Ketul R. Chaudhary

Overexpression of CYP2J2 provides protection against doxorubicin-induced cardiotoxicity

Human cytochrome P-450 (CYP)2J2 is abundant in heart and active in biosynthesis of epoxyeicosatrienoic acids (EETs). Recently, we demonstrated that these eicosanoid products protect myocardium from ischemia-reperfusion injury. The present study utilized transgenic (Tr) mice with cardiomyocyte-specific overexpression of human CYP2J2 to investigate protection toward toxicity resulting from acute (0, 5, or 15 mg/kg daily for 3 days, followed by 24-h recovery) or chronic (0, 1.5, or 3.0 mg/kg biweekly for 5 wk, followed by 2-wk recovery) doxorubicin (Dox) administration. Acute treatment resulted in marked elevations of serum lactate dehydrogenase and creatine kinase levels that were significantly greater in wild-type (WT) than CYP2J2 Tr mice. Acute treatment also resulted in less activation of stress response enzymes in CYP2J2 Tr mice (catalase 750% vs. 300% of baseline, caspase-3 235% vs. 165% of baseline in WT vs. CYP2J2 Tr mice). Moreover, CYP2J2 Tr hearts exhibited less Dox-induced cardiomyocytes apoptosis (measured by TUNEL) compared with WT hearts. After chronic treatment, comparable decreases in body weight were observed in WT and CYP2J2 Tr mice. However, cardiac function, assessed by measurement of fractional shortening with M-mode transthoracic echocardiography, was significantly higher in CYP2J2 Tr than WT hearts after chronic Dox treatment (WT 37 +/- 2%, CYP2J2 Tr 47 +/- 1%). WT mice also had larger increases in beta-myosin heavy chain and cardiac ankryin repeat protein compared with CYP2J2 Tr mice. CYP2J2 Tr hearts had a significantly higher rate of Dox metabolism than WT hearts (2.2 +/- 0.25 vs. 1.6 +/- 0.50 ng.min(-1).100 microg protein(-1)). In vitro data from H9c2 cells demonstrated that EETs attenuated Dox-induced mitochondrial damage. Together, these data suggest that cardiac-specific overexpression of CYP2J2 limited Dox-induced toxicity.

Epoxyeicosatrienoic acids limit damage to mitochondrial function following stress in cardiac cells

Epoxyeicosatrienoic acids (EETs) are polyunsaturated fatty acids synthesized from arachidonic acid by CYP2J2 epoxygenase and inactivated by soluble epoxide hydrolase (sEH or Ephx2) to dihydroxyeicosatrienoic acids. Mitochondrial function following ischemic insult is a critical determinant of reperfusion-induced cell death in the myocardium. The objectives of the current study were to investigate the protective role of EETs in mitochondrial function. Mice with the targeted disruption of the Ephx2 gene, cardiomyocyte-specific overexpression of CYP2J2 or perfused with EETs all have improved postischemic LVDP recovery compared to wild-type (WT). Perfusion with the mPTP opener, atractyloside, abolished the improved postischemic functional recovery observed in CYP2J2 Tr, sEH null and EET perfused hearts. Electron micrographs demonstrated WT hearts to have increased mitochondrial fragmentation and T-tubule swelling compared to CYP2J2 Tr hearts following 20 min global ischemia and 20 min reperfusion. Direct effects of EETs on mitochondria were assessed in isolated rat cardiomyocytes and H9c2 cells. Laser-induced loss of mitochondrial membrane potential (DeltaPsi(m)) and mPTP opening was significantly reduced in cells treated with 14, 15-EET (1 microM). The EET protective effect was blocked by the putative EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (1 muM, 14, 15-EEZE), paxilline (10 microM, BK(Ca) inhibitor) and 5HD (100 microM, K(ATP) inhibitor). Our studies show that EETs can limit mitochondrial dysfunction following cellular stress via a K(+) channel-dependent mechanism.

Inhibition of Soluble Epoxide Hydrolase by trans-4- ½4-(3-adamantan-1-yl-ureido)-cyclohexyloxy-benzoic acid Is Protective Against Ischemia-Reperfusion Injury

Arachidonic acid, a polyunsaturated fatty acid, can be metabolized to cardioprotective epoxyeicosatrienoic acids (EETs) by cytochrome P450 epoxygenases, which are subsequently hydrolyzed to less bioactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). To study the effects of pharmacological inhibitor of sEH (sEHi), C57BL6 mice hearts were perfused in Langendorff mode for 40 minutes of baseline and subjected to 30 minutes of global no-flow ischemia followed by 40 minutes of reperfusion. Hearts were perfused with the sEHi, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 0.05, 0.1, 0.5, and 1 μM). To study the mechanism(s), hearts were perfused with 0.1 μM t-AUCB in the presence or absence of putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM) or phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin (200 nM) or LY294002 (5 μM). Infarct size was determined at the end of 2-hour reperfusion by 2,3,5-triphenyltetrazolium chloride staining. Inhibition of sEH by t-AUCB significantly improved postischemic left ventricular developed pressure (LVDP) recovery and reduced the infarct size after ischemia and reperfusion, as compared with control hearts. Perfusion with 14,15-epoxyeicosa-5(Z)-enoic acid, wortmannin or LY294002 before ischemia abolished the cardioprotective phenotype; however, coperfusion of both t-AUCB and 11,12-EET did not result in an additive effect on improved LVDP recovery. Together, our data suggest that pharmacological inhibition of sEH by t-AUCB is cardioprotective.

Cardioprotective effect of a dual acting epoxyeicosatrienoic acid analogue towards ischaemia reperfusion injury

BACKGROUND AND PURPOSE Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid that are metabolized into dihydroxyepoxyeicosatrienoic acids (DHET) by soluble epoxide hydrolase (sEH). The current investigations were performed to examine the cardioprotective effects of UA‐8 (13‐(3‐propylureido)tridec‐8‐enoic acid), a synthetic compound that possesses both EET‐mimetic and sEH inhibitory properties, against ischaemia‐reperfusion injury.

Cytochrome P450 Enzymes and the Heart

The cytochrome P450 monooxygenase system (CYP) is a multigene superfamily of heme‐thiolate enzymes, which are important in the metabolism of foreign and endogenous compounds. Genetic variations, drug interactions, or pathophysiological factors can lead to reduced, absent, or increased enzymatic activity. This altered CYP activity greatly influences an individuals response to therapeutic treatment. What is not known is the impact of these changes on the many functional roles of CYP in physiological and pathophysiological processes of the heart. Many extrahepatic tissues, like heart, contain active P450 enzymes but lack information regarding their role in cellular injury or homeostasis. Much of our current knowledge about cardiac CYP has been limited to studies investigating the role of fatty acid metabolites in heart. Traditional risk factors including diabetes, smoking, and hypertension have well established links to cardiovascular disease. And new evidence strongly suggests exposure to chemicals and other environmental agents has a profound impact on the cardiovascular system. These risk factors can independently affect the expression and activity of CYP enzymes. Therefore, altered CYP activity is important from a detoxification as well as a bioactivation perspective. Considering CYP, interactions are greatly dependent on inherited differences or acquired changes in enzyme activity further research into their potential impact on pathogenesis, risk assessment, and therapy of heart disease is warranted. This review explores the expression of CYP isoforms, their functional roles, and the effects of genetic variation in the heart.

Role of B-type natriuretic peptide in epoxyeicosatrienoic acid-mediated improved post-ischaemic recovery of heart contractile function

AIMS This study examined the functional role of B-type natriuretic peptide (BNP) in epoxyeicosatrienoic acid (EET)-mediated cardioprotection in mice with targeted disruption of the sEH or Ephx2 gene (sEH null). METHODS AND RESULTS Isolated mouse hearts were perfused in the Langendorff mode and subjected to global no-flow ischaemia followed by reperfusion. Hearts were analysed for recovery of left ventricular developed pressure (LVDP), mRNA levels, and protein expression. Naïve hearts from sEH null mice had similar expression of preproBNP (Nppb) mRNA compared with wild-type (WT) hearts. However, significant increases in Nppb mRNA and BNP protein expression occurred during post-ischaemic reperfusion and correlated with improved post-ischaemic recovery of LVDP. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid prior to ischaemia reduced the preproBNP mRNA in sEH null hearts. Inhibitor studies demonstrated that perfusion with the natriuretic peptide receptor type-A (NPR-A) antagonist, A71915, limited the improved recovery in recombinant full-length mouse BNP (rBNP)- and 11,12-EET-perfused hearts as well as in sEH null mice. Increased expression of phosphorylated protein kinase C epsilon and Akt were found in WT hearts perfused with either 11,12-EET or rBNP, while mitochondrial glycogen synthase kinase-3beta was significantly lower in the same samples. Furthermore, treatment with the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin abolished improved LVDP recovery in 11,12-EET-treated hearts but not did significantly inhibit recovery of rBNP-treated hearts. CONCLUSION Taken together, these data indicate that EET-mediated cardioprotection involves BNP and PI3K signalling events.

Mitochondria and the aging heart.

The average human life span has markedly increased in modern society largely attributed to advances in medical and therapeutic sciences that have successfully reduced important health risks. However, advanced age results in numerous alterations to cellular and subcellular components that can impact the overall health and function of an individual. Not surprisingly, advanced age is a major risk factor for the development of heart disease in which elderly populations observe increased morbidity and mortality. Even healthy individuals that appear to have normal heart function under resting conditions, actually have an increased susceptibility and vulnerability to stress. This is confounded by the impact that stress and disease can have over time to both the heart and vessels. Although, there is a rapidly growing body of literature investigating the effects of aging on the heart and how age-related alterations affect cardiac function, the biology of aging and underlying mechanisms remain unclear. In this review, we summarize effects of aging on the heart and discuss potential theories of cellular aging with special emphasis on mitochondrial dysfunction.

Role of PI3Kα and sarcolemmal ATP-sensitive potassium channels in epoxyeicosatrienoic acid mediated cardioprotection

AIMS Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid that have known cardioprotective properties. While the mechanism(s) remains unknown, evidence suggests that phosphoinositide 3-kinase (PI3K) and sarcolemmal ATP-sensitive potassium channels (pmK(ATP)) are important. However the role of specific PI3K isoforms and corresponding intracellular mechanisms remains unknown. METHODS AND RESULTS To study this, mice hearts were perfused in Langendorff mode for 40 min of baseline and subjected to 20 or 30 min of global no-flow ischemia followed by 40 min of reperfusion. C57BL6 mice perfused with 11,12-EET (1 μM) had improved postischemic recovery, whereas co-perfusion with PI3Kα inhibitor, PI-103 (0.1 μM), abolished the EET-mediated effect. In contrast, blocking of PI3Kβ or PI3Kγ isoforms failed to inhibit EET-mediated cardioprotection. In addition to the improved post-ischemic recovery, increased levels of p-Akt, decreased calcineurin activity and decreased translocation of proapoptotic protein BAD to mitochondria were noted in EET-treated hearts. Perfusion of 11,12-EET to Kir6.2 deficient mice (pmK(ATP)) failed to improve postischemic recovery, decrease calcineurin activity and translocation of proapoptotic protein BAD, however increased levels of p-Akt were still observed. Patch-clamp experiments demonstrated that 11,12-EET could not activate pmK(ATP) currents in myocytes pre-treated with PI-103. Mechanistic studies in H9c2 cells demonstrate that 11,12-EET limits anoxia-reoxygenation triggered Ca(2+) accumulation and maintains mitochondrial ΔΨm compared to controls. Both PI-103 and glibenclamide (10 μM, pmK(ATP) inhibitor) abolished EET cytoprotection. CONCLUSION Together our data suggest that EET-mediated cardioprotection involves activation of PI3Kα, upstream of pmK(ATP), which prevents Ca(2+) overload and maintains mitochondrial function.

Differential effects of soluble epoxide hydrolase inhibition and CYP2J2 overexpression on postischemic cardiac function in aged mice

Cardioprotective effects of epoxyeicosatrienoic acids (EETs) have been demonstrated in models of young mice with either the cardiomyocyte specific over-expression of cytochrome P450 2J2 (CYP2J2 Tr) or deletion of soluble epoxide hydrolase (sEH null). In this study we examined differences in EET-induced cardioprotection in young (2 months) and aged (12 months) CYP2J2 Tr and sEHnull mice using Langendorff isolated perfused heart model. Improved postischemic functional recovery was observed in both young and aged sEH null mice compared to age matched WT. Conversely, the cardioprotective effect observed in young CYP2J2 Tr was lost in aged CYP2J2 Tr mice. The loss of cardioprotection in aged CYP2J2 Tr was regained following perfusion with the sEH inhibitor t-AUCB. Data demonstrated increased levels of leukotoxin diol (DiHOME) and oxidative stress as well decreased protein phosphatase 2A (PP2A) activation in aged CYP2J2 Tr. In conclusion, inhibition of sEH and EET-induced cardioprotection is maintained in aged mice. However, the loss of protective effects observed in aged CYP2J2 Tr might be attributed to increased levels of DiHOME, oxidative stress and/or decreased PP2A activity.

Effect of ischemia reperfusion injury and epoxyeicosatrienoic acids on caveolin expression in mouse myocardium.

Background: Caveolins (Cav) are structural proteins that insert into the plasma membrane to form caveolae that can bind molecules important in cardiac signal transduction and function. Cytochrome P450 epoxygenases can metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have known cardioprotective effects. Subsequent metabolism of EETs by soluble epoxide hydrolase reduces the protective effect. Aims: (1) To assess the effect of ischemia–reperfusion injury on expression and subcellular localization of caveolins. (2) To study the effect of EETs on caveolins. Methods: Hearts from soluble epoxide hydrolase null (KO) and littermate control (WT) mice were perfused in Langendorff mode and subjected to 20 minutes ischemia followed by 40 minutes reperfusion. Immunohistochemistry, immunoblot, and electron microscopy were performed to study localization of caveolins and changes in ultrastructure. Results: In WT heart, Cav-1 and Cav-3 were present in cardiomyocyte and capillary endothelial cell at baseline. After ischemia, Cav-1 but not Cav-3, disappeared from cardiomyocyte; moreover, caveolae were absent and mitochondrial cristae were damaged. Improved postischemic functional recovery observed in KO or WT hearts treated with 11,12-EET corresponded to higher Cav-1 expression and maintained caveolae structure. In addition, KO mice preserved the Cav-1 signaling after ischemia that lost in WT mice. Conclusions: Taken together, our data suggest that ischemia–reperfusion injury causes loss of Cav-1 and caveolins, and EETs-mediated cardioprotection involves preservation of Cav-1.