized with treadmill exercise over a 1- to 2-wk period. Treadmill performance tests were administered to each animal. Pigs of each sex were then randomly divided into two groups. One group (Ex; n = 16, 6 females, 10 males) underwent a progressive treadmill training program used previously in our laboratory (7, 8, 14). The second group of pigs was restricted to their pens (6 × 12 ft) for 16-20 wk and served as sedentary controls (Sed; n = 15, 7 females, 8 males). Animal protocols were approved by the University of Missouri Animal Care and Use Committee in accordance with the "Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training."
Training procedures and treadmill performance tests. Ex pigs underwent a 16- to 20-wk treadmill endurance program followed by treadmill performance tests as described previously (8, 9, 14, 24). During the final 4-8 wk of training, a typical training session consisted of the following 85-min workout: 1) 5-min warm-up run at 2.5 miles/h (mph), 2) 15-min sprint at speeds of 5-8 mph, 3) 60-min endurance run at 4-5 mph, and 4) 5-min cool-down run at 2 mph. Treadmill performance tests were administered before and at the completion of the training (Ex) or pen confinement (Sed).
Skeletal muscle oxidative enzyme activity. At the time of death, muscle samples were taken from the medial and lateral heads of the triceps brachii and deltoid, frozen in liquid nitrogen, and stored until processed. Citrate synthase activity was measured spectrophotometrically from whole muscle homogenates (34).
Preparation of coronary arteries. Pigs 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). Hearts were removed and placed in iced (4°C) Krebs bicarbonate solution during vessel isolation. Main right conduit arteries (1.0-mm ID) were dissected free from the heart beginning ~2 cm distal to the ostia and proceeding to the origin of the posterior descending artery and cleaned of connective and adipose tissue at 4°C in physiological saline solution (PSS) containing (in mM) 2 CaCl2, 138 NaCl, 1 MgCl2, 5 KCl, 10 HEPES, and 10 glucose, pH 7.4. Similarly, epicardial resistance vessels 175-225 μm ID were obtained from the apex region of the anterior left ventricular free wall at 4°C in PSS.
Cell dispersion. All experiments were performed on freshly dispersed cells by using methods modified from those described previously (35-37, 39). Arteries were incubated in enzyme solution consisting of low Ca2+ (0.5 mM) physiological solution plus 294 U/ml collagenase (CLS II, Worthington Biochemical, Lakewood, NJ), 6.5 U/ml elastase (Worthington), 2 mg/ml bovine serum albumin (fraction V, Sigma Chemical, St. Louis, MO), 1 mg/ml soybean trypsin inhibitor (type I-S, Sigma Chemical), and 0.4 mg/ml DNase I (type IV, Sigma Chemical) for 45-60 min at 37°C. The enzyme solution was then immediately replaced with enzyme-free low-Ca2+ solution, and isolated single cells were obtained with gentle trituration. Cell suspensions were stored in low-Ca2+ (0.5 mM) buffer at 4°C until use (0-6 h).
Whole cell voltage clamp. Whole cell currents were determined by using standard voltage-clamp techniques as used routinely (36-38). Cells were initially superfused with PSS during gigaohm seal formation. After whole cell configuration was obtained, the superfusate was switched to PSS with the following substitutions: tetraethylammonium chloride (TEACl; 75 mM) substituted isomolar for NaCl and 10 mM Ba2+ for Ca2+ as the charge carrier. The pipette solution contained (in mM) 120 CsCl, 10 TEACl, 1 MgCl2, 20 HEPES, 5 Na2ATP, 0.5 Tris-GTP, and 10 EGTA, pH 7.1. TEACl was included in both the superfusate and pipette solution to eliminate competing outward K+ current. Ionic currents were amplified by an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). Whole cell currents were low-pass filtered with a cutoff frequency of 1,000 Hz, digitized at 2.5 kHz, and stored on computer. Current densities (pA/pF) were obtained for each cell by normalization of whole cell current to cell capacitance to account for differences in cell membrane surface area. Capacity currents were measured for each cell during 10-ms pulses from a holding potential of 80 mV to a test potential of 75 mV. Capacity currents were filtered at a low-pass cutoff frequency of 5 kHz. Leak subtraction was not performed. Data acquisition and analysis were accomplished by using pCLAMP 7.0 software (Axon Instruments). Current-voltage (I-V) relationships were determined by measuring peak inward current during a 500-ms step depolarization to membrane potentials from 60 to 70 mV in 10-mV increments (holding potential = 80 mV). Voltage-dependent activation was determined as g/gmax = peak IBa/[gBa (V Erev)], where gBa is the maximal conductance obtained from the linear regression of the positive limb of the I-V relationship through the apparent reversal potential (Erev), V is the step potential, and IBa is the peak inward current at the corresponding step potential. Activation data were fit to a conventional Boltzmann distribution equation, 1/{1+exp[(V0.5 V)/k]}, where V0.5 is the membrane potential producing half-maximal activation and k is the slope factor. For voltage dependent inactivation, peak inward current during a 500-ms step depolarization to 20 mV was determined after a 4-s prepulse at membrane potentials from 80 to +50 mV in 10-mV increments. Relative peak current (I/Imax) data were fit to a conventional Boltzmann distribution equation, 1/{1 + exp[(V0.5 V)/k]}. All experiments were conducted at room temperature (22-25°C). Cells were continually superfused under gravity flow.
Statistics. Data are expressed as means ± SE with each cell (voltage-clamp) or animal (training efficacy variables) counted as an observation (n). For voltage-clamp experiments, three to five cells were examined for each vessel from each animal (6-10 animals per group). Repeated-measures ANOVA was used for comparisons of I-V relationships, utilizing a Greenhouse-Geisser adjustment for within-subjects factors and Sidak adjustment for comparison of between-subjects effects (27). Multivariate ANOVA was used for comparison of single variables among groups. A P value <0.05 was set as the criterion for significance in all comparisons.
RESULTS
Efficacy of exercise training. Male swine tended to have greater body and heart weights compared with females; however, heart weight-to-body weight ratio, treadmill endurance time, and skeletal muscle citrate synthase activity levels were similar in sedentary male and female pigs (Table 1). Consistent with previous reports using the treadmill-trained miniature swine model (24), Ex animals of both sexes demonstrated overall similar adaptations to endurance training, including an increase in citrate synthase activity in the medial and lateral heads of the triceps brachii, increased treadmill endurance time, and increased heart weight-to-body weight ratio (Table 1).
Gender and cell size. In both conduit and resistance arteries, CSM cells from male animals were significantly larger than cells from female animals, as indicated by a greater cell membrane surface area (i.e., cell capacitance, pF; Table 2). Cell size was unaffected by exercise training with the exception of a smaller mean cell size in resistance arterioles of Ex compared with Sed males. Whether the smaller cell size in resistance arteries of Ex males is an effect of training or due to unintentional bias is unclear; however, the latter is unlikely as the experimenter was blinded to the treatment of the animal. Furthermore, a similar trend was noted for Ex females in this and a previous study (8). Therefore, it is likely that this is a true response to exercise training and may indicate vascular remodeling. Due to variations among groups in cell membrane surface area, all ion currents were normalized to cell capacitance and presented as current density (pA/pF). Series resistance during voltage clamp was minimal (<10 M) and similar in all groups (data not shown).
ICa in CSM. Figure 1, A and B, shows representative peak inward currents during step depolarizations from a holding potential of 80 mV to 40, 0, and 20 mV in CSM from conduit (A) and resistance (B) arteries of a sedentary male swine. Depolarization steps produced characteristic voltage- and time-dependent inward currents with slow inactivation. Only traces from sedentary male swine CSM, presented as currents, were similar in Ex males. Similar data have been published previously for Sed and Ex female swine (8). Vascular smooth muscle from other arterial beds has been reported to contain two distinct types of voltage-gated Ca2+ currents, L- and T-type, with the relative contribution of each type dependent on both vascular bed and species (3, 5, 38). The channel subtype responsible for whole cell currents can be distinguished by several characteristics, including dihydropyridine sensitivity and time- and depolarization-dependent inactivation characteristics. L-type channels are highly sensitive to inhibition by dihydropyridines (3, 32), whereas T-type channels are insensitive to this class of drugs (32). Furthermore, T-type channels are activated by small depolarizations and inactivate quickly, whereas L-type channels require greater depolarization for activation and inactivate slowly (5). As shown previously for female porcine CSM (7, 8), inward currents in CSM in the present study were completely inhibited by nifedipine (Fig. 1, C and D) and showed characteristic L-type voltage dependence of activation and slow inactivation (3, 32, 38). Together, these data indicate that L-type Ca2+ channels are the predominant channel type in CSM from both male and female porcine coronary conduit and resistance arteries. Furthermore, in agreement with data shown previously for females (8), endurance training in males does not alter this L-type Ca2+-current predominance.
Gender, exercise training, and ICa I-V relationship. The effect of gender and exercise training on Ca2+ I-V relationships for conduit and resistance arteries are shown in Fig. 2. Exercise training, gender, and arterial size all exert significant effects on the I-V relationship. With regard to gender, ICa density was greater in males compared with females in CSM from both conduit and resistance arteries. As described previously (7), we observed a greater ICa density in CSM of resistance arteries compared with conduit arteries, confirming the significant effect of vessel size on CSM ICa density. Exercise training significantly increased ICa density in both conduit and resistance arteries of females; however, this effect was gender specific, as exercise training had no effect on ICa in either artery from male swine. Figure 3 shows the maximum peak ICa obtained, irrespective of membrane potential. Similar to effects on the I-V relationship, peak ICa density in males was greater than in females in both conduit and resistance coronary arteries. Exercise training significantly increased peak ICa density in both conduit and resistance arteries from females, with no effect in males. In addition, peak ICa density was greater in resistance vessels compared with conduit arteries in both sedentary and exercise trained groups of both sexes.
Voltage dependence of ICa. VGCCs switch between conducting (open) and nonconducting (closed) states primarily in response to changes in membrane potential (32). Whereas the open probability (Po) of VGCCs is increased by membrane depolarization, steady-state depolarization also results in channel inactivation. The relative number of open vs. inactivated channels determines steady-state Ca2+ conductance at a given membrane potential. Thus changes in the voltage dependence of channel activation or inactivation can significantly impact the steady-state Ca2+ influx in the cell. Figure 4 depicts the effect of gender, exercise, and arterial size on voltage-dependent activation of ICa in CSM. Exercise training had no effect on voltage-dependent activation of ICa in arteries of either female or male swine. However, both gender and arterial size influenced voltage-dependent activation. Half-maximal activation voltages (V0.5) derived from the Boltzmann equation fits to activation curves are shown in Table 3. In addition to a higher ICa density in males, voltage-dependent activation was shifted approximately 2-3 mV negative compared with females. Steady-state voltage-dependent inactivation is shown in Fig. 5, with corresponding V0.5 values provided in Table 3. Exercise training had no effect on voltage-dependent inactivation. However, gender influenced channel inactivation, as indicated by an approximate 5-mV negative shift in V0.5 for both resistance and conduit arteries in males compared with females. Neither exercise training, gender, nor arterial size had any effect on the slope of either voltage-dependent activation or inactivation (data not shown).
DISCUSSION
The present study provides the first direct evidence that gender plays a significant role in determining both basal ICa in CSM and the adaptive response of this current to endurance exercise training. Voltage-clamp data demonstrate that CSM from male swine exhibits both an increased ICa density and a negative shift in voltage-dependent activation and inactivation compared with female swine. A second novel and important finding of the present study is the influence of gender on the adaptive response of ICa to exercise training. Exercise training increased current density in CSM from both conduit and resistance arteries in a gender-specific manner [i.e., an increase in females as previously demonstrated (8, 14) with no effect in males]. This gender influence on ICa density may play an important role in determining previously reported gender-related differences in vascular reactivity and, perhaps, the incidence and severity of cardiovascular disease.
Gender and ICa. On the basis of existing epidemiological and experimental evidence, the initial hypothesis was that CSM from males would exhibit increases in ICa density and/or a negative shift in voltage-dependent activation compared with females. This original hypothesis was based on epidemiological studies that demonstrated a greater incidence of hypertension and CHD in men and postmenopausal women compared with premenopausal women (1, 20) and the proposed role of Ca2+ influx via VGCCs in the etiology of both diseases (18). Furthermore, in vitro studies have demonstrated increased contractile responses and 45Ca2+ influx in aortic smooth muscle from males compared with females (12), providing indirect evidence for an increased VGCC activity in aortic smooth muscle of males compared with females. The present findings in porcine coronary artery provide the first direct evidence to support this hypothesis. Smooth muscle ICa from both conduit and resistance coronary arteries was significantly greater in Sed male swine compared with Sed female swine. In addition, voltage-dependent activation and inactivation of ICa in CSM from male swine demonstrated a negative shift compared with female swine. This can be interpreted as a negative shift in the "window current" of ICa (i.e., the range of membrane potential over which Ca2+ channels are active) in males compared with females. Knot and Nelson (21) have demonstrated a dihydropyridine-sensitive, steep relationship between membrane potential, intracellular Ca2+, and diameter in cerebral microvessels, indicating that small changes in membrane potential can have significant effects on arteriolar diameter. Thus, although small, negative shifts in voltage-dependent activation in males may have a substantial impact on Ca2+ influx and vasomotor tone. The Boltzmann distribution fits derived in this study predict that a 3-mV negative shift in V0.5 would increase relative VGCC conductance (i.e., Po) at a given membrane potential by ~50%. All other factors being equal (e.g., Ca2+ buffering, extrusion), both the increased ICa density and negative shift in voltage-dependent activation in arteries of males would increase Ca2+ influx via VGCCs in response to vasoactive agonists, which directly or indirectly activate VGCCs, resulting in an increased contractile response. In addition, long-term increases in Ca2+ influx in CSM of males may contribute to increases in the incidence and severity of cardiovascular disease such as atherosclerosis and/or hypertension (18, 28).
Gender and exercise training. Previously, exercise training has been shown to increase L-type ICa in conduit, small artery, and arteriolar CSM of female miniature swine (8, 14). A primary purpose of the present study was to determine whether similar training adaptations occur in males. In contrast to females, exercise training did not affect ICa in conduit or resistance arteries from males, indicating that the adaptation of CSM ICa to endurance training is gender specific. As noted previously (8), the increased ICa density in females appears paradoxical within the context of VGCC-dependent Ca2+ influx as a mediator of increased vasomotor activity and cardiovascular disease (as discussed above for gender differences in sedentary individuals). This "ICa paradox" is perhaps resolved by the finding of Heaps et al. (14) that increased L-type Ca2+ influx in CSM of Ex female swine is compensated such that cytosolic Ca2+ responses are unchanged. Thus the increased ICa density that occurs with training may be a single component in a coordinated adaptation in CSM Ca2+ regulation involving sarcoplasmic reticulum Ca2+ unloading, coupled K+-channel activation, and depressed contractile responses to vasoactive agonists (6, 9, 10, 23). Similar potential adaptations in cellular Ca2+ regulation following exercise training may occur in males to reduce sensitivity of coronary arteries to vasoactive agents separate from changes in ICa (17). In pathological conditions, such as hypertension, increases in ICa may develop separately from the associated compensating mechanisms that occur with exercise training, resulting in increased cytosolic Ca2+ and contractile responses (12, 30) and, in the long term, contribute to increased vascular disease.
Potential mechanisms. As whole cell ICa is the product of the number and Po of active channels, differences in ICa density due to gender, training, and arterial size must result from differences in synthesis and membrane targeting of functional channels and/or modulation of channel Po. It is reasonable to speculate, especially for gender differences, that hormones may be responsible for ICa differences. Testosterone, estrogen, glucocorticoids, catecholamines, and aldosterone have been shown to influence L-type Ca2+-channel synthesis and activity in cultured vascular smooth muscle and other cell types (4, 13, 33). Recent sequencing of the Cav1.2 gene promoter region has provided evidence for a hormone response element strongly activated by testosterone (26). Conversely, estrogen inhibits L-type Ca2+ channels (31), whereas L-type Ca2+ channel expression in cardiac muscle is increased in estrogen-receptor knockout mice (16). If estrogen acts in vivo to decrease ICa, this would provide a logical candidate mechanism for the lower ICa in females compared with males. However, it is unlikely that training-induced reductions in circulating estrogens is a mechanism for increased ICa in exercise-trained female swine, as this training model does not alter estrous cycle length or estrogen or progesterone levels (24). Another consideration regarding this model is that male swine have higher circulating levels of 17-estradiol than females (2, 24). Whether the effect of higher estrogen levels in males is overridden by a dominant effect of testosterone, as has been proposed for sex hormone effects on endothelin receptors in porcine CSM (2), is unknown. Clarification of the role of either androgens or estrogens in determining gender-related difference in ICa will require additional studies (e.g., hormone replacement in gonadectomized animals of both sexes).
Apart from increased channel synthesis, changes in channel Po may account, in part or whole, for the ICa differences observed. As phosphorylation plays a dominant role in regulating Po of VGCCs, kinase/phosphatase regulation may be central to the observed gender and exercise-training differences in ICa. Accordingly, an intriguing candidate is protein kinase C (PKC). Recently, Kanashiro and Khalil (19) reported increased PKC-, -, and - levels in aorta of male, compared with female, rats and PKC activation increased L-type ICa in porcine CSM (15). Furthermore, exercise training has been reported to increase PKC- levels in porcine coronary arterioles (22). Whether PKC-dependent increases in VGCC activity contribute to gender and training-induced differences in coronary ICa remains to be determined.
In conclusion, the present study provides the first direct evidence that gender significantly influences CSM L-type Ca2+-channel activity and the subsequent adaptive response to endurance exercise training. Future studies directed at understanding the underlying mechanisms and functional consequences of these differences may aid in determining the basis for gender-related differences in cardiovascular disease and the cardioprotective effect of endurance training.
ACKNOWLEDGEMENTS
The authors thank Cathy Galle for invaluable technical assistance in this project and Dr. Cris Heaps and Joyce Warwick for critical reading.
FOOTNOTES
This work was supported by National Heart, Lung, and Blood Institute Grant HL-52490.
Original submission in response to a special call for papers on "Genome and Hormones: Gender Differences in Physiology."
Address for reprint requests and other correspondence: D. K. Bowles, E102 Veterinary Medicine, Univ. of Missouri, Columbia, MO 65211 (E-mail: bowlesd@missouri.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 June 2001; accepted in final form 14 August 2001.
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