Investigating LMNA-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Figure 1. The LMNA-mutated patients. (A) Pedigree of the family with the LMNA gene mutation. Solid symbols: affected individuals; Solid symbols with a “+”: an affected individual with a proven mutation; Open symbols: clinically unaffected; Crossed symbol: deceased. Red arrow: a 63-year-old (at time of biopsy) DCM male patient, who underwent heart transplantation at age 56 for his severe heart failure (ejection fraction of 19%). His brother died at the age of 44 from the same disease, while awaiting heart transplantation. Green arrow: The 35-year-old son (at time of biopsy) of the 63-year-old patient was asymptomatic, but showed ECG abnormalities at the time of diagnosis at age 34. Biopsies were obtained from both the father and son. (B) Twelve-lead ECG of the son at age 34 demonstrating first-degree AV block and left anterior fascicular block.

Figure 2. The LMNA-mutated patients iPSCs characteristics. (A) Father, son and control iPSCs showing normal karyotype. (B) Pluripotency staining of LMNA-mutated and control iPSCs. Immunofluorescence staining of typical pluripotent markers Tra1-81 (red), Tra1-60 (red), Oct4 (red), SSEA4 (green) and Nanog (green) in iPSCs generated from father, son and control. Nuclei are stained with DAPI (blue). Scale bar represents 100 µm. (C) Teratoma histological analysis obtained from in vivo differentiated LMNA-mutated and control iPSCs. Teratomas were obtained from SCID-NOD mice injected with either father, son or control iPSCs which differentiated in vivo into derivatives of all three germ layers: ectoderm, endoderm and mesoderm. n—Neuronal tissue represents ectodermal lineage. e—Endodermal epithelium with prominent mucus-producing cells representing endoderm formation. c—Cartilage as well as the chondrocyte area and muscle representing mesoderm formation. Scale bar represents 100 µm.

Figure 3. Spontaneous beat rate and If in control, father and son iPSC-CMs. (A) Representative spontaneous action potentials recorded from control, father and son iPSC-CMs. (B) Mean beat rate of control, father and son iPSC-CMs. control (FSE-5 m and 24.5), n = 30; son, n = 14; father, n = 27. One-way ANOVA was performed followed by Holm–Sidak post hoc analysis. * p < 0.05. (C) Representative recordings of If in control, father and son iPSC-CMs. (D) If current–voltage (I–V) relations in control and LMNA-mutated iPSC-CMs. Control (FSE-5 m), n = 8; son, n = 10; father, n = 18. Two-way ANOVA was performed followed by Holm–Sidak post hoc analysis. * p < 0.05 vs. control; # p < 0.05 vs. son.

Figure 4. Action potential characteristics and ICa,L in control, son and father iPSC-CMs. (A) Representative APs of control, son and father iPSC-CMs. (B–H) Summary of AP parameters in control and LMNA-mutated iPSC-CMs: (B) APA, (C) AP peak, (D) dV/dtmax, (E) APD20, (F) APD50, (G) APD90 and (H) MDP. Control (FSE-5 m and 24.5), n = 30; son, n = 14; father, n = 27. One-way ANOVA was performed followed by Holm–Sidak post hoc analysis. * p < 0.05. (I) Representative recordings of the ICa,L in control, son and father iPSC-CMs. (J) ICa,L current–voltage (I–V) relations in control and LMNA-mutated iPSC-CMs (n = 13 control (FSE-5 m); n = 8 son; n = 13 father). Two-way ANOVA was performed followed by Holm–Sidak post hoc analysis. * p < 0.05 father vs. control; # p <0.05 son vs. control.

Figure 5. Afterdepolarizations and increased BRV in son and father LMNA-mutated iPSC-CMs. (A–C) Representative recordings showing afterdepolarization-free spontaneous activity in control, son and father cardiomyocytes. (D,E) Spontaneous AP recordings demonstrating DADs in son iPSC-CMs. (F,G) Spontaneous AP recordings demonstrating afterdepolarizations in father iPSC-CMs. (H) Percentage of experiments with afterdepolarization/s in control, son and father cardiomyocytes (n = 30 control (FSE-5 m and 24.5); n = 14 son; n = 28 father). (I) Representative IBI histograms in control, son and father cardiomyocytes. (J) Superimposed representative IBI vs. time plots of control, son and father iPSC-CMs. (K) Superimposed representative Poincaré plots of control, son and father iPSC-CMs. (L) COV analysis in control, father and son cardiomyocytes. (M) SD1 analysis in control, father and son cardiomyocytes. (N) SD2 analysis in control, father and son cardiomyocytes. Control (FSE-5 m and 24.5), n = 30; son, n = 14; father, n = 27. One-way ANOVA was performed followed by Holm–Sidak post hoc analysis. * p < 0.05.

Figure 6. Abnormal response of LMNA-mutated cardiomyocytes to β-adrenergic stimulation and rapid pacing. (A) AP recordings from control, son and father cardiomyocytes showing a positive chronotropic response to isoproterenol. (B) Representative AP recordings from father and son cardiomyocytes showing afterdepolarization/s and cessation of spontaneous AP firing in response to isoproterenol. (C) Percentage of cells showing positive chronotropic response to isoproterenol (n = 7 control; n = 7 son; n = 7 father). (D) Percentage of cells showing electrophysiological abnormalities in response to isoproterenol (n = 7 control; n = 6 son; n = 7 father). (E) Representative recordings of trains protocol in control, son and father cardiomyocytes. (F–H) Percentage of cells showing electrical abnormality(ies) following stimulation at (F) 1 Hz (n = 6 control (FSE-5 m and 24.5); n = 4 son; n = 4 father), (G) 1.5 Hz (n = 8 control (FSE-5 m and 24.5); n = 4 son; n = 4 father) and (H) 2 Hz (n = 8 control (FSE-5 m and 24.5); n = 4 son; n = 3 father).

Figure 7. Effects of KB-R7943 and ranolazine in LMNA-mutated cardiomyocytes. AP recordings demonstrating different effects of KB-R7943: (A) Elimination of DADs in the son LMNA-mutated cardiomyocytes; (B) Cessation of spontaneous firing in the father LMNA-mutated cardiomyocytes showing no DADs. AP recordings from the son (C) and father (D) cardiomyocytes showing afterdepolarizations and arrhythmias before and after application of ranolazine (10 μM).

Figure 8. TEM images of LMNA-mutated and control iPSC-CMs. TEM images from control (FSE-5 m) (A–C), father (D–F) and son (G–I) iPSC-CMs. Thin red lines mark nuclear silhouettes. Black arrows point out the nucleoli. (A,B) The nuclei (n) of control iPSC-CMs show a smooth contour and scarce heterochromatin, mostly associated with the nuclear envelope (NE). Heterochromatin clumps are also visible inside the nucleus. (B) The nucleolus (arrow) with heterogeneous appearance is associated with peripheral heterochromatin. (C) Higher magnification shows nuclear lamina (NL) (arrowhead) attaching the heterochromatin (h) to the NE (arrows). (D,E) The nuclei (n) of father iPSC-CMs show an irregular contour and indentations. The heterochromatin is limited to the nuclear periphery. (E) The nucleolus (arrow) is densely packed. (F) Higher magnification shows short segments of NL (arrowhead). The heterochromatin (h) is scarcely attached to the NE (arrows). (G,H) The nuclei (n) of son iPSC-CMs show highly indented nuclear contour. (H) The heterochromatin is almost absent in the nucleus and the nucleolus (arrow) is large with numerous visible fibrillary centers. (I) Higher magnification shows the membranes of…

Figure 9. Altered gene expression in LMNA-mutated iPSC-CMs. (A) Expression of genes involved in Ca2+ handling in LMNA-mutated iPSC-CMs compared to control. * padj < 0.05 LMNA-mutated vs. control. (B) Schematic illustration of a LMNA-mutated cardiomyocyte showing the expected effect of its altered gene expression on key Ca2+ handling proteins: LTCC, plasma membrane Ca2+ ATPase (PMCA), NCX, RyR2, calsequestrin 2 (CASQ2), triadin (TRDN), junctin (JCN), calstabin2, SERCA2, phospholamban (PLN) and mitochondrial Ca2+ uniporter (MCU). Green indicates upregulation; Red indicates downregulation. White indicates gene expression alteration which is not statistically significant.