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Am J Physiol Heart Circ Physiol 288: H1233-H1241, 2005. First published November 4, 2004; doi:10.1152/ajpheart.00732.2004
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Diabetic dyslipidemia and exercise affect coronary tone and differential regulation of conduit and microvessel K+ current

E. A. Mokelke,1,3,6 N. J. Dietz,1,3 D. M. Eckman,4,5 M. T. Nelson,5 and M. Sturek1,2,3,6

Departments of 1Medical Pharmacology and Physiology and 2Internal Medicine, School of Medicine, and 3Center for Diabetes and Cardiovascular Health, University of Missouri, Columbia, Missouri; 4Division of Neonatology, Department of Pediatrics, Wake Forest University Health Sciences, Winston-Salem, North Carolina; 5Department of Pharmacology, University of Vermont, Burlington, Vermont; and 6Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana

Submitted 21 July 2004 ; accepted in final form 27 October 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Spontaneous transient outward K+ currents (STOCs) elicited by Ca2+ sparks and steady-state K+ currents modulate vascular reactivity, but effects of artery size, diabetic dyslipidemia, and exercise on these differentially regulated K+ currents are unclear. We studied the conduit arteries and microvessels of male Yucatan swine assigned to one of three groups for 20 wk: control (C, n = 7), diabetic dyslipidemic (DD, n = 6), or treadmill-trained DD animals (DDX, n = 7). Circumflex artery blood flow velocity obtained with intracoronary Doppler and lumen diameters obtained by intravascular ultrasound enabled calculation of absolute coronary blood flow (CBF). Ca2+ sparks were determined in pressurized microvessels, and perforated patch clamp assessed K+ current in smooth muscle cells isolated from conduits and microvessels. Baseline CBF in DD was decreased versus C. In pressurized microvessels, Ca2+ spark activity was significantly lower in DD versus C and DDX (P < 0.05 vs. DDX). STOCs were pronounced in microvessel (~35 STOCs/min) in sharp contrast to conduit cells (~2 STOCs/min). STOCs were decreased by 86% in DD versus C and DDX in microvessels; in contrast, there was no difference in STOCs across groups in conduit cells. Steady-state K+ current in microvessels was decreased in DD and DDX versus C; in contrast, steady-state K+ current in conduit cells was decreased in DDX versus DD and C. We conclude that steady-state K+ current and STOCs are differentially regulated in conduit versus microvessels in health and diabetic dyslipidemia. Exercise prevented diabetic dyslipidemia-induced decreases in baseline CBF, possibly via STOC-regulated basal microvascular tone.

Yucatan swine; Doppler flow; intravascular ultrasound; vasoreactivity; spontaneous transient outward K+ currents; coronary blood flow

DIABETES is a major independent cardiovascular risk factor for accelerated atherosclerosis (29). The mechanisms for accelerated coronary artery disease are unclear; however, there is increasing evidence that diabetes alters intracellular Ca2+ (Ca) handling in isolated vascular smooth muscle cells from diabetic subjects (22, 25, 26, 34, 44, 59, 63, 65–67, 70). Altered Ca handling could have profound implications in vascular reactivity because Ca is tightly coupled to vascular tone. A well-known mechanism is activation of Ca2+-sensitive potassium channels (KCa), which causes membrane hyperpolarization and subsequent vasodilation (46) and increased coronary blood flow (CBF) in some conditions (30). Thus diabetes-induced alterations in Ca regulation and KCa channel activity could dramatically shift the functional balance between contraction and KCa channel-sensitive relaxation.

The expression and activity of K+ channels are affected in several disease states (17, 36–39, 42). In hypertension, increased steady-state KCa channel current, spontaneous transient outward K+ currents (STOCs) elicited by localized Ca2+ release from the sarcoplasmic reticulum, and protein expression of the KCa channel have been interpreted as compensatory responses to the elevated global Ca(37, 38). Similarly, in smooth muscle cells of conduit coronary artery, there is an increase in KCa channel current and altered functional coupling of Ca and the KCa channel in diabetic dyslipidemia (42). It is well established that localized Ca2+ signals ("Ca2+ sparks"), not global Ca, regulate STOCs and tone of healthy resistance arteries (15, 46). However, there are no studies examining the effect of diabetic dyslipidemia on the relationship among Ca2+ sparks, KCa channels, and coronary microvessel reactivity. This is critically important, because the microvasculature, as opposed to conduit segments of the vascular tree, is the major regulator of CBF (15, 53). Recent findings from this laboratory and others have shown that the heterogeneity of the coronary vasculature, including functional expression of voltage-gated Ca2+ channels (10) and other proteins (33, 41) and vasomotor responses (47, 51), are primarily diameter dependent and may also be modulated by disease (4). An additional level of complexity is the possibility that these diameter-dependent regulatory pathways respond differently to interventions or disease states. A better understanding of these relationships could help elucidate potential therapeutic targets for the treatment of the cardiovascular complications resulting from diabetes.

Endurance exercise is generally thought to provide cardioprotection and has several well-documented effects on coronary smooth muscle cells that can be summarized as an improved vasodilation and blunted vasoconstriction (9, 18, 52). Although the exact mechanisms for this protection are unclear, altered Ca2+ regulation is a potential candidate because of its critical role in contraction and/or relaxation and, most importantly, Ca2+ homeostasis is affected by chronic endurance exercise (11, 12, 23, 24, 28, 44, 54, 55, 60, 61, 70). The close functional relationship of Ca2+ movement and/or current and the activity of K+ channels make alterations in K+ channel activity another potential candidate for the observed effect of exercise-induced cardioprotection. Exercise training has been shown to prevent the increase in whole cell K+ current (IK) and alterations in the functional coupling of Ca2+ release and KCa channels in conduit coronary smooth muscle of diabetic dyslipidemic pigs (42).

In this study, we used a porcine model of diabetic dyslipidemia to determine whether the vasculature is affected in a diameter-dependent manner by addressing several hypotheses in conduit arteries and microvessels. First, diabetic dyslipidemia would result in enhanced constriction and decreased dilation of microvessels in vivo and in vitro. Second, steady-state K+ current and STOCs in freshly dispersed smooth muscle cells from coronary conduit arteries and microvessels would show differential expression. Finally, diabetic dyslipidemia-induced changes in vasoreactivity, STOCs, and Ca2+ sparks would be prevented by endurance exercise training.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Porcine model. All procedures 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." Male miniature Yucatan (Sinclair Research Center, Columbia, MO) swine were randomly assigned to three experimental groups: control animals fed minipig chow (C, n = 7; Purina Mills, St. Louis, MO), diabetic animals fed a high-fat/high-cholesterol minipig chow (diabetic dyslipidemic, DD, n = 6), and diabetic dyslipidemic animals that were treadmill trained (DDX, n = 7). High-fat/high-cholesterol chow was composed of minipig chow supplemented with 1.8% cholesterol, 0.6% sodium cholate, and 18.6% sucrose. The DD and DDX groups were maintained at similar 3% body weight gain per week by giving additional increments of feed and insulin injections as necessary for contrast to the normal developmental 1% body weight gain per week in controls (8). Diabetes was induced in the DD and DDX animals by injecting alloxan (125 mg/kg, Sigma Chemical, St. Louis, MO) into the venous circulation via a vascular access port. We maintained a "diabetic milieu" during the 20-wk period that has been found to be highly atherogenic in humans (5, 7, 14, 35), by maintaining the animals at a fasting blood glucose between 300 and 400 mg/dl and total cholesterol >300 mg/dl (8). A unique aspect of this model is the maintenance of hyperglycemia and dyslipidemia in the DDX group to prevent any differences in blood glucose or lipid levels from confounding the interpretation of the effects of exercise. Plasma was directly assayed for total cholesterol and triglyceride levels using a standard enzymatic kit (Sigma-Aldrich, St. Louis, MO), and low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were determined by separation of plasma samples with fast protein liquid chromatography (19, 20).

Treadmill training. The treadmill exercise protocol has been published elsewhere (8, 42). In brief, DDX animals were acclimated to a motorized treadmill (Good Horsekeeping; Ash Grove, MO) over a 2-wk period. The proper exercise protocol consisted of 10-min of warm up, followed by 30 min of walking at the target heart rate, followed by 5-min of cool down. This protocol was followed 4 days/wk.

In vivo coronary procedures. The right femoral artery was accessed by arterial cutdown. An 8-Fr sheath was inserted, and then a Cordis 8-Fr Amplatz L (sizes 0.75–2.0) guiding catheter (Guidant Sales, Temucula, CA) was advanced into the aortic arch over a 0.035-in. guidewire. The guidewire was removed and a manifold was attached to allow direct measurement of arterial blood pressure, injection of contrast media, and application of various experimental solutions (e.g., adenosine, bradykinin, and prostaglandin F2{alpha}). The ostium of the left main artery was engaged with the guiding catheter, and a 0.014-in.-diameter Doppler flow wire (JoMed, Rancho Cordova, CA) was advanced down the circumflex artery (CFX). After angiography-aided placement of the flow wire in a nonbranching section of the CFX, flow velocity signals were allowed to stabilize for several minutes. The analog Doppler signals were continuously digitized both as instantaneous peak velocity (IPV) and averaged peak velocity (APV) values. Each APV value was calculated online by the Cadiometrics FloMap (JoMed) as an average of IPV over two consecutive cardiac cycles. All flow data were stored on videotape and a personal computer for further offline analysis.

A 3.2-Fr intravascular ultrasound (IVUS) catheter (35 MHz, Ultracross, Boston Scientific/SciMed; Maple Grove, MN) was advanced over the flow wire and positioned in a proximal segment of the CFX. Figure 1A shows the relative positioning of flow wire and IVUS catheter in the CFX for data collection. Figure 1B is a representative IVUS image demonstrating luminal boundary necessary for determination of artery area, which is used for calculating absolute CBF values using the following equation: CBF (in ml/min) = (artery area in cm2) x (velocity in cm/s) x 0.5 (16). Baseline CBF velocity was recorded, and all pharmacological interventions were performed once baseline blood flow velocity was reestablished. Blood flow velocity responses to intracoronary adenosine (1 µg/kg) and bradykinin (4 ng/kg) applications via the guiding catheter were evaluated. Figure 1C shows representative raw data of APV plotted versus time in response to intracoronary injection of adenosine (1 µg/kg), which is typically used for determination of coronary flow reserve (CFR) in the clinical setting (31, 32, 45). The response is a transient peak increase in APV, which when divided by the preadenosine APV results in the CFR. The insets in Fig. 1C show IPV recordings with typical cyclic flow variations in systole and diastole at baseline, peak response to adenosine, and recovery from adenosine.



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  		Fig. 1. Intravascular ultrasound (IVUS) imaging and blood flow velocity methodology. A: schematic representation of IVUS and flow catheters in left circumflex (CFX) coronary artery. Placement of flow wire at the IVUS imaging transducer was necessary to obtain simultaneous measures of flow velocity and conduit artery lumen area. RC, right coronary artery; LAD, left anterior descending coronary artery. B: representative IVUS image identifying IVUS catheter, flow wire artifact, and luminal boundary. C: typical adenosine-induced blood flow velocity response in a control animal. Average peak velocity (APV, cm/s) response to adenosine (1 µg/kg) was based on running values of each cardiac cycle, and the value for each point was derived from the two most recent successive cardiac cycles plotted versus time. Adenosine was infused in bolus at the time point represented by the vertical arrowhead (30 s). As expected, the APV increased transiently and rapidly recovered to baseline within 20 s of application. Insets: selected instantaneous peak velocity recordings (IPV, cm/s, recorded at 60 Hz, each 2 s, or ~2.5 cardiac cycles, in length) during systole (S) and diastole (D) are shown for each phase of the adenosine response (baseline, peak, recovery).

 
After dilator responses to adenosine were obtained, a bolus of PGF2{alpha} (8 µg/kg) was injected into the left main artery to determine in vivo coronary constriction. Exactly 2.5 min after injection of PGF2{alpha}, the endothelium-dependent vasodilator bradykinin (4 ng/kg) was injected into the same location. The luminal responses to both PGF2{alpha} and bradykinin were recorded on videotape for offline analysis. Offline digitization of IVUS images was accomplished using MGI VideoWave software (version 3, MGI) and an ATI Radeon video capture board (ATI Technologies, Ontario, Canada). The lumen area in each chosen frame was determined by the autotrace function in ImagePro analysis software (version 4.0, Media Cybernetics; Silver Spring, MD). Offline analyses of blood flow velocity were performed on personal computers using custom-made (Dr. N. Dietz, University of Missouri) and commercially available software (Windaq Data Acquisition, Dataq Instruments; Akron, OH). Upon completion of in vivo studies, the heart was removed with the animal under deep anesthesia, and the right coronary artery and myocardial sections from the apex and left ventricle were removed and immediately placed in sterile containers with ice-cold physiological saline solution (PSS) consisting of (in mM) 118.5 NaCl, 4.7 KCl, 24 NaHCO3, 1.18 KH2PO4, 2.5 CaCl2, 1.2 MgCl2, 0.023 EDTA, and 11 glucose.

In vitro elemental Ca2+ release events (Ca2+ sparks) in pressurized microvessels. Posterior apical sections of the heart were immediately placed in ice-cold PSS after termination of in vivo procedures. These samples were sent via airmail to the University of Vermont for in vitro experiments. Tissue was used within 24 h after removal from the heart, and only microvessel preparations that exhibited spontaneous tone were used for in vitro procedures. For confocal imaging, intact microvascular arteries branching from the CFX (75–150 µm inner diameter) were placed into PSS containing 10 µM fluo 4-AM (Molecular Probes; Eugene, OR) and 0.05% pluronic acid (Molecular Probes) for 60 min at room temperature. Arterial segments were cannulated on glass pipettes, mounted in a 5-ml myograph, and placed on the stage of a Nikon TE300 microscope where fluo 4-AM was allowed to deesterify in PSS for 30 min at 37°C. Arteries were pressurized to either 10 or 40 mmHg and imaged using a Noran Oz laser scanning confocal microscope combined with a x60 water-immersion lens (1.2 numerical aperture) by illuminating with a krypton-argon laser at 488 nm. Images of the vessel wall (56.3 µm x 52.8 µm or 256 pixels x 240 pixels) were recorded every 8.33 or 16.7 ms (120 or 60 images/s, respectively). Under each pressure two different representative areas of the same artery were scanned for 10 s. The same area of artery was not scanned more than twice to avoid any laser-induced changes in Ca2+ signaling, and effects of pharmacological interventions were measured in paired experiments. Bicarbonate-buffered PSS was warmed to 37°C, gassed with 95% O2-5% CO2, and superfused (~3 ml/min) over the vessel.

Ca2+ sparks were analyzed in smooth muscle cells using custom analysis software written by Drs. M. T. Nelson and A. D. Bonev (Univeristy of Vermont) using IDL 5.0.2 (Research Systems; Boulder, CO). Detection of Ca2+ sparks was performed by dividing an area 1.54 µm (7 pixels) x 1.54 µm (7 pixels) (i.e., 2.37 µm2) in each image (F) by a baseline (F0), which was determined by averaging 20 images without Ca2+ spark activity. Ca2+ spark amplitude was calculated as F/F0.

Whole cell patch-clamp electrophysiology. Dispersion of smooth muscle cells from the right coronary conduit artery (typically ~4-mm internal diameter, Fig. 1) has been described in detail elsewhere (e.g., 42, 54, 55). In brief, artery segments were incubated in dispersion solution containing collagenase and elastase for 45–60 min in a shaking water bath (100-Hz shaking cycle) at 37°C, and the tissue was gently triturated to displace cells. The cell suspension was centrifuged at 900 rpm for 4 min, and the remaining cell-rich pellet was resuspended in a low Ca2+-containing PSS.

Microvascular smooth muscle cells were dispersed from vessels having 50- to 150-µm inner diameter harvested from posterior apical segments. Enzymatic dispersion of microvascular cells was similar to conduit artery dispersion described above, except that the microvessels were freely suspended in the enzyme solution in a microcentrifuge tube during gentle agitation in the warm water bath, and further dispersion of fresh cells from the tissue was typically accomplished within 60–90 min of initial incubation with enzyme with the aid of gentle repeated aspiration.

Whole cell IK measurement performed routinely in our laboratory (13, 37, 42, 54–56, 58) utilized the amphotericin-perforated, patch-clamp configuration (13, 42). All patch-clamp experiments on conduit and microvascular cells were accomplished within 48 h of euthanasia. Cells were superfused with PSS as described above except that bicarbonate was not included. Microelectrode pipettes were fashioned with a pipette puller (Narishige; Tokyo, Japan) from glass capillary tubes (Fisher Scientific; Atlanta, GA) and fire polished to a final tip resistance of 2–10 M{Omega}. Pipettes were dipped in an amphotericin-free intracellular solution that was composed of (in mM) 75 K2SO4, 45 KCl, 10 NaCl, 8 MgSO4, and 10 HEPES; pH 7.10. The pipette was backfilled with an amphotericin B-containing (240 mg/ml, Sigma Chemical) intracellular solution. All experiments were performed using either a Dagan 8900 patch-clamp amplifier (Minneapolis, MN) or a List patch-clamp amplifier (Lambrecht-Pfalz). The amplifier was connected in series to a Labmaster analog-to-digital converter and personal computer equipped with AxoBASIC 1.0 software (Axon Instruments; Foster City, CA) for data acquisition. Data were digitized at 495-ms intervals following filtration through an eight-pole low-pass filter. Membrane K+ currents were measured following adequate incorporation of amphotericin B into the cell membrane, as indicated by a series resistance of 25 M{Omega} or less. Current-voltage relationships (270-ms step pulses, –80 to +60 mV in 10-mV increments, from –80-mV holding potential) were determined (13, 42, 68) and normalized to cell capacitance. We also subjected the cells to a 10-min protocol consisting of 270-ms test pulses from –60 mV (approximating resting membrane potential) to –30 mV at 1 Hz (1/s, approximating resting heart rate of 60 beats/min) to mimic the previously described influence of cardiac myocyte-generated electrophysiological fields on coronary artery in vivo (40).

Statistics. One-way ANOVA was used for multiple groups, and the Bonferroni or Tukey post hoc tests were used to determine pairwise differences. All variables were checked for normality to determine when the use of parametric statistical methods was appropriate. When normality was not satisfied the nonparametric Kruskal-Wallis one-way ANOVA on ranks was used. In an attempt to avoid a type II interpretive error (a false negative), significance was reported at both the P < 0.05 and P < 0.10 levels (69).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Porcine characteristics. Table 1 shows that the difference in body weight between the control and DDX groups and between DD and control was statistically significantly at the P < 0.05 and P < 0.1 levels, respectively. By design, both DD and DDX groups had significantly greater fasting blood glucose, total cholesterol, LDL cholesterol, and LDL/HDL compared with the control group. Efficacy of the treadmill training protocol was confirmed by resting bradycardia. The heart rate response to an absolute workload (treadmill grade) was significantly lower (P < 0.05) in the final week of treadmill training than in the initial week, also indicating an increase in maximal work capacity (cardiorespiratory fitness). Heart weight-to-body weight ratio was significantly lower in DD animals compared with DDX animals (P < 0.05), and furthermore, it was lower than control animals (P < 0.1). Heart weight in the DDX animals was significantly greater compared with the DD animals (P < 0.05) and additionally greater than the control animals (P < 0.1). Comparable results have been published by our laboratory, which highlight some of the characteristics of the porcine model of diabetic dyslipidemia and the similarities to humans (19, 20, 42, 43, 63, 70).


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  		Table 1. Characteristics of experimental animals

 
In vivo CBF and CFR. There were no differences in mean arterial pressure, baseline conduit diameter, and rate pressure product across groups (Table 2). Under these stable conditions the calculated absolute CBF in the DD animals was significantly lower compared with the control, and exercise training prevented the decrease in CBF (Fig. 2) (P < 0.05). In PGF2{alpha}-constricted arteries, dilation as a result of intracoronary bradykinin application measured as percent dilation was depressed in the DD animals compared with the control and DDX, which was statistically significant (P < 0.1, Table 2). Similarly, the CBF responses to the bolus intracoronary application of adenosine (largely endothelium-independent, smooth muscle action) was attenuated in the DD animals compared with the control and DDX (P < 0.1) animals. Finally, the CBF responses to the bolus intracoronary application of bradykinin (largely endothelium dependent) in both DD and DDX groups were significantly lower compared with the control group (P < 0.05) (Table 2).


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  		Table 2. In vivo cardiac and vasomotor responses to intracoronary application of PGF2{alpha}, bradykinin, and adenosine

 


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  		Fig. 2. Baseline coronary blood flow (CBF) was significantly depressed in diabetic dyslipidemic (DD) animals. Exercise training prevented the impairment in CBF. n = 6 for control (C), DD, and treadmill-trained animals. *Significantly different from DD, P < 0.05.

 
Ca2+ release events in pressurized microvessels. Figure 3. contains representative images of Ca2+ spark events per 10 s from all groups as well as corresponding spark analysis results (Figs. 3, A–C). Summary data for all groups are shown in Fig. 3D. Elevation of intravascular pressure from 10 to 40 mmHg increased Ca2+ spark frequency in the coronary microvessels from all groups, although the effect was not statistically significant. However, in the microvessels from the DD animals, there was a diminution in Ca2+ spark frequency compared with the control and DDX at both 10 and 40 mmHg. These differences were significant only between DD versus DDX at both pressure conditions (Fig. 3D).



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  		Fig. 3. DD reduced Ca2+ spark events at 10 and 40 mmHg. A: image of Ca2+ sparks in pressure-perfused microvessels (40 mmHg) in C animals. Red crosses represent sparks as defined as transient local fractional fluorescence (F/Fo) increases greater than 1.2. Adjacent to each image is the analysis for spark activity for one preparation. B and C: image of Ca2+ sparks in pressure-perfused microvessels (40 mmHg) in DD and DDX animals, respectively. D: quantification of Ca2+ sparks indicated a trend for a reduction in the DD group compared with the C group, although this difference did not reach statistical significance. Exercise training enhanced Ca2+ spark activity compared with the DD group (*P < 0.05). This relationship was maintained at both intravascular pressures of 10 and 40 mmHg. n = 3 animals for C and DD and 4 animals for DDX.

 
Whole cell patch-clamp electrophysiolgy. Figure 4 contains summary data comparing steady-state IK normalized to cell capacitance (Fig. 4A) and STOCs (Fig. 4B) in cells from conduit and microvessel arteries across groups. In microvessel steady-state IK there were treatment effects at membrane potentials of –10 mV to +20 mV (P < 0.05). Post hoc analysis indicated pairwise differences at the P < 0.05 level in control versus DD (0 mV to +20 mV) and DDX (10 mV) and at the P < 0.1 level in control versus DDX at +20 mV. In conduit steady-state IK, there were treatment effects at membrane potentials of –10 mV to +30 mV (P < 0.05) and +40 mV (P < 0.1). Post hoc analysis indicated pairwise differences at the P < 0.05 level in DDX versus DD and control (0 mV and +10 mV) and at the P < 0.1 level in DDX versus DD and control at –10 mV and +20 mV.



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  		Fig. 4. Spontaneous transient outward currents (STOCs) were attenuated in smooth muscle cells from microvessels obtained from DD animals but not in cells from conduit arteries. A: whole cell steady-state K+ current (IK) density-voltage relations resulting from step depolarizations from –70 to +60 mV from a holding potential of –80 mV in cells from microvascular (left) and conduit (right) coronary smooth muscle cells. Steady-state current density was generally decreased in DD (n = 19) and DDX (n = 34) versus C (n = 34). Pairwise differences occurred at the P < 0.05 level in C vs. DD (0 mV to +20 mV) and DDX (10 mV) and at the P < 0.1 level in C vs. DDX at +20 mV. Conduit cell current densities were decreased in DDX (n = 42) versus C (n = 24) from –10 to +40 mV, whereas DDX was less than DD (n = 33) at voltages greater than –20 mV. Pairwise differences occurred at the P < 0.05 level in DDX vs. DD and C (0 mV and +10 mV) and at the P < 0.1 level in DDX vs. DD and C at –10 mV and +20 mV. B: inset illustrates that step depolarization from a holding potential of –60 mV to –30 mV for 270 ms results in a small steady-state potassium current, frequently accompanied by STOCs (3 in this case). STOC frequency in smooth muscle cells from coronary microvessels of DD (n = 19) was significantly lower than STOC frequency in C (n = 35) and DDX (n = 34) (*P < 0.05). STOC frequency in cells from conduit vessels, which exhibit low frequency of events when compared with control microvessels, was not different across groups.

 
The data in Fig. 4B emphasize the differences in STOC events between cells obtained from conduit and microvessel arteries. In control and DDX animals, the number of STOC events in cells from microvessels was 10-fold greater than those events in the cells from conduit arteries. Because of the severe reduction in STOC activity in the microvessels from the DD group, the STOC number was not different between cells from conduit arteries. Importantly, the STOC events were significantly lower in cells from microvessels from DD animals compared with the control animals. Finally, exercise training prevented the diabetic dyslipidemia-induced reduction in STOC events in microvessels. There was no difference in STOC events across groups in the cells from the conduit arteries.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study to directly compare the effects of diabetic dyslipidemia and exercise training on in vivo blood flow measurements followed by in vitro measurements of IK, STOC, and Ca2+ spark activity in conduit and microvessels from the same animal model. The major findings are 1) baseline CBF was impaired in diabetic dyslipidemia, and this impairment was prevented with exercise; 2) the most profound effects were decreased Ca2+ spark activity and STOCs in microvessel smooth muscle cells of diabetic dyslipidemic animals and prevention of these decreases by exercise; and 3) the frequency of Ca2+ spark activity and STOCs in healthy animals is inversely related to the diameter of the artery. Taken together, these data suggest that Ca2+ sparks and STOCs play a larger role in maintaining tone in the microvessels compared with conduit arteries.

Porcine model of diabetic dyslipidemia and exercise training. As shown previously, chronic diabetic dyslipidemia in swine results in hypercholesterolemia, elevated LDL, elevated LDL/HDL, accelerated atheroma, enhanced coronary vasoreactivity, and alterations in ionic currents and global Ca2+ regulation (19, 20, 25, 26, 34, 42, 44, 62, 63, 70). Furthermore, mild to moderate exercise prevented many of these diabetic dyslipidemia-induced alterations (42–44, 62, 70). Additionally, the efficacy of the mild exercise protocol is indicated by the resting bradycardia in the DDX animals compared with the DD animals and decreased submaximal heart rate in DDX at the end versus the beginning of the study. These results further reinforce the importance of the porcine model as one that closely mimics individuals with diabetic dyslipidemia (27, 48, 50).

Absolute CBF and in vivo responses to constrictors and vasodilators. Simultaneous measures of conduit artery diameter and blood flow velocity provide a powerful tool for determining absolute blood flow. We noted decreased baseline absolute CBF in anesthetized animals in the DD group compared with the control group (Fig. 2). The reduction in baseline CBF was completely prevented with exercise training (Fig. 2). Importantly, the impaired resting CBF in the DD group was not due to decreased blood pressure or conduit coronary artery size because arterial pressure and artery diameter were not different across groups. Additionally, the rate times pressure product was not different across groups, suggesting that the myocardial oxygen consumption was similar across groups (Table 2). These observations support the concept that intrinsic microvascular dysfunction resulted from chronic diabetic dyslipidemia, which was improved by exercise.

The increase in percent constriction of conduit artery in response to intracoronary application of PGF2{alpha} was 2.7-fold greater in DD animals compared with control; however, the differences did not reach statistical significance. Importantly, the in vivo results are in line with in vitro results from this laboratory (20, 43) and others (1, 2, 21) showing enhanced reactivity to vasodilators and/or constrictors. Endurance exercise training prevented the heightened vasoreactivity resulting from chronic diabetic dyslipidemia. The lack of statistical significance in this variable can be most likely attributed to a low number of experimental animals. It is noteworthy that despite the low number of experimental animals, we report statistical significant differences in variables that support our original hypotheses. Intracoronary application of the endothelium-dependent vasodilator bradykinin resulted in nearly complete vasorelaxation of the artery to the pre-PGF2{alpha} level in both the control and DDX groups. The effect of bradykinin to dilate the PGF2{alpha}-constricted artery was significantly impaired in the DD group (P < 0.1). Although the exact mechanism(s) for the enhanced ability to relax as an adaptive response to exercise training is still unresolved, there is evidence that exercise training does increase endothelium-dependent vasodilation (64) and upregulate endothelial nitric oxide synthase mRNA (71). The levels of nitric oxide synthase protein were not quantified in this study.

The ability of the intracoronary application of adenosine to transiently increase CBF above basal levels was significantly impaired in the DD group compared with the control group (P < 0.1). Exercise training did not improve the CBF to adenosine application. Similarly, the ability of the intracoronary application of bradykinin to increase CBF above basal levels was impaired in the DD group (P < 0.1), and exercise training did not prevent this impairment. It is possible that the modest intensity of exercise was not sufficiently high to stimulate compensatory mechanisms to overcome the diabetic dyslipidemia-induced impairment to increase blood flow maximally. An additional possibility is that the relative amount of adenosine or bradykinin administered to the animals (which was normalized to body weight) in the DDX group was less than to the control and DD groups if based on heart weight. We contend that this is the first study where a humanlike model of diabetic dyslipidemia has been used to directly measure these effects.

In vitro whole cell IK, STOCs, and Ca2+ sparks. In the present study, steady-state IK was not significantly greater in DD compared with control in contrast to what we have shown previously (42). A potential explanation for this result could be that the animals in this present study received a different diet and were mildly obese compared with our previous studies. It is well known that dietary factors can affect membrane composition and subsequent membrane current characteristics (17). Interestingly, consistent with our previous study, endurance exercise resulted in a reduction in steady-state IK in cells from conduit arteries but not from microvessels.

Although Ca2+ sparks have little effect on global intracellular Ca2+ levels in vascular smooth muscle cells, one Ca2+ spark can activate a small number (10–30) of nearby KCa channels, giving rise to a STOC (6, 46). The activation of the resultant IK can cause membrane hyperpolarization, reduced intracellular Ca2+, and subsequent relaxation. Because Ca2+ sparks activate STOCs, STOC events can be used as a bioassay for Ca2+ sparks (55–57). In the pressurized microvessels in this study, Ca2+ spark frequency was depressed in the DD animals compared with control animals, although this did not reach statistical significance. The reduction in Ca2+ spark frequency in the DD animals was significantly lower when compared with DDX animals (Fig. 3, B and D). Using another index of Ca2+ spark activity, we found STOCs significantly lower in microvessel cells isolated from DD pigs compared with control pigs (Fig. 4B). Exercise training prevented the reduction in STOC events in DD. The relationships of the responses in Ca2+ spark frequency and STOC events to our experimental conditions across groups were very similar in the microvessels. In contrast to the microvessel data, Ca2+ sparks were basically absent from cells of conduit arteries. One possible explanation for the slight discrepancy in the statistical tests between Ca2+ sparks in pressurized microvessels versus STOCs in CSM cells isolated from microvessels is that the measurements were made using different preparations. It is indeed remarkable that the relationship between Ca2+ sparks and STOCs in microvessels withstood the difference in experimental protocols.

There is a large body of literature regarding alterations in the KCa channel both contributing to and compensating for pathophysiological conditions (37, 38, 42, 49). For example, increases in KCa current have been reported in hypertension as well as in a similar model of porcine diabetic dyslipidemia (37, 38, 42, 49). In a rat model of hypertension, Amberg et al. (3) have shown a reduction in KCa channel activity. Finally, we have reported (42) an increased coupling between Ca2+ release and KCa channel activation in conduit coronary artery in diabetic dyslipidemia. The discordance in these findings can possibly be attributed to the different animal models, different sizes of arteries, and different pathologies studied. This strongly suggests that the roles of IK, STOCs, and Ca2+ sparks in vasoregulation is likely dependent on the diameter of the vessel as well as the origin of the artery.

Summary and conclusions. To our knowledge, this is the first study assessing coronary conduit and microvessel vasoreactivity in vivo complemented by patch-clamp recordings of KCa current and confocal microscope measurements of microvascular Ca2+ sparks in vessels from the same animal model. In vivo microvascular tone was greater in animals that were diabetic dyslipidemic, and this was prevented by exercise training. STOC events were decreased in cells from DD microvessels. Exercise training prevented the reduction in STOCs and Ca2+ sparks in microvessels. Together these data support the idea that Ca2+ spark-mediated activation of KCa channels may contribute to an elevation of coronary microvascular tone and a decrease in coronary blood flow. Perhaps more importantly, the present comparison of conduit versus microvessel smooth muscle from the same vascular bed (coronary), the same animal, and the same laboratory using the same methods is compelling evidence that the contribution of Ca2+ sparks and STOCs to vasoregulation is dependent on artery diameter.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health (NIH) Grants RR-13223 and HL-62552 and the Rosebud Foundation (Kansas City, MO) to M Sturek; NIH Grants HL-44455 and HL-63722 and Totman Trust for Cerebrovascular Research to M. T. Nelson; and Individual National Research Service Award HL-10474 to E. A. Mokelke.


   ACKNOWLEDGMENTS
 
We thank members of our animal support staff, Robert Boullion, Jerilyn Giles, and James Wenzel for exemplary care and maintenance of the Yucatan swine; James Vuchetich for anesthesia; Meifang Wang for technical assistance; the School of Veterinary Medicine, University of Missouri, for access to the Research Animal Angiography Laboratory; and Dr. Shawn Kaser for critical review of the paper.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Sturek, Indiana Univ. School of Medicine, 635 Barnhill Dr., MS 309, Indianapolis, IN 46202-5120 (E-mail msturek{at}iupui.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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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