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Influenza A virus modulates ACE2 expression and SARS-CoV-2 infectivity in human cardiomyocytes

Influenza A virus (IAV) and SARS-CoV-2 virus are both acute respiratory viruses currently circulating in the human population. This study aims to determine the impact of IAV infection on SARS-CoV-2 pathogenesis and cardiomyocyte function. Infection of human bronchial epithelial cells (HBEC), A549 cells, lung fibroblasts (HLF), monocyte derived macrophages (MDMs), cardiac fibroblasts (HCF) and hiPSC-derived cardiomyocytes with IAV enhanced the expression of ACE2, the SARS-CoV-2 receptor. Similarly, IAV infection increased levels of ACE2 in the lungs of mice and humans. Of interest, we detected heavily glycosylated form of ACE2 in hiPSC-CMs and poorly glycosylated ACE2 in other cell types. Also, prior IAV infection enhances SARS-CoV-2 spike protein binding and viral entry in all cell types. However, efficient SARS-CoV-2 replication was uniquely inhibited in cardiomyocytes. Glycosylation of ACE2 correlated with enzymatic conversion of its substrate Ang II, induction of eNOS and nitric oxide production, may provide a potential mechanism for the restricted SARS-CoV-2 replication in cardiomyocytes.


INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, and the resulting disease known as COVID-19, have led to millions of deaths worldwide. Although COVID-19 is primarily a respiratory infection, extra pulmonary complications have become well characterized in severe cases. Indeed, cardiovascular damage caused by infection plays a critical role in morbidity and mortality. Numerous studies have reported an elevated level of cardiac troponin, an indicator of cardiac damage, and abnormalities in heart function in COVID-19 patients. [1][2][3] Other cardiovascular complications associated with COVID-19 include myocarditis, acute myocardial infarction, heart failure, and arrhythmias. 4,5 The mechanisms underlying such pulmonary and cardiac tissue injury are poorly understood.
In this regard, the extent to which other circulating infectious agents can influence the disease features of COVID-19 is also largely unknown. To date, there is limited evidence for co-infections with respiratory bacteria in severe COVID-19 patients. 6 By contrast, viral coinfections have been more commonly detected, including infections with rhinovirus, respiratory syncytial virus, and common cold coronaviruses. 7 Coinfection of influenza A virus (IAV) has also reported in several COVID-19 cases with poor outcome. 8,9 In a mouse model, IAV significantly elevated expression of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), in the lungs, thereby promoting infection. 10,11 ACE2 is expressed in many tissues throughout the body, potentially allowing SARS-CoV-2 to infect a wide range of cell types, including type 2 pneumocytes in the lung as well as cardiac fibroblasts and cardiomyocytes in the heart. [12][13][14] Also, we have demonstrated that IAV disseminates to the heart tissue and causes cardiac fibrosis and electrical dysfunction. 15 Thus, we wanted to determine whether IAV infection regulates ACE2 and consequent SARS-CoV-2 infectivity in human cardiac cells. Understanding the mechanism of IAV coinfection and SARS-CoV-2 pathogenesis in cardiac cells might help explain some cases of COVID-19 that involve cardiac problems and might provide the opportunity to protect the heart from SARS-CoV-2 infection.
ACE2 plays a major role in the renin-angiotensin system (RAS), in which it regulates levels of angiotensin II (Ang II) through proteolytic processing of Ang II to angiotensin (1-7), which serves as an Ang II antagonist. 16 Ang II binds primarily to receptors, affecting signaling cascades that regulate tissue remodeling, acute vasoconstriction, fibrosis, apoptosis, inflammation and hypertrophy. 17,18 ACE2 glycosylation is essential for the enzymatic conversion of Ang II into Ang (1)(2)(3)(4)(5)(6)(7). ACE2 can be heavily glycosylated in cells, with seven potential N-linked glycosylation sites and one O-linked glycosylation site 19 and molecular dynamic simulation studies indicate that two of the glycosylation sites, N90 and N332, have opposite effects on binding of the spike receptor domain (RBD). 20 Glycosylation of N90 partially covers and interferes with binding at the spike-ACE2 interface. Although studies of ACE2 in transfected human embryonic kidney (HEK293) cells have outlined the role of its N-glycans in SARS-CoV-2 interactions, glycosylation and effects of this modification has not been well characterized in pulmonary and cardiac cell types.
In this study we identify the mechanism by which IAV pre-infection modulates ACE2 expression and its glycosylation state, thereby dictating susceptibility to SARS-CoV-2 in various cell types. We show that as a result of ACE2 glycosylation, downstream events including peptidase activity, eNOS expression and nitric oxide production lead to the inhibition of SARS-CoV-2 replication in cardiomyocytes, but not in the lungs. This data strongly suggests that IAV infection may enhance the severity of SARS-CoV-2 infection in the lungs, posing a severe risk to public health.

Influenza A virus infection enhances expression of ACE2
We first determined whether IAV enhances ACE2 or TMPRSS2 expression, host proteins that are required for SARS-CoV-2 entry into lung and cardiac cells. These cells included a human pneumocyte-like epithelial cell line (A549), human lung fibroblast cells (HLF), human primary cardiac fibroblasts (HCF), and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Western blotting for ACE2 showed that ACE2 is poorly expressed in uninfected A549, HCF and HLF cells, but highly up-regulated by IAV in A549 ( Figure 1A), HCF ( Figure 1B) and HLF ( Figure S1A). Furthermore, we found that hiPSC-CMs have a basal level of ACE2 that is further increased following IAV infection ( Figure 1C). ACE2 levels quantified by Image J software are presented in the bottom panel.
Because lung tissue, the primary site of SARS-CoV-2 infection, is comprised principally of epithelial cells but also houses a large number of fibroblasts and macrophages, we separately assessed primary human bronchial epithelial cultures (HBEC), human fibroblasts (HLF) and human monocyte derived macrophages (hMDMs) to determine whether IAV infection enhances ACE2 expression in these cell types. Similar to hiPSC-CMs, both HBEC and hMDMs had low levels of ACE2 expression, which were increased by IAV infection ( Figure S1). IAV-induced cytotoxicity was quantified with the lactose dehydrogenase assay of the cell culture supernatants from IAV-infected HCF and hiPSC-CM cells. We found that IAV infection induced the death of HCF cells (28% at 48h and 42% at 72h). In contrast, IAV did not cause cytotoxicity in hiPSC-CMs until 72h post infection ( Figure S2), suggesting that hiPSC-CMs are more resistant to IAV infection or IAV-induced cytotoxicity.
We next examined whether IAV infection alters the expression of TMPRSS2 in A549, HCF, and hiPSC-CMs. We found that IAV infection did not change the level of TMPRSS2 expression in any of these three cell types ( Figure S3). To test whether the ACE2 induction is specific to influenza viruses, we exposed A549 cells to human metapneumovirus (MPV), or Sendai virus (SeV), or IAV and quantified ACE2 protein levels. IAV and MPV induced ACE2 expression, however, SeV did not ( Figure S4).
We inoculated A549 cells with a variety of influenza strains (PR8, PR8-DNS1, H1N1, H3N2 and IBV) and examined ACE2 expression. We found that the highly pathogenic influenza strains (H1N1 and H3N2) are capable of inducing the ACE2 protein (Figue S4). Although, ACE2 is primarily localized in the plasma membrane, cleaved ACE2 can be internalized and localized in the cytoplasm, and viral infection can enhance the endocytosis of cleaved ACE2. 21 Thus, we determined the cellular localization of IAV-induced ACE2. We performed immunofluorescence microscopy using ACE2 specific antibody. Confocal microscopy of A549 ( Figure 1D) and HCF cells ( Figure 1E) confirmed that most of the ACE2 immunostaining was restricted to the cell surface. However, in hiPSC-CM cells ( Figure 1F), ACE2 resides both in the outer membrane and intracellularly. Furthermore, we found that an increased expression of ACE2 in both IAV infected (high magnitude) and bystander cells (less magnitude). To confirm IAV infections in these experiments, we stained the cells with anti-Flu nucleoprotein antibody (green). The graphs shown in Figures 1G-1I  To further examine whether IAV infection induces the expression of ACE2 and dACE2 mRNA, we measured the mRNA levels of ACE2 and dACE2 using previously described specific primers for the two isoforms. 22 Our results indicate that IAV infection induces the expression of both ACE2 and dACE2 mRNA in A549 ( Figures S5A and S5B), HCF cells ( Figures 5C and 5D) and in hiPSC-CMs ( Figures 5E and 5F). Although IAV induced both forms of ACE2 in these cells, expression of dACE2 mRNA was 5-8 fold higher than ACE2 mRNA ( Figure 5). However, dACE2 mRNA is reportedly not translated into a stable protein. 23 Nonetheless, we observed that dACE2 and ACE2 are both transcriptionally upregulated on IAV infection.
Full length, fully glycosylated ACE2 is constitutively expressed in hiPSC-CM and vero cells Recently, a novel isoform of ACE2, designated delta-ACE2 (dACE2) was described that lacks the 356 N-terminal amino acids of ACE2 and is, for that reason, defective in its carboxypeptidase activity and unable to bind SARS-CoV-2. [22][23][24] To avoid detection of the non-functional form in our immunoblot analyses, we used an antibody against the N-terminal region of ACE2, which does not detect the N-terminally deleted ACE2 species. Using this antibody, we detected the 110 kDa ACE2 protein in hiPSC-CM and Vero cells, both before and after IAV infection ( Figure 1).
Non-glycosylated ACE2 is predicted to have a molecular mass of 78 kDa. The ACE2 sequence has seven N-linked glycosylation sites and one O-linked glycosylation site. 19 To confirm that the higher molecular mass of ACE2 in Vero and hiPSC-CMs is because of glycosylation, we incubated cell lysates with PNGase F before immunoblotting. The enzymatic deglycosylation decreased the molecular mass of ACE2 in Vero and hiPSC-CM cells ( Figures 2B and 2C) to a size similar to that observed in IAV-infected A549 and HCF cells. Together this data strongly indicates that the larger molecular weight of ACE2 in Vero and hiPSC-CM cells is because of glycosylation and that most, if not all, of the predicted glycosylation sites are used. Of interest, neither A549 cells nor HCF cells expressed detectable ACE2 protein until they were infected by IAV. Furthermore, the ACE2 protein they expressed was 78 kDa, not 110 kDa, suggesting either that it was truncated, not glycosylated, or both. We also found ACE2 migrated as a slightly smaller species in IAV-infected hiPSC-CM compared to uninfected cells ( Figure 2A). This reduction in size is likely because of the IAV neuraminidase glycoprotein which enzymatically removes the terminal sialic acid residues from the glycan's on glycoproteins.
Next, we examined whether glycosylation of ACE2 is critical for its peptidase activity. The cell lysates from uninfected and IAV-infected A549, HCF, Vero (susceptible to SARS-CoV-2) and hiPSC-CMs were subjected to an enzymatic peptidase assay using synthetic MCA based substrate. We found that cell lysates from Vero and hiPSC-CMs showed peptidase activity, correlating with the expression level of the highly glycosylated form of ACE2 ( Figure 2D). Furthermore, hiPSC-CMs displayed higher ACE2 enzymatic activity after IAV infection suggesting that IAV upregulated the glycosylated ACE2. In contrast, we did not detect ACE2 enzymatic activity in A549 or HCF lysates even when derived from IAV-infected cells ( Figure 2D), consistent with the idea that glycosylation is required for efficient peptidase activity. 25 Because ACE2 in IAV-infected A549 and HCF cells is poorly glycosylated and lacks peptidase activity, we next determined whether ACE2 in IAV-infected A549 and HCF cells are capable of binding to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. A549 and HCF cells were infected with IAV or left uninfected for 48 h and then incubated with biotinylated-RBD, followed by AF488 conjugated streptavidin and examined by confocal microscopy. IAV infection increased the expression of ACE2 ( Figures 2E and 2F), and increased binding of RBD. Both appeared to bind to the cell surface. These findings suggest that ACE2 glycosylation is not required for the RBD of the spike protein to bind to ACE2.

IAV infection enhances the expression of ACE2 in the lungs and hearts of mice
To determine if IAV induces ACE2 expression in vivo, we examined the expression of ACE2 in lungs and heart tissue from mice infected with IAV. Confocal microscopy imaging showed that the expression of ACE2 was enhanced in lung sections from IAV-infected mice ( Figure 3A). Furthermore, to identify the cell types, we co-stained the lung sections with SP-C antibody (marker for type II alveolar epithelial cells) and F4/80 (macrophages). Our results indicate that IAV infection in mice enhanced the expression of ACE2 primarily in type II epithelial cells ( Figure 3A) but also in macrophages to some extent ( Figure S6). Because we have previously shown that intranasal delivery of IAV disseminated into the heart tissue and caused cardiac fibrosis and electrical dysfunction. 15 Next, we examined the expression of ACE2 mRNA in heart tissue. Total RNA was extracted from IAV-infected and uninfected mice and ACE2 was quantified iScience Article by qRT-PCR. Our results revealed that IAV infection enhanced the expression of ACE2 mRNA in the heart ( Figure 3C). To further validate the mRNA data, we examined the level of ACE2 protein expressed in heart tissue by immunoblotting with ACE2 antibody. As with the mRNA levels, the ACE2 protein level was increased by IAV infection of the heart tissue ( Figure 3D). The band intensities were quantified using ImageJ software 26 ( Figure 3E).
Next, to test the contribution of virus on ACE2 expression, we performed a time course of IAV infection in mice. We infected the mice with IAV and determined the ACE2 expression in the lungs (confocal microscopy) and heart (qRT-PCR) for 16 days, at 4 day intervals. Lung sections were stained with ACE2 antibody and EpCam antibody and analyzed by confocal microscopy ( Figure S7A). The mean fluorescent intensity of ACE2 was measured using Image-J software and the results showed that ACE2 expression gradually increased, and peaking at day 8, and decreasing to basal level by day 16 ( Figure S7B). Total RNA was extracted from the hearts of IAV-infected mice (day 0, 4, 8, 12 and 16) and used to determine the ACE2 mRNA levels by qRT-PCR. Similarly, we found that ACE2 mRNA levels increased to a maximum by day 8 and returned to the basal level by day 16 ( Figure 7C). To test whether IAV induced ACE2 expression in heart tissue depends on IFN-b, we quantified IFN-b mRNA by qRT-PCR and found that IFN-b mRNA levels peaked at day 8 (severe infection) and returned to normal by day 12 (recovery) (Figure 7D). These data suggest that IAV induced IFNs may play critical role in upregulating ACE2 expression in the heart. Peptidase activity was normalized to the protein concentration. The data shown in the graph are representative of two independent experiments in which peptidase activity was measured in triplicate samples. A two-tailed Student's t test was used to analyze the data (mean G SEM; ***p< 0.0005).
(E and F) Representative confocal images of A549 and HCF cells infected with IAV for 24 h or left uninfected. A549 and HCFcells were incubated with biotynylated receptor-binding domain (RDB) of SARS-CoV-2 spike protein followed by AF488 (green) conjugated streptravidin. The cells were fixed and stained with anti-ACE2 antibody followed with AF594 (red) conjugated secondary antibody. The nuclei were stained with DAPI (scale bar = 100mm).

OPEN ACCESS
iScience 25, 105701, December 22, 2022 5 iScience Article Next, we examined human lung sections from deceased IAV and IBV patients and from patients whose deaths were unrelated to lung disease, stained for ACE2. Notably, similar to the mouse model, we observed increased ACE2 staining in IAV and IBV patients compared to patients who died of non-lung related diseases ( Figures 3F-3H). The ACE2 fluorescent intensities of human lung sections ( Figure 3I) and the accumulated data correlates well with our in vitro findings ( Figure 1).
To determine whether IAV and IBV infection in human lungs increased binding of the spike protein, we exposed the human lung sections to spike-RBD staining and co-stained with ACE2 antibody. We found that spike-RBD binding to ACE2 was increased by influenza virus infections and that spike-RBD colocalized with ACE2 ( Figures 3J and 3K and 3L and 3G). (B) ACE2 mean fluorescent intensities from ten randomly selected images from 5 lung section were determined using ImageJ software (mean G SEM; ***p< 0.0005 ;). (C) ACE2 mRNA expression in the hearts of IAV infected mice determined by qRT-PCR using specific primers. (D) Heart tissue from non-infected and IAV infected were lysed and the equal amount of protein was used to determine the expression of ACE2 by western blot using ACE2 specific antibody. (E) The band intensities of ACE2 and b-actin were measured using ImajeJ software. Graph shown is band intensity normalized with beta actin (mean G SEM; *p< 0.05; **p < 0.005). Human lung section from deceased non-lung disease (F), IAV infection (G) and IBV infection patients (H) were stained with anti-ACE2 antibody followed by AF480 conjugated secondary antibody and nuclei staining with DAPI (scale bar = 50mm).
(I) The ACE2 staining (green) was quantified using Photoshop CC color range selection mode. The total intensity of the stained area was further evaluated via ImageJ using color intensity. These data are cumulative from 10 different images from 3 independent experiments. A two-tailed Student's t-test was used to analyze the data (mean G SEM; **p< 0.005; **p < 0.0005).
(J-L) Human lung sections from deceased patients with non-lung disease, IAV and IBV infections were incubated with biotinylated receptor-binding domain (RDB) of SARS-CoV-2 spike protein followed by AF488 conjugated streptravidin. The same slides were costained with anti-ACE2 antibody followed by AF594 conjugated secondary antibody. The nuclei were stained with DAPI (scale bar = 50mm). iScience Article IAV infection enhances SARS-CoV-2 entry and replication in human lung epithelial cells and cardiac fibroblasts but inhibits the viral replication in cardio myocytes A549, HCF, and hiPSC-CMs were infected with IAV for 48 hr and then inoculated with SARS-CoV-2 (USA-WA1/2020). SARS-CoV-2 infection and replication was examined by flow cytometry and qRT-PCR. We found that the SARS-CoV-2 infectivity is very low in uninfected A549 and HCF cells. However, prior IAV infection significantly increased the percentage of SARS-CoV-2 positive A549 (2.3% to 8.8%) and HCF cells (6.15% to 14.6%) ( Figures 4A-4D), strongly supporting the concept that IAV mediated upregulation of ACE2 as observed in Figure 1 enhances the binding and entry of SARS-CoV-2. This finding was further confirmed by confocal microscopy ( Figure S8). Also, a time course experiment showed that prior IAV infection favors SARS-CoV-2 replication over time in A549 and HCF cells ( Figure S9). In contrast, we discovered that prior IAV infection in hiPSC-CMs significantly reduced the SARS-CoV-2 positive cells ( Figures 4E and 4F). The graphs shown in Figures 4G-4L represent the cumulative data from 3 independent experiments, which confirm the increased infectivity in A549 and HCF cells and decreased infectivity in hiPSC-CMs. To further confirm our findings, we quantified SARS-CoV-2 viral replication in A549, HCF cells and hiPSC-CMs by qRT-PCR using two different primer sets to the SARS-CoV-2 N protein mRNA. Our data revealed that IAV infection significantly increased the level of N protein mRNA expression in A549 and HCF cells ( Figures 4G-4J). In contrast, we observed high levels of SARS-CoV-2 N protein mRNA in hiPSC-CMs without IAV infection ( Figures 4K and 4L). Unlike A549 and HCF cells, hiPSC-CMs subjected to prior infection with IAV showed substantially reduced the expression of SARS-CoV-2 N protein mRNA ( Figures 4K and 4L).
Because SARS-CoV-2 is sensitive to interferons and it is known that IAV infection enhances the interferon response, we examined the levels of IFN-b in the cell culture supernatants of IAV-infected A549, HCF and hiPSC-CMs. Our data demonstrated that IAV infection enhances IFN-b production in all three different cell types, but the magnitudes of IFN-b released from hiPSC-CM is less than that from A549 or HCF cells (Figure S10). Furthermore, we tested whether different stages of IAV infection (6 h, 24 h and 48 h) alters the SARS-CoV-2 infections in these three cell types, and we observed that there is a significant increase in SARS-CoV-2 infection in A549 cells as the IAV infection progresses ( Figure S11A). However, in HCF cells the SARS-CoV-2 viral replication is not changed at different stages of IAV infection ( Figure S11B). In hiPSC-CMs, SARS-CoV-2 replication is significantly reduced by prior IAV infection, in all stages of IAV infection ( Figure S11C).
To test whether prior IAV infection inhibits the binding or entry of SARS-CoV-2, we incubated the IAVinfected and uninfected hiPSC-CMs with SARS-CoV-2 (MOI = 20) for 2 hours and determined the % SARS-CoV-2 positive cells. Our data indicate that prior IAV infection did not affect the binding or entry of SARS-CoV-2 in hiPSC-CMs ( Figure S12). To further test whether IAV infection (48h) causes contractile dysfunction or cell death in hiPSC-CMs, we monitored the hiPSC-CMs electrical activity with a multi-electrode array system. We found that IAV infection (MOI 2.5) of hiPSC-CMs did not affect the hiPSC-CMs electrical activity, contractile function and cell viability ( Figure S13). These results suggest that prior infection of A549 and HCF cells with IAV makes the cells more susceptible to SARS-CoV-2 whereas prior IAV infection of hiPSC-CM cells renders the cells more resistant to SARS-CoV-2 replication.

IAV infection differentially regulates the expression of genes in HCF and hiPSC-CMs
Because we found that prior IAV infection enhances SARS-CoV-2 replication in HCF cells and restricts it in hiPSC-CMs, we performed total RNA sequencing of IAV-infected HCF and hiPSC-CMs and compared their gene expression patterns at 48 h post infection, using a cutoff of p-value <0.001 for statistical significance. The heat map in Figure 5A and volcano plot in Figure 5B show the pattern of differentially expressed genes in hiPSC-CMs. Of the 2808 differentially expressed genes, the IFN response genes such as ISG15, ISG20, IFITM1, IFITM2, IFITM3 IRF2, IRF7 and IFNB1 were downregulated at 48 h in hiPSC-CMs. IFNRL1 was upregulated in hiPSC-CMs compared to HCF cells ( Figure S14A), whereas expression of the inflammatory cytokines, TNF, IL6, IL1b, IL24 and IL12A, are significantly increased in HCF cells ( Figure S14B). Because we had found increased expression of glycosylated ACE2 in hiPSC-CMs, we examined the expression of genes involved in glycosylation in our RNA sequencing data set. Expression of several N-acetylglucosaminyltransferases (MGAT4A/GNTIV, MGAT3/GNTIII, MGAT2/GNTII and MGAT5/GNTV) were highly induced in hiPSC-CMs ( Figure 5C). We confirmed the gene expression profiling results from total RNA sequencing with qRT-PCR for important upregulated glycosylation genes (MGAT3 and   (Figure 4). That finding prompted us to investigate the impact of SARS-CoV-2 on cardiomyocyte contractile function, field potential and spike amplitude using a multi-electrode array system. Because we could not employ this system in the BSL3 laboratory, we instead exposed the hiPSC-CMs to heat-killed SARS-CoV-2 at an MOI of 1 and 2.5 and recorded the cardiomyocyte electrical functions over time (0-24 hours). Incubation of heat killed SARS-CoV-2 with hiPSC-CMs significantly increased the cardiomyocyte beat rate (Figures 6A and  6B) and decreased the beat period at 6 h ( Figures 6A and 6B). Furthermore, the field potential duration (FPD) in heat killed SARS-CoV-2-exposed hiPSC-CMs was also decreased ( Figure 6C). FPD represents the characteristics of action potential (AP) and QT interval of the ECG. 29,30 The shortening of FPD shows altered action potential (AP) and ultimately indicates ion channel imbalance and the presence of arrhythmias. However, the hiPSC-CM response to HK SARS-CoV-2 was transient and returned to normal contractility within 24 h of HK SARS-Co-V-2 incubation ( Figure S15), which suggests that live virus replication is required for the induction of long-term cardiomyocyte contractile dysfunction as has been reported. 31 Next, we tested whether the interaction of spike protein with ACE2 is enough to induce the cardiomyocyte contractile dysfunction. The hiPSC-CMs were incubated with recombinant spike protein or spike receptor binding domain (RDB) and the cells were monitored for cardiomyocyte function. Of interest, we found that the incubation of spike protein or its RDB did not cause any abnormalities of cardiomyocyte contractile iScience Article function: there was no difference in beat period and FPD, and the electrocardiogram traces were not different from the control without spike protein (Figures 6D-6F). Together, these data indicate that the interaction of spike protein with the receptor ACE2 is not enough to cause cardiomyocyte contractile dysfunction, suggesting that the effects on contractions require infection or at least live virus.

IAV infection increases the level of angiotensin II in A549 and HCF cells
ACE2 is a carboxypeptidase that converts the Ang II into angiotensin1-7 and thereby inhibiting the Ang IImediated pro-inflammatory response. 32 We found that IAV infection mediated upregulation of ACE2 expression in A549 and HCF cells but there was no apparent increase in peptidase activity. In contrast, IAV infection of hiPSC CMs increases the ACE2 expression and peptidase activity ( Figure 2D). These observations prompted us to look for evidence of peptidase activity by measuring the level of Ang II in the culture supernatants harvested from uninfected vs IAV-infected A549, HCF and hiPSC-CMs. Notably, we found that the Ang II levels were significantly increased in cultures of IAV-infected A549 and HCF cells iScience Article indicating that these cells produce Ang II but lack the ACE2 enzymatic activity to convert it to Ang 1-7 ( Figures 7A and 7B). However, the IAV infection significantly reduced the levels of Ang II in hiPSC-CMs ( Figure 7C). Together, our results demonstrated that the IAV mediated induction of highly glycosylated ACE2 expression in hiPSC-CMs activates it enzymatic activity ( Figures 2C and 2D), converting the ) using Griess reagent (D-F). (G) hiPSC-CMs were directly infected with SARS-CoV-2 (MOI 2) and aafter an hour of incubation the cells were exposed to media only, SNAP (400mM ) and NAP (400mM) for 24 hours. (H and I) HCF cells were infected with IAV or left uninfected for 48hs, washed and incubated with SARS-CoV-2 (MOI 2) and after one hour the cells were treated with media only, SNAP (400mM ) and NAP (400mM) for 24 hours. The cells were lysed and total RNA was used to determine the relative mRNA expression of SARS-CoV-2 N gene by qRT-PCR using N1 and N2 primers and expressed relative to the house-keeping gene Rpp30. Data shown in graph A-C are representative of three independent experiments performed in triplicate or quadruplicates. A two-tailed Student's t-test was used to analyze the data (mean G SEM; *p< 0.05; **p< 0.005). Data shown are representative of three independent experiments performed in triplicate or quadruplicates. A two-tailed Student's t-test was used to analyze the data (mean G SEM; ns-nonsignificant; *p< 0.05; **p< 0.005; ***p< 0.0005). Prior IAV infection enhances the production of nitric oxide in hiPSC-CMs, but not in A549 and HCF cells It has been previously reported that the conversion of Ang II to Ang 1-7 and the binding of Ang 1-7 to the Mas receptor leads to the activation of the PI3K/Akt pathway resulting in upregulation of NOS3 (eNOS) and the production of NO 2 -. 33 To determine if this pathway has been activated, we measured eNOS (NOS3) expression in HCF and hiPSC-CMs, and we found that eNOS expression is not induced in HCF cells by either CoV-2 alone or IAV with CoV-2, but we observed an significant increase in eNOS expression in hiPSC-CMs on CoV-2 and IA with CoV-2 ( Figure S16). Next, we measured the NO 2 À in in cell culture supernatants of A549, HCF and hiPSC-CMs. As expected from experiments above, prior IAV infection of hiPSC-CMs increased NO 2 À levels in culture supernatants because of reduced levels of AngII, but infection of HCF and A549 cells did not, because of high levels of Ang II (Figures 7D-7F). NO 2 À has been found to inhibit SARS-CoV-2 replication 34 which might explain why prior infection with IAV inhibits SARS-CoV-2 replication in hiPSC-CMs but not in A549 or HCF. To confirm that the induction of NO 2 À production inhibits the replication of SARS-CoV-2, we treated SARS-CoV-2-infected hiPSC-CMs (susceptible to SARS-CoV-2 without prior IAV infection) with S-nitroso-N-acetyl-DL-penicillamine (SNAP) (NO 2 À donor) or N-acetyl-DL-penicillamine (NAP) (control), and measured N protein mRNA levels after 24 h. SNAP treatment diminished the SARS-CoV-2 replication in hiPSC-CMs ( Figures 7G and 7D). Because the hiPSC-CMs are metabolically active and the NO 2 À might induce contractile dysfunction and cell death, we exposed hiPSC-CMs to SNAP or NAP and monitored their contractile function with the MEA system and cytotoxicity with the LDH assay. The MEA data (Figures S17A and S17B) revealed that SNAP increased the beats per minute immediately after adding, however the beat frequency returned to normal within 3 h. Furthermore, the cell morphology did not change over this time and LDH was not released (cell death) indicating that NO 2 À is not toxic for hiPSC-CMs ( Figure S17C).
Because we found that prior IAV infection enhances SARS-CoV-2 infectivity of HCF cells, we determined if SNAP treatment inhibited the SARS-CoV-2 replication in HCF cells that were infected with IAV + SARS-CoV-2. After 24 h, total RNA was extracted, and SARS-CoV-2 N protein mRNA expression was determined. We found that the addition of SNAP significantly reduced SARS-CoV-2 replication in HCF cells as measured the N protein mRNA levels ( Figures 7H and 7I). To determine whether SNAP was producing NO 2 À in HCF cells, we infected HCF cells with IAV and, after a 24 h treatment with or without SNAP and NAP, measured NO À 2 in the supernatant. SNAP treatment increased the production of NO 2 À in HCF cells ( Figure S18).
Together, our results revealed that NO À 2 plays an important role in inhibiting the SARS-CoV-2 replication for both hiPSC-MCs and HCF.

Prior IAV infection enhances the mortality of SARS-CoV-2 infected mice
To study the interaction between IAV and SARS-CoV-2 in vivo, we infected C57BL/6 mice with 1 3 10 5 PFU of mouse adapted SARS-CoV-2 strain MA10 with or without a 5-day prior infection with IAV strain PR8 (1 3 10 4 PFU) and monitored the morbidity and mortality through day 12. The survival rate of the mice (Figure 8A) and our results revealed that prior IAV infection increases the mortality of SARS-CoV-2 co-infected mice (90% of the mice were dead by day 12). In contrast, only 30% of mice infected with SARS-CoV-2 alone died by day 12. Next, we determined the virus titers in the lung and heart homogenates of the mice that survived SARS-CoV-2, alone, and IAV with SARS-CoV-2. We found an increase of SARS-CoV-2 titer in the lungs and hearts of IAV-infected mice (only 2 out of 15 mice survived), compared to SARS-CoV-2, alone, infected mice ( Figures 8B and 8C). Taken together, these data are consistent with prior IAV infection enhancing the expression of ACE2 in the lungs and hearts and promoting SARS-CoV-2 entry and replication.

DISCUSSION
The current study revealed several novel findings. First, we determined that IAV infection resulted in elevated expression of the SARS-CoV-2 receptor, ACE2, but not the cofactor TMPRSS2 in all three human cell types studied, A549, HCF and hiPSC-CMs. Furthermore, we determined that ACE2 glycosylation correlated with ACE2 peptidase activity, which facilitates the conversion of pro-inflamamtory Ang II to immunosuppressive Ang 1-7, and thereby increasing the expression of NOS3 and NO 2 À production. Of  Figure 1). The fact that the molecular weight of ACE2 in A549 and HCF cells is less than the predicated molecular weight of non-glycosylated ACE2 (85 kDa) has been suggested to be due to cleavage of ACE2 at a site in the C-terminal region by TMPRSS2 which is induced in these cells by IAV infection. 35 Of interest, the cleaved ACE2 is retained by the cell and promotes entry of the SARS-CoV-2 virus. The size of the C-terminal fragment is 13 kDa. Thus, the 72 kDa ACE2 in IAV-infected cells (A549 and HCF) is the cleaved ACE2 fragment which contains the transmembrane domain ( Figure 1). Studies from Onabajo et al., identified an ACE2 isoform that is an interferon and virus induced gene product; specifically, a truncated isoform of ACE2, designated as delta ACE2, which lacks 356 amino-terminal amino acids therefore its non-functional in binding the SARS-CoV-2 22 . Similarly, Ng et al., also demonstrated that a truncated ACE2 transcript is induced in response to IFNs, termed MIRb-ACE2, but it is unstable and would not be translated into protein. 23 These studies reported that dACE2, but not ACE2 is induced by IFNs and viruses. In contrast, our IB analysis of human primary cells and cell lines showed an increased ACE2 expression (Figure 1) which indicated that IAV induces the expression of the full-length ACE2. Notably, the antibody used to detect ACE2 in our system is raised against an epitope in the N-terminal region of ACE2 and does not detect dACE2. This conclusion is supported by experiments with IAV infection in A549 and HCF cells in which qRT-PCR analysis revealed that IAV infection induces the expression of both full and dACE2 forms ( Figures S2A-S2D). Our findings of IAV mediated upregulation of ACE2 in cell lines was also supported by in vivo mouse and human infections ( Figure 3) and supported by Bai et al. 11 ACE2 expression is regulated transcriptionally and post-transcriptionally and involves an epigenetic process. The has-miR-125a-5p and miR200 families of microRNA are known to target the 3 0 UTR of the ACE2 mRNA and regulate ACE2 expression at the post-transcriptional level. 36 The ACE2 promoter has proximal and distal regulatory regions and the relative use of the two promoter components varies among the tissues. 37 Chromatin immunoprecipitation sequencing (ChIP-Seq) studies have revealed that ACE2 gene expression is regulated by several transcriptional factors including MYBL2, USF1, TAED4, EP300, SP1, HNF4A, CEBP, MAFF and GATA3. 38 Furthermore, Kuan et al demonstrated that angiotensin II induces the expression of ACE2 in human cardiac fibroblasts. 39 We have found that the Ang II level is high in IAVinfected A549, HCF and hiPSC-CMS ( Figure 8). Also, several studies demonstrated that ACE2 is an IFN stimulated gene and the transcription factors STAT1, STAT3, IRF8 and IRF1 bind to the ACE2 promoter iScience Article region, 40,41 suggesting ACE2 induction in these cells. Further detailed studies are required to identify the exact mechanism and transcription factors involved in the induction of ACE2 on IAV infection. The host immune cells respond to IAV infection by producing type I and III IFNs. The released IFN binds to its receptor and induces various IFN response genes, which provide a first line of defense against IAV and encounters with the invading virus. 42 ACE2 has been described as an interferon response gene in human airway epithelial cells and other cell types in the body. 40 Studies from Shajahan et al., have shown that human ACE2 contains seven N-glycosylation sites and one O-glycosylation site, 19 and the glycosylation of ACE2 is essential for it peptidase activity. 43 Remarkably, we observed that the IAV-induced ACE2 in A549 and HCF cells is not glycosylated; whereas IAV-induced ACE2 in hiPSC-CMs is glycosylated (Figures 2A-2C) and exhibits peptidase activity 43 ( Figure 2D). Furthermore, our data showed that IAV-induced ACE2 is able to bind the spike protein in A549 and HCF cells ( Figures 2E and 2F), which suggests that the 72 kDa non-glycosylated ACE2 is capable of engaging the spike protein on SARS-CoV-2. Because enhanced binding of spike protein to IAV-infected cells does not necessarily lead to enhanced pathogenicity of a SARS-CoV-2 infection, we were prompted to determine whether IAV-induced ACE2 expression enhances SARS-CoV-2 infectivity in these cells. Therefore, we inoculated IAV-infected A549, HCF and hiPSC-CMs with SARS-CoV-2 and monitored its replication and infectivity. Consistent with a recent report from Bai et al. 11 , we observed increased infectivity in A549 and HCF cells pre-infected with IAV. By contrast, we observed that in hiPSC-CMs, IAV pre-infection significantly diminished the SARS-CoV-2 replication, without affecting the entry of SARS-CoV-2 (Figures 4  and S6). However, we found that the ACE2 induced in hiPSC-CMs by IAV that was highly glycosylated and showed peptidase activity, whereas the ACE2 induced in A549 and HCF cells by IAV infection was non-glycosylated and lacked peptidase activity (Figures 1 and 2).
Importantly, in this manuscript we showed that exposure of hiPSC-CMs to heat killed SARS-CoV-2 significantly increased cardiomyocyte contractility and shortened the T wave ( Figure 7). However, incubation of spike protein and spike RDB with hiPSC-CMs did not affect CM contractile function, suggesting that the interaction of spike protein with ACE2 is not involved in the dysregulation of cardiomyocyte function. We speculate that SARS-CoV-2 replication is required for the induction of the CM contractile dysfunction. Consistent with our studies, Yang et al., demonstrated that hiPSC-CMs express ACE2 that enables the entry of SARS-CoV-2 pseudo virus. 27 Similarly, Sharma et al., demonstrated that hiPSC-CMs are susceptible to SARS-CoV-2 infection, and on infection activate genes for antiviral responses while suppressing those involved in cardiomyocyte contractility, which collectively leads to cardiomyocyte contractile dysfunction. 44 Therefore, these cells provide a valuable model to study cardiac dysfunction during SARS-CoV-2 infection.
ACE2-catalyzed conversion of Ang II into Ang (1-7) and the consequent anti-inflammatory effects have been well established. 17,18 Remarkably, we observed an increased level of Ang II in IAV-infected A549 and HCF cells. In contrast, the level of Ang II in hiPSC-CMs was significantly reduced (Figure 7), suggesting that increased expression and glycosylation of ACE2 in hiPSC-CMs exhibits an increased enzymatic activity that converts Ang II into Ang (1-7). Binding of Ang (1-7) to MasR activates the PI3K/Akt signaling pathway to induce the expression of eNOS. 45 Also, Ang (1-7) has been shown to function protectively in the heart during ischemic injury by enhancing the function of endothelial cells, vasodilation, expression of SERCA2, NOS3 and NO 2 À . 33 Notably, we observed elevated levels of NO 2 À in hiPSC-CMs ( Figures 7D-7F), which correlated with the decreased SARS-CoV-2 viral load in IAV + SARS-CoV-2 co-infected cells ( Figure 5). It had been shown previously that SARS-CoV-2 is sensitive to interferons 46 and IAV infection is known to induce an interferon response, thus it is possible that IAV-induced IFN is responsible for the inhibition of SARS-CoV-2 replication in hiPSC-CMs. However, we found similar levels of IFN-b in A549, HCF and hiPSC-CM cells infected with IAV ( Figure S10), yet we found a significant decrease in SARS-CoV-2 replication/load in CMs, which suggests that the suppression of SARS-CoV-2 replication in hiPSC-CM cells is independent of IAV-induced IFNs. Collectively, our data revealed that the control of SARS-CoV-2 in hiPSC-CMs previously infected with IAV is because of increased expression of NOS3 and NO 2 À production.
In contrast, in A549 and HCF cells, co-infection with IAV and SARS-CoV-2 failed to increase NOS3 expression and NO 2 À production and thereby failed to control SARS-CoV-2 replication. Nitric oxide has a broadrange antimicrobial effect against various pathogens including both DNA and RNA viruses. [47][48][49][50] Likewise, and similar to our results, the SNAP-mediated inhibition of SARS-CoV-2 replication was also reported by Akaberi et al. 34 Notably, our findings revealed that an increased state of glycosylation and NO production in hiPSC-CMs enhanced the inhibition of IAV replication in our co-infection model ( Figures 1C, 2C iScience Article and 8). In contrast, the increased expression of non-glycosylated ACE2 in A549 and HCF cells enhances the replication of SARS-CoV-2, which may be because of the lack of ACE2 enzymatic activity and NO 2 À production ( Figures 1A, 1B, 2D, and 8). Furthermore, our mouse co-infection studies revealed that prior IAV infection enhances the presence of SARS-CoV-2 in the lung and heart and subsequently increases the mortality (Figure 8). Although, we predicted that prior IAV infection may protect the heart from virus replication, because of the expression of glycosylated ACE2 and its enzymatic activity, we found increased virus titers in the hearts. The reasons for the increased virus titer in the heart could be the cellular composition of the heart and induction of unglycosylated ACE2 in cardiac fibroblasts, further work is needed to determine the cell type infected by SARS-CoV-2 in the heart. Our results are strongly supported by clinical data from COVID-19 patients with flu-like symptoms demonstrating the presence of SARS-CoV-2 in the heart tissue but not specifically in the cardiomyocytes. 51 A multi-organ autopsy study by Puelles et al., also showed the presence of SARS-CoV-2 viral particles in heart tissue. 52 However, the cell type harboring the virus was not identified. It is important to note that we tested whether inhibition of glycosylation by tunicamycin enhanced the infectivity of SARS-CoV-2 in hiPSC-CMs and Vero-E6 cells. Surprisingly, we found that treatment with a glycosylation inhibitor (tunicamycin) significantly reduced SARS-CoV-2 replication, which may be due to the inhibition of glycosylation of the viral glycoproteins (data not shown). Thus, our studies suggest that cardiomyocytes are susceptible to SARS-CoV-2 infection and that co-infection with IAV primes these cells by inducing the expression of IFN response genes including ACE2 and its glycosylation, which may protect cardiomyocytes from SARS-CoV-2.
In summary, we showed that lung epithelial cells and cardiac fibroblast succumb to SARS-CoV-2 when preinfected with IAV; whereas, cardiomyocytes are resistant to SARS-CoV-2 infection. The cardiomyocytes offer protection by way of upregulating the expression of glycosylated forms of ACE2 and subsequent upregulation of nitric oxide. We hypothesize that nitric oxide upregulation by hiPSC-CMs should help control SARS-CoV-2 infection in the local cardiac microenvironment, which includes other heart cells. Co-infection of SARS-CoV-2 and IAV enhances SARS-CoV-2 infection in vivo in human ACE2 transgenic mice, 11 concurring with our in vitro findings.
Our data suggest that the heart, but not the lung, may be protected during SARS-CoV-2 infection by a previous infection of IAV. It is plausible that flu vaccine may offer cardio protection by enhancing the glycosylated ACE2 expression and also protect the lungs by enhancing the IFN response which controls the SARS-CoV2 pathogenesis in the lungs. However, flu vaccine may give an opportunity to concentrate and treat lung infection in COVID-19 patients, without worrying about a cardiac dysfunction or in patients with preexisting cardiovascular conditions. Importantly, pharmacological agents that specifically enhance ACE2 protein glycosylation and NO donors might be targets for treating lung infections with IAV and SARS-CoV-2.

Limitations of the study
Though our study identifies the basic mechanism of how IAV co-infection restricts SARS-CoV2 replication in hiPSC-CMs by enhancing the glycosylation of ACE2 and its downstream activation of NO production, the role of glycosylated ACE2 on SARS-CoV2 pathogenesis is not clearly identified. Our RNA sequencing data revealed that IAV infection induces the expression of several N -glycosylation genes (MGAT3 and MGAT4A, etc.) in cardiomyocytes only ( Figure 5). To test whether these glycosylation genes are responsible for the glycosylation of ACE2 in cardiomyocytes, we will knockdown these specific genes (MGAT3 and MGAT4A etc.) in hiPSCs and differentiate them into hiPSC-CMs and study their role in IAV + SARS-CoV-2 infectivity. Furthermore, the current manuscript does not address the cells that harbor SARS-CoV2 in IAV co-infected animal hearts. To identify the cell types, we will stain the heart sections with cell specific markers with SARS-CoV2 NP antibody. Although, we have shown that other viral strains such as IBV and MPV, induces ACE2 expression, our studies are limited to PR8 influenza strain rather than the contemporary IAV strains such as H1N1, H3N2 etc. Thus, additional experiments and further investigation are ongoing to clearly dissect the mechanism of IAV mediated SARS-CoV2 pathogenesis.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Biosafety
All work with live SARS-CoV-2 was performed at Biosafety Level 3 (BSL3) according to standard operating procedures approved by the Ohio State University BSL3 Operations Group and Institutional Biosafety Committee. Samples were removed from the BSL3 facility for flow cytometry analysis and confocal microscopy only after decontamination with 4% paraformaldehyde for a minimum of 1 h, according to an in-house validated and approved method of sample decontamination. Total RNA from SARS-CoV-2-infected cells was extracted and purified with the QIAGEN RNeasy Mini Kit.

QUANTIFICATION AND STATISTICAL ANALYSIS
The data was statistically analyzed using the Graph Pad Prism version 9.0 software. The numerical results are presented as mean standard deviation. Quantitative data in histograms, line charts and individual data points were presented as mean G SEM. Statistical analyses were performed using two-tailed unpaired Student's t-tests. p < 0.05 was the criterion for statistically significant group differences.