Rapid stimulation causes electrical remodeling in cultured atrial myocytes

Abstract-Angiotensin II (Ang II) is involved in the pathogenesis of atrial fibrillation (AF). L-type calcium channel (LCC)expression is altered in AF remodeling. We investigated whether Ang II modulates LCC current through transcriptional regulation, by using murine atrial HL-1 cells, which have a spontaneous calcium transient, and an in vivo rat model. Ang II increased LCC ␣1C subunit mRNA and protein levels and LCC current density, which resulted in an augmented calcium transient in atrial myocytes. An Ϸ2-kb promoter region of LCC ␣1C subunit gene was cloned to the pGL3 luciferase vector. Ang II significantly increased promoter activity in a concentration-and time-dependent manner.Truncation and mutational analysis of the LCC ␣1C subunit gene promoter showed that cAMP response element (CRE) (Ϫ1853 to Ϫ1845) was an important cis element in Ang II-induced LCC ␣1C subunit gene expression. Transfection of dominant-negative CRE binding protein (CREB) (pCMV-CREBS133A) abolished the Ang II effect. Ang II (1 mol/L, 2 hours) induced serine 133 phosphorylation of CREB and binding of CREB to CRE and increased LCC ␣1C subunit gene promoter activity through a protein kinase C/NADPH oxidase/reactive oxygen species pathway, which was blocked by the Ang II type 1 receptor blocker losartan and the antioxidant simvastatin. In the rat model, Ang II infusion increased LCC ␣1C subunit expression and serine 133 phosphorylation of CREB, which were attenuated by oral losartan and simvastatin. In summary, Ang II induced LCC ␣1C subunit expression via a protein kinase C-, reactive oxygen species-, and CREB-dependent pathway and was blocked by losartan and simvastatin. [2][3][4] Moreover, blockade of the renin-angiotensin system has been shown to be an effective treatment of AF. 5 Ang II increases spontaneous calcium sparks 6,7 and L-type calcium channel (LCC) current (I CaL ) in cardiomyocytes. 7,8 However, downregulation of I CaL 9 and channel expression 10,11 are observed in AF. Whether Ang II increases or decreases I CaL channel subunit expression in atrial myocytes and the detailed signaling mechanisms are unknown. Accordingly, in the present study, we explored the chronic effect of Ang II on the transcriptional regulation of LCC channel subunits in atrial myocytes. We used a murine atrial cell line HL-1, which is the only available atrial myocyte cell line that continuously divides and maintains a differentiated cardiac phenotype with spontaneous depolarization. 10 We also used an in vivo rat model of Ang II infusion to verify the results obtained in the cellular study. We first found that Ang II increased LCC channel ␣1C subunit expression and augmented I CaL and calcium transient amplitude in HL-1 atrial myocytes. The detailed signaling mechanisms by which Ang II regulates the expression of LCC ␣1C subunits were also studied. Materials and Methods Culture and Transfection of HL-1 CardiomyocytesHL-1 myocytes were cultured in the Claycomb medium. Transient transfection of the HL-1 myocytes was performed using Lipo-

Section: Limitationsmentioning

confidence: 93%

Angiotensin II Increases Expression of α1C Subunit of L-Type Calcium Channel Through a Reactive Oxygen Species and cAMP Response Element–Binding Protein–Dependent Pathway in HL-1 Myocytes

Tsai

Wang

Chen

et al. 2007

“…9,11,20 HSP expression was increased by preexposure to GGA or heat shock (Figure 1). Cell tachypacing reduced CaT and contractile function, effects prevented by HSP induction (Figure 2A and 2B).…”

Section: Effect Of Hsp Induction On Tachypaced Hl-1 Myocytesmentioning

confidence: 95%

Induction of Heat Shock Response Protects the Heart Against Atrial Fibrillation

Brundel

Shiroshita-Takeshita

et al. 2006

153

191

Abstract-There is evidence suggesting that heat shock proteins (HSPs) may protect against clinical atrial fibrillation (AF).We evaluated the effect of HSP induction in an in vitro atrial cell line (HL-1) model of tachycardia remodeling and in tachypaced isolated canine atrial cardiomyocytes. We also evaluated the effect of HSP induction on in vivo AF promotion by atrial tachycardia-induced remodeling in dogs. Tachypacing (3 Hz) significantly and progressively reduced Ca 2ϩ transients and cell shortening of HL-1 myocytes over 4 hours. These reductions were prevented by HSP-inducing pretreatments: mild heat shock, geranylgeranylacetone (GGA), and transfection with human HSP27 or the phosphorylation-mimicking HSP27-DDD. However, treatment with HSP70 or the phosphorylation-deficient mutant HSP27-AAA failed to alter tachycardia-induced Ca 2ϩ transient and cell-shortening reductions, and downregulation (short interfering RNA) of HSP27 prevented GGA-mediated protection. Tachypacing (3 Hz) for 24 hours in vitro significantly reduced L-type Ca 2ϩ current and action potential duration in canine atrial cardiomyocytes; these effects were prevented when tachypacing was performed in cells exposed to GGA. In vivo treatment with GGA increased HSP expression and suppressed refractoriness abbreviation and AF promotion in dogs subjected to 1-week atrial tachycardia-induced remodeling. In conclusion, our findings indicate that (1) HSP induction protects against atrial tachycardia-induced remodeling, (2) the protective effect in HL-1 myocytes requires HSP27 induction and phosphorylation, and (3) the orally administered HSP inducer GGA protects against AF in a clinically relevant animal model. These findings advance our understanding of the biochemical determinants of AF and suggest the possibility that HSP induction may be an interesting novel approach to preventing clinical AF.

“…Previous in vitro models of atrial tachycardia remodeling have included rapidly paced HL-1 cells derived from a mouse atrial tumor cell line, 38 and paced human atrial tissue slices. 25 The HL-1 cell model displays important features of atrial remodeling but is limited by a low expression rate of I CaL (Ͻ20% of cells display prominent I CaL ) and K ϩ channel remodeling (unchanged I to , increased I Kr ) 38 that differs from the established pattern of in vivo atrial tachycardia remodeling (reduced I to , unchanged I Kr ).…”

Section: Circulation Research October 10 2008mentioning

confidence: 99%

“…25 The HL-1 cell model displays important features of atrial remodeling but is limited by a low expression rate of I CaL (Ͻ20% of cells display prominent I CaL ) and K ϩ channel remodeling (unchanged I to , increased I Kr ) 38 that differs from the established pattern of in vivo atrial tachycardia remodeling (reduced I to , unchanged I Kr ). 4,20,37 The human tachypaced atrial slice model reproduces some biochemical features seen in AF patients, but its electrophysiological properties have not been established, and atrial tissue superfusion is known to cause important functional alterations and viability problems.…”

Section: Circulation Research October 10 2008mentioning

confidence: 99%

Cellular Signaling Underlying Atrial Tachycardia Remodeling of L-type Calcium Current

Yeh

Xiao

et al. 2008

176

132

Abstract-Atrial tachycardia (AT) downregulates L-type Ca 2ϩ current (I CaL ) and causes atrial fibrillation-promoting electric remodeling. This study assessed potential underlying signal transduction. Cultured adult canine atrial cardiomyocytes were paced at 0, 1, or 3 Hz (P0, P1, P3) for up to 24 hours. Cellular tachypacing (P3) mimicked effects of in vivo AT: decreased I CaL and transient outward current (I to ), unchanged I CaT , I Kr , and I Ks , and reduced action potential duration (APD). I CaL was unchanged in P3 at 2 and 8 hours but decreased by 55Ϯ6% at 24 hours. Tachypacing caused Ca 2ϩ i accumulation in P3 cells at 2 to 8 hours, but, by 24 hours, Ca 2ϩ i returned to baseline. Ca v 1.2 mRNA expression was not altered at 2 hours but decreased significantly at 8 and 24 hours (32Ϯ4% and 48Ϯ4%, respectively) and protein expression was decreased (47Ϯ8%) at 24 hours only. Suppressing Ca 2ϩ i increases during tachypacing with the I CaL blocker nimodipine or the Ca 2ϩ chelator BAPTA-AM prevented I CaL downregulation. Calcineurin activity increased in P3 at 2 and 8 hours, respectively, returning to baseline at 24 hours. Nuclear factor of activated T cells (NFAT) nuclear translocation was enhanced in P3 cells. Ca 2ϩ -dependent signaling was probed with inhibitors of Ca 2ϩ /calmodulin (W-7), calcineurin , and NFAT (INCA6): each prevented I CaL downregulation. Significant APD reductions (Ϸ30%) at 24 hours in P3 cells were prevented by nimodipine, BAPTA-AM, W-7, or FK-506. Thus, rapid atrial cardiomyocyte activation causes Ca 2ϩ loading, which activates the Ca 2ϩ -dependent calmodulin-calcineurin-NFAT system to cause transcriptional downregulation of I CaL , restoring Ca 2ϩ i to normal at the cost of APD reduction. These studies elucidate for the first time the molecular feedback mechanisms underlying arrhythmogenic AT remodeling. (Circ Res. 2008;103:845-854.)Key Words: atrial fibrillation Ⅲ electrophysiological remodeling Ⅲ arrhythmia mechanisms Ⅲ antiarrhythmic therapy A trial fibrillation (AF) is the most common clinical tachyarrhythmia, with an incidence that increases with age and a significant association with cardiovascular morbidity and mortality. 1 AF causes electrophysiological changes, primarily resulting from the rapid atrial activation rates, that promote arrhythmia perpetuation. 2,3 Atrial action potential (AP) duration (APD) shortening is a major contributor to refractoriness abbreviation, which is, in turn, a primary factor in AF promotion. 4,5 Loss of APD and impaired APD rate adaptation are largely attributable to the associated I CaL reduction. 4,6 Atrial remodeling is believed to have important therapeutic implications, and there is interest in developing antiremodeling therapies, 7 but this approach is limited by an insufficient understanding of underlying mechanisms to allow for the definition of molecular targets. There is indirect evidence for a role of Ca 2ϩ overload in the remodeling caused by atrial tachycardia (AT), 8,9 and transcriptional downregulation of the Ca v 1.2 I CaL ␣-subunit...