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Refereed/Peer-reviewed Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor β (PDGFRβ) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models.

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory ‘cytokine-storm’, a cocktail of interferon gamma, interleukin 1β and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids and hearts of SARS-CoV-2 infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCO and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the FDA breakthrough designated drug apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage., A combination of phosphoproteomics, drug screening and single-cell sequencing approaches identifies how cytokines elevated in COVID-19 patients drives cardiac dysfunction, with BET inhibitors serving as potential lead candidates decrease ACE2 cardiac expression and infection.

SUMMARYCardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory ‘cytokine-storm’, a cocktail of interferon gamma, interleukin 1β and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids and hearts of SARS-CoV-2 infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCO and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the FDA breakthrough designated drug apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or ‘cytokine-storm’ induced dysfunction. To identify mechanisms and discover cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with high throughput phosphoproteomics and single nuclei RNA sequencing. We identify that ‘cytokine-storm’ induced diastolic dysfunction can be caused by a cocktail of interferon gamma, interleukin 1β and poly(I:C) and also human COVID-19 serum. Bromodomain protein 4 (BRD4) is activated along with pathology driving fibrotic and induced nitric oxide synthase genes. BRD inhibitors fully recover function in hCO and totally prevent death in a cytokine-storm mouse model. BRD inhibition decreases transcription of multiple genes, including fibrotic, induced nitric oxide synthase and ACE2, and prevention of cardiac infection with SARS-CoV2. Thus, BRD inhibitors are promising candidates to prevent COVID-19 mediated cardiac damage. Funding: We acknowledge grant and fellowship support from the National Health and Medical Research Council of Australia (J.E.H., M.J.S., C.R.E., T.B.), Heart Foundation of Australia (J.E.H.), QIMR Berghofer Medical Research Institute (J.E.H.), The Stafford Fox Foundation (E.R.P.), the Royal Children’s Hospital Foundation (E.R.P.), Australian Research Council Strategic Initiative in Stem Cell Science (Stem Cells Australia) (E.R.P. and J.E.H.) and the Medical Research Future Fund (MRFF9200008) (J.E.H., T.B., M.J.S., K.P.A.MD., C.R.E., E.R.P.). M.J.S. is supported by Health and Medical Research Council of Australia Program (APP1132519) and Investigator (APP1173958) grants. A.S. is also supported by Investigator grant (APP1173880). The Murdoch Children’s Research Institute is supported by the Victorian Government’s Operational Infrastructure Support Program. This project received support from Dynomics Inc. J.E.H. is supported by a Snow Medical Fellowship. Conflict of Interest: R.J.M., J.E.H., G.A.Q.-R., D.M.T. and E.R.P. are listed as co-inventors on pending patents held by The University of Queensland and QIMR Berghofer Medical Research Institute that relate to cardiac organoid maturation and putative cardiac regeneration therapeutics. J.E.H. is a coinventor on licensed patents held by the University of Goettingen. R.J.M, E.R.P., D.M.T., B.G. and J.E.H. are co-founders, scientific advisors and stockholders in Dynomics Inc. D.M.T. and B.G. are employees of Dynomics Inc. /Dynomics Pty Ltd. QIMR Berghofer Medical Research Institute has filed a patent on the use of BRD inhibitors. Ethical Approval: Animal work was approved by the QIMR Berghofer Medical Research Institute Animal Ethics Committee. Ethical approval for the use of human embryonic stem cells (hESCs) was obtained from QIMR Berghofer’s Ethics Committee and was carried out in accordance with the National Health and Medical Research Council of Australia (NHMRC) regulations. Procedures complied with standards set under Australian guidelines for animal welfare and experiments were subject to Monash University animal welfare ethics review (Approval #MARP/2019/13606).