The Macrophage Landscape Across the Lifespan of a Human Cardiac Allograft.

BACKGROUND: Much of our knowledge of organ rejection after transplantation is derived from rodent models. METHODS: We used single-nucleus RNA sequencing to investigate the inflammatory myocardial microenvironment in human pediatric cardiac allografts at different stages after transplantation. We distinguished donor- from recipient-derived cells using naturally occurring genetic variants embedded in single-nucleus RNA sequencing data. RESULTS: Donor-derived tissue resident macrophages, which accompany the allograft into the recipient, are lost over time after transplantation. In contrast, monocyte-derived macrophages from the recipient populate the heart within days after transplantation and form 2 macrophage populations: recipient MP1 and recipient MP2. Recipient MP2s have cell signatures similar to donor-derived resident macrophages; however, they lack signatures of pro-reparative phagocytic activity typical of donor-derived resident macrophages and instead express profibrotic genes. In contrast, recipient MP1s express genes consistent with hallmarks of cellular rejection. Our data suggest that recipient MP1s activate a subset of natural killer cells, turning them into a cytotoxic cell population through feed-forward signaling between recipient MP1s and natural killer cells. CONCLUSIONS: Our findings reveal an imbalance of donor-derived and recipient-derived macrophages in the pediatric cardiac allograft that contributes to allograft failure.

Hippo-Yap Signaling Maintains Sinoatrial Node Homeostasis.

BACKGROUND: The sinoatrial node (SAN) functions as the pacemaker of the heart, initiating rhythmic heartbeats. Despite its importance, the SAN is one of the most poorly understood cardiac entities because of its small size and complex composition and function. The Hippo signaling pathway is a molecular signaling pathway fundamental to heart development and regeneration. Although abnormalities of the Hippo pathway are associated with cardiac arrhythmias in human patients, the role of this pathway in the SAN is unknown. METHODS: We investigated key regulators of the Hippo pathway in SAN pacemaker cells by conditionally inactivating the Hippo signaling kinases Lats1 and Lats2 using the tamoxifen-inducible, cardiac conduction system-specific Cre driver Hcn4CreERT2 with Lats1 and Lats2 conditional knockout alleles. In addition, the Hippo-signaling effectors Yap and Taz were conditionally inactivated in the SAN. To determine the function of Hippo signaling in the SAN and other cardiac conduction system components, we conducted a series of physiological and molecular experiments, including telemetry ECG recording, echocardiography, Masson Trichrome staining, calcium imaging, immunostaining, RNAscope, cleavage under targets and tagmentation sequencing using antibodies against Yap1 or H3K4me3, quantitative real-time polymerase chain reaction, and Western blotting. We also performed comprehensive bioinformatics analyses of various datasets. RESULTS: We found that Lats1/2 inactivation caused severe sinus node dysfunction. Compared with the controls, Lats1/2 conditional knockout mutants exhibited dysregulated calcium handling and increased fibrosis in the SAN, indicating that Lats1/2 function through both cell-autonomous and non-cell-autonomous mechanisms. It is notable that the Lats1/2 conditional knockout phenotype was rescued by genetic deletion of Yap and Taz in the cardiac conduction system. These rescued mice had normal sinus rhythm and reduced fibrosis of the SAN, indicating that Lats1/2 function through Yap and Taz. Cleavage Under Targets and Tagmentation sequencing data showed that Yap potentially regulates genes critical for calcium homeostasis such as Ryr2 and genes encoding paracrine factors important in intercellular communication and fibrosis induction such as Tgfb1 and Tgfb3. Consistent with this, Lats1/2 conditional knockout mutants had decreased Ryr2 expression and increased Tgfb1 and Tgfb3 expression compared with control mice. CONCLUSIONS: We reveal, for the first time to our knowledge, that the canonical Hippo-Yap pathway plays a pivotal role in maintaining SAN homeostasis.