Abstract NBT204:
Mitochondrial GRK2 dependent regulation of myocardial cellular and transcriptional composition after ischemic injury
Slone, S; Chuprun, K; Tucker, N; Haynes, N; Kayki Mutlu, G; Roy, R; Gao, E; Koch, W
Published in: Circulation Research
Myocardial infarction (MI), an obstruction of coronary blood flow and subsequent adverse remodeling leads to chronic heart failure (HF) thus, pathological remodeling post-MI remains a prevalent etiology of HF. GRK2 [G protein–coupled receptor kinase 2], essential for desensitization of activated GPCRs, associates with poor cardiac function and therefore, a target for reducing HF. Our lab has previously shown phosphorylated GRK2 at Ser670 is required for translocating GRK2 to the mitochondria in myocytes after ischemia promoting myocyte death, however, inducing a GRK2-S670A knock-in (KI) exhibited mitochondria and cardio-protection post-MI. Interestingly, KI mice displayed improved cardiac function and remodeling post-MI compared to wild-type (WT) mice out to 4 weeks then showing equal dysfunction at 8 weeks.
To determine the GRK2-S670A-driven mechanism in non-myocyte cellular population(s) and gene expression in the eventual loss of cardio-protection post-MI.
S670A and WT 10–12 week old male mice were subjected to MI or Sham procedure and hearts were collected for single nucleus (sn) RNA sequencing (10x Genomics). Isolation and culturing of adult cardiac fibroblast (ACFs) and bone marrow-derived macrophages (BMDM) were collected from S670A and WT mouse hearts and femur/tibia bones, respectively. ACFs were stimulated with TGFβ (10ng/ml) for up to 72 hours and BMDMs were stimulated with LPS or IL-4 at 100ng/ml for up to 48 hours in cell culture followed by RNA and protein isolation for molecular analysis.
sn RNA sequence analysis from 1-week post-MI hearts shows preservation of cardiomyocytes in the KI mice and displayed highly divergent gene expression and ontology in major cell types including macrophages, cardiomyocytes, and fibroblast compared to WT mice. Our
data show LPS-primed KI BMDMs have enhanced repolarization capacity and reduced reactive oxygen species post-LPS/IL-4 treatment compared to WT BMDM. Additionally, KI ACFs show enhanced fibrotic gene expression compared to WT ACFs 24 hours post-TGFβ stimulation.
snRNA-seq suggests that alternative myocardium cell types, including fibroblasts and macrophages, contain highly divergent gene expression in GRK2-S670A KI mice compared to WT mice. Additionally, repolarized KI BMDMs show an enhanced anti-inflammatory profile compared to WT BMDMs. Finally, KI ACFs show enhanced expression of fibrotic factors post-activation compared to WT ACFs.
