Pathophysiological significance of Stim1 mutation in sympathetic response to stress and cardiovascular phenotypes in SHRSP/Izm: In vivo evaluation by creation of a novel gene knock-in rat using CRISPR/Cas9

ABSTRACT

Genetic approach using rat congenic lines between SHRSP/Izm and WKY/Izm identified stromal interaction molecule 1 (Stim1), an essential component of store-operated Ca²⁺ entry (SOCE), as a promising candidate gene responsible for the exaggerated sympathetic response to stress in SHRSP. Since SHRSP has a nonsense mutation in Stim1 resulting in the expression of a truncated form of STIM1 that caused reduction of SOCE activity in primary cultured cerebral astrocytes, we created SHRSP/Izm knocked-in with the wild-type Stim1 (KI SHRSP) by the CRISPR/Cas9 method to investigate whether the functional recovery of STIM1 would mitigate sympatho-excitation to stress in vivo in SHRSP. No potential off-target nucleotide substitutions/deletions/insertions were found in KI SHRSP. Western blotting and fluorescent Ca²⁺ imaging of astrocytes confirmed wild-type STIM1 expression and restored SOCE activity in astrocytes from KI SHRSP, respectively. Blood pressure (BP) measured by the tail-cuff method at 12, 16, and 20 weeks of age did not significantly differ between SHRSP and KI SHRSP, while the heart rate of KI SHRSP at 16 and 20 weeks of age was significantly lower than that of age-matched SHRSP. Unexpectedly, the sympathetic response to stress (evaluated with urinary excretion of norepinephrine under cold stress and BP elevation under cold/restraint stress) did not significantly differ between SHRSP and KI SHRSP. The present results indicated that the functional deficit of STIM1 was not a genetic determinant of the exaggerated sympathetic response to stress in SHRSP and that it would be necessary to explore other candidates within the congenic fragment on chromosome 1.

KEYWORDS:

• SHRSP
• CRISPR/CAS9
• Stim1
• store-operated Ca²⁺ entry (SOCE)
• sympathetic response
• stress

Acknowledgments

The authors thank Kohei Kawakami and Hiroyuki Matsuo (Department of Experimental Animals, Shimane University) for their technical assistance and Satoko Mishima (Department of Functional Pathology, Shimane University) for her secretarial work.

Author contributions

HO and TN: conceived, designed, supervised the study, and wrote the manuscript. BO: performed telemetry experiments and analyzed the data. DN: performed urinary NE experiments. TK, YK, TM: created Stim1 KI SHRSP. HO: performed experiments, and analyzed the data.

Disclosure of interest

The authors report no conflict of interest.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was partly supported by JSPS KAKENHI [Grant numbers 17K08787 (to HO) and 26293086 (to TN)], - the Takeda Science Foundation (to HO) and Shimane University “WAKATE” supporting project (to HO).