B-type natriuretic peptide limits infarct size in rat isolated hearts via KATP channel opening

B-type natriuretic peptide (BNP) has been reported to be released from the myocardium during ischemia. We hypothesized that BNP mediates cardioprotection during ischemia-reperfusion and examined whether exogenous BNP limits myocardial infarction and the potential role of ATP-sensitive potassium (K(ATP)) channel opening. Langendorff-perfused rat hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. The control infarct-to-risk ratio was 44.8 +/- 4.4% (means +/- SE). BNP perfused 10 min before ischemia limited infarct size in a concentration-dependent manner, with maximal protection observed at 10(-8) M (infarct-to-risk ratio: 20.1 +/- 5.2%, P < 0.01 vs. control), associated with a 2.5-fold elevation of myocardial cGMP above the control value. To examine the role of K(ATP) channel opening, glibenclamide (10(-6) M), 5-hydroxydecanoate (5-HD; 10(-4) M), or HMR-1098 (10(-5) M) was coperfused with BNP (10(-8) M). Protection afforded by BNP was abolished by glibenclamide or 5-HD but not by HMR-1098, suggesting the involvement of putative mitochondrial but not sarcolemmal K(ATP) channel opening. We conclude that natriuretic peptide/cGMP/K(ATP) channel signaling may constitute an important injury-limiting mechanism in myocardium.     

Effect of classic preconditioning on the gene expression pattern of rat hearts: a DNA microarray study

With the aim to enhance the plant vitamin E content, the barley gene encoding 4-hydroxyphenylpyruvate dioxygenase was overexpressed in tobacco plants under control of the 35S promoter. Transgenic lines have a higher capacity for homogentisate biosynthesis as evident by a more than 10-fold higher resistance towards the bleaching herbicide sulcotrione. Seeds from transgenic lines have an up to two-fold enhanced level of vitamin E without a change in the ratio of γ-tocopherol and γ-tocotrienol. While the vitamin E content is not affected in leaves, the level of plastoquinone is enhanced in leaves of transgenic lines during leaf senescence.     

Interaction between Connexin 43 and nitric oxide synthase in mice heart mitochondria

Connexin 43 (Cx43), which is highly expressed in the heart and especially in cardiomyocytes, interferes with the expression of nitric oxide synthase (NOS) isoforms. Conversely, Cx43 gene expression is down‐regulated by nitric oxide derived from the inducible NOS. Thus, a complex interplay between Cx43 and NOS expression appears to exist. As cardiac mitochondria are supposed to contain a NOS, we now investigated the expression of NOS isoforms and the nitric oxide production rate in isolated mitochondria of wild‐type and Cx43‐deficient (Cx43^{Cre‐ER(T)/fl}) mice hearts. Mitochondria were isolated from hearts using differential centrifugation and purified via Percoll gradient ultracentrifugation. Isolated mitochondria were stained with an antibody against the mitochondrial marker protein adenine‐nucleotide‐translocator (ANT) in combination with either a neuronal NOS (nNOS) or an inducible NOS (iNOS) antibody and analysed using confocal laser scanning microscopy. The nitric oxide formation was quantified in purified mitochondria using the oxyhaemoglobin assay. Co‐localization of predominantly nNOS (nNOS: 93 ± 4.1%; iNOS: 24.6 ± 7.5%) with ANT was detected in isolated mitochondria of wild‐type mice. In contrast, iNOS expression was increased in Cx43^{Cre‐ER(T)/fl} mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43^{Cre‐ER(T)/fl} mitochondria (0.14 ± 0.02 nmol/min./mg protein) in comparison to wild‐type mitochondria (0.24 ± 0.02 nmol/min./mg). These are the first data demonstrating, that a reduced mitochondrial Cx43 content is associated with a switch of the mitochondrial NOS isoform and the respective mitochondrial rate of nitric oxide formation.     

Rapid pacing-induced preconditioning is recaptured by farnesol treatment in hearts of cholesterol-fed rats: Role of polyprenyl derivatives and nitric oxide

Sodium selenate, administered intraperitoneally (i.p.), resulted in an improvement in glucose tolerance in treated diabetic rats. Fed rat plasma glucose levels were reduced by selenate treatment in streptozotocin diabetic rats. The lowest values of blood glucose were reached within 3 weeks of beginning the treatment. Food and fluid consumption was reduced in treated compared to untreated diabetic rats. Diabetic treated rats did not release insulin in response to a glucose challenge and insulin release in response to a challenge was markedly reduced in control treated rats. Assessment of heart function using a working heart apparatus showed that treated diabetic rats with improved blood glucose levels had normal heart function at 8 weeks of diabetes in contrast to hearts from non-treated diabetics. This study extends previous observations on the in vivo insulin-like effects of sodium selenate.     

Cholesterol diet leads to attenuation of ischemic preconditioning-induced cardiac protection: the role of connexin 43

Cardioprotection by ischemic preconditioning (IP) was abolished in connexin 43 (Cx43)-deficient mice due to loss of Cx43 located in mitochondria rather than at the sarcolemma. IP is lost in hyperlipidemic rat hearts as well. Since changes in mitochondrial Cx43 in hyperlipidemia have not yet been analyzed, we determined total and mitochondrial Cx43 levels in male Wistar rats fed a laboratory chow enriched with 2% cholesterol or normal chow for 12 wk. Hearts were isolated and perfused according to Langendorff. After a 10-min perfusion, myocardial tissue cholesterol, superoxide, and nitrotyrosine contents were measured and Cx43 content in whole heart homogenate and a mitochondrial fraction determined. In the cholesterol-fed group, tissue cholesterol and superoxide formation was increased (P < 0.05), while total Cx43 content remained unchanged. Mitochondrial total and dephosphorylated Cx43 content decreased. Hearts were subjected to an IP protocol (3 × 5 min ischemia-reperfusion) or time-matched aerobic perfusion followed by 30-min global ischemia and 5-min reperfusion. IP reduced infarct size in normal but not in cholesterol-fed rats. At 5-min reperfusion following 30-min global ischemia, the total and dephosphorylated mitochondrial Cx43 content was increased, which was abolished by IP in both normal and high-cholesterol diet. In conclusion, loss of cardioprotection by IP in hyperlipidemia is associated with a redistribution of both sarcolemmal and mitochondrial Cx43.     

Nitrate tolerance does not increase production of peroxynitrite in the heart

Clinical studies have suggested that long-term nitrate treatment does not improve and may even worsen cardiovascular mortality, and the possible role of nitrate tolerance has been suspected. Nitrate tolerance has been recently shown to increase vascular superoxide and peroxynitrite production leading to vascular dysfunction. Nevertheless, nitrates exert direct cardiac effects independent from their vascular actions. Therefore, we investigated whether in vivo nitroglycerin treatment leading to vascular nitrate tolerance increases cardiac formation of nitric oxide (NO), reactive oxygen species, and peroxynitrite, thereby leading to cardiac dysfunction. Nitrate tolerance increased bioavailability of NO in the heart without increasing formation of reactive oxygen species. Despite elevated myocardial NO, neither cardiac markers of peroxynitrite formation nor cardiac mechanical function were affected by nitroglycerin treatment. However, serum free nitrotyrosine, a marker for systemic peroxynitrite formation, was significantly elevated in nitroglycerin-treated animals. This is the first demonstration that, although the systemic effects of nitroglycerin may be deleterious due to enhancement of extracardiac peroxynitrite formation, nitroglycerin does not result in oxidative damage in the heart.     

Preconditioning decreases ischemia/reperfusion-induced release and activation of matrix metalloproteinase-2

Release and activation of matrix metalloproteinases (MMPs) significantly contribute to myocardial stunning injury immediately after ischemia and reperfusion, however, their role in preconditioning remains unknown. We therefore examined the effects of preconditioning and subsequent ischemia/reperfusion on MMP activity in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three consecutive 5-min periods of global ischemia interspersed with 5 min of reperfusion) followed by 30 min ischemia and 5 min reperfusion. To measure MMP release, coronary effluent was collected: (a) during aerobic perfusion, (b) in reperfusion following each preconditioning ischemia, and (c) during the final reperfusion following test ischemia. MMP-2 activities could be detected by gelatin zymography in the ventricles and coronary effluent samples from the perfused hearts. The levels of MMP-2 activity in the effluent were markedly increased in effluent following test ischemia from control hearts without preconditioning. This was accompanied by a decrease in corresponding tissue MMP activities. Preconditioning significantly decreased the MMP-2 activity in the coronary effluent following test ischemia/reperfusion and preserved the MMP-2 protein content and activity in the myocardium. Our results demonstrate that classic preconditioning inhibits ischemia/reperfusion induced release and activation of MMP-2. These results suggest that preconditioning may exert part of its cardioprotective effects through the reduction of MMP-2 release.