Preprint Cardiovascular symptoms of PASC are associated with trace-level cytokines that affect function of stem cell derived cardiomyocytes, 2024, Sinclair+

SNT Gatchaman · Apr 13, 2024

Cardiovascular symptoms of PASC are associated with trace-level cytokines that affect the function of human pluripotent stem cell derived cardiomyocytes
Jane Sinclair; Courtney Vedelago; Feargal Ryan; Meagan Carney; Meredith Redd; Miriam Lynn; Branka Grubor-Bauk; Yuanzhao Cao; Anjali Henders; Keng Yih Chew; Deborah Gilroy; Kim Greaves; Larisa Labzin; Laura Ziser; Katharina Ronacher; Leanne Wallace; Yiwen Zhang; Kyle Macauslane; Daniel Ellis; Sudha Rao; Lucy Burr; Amanda Bain; Benjamin L. Schulz; Junrong Li; David J. Lynn; Nathan Palpant; Alain Wuethrich; Matt Trau; Kirsty Short

Globally, over 65 million individuals are estimated to suffer from post-acute sequelae of COVID-19 (PASC). A large number of individuals living with PASC experience cardiovascular symptoms (i.e. chest pain and heart palpitations) (PASC-CVS). The role of chronic inflammation in these symptoms, in particular in individuals with symptoms persisting for >1 year after SARS-CoV-2 infection, remains to be clearly defined.

In this cross-sectional study, blood samples were obtained from three different sites in Australia from individuals with i) a resolved SARS-CoV-2 infection (and no persistent symptoms i.e. Recovered), ii) individuals with prolonged PASC-CVS and iii) SARS-CoV-2 negative individuals.

Individuals with PASC-CVS, relative to Recovered individuals, had a blood transcriptomic signature associated with inflammation. This was accompanied by elevated levels of pro-inflammatory cytokines (IL-12, IL-1beta;, MCP-1 and IL-6) at approximately 18 months post-infection. These cytokines were present in trace amounts, such that they could only be detected with the use of novel nanotechnology. Importantly, these trace-level cytokines had a direct effect on the functionality of pluripotent stem cell derived cardiomyocytes in vitro. This effect was not observed in the presence of dexamethasone. Plasma proteomics demonstrated further differences between PASC-CVS and Recovered patients at approximately 18 months post-infection including enrichment of complement and coagulation associated proteins in those with prolonged cardiovascular symptoms.

Together, these data provide a new insight into the role of chronic inflammation in PASC-CVS and present nanotechnology as a possible novel diagnostic approach for the condition.

Link | PDF (Preprint: BioRxiv) [Open Access]

Hutan · Apr 13, 2024

RNA sequencing for gene expression
25 PASC (persistent symptoms)
11 Recovered
14 healthy controls negative for SARS-CoV-2 spike protein (HC)
(note that these are already rather small numbers)

samples collected from healthy controls (HC) separated from those with any past COVID-19 (‘Any COVID-19’). However, samples from PASC/Recovered individuals tended to cluster together (Figure 1 A).
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So people with a history of Covid-19 looked different to healthy controls.
Figure 1A is blurred in this preprint, but it is clear that PASC and Recovered are not separated in the Principal Component Analysis.

At 44 weeks after infection, PASC had more than 400 differentially expressed genes while Recovered had less than 200 differentially expressed genes, compared to healthy controls.
At 68 weeks after infection, both PASC and Recovered had around 200 differentially expressed genes.

Gene set enrichment analysis - to identify affected pathways

To investigate which pathways, modules or biological processes were enriched among the DEGs, we performed a comprehensive gene set enrichment analysis (GSEA) using the molecular signatures database (mSigDB), focusing on gene-sets from the KEGG, REACTOME and Gene Ontology (GO) databases together with pre-defined blood transcriptional modules (BTMs)29.
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Comparing HCs and any individuals who had had Covid-19:

any COVID-19 individuals showed an up-regulation of pathways related to RNA processing, transcription and translation, signatures which were driven by the upregulation of ribosomal RNA (rRNA genes). (Figure 1C).
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Comparing PASC and Recovered:

Relative to Recovered individuals, PASC individuals had an up-regulation of the same translation and ribosomal signatures observed in any COVID-19 individuals at 44 wpi, but not 68 wpi (Figure 1D). At 68 wpi, the most significantly enriched Reactome pathway and Gene Ontology terms were related to erythrocytes and the production of antimicrobial peptides (Figure 1D). T cell and NK cell related BTMs were also found to be up-regulated in PASC compared to Recovered individuals (Figure 1D).
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PASC-CVS (PASC with cardiovascular symptoms) - compared with Recovered

A subset of the PASC group was considered as PASC-CVS based on self-reported chest pain (n=4 at each timepoint) (Figure 1E). Compared to Recovered individuals, PASC-CVS individuals had a more pronounced up-regulation inflammation related pathways/terms including the neutrophil degranulation, antimicrobial peptides, and response to bacterium pathways/terms at both 44 and 68 wpi and interferon signalling and at 44 wpi only (Figure 1E). BTMs related to monocytes, DCs, and immune activation were also up-regulated (Figure 1E). Many of these pathways were also significant when comparing PASC-CVS to other PASC individuals (Figure 1D&E).
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To reiterate - there were only 4 individuals at each timepoint with PASC-CVS. It's not clear to me yet whether they were even the same 4 individuals at each timepoints. So, it seems a bit of an odd focus of the paper and rings alarm bells for cherry-picking.

Hutan · Apr 13, 2024

But wait - there's more!
More PASC-CVS compared with Recovered

To determine whether the observed transcriptional differences in the blood of PASC-CVS donors could be replicated in an independent cohort, blood samples were additionally collected from age and sex-matched PASC-CVS (n=5) and Recovered (n=4) participants from Cohort 2 at >520 days post infection (Supplementary Table 2). Bulk RNASeq was independently performed on these whole blood samples (mean 55.9 million 2x150bp reads per sample).GSEA with pre-defined BTM identified numerous pathways were significantly enriched in PASC-CVS samples, predominately those associated with the immune response (e.g. ‘neutrophil degranulation’, ‘humoral immune response’ ‘interferon alpha/beta signalling’ and ‘inflammatory molecules’) (Figure 2). Pathways such as ‘platelet activation’ and blood coagulation were also enriched in PASC-CVS samples (Figure 2).
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They found an independent cohort of 5 PASC-CVS and 4 Recovered at >520 days post-infection. So, again, very small numbers. It sounds as though similar pathways were identified as in the first PASC-CVS versus Recovered analysis, but it's a bit vague.

Figure 2 does look quite convincing, but, you know, ...5 PASC-CVS and 4 Recovered. Are we just seeing the difference between people with and without heart disease? Do the PASC-CVS actually have much to do with PASC with fatigue?

And there's still more, but taking a break.

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Preprint Cardiovascular symptoms of PASC are associated with trace-level cytokines that affect function of stem cell derived cardiomyocytes, 2024, Sinclair+

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Preprint Cardiovascular symptoms of PASC are associated with trace-level cytokines that affect function of stem cell derived cardiomyocytes, 2024, Sinclair+

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