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.

Cytochrome P450 2J2 is protective against global cerebral ischemia in transgenic mice.

Cytochrome P450 epoxygenase metabolites of arachidonic acid, EETs, have multiple cardiovascular effects, including reduction of blood pressure, protection against myocardial ischemia-reperfusion injury, and attenuation of endothelial apoptosis. This study investigated the hypothesis that transgenic mice with endothelial overexpression of CYP2J2 (Tie2-CYP2J2-Tr) would be protected against global cerebral ischemia induced by bilateral common carotid artery occlusion (BCCAO) and action mechanisms of EETs on cerebral ischemia in cultures of astrocytes exposed to oxygen-glucose deprivation (OGD). Tie2-CYP2J2-Tr mice had significantly increased CYP2J2 expression, increased 14,15-EET production, increases regional cerebral blood flow (rCBF) and microvascular density, decreased ROS production, decreased brain infarct size and apoptosis after ischemia compared to wild type mice, these were associated with increased activation of the PI3K/AKT and apoptosis-related protein in ischemic brain. Addition of exogenous EETs or CYP2J2 transfection attenuated OGD-induced apoptosis in astrocytes via activation of PI3K/AKT and anti-apoptosis pathways. However, these effects were reduced by pretreatments with inhibitor of the PI3K (LY294002) and 14,15-EET (14,15-EEZE), respectively. These results indicate that CYP2J2 overexpression exerts marked neuroprotective effects against ischemic injury by a mechanism linked to increased level of circulating EETs and increases CBF and reduction of apoptosis.

Decreased tetrahydrobiopterin and disrupted association of Hsp90 with eNOS by hyperglycemia impair myocardial ischemic preconditioning.

Cardioprotection by ischemic preconditioning (IPC) is impaired during hyperglycemia, but the mechanisms underlying this phenomenon are poorly understood. This study investigated the role of hyperglycemia to adversely modulate tetrahydrobiopterin (BH(4)) and heat shock protein 90 (Hsp90) during cardioprotection by IPC. Rabbits or mice underwent 30 min of coronary occlusion followed by reperfusion with or without IPC in the presence or absence of hyperglycemia. IPC significantly (P < 0.05) decreased myocardial infarct size (46 ± 1 to 19 ± 2% of the area at risk in control and IPC rabbits, respectively) and increased BH(4) concentrations (HPLC; 7.6 ± 0.2 to 10.2 ± 0.3 pmol/mg protein, respectively), Hsp90-endothelial nitric oxide synthase (eNOS) association (coimmunoprecipitation and Western blotting in mice; 4.0 ± 0.3 to 5.4 ± 0.1, respectively), and the ratio of phosphorylated eNOS/total eNOS. These beneficial actions of IPC on infarct size, BH(4), Hsp90/eNOS, and phosphorylated eNOS were eliminated by hyperglycemia. Pretreatment of animals with the Hsp90 inhibitor geldanamycin (0.6 mg/kg) or the BH(4) synthesis inhibitor diamino-6-hydroxypyrimidine (1.0 g/kg) also eliminated cardioprotection produced by IPC. In contrast, the BH(4) precursor sepiapterin (2 mg/kg iv) restored the beneficial effects of IPC on myocardial BH(4) concentrations, eNOS dimerization, and infarct size during hyperglycemia. A-23871 increased Hsp90-eNOS association (0.33 ± 0.06 to 0.59 ± 0.3) and nitric oxide production (184 ± 17%) in human coronary artery endothelial cells cultured in normal (5.5 mM) but not high (20 mM) glucose media. These data indicate that hyperglycemia eliminates protection by IPC via decreases in myocardial BH(4) concentration and disruption of the association of Hsp90 with eNOS. The results suggest that eNOS dysregulation may be a central mechanism of impaired cardioprotection during hyperglycemia.

Polymorphisms of MMP-3 and TIMP-4 genes affect angiographic coronary plaque progression in non-diabetic and type 2 diabetic patients.

BACKGROUND: This study examined whether genetic variants of matrix metallopeptidases (MMPs) and their tissue inhibitors (TIMPs) were associated with angiographic coronary plaque progression (PP) in type 2 diabetic and non-diabetic patients. METHODS: Four hundred and ninety-nine patients were grouped, who underwent coronary angiography and received repeat examinations after 1-y follow-up. Twelve functional polymorphisms of MMPs and TIMPs were characterized. RESULTS: Genotype distribution and allele frequency of -1612 5A/6A MMP-3 and 3'UTR C/T TIMP-4 differed between patients with PP and those without in both diabetic and non-diabetic groups after Bonferroni's correction (all P<0.0041667, except for allele frequency of MMP-3 [P=0.007] and genotype/allele frequency of TIMP-4 [P=0.04 and P=0.016, respectively] in diabetes). MMP-3 and TIMP-4 polymorphisms were associated with changes in percent diameter stenosis and minimal lumen diameter in diabetic patients, and changes in cumulative coronary obstruction in both diabetic and non-diabetic patients (all P<0.05). Multivariable regression analysis revealed that hypertension, low HDL-C and genotypes of MMP-3 and TIMP-4 were independent determinants of PP in the whole patients, with these 2 genetic factors being associated with PP in diabetic and non-diabetic subgroups. CONCLUSION: This study demonstrated that MMP-3 and TIMP-4 polymorphisms affect angiographic coronary PP in type 2 diabetic and non-diabetic patients.

Ischemic postconditioning in pigs: no causal role for RISK activation.

Ischemic postconditioning (IPoC) reduces infarct size following ischemia/reperfusion. Whether or not phosphorylation of RISK (reperfusion injury salvage kinases) (AKT, ERK1/2, P70S6K, GSK3beta) is causal for protection by IPoC is controversial. We therefore studied the impact of RISK on IPoC in anesthetized pigs subjected to 90 minutes of left anterior descending coronary artery hypoperfusion and 120 minutes of reperfusion. In protocol 1, IPoC, by 6 cycles of 20/20 seconds of reperfusion/reocclusion (n=13), was compared with immediate full reperfusion (IFR) (n=15). In protocol 2, IPoC (n=4) or IFR (n=4) was performed with pharmacological RISK blockade by IC coinfusion of Wortmannin and U0126. Infarct size was determined by TTC staining, and the expression of phosphorylated RISK proteins by Western blot analysis in biopsies. In protocol 1, infarct size was 20+/-3% (percentage of area at risk; mean+/-SEM) with IPoC and 33+/-4% (P<0.05) with IFR. RISK phosphorylation increased with reperfusion but was not different between IPoC and IFR. In protocol 2, Wortmannin and U0126 blocked the increases in RISK phosphorylation during reperfusion, but infarct size was still smaller with IPoC (15+/-7%) than with IFR (35+/-6%; P<0.05).

Association of paraoxonase activity and coronary collateral flow.

OBJECTIVES: Paraoxonase is a high-density lipoprotein-bound antioxidant enzyme that inhibits atherosclerosis and endothelial dysfunction. Coronary collateral flow is a crucial clinical entity with significant impact on the cardiovascular morbidity and mortality. This study sought to determine the relationship between the degree of angiographically visible coronary collateral circulation and serum paraoxonase activity. METHODS: The study population included 98 patients (mean age=57.9+/-10.1 years, 65 men) with angiographically documented total occlusion in one of the major coronary arteries. Development of collaterals was classified by Rentrop's method. Patients were defined as having poorly developed collaterals for Rentrop grades 0 and 1 or well-developed collaterals for Rentrop grades 2 and 3. Serum paraoxonase and arylesterase activities were measured spectrophotometrically. RESULTS: Statistically significant differences between well and poorly developed collateral groups in respect to serum low-density lipoprotein cholesterol level (P=0.046), and serum paraoxonase (P=0.001), and arylesterase (P=0.014) activities were present. Serum low-density lipoprotein cholesterol level (chi=4.15, beta=-0.347, P=0.032) and serum paraoxonase activity (chi=10.43, beta=0.008, P=0.022) were independent predictors of well-developed coronary collateral flow. Serum paraoxonase activity gradually increased from collateral grade 0 to collateral grade 3 (analysis of variance P=0.003). Serum paraoxonase (r=0.362 and P<0.001) and arylesterase (r=0.245 and P=0.015) activities were both correlated with collateral flow grade. CONCLUSION: Findings of this study suggest that serum paraoxonase activity is independently associated with the degree of coronary collateral flow and reduced serum paraoxonase activity might represent a biochemical marker of impaired coronary collateral flow.