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. 2018 Oct 17;13(10):e0205719.
doi: 10.1371/journal.pone.0205719. eCollection 2018.

Functional abnormalities in induced Pluripotent Stem Cell-derived cardiomyocytes generated from titin-mutated patients with dilated cardiomyopathy

Revital Schick  1   2   3 , Lucy N Mekies  1   2   3 , Yuval Shemer  1   2   3 , Binyamin Eisen  1   2   3 , Tova Hallas  1   2   3 , Ronen Ben Jehuda  1   2   3   4 , Meital Ben-Ari  1   2   3 , Agnes Szantai  1   2   3   5 , Lubna Willi  1   2   3 , Rita Shulman  1   2   3 , Michael Gramlich  6 , Luna Simona Pane  7 , Ilaria My  8 , Dov Freimark  9 , Marta Murgia  10   11 , Gianluca Santamaria  12 , Mihaela Gherghiceanu  13 , Michael Arad  9 , Alessandra Moretti  8   14 , Ofer Binah  1   2   3
Affiliations

Functional abnormalities in induced Pluripotent Stem Cell-derived cardiomyocytes generated from titin-mutated patients with dilated cardiomyopathy

Revital Schick et al. PLoS One. .

Erratum in

Abstract

Aims: Dilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in the same outcome-decreased ventricular function. Titin (TTN)-a giant protein, expressed in cardiac and skeletal muscles, is an important part of the sarcomere, and thus TTN mutations are the most common cause of adult DCM. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CM) generated from patients, recapitulate the disease phenotype. The hypothesis was tested by 3 Aims: (1) Investigate key features of the excitation-contraction-coupling machinery; (2) Investigate the responsiveness to positive inotropic interventions; (3) Investigate the proteome profile of the AuP cardiomyocytes using mass-spectrometry (MS).

Methods and results: iPSC were generated from the patients' skin fibroblasts. The major findings were: (1) Sarcomeric organization analysis in mutated iPSC-CM showed defects in assembly and maintenance of sarcomeric structure. (2) Mutated iPSC-CM exhibited diminished inotropic and lusitropic responses to β-adrenergic stimulation with isoproterenol, increased [Ca2+]out and angiotensin-II. Additionally, mutated iPSC-CM displayed prolonged recovery in response to caffeine. These findings may result from defective or lack of interactions of the sarcomeric components with titin through its kinase domain which is absent in the mutated cells.

Conclusions: These findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies, expressed as blunted inotropic response.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Action potential (AP) characteristics of healthy and mutated (IsP and AuP) iPSC-CM.
[A-C] Recordings of spontaneous AP from healthy [A], IsP [B] and AuP [C] iPSC-CM. [D-J] Spontaneous AP parameters of healthy (38–77 day-old; black), IsP (54–77 day-old; red) and AuP (38–49 day old; blue) iPSC-CM: [D] beat rate, [E] action potential amplitude (APA), [F] maximum rate of phase 0 depolarization (dV/dtmax), [G] and [I] action potential duration at 50% (APD50) and 90% (APD90) repolarization, respectively. [H] and [J] Corrected action potential duration at 50% (cAPD50) and 90% (cAPD90) repolarization, respectively. Healthy (clone KTI n = 23, clone KTN3 n = 30, n = 53); IsP (clone 23.2 n = 9, clone 23.10 n = 3, n = 12); AuP (n = 9). One-way ANOVA (on APA, dV/dtmax, beat rate, APD50 and APD90) was performed followed by Holm-Sidak test, *P<0.05, **P<0.01 and ***P<0.001 vs healthy.
Fig 2
Fig 2. The chronotropic response to isoproterenol on spontaneous beat rate of healthy (black symbols), IsP (red symbols) and AuP (blue symbols) EBs.
Panel [A] shows representative spontaneous electrogram recordings from healthy (left), IsP (middle) and AuP (right) EBs in the absence (upper) and presence (middle) of isoproterenol. The positive chronotropic effect of isoproterenol is blocked by the β-blocker metoprolol (lower). [B] The spontaneous beating rate of healthy (clone 24.5 n = 6, clone KTN3 n = 5, clone KTI n = 7, n = 18), IsP (clone 23.10 n = 5) and AuP (n = 3) EBs. [C] Summary of the response to isoproterenol of healthy (black), IsP (red) and AuP (blue) EBs. Results are expressed as Mean±SEM. There is no statistically significant difference between the groups in Two-way ANOVA test. Iso–isoproterenol, MP–metoprolol.
Fig 3
Fig 3. BRV characterization of EBs generated from healthy, IsP and AuP iPSC.
[A] Representative electrogram recording from healthy, IsP and AuP iPSC-CM. [B-D] Representative IBIs time series of healthy (black), IsP (red) and AuP (blue) iPSC-CM and [E] combined IBIs time series. [F-H] Poincaré plots and [I] combined Poincaré plots of healthy (black), IsP (red) and AuP (blue) iPSC-CM. [J-L] histogram distribution of IBIs. [M-O] Summary of Coefficient of variation (COV) of IBIs (IBI CV) [M], SD1 [N] and SD2 [O] of Poincaré plots in healthy (clone 24.5 n = 6, clone KTN3 n = 5, clone KTI n = 6, n = 17), IsP (clone 23.10 n = 5) and AuP (n = 3) EBs. One-way ANOVA was performed followed by Holm-Sidak test, *P<0.05, **P<0.01 and ***P<0.001 vs. healthy.
Fig 4
Fig 4. The [Ca2+]i transient and contraction characteristics in healthy and mutated (IsP and AuP) iPSC-CM.
[A] Simultaneous recording of [Ca2+]i transient (orange) and contraction (green) measured from healthy (left), IsP (middle) and AuP (right) iPSC-CM. [B-D] [Ca2+]i transient healthy (clone 24.5 n = 32), IsP (clone 23.2 n = 8, clone 23.10 n = 12 n = 20), (AuP n = 9) amplitude and maximal rates of [Ca2+]i rise and decay, respectively. [E-G] Contraction healthy (clone 24.5 n = 27), IsP (clone 23.2 n = 16, clone 23.10 n = 19, n = 35), AuP (n = 14) amplitude and maximal rates of contraction and relaxation, respectively. Next to each column of individual values, the Mean+SEM (filled symbol) is shown. One-way ANOVA was performed followed by Holm-Sidak test, *P<0.05, **P<0.01 and ***P<0.001 vs. healthy.
Fig 5
Fig 5. Effect of isoproterenol on contraction and [Ca2+]i transient parameters of healthy and mutated iPSC-CM.
[A-C] Representative contractions (LAmplitude) from healthy [A], IsP [B] and AuP [C] iPSC-CM in the absence and presence of isoproterenol. Panels [D-F] display the summary of [Ca2+]i transient parameters of mutated (red–IsP, clone 23.2 n = 2, clone 23.10 n = 1, n = 3; blue–AuP, n = 8) and healthy (black–clone 24.5 n = 6) iPSC-CM: amplitude [D], and maximal rates of [Ca2+]i rise [E] and decay [F]. Panels [G-I] show the reduced response of mutated iPSC-CM (red–IsP, clone 23.2 n = 6, clone 23.10 n = 2, n = 8; blue–AuP, n = 8) compared to healthy (black–clone 24.5 n = 6) iPSC-CM in all contraction parameters: amplitude [G], and maximal rates of contraction [H] and relaxation [I]. Results are expressed as percent change from control values in Tyrode’s solution. Two-way ANOVA was performed followed by Holm-Sidak test. Two-way ANOVA showed a statistically significant difference in all 3 contraction parameters between healthy and IsP groups (P<0.05). Statistically significant difference was also seen in maximal rate of [Ca2+]i rise and relaxation between the 2 groups. For specific isoproterenol concentrations: *P<0.05, **P<0.01, ***P<0.001 vs Healthy. ISO–isoproterenol; Tyr–Tyrode’s solution.
Fig 6
Fig 6. Effect of increasing [Ca2+]out on contraction and [Ca2+]i transient parameters of healthy and mutated iPSC-CM.
[A-C] Representative contractions (LAmplitude) from healthy [A], IsP [B] and AuP [C] iPSC-CM in presence of increased [Ca2+]out. Panels [D-F] display the summary of [Ca2+]i transient parameters of mutated (red–IsP, clone 23.2 n = 3, clone 23.10 n = 4, n = 7; blue–AuP, n = 8) and healthy (black–clone 24.5 n = 7) iPSC-CM: amplitude [D], and maximal rates of [Ca2+]i rise [E] and decay [F]. Panels [G-I] show the reduced response of mutated iPSC-CM (red–IsP, clone 23.2 n = 7, clone 23.10 n = 11, n = 18; blue–AuP, n = 11) compared to healthy (black–clone 24.5 n = 12) iPSC-CM in all contraction parameters: amplitude [G], and maximal rates of contraction [H] and relaxation [I]. Results are expressed as percent change from control values at 2 mM. Two-way ANOVA was performed followed by Holm-Sidak test. Two-way ANOVA showed a statistically significant difference in all 3 contraction parameters between healthy and AuP groups and between healthy and IsP groups (P<0.05). Statistically significant difference was also seen in maximal rate of [Ca2+]i relaxation between all 3 groups. For specific [Ca2+]out concentrations: *P<0.05 vs Healthy, **P<0.01, ***P<0.001.
Fig 7
Fig 7. Effect of AT-II on contraction and [Ca2+]i transient parameters of healthy and mutated iPSC-CM.
[A-C] Representative contractions (LAmplitude) from healthy [A], IsP [B] and AuP [C] iPSC-CM in the absence and presence of AT-II. Panels [D-F] display the summary of [Ca2+]i transients parameters of mutated (red–IsP, clone 23.2 n = 2, clone 23.10 n = 8, n = 10; blue–AuP, n = 5) and healthy (black–clone 24.5 n = 8) iPSC-CM: amplitude [D], and maximal rates of [Ca2+]i rise [E] and decay [F]. Panels [G-I] show the reduced response of mutated iPSC-CM (red–IsP clone 23.2 n = 3, clone 23.10 n = 6, n = 9; AuP–blue, n = 5) compared to healthy (black–clone 24.5 n = 11) iPSC-CM in all contraction parameters: amplitude [G], and maximal rates of contraction [H] and relaxation [I]. Results are expressed as percent change from control values in Tyrode’s solution. Two-way ANOVA was performed followed by Holm-Sidak test. Two-way ANOVA showed a statistically significant difference in all 3 contraction parameters between healthy and IsP groups (P<0.001). Statistically significant difference was also seen in [Ca2+]i transient amplitude between the 2 groups. For specific AT-II concentrations: **P<0.01, ***P<0.001 vs Healthy. AT-II–angiotensin-II; Tyr–Tyrode’s solution.
Fig 8
Fig 8. Levels of sarcomeric organization in control and IsP iPSC-CM.
Panel [A] shows immunofluorescence images of α-actinin and cardiac troponin-T (cTNT) in healthy and IsP mutated single cardiomyocytes, illustrating three different levels of sarcomeric organization—fully, peripherally and perinuclear organized (upper, middle and lower panels, respectively). Panel [B] presents percentage of cells with different levels of sarcomeric organization. Statistic difference was tested using the chi-squared test Healthy (clone 24.5 n = 274), IsP (clone 23.2 n = 160, clone 23.10 n = 160, n = 320; ****P<0.001 Healthy vs IsP iPSC-CM). Scale bars = 25 μm.
Fig 9
Fig 9. Effect of caffeine on [Ca2+]i cycling of healthy and mutated iPSC-CM.
Representative [Ca2+]i transients from healthy [A], IsP [B] and AuP [C] iPSC-CM under caffeine administration (indicated by red arrow). The IsP cardiomyocytes display two-phase decline in [Ca2+]i level: fast decline as in healthy cells, and gradual slow decline until reaching [Ca2+]i basal level [B]. [D] Average recovery time from maximum of caffeine peak phase until the beginning of departing phase of the first measurable [Ca2+]i transient post-caffeine insertion of healthy (24.5 clone n = 11), IsP (clone 23.2 n = 2, clone 23.10 n = 4, n = 6) and AuP (n = 8) iPSC-CM. [E] display the percent change in fold change in area of caffeine-induced [Ca2+]i signal compared to the pre-caffeine [Ca2+]i transient of healthy (clone 24.5 n = 11), IsP (clone 23.2 n = 2, clone 23.10 n = 4, n = 6) and AuP (n = 8) iPSC-CM. One-way ANOVA was performed followed by Holm-Sidak test, ***P < 0.001 (vs Control and AuP).
Fig 10
Fig 10. Mass spectrometry-based proteomic analysis of AuP and healthy cardiomyocytes.
[A] Unsupervised hierarchical clustering of 301 proteins with significantly different expression in healthy (clone 24.5) and AuP cardiomyocytes (FDR 0.05, S0 0.5). Technical triplicates are shown for each sample. Healthy 1 and 2 indicate two unrelated control individuals. [B] Bar graphs showing the fold increase of the top three annotation enrichments in the cluster of proteins that are upregulated (red bars) and downregulated (green bars) in the AuP cardiomyocytes. The corresponding p value is reported next to each bar. [C] Volcano plot of statistical significance against fold-change (FDR 0.05, S0 0.5), highlighting the proteins with higher expression in control (black) and AuP (blue), respectively. [D] Bar plots depicting the log2 fold change expression of selected cardiac Ca2+ handling proteins that are differentially expressed between AuP and both healthy control cardiomyocytes.
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Grants and funding

This work was supported by the Israel Science Foundation (ISF) [grant number 292/13]; the Israeli Ministry of Science, Technology and Space [grant number 7-10772]; the Niedersachsen Ministry of Science and Culture [grant number 11-76251-99-16/14]; and the DZHK (German Centre for Cardiovascular Research - partner site Munich Heart Alliance – grant number 81Z7600671).