Background This investigation examined the mechanisms by which coronary perivascular adipose tissue (PVAT)-derived factors influence vasomotor tone and the PVAT proteome in lean vs. obese swine. Methods and Results Coronary arteries from Ossabaw swine were isolated for isometric tension studies. We found that coronary (P=0.03) and mesenteric (P=0.04), but not subcutaneous adipose tissue, augmented coronary contractions to KCl (20 mM). Inhibition of CaV1.2 channels with nifedipine (0.1 μM) or diltiazem (10 μM) abolished this effect. Coronary PVAT increased baseline tension and potentiated constriction of isolated arteries to PGF2α in proportion to the amount of PVAT present (0.1–1.0 g). These effects were elevated in tissues obtained from obese swine and were observed in intact and endothelium denuded arteries. Coronary PVAT also diminished H2O2-mediated vasodilation in lean, and to a lesser extent in obese arteries. These effects were associated with alterations in the obese coronary PVAT proteome (detected 186 alterations) and elevated voltage-dependent increases in intracellular [Ca2+] in obese smooth muscle cells. Further studies revealed that a Rho-kinase inhibitor fasudil (1 μM) significantly blunted artery contractions to KCl and PVAT in lean, but not obese swine. Calpastatin (10 μM) also augmented contractions to levels similar to that observed in the presence of PVAT. Conclusions Vascular effects of PVAT vary according to anatomic location and are influenced by an obese phenotype. Augmented contractile effects of obese coronary PVAT are related to alterations in the PVAT proteome (e.g. calpastatin), Rho-dependent signaling, and the functional contribution of K+ and CaV1.2 channels to smooth muscle tone.

BackgroundIn humans there is a positive association between epicardial adipose tissue (EAT) volume and coronary atherosclerosis (CAD) burden. We tested the hypothesis that EAT contributes locally to CAD in a pig model.MethodsOssabaw miniature swine (n = 9) were fed an atherogenic diet for 6 months to produce CAD. A 15 mm length by 3–5 mm width coronary EAT (cEAT) resection was performed over the middle segment of the left anterior descending artery (LAD) 15 mm distal to the left main bifurcation. Pigs recovered for 3 months on atherogenic diet. Intravascular ultrasound (IVUS) was performed in the LAD to quantify atheroma immediately after adipectomy and was repeated after recovery before sacrifice. Coronary wall biopsies were stained immunohistochemically for atherosclerosis markers and cytokines and cEAT was assayed for atherosclerosis-related genes by RT-PCR. Total EAT volume was measured by non-contrast CT before each IVUS.ResultsCircumferential plaque length increased (p < 0.05) in the proximal and distal LAD segments from baseline until sacrifice whereas plaque length in the middle LAD segment underneath the adipectomy site did not increase. T-cadherin, scavenger receptor A and adiponectin were reduced in the intramural middle LAD. Relative to control pigs without CAD, 11β-hydroxysteroid dehydrogenase (11βHSD-1), CCL19, CCL21, prostaglandin D2 synthase, gp91phox [NADPH oxidase], VEGF, VEGFGR1, and angiotensinogen mRNAs were up-regulated in cEAT. EAT volume increased over 3 months.ConclusionIn pigs used as their own controls, resection of cEAT decreased the progression of CAD, suggesting that cEAT may exacerbate coronary atherosclerosis.

Metabolic syndrome (MetS) doubles the risk of adverse cardiovascular events. Glucagon-like peptide 1 (GLP-1) receptor agonists induce weight loss, increase insulin secretion, and improve glucose tolerance. Studies in healthy animals suggest cardioprotective properties of GLP-1 receptor agonists, perhaps partially mediated by improved sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity. We examined the acute effect of GLP-1 receptor agonists on coronary smooth muscle cells (CSM) enzymatically isolated from lean, healthy Ossabaw miniature swine. Intracellular Ca2+ handling was interrogated with fura-2. The GLP-1 receptor agonist exenatide activated SERCA but did not alter other Ca2+ transporters. Further, we tested the hypothesis that chronic, in vivo treatment with GLP-1 receptor agonist AC3174 would attenuate coronary artery disease (CAD) in swine with MetS. MetS was induced in 20 swine by 6 months' feeding of a hypercaloric, atherogenic diet. Swine were then randomized (n = 10/group) into placebo or AC3174 treatment groups and continued the diet for an additional 6 months. AC3174 treatment attenuated weight gain, increased insulin secretion, and improved glucose tolerance. Intravascular ultrasound and histology showed no effect of AC3174 on CAD. MetS abolished SERCA activation by GLP-1 receptor agonists. We conclude that MetS confers vascular resistance to GLP-1 receptor agonists, partially through impaired cellular signaling steps involving SERCA.

Coronary smooth muscle (CSM) phenotype is affected by intracellular Ca2+ regulation and we have previously correlated changes in CSM Ca2+ signaling from Ossabaw swine with varying stages of coronary artery disease (CAD). Ossabaw swine are a model of metabolic syndrome (MetS) and CAD. Three year old pigs were fed an excess‐calorie, atherogenic or control diet for 6 months. CAD progression, coronary artery function, and CSM Ca2+ signaling were assessed. MetS coronaries had advanced, lipid‐laden atherosclerotic lesions containing calcium deposits, while lean had no CAD. In isometric tension studies diseased coronary rings generated significantly less tension than healthy rings in response to depolarization with 20 mM K+. CSM were enzymatically isolated and loaded with the Ca2+ indicator, fura‐2 AM. Diseased CSM showed less K+‐induced Ca2+ influx compared to healthy cells. Diseased cells recovered less from the cytosolic Ca2+ increase back to baseline, suggesting impaired Ca2+ buffering. In physiologic salt solution (PSS), there was no difference in peak Ca2+ transient elicited by caffeine‐induced intracellular Ca2+ store release. The caffeine‐induced Ca2+ transient in Ca2+‐free PSS, which represents exclusively sarcoplasmic reticulum (SR) Ca2+ release, was decreased in diseased cells. Taken together, these data suggest that lesser SR Ca2+ release elicited greater Ca2+‐activated Ca2+ influx in diseased CSM. After caffeine challenge healthy CSM buffered Ca2+ significantly below basal Ca2+ compared to diseased CSM. Overall, the data suggest impaired CSM SR Ca2+ buffering, decreased SR Ca2+ store, and increased Ca2+‐activated Ca2+ influx in CAD. Grant Funding Source: Supported by NIH HL062552, HL092245, NIH/NCATS CTSI TL1 TR000162

Chronic effects of glucagon‐like peptide (GLP‐1) receptor agonists were studied in Ossabaw swine with metabolic syndrome (MetS). MetS was induced by hypercaloric atherogenic diet for 6 months. Subcutaneous injections were given twice daily for 6 months after induction of MetS. Consistent with clinical effects, food consumption and weight gain were attenuated. MetS increases coronary artery disease (CAD) and coronary smooth muscle (CSM) Ca2+ dysfunction. CSM cells were isolated enzymatically from the coronary arteries and Ca2+ was digitally imaged with fluorescent fura‐2. In normal physiological salt solution we released sarcoplasmic reticulum (SR) stores with caffeine. Treated cells showed a larger Ca2+ transient, ΔF360/380=0.26±0.02, compared to placebo cells, ΔF360/380=0.16±0.01 (p<0.001). In lean CSM, acute exposure to exenatide markedly increased activity of the sarcoplasmic‐endoplasmic reticulum Ca2+ ATPase (SERCA). This activity was confirmed by cyclopizaonic acid block. The increased SERCA activity may explain increased SR Ca2+ store in chronic MetS CSM. Acute exposure of placebo MetS CSM to exenatide has shown no effect on SERCA activity (n=43). Evidence for myocardial resistance to GLP‐1 in MetS is consistent with the lack of acute response. Our interpretation is that chronic exposure attenuates the decline in CSM Ca2+ handling associated with more severe CAD and dedifferentiation of CSM.(Support: NIH HL062552, TL1 TR000162, Amylin Pharmaceuticals Inc.)

Myocardial ischemia activates AMP kinase (AMPK), which modulates electrophysiology. A spontaneous point mutation from Val199→Ile in the AMPK γ3 subunit of Ossabaw swine impairs AMPK function. We hypothesized that ischemia would elicit greater electrocardiographic ST segment elevation (STE) in AMPK mutant compared to non‐mutant swine. Ischemia induced by balloon occlusion of the circumflex artery was verified by angiography. AMPK mutants showed significant STE within 3 min of ischemia, while non‐mutants showed an STE of ~2mm during 15 min of ischemia. Percent survival to the STE criterion was greater in non‐mutants vs. mutants (p<0.05). Intracoronary infusion of the AMPK activator AICAR (0.5 mM) for 10 min before coronary occlusion reduced profound STE in AMPK mutants, but completely prevented STE in non‐mutants. Percent survival to the STE criterion again was greater in non‐mutants vs. mutants during ischemia. Compound C, an inhibitor of AMPK, trended to decrease time to reach STE criterion compared to AICAR. Metabolic syndrome induced by hypercaloric atherogenic diet was similar in mutants and non‐mutants. Mutants reached the STE criterion in 4 min, but the non‐mutants did not in the 15 min occlusion, thus showing a striking cardiac phenotype. In conclusion, a functional cardiac AMPK and pharmacological AMPK activation decrease ischemia‐induced ST segment elevation. (Support: NIH HL062552)

Coronary artery disease (CAD) is the accumulation of atherosclerotic plaque within the lumen of large epicardial conduit arteries. Past research has shown strong positive association between coronary epicardial adipose tissue (cEAT) and CAD. We tested a contributory role for cEAT in CAD by surgical excision of cEAT and longitudinal measures of CAD with intravascular ultrasound (IVUS). Ossabaw swine (N=8) were fed an atherogenic diet for 6 months to produce metabolic syndrome and CAD. IVUS was performed prior to an adipectomy of the cEAT on the proximal half of the left anterior descending (LAD) coronary artery, while the circumflex (CFX) was left as a sham control. Recovery of 3 months was allowed on the same diet, followed by IVUS measures of the LAD and CFX to determine atheroma changes. As predicted, there was an increase in CAD during recovery (p<0.05). The increase in the proximal LAD was only 10% of that in the CFX. Likewise an increase in the proximal and middle LAD combined was 38% of the same CFX region (p<0.05). Computed tomography (CT) showed increases in pericardial fat and association with CAD during recovery, but cEAT at the adipectomy site could not be quantified with CT. We conclude these data suggest cEAT removal decreased the progression of coronary atherosclerosis. (Support: NIH HL062552, Cardiometabolic Disease Research Foundation, Memphis, TN)

Coronary smooth muscle (CSM) cell Ca2+ regulation was studied in the Ossabaw swine model of metabolic syndrome (MetS) and coronary artery disease (CAD). MetS was induced by excess calorie atherogenic diet for 9 or 12 months and compared to lean controls on normal diet. Isometric tension studies on isolated coronary arteries revealed that 9 months of MetS increased tension development to KCl depolarization vs. lean controls, whereas 12 months reduced tension vs. control. MetS increases CAD and CSM Ca2+ dysregulation. CSM were isolated enzymatically and digitally imaged with the fluorescent Ca2+ indicator fura‐2. There was no difference in basal Ca2+ levels between groups. We released sarcoplasmic reticulum (SR) Ca2+ stores with caffeine. The peak Ca2+ release was increased in 9 month and decreased in 12 month MetS vs. lean. This Ca2+ response largely represents the caffeine‐sensitive SR Ca2+ store capacity. After the SR store depletion, a sustained Ca2+ signal above basal levels remained in the 9 month MetS group, reflecting store‐operated Ca2+ entry and decreased Ca2+ extrusion ability. Intravascular ultrasound and micro‐computed tomography imaging showed greater atherosclerosis and extracellular calcification in 12 vs. 9 month MetS. Collectively, these data suggest that CSM undergo dedifferentiation from a contractile to an osteogenic phenotype with progressively increasing CAD. (NIH HL062552, HL092245)