Modeling of LMNA-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cells

Abstract

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure and heart transplantation. A portion of familial DCM is due to mutations in the LMNA gene encoding the nuclear lamina proteins lamin A and C and without adequate treatment these patients have a poor prognosis. To get better insights into pathobiology behind this disease, we focused on modeling LMNA-related DCM using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Primary skin fibroblasts from DCM patients carrying the most prevalent Finnish founder mutation (p.S143P) in LMNA were reprogrammed into hiPSCs and further differentiated into cardiomyocytes (CMs). The cellular structure, functionality as well as gene and protein expression were assessed in detail. While mutant hiPSC-CMs presented virtually normal sarcomere structure under normoxia, dramatic sarcomere damage and an increased sensitivity to cellular stress was observed after hypoxia. A detailed electrophysiological evaluation revealed bradyarrhythmia and increased occurrence of arrhythmias in mutant hiPSC-CMs on β-adrenergic stimulation. Mutant hiPSC-CMs also showed increased sensitivity to hypoxia on microelectrode array and altered Ca²⁺ dynamics. Taken together, p.S143P hiPSC-CM model mimics hallmarks of LMNA-related DCM and provides a useful tool to study the underlying cellular mechanisms of accelerated cardiac degeneration in this disease.

Keywords: LMNA; Lamin A/C; dilated cardiomyopathy; hypoxia; induced pluripotent stem cell; microelectrode array and calcium imaging.

Figure 1

Characterization of lamina structure in human induced pluripotent stem cell (hiPSC) derived cardiomyocytes. (A) Dissociated control and dilated cardiomyopathy (DCM) hiPSC-cardiomyocytes (CMs) were cultured either in normal culture conditions or exposed to ischemic stress for 3 h, fixed and stained for lamin A (LA, green), α-actinin (magenta) and DNA (DAPI; blue). Representative maximum projections of Z-stack sections (merged) and single mid-plane confocal sections (LA, DAPI) from control1 and DCM2 CMs are shown. Scale bar 10 µm. Fluorescent intensity values are illustrated below the image with nucleoplasm/lamina (N/L) ratio numbers. (B) Fluorescence intensities at the lamina region and in the nucleoplasm were determined from mid-plane confocal sections of 20–30 randomly selected cells and the average ratios of the signals (nucleoplasm/lamina) were plotted. (***) p < 0.001. (C) Quantification of nuclear circularity. (**) p < 0.01 and (***) p < 0.001. (D) Example images of different circularity values corresponding to the calculated average values (Perfect circle = 1.0).

Figure 2

Functional characterization of hiPSC derived cardiomyocytes. (A) Table shows baseline electrophysiological characteristics by microelectrode array (MEA) from spontaneously beating cardiomyocyte clusters. (B) Representative field potential traces from control, DCM1 and DCM2 cardiomyocyte clusters at baseline. (C–E) The bar charts show chronotropic responses of control, DCM1 and DCM2 hiPSC-CM clusters to distilled water used as vehicle (n = 23, 16, 16 respectively); 100 nM adrenaline (n = 24, 26, 18 respectively) and 1µM adrenaline (n = 18, 16, 13 respectively) with black and red bars showing the beating frequency at baseline and under vehicle/adrenaline respectively. Combined results from Control1 and Control2 are shown. Data is presented as mean ± s.e.m., (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001.

Figure 3

Arrhythmia occurrence in hiPSC-CM beating aggregates on MEA. (A) Percentage of arrhythmic aggregates compared to baseline after the addition of a) vehicle (Control, DCM1 and DCM2, n = 41, 26 and 16 respectively), b) 100nM adrenaline (Control, DCM1 and DCM2, n = 32, 28 and 16 respectively) and c) 1µM adrenaline (Control, DCM1 and DCM2, n = 23, 24 and 13 respectively). (B) Representative MEA extracellular recordings from DCM hiPSC-CMs after application of adrenaline. A 60 second MEA trace is shown on left and a magnified view of the corresponding signal (red dotted lines) on the right. The representative traces showing a normal beating rhythm (a), alternations (b), dysrhythmia (c), premature beat (d) and ventricular-tachycardia like arrhythmia (e–g). The red arrows indicate arrhythmia other than alternations or dysrhythmia. (C) An irregular beating pattern observed in DCM1 and DCM2 aggregates was quantified for ≥30 consecutive beats by the formula for short term variation (STV) of inter-beat interval (a–c) and STV of field potential duration (d–f). BL signifies baseline, veh is vehicle and Adr is adrenaline. Data is presented as mean ± s.e.m. ANOVA non-parametric tests and a paired t-test were used in statistical analysis; (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001. Combined results from Control1 and Control2 are shown.

Figure 4

Confocal microscopy analysis of hiPSC-CM sarcomere structure under normal and hypoxic conditions. (A) Control and DCM CMs were cultured under normal culture conditions or exposed to ischemic stress, fixed and stained for α-actinin, LA and DAPI. Representative maximum projections of Z-stack sections from control1 and DCM2 are shown. Scale bar: 10 µm. (B) TT power analyses of the sarcomere length and organization were carried out with TTorg plugin in ImageJ. TTorg workflow of the example images: Magnification of an original image, 2D fast Fourier transformation (FFT) spectrum of the image, grey level profile of the FFT spectrum and analysis results (n = 20, AU = Arbitrary Units). Data is expressed as mean ± s.e.m.,* p < 0.05. (C) Transmission electron microscopy images of control1 and DCM2 CMs under normal culture conditions and ischemic stress are shown. Scale bar: 1 µm.

Figure 5

DCM hiPSC-CMs show increased elevated cellular stress. (A) Western blot analysis of lamin A/C, phospho-eIF2α (peIF2α), Hsp90, Hsp70, Hsp60, phospho-ERK1/2 (pERK1/2) and cleaved caspase-3 under normal culture conditions and after exposure to ischemic stress for 3 h. Actin was used as a loading control. The average numerical values of signal intensities relative to the loading control (actin) are shown below each blot. n = 2 individual experiments. Control2 hiPSC-CMs were not qualified for analysis due to lower differentiation efficiency compared to other lines. (B) Confocal microscopy analysis of Hif-1α intensity. Control1, DCM1 and DCM2 hiPSC-CMs were cultured either in normal culture conditions or exposed to ischemic stress for 2 h, 3 h, 4 h and 5 h, fixed and stained for Hif-1α, cardiac marker cTnT and DNA (DAPI). A 3 h time point is shown. Scale bar 20 µm. (C) The fluorescence intensities of Hif-1α were determined from all the confocal sections of >15 randomly selected cells at different time points and the average normalized signals were plotted. (D) Control1 and DCM2 hiPSC-CMs were cultured under normal culture conditions, fixed and stained for γH2AX, cTnT and DNA (DAPI). (E) γH2AX positive cells from control1 and DCM2 were counted and plotted (n = 500). (F) Effect of three repeated 3 h cycles of hypoxia (1% O₂) shown as H1, H2 and H3 and overnight re-oxygenation (19% O₂) on beat rate of hiPSC-CMs recorded on MEA. Control data presented in F is combined from Control1 and 2. Data is expressed as mean ± s.e.m., (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001.

Figure 6

Analysis of intracellular calcium dynamics by calcium (Ca2⁺) imaging in hiPSC-CMs. (A) Measurement of Ca²⁺ transient parameters ∆F/F0, peak duration, rise time, decay time and decay tau for the calcium imaging data. (B) Ca²⁺ transient characteristics recorded from hiPSC-CMs at baseline. (C) Calcium imaging parameters for control, DCM1 and DCM2 CMs at baseline are shown as ∆F/F0 (n = 34, 40 and 42 respectively), decay tau (n = 31, 41 and 41 respectively), rise time (n = 32, 39 and 40 respectively) and decay time (n = 31, 34 and 35 respectively). (D) Calcium imaging parameters at baseline (BL) and in the presence of 10 nM adrenaline. (E) Representative regular (a) and arrhythmic Ca²⁺ transients (b–f) detected under baseline or under 10 nM adrenaline in control, DCM1 and DCM2 CMs. Dysrhythmia (b) and alternations (c) were categorized as minor arrhythmias and oscillations (d), extra peaks (e) and plateau abnormalities (f) as major arrhythmias. The red arrows represent the Ca²⁺ abnormalities. (F) Frequency of major arrhythmias in control, DCM1 and DCM2 single dissociated hiPSC-CMs at baseline (BL) and under 10 nM adrenaline (Adr). Data is presented as mean ± s.e.m and ANOVA or non-parametric pair test was used for statistical analysis. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001. Control data presented here is combined from Control1 and Control2.