Poster T17 - Uniaxial mechanical and electrical conditioning of hiPSC-derived cardiomyocyte based bioelectric threads for electrical coupling of engineered cardiac tissues

Introduction: Over 800,000 myocardial infarctions occur annually in the United States alone, 25 % of which represent a repeat occurrence further compounding ischemia in the myocardium. With up to 1 billion cardiomyocytes lost during a single heart attack and only 1-3 % cardiomyocyte turnover per year, this massive cell loss can lead to maladaptive remodeling and eventual heart failure. Engineered cardiac tissues offer a promising avenue for replacing lost cardiomyocytes and supplementing cardiac function to prevent future heart failure. However, the field is currently limited by a lack of electrical coupling between surface-implanted engineered cardiac tissues and the host myocardium, undermining their potential therapeutic benefit. Although groups have demonstrated potential mechanical entrainment, no group has successfully demonstrated electrical coupling for host-pacing of the implanted tissue. We’ve developed a bioelectric thread, leveraging an injection based delivery device to introduce a cell-based electrical bridge between engineered cardiac and host tissues. Our previous work demonstrates successful in vitro coupling and in vivo delivery, but conduction velocities insufficient to match physiologically relevant beat rates, resulting in conduction blocks and limited benefit. Applied tension along a defined axis is well-document to induce cell alignment, subsequently increasing conduction velocity. We hypothesize that tensile conditioning combined with similar alignment of electrical stimulation across a defined axis, using point stimulation in place of the current standard of field stimulation of the entire media reservoir, will align gap junctions to cell ends, enabling efficient propagation to further increase conduction velocity and match physiologically relevant heart rates.

Materials and

Methods: Cardiomyocytes are differentiated from human induced pluripotent stem cells (hiPSCs) using an established biphasic wnt activation protocol. 95 % hiPSC-CMs are combined with 5% human cardiac fibroblasts, 20 mg/mL fibrinogen and 10 % gelatin and extruded to produce bioelectric threads. A custom, fully modular combined point stimulation and pulsatile stretch culture bioreactor, utilizing concentric cylinder point stimulation electrodes and alternating current polarity to minimize electrolysis along with an Arduino controlled stepper motor to apply pulsatile tension (Figure 1A) is used to culture the bioelectric threads for 2 weeks following fabrication. Bioelectric thread culture conditions include pulsatile stretch, static stretch, and no stretch. As electrical pacing is a known requirement for electrical maturation and development, each mechanical conditioning program is combined with point stimulation pacing or, the current field standard, field pacing. Immunohistochemistry and fluorescent imaging of alpha actinin, labeling actin filaments, cx43, labeling gap junctions, and Hoechst, labeling nuclei, is used to assess both nuclear and filament alignment of cardiomyocytes as well as gap junction expression and intracellular location between all groups (Figure 1B). In order to assess electrophysiological function and quantify conduction velocity, all bioelectric threads will be optical mapped to compare maximum capture rate, action potential duration, and conduction velocity.

Results, Conclusions, and Discussions: Data collection is still ongoing, however, preliminary data comparing static stretch culture for two weeks to no stretch groups demonstrates an increase in gap junction expression via cx43 quantity, actin filament organization, and sarcomere development. Pulsatile stretch is expected to further align cardiomyocyte actin filaments and nuclei, with point stimulation polarizing gap junction expression to cell ends. Subsequently, conduction velocity and maximum capture rate are expected to increase with both mechanical and electrical defined-axis stimuli. This, combined with our previous work demonstrating electrical coupling of bioelectric threads and engineered cardiac tissues, has the potential to facilitate electrical coupling between host myocardium and engineered tissues, an obstacle currently undercutting therapeutic potential of all engineered cardiac tissues both in research and clinical trials.

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