Cristine L. Heaps

Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

Background Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs). Methodology/Principal Findings The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10−8 to 10−5 M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium. Conclusion GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.

Effects of exercise training and hypercholesterolemia on adenosine activation of voltage-dependent K+ channels in coronary arterioles

Coronary arterioles from hypercholesterolemic swine display attenuated adenosine-mediated vasodilatation that is attributable to the elimination of voltage-dependent K(+) (Kv) channel stimulation. For the present study, we tested the hypotheses that exercise training would correct impaired adenosine-induced dilatation in coronary arterioles from hypercholesterolemic pigs through restoration of adenosine activation of Kv channels and that vasodilatation to the receptor-independent adenylyl cyclase activator, forskolin, would also be attenuated in arterioles from hypercholesterolemic pigs. Pigs were randomly assigned to a control (NC) or high-fat, high-cholesterol (HC) diet for 20 wk. Four weeks after the diet was initiated, pigs from both groups were assigned to exercise training (Ex; 5 days/wk for 16 wk) or sedentary (Sed) protocols, resulting in four groups of pigs: NC-Sed, NC-Ex, HC-Sed, and HC-Ex. Arterioles ( approximately 150 mum) from both HC-Sed and HC-Ex pigs displayed impaired adenosine-mediated dilatation that was attributable to the elimination of 4-aminopyridine (4-AP; 1 mM)-sensitive Kv channel activation compared with NC counterparts. Arteriolar smooth muscle whole cell Kv currents were significantly reduced in HC-Sed compared with NC-Sed, although HC-Ex and NC-Ex did not differ. Forskolin-mediated dilatation was attenuated by 4-AP (1 mM) and in a concentration-dependent manner by tetraethylammonium (TEA; 0.1-1 mM) in NC-Sed but not HC-Sed. Further, TEA-sensitive Kv currents were diminished in cells of HC-Sed compared with NC-Sed pigs. Quantitative RT-PCR revealed similar expression levels of Kv3.1 and 3.3 in arterioles of NC-Sed and HC-Sed swine with undetectable expression of Kv1.1, 3.2, and 3.4. Taken together, these results suggest that hypercholesterolemia-mediated attenuation of adenosine-induced vasodilatation in coronary arterioles is not corrected by exercise training and is likely attributable to an impairment in the pathway coupling adenylyl cyclase with a highly TEA-sensitive Kv channel isoform(s).

Exercise training enhances multiple mechanisms of relaxation in coronary arteries from ischemic hearts

Exercise training of coronary artery disease patients is of considerable interest, since it has been shown to improve vascular function and, thereby, enhance blood flow into compromised myocardial regions. However, the mechanisms underlying exercise-induced improvements in vascular function have not been fully elucidated. We tested the hypothesis that exercise training increases the contribution of multiple mediators to endothelium-dependent relaxation of coronary arteries in the underlying setting of chronic coronary artery occlusion. To induce gradual occlusion, an ameroid constrictor was placed around the proximal left circumflex coronary artery in Yucatan miniature swine. At 8 wk postoperatively, pigs were randomly assigned to sedentary or exercise (treadmill, 5 days/wk) regimens for 14 wk. Exercise training significantly enhanced the contribution of nitric oxide, prostanoids, and large-conductance Ca(2+)-dependent K(+) (BKCa) channels to endothelium-dependent, bradykinin-mediated relaxation in nonoccluded and collateral-dependent arteries. Combined nitric oxide synthase, prostanoid, and BKCa channel inhibition ablated the enhanced relaxation associated with exercise training. Exercise training significantly increased nitric oxide levels in response to bradykinin in endothelial cells isolated from nonoccluded and collateral-dependent arteries. Bradykinin treatment significantly increased PGI2 levels in all artery treatment groups and tended to be further enhanced after nitric oxide synthase inhibition in exercise-trained pigs. No differences were found in whole cell BKCa channel currents, BKCa channel protein levels, or arterial cyclic nucleotide levels. Although redundant, upregulation of parallel vasodilator pathways appears to contribute to enhanced endothelium-dependent relaxation, potentially providing a more refined control of blood flow after exercise training.

Ca2+ sensitization and PKC contribute to exercise training-enhanced contractility in porcine collateral-dependent coronary arteries

Exercise training enhances endothelium-dependent coronary vasodilatation, improving perfusion and contractile function of collateral-dependent myocardium. Paradoxically, studies from our laboratory have revealed increased Ca(2+)-dependent basal active tone in collateral-dependent arteries of exercise-trained pigs. In this study, we tested the hypothesis that exercise training enhances agonist-mediated contractile responses of collateral-dependent arteries by promoting Ca(2+) sensitization. Ameroid constrictors were surgically placed around the proximal left circumflex coronary (LCX) artery of female Yucatan miniature pigs. Eight weeks postoperatively, pigs were randomized into sedentary (pen confined) or exercise-training (treadmill run; 5 days/wk; 14 wk) groups. Arteries (∼150 μm luminal diameter) were isolated from the collateral-dependent and nonoccluded (left anterior descending artery supplied) myocardial regions, and measures of contractile tension or simultaneous tension and intracellular free Ca(2+) concentration levels (fura-2) were completed. Exercise training enhanced contractile responses to endothelin-1 in collateral-dependent compared with nonoccluded arteries, an effect that was more pronounced in the presence of nitric oxide synthase inhibition (N(ω)-nitro-l-arginine methyl ester; 100 μM). Contractile responses to endothelin-1 were not altered by coronary occlusion alone. Exercise training produced increased tension at comparable levels of intracellular free Ca(2+) concentration in collateral-dependent compared with nonoccluded arteries, indicative of exercise training-enhanced Ca(2+) sensitization. Inhibition of PKC (calphostin C; 1 μM), but not Rho-kinase (Y-27632, 10 μM; or hydroxyfasudil, 30 μM), abolished the training-enhanced endothelin-1-mediated contractile response. Exercise training also increased sensitivity to the PKC activator phorbol 12,13-dibutyrate in collateral-dependent compared with nonoccluded arteries. Taken together, these data reveal that exercise training enhances endothelin-1-mediated contractile responses in collateral-dependent coronary arteries likely via increased PKC-mediated Ca(2+) sensitization.

Fibronectin increases the force production of mouse papillary muscles via α5β1 integrin

The extracellular matrix (ECM) protein-integrin-cytoskeleton axis plays a central role as a mechanotransducing protein assemblage in many cell types. However, how the process of mechanotransduction and the mechanically generated signals arising from this axis affect myofilament function in cardiac muscle are not completely understood. We hypothesize that ECM proteins can regulate cardiac function through integrin binding, and thereby alter the intracellular calcium concentration ([Ca(2+)](i)) and/or modulate myofilament activation processes. Force measurements made in mouse papillary muscle demonstrated that in the presence of the soluble form of the ECM protein, fibronectin (FN), active force was increased significantly by 40% at 1 Hz, 54% at 2 Hz, 35% at 5 Hz and 16% at 9 Hz stimulation frequencies. Furthermore, increased active force in the presence of FN was associated with 12-33% increase in [Ca(2+)](i) and 20-50% increase in active force per unit Ca(2+). A function blocking antibody for α5 integrin prevented the effects of the FN on the changes in force and [Ca(2+)](i), whereas a function blocking α3 integrin antibody did not reverse the effects of FN. The effects of FN were reversed by an L-type Ca(2+) channel blocker, verapamil or PKA inhibitor. Freshly isolated cardiomyocytes exhibited a 39% increase in contraction force and a 36% increase in L-type Ca(2+) current in the presence of FN. Fibers treated with FN showed a significant increase in the phosphorylation of phospholamban; however, the phosphorylation of troponin I was unchanged. These results demonstrate that FN acts via α5β1 integrin to increase force production in myocardium and that this effect is partly mediated by increases in [Ca(2+)](i) and Ca(2+) sensitivity, PKA activation and phosphorylation of phospholamban.

Exercise Training-Induced Adaptations in Mediators of Sustained Endothelium-Dependent Coronary Artery Relaxation in a Porcine Model of Ischemic Heart Disease

The aim of this study was to test the hypothesis that exercise training enhances sustained relaxation to persistent endothelium‐dependent vasodilator exposure via increased nitric oxide contribution in small coronary arteries of control and ischemic hearts.

Exercise Training-Enhanced, Endothelium-Dependent Dilation Mediated by Altered Regulation of BKCa Channels in Collateral-Dependent Porcine Coronary Arterioles

Test the hypothesis that exercise training increases the contribution of BKCa channels to endothelium‐mediated dilation in coronary arterioles from collateral‐dependent myocardial regions of chronically occluded pig hearts and may function downstream of H2O2.

Myosin phosphatase isoforms and related transcripts in the pig coronary circulation and effects of exercise and chronic occlusion.

Myosin phosphatase (MP) is a key target of signaling pathways that regulate smooth muscle tone and blood flow. Alternative splicing of MP targeting subunit (MYPT1) exon 24 (E24) generates isoforms with variable presence of a C-terminal leucine zipper (LZ) required for activation of MP by NO/cGMP. Here we examined the expression of MP and associated genes in a disease model in the coronary circulation. Female Yucatan miniature swine remained sedentary or were exercise-trained beginning eight weeks after placement of an ameroid constrictor around the left circumflex (LCX) artery. Fourteen weeks later epicardial arteries (~1mm) and resistance arterioles (~125 μm) were harvested and assayed for gene expression. MYPT1 isoforms were distinct in the epicardial arteries (E24-/LZ+) and resistance arterioles (E24+/LZ-) and unchanged by exercise training or coronary occlusion. MYPT1, CPI-17 and PDE5 mRNA levels were not different between arteries and arterioles while Kir2.1 and eNOS were 6.6-fold and 3.9-fold higher in the arterioles. There were no significant changes in transcript abundance in epicardial arteries of the collateralized (LCX) vs. non-occluded left anterior descending (LAD) territories, or in exercise-trained vs. sedentary pigs. There was a significant 1.2 fold increase in CPI-17 in collateral-dependent arterioles, independent of exercise, and a significant 1.7 fold increase in PDE5 in arterioles from exercise-trained pigs, independent of occlusion. We conclude that differences in MYPT1 E24 (LZ) isoforms, eNOS, and Kir2.1 distinguish epicardial arteries and resistance coronary arterioles. Up-regulation of coronary arteriolar PDE5 by exercise and CPI-17 by chronic occlusion could contribute to altered vasomotor responses and requires further study.

Adaptations of the Endothelin System After Exercise Training in a Porcine Model of Ischemic Heart Disease

To the test the hypothesis that exercise training would increase endothelin‐mediated vasoconstriction in collateral‐dependent arteries via enhanced contribution of ETA.

Mesenteric Lymphatic Vessels Adapt to Mesenteric Venous Hypertension by Becoming Weaker Pumps

Lymphangions, the segments of lymphatic vessels between two adjacent lymphatic valves, actively pump lymph. Acute changes in transmural pressure and lymph flow have profound effects on lymphatic pump function in vitro. Chronic changes in pressure and flow in vivo have also been reported to lead to significant changes in lymphangion function. Because changes in pressure and flow are both cause and effect of adaptive processes, characterizing adaptation requires a more fundamental analysis of lymphatic muscle properties. Therefore, the purpose of the present work was to use an intact lymphangion isovolumetric preparation to evaluate changes in mesenteric lymphatic muscle mechanical properties and the intracellular Ca(2+) in response to sustained mesenteric venous hypertension. Bovine mesenteric veins were surgically occluded to create mesenteric venous hypertension. Postnodal mesenteric lymphatic vessels from mesenteric venous hypertension (MVH; n = 6) and sham surgery (Sham; n = 6) animals were isolated and evaluated 3 days after the surgery. Spontaneously contracting MVH vessels generated end-systolic active tension and end-diastolic active tension lower than the Sham vessels. Furthermore, steady-state active tension and intracellular Ca(2+) concentration levels in response to KCl stimulation were also significantly lower in MVH vessels compared with those of the Sham vessels. There was no significant difference in passive tension in lymphatic vessels from the two groups. Taken together, these results suggest that following 3 days of mesenteric venous hypertension, postnodal mesenteric lymphatic vessels adapt to become weaker pumps with decreased cytosolic Ca(2+) concentration.