Histone-deacetylase inhibition reverses atrial arrhythmia inducibility and fibrosis in cardiac hypertrophy independent of angiotensin

Abstract

Atrial fibrosis influences the development of atrial fibrillation (AF), particularly in the setting of structural heart disease where angiotensin-inhibition is partially effective for reducing atrial fibrosis and AF. Histone-deacetylase inhibition reduces cardiac hypertrophy and fibrosis, so we sought to determine if the HDAC inhibitor trichostatin A (TSA) could reduce atrial fibrosis and arrhythmias. Mice over-expressing homeodomain-only protein (HopX(Tg)), which recruits HDAC activity to induce cardiac hypertrophy were investigated in 4 groups (aged 14-18 weeks): wild-type (WT), HopX(Tg), HopX(Tg) mice treated with TSA for 2 weeks (TSA-HopX) and wild-type mice treated with TSA for 2 weeks (TSA-WT). These groups were characterized using invasive electrophysiology, atrial fibrosis measurements, atrial connexin immunocytochemistry and myocardial angiotensin II measurements. Invasive electrophysiologic stimulation, using the same attempts in each group, induced more atrial arrhythmias in HopX(Tg) mice (48 episodes in 13 of 15 HopX(Tg) mice versus 5 episodes in 2 of 15 TSA-HopX mice, P<0.001; versus 9 episodes in 2 of 15 WT mice, P<0.001; versus no episodes in any TSA-WT mice, P<0.001). TSA reduced atrial arrhythmia duration in HopX(Tg) mice (1307+/-289 ms versus 148+/-110 ms, P<0.01) and atrial fibrosis (8.1+/-1.5% versus 3.9+/-0.4%, P<0.001). Atrial connexin40 was lower in HopX(Tg) compared to WT mice, and TSA normalized the expression and size distribution of connexin40 gap junctions. Myocardial angiotensin II levels were similar between WT and HopX(Tg) mice (76.3+/-26.0 versus 69.7+/-16.6 pg/mg protein, P=NS). Therefore, it appears HDAC-inhibition reverses atrial fibrosis, connexin40 remodeling and atrial arrhythmia vulnerability independent of angiotensin II in cardiac hypertrophy.

Fig. 1

Atrial fibrosis is reversed by trichostatin A. Masson’s trichrome staining reveals increased atrial interstitial fibrosis in HopX transgenic mice (HopX^Tg) compared with wild-type controls (WT), while treatment with trichostatin A (TSA-HopX) resulted in complete reversal of atrial fibrosis. Note there is no obvious ventricular interstitial fibrosis in the four groups shown. Scale Bar: 40X images = 1 mm; 400X images = 100 μm. Percentage of interstitial fibrosis. There was a significant increase in atrial fibrosis (left panel) in the HopX^Tg group compared with wild-type controls, while treatment with TSA resulted in a significant reduction of atrial fibrosis. There was no significant ventricular fibrosis (right panel) detected in any the groups examined. *p<0.01 compared to WT; ^†p<0.01 compared to HopX^Tg; ^§p<0.01 compared to TSA-WT.

Fig. 2

Atrial arrhythmias induced in HopX transgenic mice. (A) Representative surface electrocardiograms from a HopX transgenic (TG) and wild-type (WT) littermate mouse demonstrating normal electrocardiographic morphologies in both animals. (B) Shown are recordings from lead I (I), the high right atrium (HRA) and the right ventricular apex (RVA) demonstrating a run of atrial fibrillation induced by burst stimulation (S) in a HopX transgenic mouse. Irregularly irregular intervals between ventricular (V) beats can be seen on both the intracardiac leads and surface lead. Sinus beats are shown upon termination of the episode with normal atrial (a) and ventricular (v) intracardiac electrograms.

Fig. 3

Atrial angiotensin and cytokines are not elevated by HopX. (A) ELISA shows no difference in angiotensin II levels in WT and HopX^Tg hearts. Immunoblots shows atrial levels of (B) phospho-JNK1/2 (46- and 54-kDa isoforms), (C) phospho-p38-MAPK (42-kDa), (D) phospho-ERK1/2 (42- and 44-kDa isoforms), (E) active TGF-β1 (25-kDa) and (F) acetylated histone H3 (Ac-H3, 17-kDa). Below the blots are band signal intensities normalized to GAPDH (both isoforms of ERK and JNK1/2 were averaged together for quantitative analysis). Active TGF-β1 is lower in HopX^Tg mice relative to control and further decreased in TSA-HopX^Tg mice. Phospho-ERK1/2 is also lower in HopX^Tg mice relative to control but increased in TSA-HopX^Tg mice. Phospho-JNK1/2 and phospho-p38-MAPK were not different between HopX^Tg, TSA-HopX^Tg or control mice. *P<0.05 compared to WT; ^†P<0.05 compared to TSA-HopX^Tg.

Fig. 4

Atrial connexin40 expression is reduced in left ventricular hypertrophy and normalized by trichostatin A. Shown is staining for connexin40 in (A) WT, (B) HopX^Tg and (C) TSA-HopX^Tg mice and for connexin43 in (D) WT, (E) HopX^Tg and (F) TSA- HopX^Tg mice. Scale bar = 100 μm. Panel (G) shows immunoblot analysis of atrial connexin40 that is lower in HopX^Tg mice and normalized by TSA, while panel (H) show the same analysis for connexin43 that is not affected by HopX or TSA. Blots are representative of three separate experiments. Panel (I) shows the normalized size distribution of connexin40 gap junctions with the same analysis of connexin43 gap junctions in panel (J). The expression of total connexin40 gap is lower in the atrium HopX^Tg mice and almost completely normalized by TSA Data averaged from 10 different sections using 3 separate atria in each group. *P<0.05 compared to WT; ^†P<0.05 compared to TSA-HopX^Tg.