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Biochemical and Biophysical Research Communications

"Funny" channels in cardiac mitochondria modulate membrane potential and oxygen consumption

Abstract

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by a family of four genes (HCN1-4). All isoforms are expressed in the heart, HCN4 being the most abundant in the sinoatrial node (SAN). HCN channels are responsible for the "funny" current ( I f ) associated with the generation and autonomic control of the diastolic depolarization phase of cardiac action potential. In this work we performed a proteomic analysis of HCN4 transfected in HEK293 cells. Most of the identified proteins in the HCN4 network belonged to mitochondria. The subcellular localization of HCN channels was predicted in plasma membrane, mitochondria and nucleus. Experimentally, HCN2 (full-length, truncated), HCN3 (full-length, truncated) and HCN4 (truncated) were detected in rat heart mitochondria by immunoblotting. I f sensitive to ZD7288, was recorded by patch-clamp in mitoplasts from cardiomyocytes. Mitochondrial membrane potential (ΔΨm) assessment in H9c2 cells revealed that ZD7288 induced almost 50% higher hyperpolarization respect to control at 30 min. Furthermore, ZD7288 reduced oxygen consumption attributed to ATP synthesis in H9c2 cells. In conclusion, we identify for the first time functional HCN channels in mammalian cardiac mitochondria and demonstrate their impact on ΔΨm and respiration.

Introduction

The contractile function of the heart depends on myocardial electrical activity. Cardiac cycle begins with the action potential originating in the sinoatrial node (SAN), propagating through atria to the atrioventricular node. The mammalian SAN expresses channels that conduct inward currents triggered by hyperpolarization in the diastolic range of voltages, contributing to the generation of the rhythmic cardiac activity. The inward current was originally named "funny" (I f) because of its unusual characteristics: mixed Na+ and K+ permeability, activation by hyperpolarization and slow activation and deactivation kinetics [1].

The molecular determinants of I f are the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels encoded by a family of four genes (HCN1-4). In addition to SAN pacemaker cells, I f has been recorded in Purkinje fibers, atrioventricular node, atrium and ventricles. I f is activated around −60 to −90 mV, but in ventricles at more negative potentials [2]. Inhibition of cardiac I f by bradycardic agents such as ivabradine has been useful to reduce incidence of cardiovascular mortality and hospitalization for some cases of heart failure [3].

Heart is one of the most energy demanding tissues because its continuous contraction and relaxation is dependent on ATP produced by the mitochondrial oxidative phosphorylation (OXPHOS). The electron transport chain and ATP synthase, located in the inner mitochondrial membrane (IMM), are essential components for the generation and preservation of energy metabolism. The high negative value of the IMM potential (ΔΨm) required for OXPHOS is a powerful driving force for mitochondria K+ uptake. Mitochondria also play an active role in Ca2+ homeostasis, reactive oxygen species (ROS) production and apoptosis [4].

Several K+ channels have been identified in heart mitochondria: ATP-sensitive ROMK (mitoKATP) [5,6], Ca2+-sensitive (mitoBKCa, SKCa) [7,8], KNa1.2 [9] and Kv7.4 [10]. Paradoxically, all these K+ channels are closed in physiological conditions. MitoKATP has been the most widely K+ channel studied as a mediator of heart protection in ischemic preconditioning [11]. Pharmacological activation of mitoKATP by diazoxide reduces mitochondrial Ca2+ uptake, inhibits the permeability transition pore opening [12] and increases ROS production by the electron transport chain [13].

In a previous report, we presented evidence of the functional expression of HCN3 channel in kidney mitochondria [14]. The aim of this work was to study the presence and physiological role of HCN channels in cardiac mitochondria.

Section snippets

Reagents and antibodies

Most reagents used in this study were purchased from Sigma-Aldrich (MO, USA). ZD7288 was obtained from Tocris (MN, USA); 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, T3168) was purchased from Invitrogen (Thermo Fisher Scientific, MA, USA). For more information about reagents see [14]. Voltage-dependent anion channel (VDAC) antibody (ab34726) was from Abcam (Cambridge, United Kingdom) and α-tubulin antibody (t9026) was from Sigma-Aldrich. Anti-HCN1

Proteomic analysis of HCN4 channel

Since HCN4 is the predominant isoform expressed in SAN, we performed a proteomic assay to establish its protein-protein interaction network. Mitochondrial proteins were the most prominent protein family associated to HCN4 (Table 1) as it was for HCN3 in our previous study [14].

Subcellular localization prediction of HCN channels and its identification in heart mitochondria

As expected, all HCN channels were found in plasma membrane. However, they exhibit other subcellular locations. Besides plasma membrane, HCN1, HCN2 and HCN4 showed a predominant nuclear location, whereas HCN3 was more

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgments

Teresa Padilla-Flores is a student of "Programa de Doctorado en Ciencias Biomédicas" at Universidad Nacional Autónoma de México (UNAM) and received a scholarship (289190) from Consejo Nacional de Ciencia y Tecnología (CONACYT, México). Grants from Dirección General de Asuntos del Personal Académico (DGAPA, UNAM) IN224919 and CONACYT A1-S-8731 (L.I.E.). Postdoctoral Scholarship Program DGAPA at UNAM (D.L.A.). Fulbright-Garcia Robles grant (L.V.). The authors thank Dr. Emmanuel Ríos Castro,

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