The human heart beats over a billion times during a human lifespan, rarely skipping more than a beat. This fascinating organ can readily adapt its rhythm and contractility to physiological changes, e.g. a fight-or-flight reaction. Most of the changes in contractility and frequency are mediated by effectors such as epinephrine or acetylcholine. The rhythm of the human heart is usually dictated by the primary pacemaker, which consists of a group of cells termed the “pacemaker” cells, located in the in the Sinoatrial Node. The reason the primary pacemaker dictates the rhythm is mainly because it has the fastest frequency, while the backup/alternative systems such as the atrioventricular node and the Purkinje fibers operate at a slower rhythm.

Biology

Several pacemaker cells can be found throughout the body. The most well-known pacemaker cells reside in the heart, located in the sinoatrial node, the atrioventricular node and in Purkinje fibers. Other oscillating cells can be found throughout the central nervous system, where the frequency of de- and repolarization is often higher.

Our team was intrigued by the way that such a tiny group of cells could have such a rapid and profound impact on the frequency of the heart. These properties inspired us to recreate a system based on the remarkable pacemaker cells.

Sinoatrial cell

The origin of stable oscillations in sinoatrial cells is a topic that is widely debated. One hypothesis states that the internal calcium cycling is the most important factor contributing to the stable rhythm, while others suggest that the oscillating membrane potential has the biggest influence on the pacing. Experimental data as well as computational simulations support both hypotheses, depending on the experimental setup. Current understanding suggests that both systems are simultaneously responsible for maintaining a stable rhythm.

The slow upstroke is mediated by both HCN and calcium channels, while the fast upstroke is mediated mostly by calcium channels. On the other hand, the repolarization is mainly mediated by potassium outflux.

Purkinje fibers

The main function of Purkinje fibers is to rapidly propagate a depolarization across the ventricles, namely, to achieve a uniform contraction across the heart. However, the Purkinje fibers also serve as a last-resort pacemaker system. It is characterized by a slow rate that ranges between 15-40 beats per minute. The most important current for the pacemaker activity is the HCN current, or the so-called funny current (If). The rapid upstroke is mediated by a voltage-sensitive sodium and calcium channel. The depolarization is followed by a plateau phase, mediated by a balance between calcium influx and potassium outflux. The repolarization occurs due to an increase in potassium outflux compared to the calcium influx, which lowers the membrane potential until the cycle repeats itself.

Neuronal pacemaker cells

Neuronal pacemaker cells often have a faster rhythm, due to the properties of the expressed HCN channels. These cells express HCN1 and HCN2, as opposed to HCN2 and HCN4, present in the human heart. HCN1 has a quicker activation rate than HCN4, which partially explains the difference in rhythm present in both cells. The duration of the depolarization is also shorter, due to the difference in ion channels responsible for the action potential. Oscillating neurons use a fast voltage-sensitive sodium channel for depolarization and a fast voltage-sensitive potassium channel for repolarization.

Engineering

Cell type

Since our aim was to create a diverse system in oscillating cells, we searched for cells that grew quickly and could easily be genetically manipulated. Cells such as Human Embryonic Kidney Cells (HEK cells) quickly came to mind. Furthermore, HEK cells are often used for patch-clamp and ion channel research. These cells provide a proof-of-concept which could be applied to other cell types. We chose not to use primary cells such as sinoatrial cells or cardiomyocytes, since primary cells often react to a variety of effectors and contain many different ion currents. Our goal was to create a biological pacemaker from scratch, to show that an oscillation can be recreated in a common, non-excitable cell line such as the HEK cell line.

Ion channels Keeping the previous information in mind, we aim to create an oscillating cell using only three ion channels to keep the complexity to a minimum. We need at least one HCN channel for slow depolarization and stable rhythm generation, a fast voltage-sensitive channel for depolarisation and a fast voltage-sensitive channel for repolarization.

Mathematical model

We created our own mathematical model with the previous selected ion channels. This computational set-up allowed us to perform experiments in silico.

Final decision

We decided to transfect the HEK cells with HCN2, α1G and hERG, mostly due to imaging capabilities, transfection efficiency and availability. Furthermore, our mathematical model showed oscillations which supported our final choice of ion channels.

Theory

The final goal of our HEKcite project is to create a biological sensor using self-designed oscillating cells. We aim to create a cellular system that can vary its oscillation frequency depending on the concentration of a certain molecule. The ultimate goal is to create a device that measures this frequency and thus indirectly measures the concentration of the molecule of interest. One of the potential applications we’ve been looking at is therapeutic drug monitoring. Drugs such as anti-epileptics, immunosuppressants and anti-psychotics would benefit greatly from continuous therapeutic monitoring. Furthermore, our system allows to measure the drug dynamics, rather than the absolute concentration which you often measure using conventional methods. Drugs that bind tightly to transporter molecules such as albumin often do not have any clinical effects and should not be measured. Patients with certain kidney diseases or liver diseases can have a fluctuating albumin concentration, which changes the free drug concentration.

Our proposed system allows to only measure this clinically relevant free concentration.

Versatility

With the design of our system, versatility was kept in mind. Molecules of interest can influence the rhythm either directly by interacting with one of the ion channels, or indirectly by second messengers or a genetic link. Both the cells and relevant ion channels could be changed when considering a new application. The possibilities of the concept are endless and only limited by the imagination of the researchers.

Practice

Members of the HEKcite team consulted with patients and doctors to assess current needs in the area of therapeutic drug monitoring. Patient preferences was taken into consideration when designing the experimental setup and hardware layout. The medical community is mostly interested in monitoring drugs that need a long-term stable concentration, such as anti-epileptics, immunosuppressants and anti-psychotics. When adapting to a new regime, many of these drugs need frequent blood samples in the first few months. Blood samples however, give information only about that specific time-frame, while continuous monitoring would allow for a easier and more precise follow-up and treatment in patients.