Electrophysiological Stimulation of Whole Heart Constructs in an 8-Pole Electrical Field

Today 2D and 3D electrophysiological stimulation represents a well established concept to enhance myocardial development and maturation in tissue-engineered constructs. However, electrical field stimulation has never been adapted to complex whole heart constructs (WHC). This study demonstrates the impact of three-dimensional electrophysiological stimulation of tissue-engineered WHC in a custom made eight-pole electrical field stimulation system by short model cultivations with neonatal rat cardiomyocytes (CM). Therefore, WHC were generated by repopulation of decellularized rat hearts with neonatal CM and subjected to perfusion based cultivation with or without additional biophysicalstimulation for 96 h. Spontaneous electrophysiological (EP) activity of the processed WHC was analyzed by qualitative evaluation of multielectrode assay (MEA) signal sequences, descriptive comparative spike sorting, and direct contrasting assessment in simple numerical quantities complemented by impulse response tests after phasing out spontaneous EP activity. As strong reduction of voltage signals by the decellularized extracellular matrix (ECM) component of WHC was observed, the active principle was determined and used to estimate the spectrum of source signals to recorded values by calculative elimination. Western blotting of key myocardial markers was employed to substantiate the functional EP evaluation by classical biochemical analysis. We observed stable spontaneous EP activity showing clear R and S, but predominantly rS patterns, for both stimulated WHC and non-stimulated controls. By the impact of stimulation, mean voltage amplitudes and beating frequencies could be significantly increased. The active principle of signal reduction in decellularized ECM could be shown to follow a nonlinear damping function with remarkable accuracy, illustrating that recorded signals of moderate voltage amplitudes can also represent far-field measurements of strong signals that are emitted in distant depths of the ECM while small amplitudes are limited to actually represent also rather weak source-signals. After phasing out spontaneous activity, both stimulated WHC and non-stimulated controls could be excited again to emit immediate impulse responses. The observed beneficial impact of 8-pole field stimulation on functional EP activity could finally be validated on the biochemical level by showing increased ratios for myosin heavy chain, cardiac tropnin T, desmin, and connexin 43 for stimulated WHC by Western blot analysis. In conclusion, we found that although electrophysiological stimulation has been incorporated into the whole heart tissue-engineered concept from the very beginning, this study presents for the first time a concept for the transfer of electrical field stimulation to the whole heart tissue-engineered approach. Furthermore to the best knowledge of the authors, this is the first control-based study showing a comparative investigation of electrophysiological stimulation of whole heart constructs.