AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 280: H2665-H2673, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 6, H2665-H2673, June 2001

Effects of deletion of muscle LIM protein on myocyte function

Zhi Su1,*, Atsushi Yao1,*, Iram Zubair1, Kazuro Sugishita1, Michael Ritter1, Fenghua Li1, John J. Hunter2, Kenneth R. Chien2, and William H. Barry1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 delta -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.


    MATERIAL AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ) 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 (tau ) value approx  500 ms under our experimental conditions. The current values at greater than five times tau  (approx 2,500 ms) from the peak of the current were used as a baseline for current transient integration.

For analysis, all records of [Ca2+]i transients, currents, and pacing signals were simultaneously digitized and acquired at the sampling rate of 1 kHz (Axo Scope, Axon Instruments). Currents were analyzed with pCLAMP6.0 software (Axon Instruments), and digitized [Ca2+]i transients were analyzed with Origin (Microcal).

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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  		Table 1.   Evidence of cardiac enlargement and failure in MLPKO mice

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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  		Fig. 1.   Organization of actin filaments. Actin filaments were stained with tetramethylrhodamine isothiocyanate-labeled phalloidin and imaged with a confocal microscope. Compared with wild-type (WT) myocytes (A and B), muscle LIM protein (MLP) knockout (MLPKO) myocytes (C and D) show less distinct sarcomeres and less aligned Z bands. Calibration white bars in A and C and in B and D are 10 and 2 µm, respectively.

[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. tau  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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  		Fig. 2.   Examples of simultaneous recordings of intracellular Ca2+ concentration ([Ca2+]i) transients and cell shortenings. In a MLPKO myocyte (right), the amplitude of [Ca2+]i transients is increased relative to that of the WT cell (left). The fractional shortening (FS) was similar in WT and MLPKO myocytes.


                              
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  		Table 2.   Characteristics of [Ca2+]i transients

The decline of [Ca2+]i transients has been shown to be dependent on the peak [Ca2+]i (5). It is, therefore, possible that the acceleration of decline of [Ca2+]i transients in MLPKO myocytes is the result of the higher peak [Ca2+]i. To elucidate this issue, we examined the relationship between tau  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 tau  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 tau  as a function of the peak [Ca2+]i. As shown by the scattered open circles in Fig. 3, tau  tended to be shorter in WT cells as the peak [Ca2+]i was increased. The mean values of tau  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 tau  in MLPKO myocytes was similar to the average value for WT cells at a comparable [Ca2+]i. This suggests that the smaller tau  in MLPKO myocytes is due primarily to higher peak systolic [Ca2+]i levels.


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  		Fig. 3.   Relationships between the time constant of [Ca2+]i decline (tau ) and peak [Ca2+]i. [Ca2+]i transients were obtained from five WT myocytes during exposure to high extracellular Ca2+ (2.16 mM). The tau  of each [Ca2+]i transient was plotted as a function of the peak [Ca2+]i (open circle ) in these myocytes and tended to be shorter at higher peak [Ca2+]i. Also, the mean value of tau  was plotted as a function of the mean value of the peak systolic [Ca2+]i in WT () and MLPKO () myocytes (see the data in Table 2). The mean value for MLPKO cells fell within the range observed for WT myocytes.

Because the [Ca2+]i transient amplitude was enhanced in MLPKO myocytes, whereas FS was similar to WT myocytes as shown in Fig. 2, it appeared that the sensitivity of shortening to Ca2+ might be reduced in these myocytes. To confirm this, FS values at comparable peak [Ca2+]i values were obtained from five WT myocytes exposed to 2.16 mM of Ca2+ solution. The results are shown in Fig. 4. The mean FS in MLPKO myocytes was significantly smaller compared with the FS at a comparable peak systolic [Ca2+]i in WT myocytes. This suggests that the response of myofilaments to Ca2+ and/or the transmission of force from myofilaments to the sarcomere is reduced in MLPKO myocytes.


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  		Fig. 4.   Relationship between cell shortening and peak [Ca2+]i. The mean value of FS was plotted as a function of the mean peak [Ca2+]i in WT (4.4 ± 0.6% and 346 ± 21 nM, respectively, n = 11; ) and MLPKO myocytes (5.2 ± 0.9% and 686 ± 74 nM, respectively, n = 10; ). The mean FS at comparable peak [Ca2+]i in WT myocytes (12.8 ± 1.4% and 691 ± 11 nM, respectively, n = 5) was also plotted in this figure (open circle ) and shows significantly larger FS compared with MLPKO myocytes. *P < 0.0001.

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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  		Fig. 5.   L-type Ca2+ channel function. The current-voltage relationships of the L-type Ca2+ channel current (ICa,L) and current density are similar in WT (; n = 15) and MLPKO (; n = 6) myocytes. Inset: representative ICa,L at 0 mV in a WT (left) and MLPKO (right) myocyte.

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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  		Fig. 6.   Representative Na/Ca exchanger currents (INa/Ca). The outward reverse INa/Ca elicited by the elimination of extracellular Na+ was normalized to membrane capacitance in a WT (thin line) and MLPKO (thick line) myocyte. There was no obvious difference between the two myocytes.

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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  		Fig. 7.   Sarcoplasmic reticulum (SR) Ca2+ content measurements. The application of caffeine with a rapid switcher device released Ca2+ from the SR. Released Ca2+ was removed in exchange for Na+ via forward Na/Ca exchange with a stoichiometry of 3 Na to 1 Ca2+, causing inward currents as shown in a WT (thin line) and MLPKO (thick line) myocyte (see MATERIALS AND METHODS). The integral of the inward current normalized to cell capacitance was used as an indicator of SR Ca2+ content and was larger in the MLPKO myocyte.

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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  		Fig. 8.   Rate of quenching of fura 2 fluorescence by exposure to 10 mM Mn2+. The rate of decline in fluorescence was similar for MLPKO (n = 8) and WT (n = 8) myocytes, suggesting that sarcolemmal cation permeability is not increased in MLPKO cells.

To examine [Ca2+]i shortening relationships under more steady-state conditions than are present during a single contraction, we measured F/Fo and FS in MLPKO and WT myocytes before and after 1.5 s and 3 min of pacing at 10 Hz in the presence of 5 µM ryanodine. The results are shown in Table 3. As before, during baseline pacing at 0.25 Hz, F/Fo was significantly greater in MLPKO than WT myocytes, whereas FS was insignificantly lower in MLPKO cells. After 3 min of tetanization, there was no significant difference in F/Fo or FS between MLPKO and WT myocytes. It appears that a maximal FS at steady state is achieved in both types of myocytes at the [Ca2+]i levels obtained during rapid pacing in the presence of ryanodine.

                              
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  		Table 3.   Comparisons of peak [Ca2+]i and FS in WT and MLPKO mouse myocytes during baseline twitches and tetanus

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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  		Table 4.   Effects of dissociation in BDM on [Ca2+]i transients and shortening


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
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DISCUSSION
REFERENCES

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 beta -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.


    ACKNOWLEDGEMENTS

The authors thank Dr. Steven C. Hunt for assistance with the statistical analysis and Pamela Larson for assistance in preparation of this manuscript.


    FOOTNOTES

* 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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Am J Physiol Heart Circ Physiol 280(6):H2665-H2673
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


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