Abstract
The stroke-prone spontaneously hypertensive rat (SHRSP) develops increased left ventricular mass index (LVMI) prior to the onset of hypertension, making it a fundamental model to better understand human cardiovascular disease. We identified a quantitative trait locus (QTL) for LVMI on chromosome 14 and, by using chromosome 14 congenic strains and gene profiling, have identified osteopontin (Spp1) as a positional candidate gene. Here, we show 1) that Spp1 may promote cardiac remodelling via extracellular vesicle (EV) signalling and 2) provide phenotypic and molecular characterisation of a CRISPR/Cas9 Spp1-knockout rat on the SHRSP genetic background (SHRSP-Spp1 KO). 1) Briefly, H9c2 cells were seeded 24 hours prior to transfection with Spp1 cloned into pcDNA1 (5 ug) for 48 hours. EVs were isolated from conditioned media (CM) via ultracentrifugation, verified by NanoSight, re-suspended in PBS and placed onto fresh H9c2 cells for 48 hours. Crystal violet stained H9c2 cells were analysed using ImageJ. Cells transfected with Spp1 cDNA derived from the SHRSP rat displayed a significant increase in cell size compared with cells transfected with empty pcDNA vector (pcDNA 107.9±1.4 vs SHRSP 141.8±2.3, p 0.05). Similarly, no difference was observed in LV relative wall thickness in SHRSP-Spp1 KO compared to SHRSP (p>0.05). Cardiac fibrosis assessed by both histology and qRT-PCR showed no significant difference in SHRSP-Spp1 KO compared to SHRSP (p>0.05). Overall, these data suggest that the cardiac phenotype of the SHRSP-Spp1 KO rat is no different from SHRSP at baseline. Further studies are required to determine whether a cardiac stress will unmask a difference in phenotype in the SHRSP-Spp1 KO compared to littermate controls.
Paper version not known (
Free)
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call