A total of 56.4 million deaths occurred globally in 2015, of which non-communicable diseases (NCDs) were responsible for 39.5 million cases (70%) and 30.7 million cases occurred in low- and middle-income countries, for which approximately 48% of the deaths took place before the age of 70. Of these NCD death cases, cardiovascular diseases (CVDs) were responsible for 17.7 million (45%) of the deaths (WHO, 2014, 2016). Based on data from the World Health Organization (WHO), the total annual number of deaths from NCDs is projected to increase to 52 million by 2030 (WHO, 2014). The most well-known risk factors for CVDs include elevated blood pressure, smoking, high cholesterol and blood glucose level, poor diet, obesity, and risky use of alcohol (WHO, 2011, 2014). However, recent studies have shown that the resting heart rate is one of the major indicators of CVD morbidity and mortality in addition to the previously known factors (Reil et al., 2011). The occurrence of atherosclerosis, coronary artery disease (CAD), heart failure, hypertension, and stroke are linked with an elevated heart rate, independently of the other CVDs (Kannel et al., 1987; Gillman et al., 1993; Diaz et al., 2005; Custodis et al., 2008, 2010; Fox et al., 2008).

The occurrences of coronary atherosclerosis in patients that have undergone coronary artery bypass surgery or recurring myocardial infarction have been independently linked to intrinsically elevated heart rate as a result of increasing the mechanical load and tensile strength on the arterial wall and exposure to low endothelial shear stress (Custodis et al., 2008; Lang et al., 2010).

To date, beta blockers, calcium-channel blockers, and other drugs have been utilized to reduce the heart rate either directly or indirectly (Parker and Parker, 1998; Freemantle et al., 1999; Poole-Wilson et al., 2004; Speranza et al., 2012). These medications have been prescribed for heart failure, angina, and other CVDs. Even though these classes of drugs have irreplaceable use in a myriad of CVDs, they have been claimed to exhibit a lack of selectivity in the reduction of heart rate. They also have additional undesirable side effects and contraindications on the respiratory system, angioedema, metabolism, and other sites.

Taking the limitations of the aforementioned classes of drugs into consideration, researchers have investigated a novel target site called the funny current (I_f) or funny (f) channel, which may be useful for selectively lowering the heart rate. This site in the mammalian sinoatrial node (SAN) has been described as the pacemaker of the heart, and it is activated in phase 4 of the action potential as a result of accelerating diastolic depolarization (Speranza et al., 2012).

A number of molecules have been identified and developed as I_f channel blockers, including alinidine (ST-567), cilobradine (DK-AH-269), zatebradine (UL-FS-49), ZD7288 (ICI-D7288), and ivabradine (S-16257-2) (Figure 1). However, ivabradine (a benzocyclobutene-containing compound) is the only molecule that has passed all of the clinical trial phases and that is used currently as an anti-anginal drug (Bucchi et al., 2007).

Overall, this novel site has been continuously investigated for the treatment of a variety of CVDs since its identification almost four decades ago. However, several controversies have arisen in the literature from both experimental animal studies and randomized controlled trials, as well as from a meta-analysis on previously developed drugs that target the I_f channel, particularly ivabradine (Borer et al., 2003; Fox et al., 2008; Tardif et al., 2009; Cammarano et al., 2016), even though these drugs have been approved by some regulatory agencies and applied in some countries (FDA, 2015). Therefore, the aim of this review is to assess the current evidence and progress on targeting the I_f channel to determine the future direction and challenges for both preventive and therapeutic purposes.

In 1979, mammalian SAN cells, a specialized region of the heart, were found to express the funny channel as an inward current triggered on hyperpolarization in the diastolic range of voltages, thus influencing the contraction rate of the entire heart (Brown et al., 1979; DiFrancesco, 2008).

The name “funny current” arose because of its numerous unusual characteristics, including the mixed Na⁺ and K⁺ current permeability, activation on hyperpolarization, and slow activation and deactivation kinetics (Brown and Difrancesco, 1980; DiFrancesco, 1993; Baruscotti et al., 2005). Studies have shown that the I_f channel is sensitized at about -100 to -110 mV, and its reversal potential is about -10 to -20 mV, which indicates that it has a mixed permeability to Na⁺ and K⁺. Hence, it is defined as an inward current activated during hyperpolarization at voltages in the range of diastolic depolarization, and it contributes to the generation of rhythmic cardiac activity (Brown and Difrancesco, 1980; Freemantle et al., 1999). Another unusual feature of the I_f channel is its direct activation by cyclic adenosine monophosphate (cAMP), which activates the opening probability of the I_f channel, and it is also regulated by voltage-dependent activation (DiFrancesco and Tortora, 1991). Thus, it is evident that its function, and hence the heart rate, is influenced by the concentration of the second messenger cAMP, which will be increased and decreased by adrenergic and cholinergic stimulation, respectively.

As shown by the overview in Figure 2, the I_f activation range (black line in the top left figure) comprises the range of diastolic (pacemaker) potentials (-120 mV to ∼-40 mV), and determines the slope of diastolic depolarization, and hence the rate of the heart, under controlled conditions (black line in the top right figure). Noradrenaline causes stimulation of G proteins by stimulating the β-adrenoreceptors and hence increases the adenylate cyclase activity (red arrow), which in turn increases the intracellular cAMP concentration, and this results in extra activation of I_f by changing the I_f activation curve to more positive voltages (red line in top left figure). Thus, more inward current brings about a short diastolic depolarization period, and hence the heart rate speeds up (red line in top right figure) (DiFrancesco and Borer, 2007).

Sinoatrial node cells in the myocardium are distinguished by the incidence of a “slow diastolic” phase that produces an impulsive and recurring action potential (DiFrancesco, 1993). Currently, it is generally understood that the generation of the diastolic depolarization is caused by activation of the I_f channel at the end of an action potential. Therefore, either stimulation or inhibition of the I_f channel may trigger or suppress the heart beat by altering the diastolic time of depolarization. Hence, the activation of the I_f channels decreases the diastolic time, which subsequently produces myocardial and associated problems. Alternatively, the inhibition of the I_f channels increases the diastolic time because of the slower firing of the next action potential and beat. This, in turn, results in improved perfusion of the myocardium and reduces its oxygen demand, which is beneficial for the management and prevention of CAD, heart failure, and other CVDs.

Studies conducted prior to the identification of the I_f channel concluded that the enhancement of the K⁺ conductance of the pacemaker cells in the SAN due to the discharge of acetylcholine (Ach) slows the heart rate (Rayner and Weatherall, 1959; Danilo et al., 1978). In addition, an experimental study by Sakmann et al. (1983) suggested that K⁺ channels are responsible for the ACh-dependent K⁺ conductance increase in SAN cells, which possess distinct opening and conductance properties compared to atrial and ventricular cells. However, SAN cells have similar properties to the other ACh-activated cells in terms of resting K⁺ current. This study also found an excess of short channel openings, the reason for which was unclear (Sakmann et al., 1983) but could be the occurrence of I_f channel activation, although it was not considered in this study. After this finding, a study by DiFrancesco et al. (1989) revealed that at low (resting) or moderate vagal activities, ACh-mediated I_f current inhibition is responsible for the slowing of heart rate, while the K⁺ current is activated by a marked vagal stimulation (high ACh concentration). The result shows in a pronounced decrement of heart rate (bradycardia) in the SAN cells and atrium as well (DiFrancesco et al., 1989; DiFrancesco and Borer, 2007). These findings provide new insights into the mechanism through which the heart rate is decreased upon activation of muscarinic receptors by ACh (green lines, as shown in Figure 2).

The mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family consists of four isoforms, namely HCN1–4, which belong to the superfamily of voltage-gated potassium channels and represent the molecular α subunits of the native “funny” channels present in the heart and brain (Baruscotti and Difrancesco, 2004; Baruscotti et al., 2010a,b; Scicchitano et al., 2012). In vivo examinations have revealed that functional HCN channels can be produced in homomeric as well as heteromeric tetramers with the exception of HCN2–3 heteromers (Baruscotti et al., 2010a). The HCN4 isoform is the dominant HCN transcript in human SAN cells, followed by HCN1 and HCN2, although HCN2 is the foremost transcript in ventricles (Chandler et al., 2009; Scicchitano et al., 2012) and its locus has been recognized as a modulator of heart rate in a genome-wide association study (den Hoed et al., 2013).

The physiological role of the HCN family of channels in the central nervous system and heart is enormous (DiFrancesco and Tortora, 1991; Baruscotti et al., 2010a), and they are responsible for the I_f current in the SAN. Six-transmembrane α-helical segments (S1–S6) make up each HCN isoform, with the positively charged S4 domain acting as a voltage sensor in the pore region formed between domains S5 and S6, which acts as a conduction pathway and selectively filters binding. The presence of a glycine–tyrosine–glycine (GYG) sequence, which is typical for the pore-loop region of K⁺-permeable voltage-gated ion channel subunits, has been reported; a linking site for cAMP in the C-terminal region of the peptide is found that binds to the channels, through a direct action on the channel itself but not by phosphorylation, in the cyclic nucleotide-binding domain (CNBD) located within the C-terminus (Barbuti et al., 2007; Baruscotti et al., 2010a; Scicchitano et al., 2012). The binding of cAMP to the HCN channels increases the probability of the channel to be open during hyperpolarization by inducing a conformational change of the protein (Scicchitano et al., 2012). Different distinguishing properties of the native I_f are exhibited by the HCN isoforms, including activation by hyperpolarized membrane potentials, modulation by cAMP, permeability to Na⁺ and K⁺, and the chunk on the part of cesium (Cs⁺) (Ludwig et al., 2004).

The funny channel is a good target for the development of new drugs as it generates spontaneous activity and controls the heart rate by acting specifically on the cardiac rate control and pacemaker rhythm. Lowering the heart rate under different cardiac conditions is one means of controlling CVDs pharmacologically, even though there are different risk factors (Sulfi and Timmis, 2006).

Various experimental and epidemiological studies have shown that, in spite of the multiple identified risk factors such as hypertension, diabetes, and smoking, an increased resting heart rate is one of the key independent predictors of CAD, heart failure, and cardiovascular mortality (Umana et al., 2003; Fox et al., 2008; Busseuil et al., 2010). An increased resting heart rate has also been linked to mental stress, which, in turn, aggravates myocardial ischemia in patients with CAD and is considered to be one risk factor for hypertension and atherosclerosis (Carter et al., 2005). The consequences of increased heart rate can favor the progression of myocardial ischemia as a result of mounting myocardial oxygen use and decreased coronary blood flow due to reduced diastolic filling time (Parker and Parker, 1998).

In mouse models of hypercholesterolemia and endothelial dysfunction, the administration of ivabradine reduced the levels of vascular oxidative stress markers and atherosclerotic plaque development and restored endothelial function through the inhibition of the I_f channel (Custodis et al., 2008). An increased heart rate can cause plaque rupture and associated myocardial problems (Dominguez-Rodriguez et al., 2011).

Heart rate during acute ischemia-reperfusion is an important determinant of susceptibility to reperfusion arrhythmias, with a higher heart rate found to cause a predisposition to arrhythmias in rats (Bernier et al., 1989). Bernier et al. (1989) found that pacing either throughout the experiment or during ischemia alone led to a rate-dependent increase in the occurrence of reperfusion-induced ventricular fibrillation (VF), from 25% in the unpaced hearts to >90% when the rate was ≥420 beats/min, but pacing during reperfusion alone did not increase the occurrence of reperfusion-induced VF. These data suggest that a clinical therapeutic strategy for heart rate reduction during ischemia-reperfusion may reduce the incidence of reperfusion arrhythmias. However, it remains unclear whether an increased heart rate is associated with ventricular or reperfusion arrhythmia. To investigate this, animal experiments have been performed using ivabradine to evaluate the outcome of selective heart rate reduction during the occurrence of ischemia and reperfusion reduced reperfusion VF. The heart rate reduction as a result of acute ischemia led to a reduction in the occurrence of reperfusion arrhythmias, whereas the heart rate at reperfusion alone did not influence the occurrence of reperfusion VF, as neither a bolus of ivabradine nor pacing prior to reperfusion changed the occurrence of reperfusion VF. Hence, the anti-arrhythmic effects of ivabradine in the heart rate reduction during acute ischemia may be the result of the slower development of ischemia-induced electrophysiological alterations (Ng et al., 2013).

All of the phases of the cardiovascular continuum (Figure 3) from vascular risk factors to cardiovascular episodes and heart failure are essentially influenced by heart rate. Hence, in CVD prevention, heart rate is one of the risk indicators and therapeutic targets (Kannel et al., 1987; Gillman et al., 1993; Diaz et al., 2005; Custodis et al., 2008, 2013; Fox et al., 2008).

Patients with chronic stable angina can benefit from decreased heart rate because of the associated improved myocardial perfusion and reduced myocardial oxygen demand. The use-dependence of heart rate reducing agents is one of their distinctive attributes in which the outcome of drug application accumulates through repetitive action (Van Bogaert et al., 1990). This important drug characteristic was achieved for ivabradine as a result of the accumulated inhibition of I_f current by the activation/deactivation procedure, which is therapeutically useful and it indicates that the slowing action of the drugs will be stronger at elevated heart rates when the effect of slower heart rate is essential (Bucchi et al., 2002).

According to a study conducted on mice to evaluate the effects of chronic mental stress and heart rate on endothelial function and cerebral ischemia, chronic mental stress impairs the function of endothelial cells, increases vascular and brain oxidative stress, and enlarges the size of cerebral lesions (Custodis et al., 2011). The endothelial function, oxidative stress, and ischemic brain injury could be restored, reduced, and protected, respectively, by heart rate reduction using ivabradine (10 mg/kg per day), as a result of the reduced activity of NADPH oxidase in the aorta in addition to aortic lipid peroxidation and reduction of the expression of nitric oxide synthase (eNOS). These results suggest that heart rate reduction by I_f inhibition is a likely new target for improving the cerebrovascular function after ischemic events (Custodis et al., 2011). In a separate study, Walcher et al. (2010) reported that the chemokine-induced migration of CD4-positive lymphocytes was inhibited upon administration of ivabradine by limiting both phosphatidylinositol 3 kinase (PI3K) activity and the phosphorylation of protein kinase B (AKT).

Multiple studies have demonstrated that atherosclerosis, heart failure, CAD, stroke, and arrhythmia are self-regulating prognosticators of CVDs as a result of an elevated heart rate. Even though randomized clinical trials have presented some controversies regarding the clinical use of ivabradine for CAD, the drug provides a safe and effective means to reduce heart rate, either alone or in combination with the already existing drugs, by selectively inhibiting the I_f channel without significantly affecting the action potential, inotropic activity, or ventricular contractility of the heart and with minimal adverse effects. Currently, funny channel inhibition is being targeted for the treatment and prevention of CVDs such as atherosclerosis and stroke.

Based on the available evidence and the prospect of I_f channel targeting; it is very apparent that inhibition of this channel can be used for the prevention and treatment of cardiovascular-related mortality and morbidity. Through this strategy, it seems plausible to reduce CVD morbidity and mortality by an early screening of the at-risk population by measuring the simple vital sign of resting heart rate and providing a long-acting dosage form of the current oral or intravenous I_f inhibitors. Furthermore, the I_f channel has recently been investigated for the prevention and treatment of stroke, hypertension, atherosclerosis, myocardial infarction, and arrhythmia; fortunately, all of the in vitro studies and animal models showed positive findings. It is also suggested that the use of cell/gene-based therapy for the in situ delivery of the genes that encode the I_f channel might represent a future treatment strategy for rhythmic disturbances, which could replace the current practice of using electronic devices. Hence, in the future, I_f channel inhibitors might be clinically useful for prevention and treatment of these disorders.

HM and TT conceived the review and wrote the draft manuscript. TT, HM, and MB participated in improving the draft manuscript and writing the final manuscript. All of the authors approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.