Congenital heart disease is one of the leading causes of pediatric morbidity and mortality, thus highlighting the importance of deciphering the molecular mechanisms that control heart development. As the terminal transcriptional effectors of the Hippo–YAP pathway, YAP and TEAD1 form a transcriptional complex that regulates the target gene expression and depletes either of these two genes in cardiomyocytes, thus resulting in cardiac hypoplasia. Vestigial-like 4 (VGLL4) is a transcriptional co-factor that interacts with TEAD and suppresses the YAP/TEAD complex by competing against YAP for TEAD binding. To understand the VGLL4 function in the heart, we generated two VGLL4 loss-of-function mouse lines: a germline Vgll4 depletion allele and a cardiomyocyte-specific Vgll4 depletion allele. The whole-body deletion of Vgll4 caused defective embryo development and perinatal lethality. The analysis of the embryos at day 16.5 revealed that Vgll4 knockout embryos had reduced body size, malformed tricuspid valves, and normal myocardium. Few whole-body Vgll4 knockout pups could survive up to 10 days, and none of them showed body weight gain. In contrast to the whole-body Vgll4 knockout mutants, cardiomyocyte-specific Vgll4 knockout mice had no noticeable heart growth defects and had normal heart function. In summary, our data suggest that VGLL4 is required for embryo development but dispensable for myocardial growth.

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Cardiac injury is common in hospitalized COVID-19 patients and portends poorer prognosis and higher mortality. To better understand how SARS-CoV-2 (CoV-2) damages the heart, it is critical to elucidate the biology of CoV-2 encoded proteins, each of which may play multiple pathological roles. HCoV-NL63 is another human coronavirus with a Spike protein (NL63-S) that also engages ACE2 for virus entry but is known to only cause moderate respiratory symptoms. By comparing the pathological effects of NL63-S and CoV-2 spike protein (CoV-2-S), we tested the hypothesis that CoV-2-S damages the heart by activating cardiomyocyte (CM) innate immune responses, which is independent of ACE2.We found that CoV-2-S and not NL63-S interacted with Toll-like receptor 4 (TLR4), a crucial pattern recognition receptor responsible for detecting pathogen and initiating innate immune responses. Our data show that the S1 subunit of CoV-2-S (CoV-2-S1) interacts with the extracellular leucine-rich repeats-containing domain of TLR4 and activates NF-kB. To investigate the possible pathological role of CoV-2-S1 in the heart, we generated constructs that express membrane-localized CoV-2-S1 (S1-TM) or HCoV-NL63-S1 (NLS1-TM). AAV9-mediated, selective expression of the S1-TM and not NLS1-TM in CMs caused heart dysfunction, induced hypertrophic remodeling, and elicited cardiac inflammation. In vitro, overexpressing S1-TM in cultured neonatal rat ventricular CMs induced hypertrophy, decreased Myh6 and increased IL-6 . In the ventricular biopsy of a deceased patient with COVID-19 associated myocarditis, Spike protein and TLR4 were detected in both CMs and non-CMs, and these two proteins were absent in a healthy pre-pandemic human heart. In the ventricular biopsy of a patient who died of modified RNA vaccine-associated myocarditis, the expression of Spike protein was only detected in a small fraction of CMs, and no obvious TLR4 signal was detected in CMs. Because CoV-2-S does not interact with murine ACE2, our study presents a novel ACE2-independent pathological role of CoV-2-S. Additionally, our data suggest that modified RNA vaccine unlikely causes myocarditis by directly transfecting the CMs.