Atrial fibrillation and electrophysiology in transgenic mice with cardiac-restricted overexpression of FKBP12

Abstract

Cardiomyocyte-restricted overexpression of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice develop spontaneous atrial fibrillation (AF). The aim of the present study is to explore the mechanisms underlying the occurrence of AF in αMyHC-FKBP12 mice. Spontaneous AF was documented by telemetry in vivo and Langendorff-perfused hearts of αMyHC-FKBP12 and littermate control mice in vitro. Atrial conduction velocity was evaluated by optical mapping. The patch-clamp technique was applied to determine the potentially altered electrophysiology in atrial myocytes. Channel protein expression levels were evaluated by Western blot analyses. Spontaneous AF was recorded in four of seven αMyHC-FKBP12 mice but in none of eight nontransgenic (NTG) controls. Atrial conduction velocity was significantly reduced in αMyHC-FKBP12 hearts compared with NTG hearts. Interestingly, the mean action potential duration at 50% but not 90% was significantly prolonged in αMyHC-FKBP12 atrial myocytes compared with their NTG counterparts. Consistent with decreased conduction velocity, average peak Na⁺ current ( I_Na) density was dramatically reduced and the I_Na inactivation curve was shifted by approximately +7 mV in αMyHC-FKBP12 atrial myocytes, whereas the activation and recovery curves were unaltered. The Na_v1.5 expression level was significantly reduced in αMyHC-FKBP12 atria. Furthermore, we found increases in atrial Ca_v1.2 protein levels and peak L-type Ca²⁺ current density and increased levels of fibrosis in αMyHC-FKBP12 atria. In summary, cardiomyocyte-restricted overexpression of FKBP12 reduces the atrial Na_v1.5 expression level and mean peak I_Na, which is associated with increased peak L-type Ca²⁺ current and interstitial fibrosis in atria. The combined electrophysiological and structural changes facilitated the development of local conduction block and altered action potential duration and spontaneous AF. NEW & NOTEWORTHY This study addresses a long-standing riddle regarding the role of FK506-binding protein 12 in cardiac physiology. The work provides further evidence that FK506-binding protein 12 is a critical component for regulating voltage-gated sodium current and in so doing has an important role in arrhythmogenic physiology, such as atrial fibrillation.

Keywords: action potential duration; cardiac electrophysiology; fibrosis; ion channels; patch clamp; voltage-gated sodium current.

Fig. 1.

Protein level of FK506-binding protein 12 (FKBP12) in the atria of FKBP12 transgenic (αMyHC-FKBP12) mice. Endogenous FKBP12 (Endo) and overexpressed transgenic flag-FKBP12 (Flag) are indicated by arrows. WT, wild type. Data are expressed as means ± SE; n = 4. *P < 0.05 vs. nontransgenic (NTG) mice.

Fig. 2.

Atrial fibrillation (AF) in FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice. A: paroxysmal AF documented by ambulatory ECG. B: the diagnosis of AF was supported by the absence of P waves despite the presence of clear P waves during sinus rhythm and irregular rhythm, which was evident in the 5-min cycle length histogram. C: AF occurred spontaneously during the electrophysiological experiment (top traces). Note that the termination of AF was followed by prolonged sinus pause (bottom traces), suggesting abnormal sinus node function. AE, atrial ectopy; pECG, pseudo ECG; iECG, intracardiac electrograms.

Fig. 3.

Conduction velocity (CV) and action potential (AP) duration (APD) in the atria of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice. A: representative pictures of AP propagation in the atria by optical mapping. Scale bar = 2 mm. B: comparison of atrial CV between αMyHC-FKBP12 and nontransgenic (NTG) control mice. C: representative AP traces in single atrial myocytes. D–G: comparison of APD at 50%, 90%, and 50-70% repolarization (APD₅₀, APD₉₀, and APD_50–70, respectively) and the AP overshoot between atrial myocytes from αMyHC-FKBP12 (n = 19 cells/4 mice) and NTG control mice (n = 24 cells/4 mice). WT, wild type. Data are expressed as means ± SE. *P < 0.05 vs. NTG mice.

Fig. 4.

Analysis of Na⁺ currents (I_Na) of isolated atrial cardiomyocytes in nontransgenic (NTG) and FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice. A: representative tracings of total I_Na. B: peak I_Na density from NTG (n = 20 cells/4 mice) and αMyHC-FKBP12 (n = 15 cells/4 mice) atrial myocytes. C: current-voltage (I-V) relationship (I-V curve) for peak I_Na density. D: voltage dependence of the steady-state activation and inactivation for I_Na. For activation, the cell was held at −100 mV and depolarized for 50 ms to potentials between −90 and +10 mV in 5-mV increments. The interpulse interval was 1 s. For inactivation, the cell was held at −100 mV, and 1-s preconditioning pulses to potentials between −120 and −30 mV in 5-mV increments were applied followed by 40-ms test pulses to −25 mV. The interpulse interval was 3 s. E: time course of I_Na recovery from inactivation. Recovery was recorded by depolarizing the cell to −30 mV from the holding potential of −100 mV for 300 ms (P1) followed by a variable recovery interval and a subsequent −30-mV test pulse (P2). *P < 0.05 vs. NTG mice.

Fig. 5.

Analysis of cardiac L-type Ca²⁺ currents (I_Ca,L) from isolated atrial cardiomyocytes in nontransgenic (NTG) and FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice. A: representative traces of whole cell I_Ca,L. B: peak I_Ca,L density in NTG (n = 12 cells/4 mice) and αMyHC-FKBP12 (n = 13 cells/4 mice) atrial myocytes. C: current-voltage (I-V) relationship (I-V curve) for peak I_Ca,L. D: voltage dependence of the steady-state activation and inactivation for I_Ca,L. For activation, the cell was held at −50 mV, ramp depolarized from −80 to −50 mV over 100 ms, and step depolarized from −50 to +60 mV with 5-mV increments. The interpulse interval was 2 s. For inactivation, the cell was held at −50 mV, and 2,000-ms preconditioning pulses to potentials from −80 to −10 mV with a 5-mV increment were applied followed by a 300-ms test pulse to −10 mV. E: normalized peak I_Ca,L (I/I_max) for I_Ca,L recovery from inactivation. Inset: recording protocol. *P < 0.05 vs. NTG mice.

Fig. 6.

Analysis of cardiac transient outward K⁺ currents (I_to), sustained K⁺ currents (I_Ksus), and inward rectifier K⁺ currents (I_K1) from isolated atrial cardiomyocytes in nontransgenic (NTG) and FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice. A: representative I_to traces of NTG and αMyHC-FKBP12 myocytes. I_to was elicited from a holding potential at −50 mV with a 50-ms ramp from −80 to −40 mV followed by depolarizing pulses from −40 to +50 mV for 625 ms (in 10-mV increments). B and C: current-voltage (I-V) relationship (I-V curve) for I_to and I_Ksus. NTG: n = 21 cells/4 mice and αMyHC-FKBP12: n = 14 cells/4 mice. D: representative I_K1 traces of NTG and αMyHC-FKBP12 myocytes. I_K1 was elicited by holding the cell at −40 mV followed by 10-mV steps from −120 to 50 mV for 300 ms. E: I-V curve for I_K1. NTG: n = 22 cells/4 mice and αMyHC-FKBP12: n = 18 cells/4 mice. WT, wild type.

Fig. 7.

A and B: representative Western blots to analyze the expression of Na_v1.5 (A) and Ca_v1.2 (B) in the atria of FK506-binding protein 12 transgenic (αMyHC-FKBP12) transgenic mice as evaluated by Western blot analysis. WT, wild type. *P < 0.05 vs. nontransgenic (NTG) mice.

Fig. 8.

A–F: representative histological analysis of the atrial free wall stained with picrosirius red using conventional light (A–D) and polarized light (E and F) from nontransgenic (NTG; A, C, and E) and FK506-binding protein 12 transgenic (αMyHC-FKBP12; B, D, and F) mice. Note the larger atrial size and more abundant interstitial fibrosis in the αMyHC-FKBP12 mouse than in the NTG mouse. Bar = 20 µm. Fibrosis appears red in E and F. RA, right atria; LA, left atria.