Sri Nagarjun Batchu

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.

Epoxyeicosatrienoic acid prevents postischemic electrocardiogram abnormalities in an isolated heart model.

Cytochrome P450 epoxygenases metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) which are in turn converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). The main objective of this study was to investigate the protective effects of EETs following ischemic injury using an ex vivo electrocardiogram (EKG) model. Hearts from C57Bl/6, transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 (Tr) and wildtype (WT) littermates were excised and perfused with constant pressure in a Langendorff apparatus. Electrodes were placed superficially at the right atrium and left ventricle to assess EKG waveforms. In ischemic reperfusion experiments hearts were subjected to 20 min of global no-flow ischemia followed by 20 min of reperfusion (R20). The EKG from C57Bl/6 hearts perfused with 1 microM 14,15-EET showed less QT prolongation (QTc) and ST elevation (STE) (QTc=41+/-3, STE=2.3+/-0.3; R20: QTc=42+/-2 ms, STE=1.2+/-0.2mv) than control hearts (QTc=36+/-2, STE=2.3+/-0.2; R20: QTc=53+/-3 ms; STE=3.6+/-0.4mv). Similar results of reduced QT prolongation and ST elevation were observed in EKG recording from CYP2J2 Tr mice (QTc=35+/-1, STE=1.9+/-0.1; R20: QTc=38+/-4 ms, STE=1.3+/-0.2mv) compared to WT hearts. The putative epoxygenase inhibitor MS-PPOH (50 microM) and EET antagonist 14,15-EEZE (10 microM) both abolished the cardioprotective response, implicating EETs in this process. In addition, separate exposure to the K(ATP) channel blockers glibenclamide (1 microM) and HMR1098 (10 microM), or the PKA protein inhibitor H89 (50 nM) during reperfusion abolished the improved repolarization in both the models. Consistent with a role of PKA, CYP2J2 Tr mice had an enhanced activation of the PKAalpha regulatory II subunit in plasma membrane following IR injury. The present data demonstrate that EETs can enhance the recovery of ventricular repolarization following ischemia, potentially by facilitating activation of K(+) channels and PKA-dependent signaling.

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.

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.

Cardiovascular Properties of a Nitric Oxide Releasing Rofecoxib Analogue: Beneficial Anti-hypertensive Activity and Enhanced Recovery in an Ischemic Reperfusion Injury Model

was subsequently discovered that rofecoxib causes undesirable cardiovascular events such as myocardial infarction and stroke; this triggered its withdrawal from the market. 3] Highly selective COX-2 inhibitors, including rofecoxib, likely alter the biochemical balance in the COX pathway. In this regard, COX-2mediated biosynthesis of beneficial vasodilatory, anti-aggregatory prostacyclin (PGI2), which can improve cardiac function following ischemic reperfusion injury, is suppressed in conjunction with a contraindicated simultaneous increase in the level of the prothrombotic thromboxane A2 (TxA2), which causes vasoconstriction, decreases cardiac function, and induces platelet aggregation). Accordingly, rofecoxib tips the PGI2–TxA2 balance toward TxA2, resulting in elevated blood pressure (BP) and increased risk of adverse prothrombotic effects. In addition to the inhibition of PGI2 biosynthesis in the vascular endothelium, selective COX-2 inhibitors may also block the synthesis of renal PGs and increase sodium reabsorption, which can also contribute to an elevation in blood pressure (hypertension). Nitric oxide (NO) is an efficient vasodilation agent and inhibitor of platelet aggregation and adhesion, and it limits cardiac ischemia-reperfusion injury. 13] Hence, the incorporation of a NO donor moiety onto rofecoxib offers an attractive strategy to circumvent adverse cardiovascular side effects associated with the use of rofecoxib. In a recent investigation, we developed this drug design concept wherein the methanesulfonyl (SO2CH3) substituent in rofecoxib is replaced by a sulfohydroxamic acid (SO2NHOH) dual-function NO donor/COX-2 pharmacophore, and a para-chloro substituent was introduced at the C4 phenyl ring position to prevent formation (obstructive metabolic halogenation) of a para-hydroxy metabolite. This hitherto-unknown sulfohydroxamic acid analogue of rofecoxib (compound 2, Figure 1) showed 1) potent (COX-2 IC50: 0.28 mm) and 2) selective (COX-2 selectivity index >304) COX-2 inhibitory activities, 3) appreciable in vivo anti-inflammatory activity (ED50: 17.7 mg kg 1 po), and 4) a 43 % release of NO for a 24-hour incubation in phosphate buffer at pH 7.4; moreover, 5) a molecular modeling study indicated that N-hydroxy-4-[4(4-chlorophenyl)-5-oxo-2,5-dihydrofuran-3-yl]benzenesulfonamide (2) assumes a favorable orientation inside the COX-2 binding site which allows multiple hydrogen bonding interactions. An illustration of these important biological features is presented in Figure 2. It was therefore of interest to ascertain some cardiovascular properties of this new NO donor rofecoxib analogue 2. Herein we describe the effects of compound 2 on systolic, diastolic, and mean blood pressure (average of systolic and diastolic values), heart rate, and its ability to enhance recovery in a cardiac ischemic reperfusion injury model. Systolic blood pressure (BPsys, mm Hg), diastolic blood pressure (BPdia, mm Hg), and heart rate (HR, beats min ) were measured at 1, 3, 6, and 24 h time intervals following oral adminis-

Bioactive Compounds in Heart Disease

Dietary sources of bioactive compounds provide desirable health benefits beyond basic nutrients and can have therapeutic value in heart disease. Numerous compounds have been discovered and many demonstrate beneficial properties with regard to cardiovascular disease (CVD). The specific protective mechanisms of action involved are unclear, and much scientific research needs to be conducted. However, sufficient evidence demonstrates associated reductions in morbidity and mortality to recommend a dietary intake rich in bioactive compounds from a variety of sources such as fruits, vegetables, whole grains, oils, and nuts. This chapter highlights the biological effects of some key bioactive compounds relative to CVD.

Fatty acids and cardiac ischemia-reperfusion injury.

Abstract Fatty acids (FAs) are essential components of the body that help maintain a normal physiological function and a protective response to pathological stimuli. Small changes in the dietary intake of FAs can influence membrane integrity and cellular signaling which can affect cardiovascular disease (CVD) outcomes. Myocardial damage resulting from ischemia/reperfusion injury has a profound effect in both the short- and long-term survivability of the individual. Relatively little mechanistic information is available regarding a molecular basis for the cardioprotective and cardiotoxic effects of FAs on ischemia/reperfusion injury. However, clearly, based on experimental data, the beneficial effects of polyunsaturated fatty acids (PUFAs) include the prevention of cardiac arrhythmias, an increased oxygen efficiency, the preservation of mitochondrial function, an altered membrane lipid composition, and a membrane-domain functionality. Thus, PUFAs can favorably modulate intracellular-signaling events in response to extracellular stress stimuli. Further mechanistic studies are needed to clarify the role of FAs in ischemia/reperfusion injury. However, while saturated fatty acids and trans fatty acids tend to be detrimental, both n-3 and n-6 PUFAs play distinct roles in mediating ischemic injury. As such, maintaining the balance between n-3 and n-6 PUFAs is a critical factor influencing a CVD outcome.