Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to β-adrenergic stimulation

Abstract

Sudden cardiac death caused by ventricular arrhythmias is a disastrous event, especially when it occurs in young individuals. Among the five major arrhythmogenic disorders occurring in the absence of a structural heart disease is catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a highly lethal form of inherited arrhythmias. Our study focuses on the autosomal recessive form of the disease caused by the missense mutation D307H in the cardiac calsequestrin gene, CASQ2. Because CASQ2 is a key player in excitation contraction coupling, the derangements in intracellular Ca(2+) handling may cause delayed afterdepolarizations (DADs), which constitute the mechanism underlying CPVT. To investigate catecholamine-induced arrhythmias in the CASQ2 mutated cells, we generated for the first time CPVT-derived induced pluripotent stem cells (iPSCs) by reprogramming fibroblasts from skin biopsies of two patients, and demonstrated that the iPSCs carry the CASQ2 mutation. Next, iPSCs were differentiated to cardiomyocytes (iPSCs-CMs), which expressed the mutant CASQ2 protein. The major findings were that the β-adrenergic agonist isoproterenol caused in CPVT iPSCs-CMs (but not in the control cardiomyocytes) DADs, oscillatory arrhythmic prepotentials, after-contractions and diastolic [Ca(2+) ](i) rise. Electron microscopy analysis revealed that compared with control iPSCs-CMs, CPVT iPSCs-CMs displayed a more immature phenotype with less organized myofibrils, enlarged sarcoplasmic reticulum cisternae and reduced number of caveolae. In summary, our results demonstrate that the patient-specific mutated cardiomyocytes can be used to study the electrophysiological mechanisms underlying CPVT.

Fig 1

Generation of pluripotent stem cells from healthy and CPVT patients. (A) Primary skin fibroblasts derived from a CPVT patient skin biopsy, and a corresponding representative iPSC colony. (B) Karyotype analysis of iPSCs from a CPVT patient. (C) Immunostaining of typical pluripotent markers shown for the iPSCs derived from the CPVT patient, clone HDF7.5. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. (D) Immunostaining of 21-day-old EBs derived from the iPSCs-HDF7.5 demonstrates expression of mesodermal (SMA, CD31, vWF), endodermal (AFP, glucagon) and ectodermal (tubulin-β3, nestin, GFAP) marker proteins. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (E) Histological analysis of a representative teratoma obtained from in vivo differentiated cells of iPSCs-HDF7.6. The formed teratomas contained derivatives of all three germ layers (ectoderm, mesoderm and endoderm). (E1) Muscle (left-up of image, mesoderm) and endocrine glands (arrow, endoderm). (E2) Cartilage (arrow, mesoderm). (E3) Gut epithelium (endoderm). (E4) Neural-like tissues at arrow (ectoderm). All images were obtained from formalin-fixed and paraffin-embedded teratoma sections stained with haematoxylin and eosin. Scale bar: 50 μm.

Fig 2

Characterization of the calsequestrin D307H mutation. (A) BamHI restriction analysis of the D307H mutation for the control and CPVT iPSCs clones, and their parental cells (HDF). Human embryonic stem cells (clone H9.2) are included for comparison. The substitution at nucleotide 1183 creates a BamHI restriction site in the mutated sequence. The 495bp PCR product of genomic DNA is cleaved in the CPVT patient chromosome to 150bp and 345bp products. (B) Sequence analysis of genomic CASQ2 obtained from iPSCs derived from control subject (WT/WT) and CPVT patient (M/M) revealing a homozygous missense mutation in CASQ2 exon 9, in position 569 of the coding sequence, converting aspartic acid to histidine at position 307 of the protein.

Fig 3

Characterization of CPVT iPSCs-CMs. (A) Micro-dissected contracting areas from the control and CPVT iPSCs-CMs were stained for typical myofilament proteins. The cardiomyocytes were co-labelled with anti-cardiac troponin I (green) and anti-sarcomeric α-actinin (red). (B) Representative PCR analysis of the cardiac gene troponin-T, and the calcium associated genes CASQ2, calreticulin, RyR2, junctin, triadin, NCX1, SERCA2 and the housekeeping gene GAPDH (n = 2). (C) The spontaneous beating rate of control iPSCs-CMs (n = 10) and CPVT iPSCs-CMs (n = 20). bpm: beats per minute.**P < 0.01. Scale bar: 10 μm.

Fig 5

Transmission electron microscopy (TEM) analysis in control iPSCs-CMs (A, C, E) and CPVT iPSCs-CMs (B, D, F, G). (A) TEM of control iPSCs-CMs and (B) CPVT iPSCs-CMs shows cells with myofibrils (mf), glycogen (gly), mitochondria (m) and lipid droplets (L). N: nucleus. (C) Note large sarcomeres in control iPSCs-CMs, and (D) slender sarcomeres in CPVT iPSCs-CMs. Z: Z bands; SR: sarcoplasmic reticulum. (E–F) Ultrastructure of ‘Ca²⁺-release units’ (small arrows) formed by ryanodine receptors between peripheral SR and plasmalemma in control iPSCs-CMs (E) and CPVT iPSCs-CMs (F). Note the connection (white arrow) between caveolae (arrowheads) and SR in Control iPSCs-CMs. J: junction. (G) A CPVT-iPSCs-cardiomyocyte with normal SR, and SR with dilated (SRd) and fragmented (SRf) cisternae. Z: Z bands; gly: glycogen; L: lipid droplets; mf: myofilaments.

Fig 6

The effects of isoproterenol on the [Ca²⁺]i transients and contractions in control and CPVT iPSCs-CMs. (A) Representative contractions tracings of control iPSCs-CMs (43-day-old EB) stimulated at 0.6 Hz, in the absence (Tyrode’s) and presence of isoproterenol. (B–C) Representative contractions tracings of CPVT iPSCs-CMs (33- and 38-day-old EBs, respectively) stimulated at 0.6 Hz, in the absence (Tyrode’s) and the presence of isoproterenol. Note that after-contractions developed only in the CPVT cardiomyocytes in the presence of isoproterenol. (D) The effects of isoproterenol on contraction parameters of control iPSCs-CMs and CPVT iPSCs-CMs, *P < 0.05. (E–F) Representative [Ca²⁺]i transients of control iPSCs-CMs (40-day-old EB) and CPVT iPSCs-CMs (34-day-old EB), respectively, before and 5 min. after isoproterenol perfusion.

Fig 7

Action potential characteristics of control and CPVT iPSCs-CMs. (A) Representative spontaneous action potentials recorded from control (A) and CPVT cardiomyocytes (B). A summary of action potential duration at 20% repolarization, APD₂₀ (C), action potential duration at 50% repolarization, APD₅₀ (D), maximum diastolic potential (MDP) (E), action potential amplitude (APA) (F), and maximal upstroke velocity of phase 0 depolarization, dV/dt_max (G), in control (black) and CPVT (red) cardiomyocytes. Note that in CPVT cardiomyocytes APD₂₀ and APD₅₀ were prolonged compared to control cells (*P < 0.05).

Fig 8

Isoproterenol caused DADs and oscillatory prepotentials in CPVT iPSCs-CMs. (A) Representative spontaneous recordings from control cardiomyocytes (30-day-old EB), and (B–D) CPVT cardiomyocytes (32-, 30- and 32-day-old EBs, respectively) in the absence (Tyrode’s) and presence of isoproterenol. Note that only in CPVT cardiomyocytes, isoproterenol caused DADs (red arrows) or oscillatory prepotentials (blue arrows).

Fig 4

CASQ2 and triadin immunofluorescence expression in control and CPVT iPSCs- derived cardiomyocytes. Micro-dissected contracting areas from H9.2-CMs and from the control and CPVT iPSCs-CMs stained for (A) anti-cardiac calsequestrin (CASQ2, red) with anti-sarcomeric α-actinin (green), and (B) anti-cardiac calsequestrin (CASQ2, red) with anti-triadin (green). Nuclei were stained with DAPI (blue). Scale bar: 10 μm.