Histone deacetylase (HDAC) inhibitors attenuate cardiac hypertrophy by suppressing autophagy

Abstract

Histone deacetylases (HDACs) regulate cardiac plasticity; however, their molecular targets are unknown. As autophagy contributes to pathological cardiac remodeling, we hypothesized that HDAC inhibitors target autophagy. The prototypical HDAC inhibitor (HDACi), trichostatin A (TSA), attenuated both load- and agonist-induced hypertrophic growth and abolished the associated activation of autophagy. Phenylephrine (PE)-triggered hypertrophy and autophagy in cultured cardiomyocytes were each blocked by a panel of structurally distinct HDAC inhibitors. RNAi-mediated knockdown of either Atg5 or Beclin 1, two essential autophagy effectors, was similarly capable of suppressing ligand-induced autophagy and myocyte growth. RNAi experiments uncovered the class I isoforms HDAC1 and HDAC2 as required for the autophagic response. To test the functional requirement of autophagic activation, we studied mice that overexpress Beclin 1 in cardiomyocytes. In these animals with a fourfold amplified autophagic response to TAC, TSA abolished TAC-induced increases in autophagy and blunted load-induced hypertrophy. Finally, we subjected animals with preexisting hypertrophy to HDACi, finding that ventricular mass reverted to near-normal levels and ventricular function normalized completely. Together, these data implicate autophagy as an obligatory element in pathological cardiac remodeling and point to HDAC1/2 as required effectors. Also, these data reveal autophagy as a previously unknown target of HDAC inhibitor therapy.

Fig. 1.

Moderate pressure overload increases cardiac autophagy, which can be suppressed by the HDAC inhibitor TSA. (A and B) C57BL/6 mice (8 wk old) were subjected to sham or TAC procedures and followed for 3 wk. The autophagy marker LC3-II was tracked by immunoblotting of total protein extracts from heart tissue. (C) LC3-II levels correlate significantly with cardiac mass, r² = 0.89. LC3-II levels are elevated after just 1 wk after TAC (D), and HDACi decreased LC3-II levels back to control values (E). There was no effect of TSA on LC3-II levels in sham-operated mice. LC3-II abundance tracks with the decrease in HW/BW in TSA-treated mice (F). Pressure overload increased GFP–LC3 puncta formation in α-MHC–GFP–LC3 mice maintained on a C57BL/6 background, an effect that was suppressed by TSA, n = 5–6 (G and H). Markers of the fetal gene program (ANF, BNP, and αSMA) were quantified by qRT-PCR (I). Each was increased in the setting of TAC and suppressed by TSA.

Fig. 2.

Hypertrophic agonist-induced autophagy is suppressed by multiple, structurally distinct HDAC inhibitors. Neonatal cardiomyocytes in culture were treated with the hypertrophic agonists PE or ET1 in the presence/absence of HDAC inhibitors TSA (66 nM), sodium butyrate (SB, 3 mM), or valproic acid (VPA, 6 mM) for 48 h. Total protein lysates were subjected to LC3 immunoblot analysis (A and B). Mean data are presented (C). Cardiomyocytes infected with GFP–LC3 adenovirus manifested GFP puncta formation with PE stimulation, which was blunted by TSA (D). Growth ligand-induced increases in LC3-II were also blunted by treatment with structurally unrelated HDAC inhibitors (E–H). n = 3–6 in all experiments. Activation of the fetal gene program, measured as qRT-PCR for ANF, BNP, and αSMA, was suppressed by HDACi (I).

Fig. 3.

HDAC inhibition blocks the augmented hypertrophic growth response in α-MHC–Beclin 1 transgenic mice. Cardiac-specific Beclin 1 overexpressing transgenic mice (8–10 wk of age) were subjected to sham or TAC procedures. The animals were then randomized into four groups: sham + veh, sham + TSA (1.0 mg/kg SQ QD), TAC + veh, or TAC + TSA and followed for 3 wk. Heart mass normalized to body mass (HW/BW) increased in both genotypes but significantly more in the Beclin 1 transgenics (A). Representative images of whole hearts and four-chamber sections (B) and mean data (n = 7–11, C) are presented. HDAC inhibition elicited a significantly greater reduction in heart growth in Beclin 1 transgenics relative to WT (D).

Fig. 4.

TSA reverses severe, preexisting cardiac hypertrophy and cardiac dysfunction in Beclin 1 transgenic mice. Beclin 1 transgenic mice were subjected to sham and TAC procedures and followed for an extended period (10 wk) to provoke severe hypertrophy and contractile dysfunction (A). Then, animals were randomized to TSA or vehicle treatment and followed for an additional 8 wk. TSA treatment led to significant declines in left ventricular mass and improvement in systolic performance. Representative M-mode echocardiographic images are presented (B). Representative images of whole hearts and four-chamber sections obtained at 10 wk (sham or TAC) and following 8 wk of therapy (vehicle or TSA) are presented (C). Mean data are presented (D). n = 6–7 for all treatment groups.