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 [Ca^2+][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.
Introduction
Dilated cardiomyopathy (DCM), the most common cardiomyopathy, is a
myocardial disorder defined by ventricular chamber enlargement and
dilatation, and systolic dysfunction that can result in progressive
heart failure, supraventricular and ventricular arrhythmias.
Consequently, DCM is a major cause of morbidity and mortality
contributing significantly to health care costs [[63]1–[64]3]. The
prevalence and incidence of idiopathic DCM were estimated to be 1:2500
individuals and 6–7:100,000 respectively, but the data collected was
based on outdated diagnosis methods. In 20–50% of cases the disease is
inherited and is referred as familial DCM [[65]2,[66]3]. The most
common inheritance form of DCM is autosomal dominant transmission,
although other forms were described, such as autosomal recessive,
X-linked and mitochondrial inheritance [[67]4].
Despite extensive research in recent years, the complex pathological
mechanisms of DCM are still unclear, mainly because numerous mutations
in different gene families result in a similar outcome—depressed
ventricular function. Hence, mutations in nuclear, sarcomeric,
cytoskeletal and surface membrane, impair myocardial force generation
and relaxation, force transmission, and/or cell survival. Furthermore,
even when a mutation is identified, the link between the genetic
abnormality and cardiac dysfunction is often unclear [[68]5].
Titin, the largest sarcomeric protein, is expressed in cardiac and
skeletal muscles and is encoded by the TTN gene. The titin protein is
located within the sarcomere as a third filament around 1 μm in length
connecting between Z-line and M-line [[69]2,[70]3]. Titin functions as
a molecular bi-directional spring responsible for the passive
elasticity of the muscle, by creating a restoring force that causes the
sarcomere to return to its resting length [[71]6,[72]7]. Mutations in
titin are a frequent cause of DCM [[73]8], and were described in
several families inherited as an autosomal dominant trait.
To decipher the cellular basis for the cardiac pathology in
titin-mutated patients, we investigated the hypothesis that iPSC-CM
generated from titin-mutated DCM patients recapitulate key aspects of
the disease phenotype. To this end, we generated iPSC-CM from two DCM
patients [[74]9,[75]10] carrying different mutations in the TTN gene,
and investigated their excitation-contraction coupling (ECC) machinery
and responsiveness to common positive inotropic interventions. The
present study demonstrates that while the basal ECC machinery of
titin-mutated iPSC-CM appears intact, their responsiveness to positive
inotropic interventions is blunted compared to healthy iPSC-CM.
Methods
[76]http://dx.doi.org/10.17504/protocols.io.tvien4e
Generation of patient-specific induced pluripotent stem cells
Skin biopsies were acquired according to approval #3611 issued by the
Helsinki Committee for experiments on human subjects at the Rambam
Health Care Campus, Haifa, Israel. The biopsy [human dermal fibroblasts
(HDF)] was obtained from a 28-year-old DCM patient (denoted IsP) from
an Israeli family carrying an adenine insertion mutation, causing a
frame shift and resulting in a stop codon and protein truncation after
19,628 amino acids [[77]9]. Additional skin (HDF24) and hair (KTN3 and
KTI [[78]11,[79]12]) biopsies were obtained from healthy subjects as
control. The dermal fibroblasts generated from the skin biopsies were
reprogrammed as previously described [[80]13] using the STEMCCA
Cassette (a single lentiviral vector containing the four factors: Oct4,
Sox2, Klf4 and c-Myc) and induced Pluripotent Stem Cells (iPSC) were
generated (IsP DCM clones 23.2 and 23.10). These iPSC clones were
spontaneously differentiated into functional cardiomyocytes as
previously described [[81]11–[82]13].
Karyotype analysis
Karyotype analysis was performed using standard G-banding chromosome
analysis by the cytogenetic laboratory according to standard
procedures.
Teratoma formation
To evaluate the iPSC differentiation capacity in vivo, 5-day-old iPSC
colonies from one 6-well plate were detached using 1 mg/ml type IV
collagenase, washed 3 times in PBS and then injected into thigh muscle
of severe combined immunodeficient (SCID) mice. Teratomas were observed
8–12 weeks after injection, and images were obtained from
formalin-fixed (4%) and paraffin-embedded teratoma sections stained
with hematoxylin and eosin (H&E) [[83]14–[84]16].
Genotyping
To confirm that the mutation is preserved in the iPSC clones, sequence
analysis was performed on the titin gene both in the patients-derived
iPSC and fibroblasts by performing PCR using primers which delimit the
mutation area in the mutated gene. The PCR was performed on genomic DNA
produced from the patients' fibroblasts and iPSC using Promega DNA
purification kit, with the primers: F-5’-TATTGCCTGGGTTAAGCCGC-3’ and
R-5’-AGCTCCTGTTGTTAGTCCGC-3’.
Immunofluorescence staining
Immunofluorescence staining was performed according to standard
protocols using the following antibodies: Alexa Fluor 555 donkey
anti-rabbit (1:100; Life Technologies, Eugene, OR, USA), Alexa Fluor
488 donkey anti mouse (1:100; Life Technologies, Eugene, OR, USA), cy5
donkey anti goat (1:100; Invitrogene, Eugene, Oregon, USA), OCT3/4
(1:100; Millipore, Santa Cruz, CA, USA), SSEA4 (1:100; Millipore,
Temecula, CA, USA), TRA1-60 (1:100; Millipore, Temecula, CA,
California), Nanog (1:50; R&D, Minneapolis, MN, USA), DAPI (1:500;
Sigma Aldrich, St. Louis, MO, USA).
Action potential recordings
For action potentials (AP) recordings, spontaneously contracting areas
of EBs (control data was obtained from experiments on KTI and KTN3
clones used in Ben-Ari et al [[85]11]) were mechanically and/or
enzymatically dispersed (collagenase II 1 mg/ml; Worthington, Lakewood,
New Jersey, USA, [86]http://www.worthington-biochem.com). Small
clusters were then plated on gelatin-coated glass coverslips (13 mm
diameter) in 24-well plates. The coverslips were incubated at 37°C, and
a recovery period of at least two days was allowed before the
electrophysiological experiment was performed [[87]11]. In all
experiments, the coverslips were perfused at 37°C with an external
solution containing (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl[2], 1 MgCl[2],
10 glucose and 10 HEPES titrated to pH 7.4 with NaOH. The patch pipette
solution contained (mM): 120 KCl, 1 MgCl[2], 3 Mg-ATP, 10 HEPES, and 10
EGTA titrated to pH 7.2 with KOH and adjusted at 290 mOsm with
saccharose (all materials were purchased from Sigma-Aldrich). Axopatch
200B, Digidata 1322 and pClamp10 (Molecular Devices, Sunnyvale, CA)
were used for data amplification, acquisition and analysis. Signals
were digitized at 10 kHz and filtered at 2 kHz. Microelectrodes with
resistances of 4–7 MΩ were pulled from borosilicate glass capillaries
(Harvard Apparatus, Holliston, USA). Analysis was preformed using
MATLAB software (MathWorks, Natick, MA, USA). Corrected AP duration
(APD) was calculated by the Bazett's correction (
[MATH:
cAPD=APD60beatrate
:MATH]
).
Extracellular electrograms recorded from spontaneously contracting EBs and
analysis of Beat Rate Variability (BRV)
Extracellular electrograms were recorded from spontaneously contracting
30–60 day-old EBs using the Micro-Electrode-Array (MEA) apparatus
(Multi Channels Systems, Reutlingen, Germany) routinely used in our lab
[[88]11,[89]17]. The MEA set-up consists of a 50×50-mm glass substrate,
in the center of which is embedded a 1.4×1.4 mm matrix of 60
titanium-nitride electrodes. The electrode diameter is 30 µm and
inter-electrode distance is 200 µm. Spontaneously contracting EBs were
mechanically dissected and adhered onto the MEA dish and their
electrical activity was recorded by the MEA data acquisition software
at sampling rate of 1000 Hz which was down-sampled to 200 Hz. During
the recording, the cultures were kept in a bath-like conformation of a
glass cylinder (glued to the center of the MEA plane) filled with 500
µL of culture medium saturated with a gas mixture consisting of 5%
CO[2]+ 95% air. The temperature was kept at 37°C using a heating
element and a temperature controller.
For BRV analysis, all recordings were analyzed to detect peaks of the
activation spikes, from which inter-beat intervals (denoted ‘IBI’) were
calculated using MATLAB software (MathWorks, Natick, MA, USA). To
generate Poincaré plots, each R-R interval (IBI[n+1]) is plotted
against its predecessor (IBI[n]), creating a scattered mass of points
in a two-dimensional array. Quantitative analysis of the plot is
performed by fitting an ellipse to the group of points, with its center
coinciding with the centroid of the ellipse (the point of the average
IBI), and adjusting two perpendicular lines traversing the centroid.
The longitudinal line designated SD2, represents long-term variability
of the data (reflecting the standard deviation of the IBIs. The
perpendicular line designated SD1 represents short-term beat-to-beat
variability [[90]18].
Measurements of intracellular Ca^2+ transients and contractions
Intracellular Ca^2+ ([Ca^2+][i]) transients and contractions were
recorded from small contracting 40-70-day old embryoid bodies (EBs) by
means of fura-2 fluorescence and video edge detector, respectively,
using the IonOptix Calcium and Contractility system (Westwood, MA, USA)
as previously described [[91]19–[92]21]. In brief, spontaneously
contracting EBs were mechanically dissected and adhered onto 18 mm
diameter gelatin-coated glass slides. Subsequently, fura-2-stained (2.5
μM) contracting areas were transferred to a chamber mounted on the
stage of an inverted microscope and perfused at a rate of 1–1.5 ml/min
Tyrode’s solution at 37°C. The Tyrode’s solution contains (mmol/l): 140
NaCl, 5.4 KCl, 1 MgCl[2], 2 sodium pyrovate, 1 CaCl[2], 10 HEPES, 10
glucose (pH 7.4 adjusted with NaOH). The EBs were paced at 0.5–2.5 Hz
which corresponded to a frequency 20–50% higher than the spontaneous
beating rate. The acquisition rate of both the [Ca^2+][i] transients
and contractions was 100 points/sec. Analysis was performed using the
IonOptix designated system. To characterize the [Ca^2+][i] transients
amplitude, the differences between maximal (systolic) and minimal
(diastolic) ratio were calculated in 20 successive transients and
averaged (R[Amp]). Similarly, the contraction amplitude (L[Amp]) was
calculated from the average differences between minimal and maximal
video cursor positions of 20 successive contractions. In addition, the
maximal rates of [Ca^2+][i] rise (+d[Ca^2+][i]/dt) and decay
(-d[Ca^2+][i]/dt[Relax]), and maximal rates of contraction
(dL/dt[Contraction]) and relaxation (dL/dt[Relaxation]) were calculated
and averaged over 20 [Ca^2+][i] transients and contractions,
respectively. The data from the Israeli iPSC-derived cardiomyocytes
(iPSC-CM) are pulled from two clones.
Immunocytological analysis in iPSC-CM
iPSC-CM were fixed in 3.7% (vol/vol) formaldehyde and subjected to
immunostaining as previously reported [[93]13]. Sarcomere structure was
visualized using primary antibodies against cardiac troponin T (mouse
monoclonal clone 13–11, Lab Vision, 1:500) and α-actinin (mouse
monoclonal clone EA-53, Sigma-Aldrich, 1:300). Alexa-Fluor-488, and
-594 conjugated secondary antibodies specific to the appropriate
species were used (Life Technologies, 1:500). Nuclei were detected with
1 µg/ml Hoechst 33528. Microscopy was performed using imaging systems
(DMI6000-AF6000), filter cubes and software from Leica microsystems.
Images were assigned with pseudo-colors. Morphological analyses were
performed by investigators blinded to the genotype of the cells.
Mass Spectrometry sample preparation and data processing
iPSC-CM were resuspended in a lysis buffer containing 6M guanidinium
chloride (GCl), boiled for 5 minutes and subsequently reduced with 10
mM tris (2-carboxyethyl) phosphine (TCEP) and alkylated with 40 mM
2-chloro-N,N-diethylacetamide. Samples were diluted (10% acetonitrile,
25 mM Tris pH 8.5), first 1:3 Lysate: Buffer for LysC digestion (25°C 3
hours), then 1:10 for Trypsin digestion. Samples were incubated at 37°C
overnight under continuous shaking. The digestion was blocked by
acidifying the sample with TFA (1% total). Peptides were de-salted
using SDB-RPS StageTips as described [[94]22]. Mass-spectrometry (MS)
analysis was performed in triplicates using a nanoflow uHPLC system
(Easy1000 nLC,) coupled via a nanoelectrospray ion source to a Q
Exactive mass spectrometer (all from Thermo Fisher Scientific).
Peptides were separated on a 50 cm long column with 75 μm inner
diameter, packed in-house with ReproSil-Pur C18-AQ 1.9 μm resin (Dr.
Maisch GmbH). Column temperature was kept at 50°C. Peptide separation
was carried out by loading the peptides in buffer A (0.1% (v/v) formic
acid) and eluting them in 120 or 240 minutes with a nonlinear gradient
of 5–60% buffer B (0.1% (v/v) formic acid, 80% (v/v) acetonitrile) at a
flow rate of 250 nl/min. MS analysis of peptides was performed in a
data-dependent acquisition mode, with survey scans (300–1700 m/z,
maximum ion injection times 60 ms) acquired at a resolution of 60,000
followed by higher-energy collisional dissociation (HCD) based
fragmentation of up to 15 most abundant precursor ions. The MS/MS scans
were acquired at a resolution of 15,000 (maximum ion injection times 60
ms). Repeated sequencing of peptides was minimized by setting a dynamic
exclusion of 20 s.
Raw MS files were processed with MaxQuant (version. 1.6.1.3) [[95]23].
The false discovery rate (FDR) cut-off was set to 1% for protein and
peptide spectrum matches. Peptides were required to have a minimum
length of 7 amino acids and a maximum mass of 4600 Da. Peak list files
were searched against the UniprotKB Homo sapiens database, based on the
2018_02 release, combined with 245 common contaminants by the
integrated Andromeda search engine [[96]24]. The mass spectrometry
proteomics data have been deposited to the ProteomeXchange Consortium
via the partner repository with the dataset identifier PXD010513. Data
analysis, statistics and annotation enrichment analysis was performed
with the Perseus software package, version 1.5.4.2 [[97]25]. Protein
Groups were filtered for at least two valid values in at least one
group of triplicates. Differentially expressed proteins were identified
by t-test at a permutation-based FDR cut-off of 0.05, 250
randomizations and S0 = 0.5, which was used to determine the curves of
the volcano plot. Pathway enrichment analysis was performed using
Fisher exact test with a Benjamini-Hochberg FDR cutoff of 0.02. GOCC,
GOBP, GOMF, CORUM, Uniprot Keywords and KEGG pathway annotations were
used for the analysis. Bar plots were generated of significant protein
of interest was performed by differential enrichment analysis of
proteomic data, DEP [[98]26].
Transmission electron microscopy
Transmission electron microscopy (TEM) was performed on 31- and
56-day-old (post-plating) iPSC-CM from IsP and AuP DCM patients (n = 2
each for two clones) and from healthy controls (n = 2). The samples
were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, post
fixed in potassium ferrocyanide reduced osmium, and further processed
for epoxy resin (Agar100) embedded as previously reported [[99]27]. The
ultra-thin sections were cut with a diamond knife at 60 nm thicknesses
using an EM UC7 Leica ultramicrotome (Leica Microsystems, Wetzlar,
Germany) and double stained with 1% uranyl acetate and Reynolds lead
citrate. TEM was performed using a Morgagni 286 transmission electron
microscope (FEI Company, Eindhoven, The Netherlands) at 80 kV. Digital
electron micrographs were recorded with a MegaView III CD and iTEM-SIS
software (Olympus, Soft Imaging System GmbH, Münster, Germany). Digital
electron micrographs were recorded with a MegaView III CD. From each
sample, two separate EBs from each batch were examined under electron
microscope. Ten CMs of each EBs were photographed. The measurements
were performed to assess the length of sarcomeres and the size of the
Z-bands (sarcomeres width). The measurements were done using iTEM-SIS
software (Figs D and E in [100]S1 File (Olympus, Soft Imaging System
GmbH, Münster, Germany) and exported as Excel documents.
Statistical analysis
Results are expressed as Mean±SEM and represent mean percentage of
change (unless indicated otherwise). Data were analyzed with Sigmastat
(Systat Software Inc., Chicago, Illinois) and Prism 5.0 (GraphPad
Software, San Diego, California). P<0.05 was considered significant.
Results
The titin-mutated patients
The Israeli DCM patient (denoted IsP) was a 28-year old (at the time of
skin biopsy) affected male described by Yoskovitz et al [[101]9]. See
On-line Supplement for clinical data. The mutation is an adenine
insertion at position 86076 (c.86076dupA) causing a frameshift in the
TTN gene. The Australian DCM patient (denoted AuP) was a 62 year old
previously described by Gerull et al [[102]28]. The mutation is a 2-bp
insertion of adenine and thymidine at position 70690 (c.70690dupAT)
leading to a frameshift with premature stop codon in the TTN gene.
Clinical data are provided in the On-line Supplement.
iPSC generation, characterization and differentiation into cardiomyocytes
The IsP iPSC clones 23.2 and 23.10 displayed normal karyotype (FigA in
[103]S1 File), expressed the pluripotent markers SSEA4, Oct4, TRA1-60
and Nanog (Panels A and B of FigB in [104]S1 File) and demonstrated in
vivo pluripotency (Panels C and D of FigB in [105]S1 File). As seen in
FigC in [106]S1 File, the adenine insertion is present in the mutated
fibroblasts and mutated iPSC clones, but absent in the healthy iPSC
clone. The AuP iPSC and iPSC-CM were previously characterized by
Gramlich and co-workers [[107]10]. Table B in [108]S1 File provides
additional details on the patients (age, clinical characteristics), the
generated iPSC (number of iPSC clones) and cardiomyocytes obtained and
analyzed from each patient.
Automaticity and the excitation-contraction coupling (ECC) machinery
Transmembrane action potential characteristics
Firstly, we analyzed action potential (AP) characteristics in IsP, AuP
and healthy spontaneously firing cardiomyocytes. While AP amplitude and
maximal upstroke velocity of phase 0 depolarization (dV/dt[max]) were
similar in IsP, AuP and healthy cardiomyocytes, the spontaneous firing
rate was lower (P<0.01) in IsP iPSC-CM than control. In addition, APD
at 50% and 90% repolarization (APD[50] and APD[90], respectively) were
prolonged in IsP and AuP iPSC-CM compared to healthy iPSC-CM. However,
when corrected to beat rate, the three groups showed no significant
difference in APD[50,] and in only APD[90] of IsP iPSC-CM was slightly
(by 39 ms) prolonged compared to control ([109]Fig 1).
Fig 1. Action potential (AP) characteristics of healthy and mutated (IsP and
AuP) iPSC-CM.
[110]Fig 1
[111]Open in a new tab
[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/dt[max]), [G] and [I] action potential
duration at 50% (APD[50]) and 90% (APD[90]) repolarization,
respectively. [H] and [J] Corrected action potential duration at 50%
(cAPD[50]) and 90% (cAPD[90]) 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/dt[max], beat rate, APD[50] and APD[90]) was performed followed by
Holm-Sidak test, *P<0.05, **P<0.01 and ***P<0.001 vs healthy.
Automaticity, chronotropic β-adrenergic responsiveness and Beat Rate
Variability (BRV)
In addition to the electrophysiological measurements at the cell level
([112]Fig 1), we determined at the network level (a contracting EB) the
spontaneous beat rate and the chronotropic response to β-adrenergic
stimulation ([113]Fig 2) as well as BRV characteristics, representing
the non-linear firing patterns ([114]Fig 3). As seen by representative
electrograms ([115]Fig 2A, upper row) and the summary ([116]Fig 2B),
the mean beat rate was similar in all 3 groups, suggesting that the
titin mutation did not affect the basic mechanisms of automaticity. To
determine whether the titin mutation altered the chronotropic
responsiveness to β-adrenergic stimulation, we analyzed the effects of
isoproterenol on the spontaneous beat rate of healthy (black), IsP
(red) and AuP (blue) EBs. As illustrated in [117]Fig 2A (middle row)
and 2C, isoproterenol had a similar positive chronotropic effect in all
3 experimental groups, which was blocked by the β-blocker metoprolol
([118]Fig 2A lower row).
Fig 2. The chronotropic response to isoproterenol on spontaneous beat rate of
healthy (black symbols), IsP (red symbols) and AuP (blue symbols) EBs.
[119]Fig 2
[120]Open in a new tab
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. BRV characterization of EBs generated from healthy, IsP and AuP iPSC.
[121]Fig 3
[122]Open in a new tab
[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.
As we previously showed that defective intracellular Ca^2+ handling
augments BRV magnitude [[123]11,[124]29], we compared BRV features
between mutated and healthy EBs ([125]Fig 3). The IBI time series
([126]Fig 3B–3E) and IBIs histogram distribution ([127]Fig 3J–3L) show
that the IBI range of AuP EBs (~3000 ms) is broader than healthy (~400
ms) and IsP (~150 ms) EBs. The larger IBI dispersion of AuP EBs is also
demonstrated by the much more scattered Poincaré plot ([128]Fig 3F–3I).
Accordingly, the BRV characteristics: Coefficient of variation
([129]Fig 3M), and Poincaré plots SD1 ([130]Fig 3N) and SD2 ([131]Fig
3O) were larger in AuP than in healthy and IsP EBs.
[Ca^2+][i] transient and contraction characteristics
Because titin is a key structural protein featuring several regulatory
domains [[132]7], we investigated whether the [Ca^2+][i] transient and
contraction characteristics are altered in the mutated cardiomyocytes
representative recordings from the 3 groups are illustrated in [133]Fig
4A. Except for the following changes, all other [Ca^2+][i] transient
and contraction characteristics were similar in the 3 groups: (1) The
contraction amplitude of mutated iPSC-CM was lower (*P< 0.05, **P<
0.01; in IsP and AuP respectively) than in healthy iPSC-CM ([134]Fig
4E). (2) AuP dL/dt[Relaxation] was lower (*P< 0.05) than healthy and
IsP iPSC-CM ([135]Fig 4G). (3) IsP maximal rate of [Ca^2+][i] rise
(+d[Ca^2+][i]/dt) was higher (**P< 0.01) than in healthy iPSC-CM
([136]Fig 4C). (4) IsP minimal rate of [Ca^2+][i] rise
(-d[Ca^2+][i]/dt) was higher (*P< 0.05) than in healthy iPSC-CM
([137]Fig 4D).
Fig 4. The [Ca^2+][i] transient and contraction characteristics in healthy
and mutated (IsP and AuP) iPSC-CM.
[138]Fig 4
[139]Open in a new tab
[A] Simultaneous recording of [Ca^2+][i] transient (orange) and
contraction (green) measured from healthy (left), IsP (middle) and AuP
(right) iPSC-CM. [B-D] [Ca^2+][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 [Ca^2+][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.
The responsiveness to positive inotropic interventions
As Gramlich et al demonstrated that exposure of mice with truncated
titin to isoproterenol or angiotensin-II (AT-II) mimic typical features
of DCM—left ventricular dilatation with impaired fractional shortening
[[140]30], we investigated whether the mutated and healthy
cardiomyocytes respond differently to the following positive inotropic
interventions: (1) β-adrenergic stimulation by isoproterenol; (2)
elevated [Ca^2+][out]; (3) AT-II.
β-adrenergic stimulation
A fundamental cardiac feature is β-adrenergic positive inotropic
response caused by increased sarcoplasmic reticulum (SR) Ca^2+ release
through ryanodine receptor 2 (RyR2) channels [[141]31]. This effect is
demonstrated in healthy cardiomyocytes by a dose-dependent increase in
[Ca^2+][i] transient amplitude, maximal rate of [Ca^2+][i] rise and
decay of the [Ca^2+][i] transient, contraction amplitude and maximal
rates of contraction and relaxation (representative traces in [142]Fig
5A, black symbols in [143]Fig 5D–5I). In contrast, the mutated
cardiomyocytes (representative traces in [144]Fig 5B and [145]Fig 5C
for IsP and AuP, respectively) were barely responsive to isoproterenol
respecting all measured parameters (red and blue symbols for IsP and
AuP in [146]Fig 5D–5I, respectively).
Fig 5. Effect of isoproterenol on contraction and [Ca^2+][i] transient
parameters of healthy and mutated iPSC-CM.
[147]Fig 5
[148]Open in a new tab
[A-C] Representative contractions (L[Amplitude]) from healthy [A], IsP
[B] and AuP [C] iPSC-CM in the absence and presence of isoproterenol.
Panels [D-F] display the summary of [Ca^2+][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 [Ca^2+][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
[Ca^2+][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.
Elevated [Ca^2+][out]
To decipher whether the attenuated response to isoproterenol was due to
dysfunctional β-adrenergic cascade or alternatively–due to impaired
downstream element mediating any positive inotropic intervention, we
investigated the inotropic effect of elevating [Ca^2+][out] (2, 3, 4
and 5 mM), which augments L type Ca^2+ current (I[Ca,L]), in turn
increasing SR Ca^2+ release, thereby increasing contractile force
[[149]32]. As seen in [150]Fig 6, while control cardiomyocytes were
responsive to elevated [Ca^2+][out], that of mutated cardiomyocytes was
blunted. Specifically, all contraction parameters of IsP and AuP
iPSC-CM were significantly lower than those of healthy iPSC-CM
([151]Fig 6G–6I). In addition, there was no difference in [Ca^2+][i]
transient parameters between the 3 groups, except for maximal rate of
[Ca^2+][i] decay which was significantly higher (than control) in IsP
cardiomyocytes ([152]Fig 6D–6F).
Fig 6. Effect of increasing [Ca^2+][out] on contraction and [Ca^2+][i]
transient parameters of healthy and mutated iPSC-CM.
[153]Fig 6
[154]Open in a new tab
[A-C] Representative contractions (L[Amplitude]) from healthy [A], IsP
[B] and AuP [C] iPSC-CM in presence of increased [Ca^2+][out]. Panels
[D-F] display the summary of [Ca^2+][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 [Ca^2+][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 [Ca^2+][i] relaxation between all 3 groups. For
specific [Ca^2+][out] concentrations: *P<0.05 vs Healthy, **P<0.01,
***P<0.001.
Angiotensin-II (AT-II)
Based on the above mentioned findings we speculated that the SR–the
downstream common step mediating these positive inotropic effectors, is
defective in titin-mutated cardiomyocytes. To test this notion, we
investigated whether AT-II, also inducing its positive inotropy via
SR-Ca^2+ release [[155]21], is equally ineffective in mutated
cardiomyocytes. Indeed, while in healthy iPSC-CM AT-II caused a
prominent dose-dependent positive inotropic and lusitropic effects,
markedly augmenting both [Ca^2+][i] transient and contraction
characteristics ([156]Fig 7), mutated cardiomyocytes were unresponsive,
collectively suggesting that a down-stream step mediating the positive
inotropic and lusitropic effect is defected in the titin-mutated
cardiomyocytes.
Fig 7. Effect of AT-II on contraction and [Ca^2+][i] transient parameters of
healthy and mutated iPSC-CM.
[157]Fig 7
[158]Open in a new tab
[A-C] Representative contractions (L[Amplitude]) 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 [Ca^2+][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 [Ca^2+][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
[Ca^2+][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.
Mechanisms underlying attenuated inotropic response of mutated iPSC-CM
To determine possible mechanisms underlying the depressed inotropic
response of mutated iPSC-CM, we investigated: (1) structural features
and sarcomeric organization; (2) The RyR-mediated SR Ca^2+ release
using caffeine; (3) The proteomic profile using MS-based shotgun
proteomics.
Structural features and sarcomeric organization
To determine whether titin mutations are associated with
ultrastructural changes, the Z-width measurements and sarcomere length
analysis were performed on 30- and 60-day-old IsP (clones 23.2 and
23.10), AuP (clone T1) and healthy (clones 24.5 and KTN3) EBs. The
sarcomere length analysis showed no significant differences between
healthy and Aup and IsP cardiomyocytes, regardless of age (see Panels D
and E of FigD and FigE in [159]S1 File and raw data in Table A in
[160]S1 File). iPSC-CM showed a heterogeneous cell morphology,
round-oval cells connected with elongated and stellate cells. Also
iPSC-CM showed various amounts of primitive Z-dense bodies, nascent
sarcomeres or primitive sarcomeres forming myofibrils of variable
length. Healthy and mutated iPSC-CM contained myofibrils that showed
poor alignment or organized sarcomeric pattern on all samples,
consistent with the immature phenotype. The number of sarcomeres could
not be appreciated with a degree of satisfactory accuracy because CM in
EBs showed an irregular shape, with cellular extensions not trapped in
the 60 nm section plane. Very few myofibrils developed A- and I-bands
either at 30 days or 56 days post-plating from control and titin
mutated EBs and this aspect could not be reliable quantified on
ultrathin sections.
Sarcomeric organization of mutated and healthy iPSC-CM was determined
by co-staining of α-actinin (Z disk) and cardiac troponin-T (A-band)
[[161]10]. Briefly, single cardiomyocytes were dissociated from
spontaneously beating embryoid bodies (EBs), and analyzed 7 days later.
Cardiomyocytes were divided into 3 groups according to sarcomeric
organization extending to: (1) the whole cytoplasm ([162]Fig 8A, upper
row); (2) <50% of the whole cytoplasm with main localization in the
cell periphery ([163]Fig 8A, middle row); (3) <50% of the whole
cytoplasm with main localization in the perinuclear area ([164]Fig 8A,
lower row). As seen in [165]Fig 8B, the percentage of cells with fully
structured myofilaments occupying the entire cytoplasm is significantly
reduced in IsP compared to healthy iPSC-CM (44% vs. 80%, respectively,
P<0.0001). In contrast, in IsP iPSC-CM there is a higher percentage of
cardiomyocytes in which organized myofibrils occupy only half of the
cytoplasm or even less (P<0.0001). This group was further divided into
two sub-groups according to the localization of the few organized
myofibrils, which were visible either in the perinuclear area or at the
cell periphery ([166]Fig 8B). In the IsP group 26.6% of the cells show
a sarcomere with an organized pattern visible only in the perinuclear
area and 29.4% display an organized pattern limited at the cell
periphery. The respective percentages in the healthy iPSC-CM were 7.3%
and 12.4%, respectively. Importantly, these results are in agreement
with the findings obtained by Gramlich et al in AuP cardiomyocytes
(fully organized 54%, perinuclear 17%, and peripherally 29%) [[167]10],
suggesting the presence of titin mutation-related defects in both
myofibril reassembly and sarcomere stability.
Fig 8. Levels of sarcomeric organization in control and IsP iPSC-CM.
[168]Fig 8
[169]Open in a new tab
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.
The RyR-mediated SR Ca^2+ release using caffeine
To decipher the mechanism(s) underlying the diminished response of
titin-mutated iPSC-CM to all positive inotropic interventions operating
by increasing [Ca^2+][i], we tested the effect of a brief application
of caffeine (10 mM, serves as an opener of the RyR2 receptor [[170]12])
on the [Ca^2+][i] transient. As illustrated by the representative
experiments ([171]Fig 9A–9C) and summary ([172]Fig 9D and 9E),
titin-mutated and healthy cardiomyocytes differed in their response to
caffeine. As we previously reported [[173]12], healthy iPSC-CM
displayed an abrupt amplitude transient increase in intracellular Ca^2+
concomitant with contraction cessation, followed by a decline in the
Ca^2+ level along resumption of contractions after ~10 sec ([174]Fig
9A). In contrast, mutated iPSC-CM displayed prolonged recovery which
was divided into two phases: (1) decline in [Ca^2+][i] level as seen in
healthy cardiomyocytes, but not to the basal level; (2) a gradual slow
decline that lasts few minutes until reaching basal [Ca^2+][i] level,
during which resumption of contractions occurs ([175]Fig 9B) (in IsP
but not AuP cardiomyocytes). Consequently, IsP iPSC-CM recovery
following caffeine administration was much slower compared to healthy
iPSC-CM and the gradual decline in [Ca^2+][i] level was longer
([176]Fig 9D).
Fig 9. Effect of caffeine on [Ca^2+][i] cycling of healthy and mutated
iPSC-CM.
[177]Fig 9
[178]Open in a new tab
Representative [Ca^2+][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 [Ca^2+][i] level: fast
decline as in healthy cells, and gradual slow decline until reaching
[Ca^2+][i] basal level [B]. [D] Average recovery time from maximum of
caffeine peak phase until the beginning of departing phase of the first
measurable [Ca^2+][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 [Ca^2+][i] signal compared to the
pre-caffeine [Ca^2+][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).
Protein expression analysis
Altogether, 301 unique proteins showed significant expression changes
between AuP and healthy cardiomyocytes (151 upregulated and 150
downregulated; permutation-based FDR 0.05, S0 0.5) ([179]Fig 10A). In
the cluster of upregulated proteins, the top 3 significantly enriched
keyword categories were Extracellular matrix, Actin-binding and Muscle
protein, whereas Disease mutation, Calcium and Cardiomyopathy were
identified as top significantly enriched Keyword terms within the
downregulated proteins ([180]Fig 10B). An additional detailed analysis
of the proteome profile of the AuP cardiomyocytes revealed that indeed
the expression levels of three important cardiac Ca^2+ handling
proteins (the ryanodine receptor 2—RYR2, the sarcoplasmic reticulum
histidine-rich calcium-binding protein—HRC, and the sodium/calcium
exchanger 1—SLC8A1) were significantly reduced in patient cells
([181]Fig 10D).
Fig 10. Mass spectrometry-based proteomic analysis of AuP and healthy
cardiomyocytes.
[182]Fig 10
[183]Open in a new tab
[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 Ca^2+ handling proteins that
are differentially expressed between AuP and both healthy control
cardiomyocytes.
Discussion
To decipher the cellular mechanisms underlying the functional
abnormalities, we investigated the actual mutated cardiomyocytes
derived from DCM patients carrying titin mutations. The major findings
were: (1) diminished response to isoproterenol, elevated [Ca^2+][out]
and AT-II; (2) altered response to caffeine, compared to healthy
cardiomyocytes.
Spontaneous electrophysiological features of titin-mutated iPSC-CM
Despite the statistically significant lower firing rate of IsP iPSC-CM
and prolonged APD displayed by both IsP and AuP iPSC-CM, these
differences lack practical significance since the mean firing rate and
APD values fall well within the normal range previously described in
iPSC-CM [[184]29,[185]33]. Additionally, when corrected to beat rate,
the only significant difference in APD[90] was between IsP iPSC-CM and
control. Furthermore, this difference was less than 40 ms, leaving IsP
iPSC-CM well within the normal range and thus lacking practical
significance [[186]33].
AuP iPSC-CM exhibited markedly increased BRV indices compared to
healthy and IsP iPSC-CM. The mutation in both IsP and AuP patients
deletes the A-band segment, leading to defective sarcomerogenesis
through disruption of mechanical force transmission from myosin
[[187]34]. Currently, we do not know the reason for this difference in
BRV parameters. Perhaps it is associated with disturbed mitochondrial
Ca^2+ handling machinery, which we previously showed to affect BRV
magnitude [[188]11].
Attenuated responsiveness of mutated iPSC-CM to isoproterenol and
[Ca^2+][out]
While the basal [Ca^2+][i] transient and contraction parameters were
mostly similar, the positive inotropic response of the mutated
cardiomyocytes was markedly suppressed. While healthy cardiomyocytes
exhibited the expected positive inotropic and lusitropic effects, the
mutated cardiomyocytes displayed blunted response to isoproterenol
([189]Fig 3). These findings are in agreement with 2 recent studies:
(1) Hinson and co-workers showed that iPSC-CM generated from DCM
patient carrying the pP22582fs+/- titin mutation (different than the
AuP and IsP mutations) demonstrated diminished responses to
β-adrenergic stress using contractile function assay [[190]35]. (2)
Peng et al reported diminished response to increased [Ca^2+][out] and
isoproterenol of cardiomyocytes derived from titin M-line deficient
mice, which was attributed to reduction in expression levels of Ca^2+
handling proteins SERCA2 and PLB [[191]36]. In addition, reduction in
protein expression levels was observed also for the Ca^2+-binding
protein calmodulin, which participates in SR Ca^2+release and regulates
the activity of SERCA2 and PLB through Ca^2+/Calmodulin-dependent
kinase II (CaMKII) [[192]36]. Further details regarding the titin
kinase domain are provided below.
Next, we subjected the healthy and mutated cardiomyocytes to increased
[Ca^2+][out]. Positive inotropic and lusitropic effects in all 3
contraction parameters–amplitude, maximal rates of contraction and
relaxation—were observed in healthy cardiomyocytes, as previously
reported [[193]32,[194]36] ([195]Fig 6). Nevertheless, IsP and AuP
cardiomyocytes displayed reduced response to increased [Ca^2+][out]
compared to healthy cells. In contrast, only the maximal rate of
[Ca^2+][i] relaxation differed between the three groups.
Impaired responsiveness of mutated iPSC-CM to AT-II and caffeine
Subsequent to the findings of decreased response of titin-mutated
iPSC-CM to isoproterenol and [Ca^2+][out], we sought to decipher the
contribution of the SR to these findings. Therefore we investigated in
healthy and mutated iPSC-CM the effect of AT-II, promoting synthesis of
1,4,5-inositol triphosphate (1,4,5-IP[3]) which triggers SR Ca^2+
release [[196]21]. Similarly to isoproterenol and [Ca^2+][out], AT-II
caused positive inotropic and lusitropic effects in healthy iPSC-CM on
[Ca^2+][i] transient and contraction parameters, as opposed to lack of
response in IsP and AuP iPSC-CM ([197]Fig 7). These results are
consistent with results published by Gramlich et al [[198]30]. The
authors found that mice with truncated titin lacking part of the A-band
and the M-band regions showed no alterations in cardiac morphology and
function under normal conditions. However, when exposed to cardiac
stress by means of isoproterenol or AT-II, these agents mimicked
typical features of DCM—left ventricular dilatation with impaired
fractional shortening.
To further investigate intracellular Ca^2+ handling, we studied the
effect of a brief application of caffeine (10 mM). Caffeine, being an
opener of the RyR2 receptor, induces Ca^2+ release from the SR which
results in increased [Ca^2+][i] levels [[199]12]. IsP iPSC-CM showed
longer recovery period until resumption of contraction compared to
healthy and AuP iPSC-CM ([200]Fig 9). Moreover, when compared to
healthy cells, diastolic Ca^2+ remained elevated in patient iPSC-CM
after caffeine application, suggesting defects in SR calcium re-uptake
and/or cellular Ca^2+ efflux. Concordantly, proteomic analysis in AuP
iPSC-CM revealed reduced level of the sarcoplasmic reticulum
histidine-rich calcium-binding protein (HRC), a regulator of Ca^2+
sequestration in the SR [[201]37], and the sodium/calcium exchanger
(SLC8A1), which represents the major Ca^2+ efflux mechanism in cardiac
myocytes ([202]Fig 10D) [[203]37]. Similar results were reported for
cardiomyocytes derived from patients in a DCM family carrying a point
mutation in cardiac troponin-T gene [[204]38]. These cardiomyocytes
displayed prolonged decay time in response to caffeine, suggesting
altered function of Ca^2+ pumps in the sarcolemma and SR membranes.
Furthermore, DCM iPSC-CM displayed abnormal contractility which was
rescued following overexpression of SERCA2, indicating compromised
Ca^2+ handling in DCM cardiomyocytes due to reduced expression of
SERCA2 and other Ca^2+ related elements [[205]36,[206]38]. In addition,
we showed that in response to caffeine administration iPSC-CM generated
from catecholaminergic polymorphic ventricular tachycardia type 2
(CPVT2) patients exhibited two-phase decline in Ca^2+ level compared to
healthy cells [[207]12]. We suggested that the vast and prolonged Ca^2+
release is due to the mutated calsequestrin protein in the CPVT2
patients which result in high levels of free Ca^2+ in the SR, and not
necessarily from a total higher SR Ca^2+ levels [[208]12]. Accordingly,
altered function of Ca^2+ handling protein such as calsequestrin and
SERCA2 might be the reason for the abnormal response of IsP iPSC-CM in
response to caffeine. This aspect is further detailed below.
Structural analysis in titin-mutated cardiomyocytes
TEM analysis showed that both healthy and mutated iPSC-CM had nascent
sarcomeres and disarrayed myofibrils with clear Z bands, with no
ultrastructural differences between the three cell types in both age
groups. Several studies reported on immature sarcomere and myofibrils
structure in healthy cardiomyocytes seen in long-term culture up to 180
days [[209]20,[210]39]. Moreover, only in 360 day-old cardiomyocytes
there were first signs of M-band [[211]39]. An important study by
Hinson et al demonstrated shorter sarcomere length in titin-truncated
iPSC-CM compared to wild-type, but the authors used three-dimensional
cardiomyocytes microtissues that can be possibly more mature than our
cultured iPSC-CM [[212]35]. Perhaps immaturity of the iPSC-CM is the
reason for the presence of only clear Z bands in titin-mutated and
healthy 31- and 56-day-old cardiomyocytes. Sarcomeric organization
analysis showed defects in assembly and maintenance of a stable
sarcomeric structure in mutated IsP compared with healthy iPSC-CM
([213]Fig 8), which corresponds to the results of Gramlich et al in AuP
iPSC-CM [[214]10].
We performed sarcomeric organization analysis and found defects in
assembly and maintenance of a stable sarcomeric structure in
titin-mutated IsP iPSC-CM which lack the A-band segment. These results
corresponds to those of Gramlich et al in AuP iPSC-CM which also lack
the A-band segment [[215]10]. In addition, Chopra et al found in
titin-mutated iPSC-CM lacking A-band segment generated from DCM
patients, that titin truncation mutations lead to defective sarcomere
formation and myofibrillar assembly due to insufficient length of the
titin protein [[216]34]. Furthermore, Tonino et al showed in a mouse
model in which part of the A-band was deleted that thick filament
length was decreased in cardiac and skeletal muscles, and functional
studies revealed reduced force generation and a DCM phenotype
[[217]40]. These findings suggest that titin truncation mutations
leading to deletion of the A-band damage sarcomerogenesis through
disruption of mechanical force transmission from myosin [[218]34].
Titin kinase domain
The titin protein is located within the sarcomere as a third filament
connecting between Z-line and M-line. Between these edges, the titin is
divided between the A-band and the elastic and extensible I-band. We
performed sarcomeric organization analysis and found defects in
assembly and maintenance of a stable sarcomeric structure in
titin-mutated IsP iPSC-CM which lack the A-band segment. These results
correspond to those of Gramlich et al in AuP iPSC-CM which also lack
the A-band segment. In addition, Chopra et al found in titin-mutated
iPSC-CM lacking A-band segment generated from DCM patients, that titin
truncation mutations lead to defective sarcomere formation and
myofibrillar assembly due to insufficient length of the titin protein.
Furthermore, Tonino et al showed in a mouse model in which part of the
A-band was deleted that thick filament length was decreased in cardiac
and skeletal muscles, and functional studies revealed reduced force
generation and a DCM phenotype. These findings suggest that titin
truncation mutations leading to deletion of the A-band damage
sarcomerogenesis through disruption of mechanical force transmission
from myosin.
In addition to its structural role, titin participates in various
signal-transduction pathways through several sites, specifically the
titin kinase (TK) domain located at the M-band [[219]41,[220]42].
Several studies investigated TK and its diverse signaling pathways and
interactions, and found that it is involved in muscle mechanical
signaling [[221]42–[222]45]. An important study by Peng et al
investigated the effect of TK absence on cardiac function using M-line
deficient mice lacking TK [[223]36]. On the cellular level, there was
no difference in sarcomere structure between 30-day old control and
M-line deficient cardiomyocytes, similar to our results obtained from
ultrastructure analysis. Only after 80 days in culture, M-line
deficient cardiomyocytes displayed sarcomere disassembly. Additionally,
cardiac function was investigated, and under basal conditions there was
no difference between 30-day old control and M-line deficient
cardiomyocytes, similar to our findings of contraction parameters.
Moreover, the β-adrenergic stimulator dobutamine which causes a
positive inotropic effect, caused blunted response in M-line deficient
cardiomyocytes compared to control, in agreement with our findings in
titin-mutated cardiomyocytes [[224]36]. The authors suggested that the
mechanism underlying the reduced contractility of M-line deficient
cardiomyocytes, is associated with calcium handling machinery, and is
based on protein expression levels analysis in which early reduction of
SERCA2 and PLB levels was observed. These results may indicate that
titin serves as a contractility regulator through its TK effects on
calcium handling proteins [[225]36].
The above mentioned findings indicate TK importance in myofibrillar
mechanical signaling, contractile function and cardiac gene expression.
These findings may constitute the basis for the results obtained from
IsP and AuP titin-mutated cardiomyocytes, which lack the M-line region
and specifically the TK domain. Furthermore, our results are consistent
with results previously published by Gramlich and co-workers [[226]30].
This group found that mice with truncated titin lacking the TK show no
alterations in cardiac morphology and function under normal conditions.
However, when exposed to cardiac stress by means of isoproterenol or
AT-II, they mimic typical features of DCM—left ventricular dilatation
with impaired fractional shortening. More importantly, Gramlich et al
(2015) generated iPSC-CM from Australian DCM patient (AuP) which lack
the TK domain due to titin truncation mutation in exon 327 [[227]10].
Significant down regulation of α- and β- myosin heavy chain and cardiac
α-actin was observed in mutated cells compared with control, similar to
previously published results [[228]10,[229]45].
These findings strengthen the notion that titin plays a key role in
various cellular pathways, in addition to its role as molecular spring.
Specifically, the TK domain is crucial for sarcomere assembly and
stability, mechanosensory processes and regulation of gene expression
through interactions with diverse proteins. Truncation of TK domain in
mutant iPSC-CM may be a plausible mechanistic link to explain the
observed results; however, this hypothesis was not addressed in the
present study.
Altogether, we investigated iPSC-CMs from an Israeli (IsP) and
Australian (AuP) titin-mutated (carrying different mutations) patients.
All the findings on the IsP iPSC-CM are new. A recent study [[230]10]
by Moretti, Gramlich (co-authors on this manuscript) and co-workers
using AuP iPSC-CM demonstrated that: (1) Correction of TTN reading
frame in patient-specific cardiomyocytes derived from induced
pluripotent stem cells rescued defective myofibril assembly and
stability and normalized the sarcomeric protein expression (2) AON
treatment in TTN knock-in mice improved sarcomere formation and
contractile performance in homozygous embryos and prevented the
development of the DCM phenotype in heterozygous animals. (3)
Disruption of the TTN reading frame due to a truncating DCM mutation
can be restored by exon skipping in both patient cardiomyocytes in
vitro and mouse heart in vivo, indicating RNA-based strategies as a
potential treatment option for DCM. All other findings regarding action
potential characteristics, beat rate variability, ultrastructural
analysis, inotropic responsiveness to AT-II and [Ca ^2+][out], response
to caffeine and proteomics, are completely novel.
In summary, our findings reflect an underlying abnormal contraction and
calcium handling mechanism, which can be attributed to lack of titin
kinase domain in the DCM patients. The kinase domain is involved in
regulation of cardiac gene expression through interactions with MURF
proteins, as was reported by several studies [[231]36,[232]43–[233]45],
and this involvement might contribute to the reduced response of
titin-mutated iPSC-CM to positive inotropic interventions. Additional
research is required to decipher the specific mechanism(s) responsible
for the reduced contractility of IsP and AuP titin-mutated
cardiomyocytes.
Supporting information
S1 File. Supplement.
(DOCX)
[234]Click here for additional data file.^ (13.9MB, docx)
Acknowledgments