Attenuation of Microbiotal Dysbiosis and Hypertension in a CRISPR/Cas9 Gene Ablation Rat Model of GPER1

Abstract

G-protein-coupled estrogen receptor, Gper1, has been implicated in cardiovascular disease, but its mechanistic role in blood pressure control is poorly understood. Here, we demonstrate that genetically salt-sensitive hypertensive rats with complete genomic excision of Gper1 by a multiplexed guide RNA CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated proteins) approach present with lower blood pressure, which was accompanied by altered microbiota, different levels of circulating short chain fatty acids, and improved vascular relaxation. Microbiotal transplantation from hypertensive Gper1^+/+ rats reversed the cardiovascular protective effect exerted by the genomic deletion of Gper1. Thus, this study reveals a role for Gper1 in promoting microbiotal alterations that contribute to cardiovascular pathology. However, the exact mechanism by which Gper1 regulates blood pressure is still unknown. Our results indicate that the function of Gper1 is contextually dependent on the microbiome, whereby, contemplation of using Gper1 as a target for therapy of cardiovascular disease requires caution.

Keywords: CRISPR-Cas systems; blood pressure; genomics; hypertension; microbiota.

Figure 1.. Screening animals for CRISPR/Cas9 mediated deletion of Gper1.

(a) Representative agarose gel picture of PCR amplified tail DNA samples from pups born post-microinjection of CRISPR/Cas9 mediated deletion of Gper1. Primer A+B and C+D encompass 3’ and 5’ ends of Gper1 however Primer A+D encompasses entire gene. The inset shows schematic of position of gRNAs cutting sites and the primers used for genotyping. The first lane after DNA ladder in all three gels is wild-type S rat DNA, Animal #1 through 10 are homozygous founders which show no band in 3’ and 5’ primer ends and shorter DNA band (~750 bps) in Primer A+D compared with S band (2333 bps). (b) Representative sequencing results from the PCR products of homozygous founders shown in panel A detected 1530 bps deletion in the DNA of Gper1 mutant rats. (c) RT-PCR analysis of mRNA expression of Gper1 in the heart homogenate using 18S RNA as a housekeeping gene (RT- reverse transcriptase).

Figure 2.. Attenuation of blood pressure of Gper1^−/− rats.

(A) Females, (B) Males. Radiotelemetry measurement of mean arterial pressure of wild-type hypertensive rats (n=8 females, 10 males) and Gper1^−/− rats (n=7 females, 12 males). Rats were monitored for BP, 3 days after recovery from surgical implantation of radiotelemetry transmitters. Data plotted are the 4 hours moving average of recordings obtained every 5 min continuously for 24h. Levels of statistical significance for all data were analyzed by independent sample t-test. Blood pressure of Gper1^−/− female and male rats was significantly lower than that of wild-type hypertensive rats.(* p<0.05, ** p<0.01, *** p<0.001)

Figure 3.. Gper1^−/− female and male rats demonstrated superior vascular function.

Third order mesenteric arteries were dissected and mounted on wire myograph chamber. (A, D) Cumulative concentration response curve (CCRC) to phenylephrine (1nM-10μM) of mesenteric arteries was recorded from Gper1^+/+ (n=12) and Gper1^−/− rats (n=12). (B, E) Endothelium-dependent relaxation to ACh was assessed by adding increasing concentrations of ACh to the vessel preparation. CCRC to ACh (1nM-10μM) of mesenteric arteries was recorded from Gper1^+/+ (n=9) and Gper1^−/− rats (n=11). Relaxation of ACh was expressed as a percentage of level of pre-contraction induced by submaximal dose of phenylephrine. (C,F) Endothelium-independent relaxation to SNP was assessed by adding increasing concentrations of SNP to the vessel preparation. CCRC to SNP (1nM-10μM) of mesenteric arteries was recorded from Gper1^+/+ (n=11) and Gper1^−/− rats (n=12). Relaxation of SNP was expressed as a percentage of level of pre-contraction induced by submaximal dose of phenylephrine. The bar graphs are the maximum response recordings of respective vasoconstrictor and vasorelaxants. *p<0.05, **p<0.01

Figure 4.. The blood pressure protecting effect in Gper1^−/− rats was reversible with transplantation of cecal content from wild-type hypertensive rats.

Radio telemetry measurement of mean arterial pressure (MAP) of wild-type hypertensive rats (n= 8) and Gper1^−/− rats (n=7) (females (A) and males (B)), 21 days after transplantation of cecal content of wild-type hypertensive rats. Levels of statistical significance for all data were analyzed by independent student’s t-test. *p<0.05

Figure 5.. Microbial sequencing in fecal samples of S and Gper1^−/− rats.

Principal Coordinates Analysis (PCoA) plots at day 4 and day 28 of male rats (A). PCoA plots were used to visualize differences in weighted Unifrac distances of fecal samples from the wild-type day 4 cohort (n=9), Gper1^−/− rat day 4 cohort (n=6), wild-type day 28 (n=6) and Gper1^−/− rat day 28 (n=7) samples. Points clustered more closely together are more similar in terms of phylogenetic distance, whereas points that are distant from each other are phylogenetically distinct. To further observe differences in beta diversity between Gper1^−/− and wild-type rats within each time-point, separate PCoA plots were generated for day 4 (B) and day 28 (C). Relative abundance plots to display differences in general microbial community structure between fecal samples collected from rats at day 4 (D) and day 28 (E).

Figure 6.

Plasma circulating SCFAs level and vascular responses of Gper1^−/− vs. wild-type rats to SCFAs. A. Serum acetate level in S and Gper1^−/− rats at Day 4 and Day 28. Targeted metabolomic study demonstrated that of the three SCFAs, serum acetate level was significantly increased in the Gper1^−/− rats administered with cecal content of S rats. B. Decreased relaxation of rat small mesenteric arteries (SMAs) to 5mM sodium acetate and sodium butyrate in Gper1^−/− rats compared with S rats. Mean phenylephrine (10 μM) contraction amplitude measured are 1-minute time intervals and normalized to amplitude at 3 minutes. The solution was changed at 3 min to that containing short chain fatty acids. The relaxation plot for a) acetate, b) propionate and c) butyrate is shown and compared between wild-type and Gper1^−/− rat SMAs.