Cardiac dysfunction due to mitochondrial impairment assessed by human iPS cells caused by DNM1L mutations

Abstract

Background: DNM1L encodes dynamin-related protein 1, which plays an important role in mitochondrial and peroxisomal division. The DNM1L mutation leads to cardiac dysfunction in patients and animal models. However, the mechanism of cardiac dysfunction caused by DNM1L mutation has not been elucidated clearly at least in the studies of human cardiomyocytes.

Methods: We established human induced pluripotent stem cells (hiPSCs) from two pediatric patients with DNM1L mutation. The hiPSCs were differentiated into hiPSC-derived cardiomyocytes (hiPS-CMs). Mitochondrial morphology and function, cardiomyocyte Ca²⁺ dynamics, and contractile and diastolic function of hiPS-CMs were analyzed.

Results: The morphology of the mitochondria was abnormally elongated in patient-derived hiPS-CMs. The mitochondrial membrane potential and oxygen consumption rate were significantly decreased, resulting in reduced ATP production. In the analysis of Ca²⁺ dynamics, the 50% time to decay was significantly longer in patient-derived hiPS-CMs than in healthy control. High-precision live-imaging system analysis revealed that contractile and diastolic function was significantly impaired under isoproterenol stimulation.

Conclusion: DNM1L mutations cause mitochondrial impairment with less production of ATP in cardiomyocytes. This leads to abnormal intracellular Ca²⁺ dynamics, resulting in contractile and diastolic dysfunction.

Impact: DNM1L mutations was identified in two pediatric patients who developed cardiac dysfunction and human induced pluripotent stem cells (hiPSCs) were established from these two patients and differentiated into hiPSC-derived cardiomyocytes (hiPS-CMs). DNM1L mutations induced abnormal mitochondrial morphology, mitochondrial dysfunction, and insufficient ATP production in hiPS-CMs. In addition, hiPS-CMs with DNM1L mutation showed abnormal Ca²⁺ kinetics and impaired contractile and diastolic function. This is the first study that elucidate the mechanism of cardiac dysfunction caused by DNM1L mutations by using hiPSCs.

Fig. 1. Comparison of mitochondrial morphology in hiPS-CMs.

a Representative images taken by mitochondrial staining of hiPS-CMs with MTR. Scale bar: 25 μm. b TEM images of hiPS-CMs. Scale bar: 2 μm. Arrow heads indicate tubular, highly elongated mitochondria. c Mitochondrial length of hiPS-CMs in TEM images. The mitochondrial length was significantly elongated in patient-derive hiPS-CMs (control: 0.699 ± 0.019 μm [n = 722], patient 1: 1.127 ± 0.031 μm [n = 750], patient 2: 1.296 ± 0.036 μm [n = 788]). n = number of mitochondria in 5 cells. **p < 0.01, ****p < 0.0001. d Representative confocal microscope images of mCh-Drp1 expression and mitochondrial morphology in each patient-derived hiPS-CMs. The mitochondria were stained with MTG. Scale bar: 10 μm. e Mitochondrial morphology of mCh-Drp1-expressing hiPS-CM was significantly shorter and fragmented compared to non-expressing hiPS-CM (patient 1 mCh-Drp1(-): 4.598 ± 0.285 μm [n = 2527], mCh-Drp1(+): 2.002 ± 0.121 μm [n = 1146], patient 2 mCh-Drp1(-): 2.270 ± 0.226 μm [n = 3132], mCh-Drp1(+): 1.345 ± 0.062 μm [n = 1635]). n = number of mitochondria of 5 cells. *p < 0.05, ****p < 0.0001.

Fig. 2. Evaluation of ΔΨm and OCR of patient-derived hiPS-CMs.

a Representative fluorescence images of JC-1 stained hiPS-CMs. Aggregated JC-1 (red) and monomeric JC-1 (green) indicate polarized and depolarized mitochondria, respectively. Scale bar: 25 μm. b Ratio metric measurement (red/green fluorescence intensity) using JC-1. The ratio of red/green intensity is significantly lower in patient-derived hiPS-CMs than in control (control: 0.800 ± 0.051 [n = 15], patient 1: 0.438 ± 0.030 [n = 21], patient 2: 0.367 ± 0.063 [n = 15]). n = number of fields of view. ***p < 0.001, ****p < 0.0001, ns: no significant. c, d OCR of hiPS-CMs was measured using a high-resolution respirometry. OXPHOS of complex I was measured by adding 5 mM pyruvate and 2 mM malate, followed by 2.5 mM ADP (c). OXPHOS of complex I + II was measured by further addition of 10 mM succinate (d). The OCR is significantly reduced in patient-derived hiPS-CMs in OXPHOS complex I (control: 126.20 ± 11.33 [n = 4], patient 1: 83.83 ± 11.05 [n = 4], patient 2: 69.08 ± 8.36 [n = 4] pmol/[s × Mill]) and OXPHOS complex I + II (control: 172.10 ± 10.45 [n = 4], patient 1: 122.70 ± 13.29 [n = 4], patient 2: 101.40 ± 10.87 [n = 4] pmol/[s × Mill]), respectively. n = number of cell pellets. *p < 0.05, **p < 0.01, ns no significant.

Fig. 3. Evaluation of mitochondrial ATP production in patient-derived hiPS-CMs using MitoMAR.

a Representative fluorescence images (LUT fire) of hiPS-CMs expressing MitoMAR before and 50-min after 25 μM oligomycin administration. Scale bar: 20 μm. b Time lapse experiments of each hiPS-CMs expressing MitoMAR with oligomycin treatment (circular dots: control, triangular dots: patient 1, cross dots: patient 2). Oligomycin was added at 0 min (F0). The averages of normalized fluorescence intensity (F/F0) per single cell (n = 15 cells) are plotted at each time point. c Average F/F0 of MitoMAR after oligomycin administration. The bar graph is obtained from a data set relevant to b. The normalized FI in the hiPS-CMs of patients 1 and 2 is significantly higher than that in control (control: 0.384 ± 0.002 [n = 15], patient 1: 0.521 ± 0.001 [n = 15], patient 2: 0.681 ± 0.007 [n = 15]). n = number of hiPS-CMs. ****p < 0.0001.

Fig. 4. Evaluation of intracellular Ca²⁺ kinetics in patient-derived hiPS-CMs.

a Representative fluorescence intensity waveforms of Fluo-4, an intracellular calcium indicator, in beat rate-controlled hiPS-CMs. Pacing rates are at 100 bpm (upper) and 150 bpm (lower), respectively. b, c The ratio of peak-to-base fluorescence intensity (F/F0) and 50% time to decay (T50) of Fluo-4 fluorescence intensity in beat rate-controlled hiPS-CMs. Pacing rates are at 100 bpm (upper) and 150 bpm (lower), respectively. There is no significant difference in F/F0 between control- and patient-derived hiPS-CMs shown in b (control: 1.996 ± 0.107 [n = 18], patient 1: 1.828 ± 0.175 [n = 20], patient 2: 1.886 ± 0.146 [n = 14] at pacing rate 100 bpm; control: 1.305 ± 0.040 [n = 13], patient 1: 1.228 ± 0.027 [n = 10], patient 2: 1.203 ± 0.039 [n = 6] at pacing rate 150 bpm). The T50 is significantly longer in patient-derived hiPS-CMs shown in c (control: 193.50 ± 4.94 [n = 18], patient 1: 222.40 ± 6.37 [n = 20], patient 2: 246.3 ± 7.10 [n = 14] ms at pacing rate 100 bpm; control: 140.40 ± 4.37 [n = 14], patient 1: 162.10 ± 5.27 [n = 10], patient 2: 178.10 ± 5.75 [n = 6] ms at pacing rate 150 bpm. d, e Effects of mitochondrial inhibitors on F/F0 and T50 in beat rate-controlled control hiPS-CMs. Pacing rates are at 100 bpm (upper) and 150 bpm (lower), respectively. Cells were pretreated with rotenone, a complex I inhibitor, for 1 h and with CCCP, an OXPHOS uncoupler, for 3 h. F/F0 shown in (d) (non-treated: 4.30 ± 0.74 [n = 22], rotenone: 2.22 ± 0.14 [n = 22], CCCP: 1.73 ± 0.08 [n = 21] ms at pacing rate 100 bpm; non-treated: 2.16 ± 0.15 [n = 22], rotenone: 1.51 ± 0.07 [n = 20], CCCP: 2.10 ± 0.36 [n = 7] ms at pacing rate 150 bpm). T50 shown in (e) (non-treated: 164.60 ± 4.50 [n = 22], rotenone: 206.90 ± 8.18 [n = 22], CCCP: 216.3 ± 6.36 [n = 21] ms at pacing rate 100 bpm; non-treated: 141.10 ± 4.49 [n = 22], rotenone: 160.30 ± 6.23 [n = 20], CCCP: 182.70 ± 10.25 [n = 7] ms at pacing rate 150 bpm). n = number of CMs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 5. Evaluation of contractile and diastolic function of patient-derived hiPS-CMs.

a–c The beating of hiPS-CMs was captured and the contractile and diastolic speeds were plotted (a). The increase in beating rate with isoproterenol titration is less in patient-derived hiPS-CMs than that in control. (beating rate Pre: 58.00 ± 1.02 vs. 28.15 ± 0.94 vs. 18.79 ± 0.71; 0.03 μM: 65.50 ± 1.03 vs. 33.45 ± 0.86 vs. 54.47 ± 2.24; 0.1 μM: 136.52 ± 1.72 vs. 74.23 ± 2.06 vs. 87.14 ± 2.00; 0.3 μM: 145.98 ± 1.05 vs. 75.74 ± 1.11 vs. 90.24 ± 2.44; 1.0 μM: 133.92 ± 0.98 vs. 61.75 ± 2.25 vs. 86.34 ± 2.51 bpm (control [n = 48] vs. patient 1 [n = 48] vs. patient 2 [n = 48])). n = number of fields of view. ****p < 0.0001. Changes in MCS and MRS with isoproterenol administration (b, c). Both MCS and MRS are lower in patient-derived hiPS-CMs than those in control (MCS Pre: 50.04 ± 2.81 vs. 38.10 ± 2.22 vs. 32.62 ± 2.10; 0.03 μM: 53.77 ± 2.78 vs. 41.79 ± 2.15 vs. 38.21 ± 2.28; 0.1 μM: 75.35 ± 3.21 vs. 57.32 ± 2.49 vs. 43.82 ± 2.13; 0.3 μM: 72.73 ± 2.79 vs. 63.88 ± 2.62 vs. 54.39 ± 2.78; 1.0 μM: 81.89 ± 3.44 vs. 62.20 ± 2.77 vs. 55.06 ± 3.47 μm/s (control [n = 48] vs. patient 1 [n = 48] vs. patient 2 [n = 48]), and MRS Pre: 40.14 ± 2.73 vs. 22.28 ± 1.04 vs. 33.96 ± 2.13; 0.03 μM: 40.40 ± 2.60 vs. 23.07 ± 0.92 vs. 32.92 ± 1.47; 0.1 μM: 54.21 ± 2.72 vs. 36.03 ± 1.81 vs. 36.74 ± 1.99; 0.3 μM: 42.55 ± 2.80 vs. 42.67 ± 2.05 vs. 40.92 ± 2.24; 1.0 μM: 49.27 ± 2.74 vs. 40.33 ± 2.32 vs. 39.76 ± 2.40 μm/s (control vs. patient 1 vs. patient 2)). n = number of fields of view. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.