08 October 2014: Molecular Biology
Pioglitazone Improves Potassium Channel Remodeling Induced by Angiotensin II in Atrial Myocytes
Jun Gu BEF , Wei Hu ADG , Xu Liu ACDG
DOI: 10.12659/MSMBR.892450
Med Sci Monit Basic Res 2014; 20:153-160
Abstract
BACKGROUND: It has been demonstrated that atrial electrical remodeling contributes toward atrial fibrillation (AF) maintenance, and that angiotensin II (AngII) is involved in the pathogenesis of atrial electrical remodeling. Peroxisome proliferator activated receptor-γ (PPAR-γ) agonists have been shown to inhibit atrial electrical remodeling, but the underlying mechanisms are poorly understood. In the present study we investigated the regulating effects of PPAR-g agonist on AngII-induced potassium channel remodeling in atrial myocytes.
MATERIAL AND METHODS: Whole-cell patch-clamp technique was used to record transient outward potassium current (Ito), ultra-rapid delayed rectifier potassium (Ikur), and inward rectifier potassium current (Ik1). Real-time PCR was used to assess potassium channel subunit mRNA expression.
RESULTS: Compared with the control group, AngII reduced Ito and Ikur current density as well as amplified Ik1 current density, which were partially prevented by pioglitazone. Furthermore, pioglitazone alleviated the downregulation of Ito subunit (Kv 4.2) and Ikur subunit (Kv 1.5), as well as the upregulation of Ik1 subunit (Kir 2.1 and Kir 2.2) mRNA expression stimulated by AngII.
CONCLUSIONS: These results suggest that pioglitazone exhibits a beneficial effect on AngII-induced potassium channel remodeling. PPAR-γ agonists may be potentially effective up-stream therapies for AF.
Keywords: Atrial Remodeling - drug effects, Angiotensin II - pharmacology, Electrophysiological Phenomena - drug effects, Gene Expression Regulation - drug effects, Heart Atria - cytology, Ion Channel Gating - drug effects, Myocytes, Cardiac - physiology, Potassium Channels - metabolism, RNA, Messenger - metabolism, Real-Time Polymerase Chain Reaction, Thiazolidinediones - pharmacology
Background
Atrial fibrillation (AF) remains the most common arrhythmia in humans and causes substantial morbidity and mortality [1]. The prevalence of AF is growing in aging populations and the complications of AF are become increasingly burdensome [1]. The mechanisms underlying AF remain elusive, and atrial electrical remodeling has emerged as crucial in the onset or persistence of AF. Electrical remodeling, such as changes in major repolarized ion channels, leads to the shortening of the action potential duration (APD) and the effective refractory period (ERP), and results in an increase in atrial conduction slowing, re-entry, and, thereby, inducible AF [1]. There is considerable interest in the role of the renin-angiotensin-aldosterone system (RAAS) in the development of atrial remodeling and AF. It has been shown that atrial electrical remodeling in part is due to the activation of the RAAS. Angiotensin II (AngII) has been implicated in the process of atrial electrical remodeling characterized by ion channels remodeling as well as shortening of the APD and ERP [1–3].
Thiazolidinediones (TZDs), agonists of peroxisome proliferator-activated receptor-γ (PPAR-γ), have been proven to have anti-inflammatory and anti-proliferative effects induced by AngII in addition to their anti-diabetic activities [4–8]. Recent studies have shown that PPAR-γ agonist pioglitazone inhibited age-related [8] or congestive heart failure-induced [7] atrial electrical remodeling as well as AF promotion. Our previous study indicated that pioglitazone is capable of alleviating AngII-induced L-type calcium channel (ICa-L) remodeling in atrial myocytes [9]. The present study was designed to investigate the effects of pioglitazone on AngII-induced potassium channels remodeling, including transient outward potassium current (Ito), ultra-rapid delayed rectifier potassium (Ikur), and inward rectifier potassium current (Ik1) of atrial myocytes.
Material and Methods
CULTURE OF ATRIAL MYOCYTES (HL-1):
HL-1 cells (mouse atrial myocytes) were obtained from the laboratory of Dr. William Claycomb (Louisiana State University Health Science Center, New Orleans, LA). Cells were cultured in Claycomb medium (JRH Biosciences, USA) supplemented with 10% fetal bovine serum (JRH Biosciences, USA), 2 mM L-glutamine (Gibco, USA), 100 μM norepinephrine (Sigma, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, USA) on flasks pre-coated with fibronectin and gelatin (Sigma, USA), then incubated at 37°C, 5% CO2/95% air. The medium was changed every 24–48 h. HL-1 cells were placed in serum-free medium for 24 h before AngII (1 μM, Sigma, USA) stimulation.
WHOLE CELL PATCH CLAMP IN HL-1 CELLS:
HL-1 cells were isolated from the culture dishes after treatment with AngII and/or pioglitazone for 24 h using enzymatic dissociation for 2 min with 0.05% trypsin-EDTA (Gibco, USA). Digestion was arrested with 0.025% trypsin inhibitor and medium, and the sediment cells were used for experimentation within 6 h. Cells were observed using an inverted microscope (Nikon, JAPAN) and allowed to adhere to the bottom of the dish.
For Ito and Ikur current recording [10,11], the internal PIPette solution contained (in mM) KCl 45, K-aspartate 85, Na-pyruvate 5, MgATP 5.0, EGTA 10, HEPES 10, and glucose 11 (pH 7.4), while the bath solution contained (in mM) N-methyl-D-glucamine (NMG) 149, MgCl2 5, CaCl2 0.65, and HEPES 5. To block Ik1 and Ica, BaCl2 (200 μM) and CdCl2 (200 μM) were added to the superfusion. For Ik1 recording[12], the internal PIPette solution contained (in mM) NaCl 5, KCl 40, KF 100, EGTA 5, EDTA 3, glucose 5, K4P2O7 10, NaVO3 0.1, and HEPES 10 (pH7.4), while the bath solution contained (in mM) NaCl 132, CaCl2 1.8, KCl 20, MgCl2 10, glucose 10, and HEPES 10 (pH 7.4). Dof (5 nM), TTX (100 μM), and CdCl2 (200 μM) were added to the superfusion to block Ikr, Ina, and ICa. Tip potentials were compensated before the pipette touched the cell. After a gigaseal was obtained, the cell membrane was ruptured by gentle suction to establish the whole-cell configuration. Current signals were recorded with an EPC-10 amplifier using the pulse+pulsefit 8.53 data-acquisition system (HEKA Instruments). Signals were filtered at 5 kHz and stored on a computer. Series resistances (Rs) were 3–5 MΩ and were electrically compensated by 70–80% to minimize the capacitive surge on the current recording and voltage drop across the clamped membrane, and were maintained at a constant value during the current recording. The holding potential was kept at −80 mV. Ito was elicited by 300-ms test pulses between −40 and +50 in 10 mV increments, and verapamil (10 μM) was added to inhibit Ikur [13]. IKur stimulus consisted of 300 ms of incremental 10-mV voltage steps from −50 mV to +70 mV, preceded by a 200-ms prepulse to +30 mv to inactivate Ito. Ik1 was recorded by 300-ms test pulses between −150 and +10 mV in 20-mV increments. Peak current levels were plotted as a function of the command potential. To account for differences in cell size, all mean data are expressed as current density. The action of AngII in the presence and absence of pioglitazone was analyzed for its effects on the current-to-voltage (I–V) relationship. All experiments were performed at 25°C.
QUANTITATIVE REAL-TIME PCR:
Total RNA was extracted from HL-1 cells with TRIzol (Invitrogen, USA) and used to synthesize single-stranded complementary DNA with a high-capacity complementary DNA reverse transcription kit (Toyobo, JAPAN). Quantitative real-time RT-PCR involved the use of gene-specific primers [14] (see Table 1 for details) and SYBR kit (Takara, JAPAN). GAPDH was used as an internal control. Results are expressed as fold difference for each gene against GAPDH by the use of the 2−ΔΔCT method. A melting-point dissociation curve generated by the instrument was used to confirm that only a single product was present.
STATISTICAL ANALYSIS:
Statistical analysis was performed using SPSS 16.0 software. All data are expressed as mean ±SD. The differences between all measured values were assessed by one-way ANOVA followed by Dunnett post hoc test. A value of P < 0.05 was considered statistically significant.
Results
THE EFFECTS OF ANGII AND/OR PIOGLITAZONE ON ITO:
Figure 1 shows AngII (1 μM) reduced the peak of Ito current density from 6.3±0.6 pA/pF to 3.6±0.4 pA/pF (P<0.01) at 50 mV compared with the control group, but the addition of pioglitazone (10 μM) markedly alleviated this change (4.8±1.0 pA/pF vs. 3.6±0.4 pA/pF, P<0.05). Furthermore, AngII made the I–V curve shift downward compared with the control group, but preincubation with pioglitazone partially prevented AngII-induced alteration.
THE EFFECTS OF ANGII AND/OR PIOGLITAZONE ON IKUR:
AngII (1 μM) inhibited the peak of Ikur current density from 11.4±1.1 pA/pF to 6.9±0.8 pA/pF (P<0.01) at 70 mV compared with the control group, while pretreatment of cells with pioglitazone had an inhibitory effect (8.6±0.8 pA/pF vs. 6.9±0.8 pA/pF, P<0.05, Figure 2). Moreover, AngII made the I–V curve shift downward but pioglitazone alleviated the changes (Figure 2).
THE EFFECTS OF ANGII AND/OR PIOGLITAZONE ON IK1:
In contrast to the control group, AngII (1 μM) amplified the peak of Ik1 current density from −6.1±0.6p A/pF to −10.1±1.1 pA/pF (P<0.01) at −150 mV. However, pretreatment with pioglitazone (10 μM) markedly suppressed AngII-induced amplification of Ik1 peak current density (−7.9±0.6 pA/pF vs. −10.1±1.1 pA/pF, P<0.01, Figure 3). AngII made the I–V curve shift downward, but pioglitazone partially prevented AngII-induced change (Figure 3).
GENE EXPRESSION OF POTASSIUM CHANNELS IN ATRIAL MYOCYTES:
Since the above results revealed a functional change in potassium channel activities carrying Ito, Ikur, and Ik1 in atrial myocytes treated with AngII and/or pioglitazone, we analyzed the expression levels of the genes encoding Ito (Kv4.2), IKur (Kv1.5), and IK1 (Kir2.1 and Kir2.2). The mRNA expression of Kv4.2 and Kv1.5 in the AngII group (1 μM) was significantly decreased compared with the control group, but the mRNA expression of Kir2.1 and Kir2.2 in the AngII group (1 μM) was markedly increased compared with the control group. Pretreatment with pioglitazone (10 μM) could in part reverse the aforementioned changes (Figure 4).
Discussion
STUDY LIMITATIONS:
Our current study had some limitations that should be mentioned. First, the major limitation of the current study is that no in vivo model was used to verify the in vitro finding. Second, we did not evaluate the effect of pioglitazone on AngII-induced potassium channel remodeling in vitro in dose-dependent manners and recommended dosage was adopted according to previous studies [9,35,36]. Third, besides the I–V curve, we did not analyze the voltage dependence of potassium channel inactivation and activation curves or the time course of recovery from inactivation. Fourth, we only analyzed the mRNA level of potassium channel subunit, but the protein level was not determined.
Conclusions
Collectively, we have demonstrated that the PPAR-γ agonist, pioglitazone, significantly inhibits AngII-induced Ito, Ikur, and Ik1 remodeling. Further studies are needed to determine if pioglitazone is effective against AF.
References
1. Iwasaki YK, Nishida K, Kato T, Atrial fibrillation pathophysiology: implications for management: Circulation, 2011; 124(20); 2264-74, pmid: 22083148
2. Nakashima H, Kumagai K, Reverse-remodeling effects of angiotensin II type 1 receptor blocker in a canine atrial fibrillation model: Circ J, 2007; 71(12); 1977-82, pmid: 18037757
3. Tsai CT, Wang DL, Chen WP, Angiotensin II increases expression of alpha1C subunit of L-type calcium channel through a reactive oxygen species and cAMP response element-binding protein-dependent pathway in HL-1 myocytes: Circ Res, 2007; 100(10); 1476-85, pmid: 17463319
4. Liu T, Zhao H, Li J, Rosiglitazone attenuates atrial structural remodeling and atrial fibrillation promotion in alloxan-induced diabetic rabbits: Cardiovascular Therapeutics, 2014; 32( 4); 178-83, pmid: 24814361
5. Nanayakkara G, Viswaprakash N, Zhong J, PPARγ activation improves the molecular and functional components of I(to) remodeling by angiotensin II: Curr Pharm Des, 2013; 19(27); 4839-47, pmid: 23323617
6. Shimano M, Tsuji Y, Inden Y, Pioglitazone, a peroxisome proliferator-activated receptor-gamma activator, attenuates atrial fibrosis and atrial fibrillation promotion in rabbits with congestive heart failure: Heart Rhythm, 2008; 5(3); 451-59, pmid: 18313605
7. Kume O, Takahashi N, Wakisaka O, Pioglitazone attenuates inflammatory atrial fibrosis and vulnerability to atrial fibrillation induced by pressure overload in rats: Heart Rhythm, 2011; 8(2); 278-85, pmid: 21034856
8. Xu D, Murakoshi N, Igarashi M, PPAR-gamma activator pioglitazone prevents age-related atrial fibrillation susceptibility by improving antioxidant capacity and reducing apoptosis in a rat model: J Cardiovasc Electrophysiol, 2012; 23(2); 209-17, pmid: 21954843
9. Gu J, Liu X, Wang QX: J Mol Cell Cardiol, 2013; 65; 1-8, pmid: 24100253
10. Liu H, Jin MW, Xiang JZ, Raloxifene inhibits transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes: Eur J Pharmacol, 2007; 563(1–3); 61-68, pmid: 17337266
11. Liu Y, Zhang Y, Lin K, Protective effect of piperine on electrophysiology abnormalities of left atrial myocytes induced by hydrogen peroxide in rabbits: Life Sci, 2014; 94(2); 99-105, pmid: 24184297
12. Goldoni D, Zhao Y, Green BD, Inward rectifier potassium channels in the HL-1 cardiomyocyte-derived cell line: J Cell Physiol, 2010; 225(3); 751-56, pmid: 20568224
13. Gao Z, Lau CP, Chiu SW, Inhibition of ultra-rapid dela yed rectifier K+ current by verapamil in human atrial myocytes: J Mol Cell Cardiol, 2004; 36(2); 257-63, pmid: 14871553
14. Suzuki T, Shioya T, Murayama T, Multistep ion channel remodeling and lethal arrhythmia precede heart failure in a mouse model of inherited dilated cardiomyopathy: PLoS ONE, 2012; 7(4); e35353, pmid: 22514734
15. Nattel S, Li D, Ionic remodeling in the heart: pathophysiological significance and new therapeutic opportunities for atrial fibrillation: Circ Res, 2000; 87(6); 440-47, pmid: 10988234
16. Tsai CT, Chiang FT, Chen WP, Angiotensin II induces complex fractionated electrogram in a cultured atrial myocyte monolayer mediated by calcium and sodium-calcium exchanger: Cell Calcium, 2011; 49(1); 1-11, pmid: 21168206
17. He X, Gao X, Peng L, Atrial fibrillation induces myocardial fibrosis through angiotensin II type 1 receptor-specific Arkadia-mediated downregulation of Smad7: Circ Res, 2011; 108(2); 164-75, pmid: 21127293
18. Khatib R, Joseph P, Briel M, Blockade of the renin-angiotensin-aldosterone system(RAAS) for primary prevention of non-valvular atrial fibrillation: a systematic review and meta analysis of randomized controlled trials: Int J Cardiol, 2012; 165(1); 17-24, pmid: 22421406
19. Chilukoti RK, Mostertz J, Bukowska A: Int J Cardiol, 2013; 168(3); 2100-8, pmid: 23414741
20. Ehrlich JR, Inward rectifier potassium currents as a target for atrial fibrillation therapy: J Cardiovasc Pharmacol, 2008; 52(2); 129-35, pmid: 18670367
21. Wakili R, Voigt N, Kääb S, Recent advances in the molecular pathophysiology of atrial fibrillation: J Clin Invest, 2011; 121(8); 2955-68, pmid: 21804195
22. Greiser M, Schotten U, Dynamic remodeling of intracellular Ca2+ signaling during atrial fibrillation: J Mol Cell Cardiol, 2013; 58; 134-42, pmid: 23298712
23. Yeh YH, Wakili R, Qi XY, Calcium-handling abnormalities underlying atrial arrhythmogenesis and contractile dysfunction in dogs with congestive heart failure: Circ Arrhythm Electrophysiol, 2008; 1(2); 93-102, pmid: 19808399
24. Brundel BJ, Van Gelder IC, Henning RH, Alterations in potassium channel gene expression in atria of patients with persistent and paroxysmal atrial fibrillation: differential regulation of protein and mRNA levels for K+ channels: J Am Coll Cardiol, 2001; 37(3); 926-32, pmid: 11693772
25. Brundel BJ, Ausma J, van Gelder IC, Activation of proteolysis by calpains and structural changes in human paroxysmal and persistent atrial fibrillation: Cardiovasc Res, 2002; 54(2); 380-89, pmid: 12062342
26. Caballero R, de la Fuente MG, Gómez R, In humans, chronic atrial fibrillation decreases the transient outward current and ultrarapid component of the delayed rectifier current differentially on each atria and increases the slow component of the delayed rectifier current in both: J Am Coll Cardiol, 2010; 55(21); 2346-54, pmid: 20488306
27. Bosch RF, Zeng X, Grammer JB, Ionic mechanisms of electrical remodeling in human atrial fibrillation: Cardiovasc Res, 1999; 44(1); 121-31, pmid: 10615396
28. Gassanov N, Brandt MC, Michels G, Angiotensin II-induced changes of calcium sparks and ionic currents in human atrial myocytes: potential role for early remodeling in atrial fibrillation: Cell Calcium, 2006; 39(2); 175-86, pmid: 16303176
29. Bosch RF, Scherer CR, Rüb N, Molecular mechanisms of early electrical remodeling: transcriptional downregulation of ion channel subunits reduces I(Ca,L) and I(to) in rapid atrial pacing in rabbits: J Am Coll Cardiol, 2003; 41(5); 858-69, pmid: 12628735
30. Van Wagoner DR, Pond AL, McCarthy PM, Outward K+ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation: Circ Res, 1997; 80(6); 772-81, pmid: 9168779
31. Yue L, Melnyk P, Gaspo R, Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation: Circ Res, 1999; 84(7); 776-84, pmid: 10205145
32. Chao TF, Leu HB, Huang CC, Thiazolidinediones can prevent new onset atrial fibrillation in patients with non-insulin dependent diabetes: Int J Cardiol, 2012; 156(2); 199-202, pmid: 21930315
33. Gu J, Liu X, Wang X, Beneficial effect of pioglitazone on the outcome of catheter ablation in patients with paroxysmal atrial fibrillation and type 2 diabetes mellitus: Europace, 2011; 13(9); 1256-61, pmid: 21551479
34. Imayama I, Ichiki T, Inanaga K, Telmisartan downregulates angiotensin II type 1 receptor through activation of peroxisome proliferator-activated receptor gamma: Cardiovasc Res, 2006; 72(1); 184-90, pmid: 16938288
35. Desouza CV, Gerety M, Hamel FG, Effects of a PPAR-gamma agonist, on growth factor and insulin stimulated endothelial cells: Vascul Pharmacol, 2009; 51(2–3); 162-68, pmid: 19520186
36. Pan HW, Xu JT, Chen JS, Pioglitazone inhibits TGFbeta induced keratocyte transformation to myofibroblast and extracellular matrix production: Mol Biol Rep, 2011; 38(7); 4501-8, pmid: 21127991
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