Selectively expressing SARS-CoV-2 Spike protein S1 subunit in cardiomyocytes induces cardiac hypertrophy in mice

Abstract

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. For example, CoV-2 Spike glycoprotein (CoV-2-S) not only engages ACE2 to mediate virus infection, but also directly impairs endothelial function and can trigger innate immune responses in cultured murine macrophages. Here we tested the hypothesis that CoV-2-S damages the heart by activating cardiomyocyte (CM) innate immune responses. 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. We found that CoV-2-S and not NL63-S interacted with Toll-like receptor 4 (TLR4), a crucial pattern recognition receptor that 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 a construct that expresses membrane-localized CoV-2-S1 (S1-TM). AAV9-mediated, selective expression of the S1-TM in CMs caused heart dysfunction, induced hypertrophic remodeling, and elicited cardiac inflammation. Since CoV-2-S does not interact with murine ACE2, our study presents a novel ACE2-independent pathological role of CoV-2-S, and suggests that the circulating CoV-2-S1 is a TLR4-recognizable alarmin that may harm the CMs by triggering their innate immune responses.

Vaccination of elite athletes against SARS-CoV-2 and broader implications for public health policy

“…Isolated SARS--CoV--2 spike proteins and/or isolated spike protein constituent components, such as S1, have been observed to exert a profound and multifaceted molecular toxicity within a wide range of cell and tissue types (4,5,9,10,13,14,15,19). Manifestations of the molecular toxicity inherent to isolated SARS--CoV--2 spike proteins and/or isolated spike protein constituent components, such as S1, include, but are not limited to: lung and endothelial cell damage (5,8), thickening of the alveolar septa (5), increased tissue infiltration of mononuclear cells (5), impairment of acetylcholine--induced, endothelium--dependent vasodilation (5), altered mitochondrial dynamics, increased mitochondrial fragmentation, impaired mitochondrial function & compensatory increases in glycolysis--(also known as metabolic reprogramming) (5), down--regulation of angiotensin converting enzyme 2 (hACE2) (3,4,5,7,8,112), up--regulation of angiotensin converting enzyme (hACE) (3,4,5,7,8,112), increased levels of angiotensin II (3,4,5,7,8,112), increased levels of reactive oxygen species (ROS) generation and accompanying oxidative stress (3,4,5,7,8,112), increased platelet aggregation in response to agonists such as adenosine diphosphate (ADP) (4), increased platelet dense granule secretion in response to agonists such as ADP(4), induction of platelet integrin αIIbβ3 activation, induction of platelet P-selectin expression, markedly accelerated spreading of platelets across fibrinogen--coated surfaces (4), acceleration of clot retraction upon exposure to agonists such as ADP (4), increased thrombus formation (4,22,…”

“…Optimized mRNA genetic templates, such as demonstrated by BNT--162b2 and mRNA--1273, compel the self--synthesis of SARS--CoV--2 spike proteins far more effectively than wild--type SARS--CoV--2 genomes encoding the SARS--CoV--2 spike protein (75). 3) SARS--CoV--2 spike proteins and constituent components of SARS--CoV--2 spike proteins, such as S1, demonstrate a profound and multifaceted molecular toxicity, independent of SARS--CoV--2 infection (4,5,9,10,13,14,15,19). 4) SARS--CoV--2 nucleotide vaccine--induced, self--generated spike proteins are subject to enzymatic cleavage and are not constrained to a trans--membrane anchored existence at the site of synthesis (136).…”

“…(1.1) Introduction SARS--CoV--2, a human β--coronavirus implicated as the causative agent in the COVID--19 pandemic is one of seven coronaviruses known to infect humans (1,10). MERS--CoV and SARS--CoV--1 comprise β-coronaviruses known to cause severe respiratory disease in humans, while SARS--CoV--2 infection has been widely associated with the onset of atypical--severe acute respiratory distress syndrome (ARDS) (1,2).…”

“…The interaction between hACE2 and isolated SARS--CoV--2 spike proteins occurs with the spike proteins existing in the pre--fusion conformation (1,3,4,5,9,12). The RBD of isolated SARS--CoV--2 spike proteins or isolated S1, upon interacting with hACE2, induces a number of molecular signaling cascades, the nature of which may vary depending on the cell and/or tissue type expressing hACE2 (1,3,4,5,10,12). The binding of hACE2 with the RBD of isolated SARS--CoV--2 spike proteins and/or isolated S1 culminates in the observed tissue--specific molecular toxicity associated with isolated spike proteins and isolated S1 (1,3,4,5,10,12).…”

“…platelet secretion of α--granule inflammatory cytokines: PF4, TNF--α, IL--8 , and IL--1β (4), increased formation of leukocyte-platelet aggregates (CD45+, CD41+) (4), increased formation of monocyteplatelet aggregates (CD14+ CD41+) (4), increased formation of neutrophil--platelet aggregates (CD65+ CD41+) (4), increased formation of neutrophil extracellular trap (NET) via platelet IL--8 secretion (4,25), induction of an inflammatory secretome by cardiac pericytes which resembles the phenomena of cytokine storm and includes: TNFα, IL--6, MCP1, and IL--1β (7,9), activation of Toll--like receptor 4(TLR4)/myeloid differentiation factor--2(MD--2) signaling in cardiomyocytes (10), activation of NF--kB signaling in cardiomyocytes subsequent to TLR4/MD--2 signaling (10), cardiac inflammation, cardiac hypertrophy and reduced cardiac systolic function (10), induction of hyper-inflammatory responses in human macrophages (13,14), induction of the NLRP3 inflammasome/caspase--1 pathway in human peripheral blood mononuclear cells (10,15), induction of the p38 MAPK pathway human peripheral blood mononuclear cells (10,15), induction of the NF--κB pathway in human peripheral blood mononuclear cells (10,15), exaggerated production of TNFα, IL--6, IL--1β and IL--8 by human peripheral blood mononuclear cells (10,15), significant degradation of blood-brain barrier properties (19), a loss of blood--brain barrier integrity (19), induction of pro-inflammatory cascades in cerebral endothelial cells (19), induction of immune infiltration into the CNS (19), up--regulation of cerebral endothelial cell matrix metalloproteinase expression (19,113), induction of micro--thrombi in the vasculature of the CNS (114), onset of acute hemorrhagic necrotizing encephalopathy (115,116,117), seeding of toxic neural protein aggregates…”