Mapping signalling perturbations in myocardial fibrosis via the integrative phosphoproteomic profiling of tissue from diverse sources

Study of the molecular basis of myocardial fibrosis is hampered by limited access to tissues from human patients and by confounding variables associated with sample accessibility, collection, processing and storage. Here, we report an integrative strategy based on mass spectrometry for the phosphoproteomic profiling of normal and fibrotic cardiac tissue obtained from surgical explants from patients with hypertrophic cardiomyopathy, from a transaortic-constriction mouse model of cardiac hypertrophy and fibrosis, and from a heart-on-a-chip model of cardiac fibrosis. We used the integrative approach to map the relative abundance of thousands of proteins, phosphoproteins and phosphorylation sites specific to each tissue source, to identify key signalling pathways driving fibrosis and to screen for anti-fibrotic compounds targeting glycogen synthase kinase 3, which has a consistent role as a key mediator of fibrosis in all three types of tissue specimen. The integrative disease-modelling strategy may reveal new insights into mechanisms of cardiac disease and serve as a test bed for drug screening

Pulmonary Vein Stenosis: Incremental Knowledge Gains to Improve Outcomes

Pulmonary vein stenosis remains a considerable clinical challenge, with high mortality still present in children with progressive disease. In this review, we discuss the clinical spectrum of pulmonary vein stenosis and what is known about the etiology and potential modifying and contributing factors in progressive pulmonary vein stenosis

SHCA PHOSPHOTYROSINE DERIVED SIGNALING IS REQUIRED FOR THE MAINTENANCE OF CARDIAC FUNCTION

Background: ShcA, a scaffolding protein, generates signalspecificity by docking to activated tyrosine kinases through distinct phosphotyrosine recognition motifs, while mediating signal complexity through formation of diverse downstream phosphotyrosine complexes. Mammalian ShcA encodes 3 isoforms having a modular architecture of a PTB domain and SH2 domain, separated by a CH1 region containing tyrosine phosphorylation sites important in Ras-MAPK activation. Objective and Methods: ShcA has a necessary role in cardiovascular development^1,2. However, the role of ShcA in the adult myocardium is largely unknown, also unclear, is how ShcA uses its signaling modules to mediate downstream signaling. To this end, cre/loxP technology was employed to generate a conditional ShcA allele series. The myocardial specific ShcA KO (ShcA CKO) and myocardial restricted domain mutant KI mice were generated using cre expressed from the mlc2v locus^3 coupled with the ShcA floxed allele and in combination with the individual ShcA domain mutant KI alleles^2. Results: ShcACKO mice develop a dilated cardiomyopathy phenotype by 3 months of life, typified by depressed cardiac function and enlarged chamber dimensions. Isolated cardiomyocytes from ShcA CKO mice have preserved contractility indicating an uncoupling between global heart function and single myocyte contractile mechanics. Force-length experiments suggest that the loss of shcAmediates the uncoupling through deregulation of extracellular matrix interactions. Subsequent, analysis of the ShcA myocardial restricted domain mutant KImice suggests that ShcA requires PTB domain docking to upstream tyrosine kinases and subsequent phosphorylation of the CH1 tyrosines important for downstream signaling. Conclusion: ShcA is required for proper maintenance of cardiac function, possibly regulation of extracellular matrix interactions. References: 1. Lai KV, Pawson AJ. The ShcA phosphotyrosine docking protein sensitizescardiovascular signaling in the mouse embryo. Genes and Dev 2000;14:1132-45. 2. Hardy WR. et al. Combinatorial ShcA docking interactions supportdiversity in tissue morphogenesis. Science2007;317:251-6. 3.Minamisawa, s. et al. A post-transcriptional compensatory pathway inheterozygous ventricular myosin light chain 2-deficient mice results in lack ofgene dosage effect during normal cardiac growth or hypertrophy. J Biol Chem 1999;274:10066-70

sj-docx-1-pch-10.1177_21501351241237785 - Supplemental material for Longitudinal Evaluation of Congenital Cardiovascular Surgical Performance and Skills Retention Using Silicone-Molded Heart Models

Supplemental material, sj-docx-1-pch-10.1177_21501351241237785 for Longitudinal Evaluation of Congenital Cardiovascular Surgical Performance and Skills Retention Using Silicone-Molded Heart Models by Matteo Ponzoni, Rawan Alamri, Brandon Peel, Christoph Haller, John Coles, Rachel D. Vanderlaan, Osami Honjo, David J. Barron and Shi-Joon Yoo in World Journal for Pediatric and Congenital Heart Surgery</p

Grb2 is expressed in wild-type mouse podocytes.

<p>Immunofluorescent staining for Nephrin and Grb2 in a wild-type (A, <i>Grb2^flx/flx</i>) or mutant (B, <i>Podocin</i>-Cre;<i>Grb2^flx/flx</i>) glomerulus. Arrows indicate nephrin-expressing podocyte cells located at the periphery of the glomerular tuft. Framed areas from top panels are shown at a higher magnification below. Grb2 staining is absent from <i>Podocin</i>-Cre;<i>Grb2^flx/flx</i> podocytes. Dashed yellow arrows in B show nephrin-negative non-podocyte cells that retained Grb2 expression.</p

Conditional inactivation of the <i>Grb2</i> gene in mice.

<p>(A) Schematic representation of the <i>Grb2</i> locus targeting strategy and the resulting conditional <i>Grb2^flx</i> allele. LoxP sites are represented with white triangles and FRT sites with white circles. Genotyping primers P1 to P4 are shown. (B) Example of 2 positive ES clones targeted at the <i>Grb2</i> locus, as judged from a positive P1/P2 PCR product of 1200 bp. (C) Example of a successful FLPe-mediated excision of the SA-IRES-β<i>geo</i>-pA cassette to generate the <i>Grb2^flx</i> allele, as judged from a positive P3/P4 PCR product of 200 bp. (D) PCR analysis of Cre-mediated excision of the <i>Grb2^flx</i> allele in mouse glomeruli. A 235 bp P1/P4 PCR product confirms excision at the locus and correlates with the presence of Cre recombinase. (E) qPCR analysis of Cre-mediated excision of the <i>Grb2^flx</i> allele in FACS-sorted podocytes from Podocin-Cre; Nephrin-CFP; Grb2flx/flx (mutant, Cre+, n = 2) or Nephrin-CFP; Grb2flx/flx (control, Cre-, n = 3) mice. Amplification levels of a P3/P4 PCR product were normalized to B-actin and used to calculate relative copy numbers of the non-excised <i>Grb2</i> flx allele (Cre-: 2.00±0.37 and Cre+: 0.13±0.01). Star represents p-value of 3.8E-05. (F) Western blot showing 2 examples of <i>ROSA</i>-CreER-mediated inactivation of the <i>Grb2^flx</i> allele in MEFs, resulting in the absence of the <i>Grb2</i> protein product. Positive Cre (+) indicates treatment with OHT to activate the expression of the transgene.</p

<i>Podocin</i>-Cre;<i>Grb2^flx/flx</i> animals display normal glomerular structure and renal function.

<p>(A) Urinalysis of <i>Podocin</i>-Cre;<i>Grb2^flx/flx</i> (Cre+) and <i>Grb2^flx/flx</i> (Cre-) mice 6 and 18 months after birth. Average measurements and standard deviations were calculated for 3–5 mice from each group. (B–C) Representative examples of ultra-structural electron microscopy analysis of kidneys of <i>Podocin</i>-Cre; <i>Grb2^flx/flx</i> (A) and <i>Podocin</i>-Cre: <i>Grb2^flx/−</i> (B) animals compared to control (Cre-negative) littermates. Bar is 2 µm.</p

Genotype analysis of the progeny born from <i>Podocin</i>/<i>Nephrin</i>-Cre;<i>Grb2^flx</i> crosses.

<p>Genotype analysis of the progeny born from <i>Podocin</i>/<i>Nephrin</i>-Cre;<i>Grb2^flx</i> crosses.</p