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Am J Physiol Heart Circ Physiol 281: H2539-H2548, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 6, H2539-H2548, December 2001

Compensatory changes in Ca2+ and myocardial O2 consumption in beta -tropomyosin transgenic hearts

Guy A. MacGowan4, Congwu Du1,3, David F. Wieczorek6, and Alan P. Koretsky1,2,5

1 Pittsburgh Nuclear Magnetic Resonance Center for Biomedical Research, 2 Department of Biological Sciences, and 3 Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, 15213; 4 Cardiovascular Institute of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213; 5 Laboratory of Functional and Molecular Imaging, National Institute of Neurological Disease and Stroke, Bethesda, Maryland 20892; and 6 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transgenic mice overexpressing beta -tropomyosin have increased myofilament Ca2+ sensitivity that we hypothesized would result in altered relationships among pressure and heart rates, intracellular Ca2+, and myocardial O2 consumption. In perfused hearts from transgenic mice there was a marked negative force-frequency response between 6 and 10 Hz with a 30 ± 3% reduction in peak-positive first derivative of pressure development over time (dP/dt) compared with 14 ± 2% in wild-type mice (P < 0.001). At 8 Hz systolic pressures were normal, though peak systolic intracellular Ca2+ was significantly reduced in transgenic mice versus wild type (726 ± 61 vs. 936 ± 67 nM, P < 0.05) indicating an alteration in the pressure-Ca2+ relationship. Over a wide range of positive and negative inotropic interventions there were normal developed pressures, though marked elevations in myocardial O2 consumption (15-54%). Because pressures are normal and intracellular Ca2+ decreased and myocardial O2 consumption increased, this suggests that these abnormalities are at least in part compensatory mechanisms to the altered myofilament function.

pressure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TROPOMYOSIN HAS A CENTRAL ROLE in normal myofilament function (28). Ca2+ binding to troponin C results in displacement of tropomyosin, which exposes actin sites to myosin binding, resulting in myofilament shortening. Tropomyosin has three isoforms. There are more alpha alpha -homodimers (27) in fast-contracting cardiac muscle and more beta beta -homodimers in slow-contracting muscle. Tropomyosin 3 is expressed in slow-twitch skeletal muscle, and is not expressed in the murine heart at any stage of development (26). To study the physiological significance of the alpha - and beta -tropomyosin isoforms, Muthuchamy and colleagues (23) developed a transgenic (TG) mouse overexpressing beta -tropomoysin in the heart. There were no structural abnormalities, though physiological analysis in the working heart preparation revealed abnormal diastolic function with a reduced maximum rate of relaxation. Subsequent studies (24) in isolated fiber bundles revealed an increase in the activation of the thin filament by strongly bound cross bridges, an increase in the Ca2+ sensitivity of steady-state force, and a decrease in the rightward shift of the Ca2+ force relation induced by cAMP-dependent phosphorylation (24).

The significance of these findings with respect to the dynamics of contraction and relaxation were studied by Wolska et al. (32). Isolated myocytes from TG mice exhibited decreased maximal rates of contraction and relaxation, though no change in the absolute length of shortening. Detergent extracted fibers from TG mice exhibited significantly less maximum tension and ATPase activity than wild-type (WT) mice. The authors concluded that these changes in dynamics were related either to the increased sensitivity of the myofilament to Ca2+, or altered feedback effects of force-bearing cross bridges on activation (7).

On the basis of these studies, we hypothesized that in perfused hearts from mice overexpressing beta -tropomyosin there would be an altered force-Ca2+ relationship secondary to the increased Ca2+ sensitivity of the myofilament. Also, myofilament abnormalities would result in compensatory effects on Ca2+ handling. There is growing evidence that alterations in myofilament function lead to changes in Ca2+ handling (12, 14). Perez et al. (25) recently showed that in the spontaneous hypertension and heart failure rat, Ca2+ cycling abnormalities were secondary to myofilament dysfunction. Also, we (18) have shown that in a TG mouse expressing mutant troponin I-lacking protein kinase C phosphorylation sites there is prolongation of Ca2+ transients and reduction of intracellular Ca2+ levels with Ca2+-induced inotropy. Because the sarcoplasmic reticulum is predominantly responsible for Ca2+ handling in the mouse heart (3), we predicted that the altered Ca2+ handling would result in abnormal responses to altered heart rates. Also, because Ca2+ cycling is an important determinant of myocardial O2 consumption, we measured myocardial O2 consumption during a series of positive and negative inotropic interventions. To address this hypothesis, we studied intracellular Ca2+ concentration ([Ca2+]i), myocardial O2 consumption, and force-frequency relationships in Langendorff perfused mouse hearts.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Study design. All studies were performed on perfused mouse hearts, in age-matched male FVB WT and TG mice (range 6-32 wk). The age matching was consistent throughout all the experiments. In one group of experiments, intracellular Ca2+ was measured with the Ca2+-sensitive fluorescent probe rhod 2 along with developed pressures. In a second group of experiments, inotropic responses and myocardial O2 consumption were measured. Experiments were performed at 8 Hz, which is close to the physiological heart rate for the mouse. In a subset of the second group, measurements were taken at 6, 8, and 10 Hz. All TG studies were performed in line 10, which expresses an approximately twofold greater amount of beta -tropomyosin than alpha -tropomyosin protein (23).

Perfused mouse hearts. Anesthesia was induced with an intraperitoneal injection of pentobarbital sodium (1.5-3.0 mg). The animal was then anticoagulated with 100 units of heparin sodium. Hearts were gravity perfused with a perfusion pressure of 60 mmHg and stimulated at the physiological mouse heart rate of 8 Hz, unless otherwise stated. The left ventricular diastolic pressure was set at 0-10 mmHg with the use of a microsyringe. The modified Krebs solution used was composed of 113 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 0.5 mM Na-EDTA, 28.0 mM NaHCO3, 5.5 mM glucose, 5.0 mM pyruvate, 2.5 mM CaCl2, and 50 µM octanoate, and was oxygenated with 95% O2-5% CO2, with the pH adjusted to 7.4.

Measurement of intracellular Ca2+. The methods used to measure intracellular Ca2+ with rhod 2 in perfused hearts have been previously extensively described (9-11, 19). Rhod 2 (100 µg for 2.5 mM experiments and 200 µg for 1.5 mM experiments; Molecular Probes; Eugene, OR) was dissolved with dimethyl sulfoxide (4 µl) and dH2O (200 µl), and loaded through the coronary perfusate. After the washout period, serial measurements of fluorescence alternating with absorbance were taken (10). Fluorescence recordings were taken at high time resolution to allow quantification of changes in fluorescence during the cardiac cycle. Excitation at 524 nm and emission at 589 nm was used for fluorescence measurements. At 8 Hz, fluorescence data was recorded for 10 s at 0.008 s intervals with an integration time for each point of 0.0062 s, resulting in 16 points per beat. At 10 Hz, data were collected every 0.006 s with an integration time of 0.0042 s resulting in 17 points per beat, and at 6 Hz data were collected every 0.01 s with an integration time of 0.0082 s resulting in 17 points per beat.

Quantification of the relative amount of rhod 2 in the heart using absorbance measurements was done by taking the ratio of absorbance at 524 nm (rhod 2 sensitive) to 589 nm (rhod 2 insensitive), which eliminated the effect of motion as both wavelengths would be equally affected by motion, though only 524 reflected the concentration of rhod 2 (9, 10). These wavelengths (524 and 589 nm) were chosen because these were isosbestic points not affected by changes in absorbance of myoglobin induced by O2 desaturation (10). In solution, maximal rhod 2 absorbance is at 554 nm. However, this wavelength is affected by changes in O2 saturation, the relative absorbance compared with 524 nm is decreased due to inner filter effects, and it does not adequately correct for changes in scattering (10). Dye absorbance (Arhod2) was calculated according to the formula
<IT>A</IT><SUB>rhod2</SUB><IT>=</IT>log [(R<SUB>524</SUB><IT>/</IT>R<SUB>589</SUB>)<SUB>0</SUB><IT>/</IT>(R<SUB>524</SUB><IT>/</IT>R<SUB>589</SUB>)<SUB>rhod2</SUB>] (1)
where R524 is the reflectance intensity at the rhod 2-sensitive point of 524 nm, and R589 is the rhod 2-insensitive point, before ()0 and after ()rhod2 loading.

At the end of the perfusion protocol, maximal fluorescence, used in the calculation of [Ca2+]i, was determined by tetanizing the heart with a 20 mM bolus of Ca2+ chloride without any energy substrate, and with 10 µM cyclopiazonic acid (Sigma), which is a potent inhibitor of Ca2+-ATPase and thus blocks Ca2+ uptake by the sarcoplasmic reticulum (2). Fluorescence and pressure were monitored continuously, and the point of maximal fluorescence was taken as the point where pressure stabilized at a steady state. To account for changes in light scattering properties from the heart during tetanization, the maximal fluorescence was corrected by multiplying by the ratio of R524 pretetanization to R524 during tetanization (10).

Intracellular Ca2+ was calculated using the formula
[Ca<SUP>2+</SUP>]<SUB>i</SUB><IT>=K</IT><SUB>d</SUB>(F<SUB><IT>t</IT></SUB><IT>−</IT>F<SUB>0</SUB>)<IT>/</IT>(F<SUB>max</SUB><IT>−</IT>F<SUB><IT>t</IT></SUB>) (2)
where Kd is the dissociation constant for rhod 2 and Ca2+ [determined by in vitro calibration with rhod 2 and myoglobin by del Nido et al. (9), and confirmed by in vivo manganese quenching (11)] and is 710 nM, Ft = fluorescence at time t, Fmax = maximal fluorescence from tetanized heart, and the fluorescence from the heart, assuming rhod 2 was not Ca2+ bound, is given by F0 = Fb + a(Fmax - Fb), where Fb is the background counts from the heart before dye loading, and a is rhod 2 fluorescence in the absence of Ca2+/rhod 2 fluorescence in the presence of saturating Ca2+. For rhod 2, the value of a is ~0; thus for rhod 2, F0 was assumed to be equal to Fb.

To account for changes in dye concentration, Eq. 2 needs to be modified to account for changes in absorbance (Arhod2, Eq. 1) due to dye leakage
[Ca<SUP>2<IT>+</IT></SUP>]<SUB><IT>i</IT></SUB><IT>=K</IT><SUB>d</SUB>[(F<SUB><IT>t</IT></SUB><IT>−</IT>F<SUB>0</SUB>)<IT>/A<SUB>t</SUB></IT>]<IT>/</IT>{[(F<SUB>max</SUB><IT>−</IT>F<SUB>0</SUB>)<IT>/A</IT><SUB>max</SUB>] (3)

<IT>−</IT>[(F<SUB><IT>t</IT></SUB><IT>−</IT>F<SUB>0</SUB>)<IT>/A<SUB>t</SUB></IT>])}
where At is dye absorbance at time t, Amax is dye absorbance just before tetanizing the heart. Amax is not determined when heart has tetanized because of the marked influence of the shape change and desaturation of myoglobin on the reflectance spectrum.

At perfusate Ca2+ 2.5 mM, intracellular Ca2+ was quantified at 8 Hz alone in n = 5 for both WT and TG. Intracellular Ca2+ was also quantified at 6, 8, and 10 Hz (in alternating orders) in WT (n = 6) and TG (n = 4) mice. In addition, force-frequency pressure responses were also measured in a group of hearts without concomitant intracellular Ca2+ measurements in WT (n = 10) and TG (n = 7) mice. Ca2+ and pressure data from all of these groups are presented together where applicable because results were consistent between groups. At perfusate Ca2+ 1.5 mM, intracellular Ca2+ was quantified at 8 Hz alone in WT (n = 3) and TG (n = 2).

Myocardial O2 consumption and inotropic responses in perfused mouse hearts. Because Ca2+ handling may be an important determinant of myocardial O2 consumption, this was measured under a range of inotropic states. Also, because a decrease in the expected rightward shift of the force-Ca2+ relationship has been demonstrated in these TG mice (24), we determined the significance of this by giving the predominant beta -adrenergic agonist dobutamine. Myocardial O2 consumption was determined from influent and effluent O2 content and coronary flow rate. A baseline period, lasting ~30 min until repeated measurements of myocardial O2 consumption and developed pressures were at steady state, was followed by an inotropic intervention. The following protocols were performed: 1) baseline perfusate Ca2+ 2.5 mM, followed by the beta -adrenergic agonist dobutamine 0.9 µM (WT, n = 6; TG, n = 8), 2) baseline perfusate Ca2+ 2.5 mM, followed by perfusate Ca2+ 3.5 mM (WT and TG, n = 6), and 3) baseline perfusate Ca2+ 1.5 mM, followed by perfusate Ca2+ 0.5 mM (WT, n = 4; TG, n = 5). In addition, in n = 4 for both WT and TG, myocardial O2 consumption was measured at 6, 8, and 10 Hz.

Data and statistical analysis. Analysis of Ca2+ transients was performed after each individual fluorescence data point had been converted to [Ca2+] because the relationship between fluorescence and [Ca2+]i may not be linear (7). Ca2+ transient decay was then calculated from the peak of the transient to 90% decay, and also the decay constant tau  Ca2+ was calculated using the exponential curve fitting, Y = m1 · e-cx, where Y is intracellular Ca2+, m1 is the arbitrary constant, c is the decay time constant, and x is time. tau  was calculated from the first point after the peak of the Ca2+ transient to 90% of the transient decay. Because the asymptote is not zero, the asymptote value was subtracted out from each data point (6, 13).

Unpaired comparisons were performed using the unpaired t-test. Repeated-measures analysis of variance (ANOVA) was used for force-frequency data. Multiple comparisons were performed with ANOVA with Scheffé's test for subgroup analysis. Data are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pressures. There was a marked negative force-frequency response in TG mice, greater than that seen in WT mice (Fig. 1). No significant differences in pressures or first derivative of pressure development over time (dP/dt) were seen at 6 Hz, though there was significant elevation of diastolic pressures and reduction in peak-positive dP/dt at 8 and 10 Hz. Between 6 and 10 Hz, peak-positive dP/dt decreased by 14 ± 2% in WT mice, though by 30 ± 3% in TG mice (P < 0.001). Developed pressures (systolic-diastolic) were not significantly different at 6 or 8 Hz between WT and TG mice (6 Hz: 70 ± 1 vs. 70 ± 3 mmHg, and 8 Hz: 59 ± 2 vs. 56 ± 2 mmHg, P = not significant), though they were significantly reduced at 10 Hz in the TG mice (42 ± 1 vs. 37 ± 2 mmHg, P < 0.05). These effects at 10 Hz were not due to ischemia because coronary flow did not change during the changes in heart rate. Coronary flows in TG mice at 6, 8, and 10 Hz were 2.3 ± 0.1 ml, and in the TG mice were 2.1 ± 0.1 ml/min.


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  		Fig. 1.   Force-frequency relationships (6, 8, and 10 Hz, perfusate Ca2+ 2.5 mM) in transgenic (TG) and wild-type (WT) mice. Systolic pressure (A), diastolic pressure (B), peak-positive first derivative of pressure development over time (dP/dt) (C), and peak-negative dP/dt (D). *P < 0.01; **P < 0.005, WT vs. TG mice.

Reduced intracellular Ca2+ and altered pressure-Ca2+ relationship. At perfusate Ca2+ 2.5 mM, peak systolic intracellular Ca2+, though not diastolic Ca2+, was reduced at 6, 8, and 10 Hz in TG mice compared with WT mice (Table 1). Because this was associated with normal developed pressures at 6 and 8 Hz, at these frequencies, this indicates an alteration in the pressure-Ca2+ relationship. That Ca2+ levels are reduced and not normal and pressures are normal and not increased indicates that there are compensatory changes in Ca2+ handling to the altered myofilament function (Fig. 2). This effect is less marked at 10 Hz when both developed pressures and peak systolic Ca2+ are reduced. There were no significant differences in peak systolic, diastolic, or Delta Ca2+ by repeated-measures ANOVA [P = not significant (NS)] between heart rates within each group.

                              
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  		Table 1.   [Ca2+]i in WT and TG mice



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  		Fig. 2.   Examples of simultaneous recordings of left ventricular pressure and intracellular Ca2+ (8 Hz, perfusate Ca2+ 2.5 mM) in WT mice (A) and TG mice (B) illustrating altered pressure-Ca2+ relationship. The pressure-Ca2+ relationship (C) indicates a leftward shift in TG mice associated with the altered pressure-Ca2+ relationship.

Additional fluorescence data from the perfusate Ca2+ 2.5 mM experiments are presented in Table 2 and indicate that the fundamental aspects of the fluorescence experiments are similar between the two groups, such as background fluorescence, maximal fluorescence after tetanization, and fluorescence increase after rhod 2 loading. However, the ratio of Delta  fluorescence to diastolic fluorescence, which is a relative measure of intracellular Ca2+ (10), is significantly reduced in TG mice, and this agrees well with the ratio of Delta Ca2+ to diastolic Ca2+, which is also significantly reduced in the TG mice. These data show that the significant reduction in intracellular Ca2+ in the TG mice is not an artifact of abnormal rhod 2 loading, absorption measurements or the tetanization procedure. At perfusate Ca2+ 1.5 mM, peak systolic and Delta  systole-diastole intracellular Ca2+ were also reduced in the TG mice (Fig. 3 and Table 1).

                              
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  		Table 2.   Additional fluorescence data from rhod-2 experiments



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  		Fig. 3.   Peak systolic intracellular Ca2+ and systolic pressures at perfusate Ca2+ 1.5 and 2.5 mM. Decrease in intracellular Ca2+ in the TG mice is proportionally similar at both levels of perfusate Ca2+. *P < 0.05 and #P = 0.05, WT vs. TG.

Ca2+ transients. In WT mice, Ca2+ transient intervals shortened as heart rates increased (Fig. 4). When normalized to the length of the cardiac cycle, the transient interval is seen to increase with higher heart rates, indicating that there is less available time for relaxation. In WT mice, shortening of the transient occurred between both 6 and 8 Hz and 8 and 10 Hz, though the extent of shortening was greater between 8 and 10 Hz (tau Ca2+, 6-8 Hz: -4%; 8-10 Hz: -16%). In the TG mice, shortening of the transient also occurred with increasing heart rates, though this was at 6-8 Hz and not between 8 and 10 Hz (tau Ca2+, 6-8 Hz: -20%; 8-10 Hz: +5%). This altered pattern of Ca2+ transient shortening with increased rate resulted in a significant abbreviation of the Ca2+ transient at 8 Hz in TG mice compared with WT mice, though not at 6 or 10 Hz. Examples of perfusate Ca2+ 2.5 mM Ca2+ transients at 6, 8, and 10 Hz and normalized Ca2+ transients are shown in Fig. 5. There were no significant differences in Ca2+ transient duration at perfusate Ca2+ 1.5 mM (8 Hz) between WT and TG mice (time to 90% decay: WT, 60 ± 4 vs. TG, 60 ± 10 ms, P = not significant).


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  		Fig. 4.   Effects of rate on intracellular Ca2+ transient decay (perfusate Ca2+ 2.5 mM). A: time from peak of the transient to 90% decay. B: time from peak of the transient to 90% normalized to heart rate (R-R interval). C: tau  Ca2+. D: tau  Ca2+ normalized to R-R interval. *P < 0.05, TG vs. WT mice.



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  		Fig. 5.   Examples of averaged Ca2+ transients (perfusate Ca2+ 2.5 mM, 5-8 cycles) at 6 Hz (A), 8 Hz (B), and 10 Hz (C), and averaged normalized Ca2+ transients at 6 Hz (D), 8 Hz (E), and 10 Hz (F). Peak systolic intracellular Ca2+ is reduced in TG mice compared with WT mice, though there is no difference in diastolic Ca2+. At 8 Hz, the Ca2+ transient is abbreviated in TG mice, though at 6 and 10 Hz, there are no differences.

Increased myocardial O2 consumption and altered pressure-myocardial O2 consumption relationship. There were no significant differences in developed pressures when perfusate Ca2+ was varied from 0.5 to 3.5 mM, or with dobutamine (Fig. 6A). There were significant increases in myocardial O2 consumption at all inotropic states studied in the TG mice compared with WT mice (all P < 0.05, and P < 0.005; Fig. 6B). This difference was greater at perfusate Ca2+ 0.5 mM (54%) compared with perfusate Ca2+ 3.5 mM (15%), or with dobutamine (21%) (Fig. 6C). This indicates an alteration in the pressure-myocardial O2 consumption relationship in the TG mice. Systolic and diastolic pressures, balloon volumes, and effluent O2 saturations are detailed in Table 3. These indicate that the relationship of pressures to isovolumic volume is similar over a wide range of inotropy, with only a few exceptions. For instance, diastolic pressure was significantly elevated in TG mice only at perfusate Ca2+ 2.5 mM (P < 0.05). Also, at perfusate Ca2+ 2.5 mM only, though not other inotropic states, coronary flow and effluent PO2 were significantly lower in the TG mice (both P < 0.05). Balloon volumes were significantly lower in the TG mice in the perfusate Ca2+ range of 2.5-3.5 mM (P < 0.05), though there were no significant differences in the other experiments. There were no differences in heart weight-to-body weight ratios between WT and TG mice (WT = 0.0048 ± 0.0001, n = 16; TG = 0.0046 ± 0.0001, n = 19; P = NS).


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  		Fig. 6.   A: developed pressure during Ca2+-induced inotropy (0.5-3.5 mM) and dobutamine (0.9 µM) in WT and TG mice. B: myocardial O2 consumption during Ca2+-induced inotropy (0.5-3.5 mM) and dobutamine (0.9 µM) in WT and TG mice. *P < 0.05 and **P < 0.005 WT vs. TG. Normal developed pressures are seen at all inotropic levels in TG mice, though there is increased myocardial O2 consumption throughout. C: % difference in myocardial O2 consumption between WT and TG mice. Differences are greater at lower levels of perfusate Ca2+. D: effects of rate (6-10 Hz) on myocardial O2 consumption. There were no significant differences within each group with altered heart rates. *P < 0.05 WT vs. TG mice.


                              
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  		Table 3.   Systolic and diastolic pressures, balloon volume, coronary flow, and effluent PO2 in WT and TG with altered perfusate calcium and dobutamine

There was no significant difference in myocardial O2 consumption between 6, 8, and 10 Hz (repeated-measures ANOVA) within each group (Fig. 6D). As developed pressures were significantly reduced at 10 Hz (though not at other heart rates), this indicates a greater alteration in the pressure-myocardial O2 consumption relationship at higher heart rates. Also, because coronary flows did not change with heart rate (see above), and myocardial O2 consumption remained elevated at all heart rates, this indicates that the negative force-frequency relationship and failure to abbreviate transients from 8 to 10 Hz in the TG mice are not the result of ischemia.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that in TG mice overexpressing beta -tropomyosin there is an alteration in the force-Ca2+ relationship with reduced peak systolic intracellular Ca2+ levels though normal systolic pressures. That intracellular Ca2+ is reduced and pressures are normal, rather than normal Ca2+ and increased pressures, indicates an adaptation in Ca2+ handling in response to the altered myofilament function. There is an altered pattern of Ca2+ transient abbreviation with increasing heart rates, with greater shortening of the transient at lower heart rates in the TG mice. There is increased myocardial O2 consumption over a wide range of inotropy. All of these findings are modulated by increasing heart rates, which are associated with marked left ventricular dysfunction in TG mice. The altered force-Ca2+ relationship is less marked at 10 Hz because there are reduced developed pressures as well as reduced intracellular Ca2+. Ca2+ transients are abbreviated at 8 Hz though not at 6 Hz and 10 Hz. At 10 Hz, myocardial O2 consumption remains elevated despite significant reductions in developed pressures, indicating a greater alteration in the pressure-myocardial O2 consumption relationship.

Functional abnormalities are not due to structural change in beta -tropomyosin TG mice. The TG mice overexpressing beta -tropomyosin have been well characterized. Several studies have demonstrated functional abnormalities that appear to relate to altered myofilament function. For instance, Muthuchamy et al. (23) have shown altered diastolic function in perfused mouse hearts, though no abnormalities were detected by light or electron microscopy, or with immunological staining using a striated muscle-specific tropomyosin monoclonal antibody. Subsequently, Wolska et al. (32) demonstrated that there were reduced maximal rates of contraction and relaxation in isolated myocytes, indicating that the isolated heart findings of Muthuchamy et al. (23) and the present study are intrinsic to the cells. Furthermore, there is no difference in heart weight-to-body weight ratios in the present study, and throughout a wide range of inotropy there are only minimal differences in systolic or diastolic pressures and isovolumic balloon volumes. Furthermore, the rate dependence of the abnormalities in the present study suggests that these findings are not structural. Thus the findings in the present study are related to intracellular abnormalities.

Altered force-Ca2+ relationship and Ca2+ transients. It is important to note that our measurements of intracellular Ca2+ relate to cytosolic free Ca2+, and not the total amount of Ca2+ cycled in a cardiac cycle, which also includes Ca2+ bound to cytosolic proteins, such as troponin C and other Ca2+-buffering proteins. There are several potential explanations for the altered force-Ca2+ relationship and Ca2+ transients, including: 1) sarcoplasmic reticulum function is altered, 2) troponin C Ca2+ binding is increased because of the increased Ca2+ sensitivity, and 3) there is increased Ca2+ binding by cytosolic Ca2+ buffering proteins other than troponin C.

Altered function of the sarcoplasmic reticulum may explain most, if not all, of our findings. Indeed, in the mouse, the sarcoplasmic reticulum is responsible for >90% of Ca2+ removal from troponin C (3). The reduced peak systolic Ca2+ may relate to reduced Ca2+ release from the sarcoplasmic reticulum, presumably as a compensatory response to the increased myofilament Ca2+ sensitivity. The abbreviated Ca2+ transients at 8 Hz may be secondary to the reduced levels of peak systolic intracellular Ca2+ (4), because if less Ca2+ is released, it may not take as long to take up Ca2+ into the sarcoplasmic reticulum. However, this does not explain the observation that the Ca2+ transient is not abbreviated relative to WT with perfusate Ca2+ 2.5 mM at 6 and 10 Hz or perfusate Ca2+ 1.5 mM (8 Hz) when reduced peak systolic intracellular Ca2+ is also seen. This suggests therefore that the abbreviation of the Ca2+ transient is at least in part related to a factor other than the reduced peak systolic Ca2+, and that it is a compensatory response, of which one explanation is enhanced uptake of intracellular Ca2+ by the sarcoplasmic reticulum.

The frequency dependence of many of our findings may implicate the sarcoplasmic reticulum. Frequency-dependent changes in force of contraction are present in most mammalian species, and are also species dependent. For instance, in the guinea pig, rabbit, and human myocardium, there is an increase in force with increasing stimulation frequency, though in the rat and mouse there is a negative force frequency relationship (3). Sarcoplasmic reticulum Ca2+ ATPase and phospholamban are especially implicated in the murine heart force frequency responses (16, 21). Expression of sarcoplasmic reticulum Ca2+ ATPase and phospholamban are unchanged in these mice (23), though phosphorylation differences could account for the altered transients. It is important to note that the supply of O2 to the myocardium in the TG mice is unchanged at the higher heart rates because coronary flow does not change, so this phenomenon is not a consequence of ischemia. Also, because myocardial O2 consumption does not change between 8 and 10 Hz, this does not appear related to increased ATP utilization above that seen at 8 Hz.

The increased myocardial O2 consumption may provide indirect evidence supporting this hypothesis of altered sarcoplasmic reticulum function. During positive inotropy, there are two major determinants of myocardial O2 consumption: ATP consumption related to Ca2+ cycling and actomyosin ATPase activity (20). Recently, Brandes et al. (5) estimated that these factors each account for 50% of the energetic requirements of myocardial contraction. It is known that in these TG mice the economy of contraction related to actomyosin ATPase activity is unchanged compared with WT mice (32), so that the increased myocardial O2 consumption in the present study is likely related to increased Ca2+ cycling. The increased energy utilization may be a factor in the abbreviation in the Ca2+ transient.

The reduced peak systolic Ca2+ may be a consequence of increased troponin C Ca2+ binding. This factor alone, however, does not explain how pressures are normal and not increased, as would be expected with increased Ca2+ sensitivity of the myofilament. One possibility is that the increased troponin C Ca2+ binding is accompanied by the myofilament dysfunction, as documented by Wolska et al. (32), which might result in reduced systolic Ca2+ though normal pressures. However, Allen and Kurihara (1) have stated that increased troponin C Ca2+ binding will only have a relatively small effect on the peak of the Ca2+ transient, because only 10-20% of troponin C Ca2+ binding has occurred at the time of the transient peak. The abbreviation of the Ca2+ transient may also reflect increased troponin C Ca2+ binding. There have been variable results in the literature regarding the effects of increased Ca2+ sensitivity on intracellular Ca2+ transients. Hajjar et al. (15) showed prolonged transients in failing and nonfailing human myocardium with the Ca2+ sensitizing agents EMD-57033 and ORG-30029. However, Lee and Allen (17) have shown that with the Ca2+ sensitizing agent EMD-53998 there is abbreviation of the Ca2+ transient. Lee and Allen (17) addressed the issues that might give rise to a shortened or prolonged transient in a computer model based on the Ca2+ fluxes in and out of the cell, Ca2+ binding to troponin C, and the attachment of cross bridges. This model also predicted shortening of the Ca2+ transient when the troponin C binding constant for Ca2+ was increased by a factor of 2. However, if the feedback between tension and the troponin Ca2+ binding constant were removed, the time course of the transient decay was prolonged. The shortened Ca2+ transient was attributed to smaller Ca2+ efflux from troponin C, so that cytosolic Ca2+ falls more rapidly. However, this hypothesis does not explain the changes in the relative durations of the transient at different heart rates seen in the present study in the beta -tropomyosin TG mice.

Increased cytosolic Ca2+ binding by proteins other than troponin C may also produce similar findings, as in the present study. Indeed, it is interesting to note that in diseased myocytes, gene transfer of the Ca2+ buffer parvalbumin (that is not normally found in cardiac myocytes) corrects diastolic dysfunction with abbreviation of Ca2+ transients (31). With increased cytosolic Ca2+ binding, a blunted response to altered levels of intracellular Ca2+ would be expected. However, the experiments in the present study, with a wide range of perfusate Ca2+ and dobutamine demonstrating normal pressures relative to WT at all levels of inotropy, may not support this hypothesis.

Potential limitations. We used rhod 2 to study intracellular Ca2+, which some investigators (30) have found in mitochondria. Our previous experiments (9, 19) have shown that there is predominant cytosolic loading and it is possible that this discrepancy is the consequence of very different loading conditions in different models used by other investigators (30). Nevertheless, we cannot exclude some contribution of mitochondrial rhod-2 fluorescence to our measurements of intracellular Ca2+. Our methods do have the advantage that the same isolated perfused heart technique is used to measure pressures, myocardial O2 consumption, and intracellular Ca2+, so these measurements are all directly comparable. We currently obtain a limited number of time points for each transient (n = 16 or 17), which limits our ability to detect differences in Ca2+ transient duration, especially at 50% of the decay time when the decay is very rapid. Also, we have not studied intracellular Ca2+ over a wide range of perfusate Ca2+, due to instability of the TG hearts at the high-perfusate Ca2+ for the relatively long time required to perform these experiments.

In conclusion, perfused mouse hearts from TG mice overexpressing beta -tropomyosin demonstrate altered pressure-Ca2+ and pressure-myocardial O2 consumption relationships, and altered pressure responses and Ca2+ transient abbreviation with increased heart rates. The reduced intracellular Ca2+ and increased myocardial O2 consumption appear at least in part compensatory mechanisms to the altered myofilament function. Overexpression of beta -tropomyosin to levels greater than seen in those mice used in the present study is associated with marked cardiac enlargement and dysfunction (22). This hypertrophy can be prevented by inhibition of the Ca2+/calmodulin-dependent protein phosphatase calcineurin (29), implying a direct link with Ca2+ homeostasis. It is interesting to speculate that the abnormalities in Ca2+ homeostasis and myocardial O2 consumption demonstrated herein may directly contribute to hypertrophy when beta -tropomyosin is expressed at greater levels, as has been proposed for other TG models involving Ca2+ signaling (8).


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-03826 (to G. A. MacGowan) and HL-40354 (to A. P. Koretsky), National Institutes of Health Grant RR-03631 (to the Pittsburgh Nuclear Magnetic Resonance Center for Biomedical Research), and by the National Institute of Neurological Disorders and Stroke intramural research program.


    FOOTNOTES

Address for reprint requests and other correspondence: G. A. MacGowan, Cardiovascular Institute of the University of Pittsburgh Medical Center, S550 Scaife Hall, 200 Lothrop St., Pittsburgh, PA 15213 (E-mail: macgowanga{at}msx.upmc.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 13 December 2000; accepted in final form 27 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(6):H2539-H2548


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