Lindon H. Young

Gö 6983 Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion

Abstract: Ischemia followed by reperfusion (I/R) in the presence of polymorphonuclear leukocytes (PMNs) results in cardiac contractile dysfunction. Inhibiting protein kinase C (PKC) inhibits the release of superoxide from PMNs. The compound Gö 6983 is an inhibitor of all five PKC isoforms present in PMNs. Therefore, we hypothesized that Gö 6983 could attenuate PMN-induced cardiac dysfunction by suppression of superoxide production from PMNs. We studied isolated rat hearts following ischemia (20 minutes) and reperfusion (45 minutes) infused with activated PMNs. In hearts reperfused with PMNs and Gö 6983 (100 nM, n = 7), left ventricular developed pressure (LVDP) and the rate of LVDP (+dP/dt max) recovered to 89 ± 7% and 74 ± 2% of baseline values, respectively, at 45 minutes postreperfusion compared with I/R hearts (n = 9) receiving PMNs alone, which only recovered to 55 ± 3% and 45 ± 5% of baseline values for LVDP and +dP/dtmax, respectively (P < 0.01). Gö 6983 (100 nM) significantly reduced PMN adherence to the endothelium and infiltration into the myocardium compared with I/R + PMN hearts (P < 0.01), and significantly inhibited superoxide release from PMNs by 90 ± 2% (P < 0.01). In the presence of PMNs, Gö 6983 attenuated post-I/R cardiac contractile dysfunction, which may be related in part to decreased superoxide production.

Increased late sodium current in left atrial myocytes of rabbits with left ventricular hypertrophy: its role in the genesis of atrial arrhythmias

Left ventricular hypertrophy (LVH) is frequently associated with clinical atrial arrhythmias, but little is known about how it causes those arrhythmias. Our previous studies have shown that LVH increases the late sodium current (I(Na-L)) that plays an important role in the genesis of ventricular arrhythmias. We hypothesize that LVH may also induce an upregulation of the I(Na-L) in atrial myocytes, leading to atrial electrical abnormalities. The renovascular hypertension model was used to induce LVH in rabbits. Action potential and membrane current recordings were performed in single myocytes. At a pacing cycle length of 2,000 ms, spontaneous phase-2 early afterdepolarizations (EADs) could be recorded from the left atrial myocytes in 10 of 12 LVH rabbits, whereas no EADs could be elicited in right atrial myocytes of LVH rabbits or atrial myocytes from any of the 12 control rabbits. Spontaneous automaticity (SA) from left atrial myocytes was observed in 9 out of 12 LVH rabbits, but none in right atrial myocytes of LVH rabbits or control rabbits, at a pacing rate of 8,000 ms. The left atrial myocytes of LVH rabbits had a significantly higher density of the I(Na-L) compared with those of control rabbits (0.90 +/- 0.12 in LVH vs. 0.50 +/- 0.08 pA/pF in control, n = 8, P < 0.01). Tetrodotoxin, an I(Na-L) blocker, abolished all atrial EADs and SA at 10 microM. Our results demonstrate that LVH induction results in a significant increase of I(Na-L) in the left atrial myocytes that may render these cells susceptible to the genesis of EADs and SA. The I(Na-L) may serve as a potentially useful ionic target for antiarrhythmic drugs for the treatment of atrial arrhythmias in the setting of LVH.

The Role of Tetrahydrobiopterin and Dihydrobiopterin in Ischemia/Reperfusion Injury When Given at Reperfusion

Reduced nitric oxide (NO) bioavailability and increased oxidative stress are major factors mediating ischemia/reperfusion (I/R) injury. Tetrahydrobiopterin (BH4) is an essential cofactor of endothelial NO synthase (eNOS) to produce NO, whereas dihydrobiopterin (BH2) can shift the eNOS product profile from NO to superoxide, which is further converted to hydrogen peroxide (H2O2) and cause I/R injury. The effects of BH4 and BH2 on oxidative stress and postreperfused cardiac functions were examined in ex vivo myocardial and in vivo femoral I (20 min)/R (45 min) models. In femoral I/R, BH4 increased NO and decreased H2O2 releases relative to saline control, and these effects correlated with improved postreperfused cardiac function. By contrast, BH2 decreased NO release relative to the saline control, but increased H2O2 release similar to the saline control, and these effects correlated with compromised postreperfused cardiac function. In conclusion, these results suggest that promoting eNOS coupling to produce NO and decrease H2O2 may be a key mechanism to restore postreperfused organ function during early reperfusion.

Triacsin C, a Fatty Acyl CoA Synthetase Inhibitor, Improves Cardiac Performance Following Global Ischemia

The role of fatty acyl CoA synthetase (FACS) in ischemia/reperfusion (I/R) injury has not been well established. Our earlier studies showed that triacsin C, a selective FACS inhibitor, decreases endothelial nitric oxide synthase (eNOS) palmitoylation and increases nitric oxide (NO) in cultured human coronary endothelial cells. In the present study, we tested the hypothesis that triacsin C would reduce infarct size and improve post-reperfusion cardiac function by increasing vascular NO. In isolated rat hearts, triacsin C, given during the first 5 minutes of reperfusion, significantly reduced infarct size and attenuated cardiac dysfunction during reperfusion. N G -nitro-L-arginine methyl ester (L-NAME, a non-selective NOS inhibitor, 50 μM) completely abolished the protective effects of triacsin C. In the ischemic hind limb model, triacsin C significantly increased intravascular NO concentration during reperfusion, an effect that was blocked by LNAME or S-methyl-L-thiocitrulline (SMTC, a selective neuronal NOS inhibitor), but not by 1400W (a highly selective iNOS inhibitor). Lastly, triacsin C significantly reduced L-NAME induced leukocyte rolling, adhesion, and transmigration in rat mesenteric circulation, as measured by intravital microscopy. In summary, this study provides novel evidence showing that triacsin C reduces myocardial infarct size, attenuates loss of post-reperfusion cardiac function, increases intravascular NO concentration and inhibits leukocyte recruitment. These pharmacologic properties suggest that triacsin C may be useful as an adjunct to

Protein kinase C (PKC) isoform peptide activator/inhibitors exert cardioprotective effects in polymorphonuclear leukocyte (PMN) induced ischemia/reperfusion (I/R) injury

Introduction Myocardial I/R injury is characterized by endothelial dysfunction, enhanced PMN infiltration into the myocardium, that results in sustained cardiac contractile dysfunction [1,2]. Enhancement of endothelial basal nitric oxide (NO) release or inhibition of PMN superoxide (SO) release reduces endothelial dysfunction and attenuates PMN/endothelial interaction. PKC is a key enzyme that regulates endothelial NO release and PMN SO release [1,2]. However, selective PKC isoforms mediating these responses are not well understood. Myristoylated PKC isoform peptides (MW=1130 to1928; Genemed Synthesis, Inc.) penetrate into cells by simple diffusion. The PKC beta II (βII) (N-MyrSLNPEWNET) and delta (δ) isoform (N-Myr-HDAPIGYD) inhibitors bind to its receptor-activated C kinase region attenuating PKC translocation. By contrast, the PKC δ peptide activator (N-Myr-MRAAEDPM) augments translocation. The PKC zeta (ζ) peptide inhibitor (N-Myr-SIYRRGARRWRKL) binds to the pseudosubstrate domain, attenuating interaction with cell membrane substrates, eNOS and NADPH oxidase [1,2]. We hypothesized that selective PKC peptide isoforms would attenuate PMNinduced contractile dysfunction (i.e., left ventricular developed pressure; LVDP) after I/R when given separately or in combination (i.e., βII /ζ) during reperfusion in the isolated perfused rat heart. We also wanted to determine the mechanism of action to account for any potential cardioprotective effects following I/R.

Inhibition of long chain fatty acyl-CoA synthetase (ACSL) and ischemia reperfusion injury.

Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [(14)C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion.

Effects of Selective NADPH Oxidase Inhibitors on Real-Time Blood Nitric Oxide and Hydrogen Peroxide Release in Acute Hyperglycemia

We hypothesized that acute hyperglycemia (200 mg/dL) would increase H2O2 and decrease NO release in blood relative to saline control. By contrast, gp91ds-tat (RKKRRQRRR-CSTRIRRQL-Amide, MW=2452 g/mol, 1.2 mg/kg, Genemed Synthesis Inc., San Antonio, Tx), a cell-permeable peptide that selectively inhibits NADPH oxidase assembly/activation, would attenuate acute hyperglycemia-induced vascular dysfunction. Furthermore apocynin (MW=166 g/mol, Sigma Chemicals), another type of NADPH oxidase inhibitor, should have similar effects on H2O2 and NO blood levels as gp91ds-tat compared to hyperglycemia control, attenuating acute hyperglycemia-induced vascular dysfunction. We found that acute hyperglycemia significantly reduced blood NO compared to saline control. The addition of gp91ds-tat or apocynin with hyperglycemia significantly improved blood NO levels, similar to saline control. Meanwhile we found acute hyperglycemia maintained a higher level of H2O2 in blood compared to saline control. By contrast, gp91ds-tat or apocynin with hyperglycemia reduced blood H2O2 levels significantly compared to hyperglycemia. These results suggest that NADPH oxidase is a significant source of ROS overproduction and vascular endothelial dysfunction under acute hyperglycemic conditions, and that supplementation with gp91ds-tat or apocynin may be beneficial to attenuate hyperglycemia induced vascular endothelial dysfunction. Moreover, blood H2O2 levels in gp91ds-tat or apocynin treated groups were still significantly higher compared to that in saline groups. This may indicate that other sources of ROS exist such as in mitochondria, which will be further investigated. Results Male Sprague-Dawley rats (275 to 325g, Charles River, Springfield, MA) were anesthetized with 60 mg/kg of pentobarbital sodium with 1000 unit heparin via intraperitoneal (i.p.) injections. The jugular vein is catheterized in order to infuse intravenously with saline, 20% D-glucose, 20% D-glucose with 1.2 mg/kg gp91ds-tat, or 20% D-glucose with 14 mg/kg apocynin (see figure 3). The continuous infusion of 20% D-glucose solution is to maintain hyperglycemia at 200 mg/dL for about 180 min. gp91ds-tat and apocynin will be added to 20% glucose to reach approximately 20 μM and 1 mM in blood, respectively. Both femoral veins will be exposed and catheterized in order to place the calibrated NO and H2O2 microsensors (100μm, WPI Inc., Sarasota, FL) at random into each femoral vein (see figure 4). These microsensors will then be connected to the Apollo 4000 free radical analyzer (WPI Inc., Sarasota, FL) to measure for blood NO and H2O2 levels in real-time. NO, H2O2, and glucose levels will then be recorded at baseline and every 20 minutes intervals throughout 180 minutes infusion period. The changes of blood NO and H2O2 levels will be expressed as the relative change to the baseline. Blood NO and H2O2 recording in picoAmps pA will be converted to the concentration (nM for NO and μM for H2O2) according to the corresponding calibration curve. All the data are represented as a mean ± SEM. The data was then analyzed by ANOVA using post hoc analysis with the Student Newman Keuls. P<0.05 was considered as significant.

The Effects of Modulating Endothelial Nitric Oxide Synthese (eNOS) Activity and Coupling in Extracorporeal Shock Wave Lithotripsy (ESWL)

We hypothesize that ESWL treatment will decrease NO and increase H2O2 release in rat renal veins compared to no-ESWL controls. We further hypothesize that a postESWL i.v. bolus of PKCe+/BH4 will increase NO and decrease H2O2 release compared to ESWL + saline controls. Whereas, we expect a post-ESWL i.v. infusion of PKCe+/BH2 will decrease NO and increase H2O2 compared to ESWL + saline controls. We hypothesize that PKCegiven with either BH4 or BH2 after ESWL will increase NO and decrease H2O2 release compared to ESWL + saline controls. ESWL treatment decreased NO and increased H2O2 blood levels compared to noESWL controls. This supports our hypothesis and previous findings in this lab that ESWL causes oxidative stress and reduced NO bioavailability. Post-ESWL PKCe+/BH4 significantly attenuated the adverse effects of ESWL by increasing NO and decreasing H2O2 release compared to ESWL+saline. This suggests that this combination enhances eNOS in its coupled state. Whereas, post-ESWL PKCe+/BH2 was similar to ESWL control in H2O2 and NO, suggesting that BH2 is nearing saturation to the eNOS binding site. In contrast, post-ESWL PKCewith either BH4 or BH2 resulted in increased NO and decreased H2O2 compared to ESWL+saline. This suggests that PKCeattenuates eNOS uncoupled activity after ESWL. Potentially, this study can help to develop therapeutic uses for PKCe+/BH4 or PKCein the attenuation of vascular endothelial dysfunction following ESWL treatment and possibly eliminate or reduce the acute renal complications that may lead to chronic conditions such as hypertension. Results

Cardioprotective Effects of Selective Mitochondrial-Targeted Antioxidants in Myocardial Ischemia/Reperfusion (I/R) Injury

Cardioprotective Effects of Mitochondrial-Targeted Antioxidants in Myocardial Ischemia/Reperfusion (I/R) Injury Reactive oxygen species (ROS) generated during myocardial I/R contribute to post-reperfused cardiac contractile dysfunction. Damaged cardiomyocyte mitochondria are major sites of excess ROS generation during reperfusion. We hypothesized that reducing mitochondrial ROS formation should attenuate myocardial I/R injury and thereby improve function of isolated perfused rat hearts subjected to I(30min)/R(45min) compared to untreated I/R hearts. Mitoquinone (MitoQ, MW=579g/mol; complexed with cyclodextrin (MW=1135g/mol) to improve water solubility, total MW=1714g/mol), a coenzyme Q derivative, and SS-31 (Szeto-Schiller) peptide ((D-Arg)-Dmt-LysPhe-Amide, MW=639g/mol, Genemed Synthesis, Inc., San Antonio, TX), an alternating cationic-aromatic peptide, are selective mitochondrial ROS inhibitors which significantly improved post-reperfused cardiac function compared to untreated I/R controls in this study (p<0.05). MitoQ elicits antioxidant effects through the recycling of ubiquinone to ubiquinol, whereas SS-31 utilizes an antioxidant dimethyltyrosine residue. Improvement in postreperfused cardiac function by MitoQ or SS-31 was associated with a significant decrease in myocardial tissue infarct size compared to untreated I/R hearts (p<0.01). These results suggest mitochondrial-derived ROS are important contributors to I/R injury, and MitoQ or SS-31 when administered at reperfusion may potentially augment the benefits of angioplasty or

Apocynin Exerts Dose-Dependent Cardioprotective Effects by Attenuating Reactive Oxygen Species in Ischemia/Reperfusion

Ischemia/reperfusion results in cardiac contractile dysfunction and cell death partly due to increased reactive oxygen species and decreased endothelial-derived nitric oxide bioavailability. NADPH oxidase normally produces reactive oxygen species to facilitate cell signalling and differentiation; however, excessive release of such species following ischemia exacerbates cell death. Thus, administration of an NADPH oxidase inhibitor, apocynin, may preserve cardiac function and reduce infarct size following ischemia. Apocynin dose-dependently (40 μM, 400 μM and 1 mM) attenuated leukocyte superoxide release by 87 ± 7%. Apocynin was also given to isolated perfused hearts after ischemia, with infarct size decreasing to 39 ± 7% (40 μM), 28 ± 4% (400 μM; p < 0.01) and 29 ± 6% (1 mM; p < 0.01), versus the control’s 46 ± 2%. This decrease correlated with improved final post-reperfusion left ventricular end-diastolic pressure, which decreased from 60 ± 5% in control hearts to 56 ± 5% (40 μM), 43 ± 4% (400 μM; p < 0.01) and 48 ± 5% (1 mM; p < 0.05), compared to baseline. Functionally, apocynin (13.7 mg/kg, I.V.) significantly reduced H2O2 by nearly four-fold and increased endothelial-derived nitric oxide bioavailability by nearly four-fold during reperfusion compared to controls (p < 0.01), which was confirmed in in vivo rat hind limb ischemia/reperfusion models. These results suggest that apocynin attenuates ischemia/reperfusion-induced cardiac contractile dysfunction and infarct size by inhibiting reactive oxygen species release from NADPH oxidase.