Inhibition of Histone Deacetylases Induces K+ Channel Remodeling and Action Potential Prolongation in HL-1 Atrial Cardiomyocytes

Background/Aims: Cardiac arrhythmias are triggered by environmental stimuli that may modulate expression of cardiac ion channels. Underlying epigenetic regulation of cardiac electrophysiology remains incompletely understood. Histone deacetylases (HDACs) control gene expression and cardiac integrity. We hypothesized that class I/II HDACs transcriptionally regulate ion channel expression and determine action potential duration (APD) in cardiac myocytes. Methods: Global class I/II HDAC inhibition was achieved by administration of trichostatin A (TSA). HDAC-mediated effects on K+ channel expression and electrophysiological function were evaluated in murine atrial cardiomyocytes (HL-1 cells) using real-time PCR, Western blot, and patch clamp analyses. Electrical tachypacing was employed to recapitulate arrhythmia-related effects on ion channel remodeling in the absence and presence of HDAC inhibition. Results: Global HDAC inhibition increased histone acetylation and prolonged APD90 in atrial cardiomyocytes compared to untreated control cells. Transcript levels of voltage-gated or inwardly rectifying K+ channels Kcnq1, Kcnj3 and Kcnj5 were significantly reduced, whereas Kcnk2, Kcnj2 and Kcnd3 mRNAs were upregulated. Ion channel remodeling was similarly observed at protein level. Short-term tachypacing did not induce significant transcriptional K+ channel remodeling. Conclusion: The present findings link class I/II HDAC activity to regulation of ion channel expression and action potential duration in atrial cardiomyocytes. Clinical implications for HDAC-based antiarrhythmic therapy and cardiac safety of HDAC inhibitors require further investigation

Atrial fibrillation complicated by heart failure induces distinct remodeling of calcium cycling proteins.

Atrial fibrillation (AF) and heart failure (HF) are two of the most common cardiovascular diseases. They often coexist and account for significant morbidity and mortality. Alterations in cellular Ca2+ homeostasis play a critical role in AF initiation and maintenance. This study was designed to specifically elucidate AF-associated remodeling of atrial Ca2+ cycling in the presence of mild HF. AF was induced in domestic pigs by atrial burst pacing. The animals underwent electrophysiologic and echocardiographic examinations. Ca2+ handling proteins were analyzed in right atrial tissue obtained from pigs with AF (day 7; n = 5) and compared to sinus rhythm (SR) controls (n = 5). During AF, animals exhibited reduction of left ventricular ejection fraction (from 73% to 58%) and prolonged atrial refractory periods. AF and HF were associated with suppression of protein kinase A (PKA)RII (-62%) and Ca2+-calmodulin-dependent kinase II (CaMKII) δ by 37%, without changes in CaMKIIδ autophosphorylation. We further detected downregulation of L-type calcium channel (LTCC) subunit α2 (-75%), sarcoplasmic reticulum Ca2+-ATPase (Serca) 2a (-29%), phosphorylated phospholamban (Ser16, -92%; Thr17, -70%), and phospho-ryanodine receptor 2 (RyR2) (Ser2808, -62%). Na+-Ca2+ exchanger (NCX) levels were upregulated (+473%), whereas expression of Ser2814-phosphorylated RyR2 and LTCCα1c subunits was not significantly altered. In conclusion, AF produced distinct arrhythmogenic remodeling of Ca2+ handling in the presence of tachycardia-induced mild HF that is different from AF without structural alterations. The changes may provide a starting point for personalized approaches to AF treatment

Expression of L-type Ca²⁺ channels (LTCC) and Na+-Ca²⁺ exchanger (NCX) 1 in AF animals and in SR controls.

<p><b>a, b</b> Regulatory LTCC α₂ subunit was significantly downregulated in AF animals, whereas the pore forming α_1c subunit was not affected. <b>c</b> AF lead to significant upregulation of the NCX1 transporter. Representative Western blots and mean (± SEM) optical density data normalized to GAPDH are displayed (n = 5 animals per group). *<i>P</i> < 0.05; ***<i>P</i> < 0.001.</p

Analysis of ryanodine receptor (RyR) and phosphorylated ryanodine receptor (p-RyR) levels in SR and AF pigs.

<p><b>a</b> Total RyR protein did not differ between animal groups. <b>b, c</b> PKA-phosphorylated RyR at Ser2808 was reduced, while CaMKII-phosphorylated RyR at Ser2814 was not affected by AF. <b>d</b> Relative p-RyR_Ser2808 but not p-RyR_Ser2814 content was diminished in atrial tissue obtained from n = 5 animals per group. Original Western blots and mean normalized optical density data are provided (*<i>P</i> < 0.05).</p

AF-induced remodeling of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) δ and protein kinase A (PKA).

<p>Representative Western blots and mean optical density values normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are displayed relative to sinus rhythm (SR) controls. <b>a,</b> Absolute expression levels of the reference protein, GAPDH, were note affected by AF. <b>b, c</b> Normalized protein expression of total CaMKIIδ and phosphorylated CaMKIIδ (Thr286) in AF animals compared to SR. <b>d</b> Relative CaMKIIδ autophosphorylation at Thr286 was not different between animal groups. <b>e, f</b> Protein levels of catalytic PKA Cα/β subunits (PKA_C) and of phosphorylated regulatory RIIα subunits (PKA_RII) in AF and SR animals. Mean values obtained from five animals per group are provided ± SEM; *<i>P</i> < 0.05.</p

Alterations of sarcoplasmic reticulum Ca²⁺-ATPase (Serca) 2a and its regulator phospholamban (PLN) during AF.

<p>Total PLN (<b>a</b>) and PLN phosphorylated by CaMKII (Thr17; <b>b</b>) or PKA (Ser16; <b>c</b>) was downregulated in AF animals. <b>d</b> Phosphorylation levels relative to total PLN. <b>e</b> Serca2a protein analysis revealed a trend towards reduced protein expression associated with AF. Mean ± SEM optical density data normalized to GAPDH obtained from n = 5 Western blots per group are displayed. *<i>P</i> < 0.05; **<i>P</i> < 0.01.</p

Clinical findings in AF pigs subjected to atrial tachypacing.

<p><b>a</b> Representative ECG recordings at baseline and after atrial fibrillation (AF) induction by atrial burst pacing show sinus rhythm on day 0 and AF on day 7. <b>b</b> Mean heart rates assessed by daily ECG measurements from AF animals (n = 5). <b>c-f</b> Atrial effective refractory periods (AERP; <b>c</b>, <b>d</b>) and corrected sinus node recovery time (SNRT; <b>e</b>, <b>f</b>) at baseline and prior to euthanization at day 7 in AF pigs (n = 5). <b>g, h</b> Echocardiographic analysis of left ventricular ejection fraction (EF) and left atrial (LA) diameter. Data are given as mean ± SEM; *<i>P</i> < 0.05; **<i>P</i> < 0.01.</p

Remodeling of Ca²⁺ handling proteins during atrial fibrillation and heart failure.

<p>Changes in protein expression are indicated by arrows (orange color). Solid black arrows indicate transport directions of Ca²⁺ or Na⁺ ions, respectively. Decreased levels of Ca²⁺-calmodulin-dependent protein kinase (CaMK) IIδ and protein kinase A (PKA) causes hypophosphorylation of phospholamban (PLN) and ryanodine receptor (RyR) 2, leading to reduced Ca²⁺ uptake into the sarcoplasmic reticulum through sarcoplasmic reticulum Ca²⁺-ATPase (Serca) 2a. Increased intracellular Ca²⁺ levels and upregulation of Na⁺-Ca²⁺ exchanger (NCX) 1 expression enhance electrogenic Na⁺-Ca²⁺ exchange. This mechanism is attenuated by reduced L-type calcium channel (LTCC) expression that limits systolic Ca²⁺ influx.</p