Download The Young Mouse Heart Is Composed of Myocytes Heterogeneous

The Young Mouse Heart Is Composed of Myocytes
Heterogeneous in Age and Function
Marcello Rota, Toru Hosoda, Antonella De Angelis, Michael L. Arcarese, Grazia Esposito,
Roberto Rizzi, Jochen Tillmanns, Derin Tugal, Ezio Musso, Ornella Rimoldi, Claudia Bearzi,
Konrad Urbanek, Piero Anversa, Annarosa Leri, Jan Kajstura
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Abstract—The recognition that the adult heart continuously renews its myocyte compartment raises the possibility that the
age and lifespan of myocytes does not coincide with the age and lifespan of the organ and organism. If this were the
case, myocyte turnover would result at any age in a myocardium composed by a heterogeneous population of
parenchymal cells which are structurally integrated but may contribute differently to myocardial performance. To test
this hypothesis, left ventricular myocytes were isolated from mice at 3 months of age and the contractile, electrical, and
calcium cycling characteristics of these cells were determined together with the expression of the senescence-associated
protein p16INK4a and telomere length. The heart was characterized by the coexistence of young, aged, and senescent
myocytes. Old nonreplicating, p16INK4a-positive, hypertrophied myocytes with severe telomeric shortening were present
together with young, dividing, p16INK4a-negative, small myocytes with long telomeres. A class of myocytes with
intermediate properties was also found. Physiologically, evidence was obtained in favor of the critical role that action
potential (AP) duration and ICaL play in potentiating Ca2⫹ cycling and the mechanical behavior of young myocytes or
in decreasing Ca2⫹ transients and the performance of senescent hypertrophied cells. The characteristics of the AP
appeared to be modulated by the transient outward K⫹ current Ito which was influenced by the different expression of
the K⫹ channels subunits. Collectively, these observations at the physiological and structural cellular level document
that by necessity the heart has to constantly repopulate its myocyte compartment to replace senescent poorly contracting
myocytes with younger more efficient cells. Thus, cardiac homeostasis and myocyte turnover regulate cardiac function.
(Circ Res. 2007;101:387-399.)
Key Words: action potential profile 䡲 excitation-contraction coupling 䡲 myocyte volume 䡲 telomere length
䡲 senescence-associated proteins
T
turnover of myocytes results in a heterogeneous population of
parenchymal cells which are structurally integrated but may
contribute differently to myocardial performance. If this were
the case, senescent cells with alterations in the mechanical,
electrical, and calcium cycling properties4 may coexist with
newly formed young myocytes with enhanced contractile
behavior.5
Data in humans and animals suggest that myocyte maturation and aging6 – 8 are characterized by loss of replicative
potential, telomeric shortening, and the expression of the
age-associated protein p16INK4a. The acquisition of the senescent phenotype involves a progressive increase in the size of
the cell that reaches a critical volume beyond which cellular
hypertrophy is no longer possible.6 In this study we have
addressed the question whether myocyte aging is only partly
related to the age of the organ and organism by testing
whether young and old cells are present in the mouse heart at
he recent identification of a population of resident
progenitor cells that controls cardiomyogenesis imposes
a reinterpretation of the fundamental mechanisms of growth
and senescence of the heart.1 Traditionally, the heart was
viewed as an organ characterized by a predetermined number
of myocytes which is defined shortly after birth and is
preserved throughout life. However, BrdU administration has
documented that myocyte renewal occurs in the adult heart
and that the rate of cell regeneration is slow, heterogeneous,
and involves only a small percentage of cells at any given
time.2,3 In mammals, ventricular myocytes are replaced several times throughout life.2,3 These findings were obtained by
pulse-chase BrdU labeling assay2 and by the rate of growth
and commitment of cardiac progenitor cells.3 Collectively,
they challenge the notion that the age and lifespan of
myocytes coincide with the age and lifespan of the organ and
organism. According to the new paradigm, the continuous
Original received February 28, 2007; revision received June 13, 2007; accepted June 20, 2007.
From the Cardiovascular Research Institute (M.R., T.H., A.D.A., M.L.A., G.E., R.R., J.T., D.T., E.M., C.B., K.U., P.A., A.L., J.K.), Department of
Medicine, New York Medical College, Valhalla; and the MRC Clinical Sciences Centre (O.R.), Faculty of Medicine, Imperial College School of
Medicine, London, UK.
Correspondence to Marcello Rota, PhD, Cardiovascular Research Institute, Vosburgh Pavilion, Room 302, New York Medical College, Valhalla, New
York 10595. E-mail [email protected]
© 2007 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.107.151449
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August 17, 2007
3 months of age and whether this nonuniform cell population
is functionally heterogeneous. This possibility would
strengthen the notion of the dynamism of the heart that is
regulated by a pool of progenitor cells which dictate cell
turnover, organ homeostasis, and aging.
Materials and Methods
Myocytes were enzymatically dissociated from the heart of C57Bl6
mice. The expression of the senescence-associated protein p16INK4a
and telomere length were determined together with the electrical and
mechanical properties of the isolated myocytes. Additionally, the
expression of Kv1.2, Kv4.2 and Kv4.3 in myocytes of different size
was determined by real-time RT-PCR and immunocytochemistry.
An expanded Materials and Methods section can be found in the
online data supplement available at http://circres.ahajournals.org.
Results
INK4a
Myocyte Aging: p16
Cell Volume
, Telomere Length, and
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Left ventricular (LV) myocytes were enzymatically dissociated and the expression of p16INK4a was determined by
immunocytochemistry (Figure 1A), the length of telomeres
was evaluated by Q-FISH (Figure 1B), and myocyte volume
was assessed by confocal microscopy (Figure 1C). In mice at
3 months of age, mononucleated, binucleated and multinucleated myocytes represented 6.0⫾0.74%, 91⫾0.62%,
3.0⫾0.71%, respectively. The increase in number of nuclei
was paralleled by an increase in average cell volume (supplemental Figure IA) and a shift to the right in the myocyte
volume distribution (supplemental Figure IB). Myocytes of
different size coexisted in distinct anatomical regions (supplemental Figure IC). These data are consistent with previous
results supporting the notion that under physiological conditions local stresses do not affect significantly myocyte volume across the LV wall.9
The analysis of myocytes was restricted to the LV base and
midregion; the apex was excluded. Mononucleated myocytes
included a subset of replicating Ki67- and BrdU-positive cells
together with dividing myocytes (Figure 2A and 2B). The
recognition that small, mononucleated myocytes proliferate
and differentiate until the acquisition of the terminally differentiated phenotype3 is in agreement with recent results in the
postnatal and adult overloaded heart.6,10 The cell cycle
inhibitor p16INK4a is a reliable marker of irreversible growth
arrest and cellular senescence.8 Apoptosis of myocytes and
cardiac progenitor cells (CPCs) in animals and humans is
restricted to p16INK4a-positive cells which do not express
telomerase and typically show severe telomere shortening.6,7,11 The percentage of senescent myocytes increased
dramatically in the larger binucleated and multinucleated
cells (Figure 2C).
Lineage commitment of CPCs to cardiomyocytes involves
the generation of progenitors, precursors, and amplifying
cells6 which divide until the adult phenotype is reached.
Telomere shortening occurs in developing and mature myocytes; it reflects the replicative history of the cell together
with a time-dependent accumulation of oxidative DNA damage.12,13 Telomere attrition promotes the expression of
p16INK4a providing a mechanistic link between these 2 indices
of cellular senescence which are both related to myocyte
volume.6,8 A distribution plot showed an inverse correlation
between telomere length and cell volume (Figure 2D). Telomere length varied from 14 to 58 kbp and large old myocytes
possessed very short telomeres consistent with their senescent
phenotype. When cells were separated in 3 classes, ⬍20 000,
20 000 to 40 000, and ⬎40 000 ␮m3, telomere length was
40⫾0.73, 33⫾0.82, and 25⫾0.36 kbp, respectively (Figure
2E). In comparison with myocytes ⬍20 000 ␮m3, the fraction
of apoptotic myocytes increased ⬇5-fold and ⬇15-fold in
cells 20 to 40 000 and ⬎40 000 ␮m3, respectively (Figure
2F). Thus, the young adult mouse heart contains a heterogeneous population of myocytes of different age and cellular
aging involves an increase in cell volume, telomeric shortening, p16INK4a expression, and apoptosis.
Myocyte Aging: Cell Mechanics and
Ca2ⴙ Handling
The documentation that senescent and nonsenescent myocytes are present in the young adult mouse heart raised the
question whether structural heterogeneity is paralleled by a
similar variability in mechanical performance. Comparable
results at the physiological level would support the notion
that the heart is constantly repopulating its myocyte compartment to preserve cardiac function. Therefore, myocytes were
field-stimulated at 1 Hz and cell and sarcomere shortening
were measured. Increases in myocyte volume were coupled
with decline in contractile behavior. Bivariate distribution of
parameters of cell contractility and volume were fitted with
straight lines. Cell shortening, maximal rate of contraction
(dL/dt), and relaxation (⫹dL/dt) decreased, respectively,
0.32⫾0.058 ␮m, 5.91⫾1.20 ␮m/sec and 5.43⫾0.92 ␮m/sec
per 10 000 ␮m3 increase in volume (Figure 3A and 3B).
Relaxation was also prolonged in large myocytes but timeto-peak-shortening was similar in all cells.
The question was whether myocyte shortening, p16INK4a
expression, and telomere length could be concurrently evaluated in the same cells to provide a cause-and-effect relationship among these variables. To facilitate our task, mononucleated, binucleated, and multinucleated myocytes were
studied. Large multinucleated myocytes were p16INK4apositive, had short telomeres, and showed a severe depression
in myocyte shortening and relengthening (Figure 3C). This
complex protocol requires measurements in living cells and
subsequently in fixed preparations; 16 myocytes were evaluated in this manner. These results support the notion that
cellular aging dictated by p16INK4a and shortened telomeres is
characterized by attenuation in myocyte mechanical behavior.
To define the mechanisms of depressed myocyte performance with cellular hypertrophy-aging, intracellular Ca2⫹ and
sarcomere shortening were analyzed. Both parameters decreased with increases in myocyte volume (Figure 3D and
3E). Diastolic sarcomeric length was not affected by cell size
and averaged 1.720⫾0.003 ␮m (Figure 3E). The relative
increase of Ca2⫹ amplitude in small-young myocytes was
associated with a prolongation of time to 50% Ca2⫹ transients.
However, the timing of Ca2⫹ decay was comparable in all
cells (not shown). The Ca2⫹ present in the SR was then
measured by the rapid addition of caffeine. The amount of
Ca2⫹ in the SR was similar in cells of different size (Figure
Rota et al
Myocyte Heterogeneity
389
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Figure 1. Myocyte characteristics. A, p16INK4a (yellow,
arrows) in a binucleated and tetranucleated myocyte (␣
-sarcomeric-actin, red). Nuclei (PI, blue). B, Telomeres
(white) in a mononucleated, binucleated, and trinucleatedmyocyte. C, Three-dimensional optical section reconstruction by confocal-microscopy of mononucleated, binucleated, trinucleated, and tetranucleated myocytes.
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Myocyte Aging: ICaL and Excitation-Contraction
(EC) Coupling
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The recognition that increases in myocyte volume were
associated with a decrease in Ca2⫹ transients in the absence of
changes in Ca2⫹ load of the SR raised the possibility that Ca2⫹
current (ICaL) or its efficacy to activate the ryanodine receptors
(RyRs) was impaired in senescent myocytes.14 The release of
Ca2⫹ from the SR is triggered by the influx of Ca2⫹ from the
extracellular space through L-type channels. However, the
ability of ICaL to promote Ca2⫹ release from the SR, ie,
excitation-contraction coupling gain (ECC gain), may be
altered by spatial remodeling of ECC elements and/or open
probability of the RyRs.
Patch-clamp in voltage-clamp mode was used to measure
Ca2⫹ current in myocytes5,7,15 ⬍20 000, 20 000 to 40 000, and
⬎40 000 ␮m3. These myocytes had membrane capacitance
(Cm) ⬍98, 98 to 164, and ⬎164 pF, respectively (supplemental Figure II). Depolarizing steps showed that ICaL density and
current-voltage relations did not differ in small-young and
hypertrophied-old myocytes (Figure 4A and 4B). ICaL
activation-curve and half-maximal-activation-potential were
comparable in all myocytes. However, in small-young myocytes, ICaL inactivation curve was shifted to the right (Figure
4B). Fast and slow time constants of ICaL inactivation time
course were similar in the myocyte categories.
Subsequently, ICaL and Ca2⫹ transients were recorded simultaneously in voltage-clamped cells. Depolarizing steps
activated similar Ca2⫹ currents and led to comparable Ca2⫹
transients in all myocytes (Figure 4C and 4D). The ratio of
Ca2⫹ transient and ICaL amplitude, ie, ECC gain, was similar in
the 3 myocyte classes (Figure 4E). Thus, ICaL and ECC gain
are not affected by myocyte volume or aging and thereby do
not account for the decrease in SR Ca2⫹ release seen in
enlarged-old myocytes.
Myocyte Aging: Action Potential
Figure 2. Myocyte formation and senescence. A, Ki67 (green)
and BrdU (white) in mononucleated myocytes. B, Metaphase
chromosomes (upper-panels, DAPI, blue) in mononucleated
myocytes (lower-panels; phospho-H3⫽green). C, p16INK4apositive myocytes (MONO, n⫽115; BI, n⫽1501; MULTI, n⫽46).
D, Distribution of telomere length vs myocyte volume (n⫽512)
was fitted with a straight line (r2⫽0.387; P⬍0.0001). E, Telomere
length in myocytes ⬍20 000 (n⫽182), 20 to 40 000 (n⫽109), and
⬎40 000 ␮m3 (n⫽221). F, Apoptosis in myocytes ⬍20 000
(n⫽297⫻103), 20 to 40 000 (n⫽259⫻103), and ⬎40 000 ␮m3
(n⫽242⫻103). *P⬍0.05 vs ⬍20 000; **P⬍0.05 vs 20 to 40 000.
3F) suggesting that defects in the fractional release of Ca2⫹
from the SR developed in enlarged-old myocytes. Thus, the
young adult mouse heart contains a population of
hypertrophied-senescent myocytes with depressed contractile
behavior possibly mediated by alterations in Ca2⫹ handling.
Lack of alterations in ICaL and ECC gain pointed to changes
in action potential (AP) duration as a mechanism for the
decrease in Ca 2 ⫹ transients and shortening in
hypertrophied-old myocytes.16,17 The inotropic effect induced by AP prolongation is mediated by changes in
transmembrane Ca2⫹ influx enhancing Ca2⫹-induced Ca2⫹
release from the SR.14,16 At 1 Hz, an inverse relationship
was found between AP duration and myocyte volume
(Figure 5A). In comparison with AP in myocytes
⬍20 000 ␮m3, 90% repolarization time was 30% and 55%
shorter in myocytes 20 000 to 40 000 and ⬎40 000 ␮m3,
respectively (Figure 5B). Similar results were obtained at
10%, 30%, 50%, and 70% repolarization.
The electromechanical properties of endocardial and epicardial myocytes were evaluated in a cell subset to determine
whether the distinct anatomical localization of these cells was
partly responsible for the results discussed above. Shorter
APs and declined contractility were detected in myocytes of
increasing volume independently from their regional distribution (supplemental Figure III). Thus, the heterogeneity in
myocyte performance and electrophysiology appears to affect
all areas of the young-adult heart.
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391
To confirm that decreases in AP duration of large myocytes were a consequence of cellular aging, the electrical
properties of 1-day-old neonatal mouse myocytes were determined (Figure 5C). The AP was significantly prolonged in
these myocytes supporting the notion that cellular aging is
coupled with faster repolarization. Therefore, changes in the
profile of the AP may promote alterations in Ca2⫹ handling
and contractile performance of hypertrophied-senescent
myocytes.
Myocyte Aging: Transient Outward Kⴙ Current
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The documentation that shorter APs are present in old
myocytes suggested that changes in transmembrane ionic
currents may occur with cellular aging. The transient outward
K⫹ current Ito is responsible for the early repolarization phase
of the AP.18 Ito expression varies with ventricular loading and
hypertrophy,17,19 raising the possibility that changes in Ito alter
the electrical properties of enlarged-senescent myocytes. Ito
density was positively correlated with myocyte volume (Figure 6A and 6B).
To confirm that the expression of K⫹ channel mRNAs
changed as a function of cell volume, the K⫹ channel subunits
Kv1.4, Kv4.2, and Kv4.3 were measured by real-time RTPCR in small and large myocytes.10 The expression of Kv1.4
was lower in large than in small cells while Kv4.2 and Kv4.3
were more abundant in hypertrophied myocytes (Figure 6C
through 6E). These results at the mRNA level were accompanied by a similar difference in K⫹ channel protein for
Kv4.3 detected by immunocytochemistry (Figure 6F). Thus,
the expression of K⫹ channel subunits in combination with Ito
current density conditions AP duration in small-young and
hypertrophied-old myocytes.
Myocyte Aging: Action Potential and
Developed Ca2ⴙ
To establish whether the AP profile was the major determinant of depressed developed-Ca2⫹ in hypertrophied-senescent
cells, AP-clamp studies were performed together with measurements of Ca2⫹ transients. Action potentials with slow
(APslow), intermediate (APintermediate), and fast (APfast) repolarizing phase were recorded from myocytes with Cm 25, 112, and
207 pF, which corresponded to cells of ⬍20 000, 20 000 to
40 000, and ⬎40 000 ␮m3, respectively (Figure 7A). Subsequently, myocytes from adult and neonatal hearts were loaded
with Fluo-3 and APslow, APintermediate, and APfast waveforms were
imposed as voltage-clamp command (AP-clamp mode) to
evaluate their effects on Ca2⫹ transients. Ca2⫹ amplitude
varied as a function of the waveforms and these changes were
Figure 3. Myocyte mechanics and Ca2⫹ cycling. A, Shorteningtracings. B-F, Bivariate distribution of physiological-parameters
versus myocyte-volume (B, E, F). B: cell-shortening (r2⫽0.0973;
P⬍0.0001), maximal rate of contraction (r2⫽0.0802; P⬍0.0001)
and relaxation (r2⫽0.1104; P⬍0.0001), time-to-peak shortening
(r2⫽0.00912; P⫽0.11) and time to 90% relaxation (r2⫽0.11954;
P⬍0.0001); n⫽281. C, Shortening-tracings in MONO, BI, and
MULTI myocytes; BI and MULTI myocytes are p16INK4a-positive
(yellow) and have short telomeres. D, Ca2⫹ transients and sarcomere shortening. E, Sarcomere shortening (r2⫽0.0311; P⬍0.05;
n⫽133), diastolic sarcomere length (r2⫽0.004; P⫽0.26; n⫽271),
Ca2⫹ amplitude (r2⫽0.1452; P⬍0.0001; n⫽133) and timing of
Ca2⫹ peak (r2⫽0.0410; P⬍0.05; n⫽133). F, Ca2⫹ transient without (r2⫽0.1347; P⬍0.05) and with (r2⫽0.0267; P⫽0.36) caffeine
and computed Ca2⫹ fractional release (r2⫽0.2212; P⬍0.05);
n⫽34.
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Figure 5. AP-duration. A, APs of myocytes. B, AP-repolarization
in myocytes ⬍20 000 (n⫽14), 20 to 40 000 (n⫽46), and
⬎40 000 ␮m3 (n⫽20). *P⬍0.05 vs ⬍20 000; **P⬍0.05 vs 20 to
40 000. C, AP in neonatal mouse myocyte and AP-repolarization
in neonatal (n⫽18) and adult (n⫽80) myocytes. *P⬍0.05 vs
neonatal-myocytes.
independent from myocyte volume (Figure 7A). In comparison with APslow waveforms, APintermediate and APfast waveforms
led to a 33% and 42% decrease in developed Ca2⫹, respectively (Figure 7B).
Consistent with results in rodents,17,20 the decrease in Ca2⫹
amplitude mediated by fast repolarization was paralleled by
attenuation in Ca2⫹ influx (Figure 7C). To strengthen the role
that repolarization has in myocyte function, cells of different
size were perfused with 4-aminopyridine which blocks Ito and
prolongs the AP21; 4-aminopyridine had a profound effect on
large myocytes and a smaller impact on small cells. In the
former, 4-aminopyridine showed a marked increase in Ca2⫹
transients together with enhanced sarcomere shortening (Figure 7D and 7E). Blockade of Ito reestablished the physiological properties of old-hypertrophied myocytes by restoring in
part Ca2⫹ cycling and contractility (supplemental Figure IV).
Thus, the AP profile modulates Ca2⫹ transients and is implicated in the decrease of Ca2⫹ release from the SR with cellular
hypertrophy and aging. This defect is reversed by prolongation of the AP.
Myocyte Properties in the Senescent Mouse Heart
Figure 4. L-type Ca2⫹ current and ECC gain. A, ICaL in myocytes. B, ICaL in myocytes ⬍20 000 (n⫽26 and 28), 20 to 40 000
(n⫽22 and 25), and ⬎40 000 ␮m3 (n⫽10 and 10). Results are
mean⫾SEM. *P⬍0.05 vs ⬍20 000. C, ICaL and Ca2⫹ transients.
D, Voltage relations for ICaL and Ca2⫹-transients in myocytes
⬍20 000 (n⫽24), 20 to 40 000 (n⫽21), and ⬎40 000 ␮m3
(n⫽30). E, ECC-gain.
The recognition that young and old myocytes with distinct
mechanical and electrical properties are present in the developing heart raised the question whether chronological myocardial aging is characterized similarly by a pool of heterogeneous myocytes with different functional behavior. LV
myocytes were obtained from mice at 22 months of age and
analyzed. The senescent mouse heart contained myocytes
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which varied in volume from 1600 to 120 000 ␮m3 (supplemental Figure V). Therefore, small and large myocytes were
present in the old heart mimicking the observations in young
animals. With respect to the young heart, however, chronological age resulted in a shift to the right in the distribution of
myocyte volume. Consistent with previous results,7 the percentage of cells positive for p16INK4a and with markedly
shortened telomeres increased with chronological age (Figure
8A through 8C). Clusters of myocytes at both extremes of the
spectrum were present in the young and old heart (Figures 2C
through 2E and 8A through 8C; supplemental Figure V).
Heterogeneity in the myocyte characteristics persisted with
age although a change in the relative proportion of young and
old cells varied in the developing and senescent heart.
Measurements of sarcomere shortening, Ca2⫹ transients
and AP duration in myocytes collected from old hearts
decreased progressively with the enlargement of the cells
(Figure 8D and 8E). Conversely, transient outward K⫹ current
Ito was linearly related to myocyte volume (Figure 8F). As
anticipated, small myocytes with prolonged AP showed
enhanced contractile performance, paralleling the observations in the young heart (Figures 3A through 3E, 5A through
5C, 6A, and 6B; supplemental Figure IIIB). When myocytes
from young and old hearts were compared, chronological age
was characterized by a consistent prolongation of the AP in
cells of all classes although this difference was not statistically significant. This phenomenon suggests that a change in
the AP of myocytes in the senescent heart tends to compensate for the attenuation in the biophysical parameters late in
life in this model.
Discussion
The myocyte compartment of the young adult mouse heart
is heterogeneous and characterized by the coexistence of
cells of different age. Old myocytes are unable to replicate,
express aging-associated proteins, and show a significant
degree of cellular hypertrophy. Cellular aging is accompanied by a progressive increase in volume of myocytes
which together with the expression of p16INK4a and telomeric shortening document the complex dynamism of the
adult heart. The recognition that senescent and nonsenescent myocytes are present in the mouse heart at 3 months
of age supports the notion that these cells do not have a
common origin and are the product of a time-dependent
activation and differentiation of distinct populations of
progenitor cells. Functionally, cellular aging leads to
alterations in the electromechanical properties of myocytes
which in combination with abnormalities in Ca2⫹ handling
negatively affect contractile performance. Collectively,
these data document that by necessity the heart constantly
repopulates its myocyte compartment to replace senescent
poorly-contracting dying myocytes with younger more
efficient cells. Thus, cardiac homeostasis and myocyte
turnover regulate cardiac function and organ aging. Although similarities have been found between agedmyocytes in the young and old mouse heart, in the latter
case senescent cells are exposed to additional stresses
dictated by defects in large conductive arteries together
with humoral changes and cardiovascular diseases which
Myocyte Heterogeneity
393
occur with chronological age. These variables point to the
complexity of organ and organism aging and the difficulty
to discern the causative role of individual factors in
myocyte aging.
Myocyte Aging
So far, chronological age has formed the premise of multiple
studies in rodents and large mammals including humans.4
Comparisons have been made among groups of animals or
individuals of different age and aging effects have been
identified and interpreted on this basis. With this approach,
however, the consequences of aging of other organs and
organism cannot be separated from intrinsic aging effects on
the heart. The underlying assumption is that chronological
age and myocyte age coincide and the heart is composed of
cells of identical age with life expectancy which coincides
with that of the organism.22 Findings in the current study do
not support this traditional view of the biology of the heart but
strongly suggest that myocardial aging is mediated by alterations in cell turnover and physiological balance between
new and old myocytes. This imbalance leads, as demonstrated here, to a disproportionate accumulation of senescent
cells in combination with negative ventricular remodeling
and heart failure.7,11
The mechanisms of cellular senescence are difficult to
identify. Mutations of mitochondrial DNA, oxidative stress
with damage to proteins, lipids and nucleic acids, telomeric
shortening, and genetic modifications are all implicated in the
onset of cellular aging and the pace by which this process
reaches the final stage of senescence and impaired cell
function.12,23,24 We have elected to measure telomere length
and the expression of p16INK4a because telomere attrition
occurs in amplifying myocytes during proliferation, differentiation, and the acquisition of the adult phenotype.6,7,11
Thereafter, telomeric shortening is mediated by cumulative
oxidative stress which is a time-dependent process.13 Severe
telomere dysfunction upregulates p16INK4a and p53 and ultimately activates the apoptotic pathway.12 Myocyte volume
was found to represent a critical variable of cellular senescence together with the inevitable outcome of depressed
mechanical behavior. Therefore, myocyte aging is a selfautonomous process only partly related to chronological age.
Myocyte Size and Number
In the young-adult mouse heart, 40% of myocytes were
⬍20 000 ␮m3 in volume and comprised 20% of the LV-mass
whereas 41% of myocytes were 20 000 to 40 000 ␮m3 and
occupied 42% of the LV. Old myocytes, ⬎40 000 ␮m3 in
volume, represented 19% of the cell pool and formed 38% of
the muscle mass. In the old mouse heart, cells with volume
⬍20 000 ␮m3 accounted for 31% of myocytes and 11% of the
organ. Myocytes 20 000 to 40 000 ␮m3 were 35% of the cells
and constituted 30% of the myocardium. Myocytes
⬎40 000 ␮m3 corresponded to 34% of cells and 59% of the
tissue. Therefore, myocyte heterogeneity is present early in
life and is potentiated in the old heart. Although the relative
contribution of myocyte classes to ventricular function could
not be determined in the young or old heart, in both cases
small young myocytes have superior contractile ability and
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Figure 6. Outward transient K⫹ current. A, Ito
in myocytes. B, Ito-V relations in myocytes
⬍20 000 (n⫽9), 20 to 40 000 (n⫽10), and
⬎40 000 ␮m3 (n⫽10). *P⬍0.05 vs ⬍20 000;
**P⬍0.05 vs 20 to 40 000. C, Kv1.4, Kv4.2,
and Kv4.3 mRNA in small and large myocytes
(n⫽5). D and E, RT-PCR products had the
expected MW and nucleotide sequences. F,
Kv4.3 in small and large myocytes.
may counteract partly the depressed mechanical behavior of
hypertrophied-senescent myocytes. The electromechanical
properties of these cells classes were independent from their
anatomical location and differently affected large proportion
of the myocyte population in the young and senescent heart.
Studies in transgenic mice with cardiac restricted overexpression of IGF-1 and Akt support this possibility.5,7 In both
cases, the genetic modifications result in an increase in the
number of myocytes which are smaller in volume, possess
enhanced mechanical performance, potentiate LV function,
and delay cardiac aging.5,7,25 In a manner similar to that
observed here in small myocytes, telomeres are longer and
the fraction of p16INK4a-positive cells is markedly reduced in
IGF-1 and Akt transgenic myocytes. These cellular properties
potentiate the myocardial response of the heart to acute and
chronic ischemic damage, genetic myopathy, and
diabetes.25,26
Myocyte Physiology
Excitation-contraction coupling is modulated by changes in
the expression and phosphorylation of proteins27,28 that regulate transmembrane Ca2⫹ influx and extrusion, release, and
uptake of Ca2⫹ from the SR, and responsiveness and binding
affinity of the myofilaments to Ca2⫹. AP profile has profound
effects on calcium cycling and myocyte mechanics. A prolongation of the repolarization phase may involve an increase
in ICaL which in turn enhances Ca2⫹ release from the SR and,
thereby, myocyte contractility.17 The results here provide
strong evidence in favor of the critical role that AP duration
and ICaL play in potentiating Ca2⫹ cycling and mechanical
behavior of young-small myocytes or in decreasing Ca2⫹
transients and the performance of senescent-hypertrophied
cells. Three statements of caution have to be made. Myocyte
mechanics were assessed in unloaded cells, which has limi-
Rota et al
Myocyte Heterogeneity
395
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Figure 6. Continued
tations in the extrapolation of results to in vivo settings. The
effects of changes in AP profile on SR Ca2⫹ load were not
determined. And we cannot exclude that the isolation protocol impacted differently on cells of different size and age
affecting their electromechanical behavior.
The AP is modulated by the transient outward K⫹ current
Ito which is influenced by the expression of K⫹ channel
subunits29 as documented here at the mRNA and protein
levels. The expression of Kv1.4, Kv4.2 and Kv4.3 varied in
young and senescent myocytes and these differences may
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Figure 7. AP and Ca2⫹ transients. A, APs with slow, intermediate, and fast repolarization (upper panel) were used as voltage clamp
commands and Ca2⫹ transients recorded. B, Ca2⫹ transients with different APs (n⫽11). Neonatal myocytes (n⫽4). Traces and values
were normalized for Ca2⫹ transients after APslow. C, ICaL in AP-clamp. Ca2⫹ influx plotted vs 70% repolarization time of the respective
APs (r2⫽0.9999; P⬍0.05, n⫽4). D and E, Differential effect of 4-aminopyridine on Ca2⫹ transients and cell shortening in myocytes⬍20 000 (n⫽8) and ⬎20 000 ␮m3 (n⫽12). Traces are normalized to baseline value.
represent the molecular mechanisms responsible for the
alterations in transient outward K⫹ current and AP with
myocyte aging and hypertrophy. Changes in Ito have been
reported in myocytes as a function of chronological age.30,31
However, they are at variance with the current results because
AP prolongations in rats have been considered a successful
adaptation of old cells.20,32 Additionally, gender differences
in myocyte behavior have been reported in mice.33 Our
observations suggest that these potential differences may be
accounted for by the type of cells sampled. A large variability
has been found in relation to the size of the cells and
expression of aging-associated proteins and telomeric length.
Our results point to the need of distribution analysis of the
myocyte electromechanical properties to avoid sampling bias
and partial characterization of the physiological behavior of
aging myocytes.
Myocyte heterogeneity is not restricted to the young and
old mouse heart. Similar findings have been reported in rats,
Rota et al
Myocyte Heterogeneity
397
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dogs and humans, and pathological conditions and aging
potentiate this phenomenon.6,34 However, it is difficult to
predict whether small developing myocytes in the adult
stressed human heart6 may have a longer repolarization
phase, enhanced Ca2⫹ transients, and increased cell shortening. Human myocytes exhibit a pronounced plateau phase of
the AP which is not present in rodents.35 Whether Ito plays a
comparable or lesser role in the regulation of AP, Ca2⫹
cycling and mechanical behavior in humans is an important
unanswered question.
A comment has to be made concerning the electromechanical properties of aging myocytes. Ca2⫹ load of the SR and
ICaL are not altered. Similarly, the ability of L-type current to
trigger the activation of the RyR and Ca2⫹ release from the SR
is not impaired. This suggests that the machinery underlying
Ca2⫹ cycling is preserved in old cells and that cellular aging
is dictated by changes in electrical activity which negatively
modulate excitation-contraction coupling. In senescent myocytes, the faster repolarization of the AP leads to decreased
Ca2⫹ transients and faster time to peak Ca2⫹. However, these
abnormalities in Ca2⫹ metabolism can be reversed by prolongation of AP which restores the mechanical performance of
old myocytes. Collectively, our observations point to electrical remodeling as a key modulator of the heterogeneity of
myocyte aging and function in the adult mammalian heart.
Sources of Funding
Figure 7. Continued
Supported by NIH grants and by MRC Strategic Grant G0300395.
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Figure 8. Myocyte aging in the old heart. A, p16INK4a-positive MONO (n⫽1744), BI (n⫽608), and MULTI (n⫽420) myocytes. *P⬍0.05 vs
MONO; **P⬍0.05 vs BI. B, Distribution of telomere length vs myocyte volume (n⫽180) was fitted with a straight line (r2⫽0.312;
P⬍0.0001). C, Telomere length in myocytes ⬍20 000 (n⫽34), 20 to 40 000 (n⫽79), and ⬎40 000 ␮m3 (n⫽67). *P⬍0.05 vs ⬍20 000. D,
Bivariate distribution of Ca2⫹ transients (r2⫽0.0534; P⬍0.05) and cell shortening (r2⫽0.0861; P⬍0.05) vs myocyte volume (n⫽89). E,
AP-repolarization in myocytes ⬍20 000 (n⫽8) and ⬎20 000 ␮m3 (n⫽10). F, Ito-V relations in myocytes ⬍20 000 (n⫽6) and ⬎20 000 ␮m3
(n⫽10). *P⬍0.05 vs ⬍20 000.
Disclosures
None.
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The Young Mouse Heart Is Composed of Myocytes Heterogeneous in Age and Function
Marcello Rota, Toru Hosoda, Antonella De Angelis, Michael L. Arcarese, Grazia Esposito,
Roberto Rizzi, Jochen Tillmanns, Derin Tugal, Ezio Musso, Ornella Rimoldi, Claudia Bearzi,
Konrad Urbanek, Piero Anversa, Annarosa Leri and Jan Kajstura
Circ Res. 2007;101:387-399; originally published online June 29, 2007;
doi: 10.1161/CIRCRESAHA.107.151449
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2007 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2007/06/29/CIRCRESAHA.107.151449.DC1
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Rota et al., The Young Mouse Heart Is Composed…
Materials and Methods
Animals
A total of 60 C57Bl/6 mice at 3 months of age, 10 mice at 22 months, and 3 litters of
neonatal mice were used. In each study, a minimum of 5 mice were employed. In a group
of 5 adult mice, BrdU, 50 mg per kg body weight, was injected twice a day for three days
before sacrifice. Animals were maintained in accordance with the Guide for Care and Use
of Laboratory Animals and the experiments were approved by New York Medical
College.
Myocyte Isolation
Following chloral hydrate anesthesia (400 mg/kg body weight, i.p.), the heart was excised
and left ventricular (LV) myocytes were enzymatically dissociated (1, 2). Briefly, the
myocardium was perfused retrogradely through the aorta at 37°C with a Ca2+-free
solution gassed with 85% O2 and 15% N2. After 5 minutes, 0.1 mmol/L CaCl2, 274
units/mL collagenase (type 2, Worthington Biochemical Corp, Lakewood, NJ) and 0.57
units/mL protease (type XIV, Sigma, St. Louis, MO) were added to the solution which
contained (mmol/L): NaCl 126, KCl 4.4, MgCl2 5, HEPES 5, Glucose 22, Taurine 20,
Creatine 5, Na Pyruvate 5 and NaH2PO4 5 (pH 7.4, adjusted with NaOH). At completion
of digestion, the LV was cut in small pieces and re-suspended in Ca2+ 0.1 mmol/L
solution. In 10 hearts, the epicardium, endocardium, base and apex of the left ventricle
were dissected and myocytes obtained from these anatomical regions. Similarly, neonatal
myocytes were enzymatically dissociated from the heart of C57Bl/6 mice at 1 day after
birth. Mice were decapitated and the heart was quickly removed and placed in Ca/Mgfree Hanks’ balanced salt solution (HBSS, Sigma). After removal of the atria, the tissue
was cut in small pieces which were transferred into 10 ml dissociation medium (0.1%
trypsin, 1:250, and 0.01% DNase I in HBSS). After 25 minutes of gentle stirring at 37°C
the supernatant was collected and the cells contained in this fraction were centrifuged at
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300 g for 4 min, resuspended in Eagle’s MEM (Sigma) supplemented with 10% FBS
(Sigma) and stored on ice. Isolated cells were pre-plated in a 150-mm Petri dish for 1
hour at 37°C, and myocytes which did not attach to the dish during this time were
collected and plated in 35-mm Petri dish at low density (500/mm2) and cultured in
Dulbecco’s MEM (Sigma) with 10% FBS (3). Patch-clamp experiments were performed
utilizing 24-48 hours cultured neonatal myocytes.
Myocyte Volume
For the determination of myocyte volume, cells were isolated from 20 and 5 mice at 3
and 22 months of age, respectively. Myocytes were washed in PBS and fixed for 20
minutes in 4% paraformaldehyde (1, 4). Myocytes were stained with α-sarcomeric actin
mouse monoclonal antibody (Sigma) and connexin 43 rabbit polyclonal antibody (Sigma)
to identify cell aggregates. Nuclei were recognized by 4'-6-diamidino-2-phenylindole
(DAPI) staining (Sigma). In physiological studies, cell length and width were measured
with a computerized image analysis system and the ratio of the minor to the major axis of
the ellipse was obtained by confocal microscopy. Cell volume was then computed
assuming an elliptical cross-section with the major axis equivalent to cell width; the
minor axis was computed from the measured ratios. In morphological studies, the volume
of freshly isolated paraformaldehyde–fixed myocytes in each cell class was obtained by
three-dimensional optical section reconstruction by confocal microscopy (Bio-Rad
Radiance 2100, Hercules, CA). Three-dimensional reconstruction of isolated myocytes
was computed utilizing Imaris software (Bitplane AG, Zurich, Switzerland). These
preparations were also used for the determination of apoptosis (see below).
Immunocytochemistry
Immunocytochemistry was performed on 4% paraformaldehyde-fixed isolated myocyte
preparations. Nuclei were stained by DAPI or PI. p16INK4a (F-12 antibody, Santa Cruz
Biotechnology, Santa Cruz, CA; 2, 5, 6), Kv4.3 (Chemicon, Temecula, CA), αsarcomeric actin (Sigma), BrdU (Roche Molecular Biochemistry, Indianapolis, IN), Ki67
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(Vector, Burlingame, CA) and phospho-H3 (Upstate Biotechnology, Charlottesville, VA)
antibodies were employed. Apoptosis was detected by the TUNEL assay (BD
Pharmingen, San Jose, CA).
Telomere Length in Isolated Myocytes
Telomere length in nuclei was evaluated in 4% paraformaldehyde-fixed isolated myocyte
preparations by quantitative fluorescence in situ hybridization (Q-FISH) and confocal
microscopy. An FITC-peptide nucleic acid probe was used (2, 5-7). The fluorescent
signal measured in lymphoma cells (L5178Y) with short (7 kbp) and long (48 kbp)
telomeres (kindly provided to us by Dr. M.A. Blasco, Madrid, Spain) were utilized to
compute absolute length of telomeres. Fluorescence intensity was measured utilizing
ImagePro software (Media Cybernetics, Silver Spring, MD).
Sarcomere Shortening and Ca2+ Transients
Isolated LV myocytes were placed in a bath on the stage of an Axiovert Zeiss
Microscope (Zeiss, Germany) and IX71 Olympus inverted microscope (Olympus,
Melville, NY) for contractility, Ca2+ transients and patch-clamp measurements.
Experiments were conducted at room temperature. Cells were bathed continuously with a
Tyrode solution containing (mmol/L): NaCl 140, KCl 5.4, MgCl2 1, HEPES 5, Glucose
5.5 and CaCl2 1.0 (pH 7.4, adjusted with NaOH). Measurements were performed in fieldstimulated cells by using IonOptix fluorescence and contractility systems (IonOptix,
Milton, MA). Contractions were elicited by rectangular depolarizing pulses, 2 ms in
duration, and twice-diastolic threshold in intensity, by platinum electrodes (1, 2, 8, 9).
Cell shortening was measured by edge-track detection system. Changes in mean
sarcomere length were computed by determining the mean frequency of sarcomere
spacing by fast Fourier transform and then frequency data were converted to length. Ca2+
transients were measured by epifluorescence after loading the myocytes with 10 µM
Fluo-3 AM (Invitrogen, Carlsbad, CA). Excitation length was 480 nm with emission
collected at 535 nm using a 40x oil objective. Fluo-3 signals were expressed as
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Rota et al., The Young Mouse Heart Is Composed…
normalized fluorescence (F/F0). Caffeine–induced Ca2+ transient was evoked by rapid
application of 20 mmol/L caffeine to assess SR-Ca2+ load (1, 10). In field-stimulated
cells, the prolongation of the action potential was achieved by perfusion with 0.5 mmol/L
4-aminopyridine (11).
Patch-Clamp Experiments
Data were acquired by means of the whole-cell patch-clamp technique in voltage-clamp
mode using a Multiclamp 700A amplifier (Axon Instruments, Union City, CA). Electrical
signals were digitized using a 500 kHz 16-bit resolution A/D converter (Digidata 1322,
Axon Instruments) and recorded using pCLAMP 9.0 software (Axon Instruments) with
low-pass filtering at 2 kHz (1, 2).
L-type Ca2+ Current
L-type Ca2+ Current (ICaL) properties were assessed using a Na+-K+ free perfusing
solution (11) of the following composition (mmol/L): N-methyl-D-glucamine (NMDG)
140, CsCl 4, MgCl2 1, HEPES 5, glucose 5.5, CaCl2 1 and 4-aminopyridine 2 (pH 7.4
with CsOH). The composition of the pipette solution was (mmol/L): NMDG 10, CsCl
113, MgCl2 0.5, Tris-ATP 5, glucose 5.5, HEPES 10, EGTA 5 and TEA-Cl 20 (pH 7.2
with CsOH). The pipettes were pulled by means of a Narishige PB-7 glass microelectrode
puller (Narishige, Tokyo, Japan) and when filled had a resistance of 1-2 MΩ. ICaL
current-voltage (I-V) relation and activation properties were determined applying
depolarizing steps 300 ms in duration from holding potential (Vh) -70 mV in 10 mV
increments. For inactivation properties a 300 ms preconditioning step was applied within
the range -70 to +60 mV in 10 mV increments prior to the 300 ms test step to 0 mV. The
half maximal activation potential and the potential giving 50% inactivation for ICaL was
obtained by fitting the activation and inactivation curves with a Boltzmann equation (1,
12). Inactivation time course of ICaL was described by fitting the current traces between
the inward peak and the end of the 300 ms pulse to 0 mV (from Vh -70 mV) with a
biexponential function (1). Membrane capacitance (Cm) was calculated using a 5 mV
voltage step.
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Excitation-Contraction Coupling Gain
For excitation-contraction (EC) coupling gain measurements (13, 14), Ca2+ current and
Ca2+ transient were measured simultaneously in voltage-clamped myocyte. Myocytes
were loaded with Fluo-3 AM and bathed with a modified Tyrode solution in which KCl
was replaced with CsCl and 2 mmol/L 4-aminopyridine was added to the solution. The
composition of the pipette solution was (mmol/L): NaCl 10, CsCl 113, MgCl2 0.5, TrisATP 5, Glucose 5.5, HEPES 10 and tetraethylammonium chloride 20 (pH 7.2 with
CsOH). Fluorescence signal intensity was collected using a photomultiplier and a photon
to voltage converter (IonOptix) connected to the patch-clamp A/D converter. Cells were
depolarized for 300 ms from Vh -40 mV to a family of test potentials ranging from -30 to
+70. Each voltage-clamp was preceded by a train of 4 conditioning pulses (100 ms, 1Hz)
form -40 to 0 mV.
Action Potentials, K+ current and Action Potential-Clamp
For action potential (AP) measurements, current-clamp mode was utilized. Cells were
stimulated at 1 Hz with current pulses 1.5 times threshold. Myocyte were bathed with
Tyrode solution as described above. The composition of the pipette solution was
(mmol/L): NaCl 10, KCl 113, MgCl2 0.5, K2-ATP 5, Glucose 5.5, EGTA 5, HEPES 10
(pH 7.2 with KOH). For action potential clamp (AP-clamp) experiments a set of action
potential waveforms were utilized as voltage-clamp command in myocytes loaded with
Fluo-3 AM. Solution were similar to the one utilized for current-clamp experiments. To
assess Ca2+ influx during AP-clamp studies, specific solutions were employed to record
ICaL. Ca2+ influx was measured as the integral of the inward current over time normalized
by membrane capacitance (11). Transient outward K+ current (Ito) was assessed in
voltage-clamp mode with Tyrode solution containing 0.3 mmol/L cadmium chloride to
block ICaL. Pipette solution was similar to that utilized for AP measurements (15, 16).
Current-voltage (I-V) relations were determined applying depolarizing steps 500 ms in
duration from holding potential (Vh) -40 mV in 10 mV increments. Ito amplitude was
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measured as the difference between the peak outward current at the beginning of the step
and Isus, the current at the end of the 500 ms step (15, 16).
Real-Time RT-PCR
Isolated myocytes were separated according to their size utilizing a 20 µm mesh filter
(CellTrics, Partec, Munster, Germany) (17). After centrifugation, cell pellets were snapfrozen in liquid nitrogen for real time RT-PCR analysis of the transcripts of Kv1.2, Kv4.2
and Kv4.3 (18). RNA was extracted utilizing Trizol reagent (Sigma) and treated with
DNase I (Sigma). Reverse transcription reaction was performed with 2 µg of total RNA,
oligo(dT) primer and 200 units of SuperScript III (Invitrogen) at 50°C for 3.5 hours.
Samples were then treated with RNase H (Ambion, Austin, TX). RT-PCR was performed
in the Real Time PCR System 7300 (Applied Biosystems, Warrington, UK) utilizing
1/20th of the synthesized cDNA together with 0.625 units of AmpliTaq Gold DNA
polymerase (Applied Biosystems), 0.5 µM each of forward and reverse primers, and 0.1
µM of FAM-labeled probe for each target gene; 0.4 µg of total RNA was included as a
non-RT control sample. PCR conditions were as follows: 95°C for 10 min, 40 cycles of
95°C for 15 sec and 60°C for 1 min. FAM fluorescence was measured during the second
step at the end of each cycle. Primers and probe of each gene were designed on the same
exon. Sequences of primers and probes were:
mKv1.4
F: 5'- AGA GGC GGA TGA ACC CAC TAC -3' (59°C)
P: 5'- CAT TTC CAA AGC ATT CCA GAT GCG TTT TG -3' (70°C)
R: 5'- CGA CTG TGA TGG GCT TCA TG -3' (59°C)
amplicon size: 110 bp
mKv4.2
F: 5'- AAT GTG TCG GGA AGC CAT AGA -3' (58°C)
P: 5'- CCA ACA GCC GAT CCA GCT TAA ATG CC -3' (70°C)
R: 5'- ACA GTT TAG TTT AAC ACA CTC TTC CAT TTT -3' (58°C)
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amplicon size: 135 bp
mKv4.3
F: 5'- CAG CAC GCT CCA CAT CCA -3' (59°C)
P: 5'- CAG TCG CTC CAG CCT TAA TTT GAA AGC AGA -3' (70°C)
R: 5'- TGG TAA TCT GGG ATG TTT TGC A -3' (59°C)
amplicon size: 116 bp
The housekeeping gene β-actin was used to normalize PCR conditions:
mActb
F: 5'- AGA AGG AGA TTA CTG CTC TGG CTC -3' (58°C)
P: 5'- CTA GCA CCA TGA AGA TCA AGA -3' (72°C)
R: 5'- ACA TCT GCT GGA AGG TGG ACA -3' (59°C)
amplicon size: 126bp
The expression profile of each target gene was analyzed using Automatic Baseline of
Sequence Detection Software version 1.2.2 (Applied Biosystems). The threshold was
fixed manually at 0.05 to determine the cycle of threshold (Ct), which was then
normalized by β-actin; the Ct value of β-actin was subtracted from the Ct value of the
target genes (∆Ct). The levels of expression were presented as percentage relative to the
quantity of β-actin transcript.
Data Analysis
Data are presented as mean±S.E.M. Significance was determined by Student’s t test and
Bonferroni method (19, 20). Bivariate distribution plots were fitted by straight line
function (19). P<0.05 was considered significant.
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Supplemental Figures
Supplemental Figure I. Volume distribution of myocytes. A, Volume of mononucleated
(MONO; n=84), binucleated (BI; n=522) and multinucleated (MULTI; n=88) myocytes.
*P<0.05 vs MONO; **P<0.05 vs BI. B and C, Volume distribution of myocytes (B,
n=694; C, n=2,819).
Supplemental Figure II. Cell capacitance. Bivariate distribution of Cm and myocyte
volume (r2=0.961; P<0.0001); n=54.
Supplemental Figure III. Regional distribution of AP duration and sarcomere
shortening. A, AP-repolarization in myocytes <20,000, and >20,000 µm3 from the
endocardium (n=11 and 10) and epicardium (n=9 and 12). *P<0.05 vs small myocytes. B,
Bivariate distribution of sarcomere-shortening vs cell volume in myocytes from
endocardium (r2=0.0915; P<0.05, n=80) and epicardium (r2=0.1135; P<0.05, n=78).
Supplemental Figure IV. Ca2+-transients and shortening with 4-aminopyridine.
Supplemental Figure V. Volume distribution of myocytes at 3-months (n=2,819) and
22-months (n=613) of age.
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