Project Summary: Acute Cardiac Injuries (ACIs) occur in up to 20-25% of hospitalized COVID-19 patients and are associated with increased risks of morbidity and mortality. COVID-19 has been shown to induce immune system “overfiring” and “misfiring”, resulting in supraphysiological inflammation. Such cytokine storms have been shown to lead to organ damage through both direct cytokine insults and indirect mechanisms (e.g., recruitment of proinflammatory immune cells into organs). Myocarditis (e.g., monocyte infiltration) is a common complication in COVID-19 ACI hearts. However, the impacts of the infiltrated monocytes on COVID-19 ACI hearts remain under-defined. As previous studies have been limited to either clinical samples (e.g., peripheral blood, autopsy/biopsy tissues) or in vitro 2D co-culture experiments, these studies yield limited functional insights. 3D organotypic models provide a powerful platform to study the functional interactions between hearts and immune cells in circulation. To develop an organotypic model of human hearts for disease modeling, we developed 3D human cardiac organoids composed of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), human cardiac fibroblasts, endothelial cells, and stromal cells. Our preliminary data showed the Interleukin (IL)-1b treated organoids recapitulated key features of transcriptome, structure, and function of COVID-19 ACI hearts. As IL-1b is not a COVID-19 specific stimulus, these results laid the foundation to use serum from COVID-19 ACI patients to recapitulate the COVID-19 cytokine insults to the hearts. In addition, the exclusion of immune cells in the organoids has limited the full recapitulation of hyperinflammation in COVID-19 ACI hearts. The goal of this proposal is to leverage the serum and peripheral blood mononuclear cells (PMBCs) harvested from COVID-19 ACI patients to 1) simulate the effects of COVID-19 cytokine insults on human hearts, 2) develop the first in vitro COVID-19 myocarditis organoid model to simulate immune cell infiltration to COVID-19 ACI hearts and assess the infiltrated COVID-19 monocytes. The central hypothesis of this proposal is that infiltrated COVID-19 immune cells (e.g., monocytes) exacerbate the hyperinflammation of COVID-19 ACI hearts. This proposal is innovative in that it will, for the first time, develop an organotypic model of myocarditis to simulate immune cell infiltration into myocardium. Accordingly, we will pursue the following 2 Aims: 1) Determine the effects of COVID-19 ACI serum on human cardiac organoids, and 2) Determine the ability of COVID-19 ACI serum treated organoids to recruit immune cells from COVID-19 ACI patients. The proposed studies will lead to follow-up R01 applications focusing on the effects and mechanisms of the infiltrated monocytes in COVID-19 ACI hearts. In addition, the 3D organotypic model of myocarditis can be used for mechanistic studies of the effects of immunomodulatory drugs on hearts.