CPI-17 Regulated Calcium Sensitization Of Vascular Smooth Muscles Is Required For Obesity Related Hypertension

Smooth muscle lines the walls of various hollow organs including blood vessels,respiratory tracts and gastrointestinal system.The basic function of smooth muscle is contraction to provide enough force and maintain the physiological homeostasis.Theabnormal contractility of smooth muscle can lead to many diseases,such as hypertension,asthma and gastrointestinal disorders.It is well documented that the primary mechanisms of smooth muscle contraction are calcium dependent signaling pathway and calcium sensitization pathway.And some smooth muscle associated disorders were reported to be caused by abnormalities of calcium sensitized smooth muscle contraction.Thus,it is important to uncover the mechanisms of calcium sensitized smooth muscle contraction for deeply understanding the smooth muscle contraction regulation,as well as the pathogenesis of smooth muscle associated diseases.So far,calcium sensitized smooth muscle contraction is considered to be accomplished by inhibition of MLCP activity,which is mainly dependent on RhoA/ROCK/MYPT1 signaling pathway and PKC/CPI-17/PP1c signaling pathway.However,in our recently report,we found that MYPT1 T694 phosphorylation constitutively occurs in vivo,and MYPT1 T852 phosphorylation induced by ROCK does not function in smooth muscle contraction,indicating that MYPT1 phosphorylation of ROCK may not be contributed to calcium sensitization mechanism.Unfortunately,although there are a lot of biochemical and in vitro data supporting that PKC/CPI-17/PPlc signaling pathway participates in calcium sensitized contraction,the in vivo role in calcium sensitization as well as its significance in vascular smooth muscle contraction remains to be determined.To further investigate the role of CPI-17 in calcium sensitization signaling pathway of vascular smooth muscle contraction,we generated CPI-17 knock-out mice by CRISPR/Cas9 genome editing technology and obtained two independent lines with deletion of 116 bp and 220 bp in the coding exon,respectively.The mesenteric arteries of CPI-17 deficient mice completely lost the responsiveness to PKC activator,PDBu,suggesting CPI-17 is the unique downstream target of PKC during vasoconstriction.Additionally,CPI-17 knock-out mice had normal blood pressure,but reduced responsiveness to various constrictors including the adrenergic receptor agonists norepinephrine and phenylephrine,thromboxane receptor agonist U46619 and Endothelin 1,indicating that PKC/CPI-17 pathway is involved in a number of agonists induced vascular smooth muscle contraction.Moreover,we further assessed the role of CPI-17 in calcium sensitized contraction using a-toxin permeabilized mesenteric artery strips.Although there was no difference of the maximal force generated by pCa 4.5 and pCa-force relationship,the contractions induced by PDBu and GTPyS under pCa 6.3 were significantly reduced after CPI-17 deletion.Notably,all these data were consistent with observations of intact mesenteric arteries.Taken together,our findings provide directly evidence to corroborate the importance of PKC/CPI-17 mediated calcium sensitization in vascular smooth muscle contraction.Obesity is considered to be the main reason for human essential hypertension.However the mechanism under obesity related hypertension remains a lot to be explored.In dietary induced obese mouse model by feeding high fat diet(HFD)for 16 weeks,we found that the expression level of PKC and CPI-17 are remarkably upregulated in HFD-fed obese mice comparted to the age-matched normal diet(ND)-fed lean mice,indicating that PKC/CPI-17 mediated calcium sensitization is required for the development of obesity related hypertension.To further confirm the role of PKC/CPI-17 mediated calcium sensitization in obesity related hypertension,we fed Cpi-17+/+,Cpi-17?116/+ and Cpi-17?116/?116 mice with HFD for 16 weeks to induce obesity related hypertension.Remarkably,HFD-fed Cpi-17?116/+ and Cpi-17?116/?116 mice had the same body weight with HFD-fed Cpi-17+/+ mice,but abolished the development of obesity related hypertension.We also detected the mesenteric arteries contractility of HFD-fed Cpi-17+/+,Cpi-17?116/+ and Cpi-17?116/?116 mice.KCl and agonists induced higher vasocontraction responsiveness to HFD-fed Cpi-17+/+ mice than ND-fed Cpi-17+/+ mice,while the responsiveness were comparable between HFD-fed and ND-fed Cpi-17?116/+ or Cpi-17?116/?116 mice.All these findings indicate that PKC/CPI-17 mediated calcium sensitization signaling pathway in vascular smooth muscle is required for the development of obesity related hypertension.In conclusion,taking the advantage of genetic approach,we firstly provide the in vivo evidence supporting that CPI-17 acts as the only downstream target of PKC and CPI-17 predominantly mediates calcium sensitization during vascular smooth muscle contraction.Moreover,we discovered increased expression level of both PKC and CPI-17 in vascular smooth muscle under obesity related hypertension,indicating the important role of PKC/CPI-17 mediated calcium sensitization pathway in obesity related hypertension.Thus our data reveal the potential therapeutic target,PKC/CPI-17 pathway for management and treatment of obesity related hypertension.