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Am J Physiol Heart Circ Physiol 286: H648-H656, 2004. First published October 9, 2003; doi:10.1152/ajpheart.00374.2003
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Intracellular calcium handling heterogeneities in intact guinea pig hearts

Rodolphe P. Katra, Etienne Pruvot, and Kenneth R. Laurita

The Heart and Vascular Research Center, and The Department of Biomedical Engineering, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109-1198

Submitted 25 August 2003 ; accepted in final form 2 October 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regional heterogeneities of ventricular repolarizing currents and their role in arrhythmogenesis have received much attention; however, relatively little is known regarding heterogeneities of intracellular calcium handling. Because repolarization properties and contractile function are heterogeneous from base to apex of the intact heart, we hypothesize that calcium handling is also heterogeneous from base to apex. To test this hypothesis, we developed a novel ratiometric optical mapping system capable of measuring calcium fluorescence of indo-1 at two separate wavelengths from 256 sites simultaneously. With the use of intact Langendorff-perfused guinea pig hearts, ratiometric calcium transients were recorded under normal conditions and during administration of known inotropic agents. Ratiometric calcium transients were insensitive to changes in excitation light intensity and fluorescence over time. Under control conditions, calcium transient amplitude near the apex was significantly larger (60%, P < 0.01) compared with the base. In contrast, calcium transient duration was significantly longer (7.5%, P < 0.03) near the base compared with the apex. During isoproterenol (0.05 µM) and verapamil (2.5 µM) administration, ratiometric calcium transients accurately reflected changes in contractile function, and, the direction of base-to-apex heterogeneities remained unchanged compared with control. Ratiometric optical mapping techniques can be used to accurately quantify heterogeneities of calcium handling in the intact heart. Significant heterogeneities of calcium release and sequestration exist from base to apex of the intact heart. These heterogeneities are consistent with base-to-apex heterogeneities of contraction observed in the intact heart and may play a role in arrhythmogenesis under abnormal conditions.

optical mapping; ratiometric calcium imaging; electrophysiology; contraction

THERE IS RECENT EVIDENCE that cytosolic calcium transients exhibit regional differences under both normal (28) and pathological conditions (47). This is not surprising because heterogeneous repolarization properties have been reported between different transmural myocardial layers (2) and within the same myocardial layer from base to apex of the heart (27, 41). Normally, such repolarization heterogeneities are physiological; however, under certain conditions they can become pathophysiological and directly influence arrhythmia vulnerability (1, 30). On the other hand, relatively little is known regarding spatial heterogeneity of intracellular calcium handling. Recent studies of mRNA and protein expression (28, 45), as well as calcium transient recordings from single cells (18) and the intact heart (28), suggest that calcium handling is heterogeneous across the transmural wall of the ventricle; however, it is not known whether calcium handling is heterogeneous from base to apex. Studies of myocardial function have reported a consistent base-to-apex gradient in stress and strain (21), ejection fraction contribution (7), and segment shortening (33), suggesting the presence of a similar gradient in calcium handling. Therefore, because of electrical and contractile heterogeneities, we hypothesize that calcium handling is heterogeneous between basal and apical regions of the intact heart.

Our understanding of intracellular calcium physiology has significantly benefited from the innovation and continued development of calcium-sensitive fluorescent dyes. Theoretically, the ratio of fluorescence collected at two separate wavelengths simultaneously from a calcium-sensitive dye can be used to minimize changes in fluorescence over space (e.g., heterogeneity of excitation light and dye loading) and time (e.g., dye washout), as well as motion artifact and other factors (10, 11, 25). Such dual-wavelength (i.e., ratiometric) imaging has been extensively performed in isolated myocytes to measure intracellular calcium levels. However, because of methodological limitations, heterogeneity of calcium handling in the intact heart has previously been difficult to quantify. To test the hypothesis that calcium handling is spatially heterogeneous from base to apex, we developed and validated an optical mapping system capable of quantifying intracellular calcium at hundreds of sites simultaneously from an intact heart using ratiometric techniques.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental preparation. This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). Adult retired breeder guinea pigs (n = 23, 800–1,100 g) were anesthetized (30 mg/kg ip pentobarbital sodium), and their hearts were excised and Langendorff perfused by an aortic cannula with oxygenated (95% O2-5% CO2) Tyrode solution containing (mM) 121.7 NaCl, 25.0 NaHCO3, 2.74 MgSO4, 4.81 KCl, 5.0 dextrose, and 2.0 CaCl2 (pH 7.4, 32°C). Perfusion pressure was maintained between 50 and 70 mmHg by regulating coronary flow using a pulsatile flow system. To measure intracellular calcium transients, hearts were stained with the calcium-sensitive indicator indo-1 AM (Molecular Probes) dissolved in 0.5 ml solution of DMSO and Pluronic (20% wt/vol) at a final concentration of 5 µM. In some experiments, 10 mM of 2,3-butanedione monoxime (BDM) was used to ensure that motion artifact, if present, did not influence our result.

The perfused hearts were placed in a Lexan chamber. The mapping field was positioned over the left anterior descending artery just below its bifurcation with the diagonal coronary artery. The anterior surface of the heart was gently positioned against an imaging window using a movable piston, which has previously been shown to have no significant effect on electrophysiological properties (19). To avoid epicardial surface cooling and desiccation, the heart was immersed in the coronary effluent, which was maintained at a uniform temperature equal to the perfusion temperature with a heat exchanger located in the chamber. Heat loss measured between the imaging window and the heart was negligible (<0.1°C) and did not significantly affect electrophysiological properties as observed in the absence of an imaging window (1, 19). During the first 10 min of perfusion, slight enlargement of the heart was observed (10%); however, all optical recordings were made after 20 min to ensure constant pressure and flow. The volume-conducted electrocardiogram (ECG) was monitored by using three silver disk electrodes fixed to the chamber in positions roughly corresponding to ECG limb leads I, II, and III. ECG signals were filtered (0.3–300 Hz), amplified (1,000x), and displayed on an oscilloscope. A fine-gauge (0.003'' diameter), polytetrafluoroethylene-coated silver unipolar electrode was inserted into the left ventricular anterior wall to stimulate the ventricular endocardial surface at twice-diastolic threshold current. To ensure steady-state conditions, the preparation was paced at a constant cycle length of 400 or 300 ms, depending on the experimental protocol (see Experimental protocol). Physiological stability of the preparation was ensured by monitoring the ECG, coronary pressure, coronary flow, and perfusion temperature continuously throughout each experiment. Preparations remain viable for 4–5 h, but the entire experimental protocol typically lasted 1–3 h.

Optical mapping system. We developed a novel optical mapping system (Fig. 1) capable of recording high-fidelity fluorescent signals at two wavelengths simultaneously with high spatial and temporal resolution. Excitation light obtained from a 500-W mercury arc lamp (Thermo-Oriel) was filtered at 350 ± 10 nm (Chroma Technology) and directed through a flexible liquid light guide (Thermo-Oriel) to the preparation. Fluorescent light from the preparation was collected by a tandem lens assembly (29). The tandem lens assembly consisted of four high-numerical aperture complex Nikon photographic lenses placed facing each other. A 445-nm dichroic long-pass mirror (Chroma Technology) was positioned between the photographic lenses of the tandem lens assembly at a 45° angle to transmit all wavelengths above 445 to a 16 x 16 element photodiode array and reflect all wavelengths below 445 nm to another 16 x 16 element photodiode array (Hamamatsu). Transmitted and reflected fluorescent light was limited to 485 ± 10 and 405 ± 10 nm, respectively, by using optical interference filters (Chroma Technology). All optical components were aligned with an accuracy of 35 µm (31). Signals recorded from both arrays and ECG signals were multiplexed and digitized with 12-bit precision at a sampling rate of 1,000 Hz per channel (Microstar Laboratories). Spatial resolution (0.88 mm) was determined by the center-to-center distance between adjacent photodiodes (1.1 mm) and the optical magnification being used (1.24x) and did not account for light scatter within the tissue. To view the preparation, the dichroic mirror was rotated to reflect an image of the preparation to a CCD video camera (Pulnix).



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  		Fig. 1. Schematic diagram of the ratiometric optical mapping system. Filtered excitation light (350 nm) from a mercury arc lamp (500 W) is directed to the preparation. Fluorescence is collected by a tandem lens assembly consisting of four complex photographic lenses. A dichroic mirror (445 nm) passes fluorescence of longer wavelengths to an emission filter (485 nm) and detector array (Detector F485) and reflects fluorescence of shorter wavelengths to a second emission filter (405 nm) and detector array (Detector F405). To view the preparation, the dichroic mirror is rotated clockwise by exactly 90° to reflect an image of the preparation to a CCD video camera.

 

For all experiments, before indo-1 loading, background fluorescence representing tissue autofluorescence (e.g., NADH, cytochromes, and flavoproteins) was recorded at both emission wavelengths (485 nm and 405 nm). The heart was then loaded for 45 min with indo-1 AM at room temperature to minimize dye compartmentalization. The dye-loading period was followed by a 20-min washout period to remove unhydrolyzed or partially hydrolyzed dye (8). All recordings made after indo-1 loading were DC coupled and include fluorescence originating from the dye and the tissue (background fluorescence). For offline analysis, background fluorescence at each wavelength was subtracted out (i.e., background subtracted). Ratiometric calcium transients (CaR) were determined by dividing the background-subtracted fluorescence calcium transients at 405 nm (CaF405) by the background-subtracted fluorescence calcium transients at 485 nm (CaF485).

Experimental protocol. To test the insensitivity of CaR to changes in excitation light intensity and to account for any physiological heterogeneities, CaF405 and CaR were compared at each site under normal light conditions and during a 50% reduction in excitation light intensity (n = 7). In four experiments, CaR was measured over an extended time period (~3 h) to investigate the insensitivity of CaR to changes in fluorescence over time (e.g., indo-1 washout). These validation experiments were performed in the presence of BDM, an electro-mechanical uncoupler, and during steady-state pacing at a cycle length of 400 ms. Finally, in six experiments, CaR and left ventricular developed pressure (LVDP) were measured in the absence of BDM. LVDP was assessed by using a latex Tyrode-filled balloon that was connected to a pressure transducer and inserted in the left ventricle. The left and right atria were removed to permit the insertion of the latex balloon and eliminate competition from the sinus node during ventricular pacing. CaR and LVDP were recorded during control conditions. Recordings were also performed during administration of isoproterenol (0.05 µM) and verapamil (2.5 µM) to compare the effects of known inotropic agents on calcium handling and LVDP. These measurements were performed during steady-state pacing at a cycle length of 300 ms and after 10 min of continuous drug administration.

Calibration protocol. To determine intracellular calcium concentration, the ratiometric calcium transients were calibrated using the techniques developed by Grynkeiwicz et al. (20). The calibration parameters, Rmin and Rmax, were obtained in situ from a separate set of experiments by perfusing hearts with either a modified calcium-free, Rmin (n = 3) or a calcium-saturated, Rmax (n = 3) Tyrode solution. The modified Rmin solution contained (mM) 137 NaCl, 5.4 KCl, 1.0 MgCl2, 10 EGTA, 5 HEPES, 0.01 4-bromo-A-23817, 0.005 carbonyl cyanide n-chlorophenyl hydrazine (CCCP), 0.002 rotenone, and 10 BDM; pH 7.4. The modified Rmax solution contained (mM) 137 NaCl, 5.4 KCl, 1.0 MgCl2, 2 CaCl2, 5 HEPES, 0.01 4-bromo-A-23817, CCCP 0.005, 0.002 rotenone, and 10 BDM; pH 7.4. Before indo-1 loading, background fluorescence was recorded at both emission wavelengths (485 and 405 nm). The heart was then loaded for 45 min with indo-1 AM as described above using the normal Tyrode solution. After calcium transients were recorded with BDM, Rmin was determined by measuring fluorescence after 20 min of perfusion with the modified Rmin Tyrode solution. Rmin was determined at every site by dividing the background-subtracted fluorescence at 405 nm (CaF405) by the background-subtracted fluorescence at 485 nm (CaF485). Rmax was determined in a similar fashion by using the modified Rmax solution. In addition, because of ionophore-induced contracture, we determined an average Rmax value from the center of the mapping field. Diastolic and systolic calcium ratio levels (CaRdias and CaRsys) are defined as the minimum level before and the maximum level after the calcium transient upstroke, respectively. With the use of measured CaRdias, CaRsys, and Rmin at each recording site, a mean Rmax value, and published values of dissociation constant (Kd) = 844 nM and {beta} = 3.0 (3), absolute diastolic (Cadias), and systolic (Casys) calcium concentrations were calculated at each site using the following standard calibration equations (20)


Data analysis. The fluorescence calcium transient amplitude (CaFamp) expressed in arbitrary units (AU), ratiometric calcium transient amplitude (CaRamp) expressed in ratio units (RU), LVDP amplitude (LVDPamp), and absolute calcium transient amplitude (Caamp) are defined as the difference between their respective systolic and diastolic values. The rise times for CaR and LVDP were calculated as the time from 10% to 90% amplitude during systole. Calcium transient duration (CaR85) was calculated from the time of 10% amplitude during systole to the time of 15% amplitude during diastole. To quantify the rate of recovery of intracellular calcium to diastolic levels, the decay portion of the calcium transient (from 70% to 0% of the decline phase) was measured by the time constant (i.e., {tau}) of a single exponential fit, as previously used for uncalibrated and calibrated fluorescent signals (6, 15, 28). Basal and apical sites (4 each) were chosen from the top two and bottom two rows of the photodiode array, respectively. To display regional differences in calcium transient parameters, numerical values and corresponding XY positions were plotted as contour maps using a MATLAB (Mathworks) contour plotting algorithm. Statistical (ANOVA and t-test) and regression analysis were considered significant for P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Validation of ratiometric calcium transients. To demonstrate that CaR is insensitive to changes in excitation light and fluorescence reduction over time, CaF and CaR were measured under different excitation light intensities and over long time periods. Figure 2A shows CaF405 and CaR measured from the same site during normal and then reduced excitation light conditions to control for site-to-site variability. When light intensity was reduced by ~50%, CaFamp decreased markedly, whereas CaR did not significantly change. Similar results were observed across the entire mapping field. Figure 2B shows contour maps of CaFamp and CaRamp obtained under normal and reduced excitation light conditions. Importantly, CaFamp exhibited a circular (i.e., bull's eye) pattern that was dependent on the pattern of excitation light. The intensity of excitation light (data not shown) and CaFamp (Fig. 2B, top) were greatest at the center of the mapping field and varied by as much as ~90% across the mapping field. In contrast, CaRamp did not exhibit a bull's-eye pattern and varied to a lesser extent (~35%). Furthermore, when light intensity was reduced, CaFamp showed an overall reduction but CaRamp, in contrast, showed no such pattern and was the same as during normal excitation light. Figure 2C shows a site-by-site comparison of CaFamp (top) and CaRamp (bottom) under normal and reduced excitation light conditions. CaFamp significantly deviated (bold line, slope = 0.57) from the line of identity (dashed line). In contrast, CaRamp was superimposable (slope = 0.98) with the line of identity. Over all experiments, the average slope for CaFamp was 0.59 ± 0.04, whereas the average slope for CaRamp was 0.99 ± 0.01 (P < 0.001, n = 7). Finally, mean CaFamp was significantly reduced over time (–46 ± 8% over a 60-min period, P < 0.01). In contrast, mean CaRamp showed no significant change (–1.3 ± 1%) over a 60-min period (data not shown). Therefore, these data suggest that ratiometric calcium transients, unlike fluorescence transients, are insensitive to changes in excitation light intensity and fluorescence over time.



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  		Fig. 2. A: calcium fluorescence (CaF405) and ratio (CaR) transients from the same site during normal and reduced excitation light conditions. Reducing the intensity of excitation light reduced CaF405 but not CaR. B: contour maps of CaF amplitude (CaFamp) and CaR amplitude (CaRamp) during normal and reduced excitation light conditions. Under reduced light conditions, CaFamp was significantly decreased compared with normal light. CaFamp also exhibited a circular "bull's eye" pattern similar to the pattern of excitation light (not shown). In contrast, CaRamp did not exhibit a circular pattern and showed no change in magnitude when excitation light was reduced. C: identity plots of CaFamp (top) and CaRamp (bottom) during normal and reduced excitation light. CaFamp showed a marked deviation (bold line, slope = 0.57) from the line of identity (dashed line, slope = 1), whereas CaRamp showed no such deviation (bold line, slope = 0.98). CaF is expressed in arbitrary units (AU). CaR is expressed in ratio units (RU).

 

Base-to-apex heterogeneity of intracellular calcium handling. Figure 3A, top, shows representative basal and apical calcium transients recorded from the left ventricle of the same heart. Interestingly, basal calcium transients had significantly smaller amplitudes compared with apical calcium transients. CaRamp measured across the entire mapping field (Fig. 3A, middle) revealed a consistent and uniform gradient from base to apex. Over all experiments, CaRamp was 60% larger (P < 0.01, n = 7) near the apex (0.08 ± 0.01 RU) compared with the base (0.05 ± 0.01 RU). In addition, calcium transients (Fig. 3B, top) taken near the base had longer duration (CaR85) compared with transients taken near the apex. Figure 3B, middle, demonstrates an apex-to-base gradient across the entire mapping field. Over all experiments, CaR85 was 7.5% longer (P < 0.03, n = 7) near the base of the heart (284 ± 14 ms) compared with the apex (264 ± 18 ms). Apical calcium transients also exhibited a faster decay (i.e., smaller {tau}) and faster rise time (Fig. 3, C and D, top, respectively) compared with basal transients. Over the entire mapping field (Fig. 3, C and D, middle), an apex-to-base gradient in {tau} and rise time were observed. On average, {tau} was 14% longer (P < 0.007, n = 7) near the base of the heart (112 ± 6 ms) compared with the apex (98 ± 7 ms), and rise time was 29% longer (P < 0.001, n = 7) near the base of the heart (49 ± 9 ms) compared with the apex (38 ± 4 ms). The gradients of amplitude, rise time, and {tau} were also observed with or without BDM (data not shown). These data suggest that significant heterogeneities of calcium handling span the anterior surface of the guinea pig heart where the apex exhibits a larger amplitude as well as faster kinetics of calcium release and sequestration compared with the base.



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  		Fig. 3. Heterogeneities of CaRamp (A), CaR85 (B), tau (C), and rise time (D). Calcium transients from the apex had a larger CaRamp compared with the base (top), and the contour map (middle) revealed a uniform gradient from base to apex. Over all experiments, calcium transient amplitude was significantly larger at the apex compared with the base (bottom). In contrast, CaR85, tau, and rise time were longer near the base compare with the apex of the heart (top), and the contour maps (middle) revealed a uniform gradient from apex to base. Similar results were observed over all experiments (bottom).

 

Intracellular calcium handling was pharmacologically altered using inotropic agents to assess their influence on the spatial heterogeneity of calcium handling. As seen from a representative experiment in Fig. 4A, CaR (left) and LVDP (right) exhibited a similar increase with isoproterenol (dashed line) and decrease with verapamil (dotted line) compared with control (solid line). Over all experiments (Fig. 4B), isoproterenol significantly increased mean CaRamp (P < 0.01) and LVDPamp (P < 0.001), whereas verapamil significantly reduced mean CaRamp (P < 0.001) and LVDPamp (P < 0.004). Figure 4A also shows that isoproterenol reduced rise time for CaR and LVDP (18 and 39 ms, respectively) compared with control conditions (30 and 59 ms, respectively). In contrast, verapamil prolonged rise time for CaR and LVDP (40 and 67 ms, respectively) compared with control conditions. Finally, end-diastolic calcium levels during isoproterenol and verapamil administration decreased compared with control at a pacing cycle length of 300 ms. Similar changes were observed in end-diastolic pressure. These data suggest that CaR is a sensitive and reliable index of intracellular calcium handling under various inotropic states. To determine the influence of isoproterenol and verapamil on calcium handling heterogeneity, contour maps of CaRamp and CaRdias were compared before and during drug administration (Fig. 5A). As expected, isoproterenol (middle) increased and verapamil (right) decreased CaRamp compared with control (left); however, the gradient of CaRamp remained from base to apex regardless of the intervention. In addition, CaRdias decreased during isoproterenol (middle) and verapamil (right) compared with control (32). Over all experiments (Fig. 5B), apical CaRamp was significantly larger than basal CaRamp during control (left), isoproterenol (middle), and verapamil (right). These data indicate that the inotropic agents used in this study significantly altered intracellular calcium levels in the intact heart; however, gradient direction remained largely unaffected.



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  		Fig. 4. A: CaR (left) and left ventricular developed pressure (LVDP) (right) during control condition (solid line) and during isoproterenol (dashed line) and verapamil administration (dotted line). The numbers in parenthesis indicate rise time. For each intervention, the changes in CaR correlate well with the respective changes in LVDP. B: summarized findings over 6 experiments. CaR and LVDP were significantly greater during isoproterenol and smaller during verapamil compared with control conditions.

 


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  		Fig. 5. A: contour maps of CaRamp (top) and CaRdias (bottom) during control conditions, isoproterenol, and verapamil administration. Overall, CaRamp increased with isoproterenol and decreased with verapamil; however, the base-to-apex gradient was always present. Furthermore, CaRdias decreased for both interventions compared with control. Similar results were obtained over all experiments (B).

 

Calibrated calcium transients. To determine absolute intracellular calcium levels, ratiometric calcium transients were calibrated using the calibration parameters, Rmin and Rmax, obtained from a separate set of experiments. Rmin at each mapping site and an average Rmax from the center of the mapping field were used to calculate intracellular calcium levels across the entire mapping field. Shown in Fig. 6 are representative maps of the calcium transient amplitude expressed as ratio values (left) and as calibrated calcium values (right) obtained from the same heart. Caamp, like CaRamp, exhibited a gradient of amplitude from base to apex. Similar results were observed in each experiment (n = 3). These data provide further evidence that in the intact heart, ratiometric optical mapping can be used to measure intracellular calcium levels and that significant heterogeneities of calcium handling exist from apex to base.



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  		Fig. 6. Shown are representative contour maps of uncalibrated CaRamp (left) and calibrated Caamp (right). Both maps exhibited the same base-to-apex pattern of calcium amplitude. Caamp was derived from Casys – Cadias using CaRsys, CaRdias, and Rmin measured in the same experiment at every site, a single average Rmax value obtained from a separate experiment, and previously published values for Kd = 844 nM and {beta} = 3.0.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have developed a novel, simultaneous multisite optical mapping system to accurately quantify intracellular calcium from an intact heart by using ratiometric techniques. Using this system, we observed significant base-to-apex heterogeneities of calcium handling across the anterior surface of the guinea pig heart.

Ratiometric calcium imaging in the whole heart using optical mapping. In theory, dual-wavelength ratiometric imaging is capable of reducing fluorescence measurement artifacts associated with changes in excitation light and dye concentration over space and time. Ratiometric calcium imaging is routinely performed in single cells. However, simultaneous multisite fluorescent recordings from the intact heart present a bigger experimental challenge. Previously, ratiometric recordings have been made in the intact heart from one or two sites (9, 18) or from a limited number of sites sequentially (42). Our results confirm that dual-wavelength ratiometric imaging using optical mapping techniques can be used to accurately measure calcium handling (calcium levels and kinetics) over time and across many sites simultaneously with high spatial and temporal resolution.

Because ratiometric and nonratiometric fluorescence measurements are not necessarily linear over the entire physiological range of intracellular calcium (20), true intracellular calcium levels cannot be inferred without calibration (9). We have previously found that multisite optical mapping techniques can accurately measure calcium concentration from cuvettes containing homogeneous buffered calcium solutions of known concentration (24). However, calibration in the intact heart is a greater challenge (3, 9), mostly due to ionophore-induced contraction that causes myocardial shrinkage. Nevertheless, in separate experiments, we were able to measure the calibration parameters Rmin and Rmax. With the use of published values for Kd and {beta}, calibrated calcium transient amplitudes (Caamp) exhibited a base-to-apex gradient similar to uncalibrated calcium transient amplitudes (CaRamp). These data, however, must be interpreted with caution because of difficulties associated with measuring Rmax. Nevertheless, the values of Caamp we report are consistent with previous studies using comparable techniques (39). Nakae et al. (39) report calcium transient amplitudes of 430 nM from the epicardial surface of the beating guinea pig heart, which is slightly higher compared with the present study. This difference could be explained by a higher calcium concentration in the Tyrode solution (2.5 mM), warmer temperature (37°C), and absence of BDM, all of which are expected to increase calcium transient amplitude. If myocytes were isolated from the apical and basal regions, it is expected that apical myocytes would exhibit larger calcium transients than basal myocytes. However, values of calibrated calcium transients obtained in whole hearts are an underestimation of true cellular calcium compared with isolated myocytes because of the effects of dye trapped in extracellular compartments, cellular tension, and tissue-filtering effects.

From the complications associated with calibrating ratiometric measurements in the intact heart, many investigators have resorted to using uncalibrated ratiometric measurements (49) while acknowledging the advantages over nonratiometric methods and the shortcomings compared with calibrated ratiometric values (11, 35, 43). In our study, CaR amplitude and time course (rise time and {tau}) were consistent with LVDP under each pharmacological intervention. However, changes in LVDP did not always parallel CaR. For example, during verapamil administration, the diastolic calcium level should continue to decline throughout the interval between beats and be below baseline, even though the left ventricular pressure tracing would not. This was not observed in our study because, most likely, the rapid pacing rate (300 ms cycle length) did not allow diastolic calcium levels to continue to decline between beats. In these experiments, a higher pacing rate was used to overcome the rapid intrinsic rhythm of the heart associated with isoproterenol. Nevertheless, the decrease in diastolic calcium levels and LVDP that we observed during verapamil administration is consistent with previous findings (32). In addition, it is important to mention that changes in CaRamp when using isoproterenol were not as large as the changes in LVDP (Fig. 5). There are several reasons that can account for this observation: 1) CaR is only measured from the anterior epicardial surface, whereas LVDP reflects the contractile function of the entire left ventricle; 2) uncalibrated CaR is nonlinear and may underestimate high calcium levels, which is a property of all calcium-chelating indicators (20); 3) myofilament sensitivity to a given calcium concentration is increased at hypothermic conditions (5, 38) and will, therefore, increase LVDP more than CaRamp at 32°C. Nevertheless, our results suggest that CaR is a sensitive and reliable marker of intracellular calcium levels and time course.

Calcium handling heterogeneities in the whole heart. Our data show a gradient in calcium transient amplitude that exists from base to apex across the anterior epicardial surface of the intact guinea pig heart. We are not aware of any similar investigation of calcium transient amplitude from base to apex. Nevertheless, our finding is consistent with previous studies reporting base-to-apex heterogeneities of myocardial pressure development (16), wall thickening (36), stress and strain (7), segment shortening (22), regional ejection fraction contributions (17), as well as myocardial perfusion and metabolic rates (13). Isoproterenol and verapamil did not alter the direction of the CaR amplitude gradient. However, end-diastolic levels during isoproterenol and verapamil were lower than control. This is expected because of smaller calcium transient amplitude during verapamil and faster calcium sequestration associated with isoproterenol (34). We also observe an apex-to-base gradient in the calcium transient time course (i.e., CaR85, {tau}, and rise time). These findings are also consistent with studies of myocardial function showing faster recovery from systole and shorter duration of contraction at the apex of the heart compared with the base (48). With the use of single-wavelength fluorescence techniques, a gradient of calcium transient duration from apex to base has been reported in the guinea pig hearts (14). However, others (42) have reported spatial differences in the calcium transient duration across the surface of rabbit hearts but without a consistent gradient. This may be due to the differences in species or the methods used to measure calcium transients.

Mechanisms and implications of calcium transient heterogeneity. The underlying mechanism of calcium heterogeneities between the base and apex is unclear. However, two mechanisms could explain these findings. First, calcium regulatory proteins may be heterogeneously expressed from base to apex. For example, differences in calcium-handling proteins (45) have been previously reported between different transmural layers of myocardium where differences in calcium transients have been observed (37). In particular, calcium transient duration and decay are longer and slower, respectively, near the endocardium compared with the epicardium, presumably due to reduced sarco(endo)plasmic reticulum Ca2+ (SERCA2a) expression near the endocardium (28). Even within the epicardial layer, proteins responsible for membrane currents have been shown to be heterogeneous (4, 40). Therefore, it is possible that the expression of proteins responsible for calcium handling is also heterogeneous from base to apex. Second, the heterogeneities of calcium handling could be related to the gradient of action potential duration known to exist between base and apex in the intact heart. Calcium transient duration gradients reported in this study correlate well with gradients of the action potential duration reported previously (27), suggesting a possible mechanistic relationship between intracellular calcium and calcium-sensitive membrane currents. Further studies, however, are required to elucidate the underlying mechanisms responsible for calcium handling heterogeneities from base to apex of the intact heart.

The calcium transient heterogeneities that we observed between the base and apex are consistent with and may, therefore, underlie heterogeneities of contraction known to exist in the normal intact heart. Importantly, ratiometric imaging using optical mapping techniques may provide new mechanistic insights to regional heterogeneities in contractile function seen under various pathophysiological conditions such as hypertrophy (12), heart failure (22), and myocardial infarction (26). Furthermore, this imaging technique may also provide new insight to arrhythmogenesis. For example, in preliminary experiments we have shown that repolarization alternans, a mechanism of arrhythmogenic T-wave alternans, occurs first near the left ventricular base of the guinea pig heart (46). Interestingly, calcium transient amplitude is smaller and rise time is slower near the left ventricular base. This suggests that reduced calcium release may be a mechanism of repolarization alternans in the intact heart (23) and may explain the development of regional arrhythmogenic repolarization alternans (44, 47). However, additional studies are required to determine the exact mechanism of repolarization alternans in the intact heart.


   ACKNOWLEDGMENTS
 
GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-68877, the Whitaker Foundation, and the Ohio Valley Affiliate of the American Heart Association.

This study was presented in part at the 75th annual scientific sessions of the American Heart Association, Chicago, 2002.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. R. Laurita, Heart and Vascular Research Center, MetroHealth Campus, Case Western Reserve Univ., 2500 MetroHealth Dr., Rammelkamp 654, Cleveland, OH 44109-1198 (klaurita{at}metrohealth.org).

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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 DISCUSSION
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