INTRODUCTION

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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 (I_Ca) 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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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/g_max = peak I_Ba/[g_Ba (V  E_rev)], where g_Ba is the maximal conductance obtained from the linear regression of the positive limb of the I-V relationship through the apparent reversal potential (E_rev), V is the step potential, and I_Ba is the peak inward current at the corresponding step potential. Activation data were fit to a conventional Boltzmann distribution equation, 1/{1+exp[(V_0.5  V)/k]}, where V_0.5 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/I_max) data were fit to a conventional Boltzmann distribution equation, 1/{1 + exp[(V_0.5  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

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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).

I_Ca 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.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Voltage-dependent Ca2+ current (ICa) in coronary smooth muscle (CSM). Representative current tracings from conduit (A) and resistance (B) CSM from male swine. Currents were obtained during 500-ms step potentials from a holding potential of 80 mV to test potentials indicated (10 mM Ba2+ external). Similar data for ICa in conduit and resistance arteries from females have been published previously (8). Representative current tracings depicting effect of nifedipine (nif; 2 µM) on inward current (20-mV test potential) in conduit (C) and resistance (D) CSM. Nifedipine essentially abolished peak and sustained inward currents (>90% and ~100%, respectively) and was equally effective in sedentary (Sed) and exercise-trained (Ex) male swine. Similar findings in Sed and Ex female swine have been reported (8), indicating that L-type voltage-gated Ca2+ channels (VGCCs) contribute predominantly to whole-cell current in CSM from conduit and resistance arteries and that this is unaffected by gender or training. Scale bars indicate 100 ms (horizontal) and 5 pA/pF (vertical).

Gender, exercise training, and I_Ca 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, I_Ca density was greater in males compared with females in CSM from both conduit and resistance arteries. As described previously (7), we observed a greater I_Ca density in CSM of resistance arteries compared with conduit arteries, confirming the significant effect of vessel size on CSM I_Ca density. Exercise training significantly increased I_Ca density in both conduit and resistance arteries of females; however, this effect was gender specific, as exercise training had no effect on I_Ca in either artery from male swine. Figure 3 shows the maximum peak I_Ca obtained, irrespective of membrane potential. Similar to effects on the I-V relationship, peak I_Ca density in males was greater than in females in both conduit and resistance coronary arteries. Exercise training significantly increased peak I_Ca density in both conduit and resistance arteries from females, with no effect in males. In addition, peak I_Ca density was greater in resistance vessels compared with conduit arteries in both sedentary and exercise trained groups of both sexes.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effect of gender, exercise and arterial size on the ICa-voltage relationship in CSM. Vm, membrane potential. Current-voltage (I-V) relationship for ICa in CSM from conduit (A and B) and resistance (C and D) arteries from female (A and C) and male (B and D) Sed () and Ex () swine. Peak inward current was obtained during a 500-ms step depolarization from 80 mV to the test potentials indicated. Repeated-measures ANOVA indicated significant main effects for training, gender, and arterial size. In Sed, inward current was greater in male vs. female and resistance vs. conduit arteries. Training increased ICa in both arterial sizes from female but not male swine. Values are means ± SE.*P < 0.05 vs. Sed.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of gender and exercise on maximal ICa . Maximal peak currents obtained from I-V protocols for Sed () and Ex () conduit (A) and resistance (B) arteries from female and male swine. In both arteries, gender had a significant effect on maximal ICa, with greater current density in males vs. females. Exercise training increased current density in both arterial sizes in females but not males. The largest peak inward current in each experiment was determined irrespective of test potential. Data are means ± SE. *P < 0.05 vs. Sed. #P < 0.05 vs. female.

Voltage dependence of I_Ca. VGCCs switch between conducting (open) and nonconducting (closed) states primarily in response to changes in membrane potential (32). Whereas the open probability (P_o) 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 I_Ca in CSM. Exercise training had no effect on voltage-dependent activation of I_Ca in arteries of either female or male swine. However, both gender and arterial size influenced voltage-dependent activation. Half-maximal activation voltages (V_0.5) derived from the Boltzmann equation fits to activation curves are shown in Table 3. In addition to a higher I_Ca 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 V_0.5 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 V_0.5 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).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of gender, exercise, and arterial size on voltage-dependent activation of ICa. Voltage-dependent activation of L-type current in CSM from conduit (A and B) and resistance (C and D) arteries from female (A and C) and male (B and D) Sed () and Ex () swine. Exercise training had no effect on activation curves in either gender or artery size. Voltage-dependent activation was shifted to more negative membrane potentials in resistance arteries of males compared with females (see Table 3). Relative conductance (g/gmax) values were fit to a conventional Boltzmann distribution equation. Data are means ± SE.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effect of gender, exercise and, arterial size on voltage-dependent inactivation of ICa. Voltage-dependent inactivation of L-type current in CSM from conduit (A and B) and resistance (C and D) arteries from female (A and C) and male (B and D) Sed () and Ex () swine. Exercise training had no effect on activation curves in either gender or artery size. In both arterial sizes, half-maximal inactivation potentials were shifted to more negative membrane potentials in males vs. females (Table 3). Relative peak current (I/Imax) data were fit to a conventional Boltzmann distribution equation. Peak current was determined during step depolarization to 20 mV following 4-s prepulses at potentials (Vm) indicated. Data are means ± SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study provides the first direct evidence that gender plays a significant role in determining both basal I_Ca 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 I_Ca 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 I_Ca 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 I_Ca 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 I_Ca. On the basis of existing epidemiological and experimental evidence, the initial hypothesis was that CSM from males would exhibit increases in I_Ca 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 I_Ca 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 I_Ca 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 I_Ca (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 V_0.5 would increase relative VGCC conductance (i.e., P_o) at a given membrane potential by ~50%. All other factors being equal (e.g., Ca²⁺ buffering, extrusion), both the increased I_Ca 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 I_Ca 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 I_Ca in conduit or resistance arteries from males, indicating that the adaptation of CSM I_Ca to endurance training is gender specific. As noted previously (8), the increased I_Ca 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 "I_Ca 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 I_Ca 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 I_Ca (17). In pathological conditions, such as hypertension, increases in I_Ca 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 I_Ca is the product of the number and P_o of active channels, differences in I_Ca 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 P_o. It is reasonable to speculate, especially for gender differences, that hormones may be responsible for I_Ca 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 Ca_v1.2 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 I_Ca, this would provide a logical candidate mechanism for the lower I_Ca in females compared with males. However, it is unlikely that training-induced reductions in circulating estrogens is a mechanism for increased I_Ca 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 I_Ca will require additional studies (e.g., hormone replacement in gonadectomized animals of both sexes).