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Targeting calcium signaling as a novel therapeutic strategy for cardiac hypertrophy and failure Tamer M A Mohamed, Riham Abou-Leisa, Min Zi, Sukhpal Prehar, Florence Baudoin, Elizabeth Cartwright, Ludwig Neyses, Delvac Oceandy Cardiovascular Research Group, The University of Manchester, United Kingdom.
Ca 2+ in myocardial (patho-)physiology NCX PMCA I Ca RyR SERCA Ca 2+ Contraction/ Relaxation LTCC & RYR / SERCA & NCX Signal Transduction PMCA4/ TRPCs & IP3R
Plasma Membrane Calcium (PMCA) ATPase pump Ca 2+ PMCA PMCA is a calcium extrusion pump. There are 4 different isoforms of PMCA pump encoded by 4 different genes. PMCA1 and 4 are expressed in the heart. Ca 2+ Whether PMCA4 regulates cardiac hypertrophy and how?
Human Relevance of Plasma Membrane Calcium ATPase pump isoforms (PMCAs) PMCA1 PMCA1 Ca PMCA2 PMCA3 PMCA4 2+ Ca 2+ Ca 2+ Ca 2+ PMCA3 PMCA2 PMCA4 Nav1.5 Ca 2+ Ca 2+ Ca 2+ nnos Ca 2+ Williams, Mohamed et al, JBC (2006) Blood Pressure (Cho et al., Nat. Gen., 2009 & Levy et al., Nat. Gen., 2009) Deafness (Schultz et al., NEJM, 2005) Ataxia (Zanni et al., PNAS, 2012) Long QT Syndrome (Ueda et al., PNAS, 2008)
PMCA4 Molecular complex Inhibitor Mohamed et al,, Methods Mol Biol. 2010 Integrin Armesilla, et al, JBC 2004 caveolae Ca 2+ PMCA4 Buch et al, JBC 2005 Rassf1 Calcineurin nnos Syntrophin NO Dystrophin Schuh et al, J Cell Biol (2001) Williams, Mohamed et al, JBC (2006) Mohamed et al, JMCC 2013 Hypertrophy & contractility Oceandy, Mohamed et al., Circulation 2009 Mohamed et al., Cardiovasc.Res. In Press Hypertrophy & Carcinogenesis Wu, et al. JCI 2009 Baggott, Mohamed et al., Carcinogenisis 2012 Contractility & Hypertrophy Oceandy, et al. Circulation 2007 Mohamed, et al. JBC 2009 Mohamed, et al. JBC 2011
Plasma Membrane Calcium (PMCA) ATPase pump Ca 2+ PMCA Ca 2+ Whether PMCA4 regulates cardiac hypertrophy and how?
PMCA4 KO Mohamed et al., JBC 2011 Protected from pathological hypertrophy Wild type Sham Wild type TAC PMCA4 KO Sham PMCA4 KO TAC
Identification of the first PMCA4 specific inhibitor PMCA4 Ca 2+ ADP ATP ATA Coupled enzyme assay on membrane microsomes from PMCA4 overexpressing cells Screened 1300 compounds of medically optimised drug library Mohamed et al., Methods Mol Biol. 2010 Mohamed et al., JMCC. 2013
Aurintricarboxylic acid (ATA) is the inhibitor % inhibition first PMCA4 specific 100 80 60 40 PMCA4 PMCA1 SERCA Na + K + ATPase 20 0 IC 50 = 0.1µM 0 0.2 0.4 0.6 0.8 1 ATA concentration ( M) Mohamed et al., JMCC. 2013
ATA inhibits PMCA4 activity in situ Cardiomyocytes w 1 e 0 r μ e M in A fe T c A ted with genetically modified calcium sensor (GCaMP2) viruses Cytopla smic- GCamP 2 Perfusion with Plain Tyrode s solution followed by tyroid solution containing 10µM ATA 50% PMCA 4- GCamP 2 Monitor the calcium oscillation during electric stimulation at 1Hz PMCA4 mut- GCamP 2 Mohamed et al., JMCC. 2013
HW / TL (mg/mm) Fibrosi μ s % m2 ATA reverses existing hypertrophy in mice Sham TAC + vehicle TAC +ATA 12 10 * 600 2.5 500 2 * 8 6 4 TAC ATA - - + - + + 4 1 0.5 0 1 300 0.5 2000 TAC ATA - - + - + +
1-(dF/dFmax) New high-throughput screening assay for PMCA4 Homogeneous Time Resolved Fluorescence (HTRF) ADP 337nm FRET ADP-d2 337nm PMCA4 + EU 3+ 680nm Ca 2+ ATP 620nm 620nm 90 No FERT 80 70 60 50 40 30 PM CA4 microsomes Lac Z microsomes 20 10 0 0 5 10 15 20 25 30 Microsomal protein concentration (ng/µl) Mohamed et al J Pharm Pharmaceut Sci. 16(2) 217-230, 2013
High-throughput screening for PMCA4 inhibitors Screened 25000 compounds and identified 112 primary hits Currently under testing for efficiency and specificity 77 confirmed hits from the original solid Best inhibitor
Conclusion 1 PMCA4 inhibitor emulates the effect of PMCA4 knockout on cardiac hypertrophy
Cardiomyocytes Vs Fibroblasts Fibroblast Cardiomyocytes
PMCA4 cardiomyocyte specific knockout did not show protection against pathological hypertrophy
HW/TL (mg/mm) ATA reverses existing hypertrophy in PMCA4 cko mice 12 10 * 8 6 4 sham TAC vehicle TAC ATA 2 0 WT TG PMCA4 cko
What is the Mechanism? fibroblast cardiomyocytes macrophages Microarray analysis: PMCA4 -/- vs Wild type fibroblasts
Microarray in PMCA4 KO skin and cardiac fibroblasts showed increased expression of Wnt inhibitors (SFRP2, IGFBP4 &IGFBP5) 1 6 folds
Schematic summary of signaling molecules described to inhibit Wnt/β-catenin signaling in the cardiac compartment. Bergmann M W Circulation Research 2010;107:1198-1208
Does PMCA4 -/- fibroblast reduce the hypertrophic response in WT cardiomyocyte? Is this phenotype through upregulation of secreted sfrp2? Adult cardiac fibroblast (ACF) Neonatal Rat Cardiomyocytes (NRCM) Adult cardiac fibroblast (ACF) was plated in medium suitable for NRCM for 24 hours medium was used for Plating NRCM
Cell size arbitrary units Treatment of NRCM with conditioned medium of WT or PMCA4 -/- cardiac fibroblasts Control +PE +PE+ anti sfrp2 WT medium Red: α-actinin staining KO medium 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 * * * Scale bar =25μm WT KO WT KO WT KO Control +PE +PE+anti sfrp2 PE: Phenylephrine
n= 7 n= 7 n= 7 n= 7 Fold Expression Fold induction Fold induction Cross talk mechanism between fibroblast and cardiomyocytes XX X 1.8 mrna expression of sfrp2 bn a F sa κ l B int l r u a cif l e lu r l as c e alc a i c um tivity 6 * * 1 2.4 * * 5 4 3 1.2 1.6 1 1.4 1. 0 2.8 01.6 2 1 0. 0 8.4 0.6 0.2 0.4 5 4 0 0.2 6 WT KO 6 0 ACF ACF 0 NFkB inhibitor - WT + - KO + WT ACF ACF KO AC A F C F ACF: Adult Cardiac Fibroblasts
Cross talk mechanism between fibroblast and cardiomyocytes During hypertrophic response X P β- catenin Proteasomal degx radation
TCF/LEF luciferase activity Arbitrary unit TCF/LEF luciferase activity (β-catenin activity reporter) ACF NRCM 2.5 2 * * * 1.5 1 0.5 0 WT KO WT KO WT KO control PE +PE+anti sfrp2 PE: Phenylephrine
Conclusion 2 Cardiac fibroblasts lacking PMCA4 produces higher levels of sfrp2 which inhibits the hypertrophic response in the neighbouring cardiomyocytes.
Current work PMCA4 KO Stem cell derived cardiomyocytes are potentially for better stem cell therapy for heart failure
The multiple faces of Wnt signaling during early cardiac development. PMCA4 KO Stem cells for better stem cell therapy for heart failure Gessert S, Kühl M Circulation Research 2010;107:186-199 Copyright American Heart Association
Generation of hips-cm Human Skin Fibroblasts Emerging ips colony ips colony Beating cardiac colonies Embryonic bodies
Fold induction Fold induction Fold induction hipsc-cms Nkx-2.5 ctnt 20000 15000 10000 5000 ctnt * 1200 1000 800 600 400 200 0 a MHC 0 0 ips SF non CM CM ips SF non CM CM ips SF non CM CM * 500 400 300 200 100 MLC 2.V *
What is the relation between the heart and the male reproductive system? Viagra may prevent and improve failure heart http://www.emaxhealth.com/1/80/28217/viagra-may-prevent-and-improve-heart-failure.html
PMCA4 KO Protected from pathological hypertrophy Immotile sperms Wild type Sham Wild type TAC PMCA4 KO Sham PMCA4 KO TAC
Effect of ATA on sperm motility Control + 30uM ATA
F/F (%) ATA inhibits sperm motility through elevation of intracellular calcium in the sperm 60 40 AP2 ATA (µm) 10 3 1 0,3 0,1 20 0 0 250 500 time (s) 750 1000
Doughnut microspheres for local non- hormonal contraception Awarded $100,000
Conclusion 3 PMCA4 inhibitor emulates the effect of PMCA4 knockout on sperm motility and could be a potential non hormonal contraceptive
Acknowledgements Card iovascular Research Group, Manchester Riham AbouLeisa Min Zi Sukhpal Prehar Elizabeth Cartwright Ludwig Neyses Delvac Oceandy Mamas Mamas Sally Hammad Arfa Maqsood Nicholas Stafford Robert Little Loan Nguyen Riyad Almaimani Florence Baudoin Simon Zakeri Abigail Robertson Vera Stankovij Collaborators: Prof. Michael I. Kotlikoff, Cornel University, USA Prof. Manuela Zaccolo University of Oxford, UK Prof. Gerd Hasenfuss University of Gottingen Dr. Kaomie Guan University of Gottingen Dr. Sheraz Gul European Screening port Prof. U. B Kaupp MaxPlanck Institute, Germany Prof. Deepak Srivastava J David Gladstone Research Institutes UCSF
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