Title: Matured engineered human cardiac tissues to study autoimmune myocarditis
Description: Antibodies to tropomyosin, cardiac troponin I, myosin, and the beta-adrenergic receptors have been implicated in myocarditis, dilated cardiomyopathy, and heart failure. However, in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), there are only a few studies on how autoantibodies play a role in autoimmune mediated heart disease, despite the prevalence of these conditions. Ro52 antibodies have been implicated in fetal heart block, but their role in adult myocarditis remains elusive. In this study, we look beyond Ro52 and characterized the relevant autoantibodies in adult patients with SLE and RA myocarditis. An optimized immunoprecipitation followed by liquid chromatography mass spectrometry methodology was performed to determine putative auto-antigens in the human heart. The quantity and specificity of auto-antibodies was correlated with clinical measures of myocardial cellular infiltration, as determined by fluorodeoxyglucose (FDG)-positron emission tomography (PET) in patients with SLE and RA.

We created autoantibody profiles that are complimentary to SLE and RA patients' clinical profile. Autoantibodies that correlated with cellular infiltration included TPI1, TPM1, MYL2, XRCC6 and APOA4. We then explored methodologies for testing patient autoantibodies using engineered cardiac tissues derived from human induced pluripotent stem cells (iPSCs). These tissues are increasingly used for drug discovery, pharmacology and in models of development and disease. While there are numerous platforms with engineered cardiac tissues, they often require expensive and non-conventional equipment and utilize complex video processing algorithms. As a result, only specialized academic labs have been able to harness this technology. In addition, methodologies and tissue features have been challenging to reproduce between different groups and models. Here, we describe a facile technology (milliPillar) that covers the entire pipeline required for studies of engineered cardiac tissues: (i) platform fabrication, (ii) cardiac tissue generation, (iii) electrical stimulation, (iv) automated real-time data acquisition, and (v) advanced video analyses.

We validate these methodologies and demonstrate the versatility of the platform by showcasing the fabrication of tissues in different hydrogel materials and by using cardiomyocytes derived from different iPSC lines in combination with different types of stromal cells. We also validate the long-term culture (100 days) of tissues within the platform and provide protocols for automated analysis of force generation and calcium flux using both brightfield and fluorescent imaging. Lastly, we demonstrate the compatibility of the milliPillar platform with electromechanical stimulation to enhance cardiac tissue function. milliPillar tissues were cultured in the presence of patient autoantibodies to recapitulate the phenotype of myocardial disease, and the calcium transients and force generation were measured. Our results indicated that milliPillar tissues exhibited a decrease in force generation after 6 days in culture with SLE autoantibodies. Separately, our results indicated a prolonged calcium transient after 7 days in culture with SLE and RA autoantibodies.

Changes to the downstroke of the calcium transient correlated most with patients’ autoantibody profiles and cellular infiltration. We confirmed autoantibody binding to live tissues/cells in 25% of the patients with SLE and myocarditis. Finally, we used changes in cardiac tissue function in the presence of autoantibodies to classify patients with SLE myocarditis with an accuracy of 87.5%.