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Zinc-finger nuclease knockout of dual-specificity protein phosphatase-5 enhances the myogenic response and autoregulation of cerebral blood flow in FHH.1BN rats.

Fan F, Geurts AM, Pabbidi MR, Smith SV, Harder DR, Jacob H, Roman RJ - PLoS ONE (2014)

Bottom Line: We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background.The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats.These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America.

ABSTRACT
We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

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Comparison of the expression and activity of Dusp5 in FHH versus FHH.1BN rats.Panel A presents a comparison of the expression of Dusp5 mRNA in cerebral arteries of FHH versus FHH.1BN rats. The upper portion of the figure presents the representative images of gels showing the qPCR products and the bar graph below compares the expression levels. Panel B presents a comparison of the expression of phosphorylated-ERK1/2, total ERK1/2, phosphorylated-PKC and beta-Actin in the brain of FHH and FHH.1BN rats. The upper panel presents the representative images and the lower panel presents the relative quantitation. Panel C presents a comparison of expression of these proteins in cerebral microvessels of FHH as compared with FHH.1BN rats. All of the vessels isolated from one strain were pooled into a single sample. The upper panel presents the representative images and the lower panel presents the quantitation of the images. Mean values ± SE from 3 rats per strain are presented in Panel A and Panel B. Panel C represents the results from duplicate aliquots run from a single pooled microvessel sample isolated from 8–10 rats per strain. * indicates a significant difference from the corresponding value in FHH rats.
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pone-0112878-g003: Comparison of the expression and activity of Dusp5 in FHH versus FHH.1BN rats.Panel A presents a comparison of the expression of Dusp5 mRNA in cerebral arteries of FHH versus FHH.1BN rats. The upper portion of the figure presents the representative images of gels showing the qPCR products and the bar graph below compares the expression levels. Panel B presents a comparison of the expression of phosphorylated-ERK1/2, total ERK1/2, phosphorylated-PKC and beta-Actin in the brain of FHH and FHH.1BN rats. The upper panel presents the representative images and the lower panel presents the relative quantitation. Panel C presents a comparison of expression of these proteins in cerebral microvessels of FHH as compared with FHH.1BN rats. All of the vessels isolated from one strain were pooled into a single sample. The upper panel presents the representative images and the lower panel presents the quantitation of the images. Mean values ± SE from 3 rats per strain are presented in Panel A and Panel B. Panel C represents the results from duplicate aliquots run from a single pooled microvessel sample isolated from 8–10 rats per strain. * indicates a significant difference from the corresponding value in FHH rats.

Mentions: To determine if the C107T and C627T SNPs that altered CpG sites and the G637A SNP that caused a G155R mutation might alter the expression of Dusp5 in FHH versus FHH.1BN rats, RT-qPCR and western blot experiments were performed. The results presented in Figure 3A indicate that the expression of Dusp5 mRNA is 2-fold higher in cerebral arteries of FHH than in the FHH.1BN control rats but the expression of protein is not different between these strains (Figure 3C). Moreover, as presented in Figure 3B and 3C, the expression of p-ERK1/2, the primary substrates for dephosphorylation by Dusp5, is significantly reduced in cerebral microvessels and the brains of FHH relative to FHH.1BN rats, while total ERK1/2 levels are not significantly altered. The expression of p-PKC protein is also significantly elevated in FHH.1BN as compared to FHH rats.


Zinc-finger nuclease knockout of dual-specificity protein phosphatase-5 enhances the myogenic response and autoregulation of cerebral blood flow in FHH.1BN rats.

Fan F, Geurts AM, Pabbidi MR, Smith SV, Harder DR, Jacob H, Roman RJ - PLoS ONE (2014)

Comparison of the expression and activity of Dusp5 in FHH versus FHH.1BN rats.Panel A presents a comparison of the expression of Dusp5 mRNA in cerebral arteries of FHH versus FHH.1BN rats. The upper portion of the figure presents the representative images of gels showing the qPCR products and the bar graph below compares the expression levels. Panel B presents a comparison of the expression of phosphorylated-ERK1/2, total ERK1/2, phosphorylated-PKC and beta-Actin in the brain of FHH and FHH.1BN rats. The upper panel presents the representative images and the lower panel presents the relative quantitation. Panel C presents a comparison of expression of these proteins in cerebral microvessels of FHH as compared with FHH.1BN rats. All of the vessels isolated from one strain were pooled into a single sample. The upper panel presents the representative images and the lower panel presents the quantitation of the images. Mean values ± SE from 3 rats per strain are presented in Panel A and Panel B. Panel C represents the results from duplicate aliquots run from a single pooled microvessel sample isolated from 8–10 rats per strain. * indicates a significant difference from the corresponding value in FHH rats.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232417&req=5

pone-0112878-g003: Comparison of the expression and activity of Dusp5 in FHH versus FHH.1BN rats.Panel A presents a comparison of the expression of Dusp5 mRNA in cerebral arteries of FHH versus FHH.1BN rats. The upper portion of the figure presents the representative images of gels showing the qPCR products and the bar graph below compares the expression levels. Panel B presents a comparison of the expression of phosphorylated-ERK1/2, total ERK1/2, phosphorylated-PKC and beta-Actin in the brain of FHH and FHH.1BN rats. The upper panel presents the representative images and the lower panel presents the relative quantitation. Panel C presents a comparison of expression of these proteins in cerebral microvessels of FHH as compared with FHH.1BN rats. All of the vessels isolated from one strain were pooled into a single sample. The upper panel presents the representative images and the lower panel presents the quantitation of the images. Mean values ± SE from 3 rats per strain are presented in Panel A and Panel B. Panel C represents the results from duplicate aliquots run from a single pooled microvessel sample isolated from 8–10 rats per strain. * indicates a significant difference from the corresponding value in FHH rats.
Mentions: To determine if the C107T and C627T SNPs that altered CpG sites and the G637A SNP that caused a G155R mutation might alter the expression of Dusp5 in FHH versus FHH.1BN rats, RT-qPCR and western blot experiments were performed. The results presented in Figure 3A indicate that the expression of Dusp5 mRNA is 2-fold higher in cerebral arteries of FHH than in the FHH.1BN control rats but the expression of protein is not different between these strains (Figure 3C). Moreover, as presented in Figure 3B and 3C, the expression of p-ERK1/2, the primary substrates for dephosphorylation by Dusp5, is significantly reduced in cerebral microvessels and the brains of FHH relative to FHH.1BN rats, while total ERK1/2 levels are not significantly altered. The expression of p-PKC protein is also significantly elevated in FHH.1BN as compared to FHH rats.

Bottom Line: We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background.The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats.These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America.

ABSTRACT
We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

Show MeSH
Related in: MedlinePlus