(164ac) Monitoring the Activity of Optogenetically Engineered Cardiomyocytes Using Microelectrode Arrays Chips

We report an integrated optogenetic and bioelectronic platform for stable and long-term modulation and monitoring of cardiomyocyte function in vitro. Optogenetic inputs were achieved through expression of a photoactivatable adenylyl cyclase (bPAC), which when activated by blue light caused a dose-dependent and time-limited increase in autonomous cardiomyocyte beat rate. Bioelectronic readouts were achieved through an integrated planar multi-electrode array (MEA) that provided real-time readouts of electrophysiological activity from 32 spatially-distinct locations.

Optogenetic modulation of cellular processes can be achieved in a reversible fashion, with high specificity, and few – if any – side effects compared to pharmacological treatments. The secondary messenger cyclic adenosine monophosphate (cAMP) plays a central role in the control of cardiomyocyte force of contraction and rate of relaxation. Increased cAMP activates the protein kinase A (PKA), which phosphorylates and enhances the function of voltage-dependent calcium channels by inducing Ca²⁺ influx and membrane depolarization.

Through the expression of bPAC, intracellular cyclic AMP ([cAMP]i) can be upregulated leading to an increase in heart beating rate. Rat ventricular cardiomyocytes were transduced with an adenoviral vector, AdbPAC, carrying a cassette with the bPAC gene (featuring a cMyc tag for immunodetection) and the mCherry fluorescent reporter co-expressed through the CMV promoter. Cell transduction was performed as a multiplicity of infection (MOI) ranging between 50 – 1000. The expression of mCherry was detected by fluorescence microscopy and quantified by flow cytometry. The fraction of mCherry-positive cells was 10.2% at MOI of 50, and markedly increased to 52.5% at MOI of 500 with no difference for cells cultured at MOI of 1000. However, cell viability was decreased from 85.0% to 55.3% for MOI of 500 and 1000, respectively. Expression of the bPAC protein was confirmed by western blotting. After 30 minutes of blue light illumination, intracellular cAMP was 2.5- fold higher with the expression of bPAC, as determined by ELISA. No difference was observed between cells infected with the empty adenoviral AdeGFP vector (control group), and cells transduced with AdbPAC but no exposure to blue light. The expression and pattern of distinct cardiomyocyte proteins such as α-actinin and connexin-43 were not altered despite the expression of bPAC as revealed by confocal microscopy.

The integrated MEA consisted of circular 30-micron diameter gold recording elements and SU-8-passivated interconnects fabricated on a silicon / silicon oxide substate. Following fabrication the recording elements were electrochemically coated with porous platinum black which reduced their impedance from ca. 260 to 8.0 kΩ. Neonatal rat cardiomyocytes were seeded onto the MEA and transfected on DIV 3 (days in vitro). Bioelectronic readouts revealed spontaneous beating behavior by DIV 6 with signal-to-noise ≥ 200 dB.

MEAs provided real-time readouts of cardiomyocyte behavior in response to optical modulation. Irradiation at 24 μW/mm² resulted in a ca. 17% increase in beat rate within 20-25 minutes of irradiation. This elevated beat rate remained stable for at least 2 hours. In addition, bPAC activation could be cycled through repeated “on” and “off” states via time-limited irradiation. Multiplexed readouts revealed that wavefront propagation rates throughout the monolayer remained constant throughout both “on” and “off” states, demonstrating that optical modulation did not affect intercellular coupling. Cardiomyocytes could be modulated reproducibly over at least four days, demonstrating that bPAC expression was stable throughout that period.

Taken together, our results demonstrate the potential of optogenetic control for rational modulation of cardiac function. Real-time readouts enable assessments of cardiac function that could be further integrated into closed feedback loops. These technologies could have clinical applications for cardiac regulation including arrythmia diagnosis and intervention.