Diabetic hearts exhibit decreased responsiveness to stimulation by ␤-adrenoreceptor (␤-AR) agonists. This decrease in activity may be due to changes in expression and/or signaling of ␤-AR. Recently we showed that right atrial strips from 14-week streptozotocin (STZ)-induced diabetic rat hearts exhibit decreased responsiveness to ␤ 1 -AR agonist stimulation, but not to ␤ 2 -AR agonist. In the present study, we investigated the effects of long-term diabetes on the expression of cardiac ␤ 1 -, ␤ 2 -, and ␤ 3 -ARs and looked at whether these changes could be restored with insulin treatment. Using reverse transcription-polymerase chain reaction (RT-PCR), PAGE, and Western blot analysis, we found that ␤ 1 -AR mRNA and protein levels decreased by 34.9 ± 5.8 and 44.4 ± 5.8%, respectively, in 14 week-STZtreated diabetic rat hearts when compared with agematched controls. On the other hand, mRNA levels encoding ␤ 2 -and ␤ 3 -ARs increased by 72.5 ± 16.6 and 97.3 ± 26.1%, respectively. Although the latter translated into a proportional increase in ␤ 3 -AR protein levels (100.0 ± 17.0%), ␤ 2 -AR protein levels decreased to 82.6 ± 1.1% of control. Insulin treatment for 2 weeks, after 12 weeks of untreated diabetes, partially restored ␤ 1 -AR mRNA and protein levels to 60.1 ± 8.4 and 83.2 ± 5.0%, respectively, of control. Although insulin treatment minimally attenuated the rise in mRNA levels encoding ␤ 2 -and ␤ 3 -ARs, the steady-state levels of these proteins returned to near control values. These data suggest that the decreased responsiveness of diabetic hearts to stimulation of ␤-AR agonists may be due to a decrease in ␤ 1 -AR and an increase ␤ 3 -AR expression. Diabetes 50: [455][456][457][458][459][460][461] 2001

Prolongation of relaxation is a hallmark of diabetic cardiomyopathy. Most studies attribute this defect to decreases in sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA2a) expression and SERCA2a-to-phospholamban (PLB) ratio. Since its turnover rate is slow, SERCA2a is susceptible to posttranslational modifications during diabetes. These modifications could in turn compromise conformational rearrangements needed to translocate calcium ions, also leading to a decrease in SERCA2a activity. In the present study one such modification was investigated, namely advanced glycation end products (AGEs). Hearts from 8-week streptozotocin-induced diabetic (8D) rats showed typical slowing in relaxation, confirming cardiomyopathy. Hearts from 8D animals also expressed lower levels of SERCA2a protein and higher levels of PLB. Analysis of matrix-assisted laser desorption/ionization time-of-flight mass data files from trypsin-digested SERCA2a revealed several cytosolic SERCA2a peptides from 8D modified by single noncrosslinking AGEs. Crosslinked AGEs were also found. Lysine residues within actuator and phosphorylation domains were cross-linked to arginine residues within the nucleotide binding domain via pentosidine AGEs. Two weeks of insulin-treatment initiated after 6 weeks of diabetes attenuated these changes. These data demonstrate for the first time that AGEs are formed on SERCA2a during diabetes, suggesting a novel mechanism by which cardiac relaxation can be slowed during diabetes. Diabetes 53: [463][464][465][466][467][468][469][470][471][472][473] 2004 R eductions in rate and force of cardiac contractions are root causes for the increased incidence of morbidity and mortality among diabetic patients (1-3). Studies show that this "diabetic cardiomyopathy" is independent of coronary vascular diseases and is brought about by shifts in metabolism, cellular biochemistry, and structure (4 -8). At the molecular level, decreases in chronotropy and inotropy result from alterations in expression and/or function of several sarcolemmal membrane receptors and associated signal transduction proteins as well as other key proteins involved in regulating/maintaining intracellular ionic homeostasis (9 -11). Of particular interest is a transport protein on the sarcoplasmic reticular membrane that plays an integral role in cardiac relaxation. This protein, referred to as sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA2a), is responsible for replenishing intracellular calcium stores following release and in so doing terminate contraction.SERCA2a is a member of a large family of P-type ATPase enzymes that utilizes the energy generated from hydrolysis of terminal phosphate bond of ATP to pump calcium against its electrochemical gradient (12,13). SERCA1a is the best studied of these single polypeptides. It consists of 10 transmembrane helixes (M1 through M10) and three cytoplasmic domains, referred to as A (actuator), N (nucleotide binding) and P (phosphorylation) domains (14). Translocation of calcium ions from the cytosol to the lumen of...

Obesity is associated with marked increases in plasma leptin concentration, and hyperleptinemia is an independent risk factor for coronary artery disease. As a result, the purpose of this investigation was to test the following hypotheses: 1) leptin receptors are expressed in coronary endothelial cells; and 2) hyperleptinemia induces coronary endothelial dysfunction. RT-PCR analysis revealed that the leptin receptor gene is expressed in canine coronary arteries and human coronary endothelium. Furthermore, immunocytochemistry demonstrated that the long-form leptin receptor protein (ObRb) is present in human coronary endothelium. The functional effects of leptin were determined using pressurized coronary arterioles (<130 microm) isolated from Wistar rats, Zucker rats, and mongrel dogs. Leptin induced pharmacological vasodilation that was abolished by denudation and the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester and was absent in obese Zucker rats. Intracoronary leptin dose-response experiments were conducted in anesthetized dogs. Normal and obese concentrations of leptin (0.1-3.0 microg/min ic) did not significantly change coronary blood flow or myocardial oxygen consumption; however, obese concentrations of leptin significantly attenuated the dilation to graded intracoronary doses of acetylcholine (0.3-30.0 microg/min). Additional experiments were performed in canine coronary rings, and relaxation to acetylcholine (6.25 nmol/l-6.25 micromol/l) was significantly attenuated by obese concentrations of leptin (625 pmol/l) but not by physiological concentrations of leptin (250 pmol/l). The major findings of this investigation were as follows: 1) the ObRb is present in coronary arteries and coupled to pharmacological, nitric oxide-dependent vasodilation; and 2) hyperleptinemia produces significant coronary endothelial dysfunction.

Recent studies implicate channels of the transient receptor potential vanilloid family (e.g., TRPV1) in regulating vascular tone; however, little is known about these channels in the coronary circulation. Furthermore, it is unclear whether metabolic syndrome alters the function and/or expression of TRPV1. We tested the hypothesis that TRPV1 mediates coronary vasodilation through endothelium-dependent mechanisms that are impaired by the metabolic syndrome. Studies were conducted on coronary arteries from lean and obese male Ossabaw miniature swine. In lean pigs, capsaicin, a TRPV1 agonist, relaxed arteries in a dose-dependent manner (EC50 = 116 +/- 41 nM). Capsaicin-induced relaxation was blocked by the TRPV1 antagonist capsazepine, endothelial denudation, inhibition of nitric oxide synthase, and K+ channel antagonists. Capsaicin-induced relaxation was impaired in rings from pigs with metabolic syndrome (91 +/- 4% vs. 51 +/- 10% relaxation at 100 microM). TRPV1 immunoreactivity was prominent in coronary endothelial cells. TRPV1 protein expression was decreased 40 +/- 11% in obese pigs. Capsaicin (100 microM) elicited divalent cation influx that was abolished in endothelial cells from obese pigs. These data indicate that TRPV1 channels are functionally expressed in the coronary circulation and mediate endothelium-dependent vasodilation through a mechanism involving nitric oxide and K+ channels. Impaired capsaicin-induced vasodilation in the metabolic syndrome is associated with decreased expression of TRPV1 and cation influx.

The role of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in regulation of coronary microvascular function is widely appreciated, but molecular and functional changes underlying the deleterious influence of metabolic syndrome (MetS) have not been determined. Male Ossabaw miniature swine consumed for 3-6 mo a normal diet (11% kcal from fat) or an excess-calorie atherogenic diet that induces MetS (45% kcal from fat, 2% cholesterol, 20% kcal from fructose). MetS significantly impaired coronary vasodilation to the BK(Ca) opener NS-1619 in vivo (30-100 microg) and reduced the contribution of these channels to adenosine-induced microvascular vasodilation in vitro (1-100 microM). MetS reduced whole cell penitrem A (1 microM)-sensitive K(+) current and NS-1619-activated (10 microM) current in isolated coronary vascular smooth muscle cells. MetS increased the concentration of free intracellular Ca(2+) and augmented coronary vasoconstriction to the L-type Ca(2+) channel agonist BAY K 8644 (10 pM-10 nM). BK(Ca) channel alpha and beta(1) protein expression was increased in coronary arteries from MetS swine. Coronary vascular dysfunction in MetS is related to impaired BK(Ca) channel function and is accompanied by significant increases in L-type Ca(2+) channel-mediated coronary vasoconstriction.

The metabolic syndrome is associated with activation of the renin-angiotensin system. However, whether the coronary vascular response to ANG II is altered under this condition is unknown. Experiments were conducted in control and chronically high-fat-fed dogs with the prediabetic metabolic syndrome both in vitro (isolated coronary arterioles, 60-110 microm) and in vivo (anesthetized and conscious). We found that plasma renin activity and ANG II levels are elevated in high-fat-fed dogs and that this increase in ANG II is associated with a significant increase in ANG II-mediated coronary vasoconstriction in isolated coronary arterioles and in anesthetized open-chest dogs. The vasoconstriction to ANG II is abolished by ANG II type 1 (AT1) receptor blockade. In conscious chronically instrumented dogs, AT1 receptor blockade with telmisartan improved the balance between coronary blood flow and myocardial oxygen consumption in the high-fat-fed dogs but not in normal control dogs, i.e., the relationship between coronary venous Po2 and myocardial oxygen consumption was shifted upward, toward normal control values. Quantitative assessment of coronary arteriolar AT1 and ANG II type 2 (AT2) receptor mRNA levels by real-time PCR revealed no significant difference between normal control and high-fat-fed dogs; however, Western blot analysis showed a significant increase in AT1 receptor protein level with no change in AT2 receptor protein density. These findings indicate that AT1 receptor-mediated coronary constriction is augmented in the prediabetic metabolic syndrome and contributes to impaired control of coronary blood flow via increases in circulating ANG II and/or coronary arteriolar AT1 receptor density.

ϩ (KV) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H2O2, as responses were attenuated by the KV channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that KV channels participate in coronary reactive hyperemia and examined the role of KV channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 Ϯ 5% and inhibited hyperemic volume 32 Ϯ 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or N G -nitro-L-arginine methyl ester (L-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or L-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 Ϯ 6% block at 9 g/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 M correolide, a selective KV1 antagonist (76 Ϯ 7% and 47 Ϯ 2% block, respectively, at 1 M). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 Ϯ 6% block at 10 g/min) and by correolide in vitro (29 Ϯ 4% block at 1 M). KV current in smooth muscle cells was inhibited by 4-AP (IC50 1.1 Ϯ 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for KV1 family members was detected in coronary arteries. Our data indicate that KV channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine-and NO-induced vasodilation. ischemic vasodilation; adenosine; 4-aminopyridine; delayed rectifier potassium channel; vascular smooth muscle IN THE CORONARY CIRCULATION, a brief period of ischemia is normally followed by a large and transient compensatory increase in blood flow. This phenomenon of reactive hyperemia, different from active (also known as functional or metabolic) hyperemia, is thought to represent a repayment of blood flow debt and is attributed to the accumulation of ischemic vasodilator metabolites. Evidence supports both adenosine and nitric oxide (NO) as mediators of reactive hyperemia (2, 4, 12, 52). Importantly, however, neither block of adenosine nor NO signaling can completely abolish reactive hyperemia (56). Thus the mechanisms of reactive hyperemia remain incompletely understood. Moreover, other mediators have been suggested, and it is likely that future studies will identify additional candidates. Rather than focus on putative metabolites underlying reactive hyperemia, we have turned our attention to possible end-effectors in vascular smooth muscle. K ϩ channels are likely targets of vasodilator metabolites, because K ϩ channels determine membrane potential and thus vascular tone (27,35). Previous studies have focused on Ca 2ϩ /voltage-sensitive (BK Ca ) and ATP-dependent (K ATP ) K ϩ channels. To date, only one study suggests a role for BK C...

Decrease in cardiac contractility is a hallmark of chronic diabetes. Previously we showed that this defect results, at least in part, from a dysfunction of the type 2 ryanodine receptor calcium-release channel (RyR2). The mechanism(s) underlying RyR2 dysfunction is not fully understood. The present study was designed to determine whether non-cross-linking advanced glycation end products (AGEs) on RyR2 increase with chronic diabetes and if formation of these post-translational complexes could be attenuated with insulin treatment. Overnight digestion of RyR2 from 8-week control animals (8C) with trypsin afforded 298 peptides with monoisotopic mass (M؉H ؉ ) >500. Digestion of RyR2 from 8-week streptozotocin-induced diabetic animals (8D) afforded 21% fewer peptides, whereas RyR2 from 6-week diabetic/2-week insulin-treated animals generated 304 peptides. Using an in-house PERLscript algorithm, search of matrix-assisted laser desorption ionization-time of flight mass data files identified several M؉H ؉ peaks corresponding to theoretical RyR2 peptides with single N ⑀ -(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, or 1-alkyl-2-formyl-3,4-glycosyl pyrrole modification that were present in 8D but not 8C. Insulin treatment minimized production of some of these nonenzymatic glycation products. These data show for the first time that AGEs are formed on intracellular RyR2 during diabetes. Because AGE complexes are known to compromise protein activity, these data suggest a potential mechanism for diabetesinduced RyR2 dysfunction. Diabetes 52:1825-1836, 2003 A significant percentage of patients with diabetes (both type 1 and type 2) develop a unique cardiomyopathy that is independent of coronary atherosclerosis (1-3). This "diabetic cardiomyopathy" as it is termed starts off with asymptomatic left ventricular diastolic dysfunction (slowing of relaxation kinetics). As the disease progresses, systolic function becomes compromised, leading to an increase in incidence of morbidity and mortality (4 -6).The release of calcium ions from internal sarcoplasmic reticulum via the type 2 ryanodine receptor calciumrelease channel (RyR2) is an integral step in the cascade of events leading to cardiac muscle contraction (7). We and others have shown that expression of this protein decreases in hearts of chronic diabetic patients (8,9) as well as in the streptozotocin (STZ)-induced diabetic rats (10 -13). Using the latter model, we found that in addition to a decrease in expression of RyR2, its functional integrity is also compromised in diabetes (14,15). This dysfunction is manifested as a decrease in RyR2 ability to bind the specific ligand [ 3 H]ryanodine and a slowing in its electrophoretic mobility using denaturing SDS-PAGE.Two distinct and separate types of post-translational modifications are likely to be induced by diabetes. First, it is well known that metabolic changes brought about by diabetes increase production of reactive oxygen species (e.g., -18]). These free radical and nonradical species react with several a...