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Am J Physiol Heart Circ Physiol 275: H2064-H2071, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 6, H2064-H2071, December 1998

Thermodynamic limitation for Ca2+ handling contributes to decreased contractile reserve in rat hearts

Rong Tian1, Jessica M. Halow2, Markus Meyer3, Wolfgang H. Dillmann3, Vincent M. Figueredo2,4, Joanne S. Ingwall1, and S. Albert Camacho2,4

1 Nuclear Magnetic Resonance Laboratory for Physiological Chemistry, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; 4 Division of Cardiology, Department of Medicine, San Francisco General Hospital, and 2 Department of Medicine, University of California, San Francisco 94110; and 3 Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California 92093

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The free energy release from ATP hydrolysis (|Delta G~p|) is decreased by inhibiting the creatine kinase (CK) reaction, which may limit the thermodynamic driving force for the sarcoplasmic reticulum (SR) Ca2+ pumps and thereby cause a decrease in contractile reserve. To determine whether a decrease in |Delta G~p| results in decreased contractile reserve by impairing Ca2+ handling, we measured left ventricular pressure and cytosolic Ca2+concentration ([Ca2+]c; by indo 1 fluorescence) in isolated perfused rat hearts, with >95% inhibition of CK with 90 µmol iodoacetamide. Iodoacetamide did not directly alter SR Ca2+-ATPase activity, baseline left ventricular developed pressure, or baseline [Ca2+]c. When perfusate Ca2+ concentration was increased from 1.2 to 3.3 mM, LV developed pressure increased from 67 ± 6 to 119 ± 8 mmHg in control hearts (P < 0.05) but did not significantly increase in CK-inhibited hearts. Similarly, the amplitude of the [Ca2+]c transient increased from 548 ± 54 to 852 ± 140 nM in control hearts (P < 0.05) but did not significantly increase in CK-inhibited hearts. We conclude that decreased |Delta G~p| limits intracellular Ca2+ handling and thereby limits contractile reserve.

free energy of adenosine 5'-triphosphate hydrolysis; sarcoplasmic reticulum; creatine kinase; calcium ion

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CONTRACTION AND RELAXATION of cardiac myocytes require rapid cycling of Ca2+ into and out of the cytosol (1, 3). This process utilizes a high level of free energy released from ATP hydrolysis (|Delta G~p|) (12, 15). One reason for this high-energy requirement is the ~10,000-fold concentration gradient of Ca2+ between the cytosol and the sarcoplasmic reticulum (SR), the primary source of Ca2+ (12, 15, 28). This gradient is maintained by the SR Ca2+-ATPase reaction, which pumps Ca2+ back into the SR during relaxation. Because the energy required by the SR Ca2+-ATPase reaction is directly related to the magnitude of the Ca2+ gradient across the SR, a high level of |Delta G~p| is required for this reaction in cardiac myocytes (12, 14).

To ensure a high level of |Delta G~p|, a high ratio of ATP concentration ([ATP]) to {ADP concentration ([ADP]) × Pi concentration ([Pi])} must be maintained [|Delta G~p| = |Delta Go - RTln([ATP]/[Pi][ADP])|], where Delta G° (-30.5 kJ/mol) is the value of Delta G~p under standard conditions of molarity, temperature, pH, and Mg2+ concentration, R is a gas constant, and T is absolute temperature. Recent studies suggest that the creatine kinase (CK) reaction (PCr + ADP + H+ left-right-arrow  ATP + Cr), where PCr is phosphocreatine and Cr is creatine, is an energy reserve system that maintains a high level of |Delta G~p| for the SR Ca2+-ATPase reaction by keeping a high ATP-to-ADP ratio (16, 20, 33). Tian and Ingwall (30) previously demonstrated that inhibition of CK activity by >95% in isolated perfused rat hearts resulted in a decrease in |Delta G~p|. Furthermore, the ability of the heart to increase contractile function (i.e., the contractile reserve) was limited when the |Delta G~p| was reduced below 52-53 kJ/mol (30). However, the mechanisms by which a decrease in |Delta G~p| limits contractile reserve have not been identified.

In this study, we tested the hypothesis that decreased |Delta G~p|, due to inhibition of CK, impairs intracellular Ca2+ homeostasis and thereby limits contractile reserve. Specifically, we determined whether inhibition of CK activity impairs the ability of the myocytes to increase free cytosolic Ca2+ concentration ([Ca2+]c) in response to an inotropic stimulation. [Ca2+]c (assessed by indo 1 fluorescence) and contractile function were determined in isolated perfused rat hearts during isovolumic perfusion. Inotropic stimulation was elicited by increasing Ca2+ concentration ([Ca2+]) in the perfusate. A low dose of iodoacetamide (IA), a sulfhydryl group modifier, was used to acutely and irreversibly inhibit CK activity without affecting other ATP synthesis and utilization pathways (11, 30). To exclude the possibility that IA inhibited SR Ca2+-ATPase activity, oxalate-facilitated SR Ca2+ uptake was also measured with tissue homogenates from these control and IA-treated hearts.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolated perfused heart preparation. Male Sprague-Dawley rats, weighing 350-400 g, were anesthetized with ketamine (100 mg/kg ip) and anticoagulated with heparin (1,000 U/kg ip). Excised hearts were arrested in an ice-cold high-potassium (30 mM KCl) saline solution and immediately attached to the perfusion apparatus. The hearts were perfused via the aorta at a constant perfusion pressure of 100 mmHg at 37°C. The perfusate contained (in mM) 118.0 NaCl, 4.7 KCl, 1.2 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, and 11.0 glucose (pH 7.4 when gassed with 95% O2-5% CO2). A [Ca2+] of 1.2 mM was used here for the purpose of being consistent with the experimental conditions of our previous studies on this subject. Coronary flow was measured by an electromagnetic flow probe (Skalar). The hearts were paced at 5 Hz. A water-filled latex balloon was mounted on rigid tubing containing a high-fidelity micromanometer (Millar Instruments, Houston, TX). The balloon was inserted into the left ventricle through an incision in the left atrium for constant monitoring of left ventricular (LV) pressure, rate of pressure development (dP/dt), and heart rate. LV end-diastolic pressure (LVEDP) was set at 5-10 mmHg by filling the balloon with H2O.

Measurement of [Ca2+]c. Indo 1 fluorescence was used to determine [Ca2+]c as previously described in detail (5-7, 9). Briefly, the hearts were loaded with indo 1 by perfusion with buffer containing 6 mM of indo 1-AM (Molecular Probes, Eugene, OR) for 30 min. Residual indo 1-AM was washed out by perfusion with standard buffer for 30 min. The ratio of fluorescence at 385 nm to fluorescence at 456 nm during excitation at 350 nm was calibrated to determine [Ca2+]c. This method of determining [Ca2+]c is based on previous work that has identified and minimized potential sources of artifact with the indo 1 technique. Specifically, the effects of motion, autofluorescence, unhydrolyzed indo 1-AM, tissue filter effect, potential loading of indo 1 into endothelial cells, and noncytosolic compartmentation have been accounted for and/or minimized (5-7, 9, 27). The rate of [Ca2+]c transient decline was assessed by the time constant of monoexponential decay (tau Ca) as previously described (8). The tau Ca was obtained by fitting the declining portion of the [Ca2+]c transient between 70 and 30% of the peak [Ca2+]c.

Accounting for noncytosolic fluorescence, primarily from mitochondria, is essential for the present study because Schreur et al. (27) previously demonstrated that increasing Ca2+ in the perfusate increases both cytosolic and noncytosolic [Ca2+]. Therefore, cytosolic and noncytosolic fluorescences were dissociated by selectively quenching cytosolic fluorescence with Mn2+ as previously described (27). Briefly, quenching of cytosolic fluorescence was determined by elimination of fluorescence transients, which was accomplished by adding MnCl2 (17.5 mM) to the perfusate.

Experimental protocols. After a 15- to 20-min equilibration period, indo 1 was loaded into all hearts (n = 20). Subsequently, IA (total dose 90 µmol dissolved in H2O; n = 10 hearts) or H2O (control; n = 10 hearts) was infused over a period of 15 min at a rate of 0.5 ml/min. To determine the effects of CK inhibition during baseline perfusion, LV function and indo 1 fluorescence were measured immediately before and after the infusion of either IA or vehicle. To determine the fraction of fluorescence arising from noncytosolic compartments during baseline, cytosolic fluorescence was quenched in a subgroup of hearts with Mn2+ as described above (IA treated, n = 4 hearts; control, n = 5 hearts).

To determine the effects of CK inhibition during inotropic stimulation, the remaining five control and six IA-treated hearts were subjected to high-Ca2+ perfusion (3.3 mM [Ca2+] in the perfusate). LV function and indo 1 fluorescence were measured simultaneously during both baseline and high-Ca2+ perfusion. To determine the fraction of fluorescence arising from noncytosolic compartments during high-Ca2+ perfusion, cytosolic fluorescence was quenched with Mn2+. At the end of each experiment, the heart was taken off the perfusion apparatus, blotted, weighed, and stored at -80°C for biochemical assays and SR Ca2+ uptake assay.

Biochemical assays. CK activity was measured for each heart using the frozen tissue. Ventricular tissue (5-10 mg) was thawed and homogenized for 10 s at 4°C in potassium phosphate buffer containing EDTA (1 mM) and beta -mercaptoethanol (1 mM), pH 7.4. Aliquots were removed for assays of protein by the method of Lowry et al. (18), with bovine serum albumin as the standard, and total Cr content with a fluorometric assay (14). Triton X-100 was then added to the remaining homogenate at a final concentration of 0.1% for analysis of CK activity (26). CK activity was measured at 30°C and is expressed in international units (IU = mmol/min) per milligram of cardiac protein.

To evaluate the effect of IA on SR Ca2+-ATPase activity independent of the CK reaction, oxalate-facilitated SR Ca2+ uptake was measured in crude ventricular homogenates from four IA-treated and four control hearts, with exogenous ATP as the sole energy source. This method was chosen because it had several advantages for our purpose. First, we could measure Ca2+ uptake in the same heart in which the Ca2+ transient was measured. Second, we could easily eliminate the CK reaction in the control group and determine whether SR Ca2+-ATPase activity was different when both groups were subjected to the identical energetic and thermodynamic status.

After 100-250 mg of LV tissue were minced with a razor blade, five volumes of solution A (25 mmol/l imidazole, pH 7.0) were added and homogenized with a Polytron homogenizer (maximum speed, 3 × 20 s; PTA 7 probe). Aliquots of the homogenates (90 µl) were transferred into tubes containing 850 ml of solution B (final concentration in mM: 100 KCl, 4.5 MgCl2, 2.5 Na2ATP, 10 NaN3, 5 potassium oxalate, and 40 imidazole, pH 7.0). After 5 min at room temperature (23°C), uptake was started by the addition of 50 µl of solution C containing 25 mM CaCl2 (11 µCi 45Ca/ml) and 15.5 mM EGTA. This yields a free [Ca2+] of 7 µM in the final solution. At this [Ca2+], SR Ca2+-ATPase activity is not inhibited by phospholamban. After 2 and 6 min, respectively (each time point was analyzed in triplicate), an aliquot of the reaction medium (100 µl) was transferred on a 0.45-µm filter in a filtration apparatus to terminate 45Ca uptake. Five milliliters of ice-cold solution A were added to eliminate any residual reaction medium. Radioactivity of the filters was determined by liquid scintillation spectroscopy, and protein concentration was assayed with the Bradford assay. Ca2+ uptake was calculated from the slope of the linear regression analysis, relating 45Ca2+ uptake per milligram of total protein to reaction time. Linearity of the uptake was confirmed up to 10 min.

Statistical analysis. All results are expressed as means ± SE. Measurements made before and after the infusion of vehicle or IA were compared by paired t-test or repeated-measures ANOVA. CK activities and the oxalate-facilitated SR Ca2+ uptake rates for control and IA-treated hearts were compared by unpaired t-test. Differences in LV function and [Ca2+]c during high-Ca2+ perfusion for vehicle- and IA-treated hearts were compared by two-way ANOVA. A value of P < 0.05 was considered significant.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of IA on the activities of CK and SR Ca2+-ATPase. Tian and Ingwall (30) previously found that 90 µmol of IA infused over 15 min inhibited CK activity by ~95% in isolated perfused rat hearts. To confirm that CK was inhibited to the same extent in the present experiments, CK activity was measured in tissue homogenates prepared from the hearts used in this study. Because inhibition of CK by IA is irreversible, CK activity in tissue homogenates can be used as a measure of CK activity in perfused hearts. Mean CK activity was 7.6 ± 0.2 and 0.4 ± 0.1 IU/mg protein in the control and IA-treated groups, respectively, demonstrating that CK was inhibited by 95%. The tissue content of total Cr, a substrate for CK, was not reduced by IA [83 ± 4 and 88 ± 5 nmol/mg protein for control and IA-treated hearts, respectively; P = not significant (NS)].

To test the possibility that IA inhibits the activity of SR Ca2+-ATPase by modifications of its sulfhydryl groups, oxalate-facilitated SR Ca2+ uptake rate was measured with tissue homogenates of the control and IA-treated hearts used in this study. In this assay, Cr and PCr (substrates for the CK reaction) were excluded and an excess amount of ATP was used as the sole energy source. Compared with the control hearts, the SR Ca2+ uptake rate was unaltered in tissue homogenates of IA-treated hearts (175 ± 37 and 165 ± 40 nM · min-1 · mg protein-1 for control and IA groups, respectively; P = NS). Because the CK reaction has been excluded as an energy-regenerating system, this result suggests that, with an identical energy supply, SR Ca2+-ATPase activity is unaltered in IA-treated hearts.

Effects of CK inhibition during baseline perfusion. Figure 1 shows representative tracings of LV pressure (A) and [Ca2+]c (B) in a control and a IA-treated heart before and after infusion of either vehicle or IA. Infusion of vehicle did not alter either LV pressure or [Ca2+]c transient in the control heart. Infusion of IA resulted in a 14-mmHg increase in both LVEDP and LV systolic pressure so that LV developed pressure was unchanged. In this heart, both peak systolic and diastolic [Ca2+]c increased by 41 nM so that the [Ca2+]c transient was maintained.


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  		Fig. 1.   Representative tracings of left ventricular (LV) pressure (A) and cytosolic Ca2+ concentration ([Ca2+]c; B) in control and iodoacetamide (IA)-treated hearts before and after infusion of either vehicle or IA during baseline perfusion. CK, creatine kinase.

Table 1 shows the group data for LV function and [Ca2+]c before and after vehicle or IA treatment during baseline perfusion. There was no significant difference in LV function or [Ca2+]c in the control group after infusion of the vehicle. There was a 13-mmHg increase in LVEDP after infusion of IA (P < 0.05), suggestive of diastolic dysfunction. However, LV systolic pressure was also increased by IA, so there was no significant difference in LV developed pressure. Similarly, -dP/dt decreased by 15% while +dP/dt was maintained in IA-treated hearts. These findings suggest that baseline contractile function (i.e., systolic function) was not significantly altered by the dose of IA used in this study, whereas diastolic function was slightly impaired.

                              
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  		Table 1.   Effect of CK inhibition during baseline perfusion

In the group treated with IA, there was a modest but significant 16% increase in diastolic [Ca2+]c (P < 0.05). Because the peak systolic [Ca2+]c was unchanged, the amplitude of the [Ca2+]c transient was decreased by 16% (P < 0.05). The tau Ca was not different in the two groups (34 ± 2 and 43 ± 7 ms for control and IA-treated groups, respectively; P = NS). These data suggest that IA had a modest effect on Ca2+ handling during baseline perfusion.

Effects of CK inhibition during high-Ca2+ perfusion. Figure 2A shows representative tracings of LV pressure in a control and an IA-treated heart during baseline and high-Ca2+ perfusion. In the control heart, increasing extracellular [Ca2+] caused a significant increase in LV developed presssure. In contrast, the IA-treated heart showed no increase in LV developed pressure. Figure 3A shows the group data for LV pressure during baseline and high-Ca2+ perfusion. In the control group, mean LV developed pressure increased by 76% during high-Ca2+ perfusion (67 ± 6 to 119 ± 8 mmHg; P < 0.05). In contrast, there was no significant change in mean LV developed pressure in the IA-treated group (67 ± 5 to 73 ± 6 mmHg; P = NS). Thus, as previously observed, inhibition of CK activity prevented the recruitment of contractile reserve by high-Ca2+ perfusion.


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  		Fig. 2.   Representative tracings of LV pressure (A) and [Ca2+]c (B) in control and CK-inhibited hearts at baseline (solid lines) and during high-Ca2+ perfusion (dashed lines).


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  		Fig. 3.   Changes in LV pressure (A) and [Ca2+]c (B) elicited by high-Ca2+ perfusion for control (solid lines) and IA-treated (dashed lines) hearts. LVSP, LV systolic pressure; LVEDP, LV end-diastolic pressure; LV Dev P, LV developed pressure; [Ca2+], Ca2+ concentration. Data are means ± SE.

Figure 2B shows the [Ca2+]c transients obtained simultaneously with the LV pressure tracings shown in Fig. 2A. Increasing extracellular [Ca2+] caused a marked increase in peak systolic [Ca2+]c in the control heart. In contrast, no increase in [Ca2+]c was observed during high-Ca2+ perfusion in the heart treated with IA. Figure 3B shows the group data for [Ca2+]c during baseline and high-Ca2+ perfusion. In the control group, the amplitude of the [Ca2+]c transient (i.e., systolic minus diastolic) increased by 56% during high-Ca2+ perfusion (548 ± 54 to 852 ± 140 nM; P < 0.05). This increase in the [Ca2+]c transient was due to a significant increase in peak systolic [Ca2+]c (P < 0.05). In contrast, the [Ca2+]c transient in the IA-treated group was not significantly changed during high-Ca2+ perfusion (422 ± 29 to 465 ± 34 nM; P = NS). Thus these hearts were unable to increase contractile function in response to high-Ca2+ perfusion and were unable to increase peak systolic [Ca2+]c. The rate of [Ca2+]c decline, estimated by tau Ca, was slower in the IA-treated group compared with the control group (40 ± 3 vs. 29 ± 3 ms; P < 0.05), suggesting a limitation for Ca2+ clearance in IA-treated hearts during inotropic stimulation.

Relationship between [Ca2+]c transient and LV developed pressure. In Fig. 4, the LV developed pressure is plotted against [Ca2+]c for each individual heart in both groups during baseline and high-Ca2+ perfusion. A linear relationship between the [Ca2+]c transient and LV developed pressure was obtained with all data points from the hearts of both groups (r2 = 0.61). Importantly, data points obtained from IA-treated hearts cluster in the lower left portion of this relationship, suggesting that the inability to increase [Ca2+]c contributes to the inability to increase LV developed pressure in these hearts.


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  		Fig. 4.   LV developed pressure vs. [Ca2+]c for each individual heart in control () and CK-inhibited () hearts during baseline and high-Ca2+ perfusion. A linear relationship between [Ca2+]c transient and LV developed pressure was obtained from all data points from hearts of both groups (y = 0.086x - 33.2; r2 = 0.61).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The major finding of this study is that inhibition of CK activity by 95% impairs the ability of hearts to increase [Ca2+]c in response to inotropic stimulation. Furthermore, failure to increase [Ca2+]c was associated with failure to increase LV developed pressure during inotropic stimulation. Combined with previous observations by Hamman et al. (11) and Tian and Ingwall (30) that this degree of CK inhibition decreases |Delta G~p| and contractile reserve, these data support the hypothesis that a decrease in |Delta G~p| impairs Ca2+ handling and thereby limits contractile reserve.

The CK reaction rapidly transfers a phosphoryl group between PCr and ATP, thus maintaining a constant high [ATP] and a low [ADP], especially at high workloads (4). Because of this unique function, the CK reaction is of particular importance in maintaining a high level of |Delta G~p|: |Delta G~p| = |Delta Go - RTln([ATP]/[Pi][ADP])|. Tian and Ingwall (30) previously showed that acute inhibition of this reaction by IA results in a decrease in the ATP-to-ADP ratio, which leads to a reduced |Delta G~p| (30). Furthermore, concurrent measurement of |Delta G~p| and cardiac performance showed that a decreased |Delta G~p| was associated with decreased contractile reserve of the heart (30). The purpose of this study was to determine by what mechanism(s) a decrease in |Delta G~p| results in depletion of contractile reserve.

We postulate that contractile reserve was impaired in hearts with a decreased |Delta G~p| due to impaired Ca2+ handling. This is because the thermodynamic driving force required for the SR Ca2+ pump is determined by the [Ca2+] gradient across the SR membrane, and a high level of |Delta G~p| is required to maintain the 10,000-fold Ca2+ gradient in cardiac myocytes. Furthermore, the thermodynamic reserve for the SR Ca2+-ATPase reaction is limited under normal conditions. To maintain the normal Ca2+ gradient, the SR Ca2+-ATPase reaction requires a |Delta G~p| of at least 52 kJ/mol, 85-90% of |Delta G~p| from ATP (15). Therefore, of all the ATPase reactions in cardiac myocytes, the SR Ca2+-ATPase reaction is the most vulnerable to a decrease in |Delta G~p| (15). Our present finding that both the [Ca2+]c transient and LV developed pressure failed to increase in CK-inhibited hearts during high-Ca2+ perfusion provides direct experimental evidence to support this hypothesis. To our knowledge, these data are the first to show that reduced |Delta G~p| results in abnormal Ca2+ handling that may account for a limitation of contractile reserve.

Previous studies (16, 33) have shown that maintaining a high ratio of ATP to (ADP × Pi) and thus a high level of |Delta G~p| is critical for Ca2+ uptake by the SR. In CK-inhibited hearts, the ability to maintain a high ATP-to-ADP ratio is substantially reduced, resulting in a decreased |Delta G~p|. During high-Ca2+ perfusion, |Delta G~p| decreased to as low as 53 kJ/mol in CK-inhibited hearts, a level that is likely to limit SR Ca2+-ATPase activity. In support of this, a recent study (20) using permeabilized rat ventricular fibers showed that SR Ca2+ uptake in the presence of the CK reaction was higher than that in the absence of the CK reaction. We found that the rate of [Ca2+]c decline, unaltered at baseline perfusion when |Delta G~p| was 56 kJ/mol, was slower in the IA-treated group compared with the control group during high-Ca2+ perfusion when |Delta G~p| decreased to 53 kJ/mol, suggesting a limitation for Ca2+ uptake in CK-inhibited hearts under these conditions.

One consequence of this limitation is an impaired net SR Ca2+ accumulation, resulting in reduced SR loading that may reduce contractility and systolic [Ca2+]c in two ways: first, less SR Ca2+ available for release, and second, a lower fractional release of Ca2+ at a lower SR Ca2+ content (2). Because >90% of Ca2+ that activates the myofilaments comes from the SR in the rat heart (1, 3), decreased SR loading would be a likely mechanism to account for the inability to increase [Ca2+]c in response to inotropic stimulation during CK inhibition.

This finding has important clinical implications. Impaired Ca2+ homeostasis has been considered a hallmark of the failing myocardium (13, 22, 32). Altered Ca2+ handling by the SR may be one of the mechanisms underlying contractile dysfunction in failing hearts (19, 22, 24). Our laboratory (17, 31) and others (10) have previously shown that decreased energy reserve via the CK reaction is also a characteristic of failing hearts and may be one of the mechanisms contributing to the development of contractile dysfunction in heart failure. The results of the present study provide further evidence that altered energetics results in abnormal Ca2+ handling and contractile dysfunction. Although failing hearts show some response to increased extracellular Ca2+ in isolated perfused heart preparations, the magnitude of contraction during high-Ca2+ stimulation remains lower in failing hearts compared with control hearts and does not exceed the level reached by the IA-treated hearts in this study (31). This is consistent with our observation that contractile function is limited in hearts with impaired energetics.

Other possibilities. Because IA is a sulfhydryl modifier, other explanations for our findings should be considered. To exclude the possibility that the dose of IA applied in this study directly modified SR Ca2+-ATPase activity in the heart, we measured oxalate-facilitated SR Ca2+ uptake in tissue homogenates. We found no difference between control and IA-treated hearts. Because this assay was performed with exogeneous ATP as the sole energy source, the results obtained here suggest that SR Ca2+ pump function has not been changed in IA-treated hearts compared with control hearts under identical conditions of ATP supply. Similarly, a previous study (34) using isolated SR membrane found that incubating 1 mol IA/mol SR Ca2+-ATPase for 6 h did not affect Ca2+ transport activity. It has been shown that the dihydropyridine binding in heart sarcolemmal membrane could be modified by IA in a dose-dependent fashion (23). However, no changes in dihydropyridine binding were observed until the IA concentration reached a level 25 times higher than what we used in this study (23), suggesting that L-type Ca2+ channels remain unaltered in our study. Furthermore, the previous study by Hamman et al. (11) showed that actomyosin ATPase activity is unaltered by the dose of IA applied in this study (11). Finally, Hamman et al. and Tian and Ingwall (30) also showed that this dose of IA did not alter mitochondrial respiration or glycolytic flux (11, 30).

We observed a small increase (16%) in diastolic [Ca2+]c at baseline in IA-treated hearts, and the mechanism for this increase is unknown. Our data cannot rule out the possibility of a spontaneous release of SR Ca2+ and/or inhibition of Na+/Ca2+ exchanger in IA-treated hearts. A recent study by Prabhu et al. (25) showed that when cardiac SR Ca2+-release channels were locked open in an intact heart, the major changes in LV function were a marked decrease in velocity-based indexes. This is not the case in our study because the baseline +dP/dt was not different in control and IA-treated hearts. Although the observation that LVEDP was increased in CK-inhibited hearts is of interest, it is unlikely that the 16% increase in diastolic [Ca2+]c observed here can be responsible for the marked increase in LVEDP. Indeed, in a separate study, Tian et al. (29) showed that the mechanisms accounting for the impaired relaxation in CK-inhibited hearts are likely to be a slowing of cross-bridge dissociation due to an increase in free [ADP] and a modest increase in end-diastolic [Ca2+]c.

In summary, we found that acutely inhibiting CK activity by ~95% impairs the ability of hearts to increase [Ca2+]c in response to inotropic stimulation. Furthermore, there was a close relationship between LV developed pressure and the amplitude of the [Ca2+]c transient during high-Ca2+ perfusion. Together with our previous observation that acute inhibition of the CK reaction decreases |Delta G~p| and contractile reserve, these data support the hypothesis that a decrease in |Delta G~p| during CK inhibition impairs Ca2+ handling and limits contractile reserve.

    ACKNOWLEDGEMENTS

We thank Dr. Amy J. Davidoff for critical review of this manuscript.

    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-52350, HL-49574 (both to J. S. Ingwall), HL-08973 (to J. M. Halow), HL-54890 (to S. A. Camacho), K08-HL-02883 (to V. M. Figueredo), and HL-52946 (to W. H. Dillmann); American Heart Association Grant-in-Aid 94-6930 (to S. A. Camacho); American Heart Association California Affiliate Grant-in-Aid 95-220 (to S. A. Camacho); and Deutsche Forschungsgemeinschaft Me1477/2-1 (to M. Meyer).

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. §1734 solely to indicate this fact.

Address for reprint requests: R. Tian, NMR Laboratory for Physiological Chemistry, 221 Longwood Ave., Rm. 247, Boston, MA 02115.

Received 27 April 1998; accepted in final form 25 August 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Bassani, J. W. M., R. A. Bassani, and D. M. Bers. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J. Physiol. (Lond.) 476: 279-293, 1994[Abstract/Free Full Text].

2.   Bassani, J. W. M., W. Yuan, and D. M. Bers. Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cradiac myocytes. Am. J. Physiol. 268 (Cell Physiol. 37): C1313-C1329, 1995[Abstract/Free Full Text].

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Am J Physiol Heart Circ Physiol 275(6):H2064-H2071
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