Exercise prevents diabetes-induced impairment in superficial buffer barrier in porcine coronary smooth muscle.

In healthy coronary smooth muscle cells, the superficial sarcoplasmic reticulum (SR) buffers rise in intracellular Ca(2+) levels. In diabetic dyslipidemia, basal Ca(2+) levels are increased, yet Ca(2+) influx is decreased and SR Ca(2+) uptake is increased. Exercise prevents diabetic dyslipidemia-induced increases in basal Ca(2+) levels and decreases in Ca(2+) influx. We tested the hypothesis that diabetic dyslipidemia impairs Ca(2+) extrusion via a decrease in superficial SR and that exercise will prevent these losses. Male Yucatan swine were maintained in four treatment groups: control, hyperlipidemic, diabetic dyslipidemic, and diabetic dyslipidemic plus aerobically exercise trained. Intracellular Ca(2+) levels were measured during depolarization-induced Ca(2+) influx and caffeine-induced SR Ca(2+) release. Na(+)/Ca(2+) exchanger and plasmalemmal Ca(2+)-ATPase activity were assessed by inhibition with low extracellular Na(+) and 5,6-carboxyeosin, respectively. Superficial SR was quantified using the internal membrane dye 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)) and novel analysis techniques. We found that, in diabetic dyslipidemia, Ca(2+) extrusion was impaired and superficial SR was decreased. Exercise prevented the diabetic dyslipidemia-induced decrease in superficial SR and restored plasmalemmal Ca(2+) extrusion. On the basis of these results, we conclude exercise attenuates the diabetic dyslipidemia-induced impairment in intracellular Ca(2+) regulation.     

Exercise training prevents Ca2+ dysregulation in coronary smooth muscle from diabetic dyslipidemic yucatan swine.

Aerobic exercise training is known to have profound cardioprotective effects in disease, yet cellular mechanisms remain largely undefined. We tested the hypothesis that increased sarcoplasmic reticulum Ca(2+) buffering and increased voltage-gated Ca(2+) channel density underlie coronary smooth muscle intracellular Ca(2+) (Ca(2+)(i)) dysregulation in diabetic dyslipidemia and that exercise training would prevent these increases. Yucatan swine were maintained in 1) control, 2) alloxan-induced hyperglycemic, 3) high fat/cholesterol fed, 4) hyperglycemic plus high fat/cholesterol fed (diabetic dyslipidemic), and 5) diabetic dyslipidemic plus exercise-trained (treadmill running) conditions. After 20 wk, the heart was removed and smooth muscle cells isolated from the right coronary artery. We utilized fura-2 imaging of Ca(2+)(i) levels to separate the functional role of the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) from the Na(+)-Ca(2+) exchanger and the plasmalemmal Ca(2+)-ATPase, and whole-cell patch clamp to examine voltage-gated Ca(2+) channel current density (i.e., Ca(2+) influx). Results indicated that diabetic dyslipidemia impaired plasmalemmal Ca(2+) efflux, increased basal Ca(2+)(i) levels, increased SERCA protein and sarcoplasmic reticulum Ca(2+)(i) buffering, and elicited an approximately 50% decrease in voltage-gated Ca(2+) channel current density. Exercise training concurrent with the diabetic dyslipidemic state restored plasmalemmal Ca(2+) efflux, SERCA protein, sarcoplasmic reticulum Ca(2+)(i) buffering, and voltage-gated Ca(2+) channel current density to control levels. Interestingly, basal Ca(2+)(i) levels were significantly lower in the exercise-trained group compared with control. Collectively, these results demonstrate a crucial role for exercise in the prevention of diabetic dyslipidemia-induced Ca(2+)(i) dysregulation.     

Changes in skeletal muscle SR Ca2+ pump in congestive heart failure due to myocardial infarction are prevented by angiotensin II blockade

In order to understand the mechanisms of exercise intolerance and muscle fatigue, which are commonly observed in congestive heart failure, we studied sarcoplasmic reticulum (SR) Ca(2+)-transport in the hind-leg skeletal muscle of rats subjected to myocardial infarction (MI). Sham-operated animals were used for comparison. On one hand, the maximal velocities (Vmax) for both SR Ca(2+)-uptake and Ca(2+)-stimulated ATPase activities in skeletal muscle of rats at 8 weeks of MI were higher than those of controls. On the other hand, the Vmax values for both SR Ca(2+)-uptake and Ca(2+)-stimulated ATPase activities were decreased significantly at 16 weeks of MI when compared with controls. These alterations in Ca(2+)-transport activities were not associated with any change in the affinity (1/Ka) of the SR Ca(2+)-pump for Ca2+. Furthermore, the stimulation of SR Ca(2+)-stimulated ATPase activity by cyclic AMP-dependent protein kinase was not altered at 8 or 16 weeks of MI when compared with the respective control values. Treatment of 3-week infarcted animals with angiotensin-converting enzyme (ACE) inhibitors such as captopril, imidapril, and enalapril or an angiotensin receptor (AT1R) antagonist, losartan, for a period of 13 weeks not only attenuated changes in left ventricular function but also prevented defects in SR Ca(2+)-pump in skeletal muscle. These results indicate that the skeletal muscle SR Ca(2+)-transport is altered in a biphasic manner in heart failure due to MI. It is suggested that the initial increase in SR Ca(2+)-pump activity in skeletal muscle may be compensatory whereas the depression at late stages of MI may play a role in exercise intolerance and muscle fatigue in congestive heart failure. Furthermore, the improvements in the skeletal muscle SR Ca(2+)-transport by ACE inhibitors may be due to the decreased activity of renin-angiotensin system in congestive heart failure.     

Gender influences coronary L-type Ca(2+) current and adaptation to exercise training in miniature swine.

Endurance exercise training increases smooth muscle L-type Ca(2+) 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 Ca(2+) current density and 2) whether endurance training in males would demonstrate a similar adaptive response as females. Proximal, conduit (approximately 1.0 mm), and resistance [~200 microm (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 Ca(2+)-channel current (I(Ca)) was determined by using whole cell voltage clamp during superfusion with 75 mM tetraethylammonium chloride and 10 mM BaCl(2). Current-voltage relationships were obtained at test potentials from -60 to 70 mV from a holding potential of -80 mV, and I(Ca) 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. I(Ca) 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 I(Ca) in resistance arteries was shifted to more negative membrane potentials in males. Exercise training significantly increased I(Ca) 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 I(Ca) in CSM and the adaptive response of I(Ca) to endurance training.     

白细胞介素-2对大鼠心肌Ca2+ATPase和Na+ /K+ATPase的影响

为了探讨IL－2对心肌细胞内钙影响的可能机制,用光学法检测心肌肌浆网Ca^2 ATPase的活性,以及细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性。结果：（1）用IL－2（10、40、200、800U/ml）灌流心脏后,其肌浆网Ca^2 ATPase的活性随IL－2浓度的升高而增强;（2）在ATP浓度为0.1-4mmol/L时,Ca^2 ATPase的活性随ATP浓度的升庙则增强,由IL－2（200U／ml）灌流后的心脏获得肌浆网（SR）,其Ca^2 ATPase的活性对ATP的反应强于对照组;（3）在[Ca^2 ]为1－40μmol/L时,心脏SR Ca^2 ATPase的活性随[Ca^2 ]增加而增强,而IL－2灌流心脏后分离的SR,其Ca^2 ATPase活性在[Ca^2 ]升高时没有明显改变;（4）用nor-BNI(10nmol/L）预处理5min后,IL－2（200U／ml）灌流后不再使SR Ca^2 ATPase的活性增强;（5）用PTX（5mg/L）预处理后,IL－2对SR Ca^2 ATPase的影响减弱;（6）用磷脂酶C（PLC）抑制剂U73122（5μmol/L）处理后,IL－2不再使SR Ca^2 ATPase活性增高;（7）用IL－2直接处理从正常大鼠分离的SR后,对SR Ca^2 ATPase活性无明显影响;（8）IL－2灌流后,对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase活性没有显著。上述结果表明,IL－2灌流心脏后使心肌肌浆网Ca^2 ATPase的活性增加,心肌细胞膜上的κ－阿片受体及其下游的G蛋白和PLC介导了IL－2的作用。尽管IL－2提高SR Ca^2 ATPase对ATP的反应性,但却抑制SR Ca^2 ATPase对钙离子的敏感性。IL－2对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性无明显影响。     

KCa+ channels contribute to exercise-induced coronary vasodilation in swine

Coronary blood flow is controlled via several vasoactive mediators that exert their effect on coronary resistance vessel tone through activation of K(+) channels in vascular smooth muscle. Because Ca(2+)-activated K(+) (K(Ca)(+)) channels are the predominant K(+) channels in the coronary vasculature, we hypothesized that K(Ca)(+) channel activation contributes to exercise-induced coronary vasodilation. In view of previous observations that ATP-sensitive K(+) (K(ATP)(+)) channels contribute, in particular, to resting coronary resistance vessel tone, we additionally investigated the integrated control of coronary tone by K(Ca)(+) and K(ATP)(+) channels. For this purpose, the effect of K(Ca)(+) blockade with tetraethylammonium (TEA, 20 mg/kg iv) on coronary vasomotor tone was assessed in the absence and presence of K(ATP)(+) channel blockade with glibenclamide (3 mg/kg iv) in chronically instrumented swine at rest and during treadmill exercise. During exercise, myocardial O(2) delivery increased commensurately with the increase in myocardial O(2) consumption, so that myocardial O(2) extraction and coronary venous Po(2) (Pcv(O(2))) were maintained constant. TEA (in a dose that had no effect on K(ATP)(+) channels) had a small effect on the myocardial O(2) balance at rest and blunted the exercise-induced increase in myocardial O(2) delivery, resulting in a progressive decrease of Pcv(O(2)) with increasing exercise intensity. Conversely, at rest glibenclamide caused a marked decrease in Pcv(O(2)) that waned at higher exercise levels. Combined K(Ca)(+) and K(ATP)(+) channel blockade resulted in coronary vasoconstriction at rest that was similar to that caused by glibenclamide alone and that was maintained during exercise, suggesting that K(Ca)(+) and K(ATP)(+) channels act in a linear additive fashion. In conclusion, K(Ca)(+) channel activation contributes to the metabolic coronary vasodilation that occurs during exercise. Furthermore, in swine K(Ca)(+) and K(ATP)(+) channels contribute to coronary resistance vessel control in a linear additive fashion.     

Sarcoplasmic reticulum Ca2+ transport and gene expression in congestive heart failure are modified by imidapril treatment

This study was designed to test the hypothesis that blockade of the renin-angiotensin system improves cardiac function in congestive heart failure by preventing changes in gene expression of sarcoplasmic reticulum (SR) proteins. We employed rats with myocardial infarction (MI) to examine effects of an angiotensin-converting enzyme inhibitor, imidapril, on SR Ca(2+) transport, protein content, and gene expression. Imidapril (1 mg.kg(-1).day(-1)) was given for 4 wk starting 3 wk after coronary artery occlusion. Infarcted rats exhibited a fourfold increase in left ventricular end-diastolic pressure, whereas rates of pressure development and decay were decreased by 60 and 55%, respectively. SR Ca(2+) uptake and Ca(2+) pump ATPase, as well as Ca(2+) release and ryanodine receptor binding activities, were depressed in the failing hearts; protein content and mRNA levels for Ca(2+) pump ATPase, phospholamban, and ryanodine receptor were also decreased by approximately 55-65%. Imidapril treatment of infarcted animals improved cardiac performance and attenuated alterations in SR Ca(2+) pump and Ca(2+) release activities. Changes in protein content and mRNA levels for SR Ca(2+) pump ATPase, phospholamban, and ryanodine receptor were also prevented by imidapril treatment. Beneficial effects of imidapril on cardiac function and SR Ca(2+) transport were not only seen at different intervals of MI but were also simulated by another angiotensin-converting enzyme inhibitor, enalapril, and an ANG II receptor antagonist, losartan. These results suggest that blockade of the renin-angiotensin system may increase the abundance of mRNA for SR proteins and, thus, may prevent the depression in SR Ca(2+) transport and improve cardiac function in congestive heart failure due to MI.     

Muscle metabolic, SR Ca(2+) -cycling responses to prolonged cycling, with and without glucose supplementation.

This study investigated the effects of prolonged exercise, with and without glucose supplementation, on metabolism and sarcoplasmic reticulum (SR) Ca(2+)-handling properties in working vastus lateralis muscle. Fifteen untrained volunteers [peak O(2) consumption (Vo(2peak)) = 3.45 +/- 0.17 l/min; mean +/- SE] cycled at approximately 60% Vo(2peak) on two occasions, during which they were provided with either an artificially sweetened placebo beverage (NG) or a 6% glucose (G) beverage (~1.00 g carbohydrate/kg body mass). Beverage supplementation started at 30 min of exercise and continued every 15 min thereafter. SR Ca(2+) handling, metabolic, and substrate responses were assessed in tissue extracted from the vastus lateralis at rest, after 30 min and 90 min of exercise, and at fatigue in both conditions. Plasma glucose during G was 15-23% higher (P < 0.05) than those observed during NG following 60 min of exercise until fatigue. Cycle time to fatigue was increased (P < 0.05) by approximately 19% during G (137 +/- 7 min) compared with NG (115 +/- 6 min). Prolonged exercise reduced (P < 0.05) maximal Ca(2+)-ATPase activity (-18.4%), SR Ca(2+) uptake (-27%), and both Phase 1 (-22.2%) and Phase 2 (-34.2%) Ca(2+)-release rates during NG. The exercise-induced reductions in SR Ca(2+)-cycling properties were not altered during G. The metabolic responses to exercise were all unaltered by glucose supplementation, since no differences in respiratory exchange ratios, carbohydrate and lipid oxidation rates, and muscle metabolite and glycogen contents were observed between NG and G. These results indicate that the maintenance of blood glucose homeostasis by glucose supplementation is without effect in modifying the muscle metabolic, endogenous glycogen, or SR Ca(2+)-handling responses.     

Human muscle sarcoplasmic reticulum function during submaximal exercise in normoxia and hypoxia.

In this study, the response of the sarcoplasmic reticulum (SR) to prolonged exercise, performed in normoxia (inspired O(2) fraction = 0.21) and hypoxia (inspired O(2) fraction = 0.14) was studied in homogenates prepared from the vastus lateralis muscle in 10 untrained men (peak O(2) consumption = 3.09 +/- 0.25 l/min). In normoxia, performed at 48 +/- 2.2% peak O(2) consumption, maximal Ca(2+)-dependent ATPase activity was reduced by approximately 25% at 30 min of exercise compared with rest (168 +/- 10 vs. 126 +/- 8 micromol.g protein(-1) x min(-1)), with no further reductions observed at 90 min (129 +/- 6 micromol x g protein(-1) x min(-1)). No changes were observed in the Hill coefficient or in the Ca(2+) concentration at half-maximal activity. The reduction in maximal Ca(2+)-dependent ATPase activity at 30 min of exercise was accompanied by oxalate-dependent reductions (P < 0.05) in Ca(2+) uptake by approximately 20% (370 +/- 22 vs. 298 +/- 25 micromol x g protein(-1) x min(-1)). Ca(2+) release, induced by 4-chloro-m-cresol and assessed into fast and slow phases, was decreased (P < 0.05) by approximately 16 and approximately 32%, respectively, by 90 min of exercise. No differences were found between normoxia and hypoxia for any of the SR properties examined. It is concluded that the disturbances induced in SR Ca(2+) cycling with prolonged moderate-intensity exercise in human muscle during normoxia are not modified when the exercise is performed in hypoxia.     

Metabolic Syndrome and Coronary Artery Disease in Ossabaw Compared with Yucatan Swine

Metabolic syndrome (MetS), a compilation of associated risk factors, increases the risk of type 2 diabetes and coronary artery disease (CAD, atherosclerosis), which can progress to the point of artery occlusion. Stents are the primary interventional treatment for occlusive CAD, and patients with MetS and hyperinsulinemia have increased restenosis. Because of its thrifty genotype, the Ossabaw pig is a model of MetS. We tested the hypothesis that, when fed high-fat diet, Ossabaw swine develop more features of MetS, greater native CAD, and greater stent-induced CAD than do Yucatan swine. Animals of each breed were divided randomly into 2 groups and fed 2 different calorie-matched diets for 40 wk: control diet (C) and high-fat, high-cholesterol atherogenic diet (H). A bare metal stent was placed in the circumflex artery, and pigs were allowed to recover for 3 wk. Characteristics of MetS, macrovascular and microvascular CAD, in-stent stenosis, and Ca²⁺ signaling in coronary smooth muscle cells were evaluated. MetS characteristics including, obesity, glucose intolerance, hyperinsulinemia, and elevated arterial pressure were elevated in Ossabaw swine compared to Yucatan swine. Ossabaw swine with MetS had more extensive and diffuse native CAD and in-stent stenosis and impaired coronary blood flow regulation compared with Yucatan. In-stent atherosclerotic lesions in Ossabaw coronary arteries were less fibrous and more cellular. Coronary smooth muscle cells from Ossabaw had impaired Ca²⁺ efflux and intracellular sequestration versus cells from Yucatan swine. Therefore, Ossabaw swine are a superior model of MetS, subsequent CAD, and cellular Ca²⁺ signaling defects, whereas Yucatan swine are leaner and relatively resistant to MetS and CAD.Abbreviations: CAD, coronary artery disease; CSM, coronary smooth muscle; IVGTT, intravenous glucose tolerance test; MetS, metabolic syndrome; SERCA, sarco–endoplasmic reticulum Ca²⁺ ATPase; ET1, endothelin 1; SOCE, store-operated Ca²⁺ entryAtherosclerotic coronary artery disease (CAD) is increased at least 2-fold in patients with metabolic syndrome (MetS)²⁷ and is accompanied by marked microvascular dysfunction that further impairs coronary blood flow.¹⁰ MetS generally is diagnosed by the presence of 3 or more of the following conditions: obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension.^17,28 There is strong support for the role of the hyperinsulinemia component of MetS in increased restenosis after percutaneous coronary interventions.^74,75,84,85 Further, our group has shown that severe coronary microvascular dysfunction occurs in MetS.⁵ Because MetS (so-called ‘prediabetes’) affects as much as 27% of the United States population, is increasing dramatically in prevalence,⁹⁴ and can progress to type 2 diabetes, there is great need for basic research using animal models that accurately mimic MetS and the accompanying CAD. Clearly, there is need for study of MetS-induced CAD and in-stent stenosis and the underlying cellular and molecular mechanisms.Mice, rats, and swine are known to recapitulate MetS;^{3,12,36,60,71,72} however, none of these models fully reproduce the combined symptoms of MetS and CAD. Further, transgenic mouse models are simply not adequate for coronary vascular interventions using stents identical to those used in humans,^{18,23,38,55,57,79,83,86} a step that is essential for translation to the clinic. Yucatan and domestic swine are commonly used large animal models for study of cardiovascular disease due to their ability to mimic the neointimal formation and thrombosis observed in humans.⁸⁶ For example, several laboratories have produced severe CAD in swine,^{8,24,51,61,62,68,91} but through toxin-induced pancreatic β-cell ablation and feeding of an atherogenic diet, rather than as a natural development subsequent to MetS or diabetes. Currently, there is a paucity of large animal models that reproduce MetS and CAD.³Research on the obesity-prone Ossabaw miniature swine⁵⁹ clearly indicates that these animals develop MetS and cardiovascular disease when fed a high-calorie atherogenic diet,^{4,5,9,16,19,42,50,52,83,92} Female Ossabaw swine on this type of diet nearly doubled their percentage body fat in only 9 wk, showed insulin resistance, impaired glucose tolerance, dyslipidemia (profound increase in the ratio of low-density to high-density lipoprotein cholesterol, hypertriglyceridemia), hypertension, and early coronary atherosclerosis.¹⁶ These data contrast with those from male Yucatan miniature pigs, which did not develop MetS even after 20 wk on a comparable excess calorie atherogenic diet.^8,68,95 Yucatan swine do not develop MetS through diet manipulation, unlike Ossabaw swine, which consistently recapitulate all MetS characteristics. However, important differences in study design have not allowed direct comparison between Yucatan and Ossabaw swine.Cytosolic Ca²⁺ signaling is involved in ‘phenotypic modulation’ of coronary smooth muscle (CSM), as characterized by proliferation and migration in several in vitro cell culture models^33,35,89,90 and in vivo rodent models of the peripheral circulation (for example, reference 51). The Yucatan swine model of diabetic dyslipidemia shows altered Ca²⁺ extrusion,⁹⁶ Ca²⁺ sequestration by the sarcoplasmic reticulum,^32,34,98 and Ca²⁺ influx through voltage-gated Ca²⁺ channels.⁹⁸ Currently, Ca²⁺ signaling has not been compared directly between MetS Ossabaw and Yucatan swine CSM. Therefore, the purpose of the present study was to test the hypothesis that compared with Yucatan swine on calorie-matched standard chow (for example, Yucatan maintenance diet^8,95) and atherogenic diets, Ossabaw swine have a greater propensity to MetS and CAD with impaired coronary microvascular dysfunction and Ca²⁺ handling in CSM.     

Serine phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase in the intact beating rabbit heart.

Recent studies have demonstrated that Ca(2+)/calmodulin-dependent protein kinase phosphorylates the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum (SR) in vitro. Also, evidence from in vitro studies suggested that this phosphorylation, occurring at Ser(38), results in stimulation of Ca(2+) transport. In the present study, we investigated whether serine phosphorylation of the SR Ca(2+)-ATPase occurs in the intact functioning heart. Hearts removed from anesthetized rabbits were subjected to retrograde aortic perfusion of the coronary arteries with oxygenated mammalian Ringer solution containing (32)P(i) and contractions were monitored by recording systolic left ventricular pressure development. Following 45-50 min of (32)P perfusion, the hearts were freeze-clamped, SR isolated, and analyzed for protein phosphorylation. SDS-polyacrylamide gel electrophoresis and autoradiography showed phosphorylation of several peptides including the Ca(2+)-ATPase and Ca(2+) release channel (ryanodine receptor). The identity of Ca(2+)-ATPase as a phosphorylated substrate was confirmed by Western immunoblotting as well as immunoprecipitation using a cardiac SR Ca(2+)-ATPase-specific monoclonal antibody. The Ca(2+)-ATPase showed immunoreactivity with a phosphoserine monoclonal antibody indicating that the in situ phosphorylation occurred at the serine residue. Quantification of Ca(2+)-ATPase phosphorylation in situ yielded a value of 208 +/- 12 pmol (32)P/mg SR protein which corresponded to the phosphorylation of approximately 20% of the Ca(2+) pump units in the SR membrane. Since this phosphorylation occurred under basal conditions (i.e., in the absence of any inotropic intervention), a considerable steady-state pool of serine-phosphorylated Ca(2+)-ATPase likely exists in the normally beating heart. These findings demonstrate that serine phosphorylation of the Ca(2+)-ATPase is a physiological event which may be important in the regulation of SR function.     

Defective intracellular Ca(2+) signaling contributes to cardiomyopathy in Type 1 diabetic rats

The goal of the study was to determine whether defects in intracellular Ca(2+) signaling contribute to cardiomyopathy in streptozotocin (STZ)-induced diabetic rats. Depression in cardiac systolic and diastolic function was traced from live diabetic rats to isolated individual myocytes. The depression in contraction and relaxation in myocytes was found in parallel with depression in the rise and decline of intracellular free Ca(2+) concentration ([Ca(2+)](i)). The sarcoplasmic reticulum (SR) Ca(2+) store and rates of Ca(2+) release and resequestration into SR were depressed in diabetic rat myocytes. The rate of Ca(2+) efflux via sarcolemmal Na(+)/Ca(2+) exchanger was also depressed. However, there was no change in the voltage-dependent L-type Ca(2+) channel current that triggers Ca(2+) release from the SR. The depression in SR function was associated with decreased SR Ca(2+)-ATPase and ryanodine receptor proteins and increased total and nonphosphorylated phospholamban proteins. The depression of Na(+)/Ca(2+) exchanger activity was associated with a decrease in its protein level. Thus it is concluded that defects in intracellular Ca(2+) signaling caused by alteration of expression and function of the proteins that regulate [Ca(2+)](i) contribute to cardiomyopathy in STZ-induced diabetic rats. The increase in phospholamban, decrease in Na(+)/Ca(2+) exchanger, and unchanged L-type Ca(2+) channel activity in this model of diabetic cardiomyopathy are distinct from other types of cardiomyopathy.     

Prolonged exercise induces structural changes in SR Ca(2+)-ATPase of rat muscle.

Sarcoplasmic reticulum (SR) isolated from the deep red portion of the gastrocnemius muscle of Sprague-Dawley rats after a single bout of prolonged exercise was shown to have depressed Ca(2+)-stimulated Mg(2+)-dependent ATPase activity over a temperature range of 15 to 42.5 degrees C when compared to SR obtained from control muscle. Inclusion of the calcium ionophore, A23187, failed to restore the depressed ATPase activity from SR of exercised muscle to control values, but it did normalize the stimulatory effect of temperature on ATPase activity. This depression was also manifested as an increased activation energy when the data were converted to an Arrhenius plot. SR vesicles from both groups showed no differences or discontinuities in plots of steady-state fluorescence anisotropy. When the binding characteristics of the fluorescent probe, fluorescein isothiocyanate (FITC), were analyzed, SR vesicles prepared from exercised muscle displayed a 40% reduction in binding capacity with no apparent change in Kd. These findings support the conclusion that a single bout of exercise induces a structural change in the Ca(2+)-ATPase protein of rat red gastrocnemius muscle that is not a direct result of gross lipid alterations or increased muscle temperature.     

cADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca(2+) release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca(2+) release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs(+) current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NP(o)) of the channels in the presence of ryanodine (0.01-1 microM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 microM) and ruthenium red (40-80 microM) substantially inhibited the activity of RYR/Ca(2+) release channels. Caffeine (0.5-5 mM) markedly increased the NP(o) of these Ca(2+) release channels of the SR, but D-myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NP(o) of these Ca(2+) release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 microM) into the cis bath solution produced a 2.9-fold increase in the NP(o) of these RYR/Ca(2+) release channels. An eightfold increase in the NP(o) of the RYR/Ca(2+) release channels (0.0056 +/- 0.001 vs. 0.048 +/- 0.017) was observed at a concentration of cADPR of 1 microM. The effect of cADPR was completely abolished by ryanodine (50 microM). In the presence of cADPR, Ca(2+)-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca(2+) release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca(2+) release through the activation of RYRs on the SR of these smooth mucle cells.     

Inositol trisphosphate receptors in smooth muscle cells

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are a family of tetrameric intracellular calcium (Ca(2+)) release channels that are located on the sarcoplasmic reticulum (SR) membrane of virtually all mammalian cell types, including smooth muscle cells (SMC). Here, we have reviewed literature investigating IP(3)R expression, cellular localization, tissue distribution, activity regulation, communication with ion channels and organelles, generation of Ca(2+) signals, modulation of physiological functions, and alterations in pathologies in SMCs. Three IP(3)R isoforms have been identified, with relative expression and cellular localization of each contributing to signaling differences in diverse SMC types. Several endogenous ligands, kinases, proteins, and other modulators control SMC IP(3)R channel activity. SMC IP(3)Rs communicate with nearby ryanodine-sensitive Ca(2+) channels and mitochondria to influence SR Ca(2+) release and reactive oxygen species generation. IP(3)R-mediated Ca(2+) release can stimulate plasma membrane-localized channels, including transient receptor potential (TRP) channels and store-operated Ca(2+) channels. SMC IP(3)Rs also signal to other proteins via SR Ca(2+) release-independent mechanisms through physical coupling to TRP channels and local communication with large-conductance Ca(2+)-activated potassium channels. IP(3)R-mediated Ca(2+) release generates a wide variety of intracellular Ca(2+) signals, which vary with respect to frequency, amplitude, spatial, and temporal properties. IP(3)R signaling controls multiple SMC functions, including contraction, gene expression, migration, and proliferation. IP(3)R expression and cellular signaling are altered in several SMC diseases, notably asthma, atherosclerosis, diabetes, and hypertension. In summary, IP(3)R-mediated pathways control diverse SMC physiological functions, with pathological alterations in IP(3)R signaling contributing to disease.     

Exercise training attenuates coronary smooth muscle phenotypic modulation and nuclear Ca2+ signaling

Physical inactivity is an independent risk factor for coronary heart disease, yet the mechanism(s) of exercise-related cardioprotection remains unknown. We tested the hypothesis that coronary smooth muscle after exercise training would have decreased mitogen-induced phenotypic modulation and enhanced regulation of nuclear Ca(2+). Yucatan swine were endurance exercise trained (EX) on a treadmill for 16-20 wk. EX reduced endothelin-1-induced DNA content by 40% compared with sedentary (SED) swine (P < 0.01). EX decreased single cell peak endothelin-1-induced cytosolic Ca(2+) responses compared with SED by 16% and peak nuclear Ca(2+) responses by 33% (P < 0.05), as determined by confocal microscopy. On the basis of these results, we hypothesized that sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and intracellular Ca(2+) stores in native smooth muscle are spatially localized to dissociate cytosolic Ca(2+) and nuclear Ca(2+). Subcellular localization of SERCA in living and fixed cells revealed a distribution of SERCA near the sarcolemma and on the nuclear envelope. These results show that EX enhances nuclear Ca(2+) regulation, possibly via SERCA, which may be one mechanism by which coronary smooth muscle cells from EX are less responsive to mitogen-induced phenotypic modulation.     

Mechanisms of ischemic preconditioning effects on Ca(2+) paradox-induced changes in heart

The effects of ischemic preconditioning (IP) on changes in cardiac performance and sarcoplasmic reticulum (SR) function due to Ca(2+) paradox were investigated. Isolated perfused hearts were subjected to IP (three cycles of 3-min ischemia and 3-min reperfusion) followed by Ca(2+)-free perfusion and reperfusion (Ca(2+) paradox). Perfusion of hearts with Ca(2+)-free medium for 5 min followed by reperfusion with Ca(2+)-containing medium for 30 min resulted in a dramatic decrease in the left ventricular (LV) developed pressure and a marked increase in LV end-diastolic pressure. Alterations in cardiac contractile activity due to Ca(2+) paradox were associated with depressed SR Ca(2+)-uptake, Ca(2+)-pump ATPase, and Ca(2+)-release activities as well as decreased SR protein contents for Ca(2+)-pump and Ca(2+) channels. All these changes due to Ca(2+) paradox were significantly prevented in hearts subjected to IP. The protective effects of IP on Ca(2+) paradox changes in cardiac contractile activity as well as SR Ca(2+)-pump and Ca(2+)-release activities were lost when the hearts were treated with 8-(p-sulfophenyl)-theophylline, an adenosine receptor antagonist; KN-93, a specific Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) inhibitor; or chelerythrine chloride, a protein kinase C (PKC) inhibitor. These results indicate that IP rendered cardioprotection by preventing a depression in SR function in Ca(2+) paradox hearts. Furthermore, these beneficial effects of IP may partly be mediated by adenosine receptors, PKC, and CaMK II.     

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.     

Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart

The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac contractility and its function is abnormal in the diabetic heart. However, the mechanisms underlying SR dysfunction in the diabetic heart are not clear. Because protein phosphorylation regulates SR function, this study examined the phosphorylation state of phospholamban, a key SR protein that regulates SR calcium (Ca2+) uptake in the heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (STZ; 65 mg kg(-1) i.v.), and the animals were humanely killed after 6 weeks and cardiac SR function was examined. Depressed cardiac performance was associated with reduced SR Ca2+-uptake activity in diabetic animals. The reduction in SR Ca2+-uptake was consistent with a significant decrease in the level of SR Ca2+-pump ATPase (SERCA2a) protein. The level of phospholamban (PLB) protein was also decreased, however, the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR Ca2+-uptake was also due to a reduction in the phosphorylation of PLB by the Ca2+-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA). Although the activities of the SR-associated Ca2+-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase (PKA) were increased in the diabetic heart, depressed phosphorylation of PLB could partly be attributed to an increase in the SR-associated protein phosphatase activities. These results suggest that there is increased inhibition of SERCA2a by PLB and this appears to be a major defect underlying SR dysfunction in the diabetic heart.