H2S production in myocardial ischaemia and reperfusion

ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S190–S218

β1 (caspase-like), β2 (trypsin-like), and β5 (chymotrypsinlike). Ischemia alone in the absence of reperfusion inhibited only the β2 activity of the 20S proteasome but not the activities of any of the other 20S or the 26S proteasome subunits (n = 10, P < 0.01). These studies represent the first evidence that ischemia alone selectively inactivates the β2 activity of the 20S proteasome. Ischemia followed by 15 min reperfusion resulted in no further inhibition of the β2 activity but resulted in inhibition of the β1 and β5 activities of the 20S proteasome as well as the β1, β2 and β5 activities of the 26S (n = 5, P < 0.01). Further inhibition of the proteolytic activities of the 20S and 26S proteasomes occurred with longer reperfusion times (30 min, 1 h, 2 h, 4 h) suggesting that the free radicals produced during reperfusion alter the proteasomes ability to properly degrade substrates. A functional proteomic approach was used to delineate whether there were direct modifications of the proteasome subunits by reactive oxygen species (ROS); thus far, evidence supporting direct modifications of the proteasome complexes by ROS during ischemia is lacking, suggesting a regulatory mechanism that was independent of ROS modification of proteasome subunits. Keywords: Proteasome; Ischemia; Protein degradation doi:10.1016/j.yjmcc.2007.03.597

Cardioprotective actions of upregulated L-arginine (L-Arg) transport Kylie Venardos, Laura Willems, David M. Kaye. Wynn Dept of Metabolic Cardiology, Baker Heart Research Institute, Melbourne, Australia Endothelial nitric oxide (NO) dysfunction and reactive oxygen species (ROS) play key pathophysiological roles in ischemia–reperfusion (I–R) injury. Our hypothesis is that insufficient substrate availability (L-Arg) for NO biosynthesis is pivotal in these events. In neonatal rat ventricular cardiomyocytes (NVCM) subjected to hypoxia-reoxygenation (H-R), decreased L-Arg uptake and NO production were observed, maximal after 2 h reoxygenation (to 42 ± 2% and 71 ± 4% of control, respectively, both p < 0.01). In parallel, mitochondrial membrane potential decreased (to ∼ 66% control after 2 h reoxygenation, p < 0.01) and ROS production increased (peaking to ∼ 130% of control after 2 h reoxygenation (p < 0.01). In addition, H2O2 elicited concentration-dependent decreases in NVCM L-Arg uptake and NO production, indicative of an inverse relationship between ROS and NO production. L-Arg supplementation (1 mM) during H-R significantly improved mitochondrial membrane potential, reduced ROS production and increased NO production at all time points measured. In isolated mouse hearts subjected to I-R, we also demonstrated that endothelial-specific overexpression of the L-Arg transporter CAT1 significantly improved recovery of left ventricular developed pressure (101 ± 10 vs. 60 ± 10 mm Hg, p < 0.05) and end diastolic pressure (17 ± 6 vs. 40 ± 5 mm Hg, p < 0.05) compared

S197

with wild-type controls, along with full restoration of coronary flow. Collectively, our results strongly suggest increased L-Arg availability restores NO levels and reduces oxidative stress, thereby improving the outcome following ischemic insults. Keywords: Ischemia–reperfusion; L-Arginine: NO; Oxidative stress doi:10.1016/j.yjmcc.2007.03.598

Effects of ischemia and reperfusion on contraction, intracellular Ca2+ and cell viability in male and female myocytes J.L. Ross, S.E. Howlett. Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada Male hearts are more sensitive to ischemia–reperfusion (I/R) injury than female hearts. As elevated intracellular Ca2+ is implicated in I/R injury, these differences may reflect changes in Ca2+ homeostasis in individual myocytes. We investigated sex differences in contraction, Ca2+ homeostasis and cell viability in ventricular myocytes exposed to I/R. Male and female rat myocytes were exposed to simulated ischemia (hypoxia, hyperkalemia, hypercapnia acidosis, 0 glucose, lactate) for 20 min (37 °C) and reperfused with Tyrode's solution. Contractions and Ca2+ transients (Fura-2) were simultaneously recorded in field stimulated cells (4 Hz). Cell viabilty was measured with Trypan Blue. Contraction amplitudes decreased during ischemia in male and female cells. However, contractions recovered during reperfusion in female myocytes, but remained markedly depressed in male myocytes during reperfusion. In contrast, amplitudes of Ca2+ transients were similar in male and female myocytes throughout I/R. Furthermore, the increase in diastolic Ca2+ caused by exposure to ischemia was similar in cells from male and female rats. There was no difference in cell viability throughout I/R between male and female myocytes. Thus, although cell viability following I/R injury was similar in male and female myocytes, myocytes from male rats showed less recovery of contractile function in reperfusion compared to female cells. The sex differences in recovery of contractile function in reperfusion are not due to differences in intracellular Ca2+ homeostasis during I/R. Keywords: Ischemia and reperfusion; Cardiomyocyte; Calcium handling doi:10.1016/j.yjmcc.2007.03.599

H2S production in myocardial ischaemia and reperfusion David J. Elsey, Robert C. Fowkes, Gary F. Baxter. Royal Veterinary College, London NW1 0TU, UK Evidence supports a physiological role of enzymically produced hydrogen sulphide (H2S) within the cardiovascular system. Here, we report the effects of ischaemia–reperfusion on

S198

ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S190–S218

maximal enzyme activity of H2S production and the concentration of H2S in rat isolated hearts. Rat hearts were Langendorff-perfused as follows: 1.20 min stabilisation baseline control group; 2.35 min left main coronary artery occlusion (CAO); 3.35 min CAO followed by 15 min reperfusion. Modification of methods used by Stipanuk and Beck (Biochem J 1982; 206: 267) enabled the left ventricle to be assayed for H2S concentration and maximal H2S production.

function and reducing myocardial cell death following I/R injury. These findings begin to delineate the differential roles of LMW and HMW isoforms in I/R injury and cardioprotection, and may lead to the development of novel therapeutic FGF2 interventions in patients susceptible to or with ischemic heart disease. Keywords: Growth factors; FGF2 protein isoforms; Cardioprotection doi:10.1016/j.yjmcc.2007.03.601

Treatment

H2S conc. (μmol/g protein)

Treatment

H2S prodn. rate (μmol/g protein/min)

LV Baseline LV 35′ Isch LV 35′ Isch + reper

0.40 ± 0.10 0.65 ± 0.05* 0.87 ± 0.04**

LV 35′ Isch + reper RV 35′ Isch + reper

0.43 ± 0.10 1.40 ± 0.30#

*p < 0.05, **p < 0.01 vs. Baseline; #p < 0.05 vs. LV 35′ Isch + reper. (1 way ANOVA + Newman–Keuls). Data are mean ± SEM, n = 6 per group.

These data show a decreased production of H2S in the left ventricle during reperfusion. The right ventricle may be responsible for the increased H2S concentration observed in the left ventricle during reperfusion. Keywords: Ischaemia–reperfusion; Hydrogen sulphide doi:10.1016/j.yjmcc.2007.03.600

Role of high molecular weight isoforms of fibroblast growth factor-2 (FGF-2) in cardiac ischemia–reperfusion injury (I/R) Siyun Liao1, Gilbert Newman1, Thomas Doetschman2, Jo El J. Schultz1. 1University of Cincinnati. 2University of Arizona, Cinci, OH, USA Our lab showed that FGF2 is an important cardioprotective molecule in I/R injury. Different FGF2 isoforms (low [LMW] and high [HMW] molecular weight isoforms) from different translation start sites exist and are differentiately localized, indicating possible unique biological functions. Our lab showed that FGF2 LMW isoform plays a role in protecting the heart from myocardial dysfunction via inhibition of JNK signaling and ultimately apoptosis. The biological actions of the HMW isoforms in I/R injury remain to be elucidated. To delineate the role of FGF2 HMW isoforms in cardioprotection, murine hearts that are deficient of HMW isoforms (FGF2 HMWKO) or that overexpress the human 24 kDa HMW isoform (24 kDa FGF2 Tg) were subjected to 60 min of global, low-flow ischemia and 120 min of reperfusion. Post-ischemic recovery of contractile function was markedly reduced in the 24 kDa transgenic vs. non-transgenic hearts (p <0.05). Conversely, there was a significant improvement in post-ischemic recovery of contractile function in FGF2 HMWKO vs. wildtype (Wt) hearts (p< 0.05). There was a significant decrease in creatine kinase release in FGF2 HMWKO vs. Wt hearts. These results provide molecular evidence that the HMW isoform(s) of FGF2 plays a deleterious role in restoring cardiac

Glutathione-S-transferase-P deficiency in mice enhances myocardial sensitivity to ischemia–reperfusion Aruni Bhatnagar, Brad Hill, Yiru Guo, Russell Prough, Sumanth Prabhu, Roberto Bolli, Dan Conklin. Inst. of Mol. Cardiol., University of Louisville, Louisville, KY Glutathione-S-transferases (GSTs) are a family of isozymes that conjugate glutathione to electrophilic substrates, including aldehydes in pollutants, pharmaceuticals, and products of lipid peroxidation. Emerging evidence indicates that GSTs regulate apoptosis signaling kinase-1 (ASK-1) and c-Jun NH2-terminal kinase (JNK). The role of GSTs in regulating myocardial oxidative stress responses during ischemia has not been studied. Because oxidative stress and stress kinase activation are important determinants of ischemic injury, we examined myocardial responses to ischemia in GSTP-null mice. Cardiac expression of GSTP was abundant by Western blot, immunohistochemistry, and ethacrynic acid activity; but was absent in tissues of GSTP-null mice. No cardiac phenotype was observed in GSTP-null mice by echocardiography and cardiomyocyte size analysis. In male GSTP wild-type and null mice, 15 min of coronary occlusion and 15 min of reperfusion led to a comparable increase in phospho-JNK (n = 4, 4). In wild-type mice the infarct size produced by 30 min of coronary occlusion and 4 h of reperfusion was 55.6 ± 3.9% (n = 8), whereas infract size increased to 73.1 ± 3.7% (n = 8) in the GSTP-null mice (P < 0.01). We conclude that GSTP is a cardioprotective gene and its deficiency increases ischemia–reperfusion injury by enhancing oxidative stress without affecting JNK activation. Given that the human allelic variants of GSTP differ in their catalytic abilities, these results raise the possibility GSTP polymorphisms may be important determinants of clinical outcomes of acute myocardial ischemia. Keywords: Ischemia/reperfusion; Oxidative metabolites; Metabolism doi:10.1016/j.yjmcc.2007.03.602

Proinflammatory cytokines link TLR4 signaling to post-ischemic cardiac dysfunction Lihua Ao, John Cha, Ning Zou, David A. Fullerton, Xianzhong Meng. Dept. of Surgery, University of Colorado HSC, Denver, USA