CRISPR-Cas9 Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin--Brief Report

OBJECTIVE: Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. APPROACH AND RESULTS: 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3xFLAG or 3xHA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a protein product of approximately 150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. CONCLUSIONS: This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research

CRISPR-Cas9 Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin

Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research

Retinoid-Induced Expression and Activity of an Immediate Early Tumor Suppressor Gene in Vascular Smooth Muscle Cells

Retinoids are used clinically to treat a number of hyper-proliferative disorders and have been shown in experimental animals to attenuate vascular occlusive diseases, presumably through nuclear receptors bound to retinoic acid response elements (RARE) located in target genes. Here, we show that natural or synthetic retinoids rapidly induce mRNA and protein expression of a specific isoform of A-Kinase Anchoring Protein 12 (AKAP12β) in cultured smooth muscle cells (SMC) as well as the intact vessel wall. Expression kinetics and actinomycin D studies indicate Akap12β is a retinoid-induced, immediate-early gene. Akap12β promoter analyses reveal a conserved RARE mildly induced with atRA in a region that exhibits hyper-acetylation. Immunofluorescence microscopy and protein kinase A (PKA) regulatory subunit overlay assays in SMC suggest a physical association between AKAP12β and PKA following retinoid treatment. Consistent with its designation as a tumor suppressor, inducible expression of AKAP12β attenuates SMC growth in vitro. Further, immunohistochemistry studies establish marked decreases in AKAP12 expression in experimentally-injured vessels of mice as well as atheromatous lesions in humans. Collectively, these results demonstrate a novel role for retinoids in the induction of an AKAP tumor suppressor that blocks vascular SMC growth thus providing new molecular insight into how retiniods may exert their anti-proliferative effects in the injured vessel wall

Mediterranean G6PD Variant Rats Are Protected From Angiotensin II-Induced Hypertension and Kidney Damage, but Not From Inflammation and Arterial Stiffness

RATIONALE: Epidemiological studies suggest that individuals in the Mediterranean region with deficiency of glucose-6-phosphate dehydrogenase (G6PD) are less susceptible to cardiovascular diseases. However, our knowledge regarding the effects of G6PD deficiency on pathogenesis of vascular diseases caused by factors, like angiotensin II (Ang-II), which stimulate synthesis of inflammatory cytokines and vascular inflammation, is lacking. Furthermore, to-date the effect of G6PD deficiency on vascular health has been controversial and difficult to experimentally prove due to a lack of good animal model. OBJECTIVE: To determine the effect of Ang-II-induced hypertension (HTN) and stiffness in a rat model of the Mediterranean G6PD (G6PD) variant and in wild-type (WT) rats. METHODS AND RESULTS: Our findings revealed that infusion of Ang-II (490 ng/kg/min) elicited less HTN and medial hypertrophy of carotid artery in G6PD than in WT rats. Additionally, Ang-II induced less glomerular and tubular damage in the kidneys - a consequence of elevated pressure - in G6PD than WT rats. However, Ang-II-induced arterial stiffness increased in G6PD and WT rats, and there were no differences between the groups. Mechanistically, we found aorta of G6PD as compared to WT rats produced less sustained contraction and less inositol-1,2,3-phosphate (IP3) and superoxide in response to Ang-II. Furthermore, aorta of G6PD as compared to WT rats expressed lower levels of phosphorylated extracellular-signal regulated kinase (ERK). Interestingly, the aorta of G6PD as compared to WT rats infused with Ang-II transcribed more (50-fold) myosin heavy chain-11 (MYH11) gene, which encodes contractile protein of smooth muscle cell (SMC), and less (2.3-fold) actin-binding Rho-activating gene, which encodes a protein that enhances SMC proliferation. A corresponding increase in MYH11 and Leiomodin-1 (LMOD1) staining was observed in arteries of Ang-II treated G6PD rats. However, G6PD deficiency did not affect the accumulation of CD45 cells and transcription of genes encoding interleukin-6 and collagen-1a1 by Ang-II. CONCLUSIONS: The G6PD loss-of-function variant found in humans protected rats from Ang-II-induced HTN and kidney damage, but not from vascular inflammation and arterial stiffness

Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels

<div><p>Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed <i>Dmpk</i> and <i>Cacna1c</i> genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of <i>Srf</i> knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca²⁺ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract.</p></div

Identification of SRF-induced DMPK that regulates expression of <i>Celf1</i> and <i>Mbnl1</i>.

<p>(A) Interactions between down-regulated proteins and SRF were analyzed using Ingenuity Pathway Analysis (IPA) software. Note that experimentally known relationships are indicated by a red line and genes containing SRF binding site(s) are indicated by highlighted proteins in green. (B) A genomic map of <i>Dmpk</i> variants expressed in SMC of jejunum and colon shown on the UCSC Smooth Muscle Genome Browser. SRF binding sites and CArG boxes are indicated in upstream promoter region and intron 1. ChIP-seq data of RNA polymerase 2 and SRF from C2C12 myocytes, and of H3K4m3 and H3K27a from mouse small intestine, are shown under the genomic map. Primer sets spanning regions of the promoter (P1 and P2), promoter and exon 1 (E1), and introns (I1 and I9) of <i>Dmpk</i> are indicated. (C) ChIP qPCR data analysis of RNA polymerase 2. DNA fragments isolated from <i>Srf</i> WT and KO jejunum smooth muscle (n = 3, respectively) by RNA polymerase 2 antibody were used for ChIP qPCR performed with primer sets spanning regions of E1 and I9 of <i>Dmpk</i> shown in <i>B</i>. Mock is negative control DNA captured with IgG. (D) ChIP qPCR data analysis of SRF. SRF ChIP and qPCR were performed as the same as above except using an anti-SRF antibody and primer sets spanning regions of CArG boxes (P1, P2, and I1), and a downstream intron region (I9) in <i>Dmpk</i> as a negative control. (E) Enrichment of <i>Dmpk</i> mRNAs in SMC of jejunum and colon. Amount of mRNAs expression (normalized FPKM) was obtained from RNA-seq data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171262#pone.0171262.ref006" target="_blank">6</a>]. (F) Reduced expression of DMPK protein in <i>Srf</i> KO smooth muscle. Amount of protein expression was obtained from LC-MS/MS proteomics data.</p

Uncoupling of Ca²⁺ transients between SMC and ICC resulting in reduced intestinal contractility in <i>Srf</i> KO mice.

<p>(A) Ca²⁺ transients recorded from several longitudinal SMC (LM) and ICC-MY in WT and KO jejunum within a field of view. Note that Ca²⁺ transients in ICC-MY (overlay of ICC-MY1-3) occur almost synchronously in WT and KO. Ca²⁺ transients in ICC-MY and LM are in phase in WT muscles (overlay of ICC-MY1 and LM1). Reduced coupling and greater latency was observed between the Ca²⁺ transients in ICC-MY and SMC in KO muscles (overlay of ICC-MY2 and LM2). (B) Frequency of Ca²⁺ transients in intestinal LM, frequency of Ca²⁺ transients in ICC-MY, and latency between Ca²⁺ transients in ICC-MY and LM of WT and KO mice are shown in graphs. (C) Contractions of WT and KO duodenal and jejunal muscles measured before and after L-NNA (100 μm) and atropine (1 μm). (D) Comparison of jejunal contractile amplitudes and summary of L-NNA and L-NNA plus atropine effects on jejunal contractions in WT and KO (expressed as percentages of control activity, i.e., no drugs present; areas under the curve for WT or KO in C). * <i>p</i> ≤ 0.5, n = 5</p

Smooth muscle degeneration in inducible SMC-specific <i>Srf</i> KO intestine.

<p>(A and B) Cross sectional images of WT and KO jejunum (A) and colon (B) with H&E staining 10, 15 and 21 days post-tamoxifen injection (PT10D, PT15D, and PT21D). Note that KO smooth muscle layers (arrows) are significantly thinner at PT15D and/or PT21D compared to WT control. (C) Summary of thickness of smooth muscle layers in KO and WT. (D and E) Cryosection images of jejunum (D) and colon (E) stained with anti-SRF and anti-SMA antibodies showing gradual depletion of SRF (green, nucleus) and reduction of SMA (ACTA2, red, cytoplasm) in KO SMC compared to WT control at PT10D, PT15D, and PT21D. (F) qPCR analysis to validate reduction of <i>Srf</i> and <i>Acta2</i> in KO jejunum and colon at PT15D. <i>Ubb</i> was used as an endogenous control. Each data point (C and F) represents the mean ± SD of experiments (n = 3). * <i>p</i> ≤ <i>0</i>.<i>05</i> and <i>** p</i> ≤ <i>0</i>.<i>01</i>, WT versus KO.</p

Identification of a predominant subtype and alternative transcriptional variants of L-type calcium channels expressed in SMC.

<p>(A) Expression levels of L-type calcium channel subtypes in SMC of jejunum and colon. (B) Expression levels of <i>Cacna1c</i> variants in SMC and tissue of jejunum and colon. (C) A genomic map of <i>Cacna1c</i> variants. Five variable regions (V1-5) are indicated. Exons are numbered 1–48. (D) Magnified view of variable regions showing alternatively started or spliced exons (indicated as exon numbers). Seven exons containing alternative transcriptional start sequence are shown by a star (*). Long (L) and short (S) exons that are differentially started or spliced are indicated.</p