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Online Appendix for the following April 6 JACC article
TITLE: Improvement of Left Ventricular Dysfunction and of Survival Prognosis of
Dilated Cardiomyopathy by Administration of Calcium Sensitizer SCH00013 in a
Mouse Model
AUTHORS: Takuro Arimura, DVM, PhD, Rika Sato, MSc, Noboru Machida, DVM,
PhD, Hidenori Bando, Dong-Yun Zhan, PhD, Sachio Morimoto, PhD, Ryo Tanaka,
DVM, PhD, Yoshihisa Yamane, DVM, PhD, Gisèle Bonne, PhD, Akinori Kimura,
MD, PhD
APPENDIX
Experimental Procedures
Mice
Generation of LmnaH222P/H222P mice was reported previously (1) and their genotyping
was performed by PCR using primers 5'-cagccatcacctctcctttg-3' and 5'agcaccagggagaggacagg-3'. Mice were fed with a chow diet and housed in a barrier
facility. All care and treatment of animals were in accordance with the guidelines for
the Care and Use of Laboratory Animals published by the National Institute of Health
(NIH Publication 85-23, revised 1996) and subjected to prior approval by the local
animal protection authority in Tokyo Medical and Dental University.
Calcium sensitizer
Male and female Lmna+/+ and LmnaH222P/H222P mice were divided into 8 groups with or
without the long-term oral administration of SCH00013 [4,5-dihydro-6-[1-2hydroxy2-(4-cyanophenyl)ethyl]-1,2,5,6-tetrahydropyrido-4yl]pyridazin-3(2H)-one]
(10mg/kg/day in the drinking water) started at 2 months of age just after the first
echocardiographic study and continued until the death for LmnaH222P/H222P mice or 14
months of age for wild type (Lmna+/+) mice. To adjust the intake of SCH00013, we
measured the water intake and body weight once a week, and the concentration of
SCH00013 in the drinking water for the following week was calculated according to
its value. Several mice after two months of administration were sacrificed to obtain
the tissue and blood sample, and plasma concentrations of SCH00013 were measured
by high-performance liquid chromatography as described previously (2).
Echocardiography and blood pressure measurement
Transthoracic echocardiography was performed using a model Prosound SSD-5000
(Aloka) with a 10MHz transducer under slight anesthesia with 1 % isoflurane in O2 as
described previously (1). Parameters of left ventricle (LV) were obtained from 2Dand M-mode, and calculated from the mean of at least three separate cardiac cycles.
Systolic blood pressure and heart rate were measured in the conscious state with a tailcuff blood pressure analyzer (MK-2000, Muromachi).
Preparation of skinned fibers and force measurements
Ventricles dissected from the hearts of mice at 3 months of age were skinned with 0.5
% Brij-58 and a small fiber (about 200 µm in diameter) was dissected from left
ventricular papillary muscle and isometric force was measured as described
previously (3).
Histopathological examinations
Mice were sacrificed and freshly removed heart were fixed in 10% phosphatebuffered formalin, embedded in paraffin-wax, sectioned at 5 μm, and stained with
hematoxylin-eosin or masson’s trichrome staining by the standard methods.
Representative stained sections were photographed using a BX50-33SP light
microscope attached to a DP25 digital camera (Olympus). Images were processed
using Adobe Photoshop 7.0 (Adobe Systems). The interstitial fibrosis area was
stained with picrosirius red using sirius Red F3B (Sigma) to color collagen and
interstitial collagen density was quantified using ImageJ software. Quantitative
analysis of collagen using Semi-Quantitative Collagen Assay Kit (Chondrex) was
done according to the manufacturer‘s instructions. The TdT-mediated dUTP nick end
labeling (TUNEL) assay was performed using in situ Cell Death Detection Kit
(Roche) according to the manufacturer‘s instructions.
RNA isolation and quantitative real-time RT-PCR analysis
Total RNA was extracted from frozen LVs using RNeasy Fibrous Tissue kit (Qiagen)
and cDNA was synthesized from 1 µg of total RNA using SuperScript II Reverse
Transcriptase (Invitrogen). For each replicate in experiments, RNA from tissue
samples of different animals was used. The real-time RT-PCR reaction contained
SYBR Premix Ex Taq (Takara), 5 pmol of each primer and 0.5 µl of the cDNA
template in a 25 μl reaction volume. Amplification was carried out using primers
listed in Supplementary Table S1 and the iCycler iQ Real-Time PCR Detection
System (Bio-Rad) with an initial denaturation at 95°C for 2 min, followed by 50
cycles at 95°C for 10 s and 60°C for 30 s. Relative steady state levels of mRNA
expression were calculated using the ΔδCT method (4). Individual expression values
were normalized against the level of Gapdh mRNA.
Protein extraction and immunobloting
LVs were excised from mice and homogenized in a total protein extraction buffer (2%
SDS, 250 mM sucrose, 75 mM urea, 1 mM dithiothreitol and 50mM Tris-HCl, pH
7.5) containing a protease inhibitor cocktail (Sigma-Aldrich). After measurement of
protein concentration using BCA protein assay kit (Pierce), equal amount of proteins
were subjected to 10%SDS-polyacrylamide gel electrophoresis (SDS-PAGE),
transferred to nitrocellulose membranes (Invitrogen) and blotted with primary
antibodies against myosin light chain 2 (Mlc2), natriuretic peptide precursor A
(Nppa), Fas, Fas-L, and Gapdh (Santa Cruz). Secondary antibodies were conjugated
with horseradish peroxidase (Dako A/S). Signals were visualized by Immobilon
Western Chemiluminescent HRP Substrate (Millipore, MA, USA) and Luminescent
Image Analyzer LAS-3000mini (Fujifilm, Tokyo, Japan). Signal densities were
quantified by using Multi Gauge ver3.0 (Fujifilm, Tokyo, Japan) and the signal
obtained by using antibody against Gapdh was used as an internal control to normalize
the amounts of protein on the immunoblots.
Statistical analysis
All data were acquired and analyzed by observers who were blinded to the animals’
genotypes. Numerical data were expressed as means ± SEM. Statistical differences
were analyzed using one-way analysis of variance (ANOVA) and then evaluated
using a Tukey adjustment for post hoc multiple comparison. Survival curves were
drawn for each group with Kaplan-Meier method, and the comparison of survival
distributions among the groups was performed by a log-rank test. A p-value of less
than 0.05 was considered to be statistically significant.
Additional Discussion
DCM is an etiologically heterogenous disease (5), but genetic factors are involved in
the pathogenesis because it is known that familial inheritance is seen in ~30-40% of
DCM patients (6,7). To date, mutations in ~20 disease-genes have been discovered in
patients with DCM (8,9), and 8% of familial and sporadic DCM may be caused by
mutations in LMNA (10). LMNA encodes A-type lamins, lamin A/C, which are
localized at the internal face of the inner nuclear membrane, and mutations in LMNA
are associated with several diverse diseases often referred to as laminopathies (11).
Although the implantation of implantable cardioverter defibrillator or pacemaker can
prevent cardiac arrhythmias that occur at early stage in the disorders, affected
individuals eventually develop heart failure for which curative treatment is difficult
and cardiac transplantation is ultimately required (12-14).
In this study, we demonstrated that the long-term administration with
SCH00013 improved the cardiac dysfunction and survival prognosis in a DCM mouse
model carrying a LMNA mutation. Our result provided an initial proof for Ca2+
sensitization as a therapeutic option for DCM caused by the LMNA mutation. Ca2+
sensitizer represents a new class of inotropic agents enhancing cardiac contractility
without increasing myocardial oxygen consumption and predisposing arrhythmias
(15). This effect increases cardiac inotropism using the available free cytoplasmic
Ca2+ and therefore the risks associated with the increased intracellular Ca2+ overload,
which is often triggered by the classic inotropic agents including cardiac glycosides,
β-adrenergic agonists and PDE III inhibitors, are circumvented (15). SCH00013 was
reported as a Ca2+ sensitizer that elicited a moderate positive inotropic effect without
significant alteration of Ca2+ transients, but it did not have a positive chronotropic
effect (16).
There are many reports on the genetic etiologies of DCM and the decreased
Ca2+ sensitivity of cardiac muscle contraction was presumed to be a
pathophysiological alteration due to the genetic abnormalities of sarcomere
components, especially the troponin complex (8). In contrast, it was not investigated
whether the abnormalities in the components of nuclear envelope, which was
associated with DCM, would alter the Ca2+ sensitivity of muscle contraction or not. In
this study, we found that the Ca2+ sensitivity of cardiac muscle contraction was not
different between Lmna+/+ and LmnaH222P/H222P mice in both males and females at 3
months of age, indicating that the Lmna mutation did not affect the Ca2+ sensitivity
(Fig. S9). However, structural and functional alterations in the Ca2+ regulatory protein
could result in abnormal regulation of intracellular Ca2+, leading to the contractile
dysfunction in the failing heart caused by the various etiologies, not only by the
genetic abnormalities of sarcomere components (17). Although the molecular
mechanisms of DCM caused by the lamin A/C mutations remain to be fully
elucidated, Ca2+ signaling abnormalities in the failing heart would be a possible
explanation for the basis of beneficial effect brought by SCH00013 in the
LmnaH222P/H222P mice.
The treatment of LmnaH222P/H222P female mice with SCH00013 significantly
increased LVFS and LVEF at all stage of CHF and improved both 50% survival time
and overall mortality. In male mice, SCH00013 also significantly increased LVFS and
LVEF at initial and middle stage of CHF (4 and 6 months of age, respectively) in the
LmnaH222P/H222P mice. In addition, it significantly improved the 50% survival time in
males. It has been demonstrated that the male LmnaH222P/H222P mice displayed more
pronounced abnormalities in the onset and progress of DCM than the female
LmnaH222P/H222P mice (1). Because both the decrease in number and function of
cardiomyocytes could be associated with the end stage of CHF, it was speculated that
SCH00013 might have less beneficial effect at the end stage of severe progressive
CHF than the initial and moderated CHF in the LmnaH222P/H222P mice.
The up-regulations of extracellular matrix remodeling-related genes in the
untreated LmnaH222P/H222P mice were significantly suppressed by SCH00013. This
finding was in good agreement with the significant inhibition of interstitial fibrosis in
the hearts from treated LmnaH222P/H222P mice. It is unknown how SCH00013 inhibits
the interstitial fibrosis in the LmnaH222P/H222P hearts, but we hypothesize that the
maintenance of contractile function may be important for survival of cardiomyocytes
and that the inhibition of degeneration and/or necrosis of cardiomyocytes lead to the
suppression of cardiac fibrosis. On the other hand, we could not exclude a possibility
that SCH00013 played a direct role in the inhibition of interstitial fibrosis.
Investigation on the role of SCH00013 in the cardiac fibrosis will be required in the
future studies.
Acknowledgements: We are grateful Ms. Takako Usami for the breeding of Lmna
mice. We thank Ms. Maki Emura and Ms. Ayaka Nishimura for their technical
assistance.
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Figure S1 Body weights and water intakes of the untreated and treated Lmna+/+
and LmnaH222P/H222P mice
(A) Growth curves of male mice: untreated Lmna+/+ (n=27), treated Lmna+/+ (n=31),
untreated LmnaH222P/H222P (n=16) and treated LmnaH222P/H222P (n=16). (B) Growth
curves of female mice: untreated Lmna+/+ (n=23), treated Lmna+/+ (n=21), untreated
LmnaH222P/H222P (n=15) and treated LmnaH222P/H222P (n=15). (C) Water intakes of male
mice: untreated Lmna+/+ (n=22), treated Lmna+/+ (n=34), untreated LmnaH222P/H222P
(n=20) and treated LmnaH222P/H222P (n=23). (D) Water intakes of female mice:
untreated Lmna+/+ (n=21), treated Lmna+/+ (n=21), untreated LmnaH222P (n=21) and
treated LmnaH222P/H222P (n=20). Closed squares, open squares, closed triangles and
open triangles indicate untreated Lmna+/+, treated Lmna+/+, untreated LmnaH222P/H222P
and treated LmnaH222P/H222P mice, respectively. Data are represented as means±SEM.
Figure S2 Survival curve of the untreated and treated LmnaH222P/H222P mice
Cumulative survival curves are indicated for untreated male and female (closed
triangles and squares, n=39 in each group, respectively) and treated male and female
(open triangles and squares, n=33 and 20, respectively).
Figure S3 Left ventricular (LV) function in the untreated and treated Lmna+/+
and LmnaH222P/H222P mice
Transthoracic M-mode echocardiographic tracings obtained from untreated and
treated Lmna+/+ and LmnaH222P/H222P female mice at 8 months of age. Vertical solid
arrows indicate the end-diastolic diameter (EDD) of LV chamber. The dotted arrows
indicate the end-systolic diameter (ESD) of LV chamber.
Figure S4 Pathological findings of hearts from the untreated and treated
Lmna+/+ and LmnaH222P/H222P female mice at 8 months of age
(A) Longitudinal sections through the atria and ventricles of the mice; scale
bar=5mm. Histopathological analysis of the LV with hematoxylin-eosin staining (B)
or masson’s trichrome staining (C); scale bars= 50μm.
Figure S5 Myocardial interstitial fibrosis in the hearts from untreated and
treated Lmna+/+ and LmnaH222P/H222P female mice at 8 months of age
(A) Typical interstitial collagen staining by picrosirius red through the ventricles of
the mice; scale bar= 100μm. (B) Quantitative analysis of myocardial collagen area
fraction from 5 pictures per cross-section expressed as percentage of collagen staining
to total area. Data are represented as means±SEM for n=4-6 per group. (C)
Quantitative analysis of collagen content in the ventricles. Data are represented as
means±SEM for n=4-6 per group. *p<0.05; ***p<0.001.
Figure S6 Expression of cardiac remodeling-related genes in the LVs from
untreated and treated Lmna+/+ and LmnaH222P/H222P female mice at 8 months of
age
Quantitative real-time RT-PCR showing the mRNA expression of Nppa, Nppb, Myh7,
Myl7, Fos, Tgfb1, Tgfb2, and Col1a2. Bars indicate the fold overexpression in mRNA
level in LVs normalized to Gapdh as calculated by the ΔδCT method. Data are
arbitrarily represented as fold-inductions and the expression of each gene in a LV
from an untreated Lmna+/+ mouse was defined as 1.00 AU. Data are expressed as
means±SEM for n=4-6 per group. *p<0.05; **p<0.01; ***p<0.001.
Figure S7 Expression of natriuretic peptide precursors A (Nppa) and myosin
light chain 2 (Mlc2) in LVs from Lmna+/+, untreated and treated female
LmnaH222P/H222P mice at 8 months of age.
(A) Representative immunoblots for Nppa and Mlc2 are shown. Labeling with
antibody against Gapdh is shown as the loading control. Densitometric analysis of
blotting data obtained from (A) is shown for Nppa (B) or Mlc2 (C). Data were
represented as intensities and that for Nppa or Mlc2 in a LV from an untreated
Lmna+/+ mouse was arbitrarily defined as 1.00 AU. Data are expressed as
means±SEM for n=4-6 per groups. ** p<0.01
Figure S8 Apoptotic signal activation in LVs from Lmna+/+, untreated and
treated female LmnaH222P/H222P mice at 8 months of age.
(A) Typical TUNEL staining through the LVs of the mice; scale bar= 40μm. (B)
Representative immunoblots for Fas and Fas-L are shown. Labeling with antibody
against Gapdh is shown as the loading control.
Figure S9 Ca2+-induced force generation in skinned cardiac muscle fibers
prepared from untreated Lmna+/+ and LmnaH222P/H222P mice at 3 months of age.
Force-pCa relationships (A), Ca2+ -sensitivity (pCa50) (B) ,and maximum force (C) in
mouse skinned cardiac muscle fibers. Data are expressed as means±SEM for n=6-10
per groups.