Calponin 1 inhibits agonist‐induced ERK activation and decreases calcium sensitization in vascular smooth muscle

Abstract Smooth muscle cell (SMC) contraction and vascular tone are modulated by phosphorylation and multiple modifications of the thick filament, and thin filament regulation of SMC contraction has been reported to involve extracellular regulated kinase (ERK). Previous studies in ferrets suggest that the actin‐binding protein, calponin 1 (CNN1), acts as a scaffold linking protein kinase C (PKC), Raf, MEK and ERK, promoting PKC‐dependent ERK activation. To gain further insight into this function of CNN1 in ERK activation and the regulation of SMC contractility in mice, we generated a novel Calponin 1 knockout mouse (Cnn1 KO) by a single base substitution in an intronic CArG box that preferentially abolishes expression of CNN1 in vascular SMCs. Using this new Cnn1 KO mouse, we show that ablation of CNN1 has two effects, depending on the cytosolic free calcium level: (1) in the presence of elevated intracellular calcium caused by agonist stimulation, Cnn1 KO mice display a reduced amplitude of stress and stiffness but an increase in agonist‐induced ERK activation; and (2) during intracellular calcium depletion, in the presence of an agonist, Cnn1 KO mice exhibit increased duration of SM tone maintenance. Together, these results suggest that CNN1 plays an important and complex modulatory role in SMC contractile tone amplitude and maintenance.

phenotype than the proliferative phenotype. 9However, what role CNN1 protein plays in mediating SMC differentiation is unknown.In addition, the functional significance of CNN1 in differentiated vascular smooth muscle cells (VSMCs) is incompletely understood.
CNN1 has long been reported to regulate SMC contraction.
Previous in vitro studies showed that CNN1 inhibits SMC contraction by preventing actin-activated Mg +2 -ATPase activity of myosin. 10her studies reported that lack of CNN1 reduced smooth muscle cell sensitivity to agonist stimulation. 11,12In a recent study by Feng et al, depletion of CNN1 was shown to reduce maximal force production induced by norepinephrine, and Cnn1 knockout aortas undergo faster relaxation after removal of KCl depolarizing solutions. 13evious studies from our laboratory reported that CNN1 undergoes agonist-induced translocation from central actin filaments to the cortical region of differentiated VSMCs. 14In addition, it has been shown that CNN1 interacts with both ERK (MAP kinase) and PKC and aids in ERK translocation to the cortical region of the VSMC where ERK gets activated. 15Nevertheless, the impact of CNN1 in mediating ERK activation remains unclear.
In the present study, we aimed to gain insight into the role of CNN1 in ERK activation and the regulation of VSMC contraction and tone.Based on an original report demonstrating the key role of an intronic serum response factor-binding element called a CArG box, 16 we generated a new Cnn1 knockout mouse by introducing a single base substitution in the CArG box.The results from the current study show that in the presence of an agonist and physiologic levels of Ca +2 , CNN1 promotes tone amplitude but inhibits ERK activation.However, in the absence of Ca +2 , CNN1 inhibits prolonged tone maintenance, pointing to two different roles of CNN1 in different signalling pathways.

| Animals and aorta preparation
Animals were maintained in Boston University, and Augusta University according to the NIH guide for the care and use of laboratory animals.All procedures were performed in accordance with approved Institutional Animal Care and Use Committee protocols at Boston University (#201900004) and Augusta University (#2019-1000).Mouse aortas were quickly collected after death by isoflurane inhalation and placed in Krebs solution (120 mM NaCl, 5.9 mM KCl,

| Generation of Cnn1 knockout mice
As a first step, we performed a competitive electromobility shift assay (EMSA) as described 16 using a 32P-labelled single strand deoxyoligonucleotide (ssODN) probe of sequence agccgccgcgccttataaggcggccttggg (Coralville, IA) containing a serum response factor (SRF) binding CArG box (underlined sequence) located in the first intron of Cnn1.This intronic CArG box was shown previously to be important for in vitro transcriptional activation of the Cnn1 promoter. 16e above radiolabelled probe was combined with in vitro translated SRF in the absence or presence of excess unlabelled 'cold' ssODNs carrying single base substitutions across the labelled probe, and the nucleoprotein complexes were resolved in a nondenaturing 6% polyacrylamide gel.The gel was vacuum dried to Whatmann filter paper and exposed to x-ray film at −80 C.   (Next Advance), and total RNA isolated with TRIzol™ (Thermo Fisher Scientific).Expression analysis was conducted with a Bio-Rad CFX96 real-time qPCR system using the delta CT method.Primer sequences were Cnn1 (forward: TCATC TGC ACC TCT GCTTTG; reverse: GGGCC AGC TTG TTC TTTACT), Actb (forward: GAGGT ATC CTG ACC CTG AAGTA; reverse: CACAC GCA GCT CAT TGTAGA).

| Bulk RNA-seq
Total RNA from wild-type and Cnn1 KO mouse aorta (n = 3/genotype), less adventitia and endothelium, was isolated with RNeasy kit and submitted to the University of Rochester Genomics Research Center for RNA quality, library construction and sequencing with an llumina NextSeq 550 sequencer (San Diego, CA) at a sequencing depth of 70 million reads per replicate as described previously. 20A and volcano plots were generated using DESeq2 in RStudio.
In brief, a counts matrix was generated from Fastq files and used as input for DEseq2 normalization and differential expression analysis.
PCA plots and volcano plots were visualized from DEG-ranked lists using ggplot with a Log 2 FC cutoff of 1 and a padj value cutoff of 0.01.Raw sequence data and expression changes were deposited at the Gene Expression Omnibus (GSE #239476).

| Phenylephrine-induced stress and stiffness
The thoracic aortas of young adult (4-5 months), wild-type (WT) and Cnn1 KO mice were excised after euthanization by isoflurane inhalation.Aortas were placed in Krebs solution (120 mM NaCl, 5.9 mM KCl, replaced with standard Krebs solution, and the muscle was allowed to relax for 30 min.After relaxation, the muscle was incubated with 10 μM phenylephrine-Krebs solution to assay the SMC contraction amplitude.Stiffness was calculated using high-frequency low amplitude (HFLA) length oscillations as previously described. 21After myography data acquisition, the tissues were snap frozen in ice-cold (-80°C) acetone-dithiothreitol (DTT)-trichloroacetic acid (TCA) solution, and samples were immediately placed in a −80°C freezer for further analysis.

| Ca-independent smooth muscle tone
To assess the Ca-independent smooth muscle tone in WT and Cnn1 KO, aortas were stretched to 80% of the initial length and allowed to equilibrate for 1 h in the Krebs solution.After equilibration, aortas were initially stimulated with 51 mM KCl Krebs solution for 15 min to check the viability of the tissue.After 15 min, the KCl Krebs solution was replaced by Krebs solution, and the tissues were allowed to relax for 30 min before treatment with 10 μM phenylephrine-Krebs solution.After 15 min of phenylephrine treatment, smooth muscle tone was assessed by gradually reducing the calcium concentration in the buffer by adding EGTA to a final concentration of 3 mM.For the analysis of molecular mechanisms responsible for tone maintenance, aortas were quick-frozen in a DTT-TCA-acetone solution under the following conditions: (1) when the aortas were maintaining tone, or (2) when the aortas reached the relaxed baseline.Once quickly frozen, the aortas were maintained in a −80°C freezer.

| Polyacrylamide gel electrophoresis and Western blotting
Frozen aortic strips were washed with an ice-cold acetone-DTT solution 3-5 times before proceeding to homogenization to remove traces of TCA.Aortic strips were placed in Precellys cell lysis tubes containing 250 μL of homogenization buffer with protease and phosphatase inhibitor cocktail (Thermo Fischer Scientific Cat # 78440).
The tubes were placed in a bead beater and 5 cycles (1 cycle: 1 min on-30 s off-30 s on-3 min off-2nd cycle) and were run at 5000 rpm with an interval of 3 min between each cycle at 4°C.Lysates were cleared by centrifugation at 12.5 K rpm for 10 min at 4°C.Protein concentration was estimated using a Bio-Rad DC protein assay kit per the manufacturer's instructions.
Fluorescent signals were captured on a Li-COR or Azure imaging system and processed in Microsoft PowerPoint for visualization.

| Statistical analysis
Data were analysed by a student t-test on Graphpad Prism 8.0 and a p-Value of <0.05 was considered statistically significant.

| CRISPR-mediated single base substitution of a CArG box abolished CNN1 expression with minimal effects on the aortic transcriptome
Competitive EMSA revealed that a C > G transversion in the first base of the intronic CArG box was highly disruptive for SRF binding (Figure S1A).Accordingly, we used 3-component CRISPR to engineer the same C > G transversion in mice (Figure S1B).A simple restriction digest of ear tag DNA distinguished this single-base substitution from the wild-type sequence (Figure S1C), and Sanger sequencing confirmed the C > G transversion with no detectable ontarget collateral damage (Figure S1D, data not shown).Quantitative RT-PCR demonstrated barely detectable levels of Cnn1 mRNA in the aorta of mice homozygous for the C > G transversion (hereafter referred as Cnn1 KO); interestingly, there was only partial KO of Cnn1 mRNA in visceral SMC-containing tissues (Figure S2A).Western blot analysis showed an expected abrogation of CNN1 protein in the aorta of KO mice (Figure 1A).Confocal immunofluorescence imaging demonstrated a similar reduction in CNN1 protein in VSMCs of the aorta and smaller vessels of the heart and lung (Figure 1B).As with mRNA expression studies, confocal microscopy showed residual expression of CNN1 protein in several visceral SMC organs of Cnn1 KO mice (Figure S2B).These results validate this Cnn1 KO mouse, demonstrating a preferential loss of Cnn1 mRNA and CNN1 protein expression in VSMCs.
To assess whether loss of CNN1 has any impact on steady-state levels of global gene expression, we performed bulk RNA-seq of the aorta, enriched for VSMCs.Principal component analysis (PCA) revealed a tight grouping of wild-type aorta samples which were distinct from the more variable Cnn1 KO aorta (Figure 1C).Using a false discovery rate of p < 0.01, DEseq2 analysis showed a massive reduction in Cnn1 and only 146 differentially expressed genes (30 downregulated and 116 upregulated) (Figure 1D).These findings indicate that loss of CNN1 has only a small effect on the aortic SMC trancriptome, consistent with the known localization of CNN1 to the cytoplasm. 8

| CNN1 negatively regulates agonist-induced ERK activation
Our previous studies using aortic tissue from ferrets suggested a role for CNN1 as a scaffolding protein in mediating ERK activation. 15o test whether this function of CNN1 exists in mice, we measured agonist-induced ERK phosphorylation in the aorta of WT and Cnn1 KO mice, 15 min following stimulation with the alpha-agonist phenylephrine (PE).Western blotting showed that ERK protein phosphorylation levels increased after PE treatment in both Cnn1 KO and WT littermate control aortic strips with higher ERK phosphorylation levels in the KO compared to the WT (Figure 2).These results confirm and extend in mice our previous findings that CNN1 negatively regulates ERK activation in VSMCs.

| Cnn1 KO mice show reduced agonist-induced stress and stiffness
VSMCs of the aorta play an important role in regulating the aortic luminal diameter to accommodate the pulsatile changes of blood through regulated events of contraction and relaxation. 22,23In addition, VSMCs contribute to the stiffness of the aorta. 21,24In previous in vitro studies, CNN1 was shown to be a negative regulator of smooth muscle contraction by inhibiting the actin-activated Mg +2 ATPase activity of myosin. 10A recent study by Feng et al using aortas from Cnn1 knockout mice showed a reduction in maximal force production by norepinephrine indicating a role for Cnn1 as a positive regulator of VSMC contraction. 13In the present study, we explored the effect of Cnn1 depletion on PE-induced stress and stiffness of the mouse thoracic aorta.Results from our study demonstrate that depletion of Cnn1 reduces PE-induced stress and stiffness (Figure 3A-D).These and previously published results, 13 support the notion of CNN1 playing an important role in the regulation of VSMC contraction.

| Cnn1 knockouts show prolonged Ca-independent smooth muscle tone
VSMCs have been shown to be capable of maintaining agonistinduced smooth muscle tone under reduced intracellular calcium levels, a phenomenon termed calcium sensitization. 257][28] On the contrary, our previous studies in ferret aorta showed that VSMCs can generate force in the absence of an increase in intracellular calcium concentration or changes in ERKdependent MLC phosphorylation levels. 15To test the role of CNN1 in the maintenance of smooth muscle tone under reduced calcium conditions, we treated aortic strips from both WT and Cnn1 KO mice with PE for 15 min followed by the gradual addition of EGTA to a final concentration of 3 mM to reduce free calcium in the buffer.We then measured the time required for the force to reach baseline.The results showed that Cnn1 KO aortic strips maintain tone longer than wild type aortic strips under these conditions (Figure 4A-C).These results suggest CNN1 negatively regulates calcium sensitization in VSMCs under conditions of reduced calcium.

| CPI-17 mediated inhibition of MLCP is responsible for calcium sensitization in Cnn1 KOs
To gain insight into the molecular mechanism responsible for increased maintenance of tone in Cnn1 KO mice, we investigated the myosin light chain phosphorylation levels and found that MLC phosphorylation levels were higher in Cnn1 KO aortas compared to wild type (Figure 5A-D).These results suggest that inhibition of myosin light chain phosphatase could be responsible for the observed MLC phosphorylation levels in the Cnn1 KO.
Two different mechanisms have been reported to cause inhibition of MLCP in SMCs: (1) Rho-kinase-mediated phosphorylation of the MYPT1 subunit of MLCP inhibits MLCP targeting to myosin; and (2) Rho-kinase or PKC-mediated phosphorylation of CPI-17, which binds to MLCP and inhibits its targeting to myosin. 29,30Western blot results of MYPT1 and CPI-17 phosphorylation levels in the WT and Cnn1 KO aortic lysates treated with PE + EGTA showed that Cnn1 KOs have more CPI17 phosphorylation compared to WT with no difference in MYPT1 phosphorylation levels between WT and Cnn1 KOs (Figure 6A-D).These results are consistent with a mechanism involving inhibition of MLCP targeting by binding of CPI17.

| DISCUSS ION
The results of this study provide new insight into the function of CNN1 in vascular tissue.The extracellular signal regulated kinase was shown to play important roles in several intracellular processes in VSMCs which includes SMC contraction and proliferation.
Previous studies from our laboratory showed that CNN1 interacts with both PKC and ERK post-agonist stimulation.Further, CNN1 appears to play an important role in ERK activation by acting as a scaffolding protein bringing together the activators of ERK. 15 The results presented in the current study show that depletion of CNN1 leads to increased agonist-induced ERK activation in VSMCs (Figure 7).This finding suggests CNN1 in wild-type mice negatively regulates agonist-induced ERK activation.
Previous in vitro biochemical studies using purified calponin showed that CNN1 inhibits smooth muscle contraction by inhibiting actin-activated Mg ATPase activity of myosin. 10However, later ex vivo studies using tissues from different animal models demonstrated a regulatory role of CNN1 in SMC contraction. 11,12A recent study using aortas from a different Cnn1 KO mouse than here demonstrated a reduction in maximal force production after norepinephrine treatment. 13In the present study, we also observed that the aortas of Cnn1 KO mice showed a reduction in agonist-induced stress confirming the role of CNN1 in SMC contraction.In addition, we also found that Cnn1 KO aortas produce less agonist-induced stiffness.Previous studies from our laboratory reported that SMCs of the aorta contribute to approximately 50% of the total aortic stiffness with the remaining coming from changes in the matrix. 21ooth muscle cell contraction can be elicited by an increased intracellular calcium concentration resulting in the phosphorylation of myosin light chains (MLC) by calcium-dependent myosin light chain kinase (MLCK).Phosphorylated myosin interacts with actin filaments leading to crossbridge cycling and smooth muscle contraction. 22,31However, agonist-induced smooth muscle tone has been shown to be maintained under reduced calcium achieved by an increase in the sensitivity of myofilaments called calcium sensitization.The calcium sensitization was shown to be achieved by the inhibition of myosin light chain phosphatase which removes the phosphate group from myosin light chains. 25Inhibition of MLCP allows the muscle to maintain the tone for a longer duration.Rho kinase-dependent inhibition of MLCP has been shown to be responsible for calcium sensitization.In addition, Rho kinase or PKCmediated potentiation CPI17 was also shown to be responsible for the inhibition of MLCP. 27,28Previous studies from our laboratory showed that calcium-independent isoforms of PKC were responsible for force production in the absence of an increase in intracellular calcium levels.In addition, CNN1 was shown to interact with PKC in an agonist-dependent manner; however, the role of this interaction was unclear. 15 Calponin has three isoforms with CNN1 being specific to smooth muscle cells. 7,8The absence of change in expression of the other two calponin isoforms, CNN2 and CNN3, in the Cnn1 KO (Figure S3) indicates that the above observed effects are mediated by specific loss of CNN1 in VSMCs.The studies herein were conducted with a new Cnn1 KO mouse carrying a single base substitution in an intronic CArG box, a regulatory element that binds the ubiquitous transcription factor, SRF. 32This Cnn1 KO differs from a previous KO wherein coding exons 5 and 6 were replaced with a neomycin cassette. 33The development of KO mice through regulatory element disruption leaves the coding sequence intact allowing for ease in genetic rescue experiments.For example, it should be possible to over-ride the transcriptional silencing effect of regulatory element mutations through CRISPR activation. 34Remarkably, the single base transversion resulted in a true null Cnn1 allele in VSMCs with near complete loss of CNN1 protein, but incomplete loss of CNN1 expression in visceral SMCs of the gastrointestinal tract and lung.
Previously, we reported an identical single base transversion of a promoter CArG box in the SMC-restricted Tspan2 gene with similar abolition of Tspan2 expression in VSMCs. 20These findings are noteworthy as they are the first to reveal the critical importance of a single base in driving full expression of two SMC-restricted genes, Cnn1 and Tspan2.Importantly, not all SRF/CArG-dependent genes rely upon a single CArG box because aggressive inactivation of more than one CArG box in the Lmod1 and Srf genes had minimal effects on each gene's expression in mice. 35,36We assume that the latter two genes, which are essential for life, harbour additional regulatory elements to safeguard expression and tissue homeostasis whereas genes dispensable for life (Cnn1 and Tspan2) lack such redundancy.It will be of interest to determine whether this trend holds with additional CArG-dependent genes as well as other transcription factor-binding sites.

| CON CLUS ION
A new VSMC-restricted Cnn1 KO mouse has been developed without disrupting the coding sequence.This model was used to extend previous studies conducted in ferrets, showing that CNN1 can act as an inhibitor of ERK activation and calcium sensitization.
17 was used to generate a novel mouse carrying a single C > G transversion in the Cnn1 intronic CArG box.Briefly, a single guide RNA (Synthego) used previously to generate a more aggressive CArG mutation18 was combined with Cas9 protein (Integrated DNA Technologies) in a ribonuceloprotein complex (RNP).The RNP was incubated at room temperature for 5 min and then mixed with a ssODN containing the C > G transversion (cagagactgatggcagcgccgccccctccccccagcgccggccccagagtcgcagaggagccgccgcggcttataaggcggccttgggcagcccgggcccgcgctatataagggccggtttgctttataaagccgggc; single base substitution bold; Integrated DNA Technologies).The RNP/ssODN mix was microinjected into zygotes (strain C57BL/6J) at a ratio of 3 pmol sgRNA: 3 pmol Cas9: 11 pmol ssODN in nuclease-free M2 microinjection buffer (Sigma) and viable 2-cell stage embryos were transferred to pseudopregnant ICR mice.Pups were genotyped by SacII digestion of a PCR product generated with primers flanking the edited allele (forward primer, 5'-AGGGA ACA CTG AGG CAC TTT CTTTCCAG-3′; reverse primer, 5′-TAGCT TCA GCT CTT ATC CCA TCTTCC-3′).Founder mice were bred to C57BL/6J mice for germline transmission of the targeted allele, and Sanger sequencing confirmed sequence fidelity across the PCR product amplified with the aforementioned primers.Heterozygous intercrosses generated mice wild-type or homozygous for the C > G transversion.There were no off-targeting events in linkage disequilibrium with Cnn1 based on the CRISPOR algorithm.

2. 4 |
Quantitative RT-PCRTissues of different Cnn1 genotypes were isolated and snap frozen in liquid nitrogen, homogenized with an automated Bullet machine

11. 5
mM Dextrose, 1.2 mM NaH2PO 4 .H 2 O, 1.2 mM MgCl 2 .6H 2 O and 2.5 mM CaCl 2 ) on ice.The outer fatty layer was carefully removed in a sylgard dissection chamber containing Krebs solution supplied with 95% O 2 and 5% CO 2 .Aortic rings of 0.5 mm were cut and placed in Eppendorf tubes containing Krebs solution with added Nuc-blue cell-permeant stain to visualize the nucleus of VSMCs for calculation of medial layer thickness.The thoracic aorta was cut into two 4-5 mm long strips followed by measuring their diameter under a dissection microscope.Each strip of the aorta was placed in an organ bath containing 50 mL of Krebs solution with 100 μM N(gamma)-nitro-L-arginine methyl ester (L-NAME).The temperature of the solution was maintained at 37°C using a circulating water bath and supplied with 95% O 2 and 5% CO 2 .Aortic strips were stretched to 80% of the initial length (optimal length for force production) and allowed to equilibrate for 30-60 min.After equilibration, the standard Krebs solution was replaced with a depolarizing 51 mM KCl Krebs solution (30 mM NaCl, 51 mM KCl, 11.5 mM Dextrose, 1.2 mM NaH2PO 4 .H 2 O, 1.2 mM MgCl 2 .6H 2 O and 2.5 mM CaCl 2 ) to check the viability of the tissues.After 15 min, the KCl Krebs solution was Cnn1 KO mouse aorta.(A) Western blot of indicated proteins in aorta.(B) Confocal immunofluorescence microscopy of indicated proteins colocalized in vessels of indicated tissues.Arrows, airway SMC; smaller arrows, vascular SMC.(C) PCA plot of WT and Cnn1 KO aorta bulk RNA-seq.DeSeq2 was used to generate principal components.(D) Volcano plot of Cnn1 KO Aorta with 30,822 genes plotted and 510 differentially expressed genes.Dotted lines indicate cutoffs for p-adjusted value >0.01 (y-axis) and Log 2 FC > |1.0| (x-axis).Of note, Cnn1 was the only gene to be both padj <0.01 and Log 2 FC < −1.0.Plot was generated with EnhancedVolcano.
The current study's results indicate that CNN1 negatively regulates calcium sensitization as the Cnn1 KO aortas maintain tone for a longer duration.As the extracellular calcium was removed by adding EGTA, the observed MLC phosphorylation levels in Cnn1 KOs resulted from the inhibition of MLCP.Our further search for the mechanism responsible for the inhibition of MLCP resulted in the identification of increased phosphorylation of CPI17 which could maintain tone longer in Cnn1 KOs by inhibiting the targeting of MLCP to myosin.The lack of increase in MYPT1 phosphorylation ruled out the involvement of Rho kinase-mediated inhibition of MLCP.More CPI17 phosphorylation in the Cnn1 KOs indicates the involvement of a calcium-independent isoform of PKC whose activity could be inhibited by CNN1.

F I G U R E 2
CNN1 inhibits agonistinduced ERK activation.(A) Western blot demonstrating phospho ERK levels in wild type (WT) and Cnn1 KO before and after stimulation with 10 μm phenylephrine (PE).(B) Graph representing increased ERK phosphorylation levels in the Cnn1 KOs compared to WT. (WT, n = 9; Cnn1 KO, n = 9).Phospho 44/42 ERK levels are normalized to total 44/42 ERK levels.F I G U R E 3 A reduction in agonistinduced stress and stiffness in Cnn1 KOs.(A,B) Myographs showing changes in force with different treatments in WT and Cnn1 KO aortas.(C) Graph showing reduced agonist-induced stress in the Cnn1 KOs compared to WT. (D) Graph showing reduced agonist-induced stiffness in the Cnn1 KOs compared to WT. (kPa -Kilo Pascals).(WT, n = 6; Cnn1 KO, n = 6).F I G U R E 4 Increased duration of tone maintenance in Cnn1 KO aortic strips under reduced calcium conditions.(A) Tone maintenance in the WT after adding EGTA.(B) Increased tone maintenance in the Cnn1 KO compared to WT. (C) Graph representing the time for relaxation in wild type and Cnn1 KO after adding EGTA to the buffer.(WT, n = 6; Cnn1 KO, n = 5).F I G U R E 5 Smooth muscle tone is maintained longer due to increased myosin light chain phosphorylation in the Cnn1 KOs.(A,B) Aortic strips were snap-frozen at the same time in WT and Cnn1 KOs to investigate the molecular mechanism responsible for increased tone maintenance in Cnn1 KOs.(C) Myosin light chain phosphorylation (Phospho MLC20) in the Cnn1 KO compared to the wild type.(D) Graph representing increased Phospho MLC20 levels in the Cnn1 KO compared to WT. (WT, n = 5; Cnn1 KO, n = 5).F I G U R E 6 CPI17 inhibited the MLCP in Cnn1 KOs.(A) Blot representing MYPT1-T850 phosphorylation levels in WT and Cnn1 KO aortas after treating with PE + EGTA.(B) Graph representing relative levels of phospho MYPT1-T850 levels in WT and Cnn1 KOs normalized to vinculin with no change observed between WT and Cnn1 KO. (WT, n = 5; Cnn1 KO, n = 5).(C) Blot representing Phospho CPI17-T38 levels in WT and Cnn1 KO aortas after treating with PE + EGTA.(D) Graph representing relative levels of phospho CPI17-T38 levels in WT and Cnn1 KOs normalized to GAPDH with more phospho CPI17 observed in Cnn1 KO compared to WT. (WT, n = 3; Cnn1 KO, n = 3).

F I G U R E 7
Schematic diagram illustrating the increased ERK activation and tone maintenance in the Cnn1 KO aortas.(A) Binding of phenylephrine to alpha agonist receptor on smooth muscle cell surface results in the release of DAG (diacyl glycerol), and DAG-mediated activation of PKC (protein kinase-C).Once activated, PKC activates RAF (rapidly accelerated fibrosarcoma) which in turn activates the MEK (MAP kinase kinase).MEK activates ERK (extracellular signal regulated kinase).More ERK phosphorylation in the Cnn1 KO indicates that CNN1 negatively regulates agonist ERK activation.(B) Phenylephrine increases the intracellular calcium ion concentration, activating MLCK (myosin light chain kinase) and subsequent myosin light phosphorylation.Phosphorylated myosin interacts with central actin filaments leading to crossbridge cycling and force production.MLCP (Myosin light chain phosphatase) inhibits smooth muscle contraction by removing the phosphate group from the myosin; however, inhibition of MLCP by Rho kinase-mediated phosphorylation of myosin targeting subunit MYPT1 or inhibition of MLCP targeting to myosin by binding of phospho CPI17 (protein kinase-C dependent phosphatase-1 inhibitor of 17 kDa) leads to increased tone maintenance in the smooth muscle cells.Cnn1 KO aortas showed an increase in calcium-independent tone maintenance by CPI17-mediated inhibition of MLCP.