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Departments of 1Anesthesiology and 2Physiology, and 3Cardiovascular Research Center, Medical College of Wisconsin; 4Department of Biomedical Engineering, Marquette University; and 5Veterans Affairs Medical Center, Milwaukee, Wisconsin
Submitted 9 March 2005 ; accepted in final form 8 November 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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phase-space diagrams; cooperativity; guinea pigs; linear model
-adrenergic and dopaminergic agonists (e.g., dobutamine and dopamine) increase myoplasmic cAMP concentrations to promote protein kinase-induced phosphorylation of 1) sarcolemmal Ca2+ channels to increase Ca2+ influx, 2) sarcoplasmic reticulum Ca2+ pumps to enhance Ca2+ uptake, and 3) troponin I to reduce myofilament Ca2+ sensitivity (12, 16). In contrast, cardiac glycosides (e.g., digoxin) do not affect cAMP levels but inhibit Na+-K+-ATPase pump activity to selectively slow Na+/Ca2+ exchange. Levosimendan, a newer Ca2+ sensitizer, is thought to stabilize the Ca2+-TnCA complex without increasing Ca2+ binding affinity with TnCA (11). We recently presented novel indexes from phase-space diagrams of simultaneously measured LV pressure (LVP) vs. [Ca2+] to understand the mechanisms of positive and negative inotropic drugs in guinea pig unpaced, isolated hearts in the absence of ischemia (27). The effects of drugs on static relationships between [Ca2+] and LVP (peak values, rates of rise and fall, etc.) were presented in earlier articles (7, 10). For this study, we compared how positive inotropic drugs with different mechanisms of action alter the dynamic phase-space relationship between [Ca2+] and LVP before and after ischemia and reperfusion (IR) injury. Specific indexes from LVP vs. [Ca2+] phase-space diagrams (loops) were evaluated to determine which of these indexes best quantified dynamic Ca2+-contraction coupling.
Central and downstream mechanisms regulating Ca2+-contraction coupling are governed by cooperative interactions. This positive feedback mechanism is responsible for amplification in signal transduction, leading to muscle activation and force generation in response to the Ca2+ stimulus (20). To quantify cooperativity, we linearly modeled the phase-space relationship between the rate of LVP development (dLVP/dt) and simultaneous [Ca2+] during inotropic interventions before and after IR injury. The ability to quantify changes in dynamic Ca2+-contraction coupling and cooperativity in response to pharmacological and pathological interventions should improve our understanding of the mechanisms of cardiac force generation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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The investigation conformed to the Guide for the Care and Use of Laboratory Animals from the US National Institutes of Health (NIH no. 85–23, revised 1996). Prior approval was obtained from the Medical College of Wisconsin and Marquette University Animal Studies Committees. The preparation of guinea pig hearts at our laboratory has been described in detail (7, 9, 10, 26–28, 30–32). English short-haired guinea pigs (n = 40) were anesthetized with ketamine (30 mg intraperitoneal) and decapitated when they were unresponsive to noxious stimulation. After thoracotomy, the inferior and superior venae cavae were cut away, and the aorta was cannulated distal to the aortic valve. Each heart was immediately perfused via the aortic root with a cold oxygenated, modified Krebs-Ringer (KR) solution at a pressure head of 55 mmHg. The KR perfusate (pH 7.39 ± 0.01, PO2 620 ± 10 Torr) was filtered (5-µm pore size) in-line and had the following calculated composition in mM (nonionized): 137 Na+, 5 K+, 1.2 Mg2+, 1.25 Ca2+, 134 Cl–, 15.5 HCO3–, 1.2 H2PO4–, 11.5 glucose, 2 pyruvate, 16 mannitol, 0.05 EDTA, 0.1 probenecid, and 5 insulin (U/l). Perfusate and bath temperatures were maintained at 37.2 ± 0.1°C using a thermostatically controlled water circulator. KR CaCl2 concentration was reduced (1.25 mM) to allow for a wider range of inotropic responses at a lower control LVP. This accounts for the low control values of systolic LVP and [Ca2+].
Isovolumetric LVP was measured continuously with a transducer connected to a thin, saline-filled latex balloon inserted into the LV through the mitral valve from an incision in the left atrium. Balloon volume was adjusted initially to a diastolic LVP of 0 mmHg so that any subsequent increase in diastolic LVP reflected an increase in LV wall stiffness, i.e., diastolic contracture. Pairs of bipolar electrodes were placed in the right atrial appendage, right ventricular apex, and LV base to monitor spontaneous heart rate (HR) and atrial-ventricular conduction time. Coronary flow was measured by an ultrasonic flowmeter placed directly into the aortic inflow line.
Measurement of Myoplasmic Free Ca2+ in Intact Hearts
Experiments were carried out in a light-blocking Faraday cage. The heart was partially immobilized by hanging it from the aortic cannula, the pulmonary artery catheter, and the LV balloon catheter. The distal end of a trifurcated fiber-optic cable (optical surface area: 3.85 mm2) was placed gently against the LV epicardial surface through a hole in the bath. A rubber O ring was placed over the fiber-optic tip to seal the hole, and netting was applied around the heart for optimal contact with the fiber-optic tip.
Hearts were loaded with the Ca2+ indicator indo 1-AM (Sigma Chemical, St. Louis, MO) to a final concentration of 6 µM; this technique has been described in detail previously (7, 9, 10, 26, 27, 30–32). Fluorescence emissions at 385 (F385) and 456 nm (F456) were recorded by using a modified luminescence spectrophotometer (SLM Aminco-Bowman II, Spectronic Instruments, Urbana, IL). The LV region of the heart was excited with light from a xenon arc lamp, and the light was filtered through a 350-nm monochromator with a bandwidth of 16 nm. The arc lamp shutter was opened only for 2.5-s recording intervals for a total exposure time of 62.5 s through the entire experiment to prevent photobleaching. Emission fluorescence was collected by fibers of the remaining two limbs of the cable and filtered by square interference filters (Corion, Franklin, MA) at 385 nm (390 ± 5 nm) and 456 nm (460 ± 5 nm). Signal strength decreases as the distance from the probe through the LV wall increases. Our pilot studies showed signal attenuation of 70–80% between the epicardial and endocardial surfaces. Although both F385 and F456 decline over time, time control studies showed that the F385-to-F456 ratio (F385/F456) remained stable, indicating that effective measured [Ca2+] was unchanged (31). The F385/F456 was converted to [Ca2+] after correcting for background autofluorescence and noncytosolic fluorescence (see APPENDIX), as has been described in detail previously (7, 10, 26, 27, 30). Developed LVP after indo 1 loading was also not significantly altered over 3 h in the absence of ischemia.
Experimental Protocol and Data Collection
Hearts were randomly assigned to receive dobutamine (4 µM), dopamine (8 µM), digoxin (1 µM), or levosimendan (1 µM) (n = 8 in each group). Each inotropic drug was infused for 2 min, 30 min before global ischemia. Next, hearts were subjected to 30 min of global ischemia, and inotropic drugs were again infused for 2 min after 30-min reperfusion. The concentrations of inotropic drugs were the approximate half-maximal effective dose concentrations, as previously established (10, 27). Eight control hearts were not exposed to inotropic interventions before or after global ischemia. At the end of each experiment, MnCl2 (100 µM) was infused for 10 min to quench the myoplasmic indo 1 Ca2+ signal to correct for the nonmyoplasmic fraction (see APPENDIX). All analog signals were digitized and recorded at 125 Hz for later analysis by using MATLAB as previously described (7, 10, 26, 27).
Simultaneous recordings of [Ca2+] and LVP were obtained before and after IR injury in the control hearts (Fig. 1A) and during administration of inotropic drugs before and after IR injury (Fig. 1, B–E). LVP and fluorescence data were low-pass filtered digitally by using a fourth-order bidirectional Butterworth filter at 25 Hz. The timing of peak diastolic [Ca2+] for each cardiac cycle was obtained over the 2.5-s recordings using a simple event detection algorithm. [Ca2+], and the simultaneously obtained LVP signals between each consecutively detected Ca2+ diastolic point, were aligned and averaged on a point-by-point basis to form the averaged [Ca2+] and LVP transient signals. Care was taken to exclude any dysrhythmic beats in the averaged Ca2+ and LVP transients.
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Phasic LVP and [Ca2+], global myocardial contractility (dLVP/dtmax) and relaxation (dLVP/dtmin), and total myoplasmic [Ca2+] influx (d[Ca2+]/dtmax) and efflux (d[Ca2+]/dtmin) from all cellular and extracellular sources were computed from the averaged Ca2+ and LVP transients. Several temporal indexes were also computed: half-maximal duration of Ca2+ and LVP transients; peak delay between systolic [Ca2+] and LVP; contraction response time (delay between d[Ca2+]/dtmax and dLVP/dtmax); and relaxation response time (delay between d[Ca2+]/dtmin and dLVP/dtmin). These indexes have been described previously (7, 10, 27).
Dynamic Indexes Derived From LVP vs. Ca2+ Phase-Space Diagrams
Averaged LVP was plotted as a function of averaged myoplasmic [Ca2+] over the cardiac cycle, and the following indexes were derived: LVP vs. [Ca2+] loop area; loop orientation (
LVP/[Ca2+]); loop position vector, P, for spatial characterization (location, |P|, and direction, 
P) computed from the origin (0 nM [Ca2+], 0-mmHg LVP) to the loop centroid {LCx = (0.5 * phasic [Ca2+]) + diastolic [Ca2+], LCy = [0.5 * phasic LVP] + diastolic LVP}; and loop trajectory vectorgrams, T(t), also measured from the origin, to link signal temporal information with loop shape. The loop indexes were used to quantify the efficiency of Ca2+-contraction coupling and have been described previously (27).
dLVP/dt vs. Ca2+ Phase-Space Diagrams
Instantaneous dLVP/dt was plotted vs. [Ca2+] to diagram the phase-space relationship between [Ca2+] and the rate of LVP rise and fall before and after IR injury (Fig. 2A). The dLVP/dt vs. [Ca2+] diagram was divided into three sections: segment 1, onset of Ca2+ transient to dLVP/dtmax; segment 2, dLVP/dtmax to dLVP/dtmin; and segment 3, dLVP/dtmin to end of Ca2+ transient. A linear regression model (Fig. 2B) was applied to all three segments during drug administration before and after global ischemia.
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Statistical Analysis
Static and dynamic indexes from LVP and [Ca2+] transients and linear regression models of dLVP/dt vs. [Ca2+] obtained during administration of inotropic drugs before and after IR were compared with control using one-way ANOVA followed by Dunnett’s comparison of means post hoc test (MINITAB Statistical Software Release 13.3, Minitab, State College, PA). Control and inotropic drug data obtained after ischemia were compared with their respective values before ischemia using Student’s paired t-test. Differences among means were considered statistically significant at P < 0.05 (two-tailed). Because only one concentration of each drug was given, we did not compare drug responses among each other. All indexes were expressed as means ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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HR. HR was unchanged on reperfusion after global ischemia in control hearts (Table 1). Dobutamine and dopamine increased HR before and after ischemia. Digoxin did not alter HR. Levosimendan increased HR before, but not after, global ischemia.
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Myocardial contractility and relaxation. Each drug enhanced myocardial contractility and relaxation from control before and after ischemia (Fig. 3, A and B). This increase was lower after ischemia in all drug groups.
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Half-maximal duration of Ca2+ and LVP transients. Each drug, except levosimendan, significantly decreased the duration of the LVP transient compared with control, before and after ischemia (Table 1). Dobutamine, dopamine, and digoxin, but not levosimendan, also shortened the Ca2+ transient duration before ischemia. Dobutamine and digoxin reduced the duration of the Ca2+ transient compared with control after ischemia. The durations of LVP and Ca2+ transients were unchanged after ischemia compared with before ischemia in each group.
Peak delay. Dobutamine alone reduced the delay between peak Ca2+ and LVP from control before and after ischemia (Table 1). Peak delay was unchanged after ischemia compared with before ischemia in each group.
Temporal sensitivity of myocardial response. Each drug shortened the contraction response time before but not after ischemia (Table 1). Contraction response time was significantly shorter after ischemia than before. Dobutamine, dopamine, and levosimendan, but not digoxin, reduced the relaxation response time from control before ischemia. Relaxation response times were shorter in control and digoxin-treated hearts after ischemia than before ischemia.
Dynamic Indexes Derived From LVP vs. Ca2+ Phase-Space Diagrams
Figure 4 illustrates phase-space diagrams before and after IR injury for all control hearts. After IR injury, the LVP vs. [Ca2+] loop was flatter and wider, and the centroid was shifted rightward. These changes are quantified as unchanged loop area, decreased orientation (from 0.25 to 0.09), and increased location (P) (175–228), which signified worsened Ca2+-contraction coupling.
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Loop position vector. The loop position vector P spatially localizes the loop in the LVP-[Ca2+] domain. Dobutamine and dopamine altered both the location and direction of the loop centroid before ischemia (Fig. 7A). Loop centroid direction and location were reduced after ischemia in control hearts (Fig. 7B). Dopamine, digoxin, and levosimendan increased the angular location of the loop centroid but did not change its distance from the origin compared with control hearts after ischemia. In contrast, dobutamine increased the distance of the centroid from the origin but did not affect direction. Drug infusion after global ischemia returned the loop centroid positions toward the values observed before ischemia.
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T(t) plots: 1) onset of cardiac cycle, 2) first minimum T(t), 3) peak |T(t)|, and 4) peak T(t). The trajectory vectorgrams show shorter cycle lengths during drugs before and after ischemia, consistent with increased HR. The interval between onset of [Ca2+] rise and diastolic LVP (points 1 and 2) was prolonged after ischemia for each drug except digoxin. This interval was reduced by dobutamine before ischemia and by digoxin, dopamine, and levosimendan after ischemia. The interval between diastolic LVP and systolic [Ca2+] (points 2 to 3) was reduced after IR injury in the control group. This interval was shortened by each drug before ischemia, but the drugs had no effect after ischemia. The delay between systolic [Ca2+] and systolic LVP (points 3 to 4) was unchanged after ischemia compared with before ischemia in each group. Dobutamine alone shortened this interval before and after ischemia.
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The linear regression model for the phase-space plots of dLVP/dt and [Ca2+] had averaged correlation coefficients of 0.9 or better for all segments and all interventions. Each slope (mmHg·nM–1·s–1) was reduced after ischemia in each group (Table 2), indicating the loss of cooperativity due to ischemic injury in the binding of Ca2+ to troponin C and/or in actinomyosin cross-bridge interactions. Dobutamine and dopamine increased the cooperativity of contraction before and after ischemia. Digoxin only increased the cooperativity of contraction after ischemia. Each drug increased the slope of segment 2 before ischemia, but only dopamine and dobutamine increased this slope after ischemia. Dopamine alone increased the cooperativity of relaxation before ischemia, whereas both dobutamine and dopamine increased this index after ischemia. Levosimendan had no effect on the cooperativity of contraction or relaxation before or after ischemia compared with control hearts.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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IR Injury and Ca2+-Contraction Coupling
Myocardial damage due to coronary artery disease or cardiac surgical procedures may be reversible (stunning) or irreversible (infarction) during reperfusion, depending on the length and severity of the ischemic insult (8, 28, 34). IR injury results from several interrelated mechanisms that include 1) reduced maximal Ca2+-activated force or Ca2+ sensitivity, 2) myoplasmic Na+ and Ca2+ overload, 3) inefficient ATP synthesis and utilization associated with NADH accumulation, 4) impaired myocardial vascular perfusion, and 5) reactive oxygen species-induced damage (18, 28, 31). Our results show that IR injury decreased Ca2+-contraction coupling. This is indicated by unchanged LVP vs. [Ca2+] loop area, but reduced loop orientation concomitant with a shift in the loop position to the right and at a shallower angle. IR injury also reduced contractile response to [Ca2+], as noted from increased total [Ca2+] influx and efflux, despite unchanged contractility and reduced relaxation.
The trajectory vectorgrams indicate that IR injury prolonged the duration of LVP diastole, reduced the phase of the cardiac cycle characterized by a rapid rise in LVP immediately after the rise in [Ca2+], and increased the duration of [Ca2+] decline. Changes in duration of these phases were not accompanied by changes in peak delay or HR and thus reflected attenuated Ca2+-contraction coupling with IR injury. Decrease in slopes of the three segments defining the dLVP/dt-[Ca2+] phase-space diagram also suggests depression in the cooperative binding of Ca2+ to troponin C and cross-bridge kinetics due to IR injury. These alterations in intracellular Ca2+ homeostasis are likely responsible for the increase in diastolic LVP and the reduction in LV compliance after ischemia.
Dopaminergic and -Adrenergic Agonists and Ca2+-Contraction Coupling
Dopamine enhanced Ca2+-contraction coupling before and after ischemia, as indicated by the increases in LVP vs. [Ca2+] loop orientation and loop area, and by the repositioning of the loop at a steeper angle in the phase-space plot. Dopamine decreased relaxation response time before ischemia; this also suggests an increase in contractile response to [Ca2+]. Increases in the steepness of all three segments of the dLVP/dt vs. [Ca2+] diagrams with dopamine suggest improved cooperativity during contraction and relaxation, both before and after ischemia.
Dobutamine infusion before and after ischemia increased the LVP vs. [Ca2+] loop area, but loop orientation was unchanged before, and reduced after, ischemia; this suggests a drug-induced decrease in Ca2+-contraction coupling before and after ischemia. A shift in the loop location rightward and at a shallower angle with dobutamine before and after ischemia also supports this hypothesis. The depression in Ca2+-contraction coupling with dobutamine occurred, despite significant increases in Ca2+ flux and availability before and after ischemia; this suggests that dobutamine decreased the contractile response to [Ca2+]. Plots of dLVP/dt vs. [Ca2+] show that, whereas dobutamine increased the cooperativity of contraction before and after IR injury, the cooperativity of relaxation was increased with drug infusion only after IR injury.
Endogenous catecholamines play a key role in regulating myocardial contractility through 1-adrenergic and dopaminergic receptors in vivo. Catecholamines increase myoplasmic [Ca2+], increase Ca2+ uptake into the sarcoplasmic reticulum, and decrease myofilament Ca2+ sensitivity (12, 16). The results observed in this and previous studies (10) suggest that activation of dopaminergic and 1-adrenergic receptors by dopamine (21) has more potent effects than activation of 1-adrenoceptors by dobutamine alone.
Cardiac Glycosides and Ca2+-Contraction Coupling
Digoxin enhanced the contractile response to Ca2+ after ischemia, as indicated by increases in contractility and relaxation, with no changes in [Ca2+] influx or efflux from the postischemic control. The duration of LVP and Ca2+ transients was reduced before and after ischemia, and contraction response time was shortened before ischemia. These changes occurred independently of HR. Dynamic indexes from phase-space diagrams of LVP vs. [Ca2+] revealed that digoxin increased loop area, orientation, and P before ischemia, but only increased orientation and P after ischemia; this suggests enhanced Ca2+-contraction coupling with digoxin after and, to a lesser extent, before ischemia. Digoxin enhanced cooperativity of contraction after, but not before, ischemia. These results indicate that digoxin had more potent effects on impaired compared with normal myocardium.
Ca2+ Sensitizers and Ca2+-Contraction Coupling
At low concentrations, levosimendan acts as myofilament Ca2+ sensitizer by binding to TnCA in the presence of Ca2+, which stabilizes the Ca2+-TnCA complex without actually increasing Ca2+ binding affinity with TnCA (11). At higher concentrations, levosimendan also acts as a cardiac phosphodiesterase III inhibitor, resulting in elevated cAMP and subsequently in increased [Ca2+] (23, 24). Levosimendan increased phasic LVP and [Ca2+] before but not after ischemia. Nevertheless, levosimendan increased contractility both before and after ischemia. These results are supported by previous studies of levosimendan and LV dysfunction in dogs (25). Levosimendan shortened contraction and relaxation response times before ischemia, possibly due to the phosphodiesterase III inhibitory effect. These changes were accompanied by an increase in HR, which indicates that levosimendan alters myocardial electrophysiological properties and is, therefore, not acting as a pure Ca2+ sensitizer (33). The dynamic indexes revealed that levosimendan increased loop area before but not after ischemia. However, levosimendan did increase P after ischemia; this suggests that Ca2+-contraction coupling may be modestly improved. In contrast, levosimendan had no effect on loop orientation before or after ischemia. Levosimendan also had no effects on cooperativity of contraction or relaxation before or after IR injury. These findings suggest that this concentration of levosimendan does not substantially affect Ca2+-contraction coupling, at least in our model.
Comparison to Previous Studies
Previous studies from our laboratory (7, 10, 27) focused on static and normalized (index ratios) indexes to elucidate the differential mechanisms of negative and positive inotropic drugs on the LVP-Ca2+ relationship. In the present study, we focused on dynamic indexes derived from phase-space analysis of the LVP-Ca2+ and the dLVP/dt-Ca2+ relationships in the presence of positive inotropic drugs. These dynamic indexes demonstrate that dopamine and dobutamine had dissimilar effects on Ca2+-contraction coupling, despite having somewhat similar mechanisms of action. Digoxin improved Ca2+-contraction coupling, particularly after ischemia, and levosimendan had very little effect on Ca2+-contraction coupling.
Backx et al. (2) and Gao et al. (13) studied the effects of normal and stunned myocardium on myofilament Ca2+ responsiveness in rat cardiac trabeculae at room temperature using instantaneous force-Ca2+ relationships. Their studies compared the steady-state relationship between intracellular Ca2+ and developed force (F) with the dynamic response during a twitch contraction. However, they did not quantify the phase-space diagrams of F vs. [Ca2+] in terms of shape, size, or temporal information, nor did they examine drug responses before and after IR injury. Our detailed characterization of the LVP vs. [Ca2+] phase-space diagrams in the intact heart at 37°C provides a mathematical approach for identifying and measuring dynamic Ca2+-contraction coupling.
Yue (35) reported that peak positive rate of rise of tension (dF/dt) was temporally related with peak estimated intracellular [Ca2+] and that the relation between positive dF/dt and [Ca2+] during the period from peak dF/dt to peak F was linear. This linear relationship was unchanged for twitch contractions of different duration and strength in ferret papillary muscles. We extended their study by examining the phase-space diagrams of dLVP/dt vs. [Ca2+] and found three linear relationships between dLVP/dt and [Ca2+] over three segments of the cardiac cycle. However, we noted that the slopes of the linear relationship between dLVP/dt and [Ca2+] were indeed sensitive to changes in contractile function due to inotropic drugs in the presence and absence of ischemia. This suggests that inotropic drugs, whatever their mode of action, affect cooperativity in the central and downstream mechanisms that regulate Ca2+-contraction coupling.
Potential Limitations
Isolated perfused hearts are used extensively to study cardiac function. They allow for the control of pre- and postload conditions and autonomic nervous system influences that can complicate in vivo studies. However, isovolumic LVP was measured globally, whereas [Ca2+] was measured only in a core of LV free wall under the optical probe.
The fluorescence technique used to estimate myoplasmic [Ca2+] is well established and superior to other Ca2+ measurement techniques (3, 22). This technique, however, is sensitive to changes in background autofluorescence and inner filtering. Although we routinely correct for background autofluorescence, it is difficult to estimate the loss of signal intensity due to inner filtering.
Because these hearts were unpaced, changes in HR with inotropic interventions likely confound some of the conclusions drawn from the static indexes of contractility, relaxation, Ca2+ influx and efflux, and all of the temporal indexes. However, only by allowing hearts to beat at their inherent rhythm can we fully understand the physiological impact of these drugs on the intact heart. Controlling the HR would lead to changes in the peak values and kinetics of the measured Ca2+ transients and thus result in LVP values that are nonphysiological.
Inotropic drugs were given before as well as after ischemia, so it is possible that the drugs induce cardiac preconditioning that could subsequently alter the static and dynamic indexes obtained after ischemia. For example, levosimendan is reported to precondition the heart and protect against subsequent IR injury (17). Conversely, digoxin is not believed to protect against IR injury and may abolish protection when coupled with ischemic preconditioning (15). The role of -adrenoceptor agonists administered before IR injury in cardioprotection remains controversial. Some researchers have reported that -adrenoceptor agonists produce a deleterious effect on IR injury (29), but others have demonstrated improved hemodynamics after IR injury when dobutamine was given before ischemia (1). In a prior study, we suggested that negative inotropic drugs, especially nifedipine, can also elicit cardiac preconditioning (7).
In summary, we demonstrate the use of phase-space analyses to examine relationships between LVP and [Ca2+] and between dLVP/dt and [Ca2+] during the cardiac cycle in guinea pig isolated hearts during administration of positive inotropic drugs before and after global ischemia. The described changes in loop shape, size, and position each furnished important information by which to quantitatively assess dynamic Ca2+-contraction coupling before and after IR injury and were used to compare the efficacies of each inotropic drug in improving cardiac function. In a related article (27a), we use the same experimental data to model mechanisms underlying LVP-[Ca2+] loop formation with respect to protein-protein interactions during cross-bridge cycling and the kinetics of TnCA association and dissociation with Ca2+. The theoretical description of the LVP-[Ca2+] relationship used the phase-space analyses described in this study to model the myofilament protein kinetics and cooperative mechanisms underlying altered Ca2+-contraction coupling by inotropic drugs before and after global ischemia.
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APPENDIX |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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The Ca2+ transient obtained from the fluorescence ratio F385/F456 is nonlinearly related to [Ca2+]. Calibration curves were derived from previously published protocols by Brandes et al. (5, 6) using modifications of a standard equation for fluorescent indicators (14).
Total intracellular [Ca2+] ([Ca2+]tot) was calculated as:
 | (1) |
/F
); S456 is F456 (for zero [Ca2+])/F456 (for saturating [Ca2+]) = 2.4; Rmax is F385 (for saturating [Ca2+])/F456 (for saturating [Ca2+]) = 5.986; Rmin is F385 (for zero [Ca2+])/F456 (for zero [Ca2+]) = 0.059; and Kd is the dissociation constant of indo 1 = 250 nM.
Nonmyoplasmic (primarily mitochondrial) [Ca2+] ([Ca2+]mito) was calculated similarly:
 | (2) |
and F
. Nonmyoplasmic fluorescence was measured at the end of each experiment after perfusing hearts with 100 µM MnCl2 for 10 min to quench fluorescence derived from the myoplasmic compartment (4). F and F were calculated at each time point by multiplying the residual mitochondrial fluorescence fractions (f385 and f456) by total end-diastolic fluorescence so that:
 | (3) |
 | (4) |
and F
, calculated at each time point by effectively subtracting mitochondrial compartment Ca2+ ([Ca2+]mito) from [Ca2+]tot and multiplying the remainder by total end-diastolic fluorescence (as in Eq. 3) so that:
 | (5) |
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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Portions of this work have appeared in abstract form in the Proceedings of the 2nd Joint EMBS/BMES conference 2002. p. 252–253.
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FOOTNOTES |
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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.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXGRANTSREFERENCES |
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postischemia vs. preischemia, all groups: P < 0.05.
