Research ArticleStem Cells

Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy

See allHide authors and affiliations

Science Translational Medicine  18 Apr 2012:
Vol. 4, Issue 130, pp. 130ra47
DOI: 10.1126/scitranslmed.3003552

Abstract

Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca2+), decreased contractility, and abnormal distribution of sarcomeric α-actinin. When stimulated with a β-adrenergic agonist, DCM iPSC–derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric α-actinin distribution. Treatment with β-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca2+ adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.

Introduction

Dilated cardiomyopathy (DCM) is a form of heart disease characterized by ventricular dilatation and systolic dysfunction (1). DCM is the most common cause of heart failure after coronary artery disease and hypertension and is the leading indication for heart transplantation (2, 3). The cost for clinical management of DCM in the United States alone has been estimated at between $4 billion and $10 billion (3). Mutations in genes encoding sarcomeric, cytoskeletal, mitochondrial, and nuclear membrane proteins, as well as proteins involved in Ca2+ metabolism, are linked to about a third to half the cases of DCM (46). Cardiac troponin T is one of the three subunits of the troponin complex (troponin T, C, and I) that regulate sarcomeric thin filament activity and contraction of cardiomyocytes. Cardiac troponin T is essential for sarcomere assembly, contraction, and force production in cardiac muscle (7). Mutations in the cardiac troponin T gene (TNNT2) often lead to DCM (8) and frequently result in heart failure at an early age and sudden cardiac death (9, 10). In vitro biochemical studies have found that decreased Ca2+ sensitivity and ATPase (adenosine triphosphatase) activity, which impair force production, may be the underlying mechanisms for certain TNNT2 mutation–induced DCM (9, 1114). Mice carrying TNNT2 mutations recapitulate the human DCM phenotype, providing extensive insights into the possible mechanisms of disease (11, 12). The contribution of mouse models to our overall understanding of DCM has been enormous. However, several important differences exist between the mouse and the human models. For example, the mouse resting heart rate is about 10-fold faster than that of human. The electrical properties, ion channel contributions, and development of mouse cardiomyocytes also differ from those of their human counterparts. Unfortunately, cardiac tissues from DCM patients are difficult to obtain and do not survive in long-term culture. With the advent of induced pluripotent stem cells (iPSCs) (15, 16), functional cardiomyocytes can be obtained by differentiation of human iPSCs derived from DCM patients (17, 18). Patient-specific cardiomyocytes derived from iPSCs from patients with cardiac defects such as long QT syndrome, Leopard syndrome, and Timothy syndrome recapitulate these human cardiovascular diseases and hence enable the testing and optimization of empirical therapies (1922). Thus, a human DCM iPSC–derived cardiomyocyte model would be an important complement to mouse models for understanding the cellular and physiological processes of DCM as well as for drug screening in human cells.

Here, we generated iPSC-derived cardiomyocytes from three generations of a family with DCM, both from DCM patients carrying a point mutation (R173W) in exon 12 of the TNNT2 gene and from unaffected healthy family members who did not carry the mutation. We studied the morphology and function of DCM iPSC–derived cardiomyocytes, made electrophysiological recordings using patch clamping and multielectrode arrays, assessed Ca2+ regulation by Ca2+ imaging techniques, and quantified contractile force using atomic force microscopy. Compared to cardiomyocytes from healthy family members, iPSC-derived cardiomyocytes from DCM patients displayed a consistently increased heterogeneous sarcomeric organization and a pronounced punctate distribution of sarcomeric α-actinin. Individual DCM iPSC–derived cardiomyocytes also exhibited altered Ca2+ handling compared to iPSC-derived cardiomyocytes from control individuals. β-Adrenergic stimulation increased the number of DCM iPSC–derived cardiomyocytes with abnormal sarcomeric α-actinin distribution, compromised contractility, and induced failure of spontaneous contraction. However, treatment with the β1-selective β-blocker (metoprolol) improved sarcomeric organization. Overexpression of sarcoplasmic reticulum Ca2+ ATPase (Serca2a) markedly increased contractile force and improved Ca2+ handling in DCM iPSC–derived cardiomyocytes. Thus, DCM iPSC–derived cardiomyocytes to some extent recapitulate features of DCM disease and could be useful for investigating disease mechanisms and for drug screening.

Results

The DCM family carries a disease-associated point mutation (R173W) in TNNT2

We recruited a cohort of seven individuals from a DCM proband carrying an autosomal dominant point mutation on exon 12 of the TNNT2 gene, which causes an arginine (R) to tryptophan (W) switch at amino acid position 173 in the protein cardiac troponin T. The potential causal effect of this particular point mutation for DCM was confirmed by genetic screening of a panel of 17 primary DCM-associated genes, in silico analysis (table S1), and genetic cosegregation studies (fig. S1). A mutation at the same amino acid position (R173G) was also reported in a completely unrelated Belgian family with DCM (23), suggesting a strong association of this particular locus with the disease. The seven recruited individuals covered three generations (I, II, and III) (Fig. 1A). Four patients (Ia, IIa, IIb, and IIIa) were confirmed to carry the TNNT2 R173W mutation in one of the two alleles by polymerase chain reaction (PCR) amplification of the genomic locus of TNNT2 and DNA sequencing; the other three individuals (Ib, IIc, and IIIb) were confirmed not to carry the mutation and served as controls (Fig. 1B and fig. S1). All four patients who carry the specific R173W mutation manifested clinical DCM symptoms with a dilated left ventricle and decreased ejection fraction and were treated medically (table S2). A 14-year-old DCM patient (IIIa) had an orthotopic heart transplant because of severe clinical symptoms.

Fig. 1

Generation of patient-specific DCM iPSC–derived cardiomyocytes. (A) Pedigree of the DCM family recruited in this study. Filled squares (male) and circles (female) represent individuals carrying the specific heterozygous R173W mutation in the TNNT2 gene. (B) The R173W point mutation was confirmed to be present on exon 12 of the TNNT2 gene in the DCM patients by PCR and DNA sequencing. CON, control. (C) A representative image of the skin fibroblasts expanded from skin biopsies from DCM and control family members. (D and E) Representative images of an (D) ESC-like and (E) TRA-1-60+ colony derived from reprogramming the skin fibroblasts with the four Yamanaka factors delivered by lentiviruses. (F) Immunofluorescence and alkaline phosphatase staining of the skin fibroblast–derived iPSCs. (G) Quantitative bisulfite pyrosequencing analysis of the methylation status at the promoter regions of Oct4 and Nanog in patient and control iPSCs. Both Nanog and Oct4 promoter regions were highly demethylated in the iPSCs. TSS, transcription start site. (H) Teratomas derived from iPSCs injected into the kidney capsule of immunodeficient mice exhibited tissues of all three embryonic germ layers, thus demonstrating that the iPSCs were pluripotent. Scale bars, 200 μm.

Generation and characterization of patient-specific iPSCs

To generate patient-specific iPSCs, we expanded skin fibroblasts from skin biopsies taken from each individual (Fig. 1C) and we reprogrammed them using the four Yamanaka factors (Oct4, Sox2, Klf4, and c-MYC) delivered by lentivirus under feeder-free conditions. Colonies with TRA-1-60+ staining and human embryonic stem cell (hESC)–like morphology (Fig. 1, D and E) were selected, expanded, and established as individual iPSC lines. For each individual, three to four iPSC lines were established for subsequent analyses. All of the DCM iPSC lines were confirmed to contain the specific R173W mutation by genomic PCR and DNA sequencing (fig. S2). All established iPSC lines expressed the pluripotency markers Oct4, Nanog, TRA-1-81, and SSEA-4, and were positive for alkaline phosphatase (Fig. 1F). Microarray analyses indicated that these iPSC lines were distinct from the parental skin fibroblasts and expressed a global gene pattern more similar to that of hESCs (fig. S3A). Quantitative bisulfite sequencing showed that the promoter regions of Oct4 and Nanog were hypomethylated in all the tested iPSC lines, indicating active transcription of the pluripotency genes (Fig. 1G). The established iPSC lines maintained a normal karyotype after extended passage (fig. S3B), with most exhibiting silencing of exogenous transgenes and reexpression of endogenous Nanog (fig. S4). iPSC lines with incomplete transgene silencing were removed from subsequent studies. These patient-specific iPSCs were able to differentiate in vitro into cells of all three germ layers (fig. S5) and subsequently formed teratomas upon injection into the kidney capsules of immunodeficient mice (Fig. 1H).

DCM iPSC–derived beating embryoid bodies exhibit normal electrophysiology at baseline

We next differentiated the DCM iPSCs into the cardiovascular lineage using a well-established three-dimensional differentiation protocol developed by Yang et al. (24). Two iPSC lines from each individual were selected for differentiation into spontaneous beating embryoid bodies and for subsequent functional analyses (table S3). Spontaneous beating was observed as early as day 8 after differentiation. The efficiency of differentiation to the cardiac lineage varied among different lines (fig. S6A and videos S1 and S2). Beating embryoid bodies derived from control and patient iPSCs contained about 50 to 60% cardiac troponin T–positive cardiomyocytes (fig. S6, B and C). Allele-specific reverse transcription–PCR (RT-PCR) of beating embryoid bodies derived from three iPSC clones from three DCM patients indicated biallelic expression of the wild-type and mutant (R173W) TNNT2 gene (fig. S7). The beating embryoid bodies from the control and DCM iPSCs 18 to 48 days after differentiation were seeded on multielectrode array probes (fig. S8A and video S3), and their electrophysiological properties were recorded (fig. S8B). Both control (n = 45) and DCM (n = 57) iPSC-derived beating embryoid bodies exhibited comparable beat frequencies, field potentials, interspike intervals, and field potential durations at baseline (table S4 and fig. S8C).

DCM iPSC–derived cardiomyocytes exhibit an increased heterogeneous sarcomeric pattern

We next dissociated the beating embryoid bodies into small beating clusters and single beating cardiomyocytes for further analysis (videos S4 to S7). The organization of myofibrils in the iPSC-derived cardiomyocytes was assessed by immunocytochemistry. Both control and DCM iPSC–derived cardiomyocytes expressed the sarcomeric proteins cardiac troponin T, sarcomeric α-actinin, and myosin light chain 2a (MLC2a), as well as the cardiac gap junction protein connexin 43 (fig. S9). However, compared to control iPSC-derived cardiomyocytes (n = 368) at day 30 after differentiation, a much higher percentage (35.1 ± 2.7%) of DCM iPSC–derived cardiomyocytes (n = 391) showed a punctate distribution of sarcomeric α-actinin over one-fourth of the total cellular area (P = 0.008) (Fig. 2, A and B, and fig. S10, A to C). There were no significant differences in cell size between control and DCM iPSC–derived cardiomyocytes (Fig. 2C) at this stage. This phenotype was consistently observed in two different DCM iPSC lines from each of the four DCM patients, suggesting a homogeneous correlation with the disease-associated R173W mutation. Sarcomeric α-actinin is an excellent marker for sarcomeric integrity and degeneration, and was used for evaluation of sarcomeric organizations in heart tissues from human patients with DCM (25). These results suggest that, compared to control individuals, an increased number of DCM iPSC–derived cardiomyocytes had a more disturbed sarcomeric organization at this stage of differentiation. Notably, cardiomyocyte clusters always showed a much less disturbed sarcomeric pattern (fig. S10, D and E). Most cardiomyocytes with punctate sarcomeric α-actinin distribution were single cells or cells at the edge of cardiomyocyte clusters (fig. S10, D and E), indicating a greater tendency for single TNNT2 R173W DCM iPSC–derived cardiomyocytes to malfunction in maintaining sarcomere integrity. To further assess the myofibrillar organization in detail, we performed transmission electron microscopy on both control and DCM iPSC–derived cardiomyocytes at day 30 after differentiation. Well-organized myofibrils with aligned Z lines and recognizable A and I bands were found in both control (n = 11) and DCM iPSC–derived cardiomyocytes (n = 12) (Fig. 2D and fig. S10F), although mitochondria and sarcoplasmic reticulum were still immature in both groups at this stage (fig. S10, G and H). However, compared to controls, DCM iPSC–derived cardiomyocytes exhibited an increased variability in the degree of sarcomeric organization, with a higher number of less well aligned Z lines and scattered patterns of condensed Z bodies (Fig. 2D and fig. S10F). Overall, these results are consistent with the sarcomeric α-actinin immunostaining in DCM cardiomyocytes shown in Fig. 2A and fig. S10, A to C.

Fig. 2

DCM iPSC–derived cardiomyocytes show an abnormal sarcomeric α-actinin distribution. (A) Immunostaining of sarcomeric α-actinin and cardiac troponin T (cTnT) at day 30 after differentiation. Single DCM iPSC–derived cardiomyocytes exhibited a punctate sarcomeric α-actinin distribution pattern, suggesting a disorganized myofilament structure. Enlarged views of the boxed areas of the merged micrographs show detailed α-actinin (red) and cardiac troponin T (green) staining patterns in the cells. Scale bars, 20 μm. (B) Compared to control iPSC-derived cardiomyocytes (n = 368), a higher percentage of DCM iPSC–derived cardiomyocytes (n = 391) showed a punctate sarcomeric α-actinin staining pattern in greater than one-fourth of the total cellular area (**P = 0.008, two-tailed Student’s t test). (C) No significant difference was observed in cell size between control (n = 36) and DCM iPSC–derived cardiomyocytes (n = 39). (D) Transmission electron microscopy images of myofibrillar organization in control and DCM iPSC–derived cardiomyocytes. Compared to control cardiomyocytes (n = 11, two iPSC lines, one each from two individuals), myofibrils in DCM cardiomyocytes (n = 12, two iPSC lines, one each from two patients) exhibited an increased variability in the degree of organization. ZL, Z line; ZB, Z bodies; A, A band; I, I band; Mt, mitochondria. Scale bars, 1 μm. (Extra transmission electron microscopy images of control and DCM iPSC–derived cardiomyocytes can be found in fig. S10, F and G.)

DCM iPSC–derived cardiomyocytes are susceptible to stress

Positive inotropic stress (increased contraction force) can induce the DCM phenotype in transgenic mouse models of DCM (26, 27) and can aggravate the disease in clinical patients (28). We next examined whether treatment with a positive inotropic reagent, such as a β-adrenergic agonist, could expedite the phenotypic response of DCM iPSC–derived cardiomyocytes. Indeed, we found that 10 μM norepinephrine treatment induced an initial positive chronotropic effect (increased rate of beating) that later became negative, eventually leading to failure of spontaneous contraction in DCM iPSC–derived beating embryoid bodies (n = 14) as reflected by recording from multielectrode array probes. By contrast, the iPSC-derived beating embryoid bodies from control individuals (n = 14) exhibited prolonged positive chronotropic activity (Fig. 3A). One week of 10 μM norepinephrine treatment in vitro markedly increased the number of cardiomyocytes with punctate sarcomeric α-actinin distribution from DCM iPSC clones (P < 0.001), with almost 80 to 90% of the DCM iPSC–derived cardiomyocytes found to have a disorganized sarcomeric pattern (Fig. 3, B and C, and fig. S11, A to E). A few single DCM iPSC–derived cardiomyocytes showed complete degeneration of myofilaments after prolonged norepinephrine treatment (Fig. 3B and fig. S11A), which was not observed in control iPSC-derived cardiomyocytes. Transmission electron microscopy indicated that, compared to control individuals (n = 6), norepinephrine-treated DCM iPSC–derived cardiomyocytes (n = 7) exhibited a more severe scattered distribution of Z bodies (Fig. 3D and fig. S11F), which was consistent with the markedly increased heterogeneous pattern of sarcomeric α-actinin staining after norepinephrine treatment. Tracking with video imaging of individual beating clusters of both control and DCM iPSC–derived cardiomyocytes treated with 10 μM norepinephrine over time showed distinct outcomes. Decreased inotropic and chronotropic activities were often observed in the DCM iPSC–derived cardiomyocytes (Fig. 3, E and F, and videos S8 to S15) compared to controls. These results suggest that DCM iPSC–derived cardiomyocytes are more susceptible to the stress induced by β-adrenergic stimulation.

Fig. 3

DCM iPSC–derived cardiomyocytes treated with norepinephrine. (A) A representative multielectrode array assay recording the chronotropic response of both control (n = 14) and DCM (n = 14) beating embryoid bodies over time after treatment with 10 μM norepinephrine (NE). Electrical signals were recorded before and after norepinephrine treatment. Beating frequencies were normalized to the values before norepinephrine treatment. (B) Representative images of sarcomeric α-actinin immunostaining of single control and DCM iPSC–derived cardiomyocytes after 7 days of norepinephrine treatment. Compared to controls, long-term norepinephrine treatment induced sarcomeric disorganization in single DCM iPSC–derived cardiomyocytes. Scale bar, 20 μm. (C) Percentage of iPSC-derived cardiomyocytes with disorganized sarcomeric α-actinin staining patterns with (control, n = 210; DCM, n = 255) or without (control, n = 261; DCM, n = 277) norepinephrine treatment. Norepinephrine treatment markedly increased the number of disorganized cardiomyocytes in the DCM group (**P < 0.001, two-tailed Student’s t test), but had less of an effect on control iPSC-derived cardiomyocytes (*P = 0.05, two-tailed Student’s t test). (D) Transmission electron microscopy images of myofibrillar organization in control and DCM iPSC–derived cardiomyocytes after norepinephrine treatment. Compared to control iPSC-derived cardiomyocytes (n = 6, two iPSC lines, one each from two individuals), myofibrils in DCM iPSC–derived cardiomyocytes (n = 7, two iPSC lines, one each from two patients) exhibited an increased scattered distribution of Z bodies. Scale bar, 1 μm. Additional transmission electron microscopy images of norepinephrine-treated control and DCM iPSC–derived cardiomyocytes can be found in fig. S11F. (E) Relative beating frequencies of cardiomyocyte clusters (n = 10) over time after norepinephrine treatment measured using video imaging. Values are normalized to the beating frequencies of cardiomyocyte clusters at the same time points without norepinephrine treatment. Data are presented as means ± SEM. (F) Tracking morphological and contractility changes of iPSC-derived cardiomyocytes over time after norepinephrine treatment by video imaging. Scale bar, 200 μm. The respective videos are in the Supplementary Material (videos S8 to S15).

Biomechanical stress generated by pressure or volume overload resulting from hypertension or myocardial injury often induces DCM and heart failure and tends to aggravate existing cardiac disease (29). We next examined the effect of mechanical stress on iPSC-derived cardiomyocytes by subjecting them to cycles of prolonged stretching. Prolonged stretching led to marked thickening and loss of obvious striation of the myofibrils in both control and DCM iPSC–derived cardiomyocytes (fig. S12, A and B). Mechanical strain also increased the number of cells with a relatively disorganized sarcomeric pattern in both control and DCM iPSC–derived cardiomyocytes. However, compared to controls, an increased heterogeneity in sarcomeric pattern was observed in DCM iPSC–derived cardiomyocytes (fig. S12C). These results suggest that DCM iPSC–derived cardiomyocytes are more susceptible to biomechanical stress.

DCM iPSC–derived cardiomyocytes exhibit altered Ca2+ handling

Cardiomyocyte contraction starts with the electrical excitation of myocytes, as reflected by generation of membrane action potentials (30). To investigate the possible underlying etiology, we assessed whether the DCM-associated R173W mutation in the TNNT2 gene affects the electrical excitation of the cardiomyocytes. We examined the electrical activities of dissociated single beating iPSC-derived cardiomyocytes by patch clamping. Three types of spontaneous action potentials (ventricular-, atrial-, and nodal-like) were observed in both control and DCM iPSC–derived cardiomyocytes (Fig. 4A). DCM ventricular-like myocytes (n = 17) exhibited normal action potentials that were comparable to those of controls (n = 18) (Fig. 4B). The average action potential duration at 90% repolarization (APD90) of the DCM iPSC–derived cardiomyocytes was not significantly different from that seen in control iPSC-derived cardiomyocytes (Fig. 4C). The average action potential frequency, peak amplitude, and resting potential were also similar between the two groups (Fig. 4, D to F). These results indicate that the electrical activities of individual control and DCM iPSC–derived cardiomyocytes were normal at baseline, consistent with the results obtained by multielectrode array analysis of beating embryoid bodies.

Fig. 4

Electrophysiological features of iPSC-derived cardiomyocytes measured by patch clamping. (A) Three types of spontaneous action potentials were observed in both control and DCM iPSC–derived cardiomyocytes (left, ventricular-like; middle, atrial-like; right, nodal-like). An estimated 70 to 80% of cells were ventricular-like cardiomyocytes, whereas the others were atrial- and/or nodal-like cells. There was no significant difference in cardiac cell fate between control and DCM iPSCs (data not shown). (B) Spontaneous action potentials in control and DCM ventricular-like myocytes using current-clamp recording. (C to F) There was no significant difference in the action potential duration (C), frequency (D), the peak amplitude (E), or the resting membrane potential (F) between control and DCM iPSC–derived cardiomyocytes at the time of measurements (days 19 to 25 after differentiation) (control, n = 18; DCM, n = 17). Statistical difference was tested with two-tailed Student’s t test.

To further investigate the underlying mechanisms of DCM disease, we measured Ca2+ handling properties (for example, Ca2+ handling of cardiomyocytes during excitation-contraction coupling, including Ca2+ transient amplitude, kinetics, and Ca2+ content of the sarcoplasmic reticulum) at the excitation-contraction coupling level by fluorescent Ca2+ imaging. DCM iPSC–derived cardiomyocytes (n = 40, five lines from three DCM patients; table S3) exhibited rhythmic frequency and timing comparable to those of control iPSC-derived cardiomyocytes (n = 87, five lines from three control individuals; table S3) (Fig. 5, A to C, E, and F). However, DCM iPSC–derived cardiomyocytes exhibited significantly smaller intracellular calcium concentration ([Ca2+]i) transient amplitudes compared to those of control iPSC-derived cardiomyocytes (P = 0.002) (Fig. 5D), indicating that the [Ca2+]i available for each contraction of DCM iPSC–derived cardiomyocytes was greatly reduced. The smaller [Ca2+]i transients observed in cardiomyocytes were consistently observed in all examined DCM iPSC lines, suggesting weaker force production in DCM iPSC–derived cardiomyocytes. To further analyze the Ca2+ handling properties of iPSC-derived cardiomyocytes, we subjected both control and DCM iPSC–derived cardiomyocytes to caffeine treatment to induce Ca2+ release from the sarcoplasmic reticulum through ryanodine receptor (RyR) Ca2+ channels (Fig. 5, G to J). Compared to controls, DCM iPSC–derived cardiomyocytes exhibited relatively smaller amplitudes, prolonged time to peak, and delayed decay time (Fig. 5, H to J), indicating that DCM iPSC–derived cardiomyocytes have relatively lower Ca2+ storage in their sarcoplasmic reticulum and altered function of Ca2+-related molecules such as Ca2+ release channels and Ca2+ pumps in the plasma and sarcoplasmic reticulum membranes.

Fig. 5

DCM iPSC–derived cardiomyocytes exhibit abnormal Ca2+ handling. (A and B) Representative line scan images (A) and spontaneous Ca2+ transients (B) in control (left) and DCM iPSC–derived cardiomyocytes (right). (C) Frequency of spontaneous Ca2+ transients in control and DCM iPSC–derived cardiomyocytes. (D) Integration of [Ca2+]i transients in control and DCM iPSC–derived cardiomyocytes showed less Ca2+ released in each [Ca2+]i transient in DCM relative to control cells (control, n = 87 cells; DCM, n = 40 cells; **P = 0.002, two-tailed Student’s t test). (E and F) There were no significant differences in the irregularity of timing (E) or irregularity of amplitude (F) of the spontaneous Ca2+ transients between control and DCM cells. (G to J) Caffeine-evoked Ca2+ release in DCM iPSC–derived cardiomyocytes. (G) Representative Ca2+ transients induced with 20 mM caffeine in Ca2+-free conditions in control and DCM iPSC–derived cardiomyocytes (control, n = 12, two iPSC lines, one each from two individuals; DCM, n = 12, two iPSC lines, one each from two patients). (H) Peak amplitude of caffeine-evoked Ca2+ transients in control and DCM iPSC–derived cardiomyocytes. The difference in the amplitude between control and DCM did not reach statistical significance. (I) Time to peak in caffeine-evoked Ca2+ transients in control and DCM iPSC–derived cardiomyocytes. (J) Decay time (50% from peak) in caffeine-evoked Ca2+ transients in control and DCM iPSC–derived cardiomyocytes. *P < 0.05; **P < 0.01, two-tailed Student’s t test.

DCM iPSC–derived cardiomyocytes exhibit impaired contractility

A deficiency in contractile force production is one of the most important mechanisms responsible for inducing DCM and heart failure (4). To investigate this further, we next measured the contraction force of iPSC-derived cardiomyocytes using atomic force microscopy, which has been used to measure contraction of cultured chicken embryonic cardiomyocytes (31). Atomic force microscopy allowed us to probe the contractile properties of iPSC-derived cardiomyocytes at the single-cell level (fig. S13 and video S16). Compared to single control iPSC-derived cardiomyocytes (n = 13), the DCM iPSC–derived cardiomyocytes (n = 17) showed a similar beat frequency and duration (fig. S14), but significantly weaker contraction forces (control, 3.56 ± 0.97 nN; DCM, 0.65 ± 0.05 nN; P = 0.001) (Fig. 6, A and B, and table S5). There was no correlation between the cell size and the contraction force for each single cell measured by atomic force microscopy (fig. S15).

Fig. 6

Overexpression of Serca2a restored contractility in DCM iPSC–derived cardiomyocytes. (A) Dot plots of mean contraction force for single iPSC-derived cardiomyocytes measured by atomic force microscopy. One-way ANOVA analysis indicated that there was a significant difference among the mean of all the groups (**P = 0.002, one-way ANOVA). Tukey’s multiple comparison test indicated that both control iPSC-derived cardiomyocytes (n = 13) (P = 0.001) and Ad.Serca2a-transduced (n = 12) (P = 0.005) DCM iPSC–derived cardiomyocytes exhibited significantly stronger contraction forces than DCM iPSC–derived cardiomyocytes transduced with control Ad.GFP (n = 17). Ad.Serca2a-transduced DCM iPSC–derived cardiomyocytes showed a comparable contraction force to that of control iPSC-derived cardiomyocytes (P = 0.578). (B) Histograms of contraction forces for all of the single iPSC-derived cardiomyocytes measured by atomic force microscopy over 100 to 400 beats. Overexpression of Serca2a restored the contraction force of DCM iPSC–derived cardiomyocytes to a level close to that of control. (C) Western blotting of Serca2a expression after adenoviral transduction of cells dissociated from iPSC beating embryoid bodies. Serca2a protein was up-regulated in cells transduced with Ad.Serca2a but not in cells transduced with Ad.GFP. (D) A representative image showing the atomic force microscopy cantilever approaching GFP-positive single beating iPSC-derived cardiomyocytes. Scale bar, 50 μm. (E) Representative spontaneous Ca2+ transients in single DCM iPSC–derived cardiomyocytes transduced with Ad.GFP and Ad.Serca2a, respectively. (F) DCM iPSC–derived cardiomyocytes transduced with Ad.Serca2a (n = 22) exhibited increased global Ca2+ transients compared to cells transduced with Ad.GFP (n = 14) (*P = 0.04, two-tailed Student’s t test). (G) Half decay time of Ca2+ transients in DCM iPSC–derived cardiomyocytes transduced with Ad.Serca2a or with Ad.GFP. (H) Percentage of iPSC-derived cardiomyocytes with a disorganized sarcomeric α-actinin staining pattern in single DCM iPSC–derived cardiomyocytes overexpressing Ad.Serca2a (n = 40) or Ad.GFP (n = 40). No significant difference was observed between the two groups (two-tailed Student’s t test). Data are the means ± SEM.

Serca2a overexpression enhanced contractility in DCM iPSC–derived cardiomyocytes

Previous studies have shown that Serca2a overexpression, a treatment investigated in a preclinical trial (32), mobilized intracellular Ca2+ and restored contractility of cardiomyocytes in failing human hearts and improved failing heart function in animal models (3335). Given our results showing smaller Ca2+ transients and compromised contractility in DCM iPSC–derived cardiomyocytes, we hypothesized that overexpression of Serca2a may rescue the disease phenotype of DCM iPSC–derived cardiomyocytes. Transduction of DCM iPSC–derived cardiomyocytes with adenoviruses carrying Serca2a coexpressing green fluorescent protein (GFP) (Ad.Seca2a) (see Materials and Methods) at a multiplicity of infection (MOI) of 100 led to overexpression of Serca2a in these cells (Fig. 6C). Coexpression of GFP together with Serca2a allowed us to recognize the individual transduced cells and measure their contractile forces by atomic force microscopy (Fig. 6D and videos S17 to S20). Forty-eight hours after transduction, overexpression of Serca2a (n = 12) restored the contractile force of single DCM iPSC–derived cardiomyocytes to a level similar to that seen in control iPSC-derived cardiomyocytes (Fig. 6, A and B, and table S5). Ca2+ imaging using the red fluorescent Ca2+ indicator Rhod-2 AM (fig. S16) indicated that DCM iPSC–derived cardiomyocytes transduced with Ad.Serca2a coexpressing GFP (n = 22) had significantly increased global [Ca2+]i transients compared to cells transduced with Ad.GFP only (n = 14) (Fig. 6, E and F) (P = 0.04), which is consistent with restoration of force production. Although Rhod-2 Ca2+ dye is not ideal for quantifying cytoplasmic Ca2+ levels (because of difficulty in calibration with using the indicator in live cells), the kinetics of Ca2+ transients and sarcomeric organization in Serca2a-transduced DCM iPSC–derived cardiomyocytes did not appear to be significantly changed (Fig. 6, G and H). On the other hand, overexpression of Serca2a in control iPSC-derived cardiomyocytes failed to produce a statistically significant increase in contractility (fig. S17), likely because the endogenous amount and function of Serca2a were already at a relatively high level in the control cells. Together, these results demonstrate that overexpression of Serca2a increased the [Ca2+]i transients and contraction force of DCM iPSC–derived cardiomyocytes and partially improved their function.

Serca2a overexpression rescued gene expression of several specific molecular pathways

Although Serca2a gene therapy is now being tested in clinical trials, the overall mechanism of individual cardiomyocyte cellular responses after Serca2a gene therapy has not been extensively studied (36). Hence, we set out to investigate the mechanisms by which Serca2a gene delivery might repair defects in DCM iPSC–derived cardiomyocytes. Gene expression profiling of Serca2a-transduced control and DCM iPSC–derived cardiomyocytes showed that different sets of genes had greater than twofold expression changes, indicating different responses to Serca2a overexpression (tables S6 and S7). There were 191 genes (65 up-regulated and 126 down-regulated) with greater than twofold expression changes in DCM iPSC–derived cardiomyocytes that were returned to expression levels similar to those in control iPSC-derived cardiomyocytes after expression of Serca2a (fig. S18A). Enriched pathway analysis indicated that several pathways, such as Ca2+ signaling, protein kinase A signaling, and G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor signaling, are involved in rescue of the DCM phenotype by Serca2a overexpression (37, 38). Several pathways not previously linked to DCM, including factors promoting cardiogenesis, integrin and cytoskeletal signaling, and the ubiquitination pathway, were also found to participate in rescuing DCM cardiomyocyte function (fig. S18B and table S8).

β-Adrenergic blockers improved sarcomeric organization in DCM iPSC–derived cardiomyocytes

Clinical studies have shown that metoprolol, a β1-selective β-adrenergic blocker, has a beneficial effect on the clinical symptoms and hemodynamic status of DCM patients (39, 40). We thus tested whether in vitro metoprolol treatment has a beneficial effect on DCM iPSC–derived cardiomyocytes carrying the TNNT2 R173W mutation. We found that 10 μM metoprolol treatment for 1 week significantly decreased the number of single DCM iPSC–derived cardiomyocytes with disorganized sarcomeric α-actinin staining (P = 0.023) (Fig. 7A). Although not statistically significant, metoprolol treatment of DCM iPSC–derived cardiomyocytes resulted in a relatively reduced chronotropic effect and increased global Ca2+ transients (Fig. 7, B to D). Metoprolol treatment also significantly reduced aggravation of DCM iPSC–derived cardiomyocytes induced by norepinephrine treatment (1 μM, P = 0.008; 10 μM, P = 0.001) (Fig. 7E). No significant effect was observed on sarcomeric α-actinin distribution in control iPSC-derived cardiomyocytes treated with metoprolol (Fig. 7F). These results suggest that blockade of the β-adrenergic pathway helped DCM iPSC–derived cardiomyocytes to resist mechanical deterioration and improved their myofilament organization.

Fig. 7

Metoprolol treatment improved sarcomeric organization of DCM iPSC–derived cardiomyocytes and alleviates the effects of norepinephrine treatment. (A) One week of 10 μM metoprolol treatment decreased the number of single DCM iPSC–derived cardiomyocytes with severe abnormal sarcomeric integrity (untreated, n = 100; treated, n = 86; *P = 0.023). (B to D) Spontaneous calcium transients in DCM iPSC–derived cardiomyocytes treated with (lower) or without (upper) 10 μM metoprolol. Compared to cells without metoprolol treatment (n = 8), cells treated with metoprolol (n = 8) had slightly increased global calcium transients (C) and slightly decreased beating rates (D). (E) Metoprolol treatment prevented the aggravation of DCM iPSC–derived cardiomyocytes induced by norepinephrine treatment. Metoprolol and norepinephrine were simultaneously added to the culture medium and refreshed daily for 1 week. Both 1 μM (n = 107, **P = 0.008) and 10 μM (n = 101, **P = 0.001) metoprolol significantly decreased the number of disorganized cells compared to those without metoprolol treatment (n = 108). (F) Metoprolol treatment (10 μM) had no significant effect on the sarcomeric integrity of control iPSC-derived cardiomyocytes (untreated, n = 88; treated, n = 75). Data are the means ± SEM. Statistical difference was tested with two-tailed Student’s t test.

Discussion

We have generated patient-specific iPSCs from a DCM family carrying a single point mutation R173W in the sarcomeric protein cardiac troponin T and derived cardiomyocytes from these iPSCs. This has allowed us to generate a large number of human DCM-specific iPSC-derived cardiomyocytes and to analyze their functional properties, explore potential underlying disease etiologies, and test effective therapies. Although the TNNT2 R173W mutation does not seem to affect other cells of the cardiovascular lineage such as endothelial cells (fig. S19), we observed marked phenotypic differences between control and DCM iPSC–derived cardiomyocytes.

Here, a higher tendency toward disturbance in sarcomeric organization was observed in DCM iPSC–derived cardiomyocytes compared to controls. An increased number of DCM iPSC–derived cardiomyocytes exhibited punctate sarcomeric α-actinin staining detected by immunocytochemistry and a more scattered distribution pattern of Z bodies as revealed by transmission electron microscopy. Notably, this phenotype was more frequently observed in single cells or cells at the edges of a cluster of cardiomyocytes than in cells on the inner side of a cluster. The heterogeneous presentation of sarcomeric organization in iPSC-derived cardiomyocytes could be explained by several factors. First, individual cardiomyocyte and cardiomyocyte clusters have different architectural matrices and physical properties for tolerating mechanical forces generated by spontaneous contractions, leading to heterogeneous sarcomeric organization. Second, cardiomyocytes seeded on culture dishes confront different environmental factors, such as the topology of surfaces and paracrine factors from surrounding cells, leading to heterogeneous myofilament organization. It is not unusual to observe heterogeneous sarcomeric organization in cultured rat neonatal cardiomyocytes as shown previously (41, 42). The increased heterogeneous presentation of sarcomeric organization in DCM iPSC–derived cardiomyocytes could be explained by their higher susceptibility to stress. Indeed, both β-agonist stimulation and cyclical stretching markedly increased the heterogeneity of sarcomeric organization in DCM iPSC–derived cardiomyocytes, indicating that they were more susceptible to positive inotropic stress.

Overall, our data are somewhat consistent with previous studies showing that neonatal cardiomyocytes from mice deficient in muscle LIM protein and embryonic heart tissue from zebrafish with mutations in nexilin (a model of DCM) were more susceptible to mechanical stress (43, 44). These results suggest that DCM iPSC–derived cardiomyocytes are less capable of maintaining their sarcomeric integrity compared to control iPSC-derived cardiomyocytes, and are more susceptible to positive inotropic and chronotropic stress. The baseline electrophysiological activities of DCM iPSC–derived cardiomyocytes were not significantly different from those of control, but these cells did show abnormal Ca2+ transients. These results suggest that DCM iPSC–derived cardiomyocytes have an impaired ability to handle Ca2+, resulting in lower contractility. Gene expression profiling using microarray analysis also indicates that DCM iPSC–derived cardiomyocytes express lower amounts of Ca2+-related key molecules (CASQ, TMEM38, NFAT, and NECAB), consistent with compromised Ca2+ handling observed by Ca2+ imaging. Finally, atomic force microscopy indicates that individual DCM iPSC–derived cardiomyocytes manifest decreased contractile force compared to controls, which is consistent with the smaller [Ca2+]i transients observed. Previous studies have also shown altered Ca2+ handling in cardiomyocytes isolated from human patients with heart failure (45). These cardiomyocytes represent a very late stage of disease, and it is still not clear whether the altered Ca2+ handling is the primary factor that contributes to the disease or merely a secondary consequence of disease progression. Our current model of DCM using iPSC-derived cardiomyocytes represents a very early stage of heart development, showing that abnormalities in Ca2+ handling occur at an early stage of development in DCM cardiomyocytes.

Although there are no biochemical data in the literature showing how the particular R173W mutation in cardiac troponin T affects Ca2+ sensitivity or ATPase activity of the myofibers, a major outcome of most of the cardiac troponin T mutations resulting in DCM is a decreased Ca2+ sensitivity in the myofilaments (9, 10, 13, 14). Decreased Ca2+ sensitivity usually suggests decreased contractility in the myofibers at physiological cytosolic Ca2+ concentrations. Indeed, our atomic force microscopy data indicated that the TNNT2 R173W DCM iPSC–derived cardiomyocytes had decreased contractility compared to controls. It is likely that decreased contraction attenuates maturation of the DCM iPSC–derived cardiomyocytes and blocks the mechanical stretch–induced gene expression of molecules associated with myofilament and Ca2+ handling (fig. S20), resulting in increased heterogeneity in sarcomeric organization under inotropic and mechanical stress. Altered Ca2+ handling also may have induced a reduction in Ca2+ transients without in vivo remodeling, further decreasing the contractility of DCM iPSC–derived cardiomyocytes in vitro. This could form a negative feedback cycle that eventually compromises overall cardiomyocyte function. Although a direct relationship between the TNNT2 R173W mutation and abnormal Ca2+ handling has not been well established, the defects in gene expression in our study suggest that the contractility and Ca2+ handling phenotypes are secondary consequences of the R173W mutation in DCM iPSC–derived cardiomyocytes. Further biochemical and molecular studies are required to better understand the disease progression and overall disease mechanisms underlying DCM that are induced by the R173W mutation in the TNNT2 gene.

We have demonstrated that prolonged treatment with the β-blocker metoprolol had a beneficial effect on the abnormal sarcomeric phenotype of DCM iPSC–derived cardiomyocytes by decreasing the number of single cardiomyocytes with abnormal sarcomeric α-actinin staining. Metoprolol treatment led to a negative chronotropic effect and improved global Ca2+ transients in DCM iPSC–derived cardiomyocytes. These results are consistent with a previous study showing that metoprolol treatment of cultured neonatal rat cardiomyocytes induced both negative chronotropic and positive inotropic effects on cells with more than 150 beats per minute (46). These results also indicate that metoprolol can reduce the number of contractions in a given time, possibly reflecting an improved contraction force. The reduction in contraction number may be beneficial and contribute to the protective effects on DCM iPSC–derived cardiomyocytes. In addition, metoprolol treatment of cultured neonatal rat cardiomyocytes up-regulates proteins of cardiac gap junction channels (47), allowing better connections and communication between cells. Together, these factors may exert beneficial effects on DCM iPSC–derived cardiomyocytes.

We have also shown in this study that overexpression of Serca2a, a gene therapy treatment for heart failure currently in clinical trials (36), can markedly improve the contractile function of DCM iPSC–derived cardiomyocytes. Delivery of exogenous Serca2a could prevent the possible negative cascades induced by the TNNT2 R173W mutation (fig. S20). Serca2a overexpression also abrogated the decreased Ca2+ storage of sarcoplasmic reticulum and contractility of DCM iPSC–derived cardiomyocytes, thus rescuing reduced contractility. Gene expression profiling further identified several new pathways that are involved in Serca2a rescue, including ubiquitination and integrin signaling. These results could help to guide further investigations of molecular mechanisms underlying DCM and to find potential therapeutic targets for treating DCM.

In summary, our data indicate that the R173W mutation in the TNNT2 gene caused impairment in myofilament regulation, Ca2+ handling, and force production in individual cardiomyocytes. These factors might be the primary reason for the eventual appearance of the DCM clinical phenotype in patients. Despite limitations in the current iPSC-derived cardiomyocyte platform such as cardiomyocyte immaturity and the lack of an in vivo environment, our overall findings demonstrate that the iPSC platform is useful for investigating disease mechanisms at an early stage of disease and for drug screening. Human iPSC-derived cardiomyocytes from DCM patients could be an important complement to biochemical and mouse models of DCM and should help to elucidate the complex etiology of DCM. Future studies could use this platform to explore the mechanisms of and treatment for not only familial DCM caused by other genetic mutations but also a variety of other hereditary cardiovascular disorders.

Materials and Methods

Patient-specific iPSC derivation, culture, and characterization

All of the protocols for this study were approved by the Stanford University Human Subjects Research Institutional Review Board. Generation of patient-specific iPSC lines was performed as previously described (48).

Cardiac differentiation of hESCs and iPSCs

Differentiation into the cardiac lineage was performed with the protocol described by Yang et al. (24). Detailed procedure is described in the Supplementary Methods.

Ca2+ imaging

Dissociated iPSC-derived cardiomyocytes were seeded in gelatin-coated four-well Lab-Tek II chambers (Nalge Nunc International) and were loaded with 5 μM Fluo-4 AM or 2 μM Rhod-2 AM (for cells expressing GFP) and 0.02% Pluronic F-127 (all from Molecular Probes) in Tyrode’s solution [140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose, 1.8 mM CaCl2, and 10 mM Hepes (pH 7.4) with NaOH at 25°C] for 15 min at 37°C. Cells were then washed three times with Tyrode’s solution. Ca2+ imaging was conducted with a confocal microscope (Carl Zeiss, LSM 510 Meta) with a 63× lens (numerical aperture = 1.4) using Zen software. Spontaneous Ca2+ transients were acquired at room temperature with line scan mode at a sampling rate of 1.92 ms/line. A total of 10,000 lines were acquired for 19.2-s recoding. For measurement of caffeine-evoked Ca2+ release, caffeine (20 mM) in Ca2+-free solution (Tyrode’s solution containing 5 mM EGTA instead of CaCl2) was used to evoke sarcoplasmic/endoplasmic reticulum Ca2+ transients in iPSC-derived cardiomyocytes.

Atomic force microscopy

iPSC-derived cardiomyocytes were seeded on glass bottom petri dishes before each experiment and switched from culture medium to warm Tyrode’s solution. Cells were maintained at 36°C for the entire experiment. Beating cells were interrogated by atomic force microscopy (MFP-3D Bio, Asylum Research) with a silicon nitride cantilever (spring constants ~0.04 N/m, BudgetSensors). For the measurement of forces, cells were gently contacted by the cantilever tip with 100 pN of force, with a typical cellular indentation of about 100 to 200 nm, with the cantilever tip remaining in the position without Z-piezo feedback for multiple sequential 2-min intervals while deflection data were collected at a sample rate of 2 kHz. Typical noise during these measurements was about 20 pN. Deflection data were converted to force by multiplying by the spring constant. Typically, 100 to 400 beats were collected for each single cell, and statistics were calculated for the forces, intervals between beats, and duration of each contraction. Forces across cells were compared with two-tailed Student’s t test.

Adenovirus transduction of iPSC-derived cardiomyocytes

First-generation type 5 recombinant adenoviruses carrying cytomegalovirus (CMV) promoter driving Serca2a plus a separate CMV promoter driving GFP (Ad.Serca2a) and adenoviruses carrying CMV promoter driving GFP only (Ad.GFP) as control were used (35). Dissociated iPSC-derived cardiomyocytes were transduced at MOI 100 overnight and then refreshed with culture medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum). Cells were used for subsequent experiments 48 hours after transduction.

Statistical analysis

Data were analyzed with either Excel or R. Statistical differences among two groups were tested with two-tailed Student’s t tests. Statistical differences among more than two groups were analyzed with one-way analysis of variance (ANOVA) tests followed by Tukey’s multiple comparison test. Significant differences were determined when P value is less than 0.05.

Supplementary Materials

www.sciencetranslationalmedicine.org/cgi/content/full/4/130/130ra47/DC1

Materials and Methods

Fig. S1. Cosegregation of the R173W mutation with the DCM patients in the family.

Fig. S2. iPSCs derived from DCM patients in the family contain the R173W mutation.

Fig. S3. Characterization of iPSC lines.

Fig. S4. Quantitative PCR of relative expression levels of total versus endogenous Yamanaka reprogramming factors.

Fig. S5. Patient-specific iPSCs can differentiate into cells from the three germ layers in vitro.

Fig. S6. Relative cardiac differentiation efficiency of the patient-specific iPSCs.

Fig. S7. Allele-specific PCR of wild-type (Wt) and mutant (R173W) TNNT2 expression in DCM and control iPSC-derived cardiomyocytes.

Fig. S8. Multielectrode arrays examining electrophysiologic properties of iPSC-derived beating embryoid bodies.

Fig. S9. iPSC-derived cardiomyocytes expressed cardiac-specific proteins.

Fig. S10. DCM iPSC–derived cardiomyocytes exhibited an increased heterogeneous sarcomeric organization.

Fig. S11. Norepinephrine treatment significantly aggravated myofilament organization in DCM iPSC–derived cardiomyocytes.

Fig. S12. Enhanced susceptibility of DCM iPSC–derived cardiomyocytes to prolonged mechanical strain.

Fig. S13. Atomic force microscopy measurement of contraction force of iPSC-derived cardiomyocytes.

Fig. S14. Beat frequency and duration of single iPSC-derived cardiomyocytes measured by atomic force microscopy.

Fig. S15. Dot plots of relative cell size versus contraction force for each single cell measured by atomic force microscopy.

Fig. S16. Ca2+ imaging of iPSC-derived cardiomyocytes transduced with Ad.Serca2a or Ad.GFP adenoviruses with red fluorescent Ca2+ indicator Rhod-2 AM.

Fig. S17. Contractility of control iPSC-derived cardiomyocytes transduced with Ad.Serca2a or Ad.GFP as measured by atomic force microscopy.

Fig. S18. Gene expression profiling of DCM iPSC–derived cardiomyocytes with Serca2a overexpression identified enriched pathways that may function in rescuing the DCM phenotype.

Fig. S19. Similar functional properties of control and DCM iPSC–derived endothelial cells.

Fig. S20. Schematic of potential mechanisms by Serca2a gene therapy in DCM iPSC–derived cardiomyocytes.

Table S1. Genetic screening of the DCM gene panel by next-generation sequencing (Illumina).

Table S2. Clinical characteristics of the R173W DCM family.

Table S3. Spreadsheet of iPSC lines used in this study and analyzed by each assay.

Table S4. Baseline electrophysiological parameters of iPSC-derived beating embryoid bodies obtained via multielectrode array recordings.

Table S5. Parameters of single DCM iPSC–derived cardiomyocytes measured by atomic force microscopy.

Table S6. List of genes with greater than twofold expression changes in DCM iPSC–derived cardiomyocytes overexpressed with Serca2a compared to DCM iPSC–derived cardiomyocytes only.

Table S7. List of genes with greater than twofold expression changes in control iPSC-derived cardiomyocytes overexpressed with Serca2a compared to control iPSC-derived cardiomyocytes only.

Table S8. Selected enriched pathways for rescued genes after Serca2a overexpression in DCM iPSC–derived cardiomyocytes.

Table S9. Primers used for real-time quantitative PCR and allelic PCR.

Video S1. iPSC-derived beating embryoid bodies 1.

Video S2. iPSC-derived beating embryoid bodies 2.

Video S3. iPSC-derived beating embryoid bodies seeded on a MED64 multielectrode array probe.

Video S4. Single beating DCM iPSC–derived cardiomyocyte.

Video S5. Single beating control iPSC-derived cardiomyocyte.

Video S6. A beating cluster of DCM iPSC–derived cardiomyocytes.

Video S7. Beating clusters of control iPSC-derived cardiomyocytes.

Video S8. Tracking morphological, inotropic, and chronotropic changes of a beating cluster of control iPSC-derived cardiomyocytes over time after norepinephrine stimulation, day 0.

Video S9. Tracking morphological, inotropic, and chronotropic changes of a beating cluster of control iPSC-derived cardiomyocytes over time after norepinephrine stimulation, day 2.

Video S10. Tracking morphological, inotropic, and chronotropic changes of a beating cluster of control iPSC-derived cardiomyocytes over time after norepinephrine stimulation, day 4.

Video S11. Tracking morphological, inotropic, and chronotropic changes of a beating cluster of control iPSC-derived cardiomyocytes over time after norepinephrine stimulation, day 6.

Video S12. Tracking morphological, inotropic, and chronotropic changes of a beating cluster of DCM iPSC–derived cardiomyocytes over time after norepinephrine stimulation, day 0.

Video S13. Tracking morphological, inotropic, and chronotropic changes of a beating cluster of DCM iPSC–derived cardiomyocytes over time after norepinephrine stimulation, day 2.

Video S14. Tracking morphological, inotropic, and chronotropic changes of a beating cluster of DCM iPSC–derived cardiomyocytes over time after norepinephrine stimulation, day 4.

Video S15. Tracking morphological, inotropic, and chronotropic changes of a beating cluster of DCM iPSC–derived cardiomyocytes over time after norepinephrine stimulation, day 6.

Video S16. Probing a single beating iPSC-derived cardiomyocyte using atomic force microscopy.

Video S17. A fluorescent single beating DCM iPSC–derived cardiomyocyte transduced with Ad.Serca2a.

Video S18. Phase-contrast video of the same cell shown in video S17.

Video S19. A fluorescent single beating DCM iPSC–derived cardiomyocyte transduced with Ad.GFP.

Video S20. Phase-contrast video of the same cell shown in video S19.

References

References and Notes

  1. Acknowledgments: We thank S. Komazaki (Department of Anatomy, Saitama Medical University) for his advice on transmission electron microscopic analyses, A. Olson (Stanford Institute for Neuro-Innovation & Translational Neurosciences) for his help with the confocal microscope, and B. Pruitt (Department of Biochemistry, Stanford University) for her help with stretch experiments. Funding: Supported by NIH New Innovator Award DP2OD004437, RC1 AG036142, R33 HL093172, R01 HL113006, CIRM RB3-05129, Burroughs Wellcome Foundation (J.C.W.), RC1 HL100490 (M.T.L. and J.C.W.), U01 HL099776 (R.C.R.), P01GM099130 (M.P.S.), Howard Hughes Medical Institute Medical Student Fellowship (A.L.), Swiss National Science Foundation PBBEP3_129803 (V.S.-F.), American Heart Association Western States Affiliate Postdoctoral Fellowship 10POST3730079 (S.H.) and 10POST3870063 (M.Y.), and the Oak Foundation Cardiovascular Fellowship (N.S.). Author contributions: N.S. performed reprogramming, established iPSCs, characterizations, differentiation, multielectrode array assays, Ca2+ imaging, project planning, experimental design, data analysis, and preparation of manuscript; M.Y. performed patch clamping, Ca2+ imaging, experimental design, data analysis, and preparation of the manuscript; E.G.N. performed multielectrode array assays, data analysis, and preparation of the manuscript; V.S.-F., S.H., L.W., L.H., R.C., A.L., and O.J.A. performed experimental work; J.L. performed atomic force microscopy; R.E.D., A.P., S.L., and M.J.B. analyzed the data and prepared the manuscript; E.A.A., R.J.H., M.P.S., M.T.L., R.C.R., and J.C.W. designed the experiments and prepared the manuscript. Competing interests: E.A.A. is a stockholder and consultant for Personalis Inc. M.J.B. has filed a patent regarding the use of atomic force microscopy to measure cardiomyocyte beat force and rate (U.S. Patent Application No. 13/307,882). The other authors declare that they have no competing interests. Data and materials availability: The microarray data can be found at Gene Expression Omnibus database with accession number GSE35108.
View Abstract

Navigate This Article