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Regular Articles |
From the Divisions of Molecular Cardiovascular Biology*
and Cardiology,
Children’s Hospital and
Research Foundation, Cincinnati, Ohio; the Division of
Cardiology,
University of Cincinnati,
Cincinnati, Ohio; and the Department of Cell Biology and
Anatomy,§
University of South Carolina,
Columbia, South Carolina
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cardiomyopathic transgenic mouse models often lack characterization of molecular and cellular changes occurring throughout various stages of pathogenesis, particularly the early phase. These investigations will be critical for understanding disease progression and determining similarities between mouse paradigms and inherited cardiomyopathic conditions in man. In human populations, phenotypic variation in progression and severity of disease complicates interpretation of the underlying etiology.4 Despite this limitation, population-based longitudinal analyses are necessary to achieve an accurate description of the progression, pathology, and variability of the cardiomyopathy as exemplified by studies such as Framingham.5,6 Transgenic mice can be created and propagated using genetically identical individuals, circumventing concerns related to genetic background heterogeneity that are inescapable in human populations. The combination of genetic homogeneity and comparable environment of the population greatly simplifies interpretation of results. Thus, studying the epidemiology of tropomodulin (Tmod)-overexpressing transgenic (TOT) mice provides valuable insight into the nature of their cardiomyopathy. Because TOTs predictably develop dilated cardiomyopathy within 2 weeks after birth,7 they provide an attractive model system to study the molecular and structural changes before and during the progressive phase of disease. Furthermore, characteristics of TOT cardiomyopathy such as loss of myofibril organization and diminished contractility are hallmarks of progressive and chronic dilated cardiomyopathy in humans.8-10
TOT utility as a paradigm of human cardiomyopathy depends on defining relationships between essential disease characteristics in man and mouse. The goal of this report was to detail the fundamental properties of TOT pathogenesis to establish the paradigm. TOT cardiomyopathy was thought to arise from Tmod-mediated myofibril degeneration,11 but the dilated phenotype apparently stems from resultant calcineurin activation driving an impaired hypertrophic response.12 The utility of TOTs in testing pharmaceutical intervention was recently demonstrated by treatment of TOTs with cyclosporin, a calcineurin inhibitor, to inhibit development of the dilated phenotype.12 Calcineurin activation is important in experimental hypertrophy13 and human cardiomyopathy,14 presumably due to chronic elevation of intracellular calcium. Results presented here indicate that calcium signaling is likely to be a primary effector in the initiation of TOT pathogenesis, leading to molecular and histological changes before development of the dilated cardiomyopathic phenotype within 1 week after birth. Concurrently and shortly thereafter, environmental factors such as maturational stress and litter size influence TOT survival, reminiscent of reported associations between environment and outcome in human cardiomyopathic conditions.6,15 Molecular and structural analyses identified mechanisms causing cardiac dilation, and population analyses demonstrated associations between survival, postnatal development, and the environment. Collectively, these findings establish fundamental parallels between the TOT paradigm and human disease, with implications not only for the development and interventional treatment of TOT cardiomyopathy, but also for dilated cardiomyopathy in general.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TOTs were established as previously described.7 The TOT population was derived from a single FVB/N line inbred to transgene homozygosity for increased production, creation of uniformly affected litters, and facilitation of population-based analyses. Mating of the founder line 65 transgenic female and nontransgenic male produced heterozygous first generation (F1) offspring. These F1 siblings were then mated to produce second generation (F2) mice with litter ratios of 1 normal: 2 heterozygotes: 1 homozygote. The heterozygotes showed elevated Tmod expression without juvenile morbidity or mortality (data not shown) and are the subject of ongoing study. F3 homozygotes developed acute dilated cardiomyopathy with variation in mortality despite consistent onset and progression of the disease.
Immunoblot Analyses
Hearts were collected at the indicated time points and washed in phosphate buffered saline, frozen in liquid nitrogen, crushed with a mortar and pestle, and reconstituted in buffer A (20 mmol/L sodium phosphate, pH 7.0, 150 mmol/L NaCl, 2 mmol/L MgCl2, 0.1% NP-40, 10% glycerol, 10 nmol/L cypermethrin, 10 nmol/L okadaic acid, 100 µmol/L phenylasine oxide, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 1 mmol/L dithiothreitol, 100 µmol/L sodium orthovanadate, 10 µg/µl pepstatin, 10 µg/µl leupeptin, 10 µg/µl aprotinin, 10 µg/µl tosyl-L-lysine chloromethyl ketone, 10 µg/µl N-tosyl-L-phenyl-alanine chloromethyl ketone). Approximately 50 µg of protein powder was added to 200 µl of buffer A and light sonication was performed to lyse remaining cells. For calmodulin immunoprecipitations, 300 µg of protein extract in buffer A was mixed with 5 µg of calmodulin antibody (Zymed, South San Francisco, CA) and 30 µl of protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and incubated at 4°C with gentle rocking for 2 hours. Samples were then gently centrifuged at 800 x g for 3 minutes in a microfuge at 4°C, at which time the supernatant was discarded and the agarose pellet was resuspended in 100 µl of buffer A and spun again. This wash was repeated two additional times. After the last spin the agarose pellet was resuspended in 20 µl of sodium dodecyl sulfate (SDS) sample loading buffer and boiled. These samples were then loaded on a polyacrylamide gel for subsequent Western blot analysis. For tropomodulin and signal transduction immunoblots, protein concentration of heart lysates was determined using the Bio-Rad DC Protein assay (#500–0116, Bio-Rad, Melville, NY). 100 µg/lane of protein sample was mixed with an equal volume of sample buffer, boiled for 5 minutes, cooled to room temperature, and loaded onto 12.5% SDS-polyacrylamide gels for electrophoresis. Separated proteins were transferred in Tris-glycine immunoblot buffer at 70V and 4°C overnight to PVDF membrane (#RPN 2020F, Hybond-P PVDF, Amersham, Arlington Heights, IL). The next day, membranes were rinsed in water and stained with 0.2% Ponceau S (#BP103-10, Fisher, Pittsburgh, PA) solution containing 3% TCA (#T-4885, Sigma, St. Louis, MO) to confirm uniform protein transfer and comparable loadings between samples. The blot was rinsed again in water, washed in TBST buffer (50 mmol/L Tris-HCl pH 7.6, 150 mmol/L NaCl, and 0.1% Tween 20), and blocked in TBST containing 5% Amersham blocking buffer (w/v). Antibodies used for the primary incubation included: anti-tropomodulin (affinity-purified polyclonal rabbit antibody produced against bacterially expressed recombinant tropomodulin), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #RDI-TRK5G4–6C5, Research Diagnostic Industries, Flanders, NJ), anti-phosphotyrosine (#P11120, Transduction Labs, Lexington, KY), anti-ERK 1 & 2, anti-phospho-p38, anti-phospho-mitogen-activated protein kinase (MAPK); (#9101S, #9213, #9211, #9211S, and #9101S, New England Biolabs, Beverly, MA) and anti phospho-JNK (#SC-6254, Santa Cruz). Membranes were incubated with primary antibodies either at 4°C overnight on a rocking platform shaker or for 2 hours at room temperature on an orbital platform shaker. After labeling, membranes were washed 3 times in TBST for 10 minutes per wash and then incubated with appropriate species-specific alkaline phosphatase-conjugated secondary antibodies for enhanced chemifluorescence as directed by the manufacturer (ECF kit, Amersham). Chemifluorescent images were scanned using a Storm 860 (Molecular Dynamics, Sunnyvale, CA).
Intracellular Calcium Analyses
Cardiomyocytes were isolated and calcium measurements were performed as previously described.16 Briefly, isolated hearts were perfused with low Ca2+ Joklik medium containing collagenase type I and type II enzyme. Measurement of intracellular free Ca2+ transients were obtained by loading isolated cells with Fura-2 a.m. dye at 37°C for 30 minutes. After loading, cells were washed and resuspended in oxygenated physiological buffer and imaged using a photo scan dual-beam spectro-photometer coupled to a microscope equipped with optics for ultraviolet viewing. Intracellular free Ca2+ was monitored as the ratio of 340:380 nm fluorescence of Fura-2, and data are reported as the 340:380 nm ratio. Two control and two TOT mice were used at 13 weeks of age, based on prior experiments which established the appropriate age for calcium measurements. Results with younger control animals show variable measurements, complicating interpretation of transgenic data (data not shown). Eight cardiomyocytes were measured from each animal.
Microscopic Analyses
Light and electron microscopy were performed as previously
described.7,17
For the confocal time course analysis,
hearts were removed from mice at 1, 5, 8, 11, 14, and 17 days after
birth, rinsed in PBS, and fixed in 2% paraformaldehyde, 10 mmol/L
HEPES, pH 7.0, overnight at 4°C on a rocking platform shaker. The
next day, hearts were transferred to PBS containing 30% sucrose and
left overnight to equilibrate at 4°C on a rocking platform shaker.
Cryoblocks were made by embedding hearts in Tissue Freezing Medium
(#H-TFM, Triangle Biomedical Sciences, Durham, NC) and freezing in
melting isopentane which was frozen over liquid nitrogen. Sections 7 to
10 µm thick were collected onto glass slides (SuperFrost Plus,
Fisher), air-dried for at least 1 hour, and used immediately or stored
at 4°C. Immunofluorescence labeling of heart sections was performed
with antibodies to Tmod (affinity purified rabbit anti-Tmod prepared
against bacterially expressed protein) and sarcomeric 
-actinin
(#A-7811, Sigma). Slides were rehydrated in PBS for 10 minutes, washed
in PBS containing 0.1 mol/L glycine for 5 minutes, rinsed briefly in
PBS, and then permeabilized in PBS containing 15% methanol for 15
minutes. After another brief rinse in PBS, sections were further
permeabilized for 20 minutes in PBS containing 0.5% Triton X-100,
washed twice in 3 minute PBS rinses, and blocked for 1 hour in PBS
containing 10% horse serum. Primary antibodies diluted in PBS
containing 10% horse serum were applied overnight at 4°C. The next
day, slides were washed 3 times in PBS for 10 minutes per wash, labeled
at room temperature in the dark with secondary antibodies diluted 1:100
in PBS (#711-095-152: anti-rabbit FITC-conjugate; #115-075-146:
anti-mouse Texas red conjugate, both from Jackson Immunoresearch, West
Grove, PA). After secondary labeling, slides were washed 3 times in PBS
for 5 minutes per wash and mounted for viewing in Vectashield medium
(Vector Labs, Burlingame, CA). Confocal images were acquired using a
Molecular Dynamics CLSM 2010 as previously described.7
Morphometric scans were taken using a 2x objective and quantitated
using Imagespace software (Molecular Dynamics).
TOT Tracking and Statistical Analysis
TOT population data were gathered and analyzed using Progeny v2.0 (Genetic Data Systems, Mishawaka, IN). Progeny combines the use of pedigree tree drawing with data entry and database relational analysis. Statistical analyses of litter survival were performed using SAS (SAS Institute, Cary, NC). Data were checked for completeness, and range and crosschecks were performed to find possible entry or transcription errors. Spearman correlation coefficients were calculated between days of survival and heart-to-body weight ratio and litter size. Subsequent analyses included analysis of covariance, where survival was the dependent variable and litter and litter size were the independent variables. This analysis was performed to account for the assumed similarity of mice from the same litter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunoblot analyses were performed to determine the accumulation
of Tmod protein during postnatal development (Figure 1)
. Protein lysates were separated by
SDS-PAGE (Figure 1A)
, transferred to nitrocellulose, and the region of
the blot corresponding to the Mr of Tmod was
excised from the blot for immunolabeling (Figure 1B)
. Tmod expression
in TOTs was estimated relative to control samples (Figure 1B
,
nontransgenic lanes) and quantitative analysis was standardized against
GAPDH (data not shown). Tmod level in control nontransgenic animals
remained constant throughout the time course investigated, so one
representative time point was shown for each experiment. Two separate
experiments (Figure 1A
, left- and right-hand gels) showed Tmod
expression was elevated over threefold at birth (Figure 1
, TOT, 0 days)
and showed a trend toward increasing expression up to almost sixfold
over the next 2.5 weeks (Figure 1
, TOT, 17 days). Comparable increases
in expression were found using the second control sample (Figure 1
,
nontransgenic, 2 days) compared with samples taken from TOTs ranging in
age (Figure 1
, TOT, 2–8 days).
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Activation of the calcium-regulated phosphatase calcineurin has
been associated with dilated cardiomyopathy in TOT
hearts.12
To quantify activated calcineurin during
postnatal development, immunoprecipitation was performed with
calmodulin-specific antibody followed by a calcineurin Western blot
(Figure 2A)
. Because activated
calcineurin is tightly associated with calmodulin, this precipitation
assay indicates the fraction of calcineurin in the activated state. The
absolute level of calcineurin and calmodulin proteins in cardiac
protein extracts was assessed by Western blot. Absolute levels of
calcineurin were increased at every time point assayed, while
calmodulin levels were invariant. Activated calcineurin was increased
1.9-fold at day 3, 7.7-fold at day 5, 4.0-fold at day 7, and 2.4-fold
at day 24. Absolute calcineurin protein levels were also up-regulated
1.6-fold at day 3, 2.4-fold at day 5, 2.6-fold at day 7, and 2.0-fold
at day 24. The increase in calcineurin protein and activated state from
days 3 through 7 before onset of dilation suggests a close association
with the initiation of TOT cardiomyopathy.
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. Total
cellular p38 MAPK level was comparable between TOTs and nontransgenic
controls and transient activation of p38 was present (Figure 2B
, upper
rows). Repeated studies of p38 activation during the postnatal period
showed these transient activity spikes vary in both intensity and
timing, occurring between postnatal days 7 and 10 and again on or after
postnatal day 13 (data not shown). Thus, p38 MAPK may participate in
normal postnatal cardiac hypertrophy, but the transient nature of
activation and intensity in the TOT samples suggests it is not a
primary effector of Tmod-induced cardiomyopathy. Analysis using
antibodies to additional hypertrophic pathway mediators ERK 1/ERK2 as
well as JNK showed no significant difference in levels of activated
protein between controls and transgenics (Figure 2B
, middle rows).
Control probing of the blot strips with antibody to GAPDH shows
equivalence of sample loadings between days and between control and TOT
samples (Figure 2B
, bottom rows). Increased Basal Level and Amplitude of Calcium Transients in TOT Cardiomyocytes
Activation of calcineurin suggested increased basal calcium level
in TOT cardiomyocytes. Intracellular calcium as determined by Fura-2
ratios in cardiomyocytes from TOTs was significantly increased 89%
(P < 0.0001) in basal (diastolic) level
compared to nontransgenic controls (Figure 3, A and B)
. The magnitude of this
increase is brought into context by noting basal calcium levels in TOT
cells are approximately equivalent to peak systolic calcium levels
(baseline + amplitude) in normal mice (1.7 versus 1.6,
respectively). Furthermore, the amplitude of the TOT transient calcium
transient is 128% greater than calcium transients in nontransgenic
cells (Figure 3C
, 1.6 versus 0.7, respectively;
P < 0.0001), raising peak systolic calcium level in
TOT cells twofold above nontransgenic levels (3.6 versus
1.6, respectively). The time to reach 80% reduction from peak calcium
level (T80) was significantly longer by 17% (P
= 0.043) in TOT cardiomyocytes, presumably due to the high systolic
calcium load (Figure 3D)
.
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The earliest observed clinical changes in TOTs symptomatic of
cardiomyopathic changes occur at least 10 days after
birth.7
Immunofluorescence analysis was performed to
examine Tmod distribution throughout the heart as well as myofibril
organization during the first 2.5 weeks after birth. Age-matched hearts
from control nontransgenics and TOTs were sectioned and labeled with
antibodies to Tmod and -actinin (Figure 4)
. TOT hearts showed intense Tmod
reactivity compared to nontransgenic controls throughout the 6 time
points sampled, ranging from 1 to 17 days after birth (Figure 4
, upper
rows). Overall, both control and TOT samples labeled uniformly
throughout the myocardium. Myofibril organization was subjectively
evaluated by using -actinin to label Z-disks followed by observation
of striation alignment and periodicity (Figure 4
, lower rows).
Sarcomeric periodicity in neonatal nontransgenics (Figure 4
, normal
fibrils, 1 and 4 days) was relatively poor, but showed progressive
improvement and clear striations at later time points (Figure 4
, 8, 11,
14, and 17 days). In comparison, myofibrils in neonatal TOTs and
nontransgenics appeared similar in degree of organization (Figure 4
, 1
and 4 days). By 8 days after birth, subtle indications of sarcomeric
dysgenesis such as poor alignment and wavy appearance were noted in the
TOT myofibrils (Figure 4
, Tg fibrils, 8 days). Progressive
deterioration of sarcomeric structure was evident in TOT sections
throughout the remainder of the time course (Figure 4
, Tg fibrils, 11,
14, and 17 days).
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where, by confocal
microscopy, myofibril integrity appeared comparable between control
nontransgenic and TOT myocardial samples. Control myofibrils at birth
showed slight curvature of Z-disk structure and myofibril alignment
(Figure 5, A and B)
, presumably due to the nascent assembly and
increased workload demands on contractile structure in the neonate.
Degenerative changes in TOT sarcomere organization were apparent at
birth relative to control nontransgenic samples. Although Z-disks were
present throughout the TOT heart sections, structures were often poorly
spaced (Figure 5C)
or unaligned (Figure 5D)
. Control myofibril
organization improved through postnatal days 3 (Figure 5E)
and 7
(Figure 5H)
of the experiment, unlike TOT myofibril organization, which
showed widening areas of Z-disk curvature and myofibril misalignment
(Figure 5, F, G, and I)
. The retention of Z-disk banding, albeit
compromised, probably accounts for why myofibril organization appeared
intact in confocal observations of early time points (Figure 4)
. The
higher resolution of electron microscopy allowed for visualization of
sarcomeric abnormalities that were not apparent at the limit of
confocal analyses.
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Actuarial data were collected from a population of 278 TOTs that
died of cardiomyopathic disease to determine trends in survival and
correlate the course of the disease with postnatal development. TOT
mortality was concentrated primarily within 6 weeks after birth (161
dead, 58% of the population), although some TOTs can live over 1 year
(Figure 6A)
. Examination of early
mortality from birth to 50 days of age showed two distinct peaks
coincident with stressful transitions in maturation of the young mouse
(Figure 6B)
. The first wave of increased mortality around 15 to 16 days
after birth (Figure 6B
, arrow 1) correlated with increased activity as
eyes opened and the mice explored their environment. The second peak of
increased mortality at 21 days after birth coincided with weaning age
(Figure 6B
, arrow 2). Mortality occurring between 30 and 50 days
(Figure 6B
, arrow 3) was associated with the development of anasarca,
which was evident in 50% of the mice that died during this time
(n = 11 of 22). Symptomatically, these edematous
and proptotic mice (Figure 7)
resemble
clinical anasarca related to secondary complications of poor heart
function reported in human cardiomyopathy,19
and mice
exhibiting this condition never survive. Collectively, one-half of the
TOT population died between 14 and 22 days after birth with losses at
days 14 and 21 accounting for 11 and 14% of all deaths, respectively.
Cumulative TOT population data indicated that survival showed a
significant association (P < 0.0001) with
litter size (Figure 8)
. Specifically,
smaller litters showed the highest mortality rate and average age at
death increased in correlation with a larger numbers of siblings. This
result is consistent with the interpretation that gestational and/or
postnatal environmental factors play a significant role in the
lethality of TOT cardiomyopathy.
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increased quickly after birth and remained high throughout
the lifetime of the mouse. A significant difference between TOT and
control nontransgenic ratios was apparent by 9 days after birth (Figure 6D
, at arrow showing bracketed region). However, increased TOT
heart:body weight ratio plateaued rapidly within 48 hours after birth
and did not change significantly throughout the study. In general,
disproportionate increases in TOT heart weight occur around 1.5 weeks
after birth, with most of the cardiac enlargement occurring in a very
short time of 2 to 3 days. The average heart:body weight ratio for TOTs
was 15.0 ± 4.0 mg/g compared to an average of 5.3 ± 0.2
mg/g for nontransgenic controls. Chamber Dilation without Increasing Wall Thickness in TOT Hearts
TOT hearts were measured to determine ventricular wall and septal
thickness as well as left ventricular chamber cross-sectional area.
Three time points of 8, 11, and 14 days were chosen to observe changes
before and after development of increased heart:body weight ratio
(Figure 6)
. Ventricular wall (Figure 9, A and C)
and septal thickness (Figure 9B)
remained comparable without
significant differences (P > 0.1) between
controls and TOTs throughout the time points investigated, consistent
with the lack of hypertrophic response previously
reported.6
Left ventricular chamber cross-sectional area
was consistently greater in TOTs than in nontransgenic controls (Figure 9D)
. The trend toward increasing left ventricular chamber size by day
11 (0.3 > P > 0.2) became significant by day 14
(P < 0.03).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. However, marked elevation of
Tmod expression at birth (Figure 1)
suggested initial stages of TOT
cardiomyopathy were underway by late gestation or early postnatal
development. Early postnatal irregularities in sarcomeric periodicity
evident by ultrastructural analysis (Figure 5)
progressed to widespread
loss of Z-disk structural integrity and myofibril organization,
apparent by confocal microscopy (Figure 4)
several days later.
Ultrastructural analysis before development of marked cardiomyopathic
changes showed the initial stages of pathogenesis before onset of
secondary compensatory effects, which complicate the interpretation of
transgene mechanism. Intracellular structural anomalies reveal the
immediate and direct effects of transgene expression, specifically that
neonatal expression of Tmod weakens sarcomere structure and compromises
myofibril alignment, eventually leading to gross pathology over 1 week
later. Based on knowledge of Tmod function in regulation of actin filament length from in vitro11,20,21 and in vivo7 studies, it is reasonable to postulate that myofibrillogenesis is impaired in the TOTs. Tmod binding to tropomyosin on the slow-growing (pointed) end of an actin filament inhibits polymerization.19 Normally present in low levels relative to actin, excess Tmod level dissociates tropomyosin from actin filaments.22 Because tropomyosin stabilizes actin filaments,23 Tmod-mediated uncoating renders actin filaments susceptible to depolymerization and breakdown. This scenario is consistent with the in vitro effect of Tmod overexpression in cardiomyocytes where decreased I-band width, sarcomere shortening, and myofibril degeneration were observed.11 Similar in vivo effects were noted after Tmod overexpression in the heart including loss of myofibril organization and thin filament disarray.7 Resultant degeneration of myofibril structure leads to impaired contractility typically associated with development of dilated cardiomyopathy. Underlying pathogenic causes of impaired contractility can be attributed to loss of myofibril integrity as in TOT,7 defective force transmission from the myofibril to myocardium as in muscle LIM protein (MLP)-deficient mice,24 or transcriptional down-regulation of unidentified target genes essential for cardiomyocyte function and/or viability as in dominant-negative cAMP response element binding protein (CREB)-overexpressing mice.25 All three lines share a similar phenotype, but the underlying causes are different: the first from overexpression of a sarcomeric component (Tmod), the second from deletion of a cytoskeletal protein component (MLP), and the third from overexpression of a mutated transcription factor (CREB). A condition similar to anasarca in TOTs, referred to as massive generalized edema, has been reported to occur in CREB-induced cardiomyopathy.25 Molecular mechanisms causing pathological changes in these transgenic lines are unrelated, but they are likely to impact on interdependent processes. Loss of myofibrils probably contributes to degeneration of the myocardial cytoskeleton and vice versa. Interdependence of myofibril formation and attachment via the cytoskeleton is observed in cardiomyocyte cultures where contractility stimulates myofibril organization26 and decreased tension results in myofibril breakdown and reorganization.27 Shared phenotypic characteristics between the TOT, CREB, and MLP mouse models will help to advance understanding of the pathogenesis of dilated cardiomyopathy. However, in comparison to CREB and MLP, a combination of biochemical, in vitro, and in vivo analyses have extensively defined the functional activity of Tmod. The apparently direct causal relationship between Tmod overexpression, myofibril degeneration, and loss of contractile function makes this an attractive model system to study the process of dilated cardiomyopathy development.
TOT cardiomyopathy was initially described as a "failed hypertrophic
response."7
However, TOT hearts show a significant
increase in heart:body weight ratio (Figure 6)
, which could be
interpreted as hypertrophic growth. Hypertrophic and dilated changes
can occur concurrently in cardiomyopathy, but TOT hearts show little
evidence of a hypertrophic phenotype. Five independent observations
point to a lack of hypertrophy in TOT hearts: i) no change in
expression of some genes associated with hypertrophy such as skeletal
actin,7
ii) activation of calcineurin signaling (a
powerful mediator of hypertrophy13
) fails to induce
typical hypertrophic changes, iii) ventricular wall thickening
typically associated with hypertrophy does not occur in TOT hearts
(Figure 9)
, iv) TOT cardiomyocytes are generally elongated without
increased width, and cardiomyocytes with increased width typical of
hypertrophic enlargement are never observed (Sussman, unpublished
observation), and v) TOT hearts progress directly to failure without
ever undergoing a transitory enhancement of contractile function, as is
sometimes observed with hypertrophic mouse models of cardiomyopathy.
The potential contribution of fluid retention in initial stages of
dilation was examined in postnatal day 12 TOTs, but dry weight
measurements indicate that contribution of fluid to heart:body weight
is comparable between postnatal day 12 TOTs and nontransgenic controls
(data not shown). Thus, we postulate that changes in TOTs arise from
cardiomyocyte elongation and possibly cell slippage, both processes
associated with increasing ventricular chamber volume characteristic of
dilation.
Because TOT cardiac morphology can be rescued by inhibition of
calcineurin without improving contractility or altering tropomodulin
level,12
TOT cardiac dilation is caused by secondary
compensatory mechanism(s) rather than a primary effect of tropomodulin
expression. However, the previous study did not establish a temporal
relationship between calcineurin activation and the onset of the
cardiomyopathic phenotype. In this report we demonstrate that
calcineurin protein levels and activation state are significantly
increased as early as 3 days after birth (Figure 2)
. Increased
expression and activation of calcineurin was maintained throughout the
progressive development phase of TOT cardiomyopathy, implicating
calcineurin as the regulator of a secondary reactive response that
leads to the dilated phenotype. This idea is supported by the
demonstration of substantially elevated diastolic and peak systolic
calcium levels in TOT cardiomyocytes (Figure 3)
. Increased calcium
levels coupled with calcineurin activation in TOT cardiomyocytes
suggests the following mechanism of cardiomyopathic pathogenesis:
altered sarcomeric architecture caused by tropomodulin overexpression
leads to compensatory increases in intracellular calcium to enhance
contractility, and chronic elevation of calcium levels activate the
calcium-sensitive phosphatase calcineurin. This hypothesis is
consistent with our previous report that calcineurin inhibitory drugs
(cyclosporin and FK506) prevent the initiation and progression of
cardiac dilation, which is characteristic of TOT hearts.12
Collectively, these findings correlate with other models of heart
disease where alterations of sarcomeric structure/function are
associated with altered calcium dynamics.28-30
However,
the enhanced basal and systolic calcium level coupled to the heart
failure phenotype of TOTs is without precedent. Although calcium level
determinations were performed on 13-week-old TOTs, it is reasonable to
expect a similar rise in calcium levels within cardiomyocytes of
younger TOTs. Calcium-dependent calcineurin activation in neonatal mice
(Figure 2)
serves as circumstantial evidence that TOT cardiomyocytes
possess elevated calcium levels within the first week after birth. The
13-week time point was chosen for calcium ratio imaging to maintain
consistency with previously published studies16
and
provide a high yield of viable cells. Technical considerations of the
collagenase perfusion prevent the use of hearts from neonatal animals
but, by confocal microscopy, the extent of myofibril degeneration
observed between individual cardiomyocytes of young (2–3 weeks) and
old (24–30 weeks) TOT mice are indistinguishable (data not shown).
Thus, at least from a structural level, there is no reason to
anticipate that cardiomyocytes from older animals would differ
significantly from those of younger mice. Pilot experiments using
cardiomyocytes from 4-week-old nontransgenic controls and TOTs showed
substantial heterogeneity of calcium handling between cells within each
preparation, perhaps as a consequence of ongoing postnatal
developmental growth. As techniques to assess intracellular calcium
levels improve, it will be interesting to examine intracellular calcium
levels in relation to neonatal growth and TOT pathogenesis. At present,
the circumstantial evidence of calcineurin activation (Figure 2)
together with calcium measurements from isolated cardiomyocytes is
consistent with a syndrome of myofibril dysgenesis leading to
compensatory calcium increases. This combination of conditions in TOTs
offers a unique paradigm to assess the mechanism underlying altered
calcium handling in dilated cardiomyopathy.
The notion that calcineurin acts as a primary regulatory pathway for
initiation of compensatory cardiomyopathic changes in TOTs is further
supported by the analysis of MAPK signaling factors. The three main
branches of the MAPK signaling cascade (p38, ERK1/2, and JNK1/2) were
analyzed during the initiation and progression of TOT cardiac dilation
and no significant association between any MAPK signaling factors and
disease was found. p38 activation apparently participates in normal
postnatal cardiac growth as seen in nontransgenic controls (Figure 2B)
,
possibly as a mediator of cardiac hypertrophy.18
Variation
in the timing and intensity of p38 activation is presumably due to
differences in maturational rate of the mice and development of the
myocardium. Overall, MAPK signaling factors are not associated with the
reactive dilated response that is characteristic of TOT hearts.
Participation of calcineurin signaling in TOT pathogenesis together
with increased calcium levels in TOT cardiomyocytes (Figure 3)
indicate
a primary role for calcium signaling pathways in the etiology of TOT
disease. Presumably, rising calcium level results from decreased
calcium uptake by the sarcoplasmic reticulum during relaxation, as
observed in the failing heart.33
Abnormal regulation of
calcium currents in cardiac hypertrophy and failure leads to
prolongation of the action potential, altering both contractile and
relaxation properties.34
As a consequence, altered
expression of many calcium transport and regulatory proteins are
observed in human and experimental mouse cardiomyopathies. For example,
sarco/endoplasmic reticulum Ca2+-ATPase mRNA expression was
significantly depressed in samples from 3-week-old TOTs.7
Altering calcium level is one mechanism to regulate inotropy, so it is
reasonable to speculate that calcium levels initially rise as a
compensatory effort to increase contractility in the TOT heart.
Activation of calcineurin within 5 days after birth suggests that
elevation of calcium levels occurs early in TOT pathogenesis before
marked changes are apparent in cardiac morphology. Subsequent
deterioration of myofibril organization and altered expression of
calcium handling proteins would provoke further elevation of
intracellular calcium. Interestingly, pathologically high calcium
levels may be necessary for TOT survival: breeding TOTs into a
phospholamban-null background, which should enhance calcium reuptake
into the sarcoplasmic reticulum, dramatically increased TOT mortality
(Delling and Molkentin, unpublished observation). Rising calcium within
TOT cardiomyocytes is likely to have consequences for multiple
calcium-dependent signal transduction pathways in addition to
calcineurin, including selected protein kinase C
isoforms35
and tyrosine kinases such as
pyk2.36
Preliminary results show pyk2 activation in TOT
hearts, which probably exacerbates loss of force transmission from
cardiomyocytes to the myocardium by affecting focal adhesion contact
organization (data not shown).
The contributory effect of normal cardiac development to postnatal cardiomyopathy should be considered when describing phenotypes, especially in view of the significant number of mouse models exhibiting pathology within the first month after birth.7,13,31,32 Rapidly changing demands on the heart at birth37,38 coupled with postnatal cardiac hyperplasia and hypertrophy39,40 require cardiac adaptation involving processes similar to those observed in conjunction with cardiomyopathy. Typically, increased heart weight and cell volume, altered myofibril organization and contractility, and chamber dilatation are considered characteristic cardiomyopathic changes when occurring late in life. The resilient postnatal heart can accommodate these normal developmental stresses. However, the TOT heart with impaired myofibrillogenesis is presumably unable to meet demands of normal postnatal hypertrophy, leading to a cyclic amplification of hypertrophic signaling, which is maladaptive and leads to decompensation. The quiescent period of comparable heart:body weight ratios between nontransgenic and TOTs through 9 days after birth was followed by a rapid onset of increased heart weight occurring within 48 hours, after which the elevated heart weight:body weight ratio was maintained at a consistently high level. The swift cardiac adaptive response and prolonged plateau of enlargement indicates that the TOT heart rapidly compensated for loss of contractility up to an inherent limit in the progression of cardiac dilation.
Initially, extended longitudinal studies of TOTs were hampered by high
mortality rates within 1 month after birth.7
However, a
small percentage of severely affected mice survived. The TOT population
was increased by breeding surviving mice, resulting in uniformly
affected homozygous litters. With the breeding program established, the
longitudinal study of TOT pathogenesis was started. Using the TOT
relational database for analysis of phenotypic trends, developmental
aspects of TOT cardiomyopathy became apparent, and early mortality at 2
weeks after birth narrowed the window for onset of TOT pathogenesis to
neonatal life. Variable mortality between genetically identical
siblings suggested environmental factors influenced TOT survival beyond
the critical 1-month period. The hypothesis of postnatal litter size
influencing survival in TOTs can be tested by removing some siblings at
birth to decrease the number of offspring. The association between TOT
survival and larger litter size (Figure 8)
may stem from developmental
retardation and slower maturation of mice reared in large litters
compared to small litters,41,42
allowing postnatal cardiac
hypertrophy to progress more gradually. This survival analysis is, to
our knowledge, the first demonstration of an environmental factor
impacting on survival in a mouse cardiomyopathic model.
Hallmarks of dilated cardiomyopathy such as poor systolic performance and increased ventricular chamber cavity8-10 are seen in several cardiomyopathic transgenic lines7,24,25 but the unique combination of features in TOTs offers a useful paradigm for understanding dilated cardiomyopathy. First, TOTs suffer from a thin filament-based sarcomeric disease, a myofibril component now receiving more attention due the discovery of actin-based familial cardiomyopathy.43 Second, TOTs exhibit dilation in the absence of the significant hypertrophic response and fibrosis often seen in transgenic cardiomyopathies.13,31 Third, the myofibril disarray and lack of ventricular wall thickening found in TOTs is reminiscent of histological reports describing some human dilated cardiomyopathies.8-10,44-46 Fourth, circumstantial evidence from genetic studies suggests Tmod as a candidate gene for human cardiomyopathy.47 Fifth, extensive molecular and biochemical characterizations defining the interaction between Tmod, tropomyosin, and actin provide a clear mechanism of action for Tmod overexpression in vivo. Last, but not least, TOT cardiomyopathy involves presence of the transgene together with compensatory failed hypertrophy, a combinatorial mechanism leading to disease which has not been addressed in previous studies and may be relevant to human cardiomyopathic disease. To elucidate how hypertrophy contributes to pathogenesis and identify molecular mechanism(s) involved in the TOT paradigm, cross-breeding of TOTs to other transgenic lines with altered postnatal cardiac development is underway.
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Acknowledgements |
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Footnotes |
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Supported by a grant-in-aid from the American Heart Association Ohio Affiliate, a Scientist Development Grant (9630047N) and a grant-in-aid (9750638N) from the American Heart Association National, and a National Institutes of Health R29 award (HL58224–01).
Accepted for publication August 24, 1999.
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-tropomyosin in hearts of transgenic mice indices changes in thin filament response to Ca2+, strong cross-bridge binding, and protein phosphorylation. J Biol Chem 1996, 271:11611-11614This article has been cited by other articles:
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  S. Siddiqi, N. Gude, T. Hosoda, J. Muraski, M. Rubio, G. Emmanuel, J. Fransioli, S. Vitale, C. Parolin, D. D'Amario, et al. Myocardial Induction of Nucleostemin in Response to Postnatal Growth and Pathological Challenge Circ. Res., July 3, 2008; 103(1): 89 - 97. [Abstract] [Full Text] [PDF]
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 J. Melendez, C. Turner, H. Avraham, S. F. Steinberg, E. Schaefer, and M. A. Sussman Cardiomyocyte Apoptosis Triggered by RAFTK/pyk2 via Src Kinase Is Antagonized by Paxillin J. Biol. Chem., December 17, 2004; 279(51): 53516 - 53523. [Abstract] [Full Text] [PDF]
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 A. M Janczewski, M. Zahid, B. H Lemster, C. S Frye, G. Gibson, Y. Higuchi, E. G Kranias, A. M Feldman, and C. F McTiernan Phospholamban gene ablation improves calcium transients but not cardiac function in a heart failure model Cardiovasc Res, June 1, 2004; 62(3): 468 - 480. [Abstract] [Full Text] [PDF]
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 E. C. Goldsmith, W. Carver, A. McFadden, J. G. Goldsmith, R. L. Price, M. Sussman, B. |
