key: cord-0818917-0q15djph
authors: Tangos, Melina; Budde, Heidi; Kolijn, Detmar; Sieme, Marcel; Zhazykbayeva, Saltanat; Lódi, Mária; Herwig, Melissa; Gömöri, Kamilla; Hassoun, Roua; Robinson, Emma Louise; Meister, Toni Luise; Jaquet, Kornelia; Kovács, Árpád; Mustroph, Julian; Evert, Katja; Babel, Nina; Miklós, Fagyas; Lindner, Diana; Püschel, Klaus; Westermann, Dirk; Mannherz, Hans Georg; Paneni, Francesco; Pfaender, Stephanie; Toth, Attila; Mügge, Andreas; Sossalla, Samuel; Hamdani, Nazha
title: SARS-CoV-2 infects human cardiac myocytes promoted by inflammation and oxidative stress
date: 2022-05-26
journal: Int J Cardiol
DOI: 10.1016/j.ijcard.2022.05.055
sha: 96321b3335a32f0331e5eb9fbbee292b6455e431
doc_id: 818917
cord_uid: 0q15djph

Introduction The respiratory illness triggered by severe acute respiratory syndrome virus-2 (SARS-CoV-2) is often particularly serious or fatal amongst patients with pre-existing heart conditions. Although the mechanisms underlying SARS-CoV-2-related cardiac damage remain elusive, inflammation (i.e. ‘cytokine storm’) and oxidative stress are likely involved. Methods and Results: Here we sought to determine: 1) if cardiomyocytes are targeted by SARS-CoV-2 and 2) how inflammation and oxidative stress promote the viral entry into cardiac cells. We analysed pro-inflammatory and oxidative stress and its impact on virus entry and virus-associated cardiac damage from SARS-CoV-2 infected patients and compared it to left ventricular myocardial tissues obtained from non-infected transplanted hearts either from end stage heart failure or non-failing hearts (donor group). We found that neuropilin-1 potentiates SARS-CoV-2 entry into human cardiomyocytes, a phenomenon driven by inflammatory and oxidant signals. These changes accounted for increased proteases activity and apoptotic markers thus leading to cell damage and apoptosis. Conclusion This study provides new insights into the mechanisms of SARS-CoV-2 entry into the heart and defines promising targets for antiviral interventions for COVID-19 patients with pre-existing heart conditions or patients with co-morbidities.

Although cardiovascular co-morbidities play a prominent role in SARS-CoV-2 infection and existing cardiovascular diseases are associated with poor outcomes, the relationship between SARS-CoV-2 and the cardiovascular system is still poorly understood [1] [2] [3] [4] . A study of 273 COVID-19 patients for markers of cardiac injury at hospital admission found myoglobin elevation in 10.6%, high-sensitivity troponin I in 9.9% and NT-proBNP elevation in 12.5% of patients. Death occurred in 24 patients (8.8%), and correlated significantly with the presence of cardiac injury at admission [2] . Furthermore, a substantial percentage of patients infected with COVID-19 (approx. 7-20%) develop additional cardiac injury [1, 3, 5] .

SARS-CoV-2 is thought to enter a cell via interaction of the viral spike (S) protein with the ACE2 cell surface receptor, initiating membrane fusion followed by uncoating and viral replication [6, 7] . Intriguingly, despite the relatively low level of ACE2 expression in the heart [8] , cardiovascular complications are frequent and often dramatic in COVID-19 patients. Post-mortem studies have found SARS-CoV-2 in myocardial tissues, demonstrating entry of the virus despite low ACE2 levels [9] . Therefore and based on the low ACE2 expression in the heart, we believe other mechanisms are involved. The mechanisms of cardiac damage due to SARS-CoV-2 infection remain speculative, but ongoing inflammation, a 'cytokine storm' and oxidative stress are possible contributory factors. Inflammatory signals attract leukocytes to a site of injury or infection and leukocytes (neutrophils and macrophages) respond by producing numerous reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion and others. Advanced age, cardiovascular risk factors and heart diseases predispose to heart failure (HF) and share a common environment of pro-inflammatory signals and oxidative stress within cardiac cells [10] [11] [12] . Epidemiological studies from China show that approximately 20% of COVID-19 patients have developed cardiovascular diseases and are more likely life threatening complications in the course of infection [13] . Even more, some patients have developed myocarditis, a cardiac disorder characterized by inflammatory cell infiltration of the heart and greater risk of deterioration of cardiac function [14] , indicating SARS-CoV2associated myocarditis or "myocarditis-like syndromes". Neutrophils express and release cytokines, which in turn amplify inflammatory reactions by several other cell types. In addition to employing and activating other cells of the immune system, neutrophils play an important role in the defence against invading pathogens.

Human neutrophils express Toll-like receptor (TLR2) and (TLR4) protein on the cell surface and both receptors regulate neutrophil activation and its life span [15] . In addition, neuropilin-1 (NPR-1) functions as an endogenous negative modulator of the TLR4-NFkB pathway and facilitates together with the receptor for advanced glycation end products (RAGE) SARS-CoV-2 cell entry and infectivity [16, 17] .

We hypothesize that oxidative stress and a pro-inflammatory environment exacerbates SARS-CoV-2-associated damage via an yet unknown mechanism.

We therefore investigated 1) whether SARS-CoV-2 is found in the heart and if cardiomyocytes are targeted by SARS-CoV-2 and 2) how inflammation and oxidative stress promote the viral entry into cardiac cells.

Furthermore, using cardiac tissue samples from SARS-CoV-2 infected patients, we analysed pro-inflammatory and oxidative stress [10] [11] [12] and its impact on virus entry and virus-associated cardiac damage.

A detailed description of methods is provided in the online supplementary methods.

All details related to the patient's characteristics are included either in the extended supplementary section or in the results section.

Whole transcriptome sequencing was performed for each sample. For details, see supplementary methods section.

Frozen LV slides were fixed, blocked and either single-or dual-stained with various antibodies, followed by appropriate secondary antibodies. Immunostained samples were analysed using confocal laser scanning microscopy [10, 12, 18] .

Left ventricle (LV) myocardial tissues were obtained from patients with end-stage HF (NYHA III-IV) (n=14; 8 males, 6 females; mean age 62±2 years) characterized by LV dilation, LV systolic dysfunction and with ejection fractions of 25 ± 2.8% suffering from hypertension and/or diabetes mellitus. Healthy LV myocardium (n=14) was obtained from healthy donor hearts that were not transplanted due to technical reasons (n=14; 8 males, 6 females mean age 58±6 years). Furthermore, we included 59 deceased patients with SARS-CoV-2 infection confirmed by qRT-PCR. All SARS-CoV-2 patients selected in this study were sorted according to their virus load within the myocardium and we have selected patients with a viral load above 1000 copies per μg RNA and the average virus load per μg RNA was 26530 ± 12432. These patients had high cardiovascular risk or established cardiovascular disease and were subsequently referred to as "SARS-CoV-2 patients". The majority of patients died as a result of pneumonia/acute respiratory distress syndrome or multi-organ failure. We also employed other autopsy hearts without SARS-CoV-2 infection to confirm that the alterations found in SARS-CoV-2 infected patients are due to the virus infection rather than the storage conditions of the tissues. Our findings suggested that the alteration maps in autopsy hearts without SARS-CoV-2 infection are similar to the alterations found in tissues from HF and donors, but different from SARS-CoV-2 infected patients, indicating that our molecular and functional findings in SARS-CoV-2 infected patients are mainly a consequence of the viral infection (Fig.S1 ).

We first assessed the effect of SARS-CoV-2 infection on cardiomyocyte function. Representative images ( Fig.1 .A-C) show skinned cardiomyocytes and accompanying original recordings of force responses to stepwise cell stretching (sarcomere length (SL) 1.8-2.3μm) in relaxing buffer and activating buffer. Ca 2+ -activated tension generated by a single cardiomyocyte was severely reduced in SARS-CoV-2 patients compared to HF patients and the healthy donor group. Remarkably, only around 10% of all SARS-CoV-2 cardiomyocytes showed any force development and those that did showed an almost 75% reduction compared to HF patients and the donor group ( Fig.1.B) . Accordingly, the rate constant of force redevelopment (ktr) was significantly lower in the remaining functional SARS-CoV-2 patient cardiomyocytes (0.10±0,0012s -1 ) compared to HF patients (0.56±0,002s -1 ) and the donor group cardiomyocytes (0.59±0,002s -1 ). Considerable degradation of cardiac myosin binding protein C (cMyBPC) and cardiac troponin I (cTnI) was in fact observed ( Fig.1 .F-G). In addition, the passive sarcomere length-tension relationship in isolated skinned cardiomyocytes from SARS-CoV-2 patients J o u r n a l P r e -p r o o f was higher compared to HF patients and the donor group ( Fig.1 .C). This may relate to high titin degradation [10, 12, [18] [19] [20] [21] [22] [23] [24] [25] , as demonstrated by Coomassie blue staining and Western blotting with specific anti-titin antibodies 

Oxidative stress can activate a range of transcription factors, which lead to the differential expression of genes involved in inflammatory pathways. The inflammation triggered by oxidative stress is the cause of many chronic diseases [11, 12, 25] . Proteases are known to be central in the inflammation process. One potential effect of SARS-CoV-2 infection on cardiomyocytes is the activation of proteolytic enzymes ( [26, 27] . Many viruses, including SARS-CoV-2, enter host cells via cleavage and activation of the S-protein by host proteases [28, 29] , and membrane fusion is dependent on proteolysis of the S-protein by host cathepsin L, as shown for SARS-CoV-1 [30, 31] . We therefore assessed the possible colocalization of spike protein with cathepsin in the heart using Duolink staining, which is a proximity ligation assay (PLA) that monitors protein-protein interaction for ranges up to 40 nm. Duolink staining showed that cathepsin and spike protein do indeed interact ( Fig.2 .E), thus suggesting that cathepsin inhibition may be a viable target in the treatment of SARS-CoV-2. The transmembrane serine protease 2 (TMPRSS2) gene level was higher in SARS-CoV-2 patients compared to non-failing hearts ( Fig.S3 .D).

Proteolytic enzymes and proteins that cause apoptosis include Bcl2-associated X (BAX) (an indicator of mitochondrial damage) and nuclear factor of activated T-cells (NFAT). We found increased expression and activity of both these proteins ( Fig.2 .G,I,F,H). Increased NFAT activity may indicate inhibition of the innate immune system by SARS-CoV-2 in HF patients. Apoptotic proteins such as caspase 3 (Fig.2 .J) and 9 ( Fig.2 .L) also showed increased activity but dramatically reduced expression levels, indicating protein cleavage and activation of apoptosis ( Fig.2.K,M) . Some other apoptotic signaling genes were altered, but others unchanged in SARS-CoV-2 patients (Fig.S3 ). In addition, we found increased calpain and calcineurin activity ( Fig.2.N,P) , but no change in expression levels ( Fig.2 .O,Q). Calpain inhibitors have been suggested as a potential therapy for J o u r n a l P r e -p r o o f SARS [32] . These findings indicate that, together with oxidative stress, apoptosis may contribute to contractile deterioration.

The activity and composition of cardiac inflammasomes in SARS-CoV-2 infected patients is still poorly understood. Figure 3 .A proposes an inflammasome-inflammation pathway. The proinflammatory mediators high mobility group box 1 (HMGB-1) protein ( Fig.3.B; Fig.S2 .A) and calprotectin ( (Fig.3.M) , and vascular cell adhesion protein-1 (VCAM1) (Fig.3.N) , likely indicating an antiviral response. In addition, tumour necrosis factor alpha (TNFα) (Fig.3.O; Fig.S2 .L) was also increased in SARS-CoV-2 patients compared to HF patients and the donor group. Interestingly, certain genes remained unchanged in SARS-CoV-2 patients (Fig.S2 .G,J,K).

Inflammation-associated oxidative stress or vice versa leads to a deterioration of cardiomyocyte function that is partly due to modification of contractile proteins [24, 36] . In cardiac tissue, an overall increase of hydrogen peroxide (H 2 O 2 ) ( Fig.3 .P) was noted, along with increased H 2 O 2 levels in the cytosol (Fig.3.Q) . Oxidative stress-J o u r n a l P r e -p r o o f induced defence mechanisms were reciprocally affected, as levels of reducing glutathione (GSH) decreased ( Fig.3 .R,S), while lipid peroxidase (LPO) production increased (Fig.3.T,U) , indicating that cardiac ROS production is elevated upon SARS-CoV-2 infection. Of note, a strong association between ROS and proinflammatory signals has previously been reported in various lung diseases, including SARS-CoV-2 infection [37] .

Neutrophils are one of the primary cell types releasing proteolytic enzymes, including neutrophil elastase and play an essential role during an inflammatory response. They are rapidly mobilized from the circulation into damaged tissues. As proteolytic enzymes are increased, we wanted to define the receptor-mediated signaling events responsible for IL-6-driven neutrophil trafficking, we investigated the entire cascade in the heart. In SARS-CoV-2 patients, IL-6 activity was significantly increased compared to HF patients and the donor group ( Fig.4.A) , while relative expression was unchanged (Fig.4.B) . Myeloperoxidase, a peroxidase enzyme abundantly present in neutrophil granulocytes, showed a significant increase in both enzyme activity (Fig.4.C) and expression (Fig.4.D) . Neuropilin-1 (NPR-1), a receptor found in the vasculature of the heart, functions as an alternative SARS-CoV-2 receptor [16] . NPR-1 activity was increased in SARS-CoV-2 compared to HF and donor group hearts (Fig.4.E) . Using Western blot, several bands of different molecular weights were detected, in addition to a cleavage product (bands at 75 kDa and 63 kDa) (Fig.4.I) , a band at 100 kDa ( Fig.4 .F,I) and a band at 135 kDa ( Fig.4.G,I) . The NPR-1 100 kDa and 135 kDa proteins were significantly reduced in SARS-CoV-2 hearts ( Fig.4 .F,G,I), while the cleaved products were entirely confined to SARS-CoV-2 patients (Fig.4 .I). These findings accord with the study which showed that NRP1 potentiates SARS-CoV-2 infectivity in human embryonic kidney 293T cells in vitro [16] . Neutrophil elastase activity was increased in SARS-CoV-2 ( Fig.4.H) , potentiating infection in the presence of other host elements. Using confocal microscopy, we detected staining of the SARS-CoV-2 spike protein with IL-6 in cardiomyocytes (Fig.4.J) . Importantly, the spike protein co-localized with NPR-1 in cardiomyocytes (Fig.4.K) .

In addition to other well-known functions, histones are important pro-inflammatory agents and act as the major pro-inflammatory component of neutrophils [38] , potentiating signaling by recruiting TLR4 to histonecontaining endosomes. Neutrophil extracellular traps (NETs), which comprise a DNA backbone coiled around histones accompanied by enzymes found in neutrophil cytoplasmic granules, are released during cell damage J o u r n a l P r e -p r o o f Journal Pre-proof (NETosis) . We therefore investigated histones and related mechanisms of cell damage following SARS-CoV-2 infection.

As NET formation is altered by specific properties of histone beads, we investigated the expression of HDAC4 and the posttranslational modification of histones. Various molecular weights were detected using Western blot ( Fig.4.P) , and the expression levels of HDAC4 245 kDa and 75 kDa proteins were significantly reduced in SARS-CoV-2 compared to HF patients and the donor group (Fig.4.L,O) , while the expression levels of the 135 kDa and 100 kDa proteins were increased (Fig.4.M,N) . Histones can be altered through various chemical modifications including acetylation, methylation, phosphorylation, ubiquitination and acylation of free Nterminal tails or globular domains that physically interact with DNA. While histone 3 showed an overall increased expression in SARS-CoV-2 ( Fig.4.Q) , the acetylation (Fig.4.R) , dimethylation (Fig.4 .S) and phosphorylation ( Fig.4 .T) of histone 3 were all significantly reduced.

Taken together, our data suggest that SARS-CoV-2 infection and the resultant oxidized microenvironment cause alterations of protein localization and expression, enzyme activity, inflammation, oxidative stress, which together lead to severe cardiomyocyte damage and subsequent cell death .

A recent study demonstrated that human iPSC cardiomyocytes are susceptible to SARS-CoV-2, with cardiomyocyte infection resulting in viral replication, cytopathy and an induction of apoptosis that was followed by a cessation of beating 72 hours after infection [39] . Viruses display considerable redundancy and flexibility so that they can exploit weak multivalent interactions to enhance affinity. Recent studies of SARS-CoV-2 entry have focused almost entirely on the ACE2 receptor, which in many organs, as well as respiratory and olfactory epithelial cells, actually shows low protein levels [40] . By contrast, our data shows that in the failing human heart an environment with increased protease activity is required to facilitate SARS-CoV-2-host cell interactions in cardiomyocytes.

Our paradigm emphasizes the role of inflammation and oxidative stress to promote SARS-CoV-2 entry to cardiomyocytes. We defined the receptor-mediated signaling events responsible for IL6-driven neutrophil trafficking. Remarkably, the inflammatory signaling pathways were highly regulated in cardiomyocytes, hence suggesting a key role for the organelle in COVID-19 and its associated inflammatory pathologies. Virus infection seems to excessively activate monocytes and macrophages leading to the development of a cytokine storm and subsequently to the appearance of acute respiratory distress syndrome [41] . Our findings are consistent with SARS and Middle East Respiratory Syndrome as the presence of "cytokine storm" may have a J o u r n a l P r e -p r o o f key role in the pathogenesis of SARS-CoV-2 [42, 43] . Infected SARS-CoV-2 cells are reprogrammed for virus particle production and die after their lytic release, potentially due to the cytosolic components release by removing cells from the system that induce the massive inflammatory reaction leading to an "over-reaction" of the immune system "cytokine storm" [44] . A number of neutrophils have been reported during inflammation, but also shown to have the capacity to produce a number of cytokines such as TNF-α, IL-6, and IL-8, all of which are involved in regulation of the immune response and inflammation. Some of the ILs initiate degranulation and the production of ROS which induce oxidative stress, which plays a key role in the pathogenesis of HF development [11, 12, 25, 45] . Oxidative stress and ROS modify lipids, proteins, carbohydrates, nucleic acids, and induce the mitochondrial permeability transition leading to autophagy, apoptosis, and necrosis. HMGB1 protein, a chromatin-binding nuclear protein and damage-associated molecular pattern molecule, is integral to oxidative stress and downstream apoptosis or survival. Accumulation of HMGB1 at sites of oxidative DNA damage can lead to DNA repair, suggesting a DNA damage in SARS-CoV-2 infected patients. A potential mechanism linking ROS and myocardial inflammation, in particular in conditions of hyperglycaemia, is the activation of NF-kB signaling, since NF-kB activation induces various pro-inflammatory genes which, in turn, lead to ROS generation. These imply the pivotal role of ROS and inflammation in activating NF-kB signaling in hyperglycemia conditions [46] and potentially in SARS-CoV-2 infection. Increased H2O2 and 3-nitrotyrosine, as shown previously [11, 12, 25] could have resulted from the uncoupling of eNOS, thereby switching the eNOS dimer to a superoxide anion-generating monomer, hypothetically the same is true for SARS-CoV-2 infected patients.

A significant invasion of neutrophils produced in the affected organs during first stage of infection [47] . Neutrophils combat pathogens either by release of their granular content leading to production of ROS, by phagocytosis, or by neutrophil extracellular traps (NETs). NETs are web-like structures formed from decondensed chromatin coated with histones and oxygenases like myeloperoxidase [48] . NETs were shown to capture and inactivate invading pathogens like bacteria and viruses [49, 50] . The NET producing neutrophils . The inflammasome generally responds to inducers of inflammation as well as infectious agents such as viruses, bacteria and fungi. Thereby, some of the caspases will promote the secretion of some inflammatory cytokines as the case in SARS-CoV-2 infected patients.

Our data provide new insights into the mechanisms of SARS-CoV-2 entry into the heart and define promising targets of antiviral interventions for COVID-19 patients suffering from pre-existing heart condition, heart failure patients and/or patients with co-morbidities.

An anti-viral drug could be combined with an immune system booster and anti-oxidants, aiming for redox balance at the cellular level via enhanced antioxidant metabolites. We could observe a reversed altered force production and stiffness of SARS-CoV-2 cardiomyocytes upon treatment with Mito-TEMPO, an antioxidant that J o u r n a l P r e -p r o o f targets mitochondrias (Fig.S4) . Remarkably, in SARS-CoV-2 infected patients Mito-TEMPO treatment significantly reduced inflammatory molecules IL-6 and 18 ( Fig.S4.D,E) and oxidative stress levels, as assessed by H 2 O 2 (Fig.S4 .F) and increased anti-oxidant GSH (Fig.S4.G) . This approach may be appropriate in high risk groups with pre-existing heart conditions or patients with co-morbidities characterized by increased oxidative stress and inflammation. Additional, and based on our study and other studies, calpain inhibitors could also be used as a treatment option for SARS infected patients and has already been suggested as a potential therapy for SARS [32] . Moreover, based on the fact that cathepsin is elevated in SARS-CoV-2 infection and positively correlated with disease course and severity, also known to cleave the SARS-CoV-2 spike protein and enhanced virus entry, it is therefore suggested that cathepsin inhibition could prevent SARS-CoV-2 infection. Indeed, this suggestion has been experimentally in vivo employed showing a significant inhibition of SARS-CoV-2 in Huh7 cells treated with cathepsin inhibitor [63] . Finally, since increased inflammation and oxidative stress is a hallmark of SARS-CoV-2. We therefore believe that a one-component therapy will not alone be sufficient in these patients. We also propose drugs or biological modulators that inhibit viral spreading and replication in recipient cells, combined with enhancer defence mechanisms that reduce oxidative stress and inflammation, which will be more effective than a single agent. An anti-viral drug could be combined with an immune system booster and anti-oxidants, aiming for redox balance at the cellular level via enhanced antioxidant metabolites, thus protecting enzyme function and preventing mitochondrial destruction. This approach may be appropriate in high risk groups with pre-existing heart conditions or patients with co-morbidities characterized by increased oxidative stress and inflammation. Finally, regarding cytokine storms sometimes reported in SARS-CoV-2 HF patients, we suggest a multi-drug cocktail could be combined with an IL-6 inhibitor. Gathering together mechanistic insights on SARS-CoV-2 cellular entry and replication, several potential targets of future antiviral therapeutics emerge for infected HF patients and patients with co-morbidities.

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Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

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All authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.

The authors declare no competing financial interests.J o u r n a l P r e -p r o o f