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1 Division of Cardiology, University of Utah Health Science Center, Salt Lake City, Utah 84132; and 2 Department of Medicine, University of California, La Jolla, California 92093
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Muscle LIM protein (MLP) may serve as a scaffold protein on the actin-based cytoskeleton, and mice deficient in this protein (MLPKO) have been recently reported to develop dilated cardiomyopathy. To determine the causes of depressed contractility in this model, we measured intracellular Ca2+ concentration ([Ca2+]i) transients (fluo 3), cell shortening, L-type Ca2+ channel current (ICa,L), Na/Ca exchanger current (INa/Ca), and sarcoplasmic reticulum (SR) Ca content in left ventricular MLPKO myocytes. ICa,L-voltage relationships, INa/Ca density, and membrane capacitance did not differ between wild-type (WT) and MLPKO myocytes. The peak systolic [Ca2+]i was significantly increased in MLPKO myocytes (603 ± 54 vs. 349 ± 18 nM in WT myocytes). The decline of [Ca2+]i transients was accelerated in MLPKO myocytes, and SR Ca2+ content was increased by 21%, indicating that SR Ca2+-ATPase function is normal or enhanced in MLPKO myocytes. Confocal imaging of actin filaments stained with tetramethylrhodamine isothiocyanate-labeled phalloidin showed disorganization of myofibrils and abnormal alignment of Z bands, and fractional shortening was significantly diminished in MLPKO myocytes compared with that in WT myocytes at comparable peak [Ca2+]i. Thus a reduced [Ca2+]-induced shortening may be involved in the pathogenesis of myocardial dysfunction in this genetic model of heart failure.
cytoskeleton; knockout mouse; dilated cardiomyopathy; Ca2+ transient; excitation-contraction coupling; 2,3-butanedione monoxime
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IT HAS RECENTLY BEEN
RECOGNIZED that mutations in the
cytoskeletal/sarcolemmal/extracellular matrix complex may also cause cardiomyopathy. Sakamoto et al. (25) reported that a
mutation in the gene for 
-sarcoglycan, which is a subcomplex of the
dystrophin-associated glycoprotein complex, is present in the autosomal
recessive cardiomyopathy of the Syrian hamster, and mutations in this
complex may produce myopathy in humans (16, 19, 21, 30).
Mutations in the dystrophin-glycoprotein complex have been reported by
Straub et al. (27) to increase sarcolemmal permeability to
Evans blue dye in skeletal muscle cells, and Sen et al.
(26) reported that 45Ca uptake by Syrian
cardiomyopathic hamster cardiac myocytes is increased. These
observations suggest that an alteration in sarcolemmal Ca2+
permeability may occur as a result of alterations in the
dystrophin-glycoprotein complex and may contribute to Ca2+
overload and myocyte dysfunction.
Olson et al. (20) reported that a mutation in cytoskeletal actin is responsible for a familial human dilated cardiomyopathy. They speculated that defective transmission of force from contractile elements to the sarcolemma may be involved in the pathogenesis of heart failure due to this abnormality in the cytoskeleton. Mutations in desmin, another cytoskeletal protein, can also cause dilated cardiomyopathy (13). Arber et al. (1) reported that disruption of the gene for muscle LIM protein (MLP), a scaffold protein of the actin-based cytoskeleton, results in structural and functional changes of dilated cardiomyopathy in the mouse. Minamisawa et al. (17) reported that Ca2+ transient amplitude is reduced in right ventricular myocytes from failing hearts in MLP knockout (MLPKO) animals and that the failure phenotype and myocyte Ca2+ transient may be "rescued" in the offspring of MLPKO animals crossed with phospholamban (PLB) knockout animals.
The purpose of our study was to examine left ventricular myocyte Ca homeostasis, contraction, and sarcolemmal permeability in myocytes from MLP-deficient mice in order to investigate possible mechanisms by which cytoskeletal abnormalities might result in cardiomyopathy and heart failure.
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MATERIAL AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MLPKO mice. MLPKO mice were produced as previously described (1). MLPKO and age-matched littermate [wild type (WT)] mice, 6 ± 1 mo old, were used for experiments.
Dissociation of adult mouse ventricular myocytes. Adult mouse myocyte isolations were enzymatically performed as previously described (33). Briefly, hearts were removed from anesthetized mice and immediately attached to an aortic cannula. After hearts were perfused with Ca2+-free modified Tyrode solution containing (in mM) 126 NaCl, 4.4 KCl, 1.0 MgCl2, 18 NaHCO3, 11 glucose, and 4 HEPES for 5 min, they were digested with 0.90 mg/ml collagenase D (Boehringer-Mannheim Biochemicals) in 25 µM CaCl2-containing modified Tyrode solution for 7-12 min, which also contained 30 mM 2,3-butanedione monoxime (BDM) and 0.13 U/ml insulin. In some experiments, BDM was omitted from the dissociation solution. The digested hearts were removed from the cannula, and the left ventricles were cut into small pieces in 100 µM Ca2+ modified Tyrode solution. The cell suspension was then centrifuged at 300 rpm for 3 min, and the pellet of the cells was resuspended in 200 µM Ca2+ and 2% albumin-Tyrode solution and allowed to settle for 20 min at 30°C. The cells were then resuspended in 0.9 mM Ca2+ culture media composed of 50% minimal essential medium (GIBCO-BRL Laboratories), 50% modified Tyrode solution without BDM, 10 mM pyruvic acid, and 6.1 mM glucose at 30°C in a 5% CO2 atmosphere until use. Isolated cells were all used for experiments within 6 h after isolation.
Measurement of intracellular Ca2+ concentration transients, intracellular Na+ concentration, and cell shortening. The intracellular Ca2+ concentration ([Ca2+]i) in isolated myocytes was measured with fluo 3 as previously described (32, 33). Myocytes attached to laminin-coated glass coverslips were incubated in a 1 µM fluo 3-AM (Molecular Probes)-containing HEPES solution (loading solution) at 30°C in the dark for 30 min.
Fluo 3-loaded myocytes were excited by a mercury arc lamp system at a 485-nm wavelength through an epifluorescence attachment (505-nm dichroic mirror, Omega) and a ×40 Fluor oil objective lens (Nikon). Fluorescence (530 nm, DF30, Omega) was detected with a photomultiplier tube (Hitachi). Calibration of the [Ca2+]i transients was performed by superfusing the myocyte with 10 µM ionomycin, 10 mM MnCl2, and 30 mM BDM-containing HEPES solution. The maximum fluorescence (Fmax) was calculated with the formula Fmax = FMn2+ × 5, where FMn2+ is the fluorescence in the presence of Mn2+. Minimum fluorescence (Fmin) was 1/40 of Fmax. Therefore, [Ca2+]i could be calculated with the following formula: [Ca2+]i = Kd × (F
Fmin)/(Fmax F). In this
formula, F is the measured fluorescence intensity of the myocyte
corrected for background and autofluorescence, and
Kd is the dissociation constant (493 nM)
(33). All experiments on [Ca2+]i
were performed at 25°C. In some experiments, we measured
intracellular fluo 3 F/F0 (where F0 is resting
fluorescence intensity) to quantitate peak
[Ca2+]i (7).
The intracellular Na+ concentration
([Na+]i) was measured with
1,3-benzenedicarboxylic acid,
4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester (SBFI-AM) as previously
described (33). After myocytes were loaded by incubation
for 120 min in 10 µM SBFI-AM, myocytes were illuminated sequentially
at 60 Hz by 340- and 380-nm excitation light passing through band-pass filters (P10-340 and P10-380, Corion) with an optical switcher (DX-1000, Solamere Technology Group), and the fluorescence at 510 nm
(P10-510, Corion) was continuously recorded. The ratio of the
fluorescence intensity during excitation with 340- and 380-nm light (R)
was used as an indicator for [Na+]. The myocyte was
sequentially exposed to three calibration solutions of 5, 10, and 15 mM
Na+, which contained (in µM) 2 gramicidin-D, 40 monensin,
and 100 strophanthidin. In each solution,
[Na+]i was equilibrated to the extracellular
Na+ concentration ([Na+]o), and
the stable fluorescence at each [Na+]i was
then obtained. Data were all digitized and directly acquired by a
computer. The relationship between R and
[Na+]i was fitted, and the fluorescence
intensity from the myocyte was then converted to
[Na+]i.
Myocytes were field stimulated with platinum electrodes with 7-ms
pulses of alternating polarity, and [Ca2+]i
transients and pacing signals were recorded on tape for further analysis. For measurement of cell shortening, an intensified
charge-coupled device camera (GenII+, Stanford Photonics) was used to
obtain the image of the myocyte simultaneously with
[Ca2+]i transients by illuminating myocytes
with low-intensity red light, which was directed to the camera via a
650-nm dichroic mirror. Shortening was measured with a video motion
detector (Cresent Electronics; Sandy, UT). In some experiments,
myocytes were paced at 10 Hz in the presence of 5 µM ryanodine to
determine [Ca2+]i and shortening during
tetanus (34).
L-type Ca2+ channel current and
Na/Ca exchanger current measurements.
L-type Ca2+ current (ICa,L) and
Na/Ca exchanger current (INa/Ca) were
consecutively measured as previously described (29, 32,
33). A myocyte was superfused with HEPES solution containing (in
mM) 126 NaCl, 1.0 MgCl2, 1.08 CaCl2, 24 HEPES,
and 13 NaOH (pH 7.4) and was then voltage clamped (Axopatch 200A, Axon
Instruments) with a suction pipette (2-3 M
) filled with
solution containing (in mM) 15 NaCl, 0.2 MgCl2, 14 EGTA,
3.0 MgATP, 5.5 glucose, and 10 HEPES. Ca2+ (3.9 mM) was
added as H2CaEGTA, providing 100 nM of free
Ca2+. The pH was adjusted to 7.1 with CsOH, and the
concentration of Cs+ in the solution was brought to 130 mM
by adding CsCl. After the myocyte was stabilized at 80 mV for 3 min,
the myocyte was held at a potential of 40 mV, and
ICa,L was then activated by depolarizing the
myocyte membrane to more positive values for 500 ms. After ICa,L was measured and after 20 min had passed
after starting dialysis, the myocyte was abruptly exposed to
Na+-free solution at a holding potential of 40 mV for
5 s with a solenoid-based rapid solution switcher, which can
change the bulk solution surrounding the myocyte within 4 ms
(31). This procedure elicits an outward current due to
reverse Na/Ca exchange (3 Na+ out, 1 Ca2+ in),
the magnitude of which can be used to estimate Na/Ca exchanger density
(29) when normalized by cell membrane capacitance.
Measurement of sarcoplasmic reticulum Ca content.
Sarcoplasmic reticulum (SR) Ca2+ content was determined by
measuring the integral of the caffeine-induced inward
INa/Ca (33). In brief, myocytes
were voltage clamped at 80 mV with a single suction pipette filled
with a solution composed of (in mM) 15 NaCI, 100 CsCl, 30 tetraethylammonium chloride, 5 MgATP, 10 HEPES, and 5.5 dextrose (pH
7.1 adjusted with CsOH). The voltage-clamped cell was then superfused
in microstream with a solution containing (in mM) 126 NaCl, 4.4 KCl,
1.0 MgCl2, 1.08 CaCl2, 2 CsCl, 0.1 BaCl2, 11 dextrose, and 24 HEPES (pH 7.4 adjusted with NaOH
to give a final [Na+]o of 140 mM). After a
train of steady-state conditioning pulses (eight 200-ms pulses to 0 mV
at 0.25 Hz), the myocyte was abruptly immersed for 6 s in an
adjacent switcher solution microstream in which 10 mM caffeine was
added to release SR Ca2+. On the basis of the stoichiometry
of the electrogenic Na/Ca exchange (3:1), the integral (nA × ms = pC) of the resulting inward INa/Ca was
converted to the amount of Ca2+ (in pmol) extruded by Na/Ca
exchange during the sustained exposure of cells to caffeine and
normalized by cell capacitance (in pF). The decline of the current
curve was well fitted with a single exponential, with a time constant
of [Ca2+]i decline (
) value 
500 ms under our experimental conditions. The current values at greater
than five times (2,500 ms) from the peak of the current were
used as a baseline for current transient integration.
Immunofluorescence and cell size analysis. To examine the organization of the contractile elements, we stained actin filaments with tetramethylrhodamine isothiocyanate (TRITC)- labeled phalloidin (Sigma). After isolation, myocytes attached on laminin-coated coverslip were fixed with 3.7% formaldehyde, permeabilized in 0.5% Triton X-100 in PBS, and blocked with 10% goat serum in PBS plus 0.1% Tween 20. Cells were then stained with TRITC-labeled phalloidin (Sigma) (40 mg/ml in PBS containing 2 mg/ml BSA, 1% DMSO, and 0.5% NP-40), and the myocytes were washed with 0.1% Tween 20 in PBS and examined with a confocal microscope (MRC 2000, Bio-Rad). Stained myocytes were excited at 488 nm with an argon laser, and 530-nm emission light was collected to create images. In other experiments, nonfixed freshly dissociated myocytes were imaged during bright field illumination with an intensified video camera (XR GEN3, Solamere Technology). Myocyte area was then calculated from measurement of cell length and width.
Sarcolemmal cation permeability. To assess permeability of the sarcolemma of MLPKO and WT myocytes to cations, we loaded cells with fura 2 by incubating them in 1 µM fura 2-AM for 30 min. Myocytes were illuminated with 360-nm light (fura 2 Ca2+-insensitive isosbestic wavelength), and fluorescence was measured at 510 nm during exposure to 10 mM MnCl2. Mn2+ quenches cytosolic fura 2 fluorescence, and thus the rate of decline of 510-nm fluorescence indicates the rate of entry of Mn2+ into the cell.
Statistical analysis. Results are expressed as means ± SE; n = the number of myocytes studied. Myocytes were obtained from at least four different hearts for every condition studied. A paired t-test was performed if the data were obtained from the same myocyte. Significance was tested by ANOVA if unpaired or multiple comparisons were made. A mixed-model ANOVA was employed, where strain is a fixed effect and the myocyte from a specific heart is the random effect. This analysis allows compensation for bias due to the fact that several myocytes from a given heart were studied by testing for an effect of isolation from a specific heart. Values of P < 0.05 were considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiac enlargement in MLPKO mice.
Data on left ventricular wet weight, lung weight, liver weight, and
body weight in WT and MLPKO mice are summarized in Table 1. The ratios of left ventricular wet
weight and lung weight to body weight were significantly increased in
MLPKO mice, confirming left ventricle enlargement and lung congestion.
This result is consistent with a previous report (1).
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Myofilament organization.
The gross appearance of intact myocytes isolated from the left
ventricle of MLPKO hearts was abnormal. Freshly isolated MLPKO cells
frequently had a slightly curved or "comma" shape. In fixed WT
myocytes, myofibrils were clearly and well developed, and Z bands were
regularly oriented through the myocyte (Fig.
1, A and B). In
contrast, in MLPKO myocytes, the sarcomere organization appeared
abnormal: the lateral alignment of Z bands between adjacent myofilaments was impaired and the Z bands themselves were broader and
less distinct than in WT cells (Fig. 1, C and D).
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[Ca2+]i transients and
contractions.
Figure 2 shows representative recordings
of simultaneously measured left ventricular myocyte
[Ca2+]i transients and fractional shortening
(FS). The mean values of systolic and diastolic
[Ca2+]i and the characteristics of the
decline of [Ca2+]i are given in Table
2. The peak systolic
[Ca2+]i and the amplitude of
[Ca2+]i transients (peak systolic minus end
diastolic [Ca2+]i) were significantly
increased in MLPKO myocytes, whereas the diastolic
[Ca2+]i was not different from that in WT
cells. was shorter in MLPKO myocytes than in WT myocytes,
suggesting an increased rate of uptake of Ca2+ by the SR
Ca2+-ATPase, although this difference was of borderline
statistical significance (P < 0.08).
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and the peak value of
[Ca2+]i transients. WT myocytes were
superfused with a high-Ca2+ (2.16 mM) solution after
initial pacing in regular solution containing 1.08 mM Ca2+.
In this procedure, the peak systolic [Ca2+]i
was progressively increased as the bathing solution was replaced with
the high-Ca2+ solution, whereas the diastolic
[Ca2+]i was increased by only <10 nM. This
stability of diastolic [Ca2+]i in the
presence of enhanced Ca2+ loading induced by high
extracellular Ca2+ has been previously reported in mouse
ventricular muscle (8). We measured and the peak
systolic [Ca2+]i of 15-20 consecutive
[Ca2+]i transients in WT myocytes after
starting superfusion of the high-Ca2+ solution and plotted
as a function of the peak [Ca2+]i. As
shown by the scattered open circles in Fig.
3, tended to be shorter in WT cells
as the peak [Ca2+]i was increased. The mean
values of as a function of the mean value of the peak systolic
[Ca2+]i in WT (closed circle) and MLPKO
myocytes (closed square) were overplotted on the same graph (Fig. 3).
The mean in MLPKO myocytes was similar to the average value for WT
cells at a comparable [Ca2+]i. This suggests
that the smaller in MLPKO myocytes is due primarily to higher peak
systolic [Ca2+]i levels.
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Function of L-type Ca2+ channels.
It is well known that the peak systolic
[Ca2+]i level depends primarily on
Ca2+ influx through L-type Ca2+ channels and
subsequent Ca2+ release from SR (4,
6). To elucidate the mechanism of the increased peak
systolic [Ca2+]i in MLPKO myocytes, we
examined the function of the L-type Ca2+ channel with a
whole cell voltage clamp. The mean value of
ICa,L density at each voltage is shown in Fig.
5. Neither the current density nor the
current-voltage relationship were altered, suggesting that the basic
function of the L-type Ca2+ channel is similar in MLPKO and
WT mice.
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Function of Na/Ca exchangers.
Recently, we (33) reported that the reverse function of
the Na/Ca exchanger can maintain Ca2+ loading in mouse
ventricular myocytes (33). In human dilated cardiomyopathy, expression of the Na/Ca exchanger has been reported to
be upregulated (28). Hence, it seemed possible that an
upregulation of Na/Ca exchange could induce an increase in peak
systolic [Ca2+]i in MLPKO myocytes despite
similar L-type Ca2+ channel function. We therefore directly
examined the function of the Na/Ca exchanger. As shown in Fig.
6, the mean value of INa/Ca density was similar between WT
(n = 15, 1.41 ± 0.06 pA/pF) and MLPKO myocytes
(n = 6, 1.40 ± 0.07 pA/pF). This indicates that
the function and density of the Na/Ca exchanger are not altered in
MLPKO myocytes.
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SR Ca content.
Because the function of L-type Ca2+ channels and the Na/Ca
exchanger did not appear to be altered, the greater peak systolic [Ca2+]i in MLPKO myocytes could be due to an
increase in Ca2+ release from SR. Therefore, we measured
releasable SR Ca2+ content. After a voltage-clamp pacing
protocol designed to ensure stable SR loading of Ca2+,
myocytes were abruptly exposed to 10 mM caffeine with a rapid solution
switcher. This procedure released Ca2+ from SR, and
subsequently the released Ca2+ was removed via Na/Ca
exchange, causing inward currents, as shown in Fig.
7. The integral of the inward current
normalized to membrane capacitance was somewhat increased in MLPKO
myocytes [10.72 ± 0.78 (n = 12) vs. 8.84 ± 0.49 pmol/µF (n = 17) in WT myocytes], but this
difference did not reach statistical significance (P = 0.09). In these voltage-clamp measurements, there was no significant difference in capacitance between WT (n = 32, 192 ± 9 pF) and MLPKO myocytes (n = 19, 199 ± 11 pF), suggesting that the cells isolated from these hearts were not
hypertrophied. This lack of hypertrophy of MLPKO myocytes was confirmed
by measurement of myocyte length, width, and area (MLPKO myocytes:
102.9 ± 1.6 µm, 24.9 ± 0.4 µm, 2,553 ± 52 µm2, n = 235; vs. WT: 111.4 ± 21.9, 26.3 ± 0.5, 2,956 ± 75, n = 176, respectively).
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Other possible causes of increased
[Ca2+]i transient.
Increased Ca2+ loading could result from increased
[Na+]i or altered sarcolemmal
Ca2+ permeability. However,
[Na+]i was not increased in MLPKO (15 ± 0.8 mM, n = 12) vs. WT myocytes (18.8 ± 1.5, means ± SE, P = not significant,
n = 21, respectively), and
[Na+]i values in MLPKO cells were similar to
those measured in mouse myocytes previously (33).
Also, as shown in Fig. 8, the rate of
quenching of intracellular fura 2 by externally applied
Mn2+ was similar in MLPKO and WT cells. This suggests that
sarcolemmal permeability is not markedly increased in MLPKO myocytes
and is consistent with observations in two animals that in vivo myocyte permeability as assessed by uptake of Evans blue dye (27)
is not increased in the MLPKO ventricle (Ross J, personal
communication).
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Effects of BDM.
Our results indicate that peak [Ca2+]i levels
are higher in MLPKO than WT myocytes, whereas Minamisawa et al.
(17) reported the opposite. This difference could be due
to differences in the dissociation techniques employed and possibly to
the fact that right ventricular myocytes were studied in the
experiments of Minamisawa et al. (17). One difference in
dissociation techniques is that we used BDM in our dissociation
procedure for mouse ventricular myocytes to improve the yield
(23). We therefore examined
[Ca2+]i transients and FS in myocytes
dissociated in the presence and absence of BDM. The results are shown
in Table 4. In MLPKO myocytes, both
F/Fo and FS were significantly greater in myocytes
dissociated in the presence of BDM. Dissociation in BDM had no effect
on F/Fo in myocytes from normal mice. It is important to
note that MLPKO myocytes dissociated without BDM had an insignificantly
reduced F/Fo compared with WT myocytes (2.4 ± 0.15, n = 24 vs. 2.7 ± 0.2, n = 26, P = not significant) but a markedly reduced FS
(2.0 ± 0.5%, n = 24 vs. 5.3 ± 0.5%,
n = 12, P < 0.0001). These findings indicate that the presence of BDM in the dissociation medium increases the amplitude of the [Ca2+]i transient in
MLPKO myocytes but not in WT myocytes and further confirm that the
sensitivity of FS to peak [Ca2+]i is reduced
in MLPKO myocytes.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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It is clear that in these transgenic mice, knockout of the cardiac muscle cytoskeletal LIM protein eventually produces a dilated cardiomyopathy phenotype. Our results demonstrate that in nonhypertrophied myocytes isolated from these hearts, two distinct abnormalities can be identified: an increase in [Ca2+]i transient amplitude in cells exposed to BDM during the dissociation process and a reduced sensitivity of shortening to [Ca2+]i.
Possible causes and significance of increased [Ca2+]i transient. An increased [Ca2+]i transient was indicated by measurements in four separate protocols in different cells. The density of the main sarcolemmal Ca2+ flux pathways, the L-type Ca2+ channel and the Na/Ca exchanger, did not appear to be altered in the MLPKO myocytes. Also, the MLPKO [Na+]i, an important regulator of Na/Ca exchange, was similar to that in WT. Thus neither altered Na/Ca exchange nor increased ICa,L-mediated Ca2+ flux appear to account for the increased [Ca2+]i transients we observed. The cause of the increase in peak [Ca2+]i remains uncertain. An elevation in the [Ca2+]i transient has also been reported in some nonmurine cardiac hypertrophy models (11, 14) and may be due to increased Ca2+ influx due to sarcolemmal stress (12, 18). It is possible that knockout of the MLP could alter sarcolemmal stresses by inducing changes in the attachment of sarcomeres to the sarcolemma. This would be consistent with the curved appearance of freshly dissociated MLPKO myocytes and the abnormal organization of sarcomeres we detected in phalloidin-stained cells. Our studies in vivo (Evans blue dye) and in vitro (Mn2+ quenching of fura 2 fluorescence) did not indicate a marked increase in sarcolemmal permeability. However, these relatively crude measures do not exclude a more subtle alteration of sarcolemmal permeability to Ca2+ in MLPKO myocytes.
The increase in [Ca2+]i transient may be of pathogenetic significance. Molkentin et al. (18) recently described a calcineurin-dependent transcriptional pathway that may be involved in cardiac hypertrophy. They demonstrated that cardiac hypertrophy can be induced by the Ca2+-dependent phosphatase calcineurin acting via the transcription factor nuclear factor (NF)-AT3. These investigators produced transgenic mice that express activated forms of calcineurin or NF-AT3 and develop cardiac hypertrophy and then heart failure. These findings indicate a potentially important role for increased [Ca2+]i in the development of hypertrophy and the subsequent progression to failure. The hearts from which we isolated cells were enlarged (Table 1). However, the cells we studied were not yet hypertrophied, as indicated by a normal membrane capacitance and no increase in cell size. This may indicate that the mass of nonmyocyte cells is markedly increased. Alternatively, there may be some myocyte hypertrophy in vivo, but only the most healthy nonhypertrophied cells survive the dissociation process, and thus may not accurately reflect in vivo function. This hypothesis is supported by the relatively lower percent yield of rod-shaped cells obtained from MLPKO (10-20%) compared with WT hearts (40-50%). Hypertrophy of myocytes induced by an increase in [Ca2+]i and/or by the increase in wall stress produced by cardiac dilatation and remodeling could eventually lead to changes in Ca2+ homeostasis due to alterations of ICa,L and/or decreased SR Ca2+-ATPase activity, with a resulting reduction in the [Ca2+]i transient (2, 3, 10). This could result in a progression of the failure phenotype.Causes and significance of reduced [Ca2+]i-induced shortening. The nonhypertrophied myocytes we studied had a reduced unloaded shortening relative to peak [Ca2+]i during a single action potential (Fig. 4 and Tables 3 and 4). This finding is consistent with the hypothesis of Olson et al. (20): that cytoskeletal mutations may produce failure by altering transmission of force from the myofilaments to the sarcolemma. Also in support of this hypothesis, Papp et al. (22) recently demonstrated that degradation of the cytoskeletal protein desmin by calpain-I decreases Ca-dependent force development in permeabilized ventricular myocytes.
In MLPKO mice, we suggest that the genetic defect in the cytoskeleton also results in a decrease in force development in the cell not due to a decrease in the [Ca2+]i transient but to altered force transmission from the contractile elements to the sarcolemma. Although under basal conditions the magnitude of unloaded shortening of MLPKO myocytes was similar to that of WT cells due to the increased magnitude of the MLPKO [Ca2+]i transient, we suspect that in the intact MLPKO heart in which myocyte loading is significant, this defect results in impaired force of contraction. This would certainly be consistent with the reduced systolic function noted in MLPKO animals by echocardiographic techniques (1, 24). In this setting, an increase in [Ca2+]i might be expected to compensate for the defect. Indeed, the increase in peak [Ca2+]i we noted may partially but insufficiently compensate in vivo. This hypothesis is supported by recent work showing that genetically engineered enhancement of SR function by
-adrenergic receptor kinase-1 (BARK-1) inhibition
(24) or by PLB ablation (17) can prevent the
development of the heart failure phenotype in MLPKO mice. It is
important to note that rescue of the failure phenotype by enhancement
of SR function does not necessarily mean that impaired SR function is
the primary cause of the initiation of the failure phenotype. Impaired
force production resulting from a variety of processes would be
expected in response to a marked increase in the magnitude of the
[Ca2+]i transient. Indeed, in preliminary a
study of 17 myocytes from 2 double knockout animals (PLB and MLP, Ref.
17) we found a peak F/Fo of 5.4 ± 0.3 and a
FS of 12.5 ± 1.2%, which contrasts with WT values of 2.6 ± 0.12 and 6.5 ± 0.8%, respectively (Table 3). Thus rescue of the
failure phenotype in these animals may require substantial enhancement
of SR function relative to normal. This idea is consistent with the
observation of Minamisawa et al. (17) that the peak left
ventricular dP/dt in double knockout animals was
significantly greater than that in WT animals.
The difference in our and Minamisawa et al. (17) findings
regarding the magnitude of [Ca2+]i transients
in MLPKO myocytes may be due in part to our use of BDM in the
dissociation process. The presence of BDM reduces myocyte injury due to
Ca2+ overload and increases myocyte yield after collagenase
dissociation of myocardial tissue (23). Thus reduced
injury of already damaged and fragile failing MLPKO myocytes might be
expected to improve the [Ca2+]i transient.
BDM is also a nonenzymatic phosphatase (9). Marx et al.
(15) recently reported that hyperphosphorylation of the ryanodine receptor in failing hearts may cause defective
Ca2+ regulation by causing dissociation of FK506-binding
protein from the ryanodine receptor. It is theoretically possible that
BDM improves SR Ca2+ release in failing hearts by its
phosphatase action. In any event, our finding of normal to enhanced SR
function in nonhypertrophied MLPKO myocytes isolated in the presence of
BDM strongly suggests that impaired SR Ca2+-ATPase function
is not the primary cause of heart failure in this genetic model.
Further studies in this model will be required to determine the
mechanisms of the increased [Ca2+]i
transient, whether it is a compensatory process or part of the
pathogenesis of failure that develops in these animals, and the nature
of secondary effects on myocytes that may contribute to progression of
heart failure.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Steven C. Hunt for assistance with the statistical analysis and Pamela Larson for assistance in preparation of this manuscript.
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FOOTNOTES |
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* Authors contributed equally to this work.
This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-53773.
Address for reprint requests and other correspondence: W. H. Barry, Div. of Cardiology, Univ. of Utah Health Science Center, 50 No. Medical Dr., Salt Lake City, UT 84132 (E-mail: whbarry{at}med.utah.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 June 2000; accepted in final form 10 January 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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) in
these myocytes and tended to be shorter at higher peak
[Ca2+]i. Also, the mean value of 
) and MLPKO
(
) myocytes (see the data in Table 
; n = 6) myocytes. Inset:
representative ICa,L at 0 mV in a WT
(left) and MLPKO (right) myocyte.
