Using RNA-seq as an unbiased approach to identify adaptive responses to mild vs. moderate chronic hypercapnia in goats
Authors: Grams Kirstyn , Neumueller Suzanne , Burgraff Nicholas , Mouradian Gary , Pan Lawrence , Hodges Matthew , Forster Hubert 
Published: May 2023

Chronic hypercapnia (CH, PaCO2 > 45mmHg) is commonly reported in chronic obstructive pulmonary disease (COPD). The severity of CH can increase acutely during frequent acute-on-chronic exacerbations and/or chronically as COPD progresses. We have shown (Buchholz et al., 2022) in awake goats that mild (PaCO2 ~55mmHg) and moderate (~65mmHg) CH elicits similar time-dependent steady-state physiologic adaptations. However, during acute-on-chronic CO2 challenges, moderate CH chronically suppressed ventilatory CO2 sensitivity whereas mild CH only transiently decreased it suggesting limitations in adaptation to CH in respiratory control. To gain mechanistic insight, we previously identified transient changes in markers of glutamate-receptor plasticity, interleukin-1 Beta signaling, and serotonergic neuromodulation within key nodes of cardiorespiratory control in mild CH. However, these changes did not appear to account for the observed physiologic changes, indicating a broader unbiased approach is needed to identify molecular shifts in pathways altered by CH. Here, utilizing bulk tissue RNA-Sequencing (RNAseq), we tested the hypothesis that mild CH alters gene expression and signaling pathways that are distinct from moderate CH in key nodes of cardiorespiratory control. Female goats were randomly assigned into 3 experimental groups (n=6/group): Group 1 (control) was exposed to room air for 14 days (d), Group 2 (Mild CH) was exposed to 6% inhaled CO2 (InCO2) for 14d and Group 3 (Moderate CH) was exposed to 6% InCO2 for 7d followed by 8% InCO2 for an additional 7d. Total RNA was extracted from flash-frozen tissue biopsies of the retrotrapezoid nucleus (RTN), nucleus tractus solitarius (NTS), and ventral respiratory column (VRC). cDNA libraries were generated for paired-end whole transcriptome sequencing. When compared to room air control, we found that sites of cardiorespiratory control in mild CH were highly enriched with upregulated genes involving innate immunity, cytokine activity, T-cell activation, and vascular function. Alternatively, moderate CH downregulates genes related to adaptive immunity, cellular function and structure, and vascular function. Both mild and moderate CH enriched pathways of antigen processing and presentation and immune response. However, the genes and their transcriptional activity in these pathways greatly differ between mild and moderate CH. RNAseq and pathway analyses reveal that varying severities of CH result in different gene expression profiles, primarily related to immune function. These contrasting findings between mild and moderate CH suggests that neuroinflammation may influence the respiratory network’s ability to adjust to physiologic challenges of varying severities to chronic and acute-on-chronic hypercapnia. National Heart, Lung, and Blood Institute Grant F31HL159908; Department of Veterans Affairs Grant BX003284. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.