Maintenance and Reversibility of Paroxysmal Atrial Fibrillation in JDP2 Overexpressing Mice

Abstract

Heart-specific overexpression of transcriptional regulator JDP2 (jun dimerization protein 2) for 5 weeks provokes paroxysmal atrial fibrillation (AF) in mice. We now investigated whether AF and atrial remodeling will be reversible upon termination of JDP2 overexpression, and whether paroxysmal AF converts to permanent AF in the presence of maintained JDP2 overexpression. Cardiac-specific JDP2 overexpression for 5 weeks, resulting in paroxysmal AF, was either continued or repressed via a tet-off system for another 5 weeks. ECGs were recorded weekly. Thereafter, heart and lung weights, and atrial mRNA and protein expression were determined. Extending JDP2 overexpression did not aggravate the AF phenotype, still paroxysmal AF, prolongation of PQ intervals, and atrial hypertrophy were present. This phenotype was completely reversible upon cessation of JDP2 overexpression. A massive downregulation of connexin40 and calcium handling proteins, including SERCA2a, calsequestrin, and ryanodine receptor, was observed in atria after prolonged JDP2 overexpression. In conclusion, atrial remodeling and paroxysmal AF under JDP2 overexpression are not sufficient to maintain or aggravate AF in the absence of JDP2. The comparison of the two groups indicates that the downregulation of calcium proteins and connexins is an important factor in the maintenance of the disease.

Keywords: ECG; JDP2; SERCA2a; atrial hypertrophy; calcium; connexin; paroxysmal atrial fibrillation; ryanodine receptor.

Figure 1

Experimental design of JDP2 overexpression. (A) Mice were fed with doxycycline diet until 5 weeks of age to suppress JDP2 overexpression in double transgenic mice. Then JDP2 overexpression was started by switching to standard diet. In group 1, JDP2 overexpression was stopped after 5 weeks, while in group 2, JDP2 overexpression was maintained over 10 weeks. WT mice received the same feeding protocols. ECG recordings were performed every week, starting at the time point of 5-week JDP2 overexpression. At the end of the experiments, hearts were removed to perform weight and expression analyses. (B) JDP2 mRNA expression was determined by real time RT-PCR (* p < 0.05 vs. WT, n = 9–13).

Figure 2

Conduction defects under prolonged JDP2 overexpression. ECGs were recorded over 30 min after 5 weeks of JDP2 overexpression, and then every week up to week 10, either in absence (group 1) or presence of JDP2 overexpression (group 2) (* p < 0.05 vs. WT, n = 9–12).

Figure 3

Paroxysmal atrial fibrillation under prolonged JDP2 overexpression. ECGs were recorded over 30 min after 5 weeks of JDP2 overexpression, and then every week up to week 10, either in absence (group 1) or presence of JDP2 overexpression (group 2). (A) Paroxysmal atrial fibrillation in mice overexpressing JDP2 for 10 weeks (upper panel), compared to sinus rhythm in WT mice (lower panel). (B) Mean duration times of atrial fibrillation (* p < 0.05 vs. WT, n = 9–13).

Figure 4

Organ weights in JDP2 mice. Lungs and hearts were harvested from mice after 5 weeks of JDP2 overexpression, followed by 5 weeks without overexpression (group 1) or another 5 weeks with JDP2 overexpression (group 2). The total heart weight (HW) and weights of left and right ventricles (LV and RV), atria, and lungs were determined and related to the body weight (BW) (* p < 0.05 vs. WT, n = 9–13).

Figure 5

Induction of hypertrophic and fibrotic mRNA expression under prolonged JDP2 overexpression. mRNA expression in atria of mice after 5 weeks of JDP2 overexpression, followed by 5 weeks without overexpression (group 1) or another 5 weeks with JDP2 overexpression (group 2) was determined by real time RT-PCR (* = p < 0.05 vs. WT, n = 9–13). The dashed line indicates the mRNA expression level in WTs.

Figure 6

Decreased mRNA expression of various calcium-handling proteins and connexins under prolonged JDP2 overexpression. mRNA expression in atria of mice after 5 weeks of JDP2 overexpression, followed by 5 weeks without overexpression (group 1) or another 5 weeks with JDP2 overexpression (group 2) by real time RT-PCR (* = p < 0.05 vs. WT, n = 9–13). The dashed line indicates the mRNA expression level in WTs.

Figure 7

No change in inflammatory marker gene expression. mRNA expression of the inflammatory marker genes in atria of mice after 5 weeks of JDP2 overexpression, followed by 5 weeks without overexpression (group 1) or another 5 weeks with JDP2 overexpression (group 2) by real time RT-PCR (n = 9–13). The dashed line indicates the mRNA expression level in WTs.

Figure 8

Expression of various calcium handling proteins with and without prolonged JDP2 overexpression. Original western blots and quantified data from atria isolated from mice after 5 weeks of JDP2 overexpression, followed by 5 weeks without overexpression (group 1) or another 5 weeks with JDP2 overexpression (group 2) (n = 8 per group; * p < 0.05, ns = non-significant). GAPDH was used as a loading control for normalization.

Figure 9

RyR expression and phosphorylation, and connexin 40 expression with and without prolonged JDP2 overexpression. Original western blots and quantified data from atria isolated from mice after 5 weeks of JDP2 overexpression followed by 5 weeks without overexpression (group 1) or another 5 weeks with JDP2 overexpression (group 2) (n = 8 per group; * p < 0.05; ** p < 0.01; **** p < 0.0001, ns = non-significant). GAPDH or vinculin was used as a loading control for normalization. Note that the GAPDH loading control shown for pS2808 is identical to the one shown in Figure 8 for CSQ, as the proteins were derived from the same membrane.