Investigating the cardiac pathology of SCO2-mediated hypertrophic cardiomyopathy using patients induced pluripotent stem cell-derived cardiomyocytes

Abstract

Mutations in SCO2 are among the most common causes of COX deficiency, resulting in reduced mitochondrial oxidative ATP production capacity, often leading to hypertrophic cardiomyopathy (HCM). To date, none of the recent pertaining reports provide deep understanding of the SCO2 disease pathophysiology. To investigate the cardiac pathology of the disease, we were the first to generate induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) from SCO2-mutated patients. For iPSC generation, we reprogrammed skin fibroblasts from two SCO2 patients and healthy controls. The first patient was a compound heterozygote to the common E140K mutation, and the second was homozygote for the less common G193S mutation. iPSC were differentiated into cardiomyocytes through embryoid body (EB) formation. To test the hypothesis that the SCO2 mutation is associated with mitochondrial abnormalities, and intracellular Ca²⁺ -overload resulting in functional derangements and arrhythmias, we investigated in SCO2-mutated iPSC-CMs (compared to control cardiomyocytes): (i) the ultrastructural changes; (ii) the inotropic responsiveness to β-adrenergic stimulation, increased [Ca²⁺ ]_o and angiotensin-II (AT-II); and (iii) the Beat Rate Variability (BRV) characteristics. In support of the hypothesis, we found in the mutated iPSC-CMs major ultrastructural abnormalities and markedly attenuated response to the inotropic interventions and caffeine, as well as delayed afterdepolarizations (DADs) and increased BRV, suggesting impaired SR Ca²⁺ handling due to attenuated SERCA activity caused by ATP shortage. Our novel results show that iPSC-CMs are useful for investigating the pathophysiological mechanisms underlying the SCO2 mutation syndrome.

Keywords: HCM; SCO2 mutation; [Ca2+]i transients and contractions; action potentials; arrhythmias; cardiomyocytes; iPSC.

Figure 1

TEM images of control and SCO2‐mutated iPSC‐CMs. (A) Normal ultrastructure of 30‐day‐old control iPSC‐CMs with organized myofibrils (mf), grouped normal mitochondria (nm) and glycogen masses (gly). NC‐nucleus. (B) Higher magnification of the square marked area in (A) shows normal mitochondria (nm) from control iPSC‐CMs, containing parallel cristae (arrowheads). (C) 30‐day‐old SCO2^G193S iPSC‐CMs contain higher glycogen (gly) content, lipid droplets (L), poor organized myofibrils (mf) and oversized mitochondria (M). The intercellular space is enlarged compared with control. NC‐nucleus. (D) Enlarged mitochondrion (M) from 30‐day‐old SCO2^G193S iPSC‐CMs (square marked area in C) shows increased number of disarrayed cristae, vacuolated cristae (red arrow) and glycogen inclusions (gly). A normal mitochondrion (nm) is visible nearby. (E) 30‐day‐old SCO2^E140K cardiomyocytes show organized myofibrils (mf), large masses of glycogen (gly), lipid droplets (L) and clusters of mitochondria (nm). NC‐nucleus. (F) Higher magnification of the square marked area in E shows normal structured mitochondria (nm) and slightly enlarged, doughnut‐shaped mitochondria (M). gly: glycogen.

Figure 2

TEM images showing mitochondria from 15‐, 30‐ and 45‐day‐old control (A–C), SCO2^G193S (D–F) and SCO2^E140K (G–I) iPSC‐CMs. Normal mitochondria show few parallel cristae (nm). The mitochondria abnormalities (M) progress with the age of SCO2^G193S iPSC‐CMs: disarray and decreased number of mitochondrial cristae (D); few tightly packed cristae and glycogen accumulation inside the mitochondrion (E); mitochondria are enlarged and contain disarrayed and highly increased number of cristae (F). SCO2^E140KiPSC‐CMs present mostly mitochondria with normal ultrastructure (nm) (G‐I) and rare doughnut‐like mitochondria (M) at 45 days (I), mf: myofibrils; NC: nuclei.

Figure 3

The effects of isoproterenol on [Ca²⁺]_i transients and contractions in control, SCO2^G193S and SCO2^E140K iPSC‐CMs. (A–C) [Ca²⁺]_i transients (R = F₃₄₀/F₃₈₀) and contractions (L _amp) from control, SCO2^G193S and SCO2^E140K iPSC‐CMs, respectively. (D) [Ca²⁺]_i transient amplitude (R _amp); (E) maximal rate of [Ca²⁺]_i rise (+d[Ca²⁺]/dt); (F) maximal rate of [Ca²⁺]_i decay (‐d[Ca²⁺]/dt); (G) maximal amplitude (L _amp); (H) maximal contraction rate (+dL/dt); (I) maximal relaxation rate (−dL/dt). The effect of isoproterenol on contraction characteristics of SCO2^G193S (n = 13), SCO2^E140K (n = 6) and control (n = 8) was expressed as per cent change. The effect of isoproterenol on the [Ca²⁺]_i transients was expressed as the per cent change in the fluorescence ratio, F₃₄₀/F₃₈₀ (n = 6, n = 5 and n = 10, for SCO2^G193S, SCO2^E140K and control respectively); Tyr: Tyrode's solution; *P < 0.05, **P < 0.001 (versus control).

Figure 4

The effects of increased [Ca²⁺]_o on the [Ca²⁺]_i transients and contractions. (A) R_amp; (B) +d[Ca²⁺]/dt; (C) −d[Ca²⁺]/dt; (D) L_amp; (E) +dL/dt; (F) −dL/dt. The effect of [Ca²⁺]_o on contraction characteristics of SCO2^G193S (n = 6), SCO2^E140K (n = 7) and control (n = 12) was expressed as per cent change. The effects of AT‐II on the [Ca²⁺]_i transients and contractions. (G) R_amp; (H) +d[Ca²⁺]/dt; (I) ‐d[Ca²⁺]/dt; (J) L_amp; (K) +dL/dt; (L) −dL/dt. The effect of AT‐II on contraction characteristics of SCO2^G193S (n = 7), SCO2^E140K (n = 8) and control (n = 10) was expressed as percent change. The effect on the [Ca²⁺]_i transients was expressed as the percent change in the fluorescence ratio, F₃₄₀/F₃₈₀ (n = 6, n = 6 and n = 7, for SCO2^G193S, SCO2^E140K and control respectively); Tyr: Tyrode's solution; *P < 0.05, **P < 0.001 (versus control).

Figure 5

The effects of caffeine on control, SCO2 ^E140K and SCO2^G193S iPSC‐CMs. [Ca²⁺]_i transients from (A) control (B–C) SCO2^E140K and (D–E) SCO2^G193S iPSC‐CMs, demonstrating the effect of caffeine. (F) The mean recovery time, calculated as the time from the peak of caffeine‐induced [Ca²⁺]_i rise to the first measurable [Ca²⁺]_i transient; (G) per cent change in area of the caffeine‐induced Ca²⁺ signal compared to the pre‐caffeine area; (H) per cent change in caffeine‐induced Ca²⁺ signal amplitude compared to the pre‐caffeine amplitude. Control iPSC‐CMs (n = 7), SCO2^G193S iPSC‐CMs (n = 11), SCO2^E140K iPSC‐CMs (n = 20), *P < 0.05. Asterisk above bars connecting columns represents significant difference between groups. Each SCO2‐mutated group was divided into subgroups FR and SR according to type of reaction.

Figure 6

Transmembrane action potential recordings from control, SCO2^G193S and SCO2^E140K iPSC‐CMs in response to increasing concentrations of isoproterenol. (A) Representative recordings from control iPSC‐CMs in the presence of Tyrode's solution and isoproterenol concentrations of 10⁻⁹ M, 10⁻⁸ M, 10⁻⁷ M and 10⁻⁶ M. Control iPSC‐CMs displayed a positive chronotropic response to increasing concentrations of isoproterenol, represented by firing rate. (B) Recordings from SCO2^G193S and (C) from SCO2^E140K iPSC‐CMs in the presence of Tyrode's solution and increasing isoproterenol concentrations. SCO2^G193S iPSC‐CMs show generation of DADs with isoproterenol. SCO2^E140K iPSC‐CMs display DADs under baseline conditions and in the presence of different isoproterenol concentrations.

Figure 7

The chronotropic response of extracellular electrograms recorded from control, SCO2^E140K and SCO2^G193S iPSC‐CMs to isoproterenol. (A) The spontaneous beating rate of control (n = 11), SCO2^E140K (n = 7) and SCO2^G193S (n = 10) iPSC‐CMs. (B) The effect of isoproterenol on the beat rate of iPSC‐CMs and the blocking effect of the β‐blocker metoprolol. (C–E) The EB was perfused initially with DMEM solution (Control) and then with isoproterenol 10⁻⁶ M in DMEM solution. The different time intervals denote the arrhythmia in the isoproterenol‐treated SCO2‐mutated EBs. The arrhythmia was blocked by the β‐blocker metoprolol. (C) Control, (D) SCO2^E140K and (E) SCO2^G193S iPSC‐CMs. Results are expressed as per cent change from control (absence of isoproterenol). The arrhythmia is marked by red bar; bpm: beats per minute; C: control conditions—DMEM solution in the absence of isoproterenol; Iso: Isoproterenol; Met: Metoprolol; *P < 0.05, **P < 0.001 (versus control).

Figure 8

The spontaneous electrical activity and BRV properties of control, SCO2^E140K and SCO2^G193S iPSC‐CMs. (A) EBs were perfused with DMEM solution, activation spikes and repolarization waves were recorded in control, SCO2^E140K and SCO2^G193S iPSC‐CMs (first recorded at 75 sec. and then at 456 sec.). Inter‐beat intervals (IBIs), histograms and Poincaré plots analysis in control (B–D), SCO2^G193S (E–G) and SCO2^E140K iPSC‐CMs (H–J). (B, E, H) IBIs time series, (C, F, I) Histogram distribution of IBIs, (D, G, J) Poincaré plots of the BRV. (K) Combined Poincaré plots of (D, G, J). (L–O) Comparisons of BRV magnitude in control (n = 8), SCO2^E140K (n = 5) and SCO2^G193S (n = 8). (L) Summary of mean IBI, (M) coefficient of variance of IBIs (IBI CV), (N) SD1 and (O) SD2 of Poincaré plots in contracting EBs. Altered time intervals were marked by red bar. *P < 0.05.

Figure 9

Schematic model of a proposed mechanism for the observed abnormalities in SCO2 iPSC‐CMs. The scheme describes the three distinct cellular pathways converging at the sarcoplasmic reticulum (SR). The scheme suggests an explanation for the delayed afterdepolarizations (DADs) and the attenuated inotropic responsiveness of SCO2‐mutated cardiomyocytes. While in control cardiomyocytes, these different pathways cause positive inotropic and lusitropic effects due to SR Ca²⁺ release, in SCO2‐mutated iPSC‐CMs, Ca²⁺ depleted SR accounts for the impaired inotropic responses.