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. 2016 Nov 1;311(5):H1139-H1149.
doi: 10.1152/ajpheart.00156.2016. Epub 2016 Sep 16.

Changes in cardiac Nav1.5 expression, function, and acetylation by pan-histone deacetylase inhibitors

Affiliations

Changes in cardiac Nav1.5 expression, function, and acetylation by pan-histone deacetylase inhibitors

Qin Xu et al. Am J Physiol Heart Circ Physiol. .

Abstract

Histone deacetylase (HDAC) inhibitors are small molecule anticancer therapeutics that exhibit limiting cardiotoxicities including QT interval prolongation and life-threatening cardiac arrhythmias. Because the molecular mechanisms for HDAC inhibitor-induced cardiotoxicity are poorly understood, we performed whole cell patch voltage-clamp experiments to measure cardiac sodium currents (INa) from wild-type neonatal mouse ventricular or human-induced pluripotent stem cell-derived cardiomyocytes treated with trichostatin A (TSA), vorinostat (VOR), or romidepsin (FK228). All three pan-HDAC inhibitors dose dependently decreased peak INa density and shifted the voltage activation curve 3- to 8-mV positive. Increases in late INa were not observed despite a moderate slowing of the inactivation rate at low activating potentials (<-40 mV). Scn5a mRNA levels were not significantly altered but NaV1.5 protein levels were significantly reduced. Immunoprecipitation with anti-NaV1.5 and Western blotting with anti-acetyl-lysine antibodies indicated that NaV1.5 acetylation is increased in vivo after HDAC inhibition. FK228 inhibited total cardiac HDAC activity with two apparent IC50s of 5 nM and 1.75 μM, consistent with previous findings with TSA and VOR. FK228 also decreased ventricular gap junction conductance (gj), again consistent with previous findings. We conclude that pan-HDAC inhibition reduces cardiac INa density and NaV1.5 protein levels without affecting late INa amplitude and, thus, probably does not contribute to the reported QT interval prolongation and arrhythmias associated with pan-HDAC inhibitor therapies. Conversely, reductions in gj may enhance the occurrence of triggered activity by limiting electrotonic inhibition and, combined with reduced INa, slow myocardial conduction and increase vulnerability to reentrant arrhythmias.

Keywords: gap junctions; romidepsin; sodium current; trichostatin A; vorinostat.

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Figures

Fig. 1.
Fig. 1.
Reduction of cardiac sodium current INa by pan-histone deacetylase (HDAC) inhibition. Families of cardiac Na+ current traces are shown for 10-mV depolarizing voltage-clamp steps from −90 to +50 mV under control (A), 100 nM trichostatin A (TSA; B), 5 μM vorinostat (VOR; C), and 10 nM romidepsin (FK228; D). E: diagram of the Vm pulse protocol for activating INa. F–H: INa current density (pA/pF)-Vm relationships under control conditions and after an 18- to 24-h treatment with 50 or 100 nM TSA (F), 1 or 5 μM VOR (G), and 10 nM FK228 (H) illustrate dose-dependent decreases in peak INa with pan-HDAC inhibitor treatments.
Fig. 2.
Fig. 2.
Shift of INa activation by pan-HDAC inhibition. A–C: the apparent shift in Vm-dependent activation present in the I–V curves in Fig. 1, F–H, was examined by calculating the Na+ conductance (gNa) and normalizing the data to the maximum gNa (Na) for each experimental group and plotting the activation curves for TSA (A), VOR (B), and FK228 (C) relative to the control activation curve. The data points were fitted with a Boltzmann function with a half-activation voltage (V½act) of −49.8 mV for control, −46.6 and −47.0 mV for 50 and 100 nM TSA, −46.0, and −42.3 mV for 1 and 5 μM VOR, and −46.2 mV for 10 nM FK228, respectively. D: Vm-dependent inactivation was assessed using a prepulse protocol illustrated in E and no shift in the half-inactivation voltage (V½inact) of −83.0 ± 0.5 mV was observed between control, 100 nM TSA, and 5 μM VOR conditions. F: comparison of the overlap between the gNa inactivation and activation curves under control and 5 μM VOR conditions illustrating the decrease in the Vm-dependent “window” for persistent activation of INa.
Fig. 3.
Fig. 3.
INa inactivation time constants. A–C: the average Vm-dependent inactivation time constants (τh) from the control, 100 nM TSA (A), 1 μM VOR (B), and 10 nM FK228 (C) INa current traces illustrate only a slight slowing of the inactivation rates at low activating potentials of −60 to −40 mV with no slowing of inactivation at fully activated potentials > −40 mV.
Fig. 4.
Fig. 4.
Effect of pan-HDAC inhibition on human INa. A and B: examples of a family of INa traces obtained from a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) under control (A) and 1 μM VOR (B) conditions. C: INa current density (pA/pF)-Vm relationships under control conditions and 1 μM VOR conditions illustrate a significant reduction in INa density after pan-HDAC inhibitor treatment, verifying previous results obtained from neonatal mouse ventricular myocyte (NMVM) cultures.
Fig. 5.
Fig. 5.
Effects of pan-HDAC inhibition on NaV1.5 expression. A: the abundance of mRNA levels were measured by RT-PCR from high density NMVM cultures under control conditions for the Cx43 (Gja1), NaV1.5 (Scn5a), N-cadherin (Cdh2), and the sodium-calcium exchanger (NCX, Slc8a) genes relative to Gapdh. B: the relative mRNA levels for these same cardiac genes after 1 μM VOR treatment were compared with control values and only Gja1 and Cdh2 levels were significantly reduced as previously reported. The Gapdh levels were not significantly different from control values (P > 0.25, paired t-test). C: representative Western blots for NaV1.5 protein from TSA- and VOR-treated NMVM samples. HDAC1 was used as loading control and acetylated α-tubulin (Ac-α-t) as an indicator of increased protein acetylation. The experiments were performed in triplicate. D: densitometric scans of the Western blot data were performed to quantify the NaV1.5 protein levels at low and high doses of TSA and VOR. NaV1.5 protein levels were significantly reduced at both doses of TSA and VOR. E: the relative mRNA levels for the Slc8a, Gja1, and Cdh2 genes were significantly reduced by 10 nM FK228 treatment compared with control values but the Scn5a levels were not significantly affected.
Fig. 6.
Fig. 6.
Romidepsin inhibition of cardiac HDAC activity and gap junction conductance. A: total HDAC activity was measured in NMVM cultures exposed to increasing concentrations of FK228 using the deacetylated Fluor-de-Lys fluorescence method. All data were normalized to the background subtracted maximum relative fluorescence of the control well. The data from three experiments were averaged and fitted with a second-order exponential decaying function and equilibrium inhibition constants (Ki) were calculated from the decay constant for each exponential component. B: the gap junction conductance (gj) was measured in NMVM cell pairs from control and FK228-treated culture dishes and a dose-dependent decrease in gj was observed, consistent with published results with TSA and VOR (71).
Fig. 7.
Fig. 7.
A: putative Nε-lysine acetylation sites on the NaV1.5 Na+ channel subunit. The full length mouse NaV1.5 amino acid sequence was analyzed for possible lysine acetylation (Ac-K) sites using the prediction algorithm available at the PHOSIDA Posttranslational Modification database (http://www.phosida.org; Ref. 18). Eight putative acetylation sites (probability >90%) were identified and mapped onto the membrane topological model for the NaV1.5 channel protein. Six of the eight predicted NaV1.5 Ac-K sites (K158, K175, K767, K863, K1362, and K1617) mapped to transmembrane or extracellular domain locations and were discounted as possible acetylation sites (lined circle). Two possible Ac-K sites, K830 and K1644, mapped to cytoplasmic domains located near the cytoplasmic COOH-terminal side of the repeat domain II and IV S4 domains (star circle). We hypothesize that acetylation of these 2 sites, which are conserved in the human NaV1.5 sequence, may account for the slight positive voltage shift (< 10 mV) in the cardiac INa activation curve associated with pan-HDAC inhibitor treatments (Fig. 2, A-C). B, left: an example of an immunoprecipitation (IP) performed on saline:DMSO (control, C) or TSA-injected (T) mouse heart ventricular lysates with anti-NaV1.5-protein G magnetic beads and immunoblotted (IB) with an anti-acetyl-lysine (Ac-K) antibody to detect protein acetylation. TSA injection for 5 days caused a dramatic increase in the acetylated NaV1.5 band. Rabbit IgG controls for control (IgGc) and TSA heart samples (IgGt) are shown in lanes 3 and 4. B, right: lanes 1 and 2 are the NaV1.5 immunoprecipitate input controls for the control and TSA heart lysates and the lanes 3 and 4 are the rabbit IgG controls for each sample. C: Normalized mean Ac-K NaV1.5 band densities for the upper [high molecular weight (MW), upper arrow] and lower (lower MW, lower arrow) from 3 control:TSA immunoprecipitation experiments as illustrated in B. Each Ac-K TSA band density was divided the control band density for each experiment and this TSA/control band ratio was normalized by dividing by the NaV1.5 input TSA/control band ratio.

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