Drapeau FrançaisCharacterization of DMD and CPVT patient-specific cardiomyocytes derived from induced pluripotent stem cells

Cardiac excitation-contraction coupling refers to a cellular cascade leading to contraction. Briefly, membrane depolarization activates voltage-sensing Ca2+ channels in the transverse tubules that in turn activate the ryanodine receptor 2 (RyR2), sarcoplasmic reticulum (SR) Ca2+ release and cell contraction. RyR2 is a macromolecular complex interacting with the channel-stabilizing subunit calstabin2 (FKBP12.6), kinases, phosphatases, phosphodiesterase and calmodulin.

Maladaptive cAMP-dependent protein kinase A (PKA)-mediated phosphorylation and redox-dependent modifications (thio-nitrosylation and oxidation) of the RyR2 contribute to impaired Ca2+ handling and contractile dysfunction in heart failure, diabetes and muscular dystrophies including the severe Duchenne muscular dystrophy (DMD). Single-point mutations of RyR2 are also linked to cardiomyopathiFig 1 Theme 2 EQ2es such as catecholaminergic polymorphic ventricular tachycardia (CPVT).

Using animal models, we have previously shown that SR Ca2+ leak mediated by RyR2 remodeling due to post-translational modifications (DMD) or single-point mutations (CPVT), can cause ventricular tachycardia, sudden cardiac death under stress conditions such as beta-adrenergic stimulation, and/or dilated cardiomyopathy leading to heart failure. However, the animal models classically used do not always recapitulate the phenotype seen in patients and it is ethically impossible to obtain fresh cardiac ventricular biopsies from patients.

Therefore, to further validate the role of RyR2 in DMD and CPVT patients, we hypothesize that human induced-pluripotent stem cells (hiPSC)-derived cardiomyocytes (CMs) from DMD and CPVT patient biopsies represent an excellent ex vivo alternative model to investigate Ca2+ handling abnormalities in CMs lacking dystrophin and in CMs harboring RyR2 single-point mutations (Fig. 1 and  2).

équipe 2 Axe 2 2Overall, the main objective of this research project is to provide a deeper knowledge about the basic mechanisms initiating and responsible for cardiomyopathies related to DMD and CPVT. Another objective is also to screen new pharmacological tools to treat those cardiac disorders by targeting specifically RyR2.

Figure 2 : representative immunochemistry of cardiac troponin T (cTnT) in human pluripotent stem cell-derived cardiomyocytes. The white scale bar represents 20 μm (adapted from Pesl, Acimovic et al, 2014, Heart Vessels).

Collaborations :

  • Department of cardiopediatry, CHRU Montpellier, France
  • Petr Dvorak and Vladimir Rotrekl, Department of Biology, Faculty of Medicine Masaryk University, Brno, Czech Republic
  • Petr Vondracek, Children Hospital, Faculty of Medicine Masaryk University, Brno, Czech Republic
  • Marwan Refaat, Department of Internal Medicine, American University of Beirut, Faculty of Medicine, Beirut, Lebanon
  • Jonathan Lu, Department of Pharmacology, Columbia University, New York (NY), USA

Financial support : 

  • FRM
  • AFM
  • ESC
  • PHC Barrande grant (French-Czech network)
  • Czech GACR grant
  • Fondation Cœur et Expérimental

Major Publications :

  1. Jelinkova S, Fojtik P, Kohutova A, Vilotic A, Markova L, Pesl M, Jurakova T, Kruta M, Vrbsky J, Gaillyova R, Valaskova I, Frak I, Lacampagne A, Forte G, Dvorak P, Meli AC, Rotrekl V. 1. (2019). Dystrophin Deficiency Leads to Genomic Instability in Human Pluripotent Stem Cells via NO Synthase-Induced Oxidative Stress.Cells 2019 Jan 15;8(1). pii: E53.
  1. Acimovic I, Refaat MM, Moreau A, Salykin A, Reiken S, Sleiman Y, Souidi M, Přibyl J, Kajava AV, Richard S, Lu JT, Chevalier P, Skládal P, Dvořak P, Rotrekl V, Marks AR, Scheinman MM, Lacampagne A, Meli AC. (2018). “Post-Translational Modifications and Diastolic Calcium Leak Associated to the Novel RyR2-D3638A Mutation Lead to CPVT in Patient-Specific hiPSC-Derived Cardiomyocytes.J Clin Med. 2018 Nov 8;7(11). pii: E423.
  1. Cheung JW, Meli AC, Xie W, Mittal S, Reiken S, Wronska A, Xu L, Steinberg JS, Markowitz SM, Iwai S, Lacampagne A, Lerman BB, Marks AR. Short-coupled polymorphic ventricular tachycardia at rest linked to a novel ryanodine receptor (RyR2) mutation: Leaky RyR2 channels under non-stress conditions. (2014) Int J Cardiol. 180C:228-236.
  1. Meli AC, Refaat MM, Dura M, Reiken S, Wronska A, Wojciak J, Carroll J, Scheinman MM, Marks AR A novel ryanodine receptor mutation linked to sudden death increases sensitivity to cytosolic calcium. (2011). Circ Res. 109: 281-290.

Coordinators :

Lacampagne Alain

 Participants : 

Amedro Pascal