The stroke-prone spontaneously hypertensive rat (SHRSP) is known to have exaggerated sympathetic nerve activity to various types of stress, which might contribute to the pathogenesis of severe hypertension and stroke observed in this strain. Previously, by using a congenic strain (called SPwch1.72) constructed between SHRSP and the normotensive Wistar-Kyoto rat (WKY), we showed that a 1.8-Mbp fragment on chromosome 1 (Chr1) of SHRSP harbored the responsible gene(s) for the exaggerated sympathetic response to stress. To further narrow down the candidate region, in this study, another congenic strain (SPwch1.71) harboring a smaller fragment on Chr1 including two functional candidate genes, Phox2a and Ship2, was generated. Sympathetic response to cold and restraint stress was compared among SHRSP, SPwch1.71, SPwch1.72 and WKY by three different methods (urinary norepinephrine excretion, blood pressure measurement by the telemetry system and the power spectral analysis on heart rate variability). The results indicated that the response in SPwch1.71 did not significantly differ from that in SHRSP, excluding Phox2a and Ship2 from the candidate genes. As the stress response in SPwch1.72 was significantly less than that in SHRSP, it was concluded that the 1.2-Mbp congenic region covered by SPwch1.72 (and not by SPwch1.71) was responsible for the sympathetic stress response. The sequence analysis of 12 potential candidate genes in this region in WKY/Izm and SHRSP/Izm identified a nonsense mutation in the stromal interaction molecule 1 (Stim1) gene of SHRSP/Izm which was shared among 4 substrains of SHRSP. A western blot analysis confirmed a truncated form of STIM1 in SHRSP/Izm. In addition, the analysis revealed that the protein level of STIM1 in the brainstem of SHRSP/Izm was significantly lower when compared with WKY/Izm. Our results suggested that Stim1 is a strong candidate gene responsible for the exaggerated sympathetic response to stress in SHRSP.

To clarify the genetic mechanisms of stroke susceptibility in the stroke-prone spontaneously hypertensive rat (SHRSP), a quantitative trait locus (QTL) analysis was performed. Using 295 F2 rats of a cross between SHRSP/Izm and SHR/Izm, 2 major QTLs for stroke latency under salt loading were identified on chromosomes (chr) 1 and 18. Evaluation of 6 reciprocal single and double congenic rats for these QTLs showed that substitution of the SHRSP for the SHR fragment at the chr 1 and 18 QTLs increased the relative risk for stroke by 8.4 and 5.0, respectively. The combined effect of the 2 QTLs was 10× greater than that of the background genome (by Cox hazard model). Blood pressure monitoring by radio telemetry indicated that the combination of the 2 QTLs had a clear effect on the salt-dependent blood pressure increase, suggesting an important role for the salt-sensitive blood pressure increase in the susceptibility of SHRSP to stroke. A haplotype analysis of 11 substrains of SHRSP and SHR using 340 simple sequence repeat markers in the chr 1 QTL suggested that the 7-Mbp fragment between D1Rat260 and D1Rat178 was most likely to harbor the responsible gene(s), which was confirmed by a study of additional subcongenic strains. This study indicated a major role for 2 QTLs on chr 1 and 18 in stroke susceptibility in SHRSP under salt loading. The salt-sensitive blood pressure increase was implied to play a key role in the stroke susceptibility.

We previously revealed that two major quantitative trait loci (QTLs) for stroke latency of the stroke-prone spontaneously hypertensive rat (SHRSP) under salt-loading were located on chromosome (Chr) 1 and 18. Here, we attempted further dissection of the stroke-QTLs using multiple congenic strains between SHRSP and a stroke-resistant hypertensive rat (SHR). Cox hazard model among subcongenic strains harboring a chromosomal fragment of Chr-1 QTL region showed that the most promising region was a 2.1 Mbp fragment between D1Rat177 and D1Rat97. The QTL region on Chr 18 could not be narrowed down by the analysis, which may be due to multiple QTLs in this region. Nonsynonymous sequence variations were found in four genes (Cblc, Cxcl17, Cic, and Ceacam 19) on the 2.1 Mbp fragment of Chr-1 QTL by whole-genome sequence analysis of SHRSP/Izm and SHR/Izm. Significant changes in protein structure were predicted in CBL-C and CXCL17 using I-TASSER. Comprehensive gene expression analysis in the kidney with a cDNA microarray identified three candidate genes (LOC102548695 (Zinc finger protein 45-like, Zfp45L), Ethe1, and Cxcl17). In conclusion, we successfully narrowed down the QTL region on Chr 1, and identified six candidate genes in this region.