Ashok K. Chaudhary

Oxidized ω–3 Fatty Acids Inhibit Pro-Inflammatory Responses in Glomerular Endothelial Cells

Background: ω–3 fatty acids have beneficial effects in chronic inflammatory diseases that are characterized by accumulation of leukocytes and leukocyte-mediated tissue injury. Accumulation of leukocytes occurs, in part, due to pro-inflammatory responses in endothelial cells, such as increase in expression of leukocyte adhesion receptors and chemokines, such as MCP-1 and IL-8. Methods: ω–3 fatty acids, such as EPA, are highly polyunsaturated and readily undergo auto-oxidation. We studied the effect of oxidized EPA and unoxidized (native) EPA on leukocyte-glomerular endothelial cell interactions using adhesion assays, ELISA assays and transmigration assays. We used electrophoresis mobility shift assays to determine the effect of oxidized and unoxidized EPA on cytokine-induced nuclear factor-ĸB (NF-ĸB) activation. Results: Oxidized EPA but not unoxidized EPA dose-dependently inhibits cytokine-induced leukocyte adhesion receptors on glomerular endothelial cells, which correlates with inhibition of leukocyte-glomerular endothelial cell interactions. Oxidized EPA but not unoxidized EPA inhibits cytokine-induced glomerular endothelial and mesangial cell expression of MCP-1, and to a lesser extent IL-8. Transmigration assays show that oxidized EPA but not unoxidized EPA inhibits leukocyte transmigration across glomerular endothelial cells. Oxidized EPA but not unoxidized EPA potently inhibited cytokine-induced activation of NF-ĸB in glomerular endothelial and mesangial cells. Conclusions: These studies show that the beneficial effects of fish oil in chronic inflammatory diseases, including IgA nephropathy, may result from the inhibitory effects of oxidized ω–3 fatty acids on pro-inflammatory events in endothelial cells via inhibition of NF-ĸB activation.

Catecholamine-Independent Heart Rate Increases Require Ca2+/Calmodulin-Dependent Protein Kinase II

Background —Catecholamines increase heart rate by augmenting the cAMP responsive HCN4 9pacemaker current9 ( I f ) and/or by promoting inward Na + /Ca 2+ exchanger current ( I NCX ), by a 9Ca 2+ clock mechanism in sinoatrial nodal cells (SANCs). The importance, identity and function of signals that connect I f and Ca 2+ clock mechanisms are uncertain and controversial, but the multifunctional Ca 2+ and calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to β-adrenergic receptor (β-AR) stimulation. The aim of this stuy is to measure the contribution of the Ca 2+ clock and CaMKII to cardiac pacing independent of β-AR agonist stimulation. Methods and Results —We used the L-type Ca 2+ channel agonist BayK 8644 (BayK) to activate the SANC Ca 2+ clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from WT control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs, but increased I Ca equally in all SANCs, indicating that increasing I Ca was insufficient and suggesting CaMKII activation was required for heart rate increases by BayK. BayK did not increase I f or protein kinase A (PKA)-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca 2+ by BayK did not augment cAMP/PKA signaling at these targets. Late diastolic intracellular Ca 2+ release and I NCX were significantly reduced in AC3-I SANCs and the response to BayK was eliminated by ryanodine in all groups. Conclusions —The Ca 2+ clock is capable of supporting physiological fight or flight responses, independent of β-AR stimulation or I f increases. Complete Ca 2+ clock and β-AR stimulation responses require CaMKII.Background—Catecholamines increase heart rate by augmenting the cAMP-responsive hyperpolarization-activated cyclic nucleotide-gated channel 4 pacemaker current (If) and by promoting inward Na+/Ca2+ exchanger current (INCX) by a “Ca2+ clock” mechanism in sinoatrial nodal cells (SANCs). The importance, identity, and function of signals that connect If and Ca2+ clock mechanisms are uncertain and controversial, but the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to &bgr;-adrenergic receptor (&bgr;-AR) stimulation. The aim of this study was to measure the contribution of the Ca2+ clock and CaMKII to cardiac pacing independent of &bgr;-AR agonist stimulation. Methods and Results—We used the L-type Ca2+ channel agonist Bay K8644 (BayK) to activate the SANC Ca2+ clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from wild-type control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs but increased L-type Ca2+ current (ICa) equally in all SANCs, indicating that increasing ICa was insufficient and suggesting that CaMKII activation was required for heart rate increases by BayK. BayK did not increase If or protein kinase A-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca2+ by BayK did not augment cAMP/protein kinase A signaling at these targets. Late-diastolic intracellular Ca2+ release and INCX were significantly reduced in AC3-I SANCs, and the response to BayK was eliminated by ryanodine in all groups. Conclusions—The Ca2+ clock is capable of supporting physiological fight-or-flight responses, independent of &bgr;-AR stimulation or If increases. Complete Ca2+ clock and &bgr;-AR stimulation responses require CaMKII.

Catecholamine-Independent Heart Rate Increases Require CaMKII

Background —Catecholamines increase heart rate by augmenting the cAMP responsive HCN4 9pacemaker current9 ( I f ) and/or by promoting inward Na + /Ca 2+ exchanger current ( I NCX ), by a 9Ca 2+ clock mechanism in sinoatrial nodal cells (SANCs). The importance, identity and function of signals that connect I f and Ca 2+ clock mechanisms are uncertain and controversial, but the multifunctional Ca 2+ and calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to β-adrenergic receptor (β-AR) stimulation. The aim of this stuy is to measure the contribution of the Ca 2+ clock and CaMKII to cardiac pacing independent of β-AR agonist stimulation. Methods and Results —We used the L-type Ca 2+ channel agonist BayK 8644 (BayK) to activate the SANC Ca 2+ clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from WT control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs, but increased I Ca equally in all SANCs, indicating that increasing I Ca was insufficient and suggesting CaMKII activation was required for heart rate increases by BayK. BayK did not increase I f or protein kinase A (PKA)-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca 2+ by BayK did not augment cAMP/PKA signaling at these targets. Late diastolic intracellular Ca 2+ release and I NCX were significantly reduced in AC3-I SANCs and the response to BayK was eliminated by ryanodine in all groups. Conclusions —The Ca 2+ clock is capable of supporting physiological fight or flight responses, independent of β-AR stimulation or I f increases. Complete Ca 2+ clock and β-AR stimulation responses require CaMKII.Background—Catecholamines increase heart rate by augmenting the cAMP-responsive hyperpolarization-activated cyclic nucleotide-gated channel 4 pacemaker current (If) and by promoting inward Na+/Ca2+ exchanger current (INCX) by a “Ca2+ clock” mechanism in sinoatrial nodal cells (SANCs). The importance, identity, and function of signals that connect If and Ca2+ clock mechanisms are uncertain and controversial, but the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to &bgr;-adrenergic receptor (&bgr;-AR) stimulation. The aim of this study was to measure the contribution of the Ca2+ clock and CaMKII to cardiac pacing independent of &bgr;-AR agonist stimulation. Methods and Results—We used the L-type Ca2+ channel agonist Bay K8644 (BayK) to activate the SANC Ca2+ clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from wild-type control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs but increased L-type Ca2+ current (ICa) equally in all SANCs, indicating that increasing ICa was insufficient and suggesting that CaMKII activation was required for heart rate increases by BayK. BayK did not increase If or protein kinase A-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca2+ by BayK did not augment cAMP/protein kinase A signaling at these targets. Late-diastolic intracellular Ca2+ release and INCX were significantly reduced in AC3-I SANCs, and the response to BayK was eliminated by ryanodine in all groups. Conclusions—The Ca2+ clock is capable of supporting physiological fight-or-flight responses, independent of &bgr;-AR stimulation or If increases. Complete Ca2+ clock and &bgr;-AR stimulation responses require CaMKII.

Free radical scavenger specifically prevents ischemic focal ventricular tachycardia

BACKGROUND Focal ventricular tachycardia (VT) in acute myocardial ischemia is closely related to triggered activity (TA), which may be blocked by scavenging reactive oxygen species (ROS). OBJECTIVE This study analyzed effects of acutely administered ROS scavenger-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) on VT in vivo and TA in vitro. METHODS Forty-three alpha chloralose anesthetized dogs with coronary artery occlusion were studied. Three-dimensional activation mapping helped to locate the origin of focal or reentrant VT. TEMPO (30 mg/kg intravenously) or vehicle was given. Endocardium excised from the site of origin of VT was studied using standard microelectrode techniques and measures of ROS. RESULTS Reentry and focal VT induction were both highly reproducible. TEMPO blocked focal VT in 6 of 11 dogs (P <.05), but 9 of 9 dogs with reentrant VT continued to have VT re-induced after TEMPO. TEMPO did not alter effective refractory period (168 +/- 3 to 171 +/- 3 ms), mean blood pressure (88 +/- 3 to 81 +/- 3 mm Hg), and size of ischemia (42% +/- 3% vs 40% +/- 4%). In vitro, TEMPO (10(-3) M, n = 14) produced no change in action potentials. Nevertheless, TA was reversibly attenuated from 5.3 +/- 1.1 to 0.4 +/- 0.4 complexes with TEMPO (n = 15, P <.05). Lucigenin-enhanced chemiluminescence and dihydroethidium staining showed increased ROS in ischemic endocardium; TEMPO dramatically reduced ROS in ischemic sites. CONCLUSION TEMPO, a scavenger of ROS, prevented triggered activity associated with focal VT during myocardial ischemia in areas of increased ROS. Antioxidant therapy may play an important role in blockade of focal VT under the conditions of myocardial ischemia.

Association among intracardiac T-wave alternans, ischemia, and spontaneous ventricular arrhythmias after coronary artery occlusion in a canine model.

T-wave alternans (TWA) has been investigated as a marker for susceptibility to lethal ventricular arrhythmia. In this article, we studied intracardiac TWA and ischemia as predictors of spontaneous ventricular tachycardia (VT) or ventricular fibrillation (VF) in a canine model of coronary artery occlusion (CAO). Anesthetized, open-chest dogs were studied. Electrograms from intracardiac bipolar electrodes (IBEs) were assessed for TWA and spontaneous VT or VF. TWA was defined on IBE as T wave voltage change on every other complex. In each heart, we examined 62 electrograms measured in the risk zone and surrounding normal sites, filtered from 3 to 1300 Hz. Ischemia was measured as percent of all IBE recorded that had QRS voltage drop >45%. Mapping localized the three-dimensional origin of spontaneous VT or VF. The data from dogs with VF (n = 5), VT (n = 8), or controls (no VT or VF, n = 8) were analyzed before left CAO, at the 20th min after CAO and times immediately preceding VT and VF. We found a correlation between intracardiac TWA and ischemia. More importantly, increases in intracardiac TWA peaked immediately preceding spontaneous VF and VT and were significantly higher compared to controls at comparable times. At VT/VF origins and adjacent sites, the mean TWA magnitude and discordance of TWA distinguished between VT/VF and controls at comparable times but not between VT and VF or between reentry and focal mechanisms. TWA was more common than ischemia at VT/VF origins. In summary, changes in intracardiac TWA and ischemia correlate with impending spontaneous VT/VF in a clinically applicable canine model of CAO.

Role of NADPH Oxidase and Xanthine Oxidase in Mediating Inducible VT/VF and Triggered Activity in a Canine Model of Myocardial Ischemia

Background: Ventricular tachycardia or fibrillation (VT/VF) of focal origin due to triggered activity (TA) from delayed afterdepolarizations (DADs) is reproducibly inducible after anterior coronary artery occlusion. Both VT/VF and TA can be blocked by reducing reactive oxygen species (ROS). We tested the hypothesis that inhibition of NADPH oxidase and xanthine oxidase would block VT/VF. Methods: 69 dogs received apocynin (APO), 4 mg/kg intraveneously (IV), oxypurinol (OXY), 4 mg/kg IV, or both APO and OXY (BOTH) agents, or saline 3 h after coronary occlusion. Endocardium from ischemic sites (3-D mapping) was sampled for Rac1 (GTP-binding protein in membrane NADPH oxidase) activation or standard microelectrode techniques. Results (mean ± SE, * p < 0.05): VT/VF originating from ischemic zones was blocked by APO in 6/10 *, OXY in 4/9 *, BOTH in 5/8 * or saline in 1/27; 11/16 VT/VFs blocked were focal. In isolated myocardium, TA was blocked by APO (10−6 M) or OXY (10−8 M). Rac1 levels in ischemic endocardium were decreased by APO or OXY. Conclusion: APO and OXY suppressed focal VT/VF due to DADs, but the combination of the drugs was not more effective than either alone. Both drugs inhibited ischemic Rac1 with inhibition by OXY suggesting ROS-induced ROS. The inability to totally prevent VT/VF suggests that other mechanisms also contribute to ischemic VT.

Angiotensin II effects on ischemic focal ventricular tachycardia are predominantly mediated through myocardial AT2 receptor

Ischemic focal ventricular tachycardia (VT) occurs in animals and humans. Angiotensin-converting enzyme inhibitors and receptor blockers reduce sudden death in patients with ischemic heart disease. In our dog model of coronary artery occlusion (CAO), we tested the hypothesis that angiotensin II (AGII) will selectively promote focal VT and that the specific AT(2) blocker PD-123319 (PD), or AT(1) blocker losartan, will affect this VT. Anesthetized dogs (n = 90) underwent CAO, followed by three-dimensional activation mapping of inducible VT. Dogs without VT in 1-3 h after CAO received AGII, and those with VT received either PD or losartan. Focal endocardium excised from ischemic sites was studied in vitro with standard microelectrode. Of 33 dogs with no inducible VT, AGII infusion resulted in sustained VT of only focal Purkinje origin in 13 (39%) compared with 0 of 20 dogs with saline. Of 26 dogs with inducible VT at baseline, given PD, reinduction was blocked in 8 of 10 (P < 0.05) focal VT, but only 1 of 15 with reentry. In contrast, of 11 dogs given losartan, reinduction of either mechanism was not blocked. In vitro triggered activity in Purkinje was blocked by PD in 13 of 19 (P < 0.05), but not by losartan in 8. Also, triggered activity was promoted by AGII, losartan, or the combination in 9 of 12 tissues. AGII promotes only focal, mainly Purkinje ischemic VT. PD, but not losartan, preferentially blocked focal VT, which is likely due to triggered activity due to delayed afterdepolarizations in Purkinje.

Catecholamine-Independent Heart Rate Increases Require Ca2+/Calmodulin-Dependent Protein Kinase IIClinical Perspective

Background —Catecholamines increase heart rate by augmenting the cAMP responsive HCN4 9pacemaker current9 ( I f ) and/or by promoting inward Na + /Ca 2+ exchanger current ( I NCX ), by a 9Ca 2+ clock mechanism in sinoatrial nodal cells (SANCs). The importance, identity and function of signals that connect I f and Ca 2+ clock mechanisms are uncertain and controversial, but the multifunctional Ca 2+ and calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to β-adrenergic receptor (β-AR) stimulation. The aim of this stuy is to measure the contribution of the Ca 2+ clock and CaMKII to cardiac pacing independent of β-AR agonist stimulation. Methods and Results —We used the L-type Ca 2+ channel agonist BayK 8644 (BayK) to activate the SANC Ca 2+ clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from WT control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs, but increased I Ca equally in all SANCs, indicating that increasing I Ca was insufficient and suggesting CaMKII activation was required for heart rate increases by BayK. BayK did not increase I f or protein kinase A (PKA)-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca 2+ by BayK did not augment cAMP/PKA signaling at these targets. Late diastolic intracellular Ca 2+ release and I NCX were significantly reduced in AC3-I SANCs and the response to BayK was eliminated by ryanodine in all groups. Conclusions —The Ca 2+ clock is capable of supporting physiological fight or flight responses, independent of β-AR stimulation or I f increases. Complete Ca 2+ clock and β-AR stimulation responses require CaMKII.Background—Catecholamines increase heart rate by augmenting the cAMP-responsive hyperpolarization-activated cyclic nucleotide-gated channel 4 pacemaker current (If) and by promoting inward Na+/Ca2+ exchanger current (INCX) by a “Ca2+ clock” mechanism in sinoatrial nodal cells (SANCs). The importance, identity, and function of signals that connect If and Ca2+ clock mechanisms are uncertain and controversial, but the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to &bgr;-adrenergic receptor (&bgr;-AR) stimulation. The aim of this study was to measure the contribution of the Ca2+ clock and CaMKII to cardiac pacing independent of &bgr;-AR agonist stimulation. Methods and Results—We used the L-type Ca2+ channel agonist Bay K8644 (BayK) to activate the SANC Ca2+ clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from wild-type control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs but increased L-type Ca2+ current (ICa) equally in all SANCs, indicating that increasing ICa was insufficient and suggesting that CaMKII activation was required for heart rate increases by BayK. BayK did not increase If or protein kinase A-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca2+ by BayK did not augment cAMP/protein kinase A signaling at these targets. Late-diastolic intracellular Ca2+ release and INCX were significantly reduced in AC3-I SANCs, and the response to BayK was eliminated by ryanodine in all groups. Conclusions—The Ca2+ clock is capable of supporting physiological fight-or-flight responses, independent of &bgr;-AR stimulation or If increases. Complete Ca2+ clock and &bgr;-AR stimulation responses require CaMKII.