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Cardiac Insulin Resistance Due to Reduced Phosphoinositide-3-kinase (p110α) Activation Increases the Heart's Susceptibility to Atrial Fibrillation
Abstracts
S131
296 3 ADRENERGIC RECEPTOR STIMULATES THE CARDIAC NA/K PUMP VIA AN NO-DEPENDENT PATHWAY AND REDUCES OXIDATIVE MODIFICATION OF THE 1 PUMP SUBUNIT
297 CARDIAC INSULIN RESISTANCE DUE TO REDUCED PHOSPHOINOSITIDE-3-KINASE (P110␣) ACTIVATION INCREASES THE HEART’S SUSCEPTIBILITY TO ATRIAL FIBRILLATION
G.A. Figtree 1 , A. Garcia 1 , C. Liu 1 , H. Bundgaard 2 , E.J.
L. Pretorius 1,2 , X.J. Du 1 , E.A. Woodcock 1 , H. Kiriazis 1 , R.C.Y. Lin 3 , S. Marasco 4 , G.A. Head 1 , A.M. Dart 1,4 , G.L. Jennings 1 , J.R. McMullen 1
Hamilton 1 ,
K.K.M.
Chia 1 ,
H.H.
Rasmussen 1
1 North Shore Heart Research Group, Kolling Institute, Univer-
sity of Sydney, Australia 2 Medical Department, The Heart Centre, Rigshospitalet, National University Hospital, University of Copenhagen, Denmark Receptor coupled activation of NADPH oxidase in cardiac myocytes induces inhibition of the Na+ –K+ pump and oxidative modification (glutathionylation) of its 1 subunit, while stimulation of nitric oxide (NO)-activated guanylyl cyclase eliminates receptor-coupled activation of NADPH oxidase and reverses pump inhibition and glutathionylation of the Na+ –K+ pump. Since the 1 subunit is glutathionylated at baseline and since glutathionylation inhibits the pump, we have tested the hypothesis that receptor-coupled NO synthesis stimulates the Na–K pump, and is associated with a decrease in glutathionylation from the baseline. Exposure of ventricular myocytes to 10 nM of 3 adrenergic agonist BRL 37344 (BRL) increased NO-sensitive DAF-2DA fluorescence. We measured pump current (Ip ) by the whole cell patch clamp technique. Ip was identified as the ouabain-induced shift in holding current. Ip (Mean ± SEM) of myocytes exposed to 10 nM BRL (0.55 ± 0.03 pA/pF, N = 6) was significantly (P < 0.01) larger than Ip of controls (controls 0.35 ± 0.002 pA/pF, N = 33). Blockade of 1 /2 adrenergic receptors with 1 M nadolol; ␣-adrenergic receptors with 1 M prazosin; or PKA by 0.5 mM H-89 had no effect on the BRL-induced increase in Ip . However, inhibition of NOS by 10 mM L-NAME or radicicol abolished pump stimulation. To examine the glutathionylation of the 1 subunit we loaded myocytes with biotin-tagged glutathione. Glutathionylated proteins were precipitated with streptavidin. 3 -AR agonists decreased 1 subunit glutathionylation. We conclude that receptor coupled activation of NO-dependent signalling induces an increase in Ip and a decrease in glutathionylation of the 1 pump subunit. doi:10.1016/j.hlc.2009.05.298
1 Baker
IDI Heart and Diabetes Institute, Melbourne, Australia of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Australia 3 Ramaciotti Centre for Gene Function Analysis, University of New South Wales, Sydney, Australia 4 Alfred Hospital, Melbourne, Australia 2 Faculty
Introduction: Atrial fibrillation (AF) is the most common sustained arrhythmia in cardiology departments worldwide. Despite being potentially life-threatening, the aetiology of AF is poorly understood. Previously it was reported that the reduction of the cardioprotective kinase phosphoinositide-3-kinase (p110␣) [PI3K] in a setting of disease (due to over-expression of mammalian sterile 20like kinase 1 [Mst1]) reduces survival and cardiac function, while concurrently increasing fibrosis, particularly in the atria. Aim: To examine whether reduced PI3K adversely affects cardiac conduction in a setting of disease. Hypothesis: Reduced PI3K activity will increase cardiac conduction abnormalities. Methods: Cardiac-specific Mst1 mice were crossed with cardiac-specific dominant negative PI3K (dnPI3K) mice. Non-transgenic, single-transgenic (dnPI3K or Mst1) and double-transgenic (dnPI3K-Mst1) mice were subjected to electrocardiography (ECG) and ambulatory telemetry studies. Atrial gene expression was examined using microarray analysis. Atrial appendages from patients undergoing mitral valve surgery (with chronic AF) or coronary artery bypass graft surgery (who did or did not develop post-operative acute AF) were collected. PI3K activity was analysed. Results: dnPI3K-Mst1 mice showed varying degrees of first, second, and third degree atrioventricular conduction blockade. Spontaneous AF occurred in 40% of anaesthetised and 100% of ambulatory dnPI3K-Mst1 mice (n = 6). Microarray analysis showed up-regulation of genes associated with fibrosis, cardiac conduction channels and metabolism in the dnPI3K-Mst1 mice compared with other groups (p < 0.05; n = 4). PI3K activity was decreased in atrial appendages from patients with acute (n = 4) or chronic AF (n = 5) compared with those that did not develop AF (p < 0.05; n = 6). Conclusion: Reduced PI3K activation increases the heart’s susceptibility to AF. doi:10.1016/j.hlc.2009.05.299
ABSTRACTS
Heart, Lung and Circulation 2009;18S:S1–S286
S131
296 3 ADRENERGIC RECEPTOR STIMULATES THE CARDIAC NA/K PUMP VIA AN NO-DEPENDENT PATHWAY AND REDUCES OXIDATIVE MODIFICATION OF THE 1 PUMP SUBUNIT
297 CARDIAC INSULIN RESISTANCE DUE TO REDUCED PHOSPHOINOSITIDE-3-KINASE (P110␣) ACTIVATION INCREASES THE HEART’S SUSCEPTIBILITY TO ATRIAL FIBRILLATION
G.A. Figtree 1 , A. Garcia 1 , C. Liu 1 , H. Bundgaard 2 , E.J.
L. Pretorius 1,2 , X.J. Du 1 , E.A. Woodcock 1 , H. Kiriazis 1 , R.C.Y. Lin 3 , S. Marasco 4 , G.A. Head 1 , A.M. Dart 1,4 , G.L. Jennings 1 , J.R. McMullen 1
Hamilton 1 ,
K.K.M.
Chia 1 ,
H.H.
Rasmussen 1
1 North Shore Heart Research Group, Kolling Institute, Univer-
sity of Sydney, Australia 2 Medical Department, The Heart Centre, Rigshospitalet, National University Hospital, University of Copenhagen, Denmark Receptor coupled activation of NADPH oxidase in cardiac myocytes induces inhibition of the Na+ –K+ pump and oxidative modification (glutathionylation) of its 1 subunit, while stimulation of nitric oxide (NO)-activated guanylyl cyclase eliminates receptor-coupled activation of NADPH oxidase and reverses pump inhibition and glutathionylation of the Na+ –K+ pump. Since the 1 subunit is glutathionylated at baseline and since glutathionylation inhibits the pump, we have tested the hypothesis that receptor-coupled NO synthesis stimulates the Na–K pump, and is associated with a decrease in glutathionylation from the baseline. Exposure of ventricular myocytes to 10 nM of 3 adrenergic agonist BRL 37344 (BRL) increased NO-sensitive DAF-2DA fluorescence. We measured pump current (Ip ) by the whole cell patch clamp technique. Ip was identified as the ouabain-induced shift in holding current. Ip (Mean ± SEM) of myocytes exposed to 10 nM BRL (0.55 ± 0.03 pA/pF, N = 6) was significantly (P < 0.01) larger than Ip of controls (controls 0.35 ± 0.002 pA/pF, N = 33). Blockade of 1 /2 adrenergic receptors with 1 M nadolol; ␣-adrenergic receptors with 1 M prazosin; or PKA by 0.5 mM H-89 had no effect on the BRL-induced increase in Ip . However, inhibition of NOS by 10 mM L-NAME or radicicol abolished pump stimulation. To examine the glutathionylation of the 1 subunit we loaded myocytes with biotin-tagged glutathione. Glutathionylated proteins were precipitated with streptavidin. 3 -AR agonists decreased 1 subunit glutathionylation. We conclude that receptor coupled activation of NO-dependent signalling induces an increase in Ip and a decrease in glutathionylation of the 1 pump subunit. doi:10.1016/j.hlc.2009.05.298
1 Baker
IDI Heart and Diabetes Institute, Melbourne, Australia of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Australia 3 Ramaciotti Centre for Gene Function Analysis, University of New South Wales, Sydney, Australia 4 Alfred Hospital, Melbourne, Australia 2 Faculty
Introduction: Atrial fibrillation (AF) is the most common sustained arrhythmia in cardiology departments worldwide. Despite being potentially life-threatening, the aetiology of AF is poorly understood. Previously it was reported that the reduction of the cardioprotective kinase phosphoinositide-3-kinase (p110␣) [PI3K] in a setting of disease (due to over-expression of mammalian sterile 20like kinase 1 [Mst1]) reduces survival and cardiac function, while concurrently increasing fibrosis, particularly in the atria. Aim: To examine whether reduced PI3K adversely affects cardiac conduction in a setting of disease. Hypothesis: Reduced PI3K activity will increase cardiac conduction abnormalities. Methods: Cardiac-specific Mst1 mice were crossed with cardiac-specific dominant negative PI3K (dnPI3K) mice. Non-transgenic, single-transgenic (dnPI3K or Mst1) and double-transgenic (dnPI3K-Mst1) mice were subjected to electrocardiography (ECG) and ambulatory telemetry studies. Atrial gene expression was examined using microarray analysis. Atrial appendages from patients undergoing mitral valve surgery (with chronic AF) or coronary artery bypass graft surgery (who did or did not develop post-operative acute AF) were collected. PI3K activity was analysed. Results: dnPI3K-Mst1 mice showed varying degrees of first, second, and third degree atrioventricular conduction blockade. Spontaneous AF occurred in 40% of anaesthetised and 100% of ambulatory dnPI3K-Mst1 mice (n = 6). Microarray analysis showed up-regulation of genes associated with fibrosis, cardiac conduction channels and metabolism in the dnPI3K-Mst1 mice compared with other groups (p < 0.05; n = 4). PI3K activity was decreased in atrial appendages from patients with acute (n = 4) or chronic AF (n = 5) compared with those that did not develop AF (p < 0.05; n = 6). Conclusion: Reduced PI3K activation increases the heart’s susceptibility to AF. doi:10.1016/j.hlc.2009.05.299
ABSTRACTS
Heart, Lung and Circulation 2009;18S:S1–S286