Title: Reduced Phosphoinositide 3-Kinase (p110α) Activation Increases the Susceptibility to Atrial Fibrillation
Abstract: Atrial fibrillation (AF) is the most common sustained arrhythmia presenting at cardiology departments. A limited understanding of the molecular mechanisms responsible for the development of AF has hindered treatment strategies. The purpose of this study was to assess whether reduced activation of phosphoinositide 3-kinase (PI3K, p110α) makes the compromised heart susceptible to AF. Risk factors for AF, including aging, obesity, and diabetes, have been associated with insulin resistance that leads to depressed/defective PI3K signaling. However, to date, there has been no link between PI3K(p110α) and AF. To address this question, we crossed a cardiac-specific transgenic mouse model of dilated cardiomyopathy (DCM) with a cardiac-specific transgenic mouse expressing a dominant negative mutant of PI3K (dnPI3K; reduces PI3K activity). Adult (∼4.5 months) double-transgenic (dnPI3K-DCM), single-transgenic (DCM-Tg, dnPI3K-Tg), and nontransgenic mice were subjected to morphological, functional/ECG, microarray, and biochemical analyses. dnPI3K-DCM mice developed AF and had depressed cardiac function as well as greater atrial enlargement and fibrosis than DCM-Tg mice. AF was not detected in other groups. Aged DCM-Tg mice (∼15 months) with a similar phenotype to dnPI3K-DCM mice (4.5 months) did not develop AF, suggesting loss of PI3K activity directly contributed to the AF phenotype. Furthermore, increasing PI3K activity reduced atrial fibrosis and improved cardiac conduction in DCM-Tg mice. Finally, in atrial appendages from patients with AF, PI3K activation was lower compared with tissue from patients in sinus rhythm. These results suggest a link between PI3K(p110α) and AF. Atrial fibrillation (AF) is the most common sustained arrhythmia presenting at cardiology departments. A limited understanding of the molecular mechanisms responsible for the development of AF has hindered treatment strategies. The purpose of this study was to assess whether reduced activation of phosphoinositide 3-kinase (PI3K, p110α) makes the compromised heart susceptible to AF. Risk factors for AF, including aging, obesity, and diabetes, have been associated with insulin resistance that leads to depressed/defective PI3K signaling. However, to date, there has been no link between PI3K(p110α) and AF. To address this question, we crossed a cardiac-specific transgenic mouse model of dilated cardiomyopathy (DCM) with a cardiac-specific transgenic mouse expressing a dominant negative mutant of PI3K (dnPI3K; reduces PI3K activity). Adult (∼4.5 months) double-transgenic (dnPI3K-DCM), single-transgenic (DCM-Tg, dnPI3K-Tg), and nontransgenic mice were subjected to morphological, functional/ECG, microarray, and biochemical analyses. dnPI3K-DCM mice developed AF and had depressed cardiac function as well as greater atrial enlargement and fibrosis than DCM-Tg mice. AF was not detected in other groups. Aged DCM-Tg mice (∼15 months) with a similar phenotype to dnPI3K-DCM mice (4.5 months) did not develop AF, suggesting loss of PI3K activity directly contributed to the AF phenotype. Furthermore, increasing PI3K activity reduced atrial fibrosis and improved cardiac conduction in DCM-Tg mice. Finally, in atrial appendages from patients with AF, PI3K activation was lower compared with tissue from patients in sinus rhythm. These results suggest a link between PI3K(p110α) and AF. Atrial fibrillation (AF) is a cardiac disorder characterized by uncoordinated atrial activation. 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The rationale for hypothesizing a link between AF and reduced PI3K(p110α) activity came from multiple lines of evidence. First, we previously demonstrated that decreasing PI3K activation alters gene expression of ion channels in ventricular tissue. 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Animal care and experimentation were approved by the Alfred Medical Research and Education Precinct Animal Ethics Committee. Cardiac-specific DCM-Tg (mammalian sterile 20-like kinase 1, line no. 28; C57BL/6 background) and cardiac-specific dnPI3K-Tg (FVB/N; provided by P. Kang, BIDMC) were generated and genotyped as described.16Shioi T Kang PM Douglas PS Hampe J Yballe CM Lawitts J Cantley LC Izumo S The conserved phosphoinositide 3-kinase pathway determines heart size in mice.EMBO J. 2000; 19: 2537-2548Crossref PubMed Scopus (528) Google Scholar, 33Yamamoto S Yang G Zablocki D Liu J Hong C Kim SJ Soler S Odashima M Thaisz J Yehia G Molina CA Yatani A Vatner DE Vatner SF Sadoshima J Activation of Mst1 causes dilated cardiomyopathy by stimulating apoptosis without compensatory ventricular myocyte hypertrophy.J Clin Invest. 2003; 111: 1463-1474Crossref PubMed Scopus (233) Google Scholar The DCM-Tg model has been considered clinically relevant because mice develop DCM as a consequence of increased apoptosis.33Yamamoto S Yang G Zablocki D Liu J Hong C Kim SJ Soler S Odashima M Thaisz J Yehia G Molina CA Yatani A Vatner DE Vatner SF Sadoshima J Activation of Mst1 causes dilated cardiomyopathy by stimulating apoptosis without compensatory ventricular myocyte hypertrophy.J Clin Invest. 2003; 111: 1463-1474Crossref PubMed Scopus (233) Google Scholar Male heterozygous DCM-Tg and female heterozygous dnPI3K-Tg were crossed to generate 1) double transgenic mice: dnPI3K-DCM, expressing both transgenes (dnPI3K+/−DCM+/−), 2) dnPI3K-Tg (dnPI3K+/−DCM−/−), 3) DCM-Tg (dnPI3K−/−DCM+/−) and 4) nontransgenic littermate controls (NTg; dnPI3K−/−DCM−/−). This study is focused on the above four genotypes. However, a small subset of DCM mice with increased PI3K activity were also generated (described below) to assess whether increasing PI3K activity could reverse some of the phenotypic characteristics of the DCM model. All mice were bred on the same genetic background (C57BL/6-FVB/N). Male heterozygous DCM-Tg and female heterozygous constitutively active (ca) PI3K-Tg (caPI3K; PI3K activity increased by 6.5-fold in the heart16Shioi T Kang PM Douglas PS Hampe J Yballe CM Lawitts J Cantley LC Izumo S The conserved phosphoinositide 3-kinase pathway determines heart size in mice.EMBO J. 2000; 19: 2537-2548Crossref PubMed Scopus (528) Google Scholar) were crossed to generate caPI3K-DCM expressing both transgenes (caPI3K+/− DCM+/−). In vivo heart function and chamber dimensions were assessed by echocardiography using a Hewlett-Packard Sonos 5500 ultrasonograph with a 15-MHz linear array transducer. Mice were anesthetized with 2,2,2-tribromoethanol (240 mg/kg, i.p.). Left ventricular (LV) wall thicknesses [LV posterior wall (LVPW) and interventricular septum (IVS)], LV chamber dimensions (LVD) at end-diastole and end-systole (LVDd and LVDs), and fractional shortening [FS = (LVDd-LVDs)/LVDd] were determined from M-mode images. Left atrial size was determined from long-axis two-dimensional images at end-systole.34Du XJ Gao XM Wang B Jennings GL Woodcock EA Dart AM Age-dependent cardiomyopathy and heart failure phenotype in mice overexpressing beta(2)-adrenergic receptors in the heart.Cardiovasc Res. 2000; 48: 448-454Crossref PubMed Scopus (83) Google Scholar Arterial pressures, LV systolic and end-diastolic pressures (LVSP, LVEDP), maximal rates of rise and fall of LV pressures (dP/dtmax, dP/dtmin) and HR were measured in anesthetized mice (ketamine/xylazine/atropine 100/10/1.2 mg/kg i.p.) using a 1.4-Fr Millar (Houston, TX) MIKRO-TIP catheter and a Powerlab system (ADInstruments, Sydney, Australia).35Du XJ Autelitano DJ Dilley RJ Wang B Dart AM Woodcock EA beta(2)-adrenergic receptor overexpression exacerbates development of heart failure after aortic stenosis.Circulation. 2000; 101: 71-77Crossref PubMed Scopus (112) Google Scholar ECG recordings were measured in anesthetized mice (2,2,2-tribromoethanol: 240 mg/kg, i.p.) using the Powerlab system and BioAmp (ADInstruments). Two pairs of 27-gauge needle electrodes were placed subcutaneously and recordings were made from a chest lead (equivalent to V5). All signals were sampled at 1 kHz for a period of 5 to 10 minutes. Averaged HRs, P-R intervals, R-R intervals, QRS intervals and amplitudes of positive R- and P-waves were measured digitally using ADInstruments (Chart 5 Pro ECG analysis module). An electrophysiology catheter (EPR-800, 1.1F; Millar) was inserted into the jugular vein for placement inside the right atrium and right ventricle of anesthetized mice (ketamine/xylazine/atropine, 100/10/1.2 mg/kg i.p.). Surface ECG recordings (lead II position) were measured simultaneously. To confirm anesthetized ECG findings, ambulatory ECGs were recorded in conscious unrestrained mice after implantation of telemeters (TA10EA-F20, Data Sciences International) with leads subcutaneously positioned at the right foreleg and left side of the chest (ie, similar position to surface leads; V5). A 7-day recovery period was allowed before recording for 24 hours. Files were recorded using ART acquisition (Data Sciences International, Minneapolis, MN) or Labview data acquisition.36Head GA Lukoshkova EV Mayorov DN van den Buuse M Non-symmetrical double-logistic analysis of 24-h blood pressure recordings in normotensive and hypertensive rats.J Hypertens. 2004; 22: 2075-2085Crossref PubMed Scopus (25) Google Scholar Body weight, tibia length, and wet weights of ventricles, atria, and lungs were measured. 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Five microliters of undiluted mouse plasma was loaded per lane. Gels were incubated at 37°C in a humidified chamber until regions of proteolysis appeared. Matrix metalloproteinase 2 (MMP-2) and MMP-9 activities in protein from mouse ventricular tissue were assessed by gelatin zymography as described.39Lekgabe ED Kiriazis H Zhao C Xu Q Moore XL Su Y Bathgate RA Du XJ Samuel CS Relaxin reverses cardiac and renal fibrosis in spontaneously hypertensive rats.Hypertension. 2005; 46: 412-418Crossref PubMed Scopus (162) Google Scholar PI3K activity was assessed in mouse ventricular tissue and human atrial tissue.16Shioi T Kang PM Douglas PS Hampe J Yballe CM Lawitts J Cantley LC Izumo S The conserved phosphoinositide 3-kinase pathway determines heart size in mice.EMBO J. 2000; 19: 2537-2548Crossref PubMed Scopus (528) Google Scholar, 20McMullen JR Shioi T Huang WY Zhang L Tarnavski O Bisping E Schinke M Kong S Sherwood MC Brown J Riggi L Kang PM Izumo S The insulin-like growth factor 1 receptor induces physiological heart growth via the phosphoinositide 3-kinase(p110α) pathway.J Biol Chem. 2004; 279: 4782-4793Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar Mouse ventricular tissue or human atrial tissue lysate (1 mg) was immunoprecipitated with an anti-p85 antibody (0.5 μl, Upstate Biotechnology, New York, NY) and subjected to an in vitro lipid kinase assay using phosphatidylinositol as a substrate. Part of the immunoprecipitated enzyme was subjected to Western blotting and probed with the anti-p85 antibody to confirm that an equal amount of enzyme was used for the assay. The study of human atrial tissue was approved by the Alfred Hospital Human Ethics committee. Tissue samples of atrial appendages were collected from patients undergoing coronary artery bypass graft (CABG) surgery (right appendage only) who did or did not develop acute AF, as well as patients undergoing mitral valve surgery (left appendages) with chronic AF. Medications (statins, β-blockers, and ACE inhibitors) were evenly distributed between groups and stopped the night before surgery. Patients with diabetes were not included in the study. Phosphorylation of Akt and extracellular signal regulated kinase (ERK) 1/2 were assessed in mouse ventricular tissue as described.16Shioi T Kang PM Douglas PS Hampe J Yballe CM Lawitts J Cantley LC Izumo S The conserved phosphoinositide 3-kinase pathway determines heart size in mice.EMBO J. 2000; 19: 2537-2548Crossref PubMed Scopus (528) Google Scholar Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and GAPDH were assessed in mouse ventricular samples as described.40McMullen JR Sherwood MC Tarnavski O Zhang L Dorfman AL Shioi T Izumo S Inhibition of mTOR signaling with rapamycin regresses established cardiac hypertrophy induced by pressure overload.Circulation. 2004; 109: 3050-3055Crossref PubMed Scopus (427) Google Scholar Gene expression profiling was perf