The development of a high-throughput, high-information content device to study and understand cardiac electrophysiology would be important for the fields of cardiac physiology, tissue engineering and drug research. More than 850,000 people are hospitalized for arrhythmias each year and ventricular fibrillation (VF) is a leading cause of cardiac death[1]. Despite the intensive research in this area, the mechanism of VF is still poorly understood[2-5].
Arrhythmia is a known side effect of commercial drugs. One of the mechanisms by which drugs can cause a potentially fatal form of ventricular tachy arrhythmia, called Torsades de pointes (Tdp), is through the prolongation of the QT interval (in an ECG the length of the ventricular action potential). It has been reported that approximately 2-3% of all prescribed drugs can cause long QT syndrome[6, 7]. A broad range of cardiovascular drugs and antibiotics also have the potential risk of causing drug induced Tdp[8, 9]. At the same time, prolongation of the QT interval does not necessarily lead to Tdp; lengthening of the QT interval could even be antiarrhythmogenic, as it is considered a mechanism of action of the class III anti-arrhythmics[8, 9]. Thus, a relatively high-throughput method to identify cardiac side effects and differentiate between arrhythmic and anti-arrhythmic effects at an early stage of drug development would have a significant impact on the field.
Gap junctions play an important role in the propagation of excitation in cardiac tissue. Changes in gap junction function affect major cardiac parameters, such as conduction velocity (CV). It has been observed in several cardiovascular diseases that the expression of connexins (protein molecules that form gap junction channels) is decreased or their distribution is changed, leading to a malfunction in gap junction coupling[10]. Understanding the pharmacological modulation of cardiac gap junction channels would further aid the drug development enterprise.
Introduction of an in vitro method for cardiac side effect testing, which has high predictive value, would have a significant impact on drug development as it could also reduce the cost, time and the number of drugs failing in clinical trials[11]. in vitro testing would also reduce the need for animal testing and could be used to study drug effects with a functional assay, but at the cellular level. Other in vitro methods, such as whole heart experiments (Langendorff heart model) or the Purkinje fiber preparation are difficult and time consuming[11]. Traditional methods used to study QT interval prolongation at the cellular level include patch-clamp experiments. However, these experiments are time intensive, require a skilled operator and cannot be used to study action potential (AP) propagation or parameters such as CV and re-entry. Moreover, evidence suggests that prolongation of QT intervals is not the best predictor of Torsades de pointes. The measurement of the length, or the variability in the length, of the refractory period after a cardiac action potential may have more relevance for predicting arrhythmic behavior[9].
Cardiac myocytes cultured on microelectrode arrays (MEA) have several benefits compared to either traditional patch clamp electrophysiology or isolated organ methods. The use of MEAs in the investigation of cardiac side effects would provide information in a relatively high-throughput and low cost manner compared to standard patch-clamp electrophysiology. However, at this time, it is still a low information content method and this has limited its use. Cardiac myocytes on MEAs have been used in a number of studies to investigate the effect of toxins, such as pesticides[12] and cardioactive drugs[13] on cardiac field potentials. A commercial system has also been introduced to measure QT intervals in a relatively high-throughput fashion[14], but, to date, it has only limited applications. However, cardiac myocytes can now be maintained over longer periods of time[15], thus chronic experiments, such as the monitoring of network remodeling for specific diseases, is now feasible. In addition, serum-free formulations for cardiac culture have also been introduced, which would increase the reproducibility of such a system[16].
All of the above mentioned studies utilized unorganized monolayers of cardiomyocytes on the MEAs. Development of a patterned cardiac myocyte layer that is aligned with the electrodes of a MEA could solve several problems associated with the random spread of excitation in a cardiac monolayer, which makes evaluation of the obtained data, such as CV, difficult. It would also enable the development of specific open-loop or closed-loop stimulation protocols to measure critical parameters, such as the length of the refractory period after the action potential. It could also be used to create a high-throughput, low-cost functional reentry model.
There are several lines of evidence indicating that not only contact interaction with the surface but the shape of the attachment area determines the physiology of cardiac myocytes[17]. Pattern geometries determine the extent of the alignment/of the long axis of cardiac myocytes, alignment determines CV[18] and other physiological and pharmacological properties of cardiac tissues[19, 20].
Several different methods have been developed for cell patterning. One category of this technique is based on direct placement of cells or extracellular matrix molecules on desired locations and includes patterning through microfluidic channels[21-23], microcontact printing[24, 25] and inkjet printing[26]. Cardiac myocytes have previously been patterned on glass using photoresist[27] as well as other techniques[15, 17, 19, 20, 25]. Another method utilized photolithography following surface modification with self-assembled monolayers (SAMs) for neurons[28-30] as well as myocytes[15, 31]. The benefit of this method is the compatibility of the technique with cheap automated silicon manufacturing steps and the ability of the cells to self-assemble after random plating.
SAMs are one molecule thick monolayers attached to a surface composed of organic molecules, which have been extensively used for surface patterning[31-33]. Surface modification with SAMs is also compatible with advanced photolithography methods[30, 34]. Studies have also shown that cells survive on these surfaces for extended periods of time[35, 36], do not migrate off the patterned areas[34] and exhibit the typical morphology and physiology of the specific cell type[16, 37].
The goal of this study was the development of patterned, rat, cardiomyocyte cultures on MEAs in a serum-free medium for the study of cardiac physiology and pharmacology utilizing a high-throughput technique, but with high information content. An adsorbed fibronectin layer was used as the foreground because it supported cardiac myocyte attachment and growth and a 2-[Methoxy(Polyethyleneoxy)Propyl]TrimethoxySilane (SiPEG) SAM was used as the cell repellent background because of its excellent protein adsorption resistant properties[38]. The measurement of CV with the patterned cardiac myocyte monolayers and the feasibility to apply different stimulation protocols to the MEA/cardiac system was demonstrated. The action of 1-Heptanol and Sparfloxacin was also assessed. This method could easily be adapted for use with human cardiac myocytes to eliminate interspecies differences in drug side effect screening and could become an alternative to the existing more complex, expensive and time consuming methods.