Histone deacetylase inhibition reduces cardiac connexin43 expression and gap junction communication

Abstract

Histone deacetylase inhibitors (HDACIs) are being investigated as novel therapies for cancer, inflammation, neurodegeneration, and heart failure. The effects of HDACIs on the functional expression of cardiac gap junctions (GJs) are essentially unknown. The purpose of this study was to determine the effects of trichostatin A (TSA) and vorinostat (VOR) on functional GJ expression in ventricular cardiomyocytes. The effects of HDAC inhibition on connexin43 (Cx43) expression and functional GJ assembly were examined in primary cultured neonatal mouse ventricular myocytes. TSA and VOR reduced Cx43 mRNA, protein expression, and immunolocalized Cx43 GJ plaque area within ventricular myocyte monolayer cultures in a dose-dependent manner. Chromatin immunoprecipitation experiments revealed altered protein interactions with the Cx43 promoter. VOR also altered the phosphorylation state of several key regulatory Cx43 phospho-serine sites. Patch clamp analysis revealed reduced electrical coupling between isolated ventricular myocyte pairs, altered transjunctional voltage-dependent inactivation kinetics, and steady state junctional conductance inactivation and recovery relationships. Single GJ channel conductance was reduced to 54 pS only by maximum inhibitory doses of TSA (≥ 100 nM). These two hydroxamate pan-HDACIs exert multiple levels of regulation on ventricular GJ communication by altering Cx43 expression, GJ area, post-translational modifications (e.g., phosphorylation, acetylation), gating, and channel conductance. Although a 50% downregulation of Cx43 GJ communication alone may not be sufficient to slow ventricular conduction or induce arrhythmias, the development of class-selective HDACIs may help avoid the potential negative cardiovascular effects of pan-HDACI.

Acetylation mediates Cx43 reduction caused by electrical stimulation

“…However, Veenstra and coworkers showed that SAHA also induced Cx43 mRNA reduction in neonatal mouse cardiomyocytes, consistently with the decreased association of Cx43 promoter with RNA Pol II and the increased association with HDACI [25]. In our model, increased acetylation was only evident for proteins with molecular weights ranging from 25 to 130 kDa, while the signals for anti-pan-Ac-K remained stable at molecular weights below 25 kDa, compatible with histones.…”

“…3) [25], thus suggesting the hypothesis that electrical stimulation could promote Cx43 acetylation. However, the adjunct of Verapamil 10 μM to SAHA treated HL-1 cells failed to rescue Cx43 localization at appositional membranes (data not shown).…”

Detrimental Effect of Class-selective Histone Deacetylase Inhibitors during Tissue Regeneration following Hindlimb Ischemia

“…Although DIs are now in clinical use as valuable anticancer therapy, cardiac adverse side effects (39,40) and cardiac hypertrophy (41) have been recently described associated with their use. Thus, the evaluation of their safety and toxicity is of particular relevance for potential therapeutic application in regenerative medicine.…”

Functional formation of heterotypic gap junction channels by connexins-40 and -43

“…Aliquots of 1-5 mg protein, [protein] determined by the Bradford method (Bio-Rad), separated by SDS-PAGE on 10% polyacrylamide gels, blotted onto Immobilon-P membranes (Millipore), and blocked in 5% nonfat milk Tris-buffered saline (TBS), pH 7.4, overnight at 4 C. Membranes were incubated for 3 h at room temperature with rabbit polyclonal antibodies directed against amino acids 363-382 of Cx43 (Sigma, #C6219) (1:10,000 dilution) or against the C-terminal domain of Cx40 (Invitrogen, #36-4900) (1:1000 dilution). 32 Blots were rinsed repeatedly in TBS and incubated for 1 h at room temperature with a peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Jackson) (Cx43, 1:5,000 dilution; Cx40, 1:5000 dilution, all in 5% nonfat milk TBS, pH 7.4). Immunoblots were developed with ECL Plus chemifluorescent reagents (GE Healthcare) and quantified using a STORM Phosphoimager.…”

“…All results are expressed relative to GAPDH and a cellular RNA sample without reverse transcription was run as a negative control to test for genomic DNA. 31,32 Immunoblot analysis Cell homogenates were harvested by scraping in ice cold phosphate-buffered saline containing a Roche mini-EDTA-free protease inhibitor tablet (per 5 ml) and 1 mM phenylmethylsulfonyl fluoride. The samples were centrifuged at 10,000 rpm for 2 min and the pellets resuspended and lysed by sonication in 25-100 ml 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 1% Triton X-100, 0.02% sodium azide, 50 mM sodium fluoride, 0.5 mM sodium orthovanadate, and one Roche mini EDTA-free protease inhibitor tablet.…”