PDK1–AKT signaling pathway regulates the expression and function of cardiac hyperpolarization-activated cyclic nucleotide-modulated channels

Abstract

Aim

The enzyme 3-phosphoinositide-dependent protein kinase-1 (PDK1) is associated with cardiac and pathological remodeling and ion channel function regulation. However, whether it regulates hyperpolarization-activated cyclic nucleotide-modulated channels (HCNs) remains unclear.

Main methods

In the atrial myocytes of heart-specific PDK1 “knockout” mouse model and neonatal mice, protein kinase B (AKT)-related inhibitors or agonists as well as knockdown or overexpression plasmids were used to study the relationship between PDK1 and HCNs.

Key findings

HCN1 expression and AKT phosphorylation at the Thr308 site were significantly decreased in atrial myocytes after PDK1 knockout or inhibition; in contrast, HCN2 and HCN4 levels were significantly increased. Also, a similar trend of HCNs expression has been observed in cultured atrial myocytes after PDK1 inhibition, as further demonstrated via immunofluorescence and patch-clamp experiments. Moreover, these results of PDK1 overexpression indicate an opposite trend compared with the previous experimental results. However, the results of PDK1 inhibition or overexpression could be reversed by activating or inhibiting AKT, respectively.

Significance

These results indicate that the PDK1–AKT signaling pathway is involved in the regulation of HCN mRNA transcription, protein expression, HCN current density, and cell membrane location.

Introduction

The protein kinase A, protein kinase G, and protein kinase C (AGC) family regulates multiple cellular functions and physiological processes [1]. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) [2] and phosphatidylinositol 3-kinase (PI3K) [3] are important signaling molecules in heart failure (HF) processing, pathologic heart remodeling; and the functioning of ion channels including sodium [4], potassium, and calcium currents in cardiomyocytes [5]. Similarly, AKT, is also important in many cardiovascular pathological processes [6]. Mice with PDK1 knockout presented with HF and sudden death within 11 weeks [7]. Sodium current downregulation and atrioventricular block might be causes of sudden cardiac death [4]. In clinical, the inhibition of PI3K–PDK1–AKT signaling represents an underexplored target for cancer therapy [8,9]. However, the cardiovascular toxic effects, including HF and arrhythmia, of targeted cancer therapies have attracted increasing attention [10]. For arrhythmia, the incidence rates of QT prolongation and torsades de pointes are on the rise due to the drugs used in cancer management [8]. For example, ibrutinib, a potent tyrosine kinase inhibitor, was associated with a higher risk of atrial fibrillation (AF) [11]. Further, patients with germline mutations of certain AGC family genes, such as PTEN [12] or CYP27A1 [13], are plagued by a cancer-predisposition syndrome and a higher risk of cardiovascular diseases.

A variety of experimental and clinical studies have found that HF, coronary artery disease, and arrhythmia are linked with an elevated heart rate [14]. An increased heart rate is one of the independent predictors of cardiovascular diseases, especially HF [15]. The hyperpolarization-activated cyclic nucleotide-modulated channel (HCN), which is regulated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] on the membrane constitutive component [16], generates spontaneous electrical activity in the heart [17], and it is suggested that increased atrial or ventricular expression of HCNs in hypertrophied and failing hearts leads to arrhythmia [18]. Thus, the inhibition of HCN with ivabradine is considered to reduce the heart rate and improve the prognosis of HF [19]. Recent research results have also supported that HCN2/4 RNA and protein expression were significantly increased in atrial myocytes of animals with atrial fibrillation [20], and ivabradine has also been found to inhibit HCNs via AKT-endothelial nitric oxide synthase signaling [21] and decrease the induction rate of AF [22]. These results suggest that changes in regional HCN expression patterns were associated with the onset and maintenance of AF [23]. It is understood that HCNs are modulated by cyclic nucleotides in cardiac nodal cells and subsidiary pacemakers [24], but whether HCNs are regulated by the AGC protein kinase family remains unclear.

Given their similar cardiovascular toxic or side effects and the inhibition of AGC protein kinases in certain antitumor drugs, electrical remodeling of HCNs has previously been associated with the onset and maintenance of HF and AF. We sought to investigate the correlation between the two mechanisms by considering the hypothesis that the PDK1–AKT signaling pathway of AGC family protein kinases is involved in the regulation of HCNs in an effort to better understand the mechanism of the cardiovascular toxicity of cancer therapies.