key: cord-1040869-oq6v94wi
authors: Amendola, Alessandra; Garoffolo, Gloria; Songia, Paola; Nardacci, Roberta; Ferrari, Silvia; Bernava, Giacomo; Canzano, Paola; Myasoedova, Veronika; Colavita, Francesca; Castilletti, Concetta; Sberna, Giuseppe; Capobianchi, Maria Rosaria; Piacentini, Mauro; Agrifoglio, Marco; Colombo, Gualtiero I; Poggio, Paolo; Pesce, Maurizio
title: Human cardiosphere-derived stromal cells exposed to SARS-CoV-2 evolve into hyper-inflammatory/pro-fibrotic phenotype and produce infective viral particles depending on the levels of ACE2 receptor expression
date: 2021-03-10
journal: Cardiovasc Res
DOI: 10.1093/cvr/cvab082
sha: bbff2f28036df1fd340d60edb81a0a5ddb9668f5
doc_id: 1040869
cord_uid: oq6v94wi

AIMS: Patients with severe respiratory syndrome caused by SARS-CoV-2 undergo cardiac complications due to hyper-inflammatory conditions. Although the presence of the virus has been detected in the myocardium of infected patients, and infection of induced pluripotent cells-derived cardiomyocytes has been demonstrated, the reported expression of ACE2 in cardiac stromal cells suggests that SARS-CoV-2 may determine cardiac injury by sustaining productive infection and increasing inflammation. METHODS AND RESULTS: We analyzed expression of ACE2 receptor in primary human cardiac stromal cells derived from cardiospheres, using proteomics and transcriptomics before exposing them to SARS-CoV-2 in vitro. Using conventional and high sensitivity PCR methods, we measured virus release in the cellular supernatants and monitored the intracellular viral bioprocessing. We performed high-resolution imaging to show the sites of intracellular viral production and demonstrated the presence of viral particles in the cells with electron microscopy. We finally used RT-qPCR assays to detect genes linked to innate immunity and fibrotic pathways coherently regulated in cells after exposure to the virus. CONCLUSIONS: Our findings indicate that cardiac stromal cells are susceptible to SARS-CoV-2 infection and produce variable viral yields depending on the extent of cellular ACE2 receptor expression. Interestingly, these cells also evolved toward hyper-inflammatory/pro-fibrotic phenotypes independently of ACE2 levels. Thus, SARS-CoV-2 infection of myocardial stromal cells could be involved in cardiac injury, and explain the high number of complications observed in severe cases of COVID-19. TRANSLATIONAL PERSPECTIVE: In the present investigation, we provide evidence that human cardiac stromal cells, a major component of the non-contractile cellular fraction in the heart can be infected by SARS-CoV-2 in vitro, in direct relationship to the extent of ACE2 receptor expression. Our work also suggests that these cells, when exposed to the virus, can evolve toward inflammatory and fibrotic phenotypes independently of ACE2. In addition to describing a novel cellular target of SARS-CoV-2 in the human heart, our study generates new hypothesis on potential mechanisms underlying cardiac complications observed in COVID-19 patients.

Since the beginning of the SARS-CoV-2 pandemic outbreak, a relatively high incidence of cardiac complications have been reported 1, 2 . These range from elevation of cardiac damage markers such as circulating troponin and BNP 3, 4 , to cardiac arrest 5 , cardiogenic shock 6 , myocarditis 7 and heart failure 8 . The susceptibility of the myocardial tissue to SARS-CoV-2 infection 6, 9 has been inferred based on the expression of the Angiotensin-Converting Enzyme-2 (ACE2) receptor in various cardiac cell types 10, 11 , and the evidence that the virus interacts with this receptor via the Spike (S) protein, as a main cellular docking/internalization site 12 .

In an attempt to explain the cardiac complications observed in patients with COVID-19, experimental studies have tried to assess the direct susceptibility of endothelial cells 13, 14 and induced pluripotent cells (iPSCs)-derived cardiac myocytes 15, 16 to SARS-CoV-2 infection, with contrasting results. Furthermore, individual variations in the level of ACE2 mRNA expression have been reported by single-cell RNA sequencing in human myocardial cells, including cardiac fibroblasts 11 , thus providing a rationale for the possible involvement of these cells in the cardiac damage observed in patients with COVID-19. Cardiac stromal cells (cSt-Cs), often also referred to as cardiac fibroblasts 17 , are non-contractile myocardial cells that fulfill an important accessory function in the heart, i.e. the renewal of the extracellular matrix and maintenance of myocardium structural integrity. These cells can be derived in culture using different isolation methods and express a variety of mesenchymal and/or fibroblast markers, likely related to different origins and maturity stages 18 . Under pathologic conditions, e.g. in response to ischemia, cSt-Cs can acquire pro-inflammatory/pro-fibrotic phenotypes, and participate in cardiac inflammation and fibrosis [19] [20] [21] [22] .

Based on these evidences we hypothesized that cardiac complications observed in COVID-19 could be due, at least in part, to the combined effects of direct infection and pro-inflammatory/pro-fibrotic conversion of cardiac stromal cells. To address this possibility, we analyzed the effects of the SARS-CoV-2 virus on cSt-Cs derived from cardiospheres 23 in vitro, in correlation to the extent of ACE2 receptor expression.

The use of human cells for in vitro experiments was approved by the local ethical committee (approval date : 19 May 2012 and subsequent renewal on 16 May 2016) and has been performed in conformity with the principles of the declaration of Helsinki. Patients gave their written consent to donate small fragments of right atrial appendage before routine coronary bypass grafting interventions. Collection of material occurred before the beginning of the SARS-CoV-2 pandemic outbreak. Experiments performed with SARS-CoV-2 in vitro did not require specific ethical authorization according to a specific instruction ("Data processing in clinical trials and medical research in the context of the COVID-19 health emergency" -article 3), published by the Italian Data Protection Authority to rule the use of patients material in case of experimental studies on COVID-19. See https://www.garanteprivacy.it/temi/coronavirus/faq#English for more information.

Cardiac stromal cells were derived using the 'cardiosphere' method 23 . Briefly, small fragments of the cardiac tissue were let to attach onto the bottom of tissue culture dishes, until an outgrowth of cells was achieved.

Following a mild digestion with Trypsin, cells were recovered and sub-cultured onto Poly-D-Lysine coated dishes for cardiosphere formation. Cardiosphere-derived cells were obtained from mature cardiospheres, typically after 3-5 wks of culture by digestion of the cell aggregates and expansion in fibronectin-coated dishes. Cells were used for experiments at passage 2-3 after derivation from cardiospheres, and characterization with RT-qPCR, Western analysis and flow cytometry for assessment of ACE2 receptor expression (see Supplementary Information).

After thawing, three cSt-Cs lines were plated at a 60% confluence in 6-well culture plates and exposed to variable amounts (0.1, 1, 10 multiplicity of infection, MOI) of SARS-CoV-2 isolates in a biosafety level (BSL) 3 facility 24 in technical quadruplicates. After 2, 24, and 72 hours, cells underwent RNA extraction and immunofluorescence staining, while culture supernatants were collected, as described in Supplementary Information.

To assess changes in cSt-Cs phenotype after exposure to SARS-CoV-2 in vitro, RT-qPCR assays were conducted on total RNA extracted from cells exposed at each viral concentration and time points. In addition, immunofluorescence staining for SARS-CoV-2 in combination with other cellular markers and transmission electron microscopy were performed. Details of the RNA analysis and microscopy methods are provided in the Supplementary Information.

To assess the expression of ACE2 receptor in cardiac stroma cells, we analyzed the levels of ACE2 protein expressed in 10 lines of human cardiospheres-derived cSt-Cs available to our laboratory (see Supplementary   Information for the methodology of isolation and expansion) 23 . Figure 1A shows the results of the ACE2 expression in the cSt-Cs by Western Blotting and RT-qPCR. A relatively high variability in the expression of the receptor was observed (Figure 1A) , with no apparent relationship with demographic characteristics (i.e. age), risk conditions (e.g. dyslipidemia, hypertension) or medication (e.g. anti-hypertensive treatment) ( Table   S1 ). On the other hand, ACE2 protein expression was highly correlated with the levels of ACE2 gene transcription, as verified by a linear regression analysis of the RNA/Protein expression data ( Figure 1A ). This indicates that the control of ACE2 expression in cST-Cs occurs at a transcriptional level. In keeping with results obtained with other primary human-derived mesenchymal cell lines, TMPRSS2 the other major receptor facilitating SARS-CoV-2 infection 25 was not expressed by cSt-Cs (data not shown).

To classify the cells for ACE2 expression, we grouped the cSt-Cs into three discrete classes (high, medium and low expression) based on the distribution of both the ACE2 protein normalized expression level (by Western Blot) and of the 2 -ΔCt gene expression data (by RT-qPCR) data, using K-means clustering ( Figure S1 ). Table S1 . The three cell lines were tested for expression of cardiac fibroblast/mesenchymal markers 26 . As shown in Figure 1B , the expression of CD29 and CD44 was very similar, while a relatively higher variability was observed for the expression of CD90 and CD105, typical markers of cardiac-resident mesenchymal cells 27 . This variability, however, remained within the limits of the general variation in expression of mesenchymal markers in cells amplified from all donors ( Figure 1B) . All these cells, finally, did not express endothelial markers CD31 and CD144 (Figure 1B) , excluding contamination by endothelial cells. To assess the susceptibility to SARS-CoV-2 infection, we exposed them to increasing amounts (multiplicity of infection, MOI: 0.1, 1, and 10; see Supplementary information for details) of SARS-CoV-2 isolates 24 , and monitored the appearance of cytopathic effects, from two to 72 hours post-infection. Visual inspection of the cells revealed potential differences according to the infection rate, with clear signs of cytopathic effects in ACE2 Hi cells already at two hours after the viral absorption, and consisting of cell rounding and wrinkling, cytoplasmic volume reduction, and detachment ( Figure 1C ). These effects were noticeable at a lower extent, in 'Mid' cells, and undetectable in ACE2 'Lo' cSt-Cs, even at 72 hours post-infection (data not shown).

In order to monitor the viral yield in the culture supernatants of the three cell lines, we performed RT-qPCR to and 'Hi' cells exhibited an increase in the curve of viral RNAs amplification, evident for 'Mid' cells at 72 hours at 10 MOI, and for 'Hi' cells at 24 and 72 hours at all the MOIs, suggesting viral production. To confirm these data more quantitatively, we assessed the copy number of the virus in the supernatant using a digital-PCR (dPCR) amplification protocol, using primers specific for the SARS-CoV-2 N2 gene region. Digital PCR methods, in our and others' hands, are more sensitive than conventional PCR to detect SARS-CoV-2 copies in biological fluids with low viral titers 28, 29 . As shown in Figures S2 and 1F , determination of the viral copy number was more precise with this technique. In particular, it was possible to appreciate that also the cell line expressing the lowest ACE2 levels produced viral particles in the supernatant (e.g. 72 hours, 10 MOI), even though their amount was almost three Log10 lower than those produced by the 'Hi' cells under the same conditions. To confirm that these viral particles are infective, we finally exposed the kidney-derived Vero E6 cell line 30 to the supernatants of the infected cSt-Cs, followed by the determination of the fifty-percent tissue culture infective dose (TCID50, see 

In order to investigate SARS-CoV-2 intracellular bioprocessing, we first assessed the temporal dynamics of E, N2 and RdRP viral transcripts in RNAs of cSt-Cs extracted cellular. In line with the previous results, clear differences were detected in the copy number of these genes in RNAs extracted from the different cell lines, with very limited number of copies/µL in 'Lo' cells at all the employed MOIs and MOI-dependent increases in 'Mid' and 'Hi' cells (Figure 2A) . To find microscopic evidences of viral intracellular replication, we then performed immunofluorescence on ACE2 'Lo' and 'Hi' infected cells using a human anti-SARS-CoV-2 serum, together with activated fibroblasts/myofibroblasts markers alpha-smooth muscle actin (αSMA) and/or Collagen-1 (Col1). associated with intense viral production ( Figure 2D) 31 , or the formation of a reticulo-vesicular ER network supporting SARS-CoV-2 replication 32 . It was also interesting to observe that when cells exhibiting SARS-CoV-2 staining were found in contact with non-infected cells (Figure 2F ), viral particles appeared to transit from the positive cell to the surrounding negative cells (see inset in Figure 2F ). This suggests that SARS-CoV-2 may transfer from infected to uninfected cSt-Cs by direct cell-to-cell transfer, the so-called 'virological synapse', one of the modalities of viral intercellular propagation inside tissues 33 .

To substantiate further the presence of the virus in cSt-Cs and reveal signs of cytotoxicity, we performed ultrastructural analysis using transmission electron microscopy (TEM). The ultrastructural features of the infected cells exhibited clear differences from those of non-infected cells (Compare panel b in Figure 3A with panel c in Figure 3B) . Namely, infected cells exhibited swelling of the rough endoplasmic reticulum (rER) with ribosomes frequently dissociated from the ER structure. This observation has been previously associated with high level of viral production 31 , and again suggests the formation of reticulo-vesicular ER networks supporting SARS-coronavirus replication 32 , as also shown in Figure 2D . Cells exposed to the virus also showed bigger multilamellar bodies (LB) visible in the cell cytoplasm compared to control cells (compare panel c in Figure 3A with panel b in Figure 3B ). SARS-CoV-2 virions were detected, alone or in clusters, predominantly in intracellular compartments (i.e. vacuoles) (Figure 3B panels c, d and 3C panel c), as previously shown in different cultured cell lines and lung cells of infected patients 34 . Finally, cells exhibiting a high number of viral particles outside intracellular structures were found (Figure 3C   panels a, b) and clear signs of s of degeneration, such as cytoplasm condensation were also observed ( Figure 3C panels a, b, d) .

CSt-Cs have a central role in cardiac healing following acute injury, as they trigger the production of inflammatory cytokines and extracellular matrix remodeling enzymes necessary for the recruitment of leukocytes and activation of the innate immunity process priming myocardium repair 20 . Since SARS-CoV-2 infection causes sharp upregulation of inflammatory cytokines in target organs through infection-dependent and innate immunity signaling mediated by Toll-like receptors 35 , cardiac inflammation observed in COVID-19 patients may result from a combination of the systemic 'cytokine storm' and a direct inflammatory response by cardiac-resident cells 36 . To assess this hypothesis, we tested the effects of the virus on the activation of inflammatory factors and genes potentially involved in cardiac fibrosis 16 . We therefore analyzed the expression of genes involved in innate immune response and cardiotoxicity using RNAs extracted from the three cell lines infected with 10 MOI SARS-CoV-2 for 2, 24 and 72 hours. To do this, we used low-density PCR arrays containing primers specific for tissue inflammation and fibrosis, as well as single RT-qPCR tests (see Supplementary Information for further details). As shown in Table S4 , 17 genes out of the 168 contained in the lowdensity arrays were significantly over/down modulated in infected cells vs. the uninfected cells at the same time point. This regulation was clearly time dependent and did not reflect differences in ACE2 expression. Unsupervised clusterization of the average fold changes ( Figure 4A ) revealed a coordinated regulation of genes that were significantly more expressed at early (HSP1, PD4, FOSL, BCL2A1, HMOX), intermediate (ITPR2, RND1, ZNF148) , and late (NEXN, SERPINE1, ZNF23, CCL7, FHL1, ICAM1, EGR1, STAT-1) time points, respectively. In particular, the genes that were significantly upregulated as early as at 2 hours post-infection indicate an early response of cSt-Cs to stress conditions determined by exposure to the virus. For example, HSPH1 (hsp110) is a heat shock protein strongly upregulated in response to coronaviruses exposure and in particular to their Envelope (E) proteins 37 , while FOSL1 is a transcription/cellular factor engaged in Interferon signaling in responses to viral infection 38 . It was remarkable to note that some of the transcripts significantly upregulated at 72 hours post-infection in response to virus encode for, i) a membrane adhesion protein involved in cell-to-cell intercellular viral transmission (e.g. ICAM1) 39 ; ii) a chemokine with potent pro-inflammatory effects (CCL7/MCP3) in COVID-19 40, 41 ; and iii) transcriptional regulators EGR1 and STAT1 involved, respectively, in SARS-CoV-related TGF-β1 signaling 42 and immune response in COVID-19 patients 43 . We finally investigated the regulation of mRNAs encoding for key factors involved in COVID-19 'cytokine storm' and cardiac inflammatory/fibrotic responses 1, 44, 45 . Results of single RT-qPCR tests clearly indicated that cells from the three cell lines responded to viral exposure with a time-dependent upregulation of pro-fibrotic genes CTGF, ACTA2, Col1A and Col3A and of inflammatory cytokines IL-1β and CCL2 (MCP1) and, to a lower extent, IL-6 mRNAs irrespective of ACE2 expression levels ( Figure 2B, C) . Together, these results highlight an additional cardiac pathogenesis mechanism by SARS-CoV-2 independent of ACE2 expression, consisting of substantial upregulation of genes involved in response to viral infection, intercellular virus transmission and related to innate immunity signaling and fibrotic activation.

This includes endothelial 13 , kidney and urogenital tract cells 46 , enterocytes 47 , and a variety of human iPSCsderived cell types 48 , including cardiac myocytes 16 . As of today, despite the numerous reports showing extensive cardiac damages consequent to infection 49 and the presence of the virus in myocardial biopsies 6, 50 , there is still uncertainty about the underlying mechanisms 36 . As outlined in various cardiology-oriented reviews on COVID-19 pathophysiology 1, 2 , the heart could be affected by cumulative effects of the cytokine storm elicited by innate immunity activation 35 , as well as of in situ cytopathic effect determined by direct infection and replication of the virus in the myocardium 6, 50, 51 . In the present study, we provide the proof-of-concept that human myocardial stromal cells are susceptible to infection and permissive for intracellular replication of SARS-CoV-2. We also show that viral infectivity and productivity are strictly related to the expression level of the ACE2 receptor, thus confirming the influence of variations in the expression of this receptor, observed in different individuals, on the different responses to SARS-CoV-2 infection, as reported elsewhere 11 . Interestingly, the expression of ACE2 in stromal cells seemed not to be associated with anti-hypertensive therapy taken by donors of the cells (Table S1) , thus excluding a correlation between the susceptibility of the cells to infection and the known modulation of the receptor determined by regulators of the Renin-Angiotensin System, as discussed recently 52 .

In our experiments, we constantly observed the presence of cells that did not exhibit SARS-CoV-2 staining nor cytopathic effects, even among those with 'Hi' ACE2, even at 72 hours post-infection (Figure 2) . (Figure 2F ).

While this may reflect an inefficient viral replication in stromal cells (such as demonstrated recently for endothelial cells 14 ) , it may also reflect a heterogeneous expression levels of ACE2 co-receptors 53 . Finally, our high-resolution confocal images (Figure 2F ) also suggest the possibility of direct transmission of the virus via cell-to-cell contacts, a modality of intracellular transmission similar to that observed for other viruses, the viral synapses 33 .

Our study suggests a second potential pathogenic mechanism that may be independent of direct penetration and replication of SARS-CoV-2 in cSt-Cs. Indeed, exposure to the virus was able per se to elicit inflammatory and pro-fibrotic responses in cSt-Cs independently of the expression of ACE2. This evidence is supported by the transcriptomic data presented in Figure 4 , where we show a similar time-dependent trend in up/down-regulation of genes related to inflammation and fibrosis. In this regard, we hypothesize that cells exposed to the virus could mount an innate immune response by activating the nuclear factor-κB (NF-κB) pathway via the interaction of the viral Spike protein with Toll-like receptors (TLRs) 54, 55 , leading to upregulation of IL6 and other proinflammatory cytokines such as IL1, IL2, TNF-α and Interferon (IFN)-γ, as well as pro-fibrotic genes.

Taken together, these data suggest that the variability of SARS-CoV-2 infection and spreading modality observed in the cardiac stroma may contribute to the puzzling scenario of COVID-19 cardiac complications. In particular, based on the data of the present report, we hypothesize that SARS-CoV-2 might contribute to myocardial damage with potentially cumulative effects of i) an intra-myocardial cytopathic effects due to viral replication in the stromal component directly connected to ACE2 expression levels and, ii) an ACE2-independent innate immunity response boosting myocardial inflammation and fibrosis 45 . Given the prevalently hypothesis-generating nature of our investigation, it is impossible at the moment to determine whether one of these two modalities, or both of them are prevalent in cardiac injury observed in COVID-19 patients.

The present work was conducted using cells derived with the cardiosphere method 23 , one of the procedures used historically to obtain cells with mesenchymal-like characteristics from the human heart 27 . Since in our experiments, cells were expanded from cellular outgrowths of right atrial appendage fragments, and through several culture passages, a potential limitation may be the selection of specific cellular phenotypes, thus determining an overall under-representation of the various stromal cells/fibroblasts subtypes present in the heart, as recently demonstrated 56 .

A second limitation of our study is the reduced sample size and the lack of specific functional studies (e.g. loss of function) allowing to correlate directly the function of ACE2 receptor with viral entry and intracellular virus packaging inside cSt-Cs.

Caution should be finally adopted in extending our findings to the situation encountered in COVID-19 patients, where still today there is a heated debate about the direct vs. the indirect effects of SARS-CoV-2 on cardiac inflammation and fibrosis, including the occurrence of actual myocarditis 57 . In this regard, future studies using cells derived from various districts of the human heart, e.g. atrial vs. ventricular fibroblasts, or combining multiple cell types, e.g. cardiac fibroblasts and iPSC-derived cardiomyocytes (which also susceptible to infection 16 ) in tissue constructs/organoids exposed to the virus, should be performed to address this important point. 

The authors declare no competing interests. 

The data contained in the present study will be available upon written request to the corresponding author (Table S1 ). ACE2 band is colored in green with a molecular weight (MW) ~86 kDa. In red the GAPDH bands (MW 37 kDa) used for data normalization. On the right side, it is indicated the result of a linear regression analysis of protein/RNA data in the same cells, showing a highly significant data correlation. In color it is indicated the 90% confidence interval. In both panels, numbers and symbols in color indicate, respectively, the data from the 'Lo' (blue), 

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As shown, expression of pro-fibrotic genes (B) exhibited variability in relationship to the viral dose used in infection experiments, especially for Col1A and CTGF. In contrast, expression of genes encoding for the pro-inflammatory cytokines (C) was more consistently upregulated in the three lines above the level of uninfected cells already at 24 hours of expression

overlapping the values of the gene expression fold changes in each cell line (color coded as in Fig 1A and Table S1) to the bar graphs indicating the average and the standard error of the data. In both panels * indicates P < 0.05 statistical significance by one-way ANOVA analysis (repeated measures) calculated on the ΔCt values for each gene at each viral concentration used for the infected vs