Shearer, G. | bioRxiv Science

bioRxiv2024

A Cell Autonomous Free fatty acid receptor 4 - ChemR23 Signaling Cascade Protects Cardiac Myocytes from Ischemic Injury

Acute myocardial infarction (AMI) causes ischemic damage and cardiac remodeling that ultimately progresses into ischemic cardiomyopathy (ICM). Coronary revascularization reduces morbidity and mortality from an MI, however, reperfusion also induces oxidative stress that drives cardiac myocyte (CM) dysfunction and ICM. Oxidative stress in CMs leads to reactive oxygen species (ROS) production and mitochondrial damage. Free fatty acid receptor 4 (Ffar4) is a GPCR for long chain fatty acids (FA) that is expressed in multiple cell types including CMs. We have recently shown that CM-specific overexpression of Ffar4 protects the heart from systolic dysfunction in the context of ischemic injury. Mechanistically, in CMs, Ffar4 increases the levels of 18-hydroxyeicosapentaenoic acid (18-HEPE), an eicosapentaenoic acid (EPA)-derived, cardioprotective oxylipin (oxidatively modified FA). 18-HEPE is the precursor for resolvin E1 (RvE1), a cardioprotective, specialized pro-resolving mediator (SPM) that activates the GPCR ChemR23. We hypothesize Ffar4 in CMs protects the heart from oxidative stress and ischemic injury through activation of a CM-autonomous, Ffar4-ChemR23 cardioprotective signaling pathway. Here, we developed an in vitro hypoxia reoxygenation (H/R) model (3 hours of hypoxia, 17 hours of reoxygenation) in adult CMs as a model for ischemic injury. In adult CMs subjected to H/R, TUG-891, an Ffar4 agonist, attenuated ROS generation and TUG-891, 18-HEPE, and RvE1 protected CMs from H/R-induced cell death. More importantly, we found that the ChemR23 antagonist -NETA prevented TUG-891 cytoprotection in adult CMs subjected to H/R, demonstrating that ChemR23 is required for Ffar4 cardioprotection. In summary, our data demonstrate co-expression of Ffar4 and ChemR23 in the same CM, that Ffar4, 18-HEPE, and RvE1 attenuate H/R-induced CM death, and that ChemR23 is required for Ffar4 cardioprotection in H/R support a CM-autonomous Ffar4-ChemR23 cardioprotective signaling pathway.

physiology↗

bioRxiv2020

The Histoplasma capsulatum DDR48 Gene Is Required For Survival Within Macrophages, Response To Oxidative Stress, And Resistance to Antifungal Drugs

Histoplasma capsulatum (Hc) is a systemic, dimorphic fungal pathogen that affects upwards of 500,000 individuals in the United States annually. Hc grows as a multicellular mold at environmental temperatures; whereas, upon inhalation into a human or other mammalian host, it transforms into a unicellular, pathogenic yeast. This manuscript is focused on characterizing the DNA damage-responsive gene HcDDR48. HcDDR48 was originally isolated via a subtractive DNA library enriched for transcripts enriched in the mold-phase of Hc growth. Upon further analysis we found that HcDDR48 is not just expressed in the mold morphotype, but both growth programs dependent upon the environment. We found that HcDDR48 is involved in oxidative stress response, antifungal drug resistance, and survival within resting and activated macrophages. Growth of ddr48{Delta} yeasts was severely decreased when exposed to the reactive oxygen species generator paraquat, as compared to wildtype controls. We also found that ddr48{Delta} yeasts were 2-times more sensitive to the antifungal drugs amphotericin b and ketoconazole. To test HcDDR48s involvement in vivo, we infected resting and activated RAW 264.7 murine macrophages with Hc yeasts and measured yeast survival 24-hours post-infection. We observed a significant decrease in yeast recovery in the ddr48{Delta} strain compared to wildtype Hc levels. Herein, we demonstrate the importance of maintaining a functional copy of HcDDR48 in order for Hc yeasts to sense and respond to numerous environmental and host-associated stressors. ImportanceHistoplasma capsulatum is an intracellular pathogen of phagocytes, where it subverts immune recognition and avoids killing by the innate immune system. Macrophages provide a permissive environment for Hc replication and killing only occurs upon the onset of the T-cell driven adaptive immune response. Hc has evolved numerous virulence factors that aid in its survival against host-derived ROS and RNS in vivo. While these virulence factors have been described in past years, only a few reports describing the regulation of these genes and how this intricate system leads to fungal survival. In this study, we characterized the stress response gene DDR48 and determined it to be indispensable for Hc survival within macrophages. HcDDR48 regulates transcript levels of superoxide dismutases and catalases responsible for detoxification of ROS and contributes to antifungal drug resistance. Our studies highlight DDR48 as a potential target to control Hc infection and decrease the severity of the disease process.

microbiology↗