R6 - Using iPSC derived engineered heart tissues to model cardiac injury following streptococcus pneumonia infection

Introduction: : Beyond the impact of acute infection, cases of severe community-acquired pneumonia (CAP) can be followed by severe complications, including cardiac disease. Nearly 30% of adults hospitalized with CAP experience a major adverse cardiac events within 30 days of admission. After recovery patients hospitalized for pneumonia also have an estimated 2-fold risk of adverse cardiac events for the following 10 years. We aim to understand the mechanisms by which pneumonia affects the long-term risk of cardiovascular diseases by modeling acute and long-term infection in an in vitro system. Streptococcus pneumoniae (Spn), is a Gram-positive bacterium and the leading cause of CAP, can invade the heart, invade and kill 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. To address these challenges, we have developed a cardiac tissue chip (CTC) platform to comprehensively evaluate post-infection events.

Materials and

Methods: : To generate 3D cardiac organoids, we produce ~300 um diameter iPSC spheroids using custom microwell inserts. These are differentiated in a multi-well plate, kept in motion on a linear shaker, through a three-phase modulation of Wnt signaling in a modified implementation of previously described protocols. Separately, molds for the CTC are prepared by bench top photolithography. These are then 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, bacteria suspension is added to the CTCs for 3 hours. The CTCs are then thoroughly washed and treated with gentamicin supplemented medium for two hours. These steps eliminate extracellular and planktonic bacteria and insure that the initial inoculum necessary to infect the CTC does not overwhelm the culture. 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 and beat amplitude (via bright field videos), action potential propagation (via voltage sensitive dyes), and fix tissues for immunofluorescent staining. We also collected supernatant medium to assess cytokine secretion.

Results, Conclusions, and Discussions:: Our platform achieves stable infection of functional cardiac tissues for over 72 hours with no visible overgrowth. We have successfully observed small clusters of bacteria within the tissue, which emulated the microlesions observed in vivo (Fig 1C). Our analysis of cytokine secretion from the tissue chips showed that infection drove increased expression of cytokines involved in immune cell recruitment, immune modulation, inflammation and apoptosis, and neutrophil attraction (Fig 1D). Our model was also able to replicate findings from in vitro mouse studies which identified that S. pneumoniae with a deficiency in zmpB, the gene encoding Zinc metalloprotease B (ZmpB), caused decreased cardiac invasion and injury. In our studies, we inoculated CTC’s with TIGR4 wild type (WT) or ZmpB deficient bacteria. We observed decreased bacteria invasion in the mutant. When we tracked the same tissues over time, we observed that the beat rate (relative to time 0) was lower in the wild type compared to the mutant, consistent with mutant causing less cardiac damage (Fig 1E). These data show how this engineered heart tissue model can compliment in vivo studies by replicating microlesion formation and allowing for functional measurements on human tissue to validate animal study findings and elucidate the mechanisms involved in this process.

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