Poster T18 - Development of an in vitro cardiac model to dissect the mechanisms of cardiac tissue injury following streptococcus pneumonia infection

Introduction: In community-acquired pneumonia (CAP), Beyond the impact of acute infection, CAP can be followed by severe complications, including cardiac diseases. Nearly 30% of adults hospitalized with CAP go on to develop adverse cardiac events within 30 days of admission, and after recovery these patients have an estimated 2-fold risk for the following 10 years. We aim to understand the mechanisms by which pneumonia affects the long-term risk of CVD. The leading cause of CAP is Streptococcus pneumoniae (Spn). Spn can invade the heart, enter cardiomyocytes, and eventually cause cardiac dysfunction. In animal models it is difficult to evaluate the molecular, cellular, and functional changes associated with infection with relevant spatial and temporal resolution. Advances in human induced pluripotent stem cell (hiPSC) technologies have expanded the possibilities of cell culture-based cardiomyocyte studies, however 2D monolayers models do not replicate 3D tissue structure or allow for creation of the distinct microlesions observed in vivo during infection. We are developing a cardiac tissues chip (CTC) platform, to comprehensively evaluate post-infection events and test the hypothesis that autophagy protects cardiomyocytes against intracellular bacteria and helps sustain cardiac function and Spn infection.

Materials and

Methods: To generate 3D cardiac organoids, we first produce ~300 um diameter iPSC spheroids using custom microwell inserts. These are differentiated in a multi-well plate, while kept in constant motion on a linear shaker to prevent further aggregation, through a three-phase modulation of Wnt signaling in a modified implementation of previously described protocols. Separately, molds for the CTC are prepared by photolithography which are cast in PDMS. The current design is a 7 x 4 array of 1 mm x 0.5 mm x 0.5 mm rectangular wells which are bonded to a thin membrane. The cardiac organoids are seeded into the wells, resulting in an array of millimeter scale tissues (Fig 1A,B). For inoculation, the CTCs are placed in a 3D printed chamber attached to a peristaltic pump (Fig 1C). Spn stock is thawed at room temperature, diluted in cardiac medium, and perfused into the CTC for 3 hours. The CTCs are then perfused with gentamicin supplemented medium for two hours, which eliminates extracellular and planktonic bacteria. The medium is replaced with antimicrobial free medium, and culture is continued under perfusion. We collect functional readouts from the tissues over time including beat rate, beat amplitude, action potential propagation (via voltage sensitive dyes), and fix tissues for immunofluorescent staining.

Results, Conclusions, and Discussions: Our platform achieves stable infection of functional cardiac tissues for over 72 hours with no visible overgrowth. We have observed small clusters of bacteria within the tissue, like the microlesions observed in vivo (Fig 1D). We confirmed that at 72 hours, infection results in increased cell death. In experiments where tissues were treated with the autophagy inhibitor bafilomycin A (Bafi), we saw increased overall cell cytotoxicity. Here, we also tracked the function of individual organoids w at each timepoint. Most infected organoids ceased contractility by 48 hours. We normalized the beat rates at each timepoint against that from time 0 (pre-infection) and observed that autophagy inhibition (Bafi treatment) resulted in decreased normalized beat rate (Fig1 E). We performed a similar analysis based on the amplitude of motion extracted from the videos. We similarly saw a decrease in normalized motion amplitude in the autophagy inhibited samples (Fig1 F).

Acknowledgements (Optional):