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A splice variant of smooth muscle myosin phosphatase regulatory subunit tunes arterial reactivity and suppresses response to salt loading
Author(s) -
Reho John,
Kenchegowda Doreswamy,
Asico Laureano D.,
Fisher Steven
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.730.1
Subject(s) - myosin light chain phosphatase , biology , protein phosphatase 1 , gene isoform , alternative splicing , microbiology and biotechnology , myosin , myosin light chain kinase , phosphatase , biochemistry , phosphorylation , gene
Nitric oxide (NO) and reactive oxygen species (ROS) through activation of cGMP‐dependent protein kinase (cGK1a) activate smooth muscle Myosin Phosphatase (MP), thereby causing de‐phosphorylation of myosin and vasorelaxation. cGK1a is targeted to the MP regulatory subunit (Mypt1) via Leucine Zipper (LZ)‐mediated hetero‐dimerization. Isoforms of Mypt1 with variable presence of the C‐terminal LZ motif are generated by alternative splicing of the 31 nt Exon24 (E24). We have demonstrated that the expression of these isoforms is vessel‐specific, developmentally regulated and modulates in disease. Based on correlations of expression and function and biochemical assays we have proposed a model in which Mypt1 E24 splicing tunes arterial sensitivity to NO/ROS mediated vasorelaxation. To directly test this model LoxP sites were inserted in introns flanking E24 and bred into smMHCCre ER for Tamoxifen‐inducible and smooth muscle‐specific deletion of E24 from Mypt1 (E24 cKO). This converts Mypt1 to the E24‐ splice variant coding for the LZ+ isoform. We generated an allelic series of mice: Cre+ with 0, 1 or 2 floxed alleles and measured blood pressure (BP) by telemetry and arterial function by wire myography. E24 cKO had significantly lower systemic BP with no difference between hetero‐ and homo‐zygotes. There was no effect on the circadian variation in BP. Vasorelaxation of 1 st order mesenteric arteries (MA1) to the NO donor DEA/NO, and its 2nd second messenger cGMP in permeabilized and calcium clamped arteries, was markedly enhanced again with no difference between hetero‐ and homo‐zygotes. The only parameter in which these mice differed was a marked suppression of force generation to the a‐adrenergic agonist phenylephrine (PE) in the homozygotes. This was specific to this signaling pathway as there were no changes in force generation to the thromboxane analogue U46619, Angiotensin II, KCl depolarization or calcium. Two weeks of salt loading (4% sodium diet) increased MA1 force generation to PE in wild type mice. This response was abrogated y in the homozygous Mypt1 E24 cKO mice. We conclude that Mypt1 E24 splice variants tune arterial sensitivity to vasodilators such as NO/ROS signaling though cGK1 activation of MP. Targeting of therapies to the contractile apparatus of microvascular smooth muscle may have salutary effects in the treatment of hypertension and other cardiovascular diseases in which increased vascular resistance has a dominant role. Support or Funding Information NIH R01‐HL066171, 7R37‐HL023081, T32‐HL072751