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Am J Physiol Heart Circ Physiol 290: H1566-H1574, 2006. First published December 3, 2005; doi:10.1152/ajpheart.00686.2005
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Calcium spark properties in ventricular myocytes are altered in aged mice

Susan E. Howlett, Scott A. Grandy, and Gregory R. Ferrier{dagger}

Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada

Submitted 23 June 2005 ; accepted in final form 23 November 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study determined whether whole cell Ca2+ transients and unitary sarcoplasmic reticulum (SR) Ca2+ release events are constant throughout adult life or whether Ca2+ release is altered in aging ventricular myocytes. Myocytes were isolated from young adult (~5 mo old) and aged (~24 mo old) mice. Spontaneous Ca2+ sparks and Ca2+ transients initiated by field stimulation were detected with fluo-4. All experiments were conducted at 37°C. Ca2+ transient amplitudes were reduced, and Ca2+ transient rise times were abbreviated in aged cells stimulated at 8 Hz compared with young adult myocytes. Furthermore, the incidence and frequency of spontaneous Ca2+ sparks were markedly higher in aged myocytes compared with young adult cells. Spark amplitudes and spatial widths were similar in young adult and aged myocytes. However, spark half-rise times and half-decay times were abbreviated in aged cells compared with younger cells. Resting cytosolic Ca2+ levels and SR Ca2+ stores were assessed by rapid application of caffeine in fura-2-loaded cells. Neither resting Ca2+ levels nor SR Ca2+ content differed between young adult and aged cells. Thus increased spark frequency in aging cells was not attributable to increased SR Ca2+ stores. Furthermore, the decrease in Ca2+ transient amplitude was not due to a decrease in SR Ca2+ load. These results demonstrate that alterations in fundamental SR Ca2+ release units occur in aging ventricular myocytes and raise the possibility that alterations in Ca2+ release may reflect age-related changes in fundamental release events rather than changes in SR Ca2+ stores and diastolic Ca2+ levels.

senescence; sarcoplasmic reticulum; Ca2+ imaging; fluorescence; excitation-contraction coupling

AGING CAUSES SIGNIFICANT CHANGES in contractile function in mammalian hearts in the absence of overt disease (21). Interestingly, whereas cardiac contractile function during exercise is compromised in older adults, contractility at rest is not altered substantially by age (21). Furthermore, myocardial contractions are prolonged and relaxation is relatively less complete in older individuals (21, 22). Interestingly, similar changes have been reported in individual ventricular myocytes isolated from hearts of aged mammals. Amplitudes of contractions and Ca2+ transients are smaller and prolonged in aged myocytes compared with young myocytes when the frequency of contractions is increased by rapid pacing or when cells are stimulated with catecholamines (24, 39). These observations suggest that defects in cardiac contractile function in aging originate, at least in part, at the level of the ventricular myocyte.

Contraction of mammalian cardiac muscle is activated by a transient rise in intracellular free Ca2+ (5). This Ca2+ transient arises primarily from release of intracellular Ca2+ stores in the sarcoplasmic reticulum (SR) (5). SR Ca2+ release is triggered by Ca2+ influx, primarily through L-type Ca2+ channels (5). In the heart, Ca2+ is released from the SR through Ca2+ release channels, which also are called type 2 ryanodine receptors (23). Because Ca2+ transients are abnormal in aging cells (24), it is possible that contractile dysfunction may reflect abnormalities in SR Ca2+ release.

Some studies have shown that ryanodine receptor protein levels are reduced in aged hearts (1, 15), whereas other studies have not reported a change (40). Furthermore, phosphorylation of ryanodine receptors by Ca2+-calmodulin dependent kinase decreases with age (40). Thus alterations in ryanodine receptor density and/or properties are thought to occur in the aging heart and may alter Ca2+ release properties.

In the young adult heart, Ca2+ influx through L-type Ca2+ channels activates clusters of ryanodine receptors and results in brief, localized release of Ca2+ called Ca2+ sparks (9, 17). When a myocyte is activated by an action potential, many Ca2+ sparks fuse to form the Ca2+ transient (7, 8, 31). In addition, in quiescent cells, spontaneous Ca2+ sparks can occur in the absence of Ca2+ influx through L channels (20, 26, 31). Spontaneous sparks occur at a low frequency in quiescent myocytes and are thought to provide a pathway for SR Ca2+ leak that limits SR Ca2+ content (4, 27). Whether changes in the characteristics and behavior of spontaneous Ca2+ sparks occur in aged myocytes is not yet known. If Ca2+ spark frequency increases in aging, contractile function might be impaired by increased Ca2+ leak from the SR. On the other hand, if Ca2+ spark frequency decreases in aging, this may provide a protective response that helps maintain SR Ca2+ load and contractility in aging. The objective of this study was to determine whether there were age-related changes in Ca2+ transients both at the whole cell level and at the level of unitary Ca2+ release in intact myocytes from young adult and aged hearts. Studies compared field-stimulated Ca2+ transients and spontaneous Ca2+ sparks in fluo-4-loaded ventricular myocytes from young adult (~5 mo old) and aged (~24 mo old) mice. All experiments were conducted at 37°C, which is close to core body temperature in the mouse (30).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation. These studies were conducted according to guidelines published by the Canadian Council on Animal Care. The Dalhousie University Committee on Laboratory Animals approved all animal care protocols. Ventricular myocytes were isolated from adult male and female mice (B6SJLF1/J) originally purchased from Jackson Laboratories (Bar Harbor, ME) and raised in the Dalhousie University animal care facility. Young adult mice were ~5 mo of age and aged mice were ~24 mo of age (ages were 22.8 ± 1.3 vs. 98.0 ± 1.6 wk for young adult and aged animals, respectively). Mice were weighed and then anesthetized with pentobarbital sodium (200 mg/kg ip) coinjected with heparin (100 U) to minimize blood coagulation. Hearts were cannulated in situ and were then perfused at 2 ml/min with oxygenated Ca2+-free buffer at 37°C. The Ca2+-free buffer had the following composition (in mM): 130 NaCl, 5 KCl, 1 MgCl2, 0.33 NaH2PO4, 25 HEPES, 20 glucose, 3.0 Na pyruvate, and 1.0 Na lactate (pH 7.4 with NaOH). After 10 min, the perfusate was switched to the Ca2+-free solution above supplemented with collagenase (24 mg/30 ml, Worthington type I, 242 U/mg), dispase II (10 mg/30 ml, Boehringer-Mannheim), trypsin (1 mg/30 ml, Sigma), and 50 µM CaCl2. Hearts were perfused for an additional 10 min. The ventricles were then cut into small pieces in a high-potassium, substrate-enriched solution of the following composition (in mM): 30 KCl, 90 KOH, 30 KH2PO4, 3 MgSO4, 50 glutamic acid, 20 taurine, 0.5 EGTA, 10 glucose, and 10 HEPES (pH 7.4 with KOH). Ventricular myocytes were released from ventricular muscle by gentle agitation, and the cell suspension was filtered through a 225-µm polyethylene mesh (Spectrum).

Ca2+ sparks. Myocytes were loaded with fluo-4 by incubation in 20 µM fluo-4 AM (Molecular Probes) for 25–30 min in the dark at room temperature. A fluo-4 AM stock was prepared in DMSO (Sigma) and Pluronic F-127 (Molecular Probes) as described in our previous studies (13, 16). Myocytes loaded with fluo-4 were placed in a 1.5-ml experimental chamber on the stage of a Zeiss LSM 510 laser scanning microscope (Axiovert 100). The bottom of the chamber was formed by a glass coverslip (24 x 50 mm, 0.08–0.13 mm in thickness; VWR Scientific) coated with natural mouse laminin (Invitrogen). Laminin (1 mg) was dissolved in 100 ml of medium 199 (Sigma), and 1-ml aliquots of solution were frozen at –70°C until use. An aliquot of laminin (300 µl) was placed on the coverslip for ~20 min and then removed with a pipette before the introduction of cells. Cells were allowed to settle on the bottom of the experimental chamber for 5 min and were then superfused at 1.3 ml/min with a 37°C solution of the following composition (in mM): 145 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with NaOH). The temperature was controlled by a water-jacketed heat exchanger positioned immediately before the bath inlet.

Ca2+ sparks were measured in quiescent myocytes only. In other experiments, myocytes were field stimulated through a pair of bipolar platinum wire electrodes that were 2–4 mm apart. Stimuli were 3-ms rectangular pulses delivered at a frequency of 2 Hz (model SD9, Grass Instruments). Changes in free Ca2+ were measured in line scan mode with excitation at 488 nm, and emission was measured at ≥525 nm (Zeiss oil immersion objective, x40/1.3 numerical aperature). Cells were repetitively scanned along the length of the cell at 1.5-ms intervals for 6 s. Each line was composed of 512 pixels. The confocal pinhole was adjusted to 54 µm to maximize x-y-z resolution of 0.26 x 0.26 x 0.75 µm. Laser intensity was reduced to ≤5% maximum to decrease cell damage and photobleaching. Emission lines were stacked in temporal order to construct line scan diagrams.

Ca2+ concentrations. In separate experiments, Ca2+ concentrations were measured in myocytes loaded with fura-2. Cells were incubated with fura-2 AM (5 µM) for 20–30 min at room temperature in the dark. Stock solutions of fura-2 AM were prepared by dissolving 50 µg of fura-2 AM in 20 µl of anhydrous DMSO. Fluorescence was measured ratiometrically with a DeltaRAM system (Photon Technologies International) and Felix software (PTI, Brunswick, NJ) as described previously (13). Fura-2 was excited alternately at 340 and 380 nm, and emission was recorded at 510 nm. Background fluorescence was subtracted for each excitation wavelength, and the ratio of emission during excitation at 340 and 380 nm was converted to Ca2+ concentration with an in vitro calibration curve determined with the same optical path used in the experiments.

Fura-2 Ca2+ transients elicited by rapid application of caffeine were used as an index of SR Ca2+ load (5). In these experiments, cells were voltage clamped to allow the delivery of trains of conditioning pulses to ensure a consistent activation history in young adult and aged myocytes. Discontinuous single-electrode voltage clamp (5–8 kHz) was conducted with high-resistance microelectrodes (18–26 M{Omega}, 2.7 M KCl) to minimize cell dialysis. Voltage clamp was conducted with pCLAMP software (version 8.0, Axon Instruments) and an Axoclamp 2B amplifier (Axon Instruments). Further details are provided in RESULTS. SR Ca2+ stores were assessed by rapid application of 10 mM caffeine for 1 s with a solution switcher device that rapidly changes the solution bathing the cell and maintains temperature at 37°C (18). Caffeine was applied 1 s after a train of ten 50-ms conditioning pulses from –80 to 0 mV, delivered at a frequency of 2 Hz. Caffeine was applied in solution with 0 mM Na+ and 0 mM Ca2+ to minimize efflux of Ca2+ through Na+-Ca2+ exchange (20). The composition of the solution was (in mM) 140 LiCl, 4 KCl, 4 MgCl2, 5 HEPES, 10 glucose, 0.3 lidocaine, and 4 4-aminopyridine (pH 7.4 with LiOH). Released Ca2+ was measured by the 340/380 fura-2 ratio, and 340/380 emission ratios were converted to Ca2+ concentrations as described above. Resting Ca2+ concentrations were measured in cells held at –80 mV. The caffeine-induced increase in peak Ca2+ concentration was measured as the peak increase in Ca2+ concentration induced by caffeine application and compared in young adult and aged myocytes.

Data analysis. Ca2+ sparks were visualized and analyzed with ImageJ (National Institutes of Health). Fluorescence signals were normalized by dividing them by the average background fluorescence intensity at rest. Ca2+ sparks were defined as local peak elevations of fluorescence intensity (F) that were ≥1.5 times the background levels (F0). The black background levels were subtracted from the data before calculating F/F0 ratios. The percentage of cells exhibiting Ca2+ sparks, called the spark incidence, was calculated for young adult and aged myocytes. The frequency of Ca2+ sparks (sparks·100 µm–1·s–1) also was calculated for each cell; cells that did not spark were included in this measure. Amplitude (F/F0), full width at half-maximum amplitude (FWHM), half-rise time, and half-decay time were calculated for individual sparks to characterize and compare sparks in young adult and aged myocytes. In field-stimulated myocytes, the intensity of each scan line (vertical axis) was averaged, and the mean intensities were plotted as a function of time to yield spatially averaged Ca2+ transients (13). F/F0, time to peak, and the time constant of transient decay were measured for Ca2+ transients recorded from young adult and aged myocytes. Time to peak was measured as the difference between the time where the transient reached its peak and the time where the upstroke of the transient began. The time constant for the decay of the Ca2+ transient was determined for each Ca2+ transient by fitting an exponential function to the decay of the transients.

Statistical analyses were conducted with Sigmastat (version 2.03, Jandel Scientific). Data are presented as means ± SE. Differences between means were tested for significance with a t-test. Differences in incidence were assessed with a {chi}2-test. Differences were considered statistically significant for P < 0.05. Nonlinear curve fitting was conducted with Sigmaplot (version 8.02, Jandel Scientific).

Chemicals. Lidocaine, HEPES buffer, EGTA, MgCl2, anhydrous DMSO, 4-aminopyridine, and caffeine were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Fura-2 AM, fluo-4 AM, and Pluronic F-127 were purchased from Molecular Probes (Hornby, ON, Canada). All other chemicals were purchased from BDH (Toronto, ON, Canada). Fura-2 AM and fluo-4 AM were dissolved in DMSO as described in Ca2+ concentrations, and all other chemicals were dissolved in deionized water.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In initial experiments, we compared Ca2+ transients recorded from young adult and aged myocytes. Figure 1A shows a representative line scan diagram recorded from a young adult myocyte field stimulated at 2 Hz. The line scan illustrates one stimulated Ca2+ transient. A similar recording from an aged myocyte stimulated at the same rate is shown in Fig. 1B. The average fluorescence intensity along each line scan was plotted as a function of time to obtain the spatially averaged Ca2+ transients shown in Fig. 1C. The Ca2+ transients recorded from young adult and aged myocytes had similar amplitudes and time courses. Fig. 1 also shows mean amplitudes and time courses of Ca2+ transients recorded from young adult and aged myocytes. Mean amplitudes of Ca2+ transients were similar in young adult and aged myocytes (Fig. 1D). Mean times to peak transient also were similar in young adult and aged cells (Fig. 1E). Figure 1F shows that the time constants for decay of the Ca2+ transient were similar in cells from young and aged hearts.


Figure 1
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  		Fig. 1. Ca2+ transients recorded from young adult and aged myocytes field stimulated at 2 Hz. A: stimulated Ca2+ transients recorded from a young adult cell in a representative line scan diagram. B: line scan diagram of a stimulated Ca2+ transient recorded from an aged myocyte. C: stimulated Ca2+ transients recorded from young and aged cells represented as spatially averaged Ca2+ transients. Amplitudes and time courses of these representative Ca2+ transients appeared similar in the two cells. D: mean amplitudes of Ca2+ transients were not significantly different in young adult and aged cells paced at 2 Hz. F/F0, fluorescence intensity/background fluorescence levels. E: mean times to peak were similar in young adult and aged myocytes. F: time constant for decay of Ca2+ transient (tau) also was similar in cells from young adult and aged hearts. n = 7–11 young adult and 5–9 aged myocytes.

 
We also compared Ca2+ transients recorded from young adult and aged myocytes stimulated at 8 Hz. Representative Ca2+ transients recorded from aged cells were visibly smaller than transients recorded from young adult cells when the stimulation frequency was increased to 8 Hz (Fig. 2, A and B). Spatially averaged Ca2+ transients are shown in Fig. 2C. These records indicate that the amplitude of the Ca2+ transient was smaller and the time to peak Ca2+ transient appeared to be shorter in the aged myocyte than in the younger cell (Fig. 2C). Fig. 2D shows that mean Ca2+ transient amplitudes were significantly smaller in aged myocytes compared with young adult myocytes. Furthermore, the time to peak transient was significantly shorter in aged myocytes than in young adult cells (Fig. 2E). However, the time constant for decay of the Ca2+ transient was similar in young adult and aged myocytes (Fig. 2F). Thus Ca2+ transients are smaller and have shorter rise times in aged myocytes when cells are paced at 8 Hz.


Figure 2
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  		Fig. 2. Amplitudes of spatially averaged Ca2+ transients are significantly smaller in aged myocytes compared with young adult myocytes when cells are paced 8 Hz. A and B: representative line scan diagrams recorded from a young adult cell and an aged cell when the stimulation rate was increased to 8 Hz. C: stimulated Ca2+ transients presented as spatially averaged Ca2+ transients in young adult and aged cells. Amplitude of Ca2+ transient was smaller in the aged cell compared with the younger cell, and time to peak transient appeared to be shorter in the aged cell. D: mean amplitudes of Ca2+ transients were significantly smaller in aged myocytes compared with young adult cells. E: time to peak Ca2+ transient was significantly shorter in aged myocytes stimulated at 8 Hz. F: time constants for decay of Ca2+ transients were similar in young adult and aged myocytes. n = 7–11 young adult and 5–9 aged myocytes. *P < 0.05.

 
These results suggest that age-related changes in Ca2+ transients depend upon the frequency of stimulation. To examine this directly, we compared the effect of frequency on Ca2+ transient parameters within each age group. These data are shown in Fig. 3. Figure 3A shows that Ca2+ transient amplitudes decreased when stimulation frequency was increased in both young and aged myocytes. Time to peak transient was prolonged in young adult myocytes when cells were stimulated at 8 Hz (Fig. 3B). In contrast, increased stimulation rate had no effect on time to peak transient in the aged cells (Fig. 3B). The time constant for decay of the Ca2+ transient was abbreviated in both young adult and aged cells when stimulation rate was increased (Fig. 3C). Thus increased stimulation frequency had similar effects on Ca2+ transient parameters in young and aged myocytes, except that time to peak transient was prolonged by increased rate in young cells but not in aged cells.


Figure 3
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  		Fig. 3. Comparison of effects of stimulation frequency on Ca2+ transient parameters recorded from young adult and aged myocytes. A: amplitudes of Ca2+ transients decreased significantly in young adult and aged myocytes when stimulation frequency was increased from 2 to 8 Hz. B: time to peak transient increased when stimulation frequency was increased in young myocytes but not in aged cells. C: time constants for decay of Ca2+ transient decreased when stimulation frequency was increased in both young adult and aged myocytes. n = 7–11 young adult and 5–9 aged myocytes. *P < 0.05.

 
In the next series of experiments, we explored whether there are changes in fundamental Ca2+ release events for SR Ca2+ in aging myocytes. We determined whether the frequency or incidence of spontaneous Ca2+ sparks was altered in myocytes from aged hearts. Fig. 4, A and B, shows representative line scan diagrams recorded from quiescent ventricular myocytes isolated from young adult and aged hearts, respectively. The line scan diagram recorded from the young adult cell shows only a single spontaneous Ca2+ spark (Fig. 4A). Figure 4B shows the same size line scan diagram recorded from a myocyte isolated from an aged mouse heart. This example shows that a higher frequency of spontaneous Ca2+ sparks was observed in the aged myocyte than in the young adult cell. Figure 4C shows that a significantly higher percentage of aged myocytes exhibited Ca2+ sparks compared with young adult cells. Indeed, close to 70% of aged myocytes exhibited Ca2+ sparks, whereas only 30% of young adult myocytes exhibited spontaneous sparks. We also determined the overall frequency of sparks occurring in the two groups. The frequency of Ca2+ sparks was markedly and significantly increased in cells isolated from aged hearts when compared with young adult hearts (Fig. 4D). When cells that did not spark were excluded from this measure, the frequency of Ca2+ sparks also was increased in aged myocytes, although this increase was not statistically significant (2.12 ± 0.96 vs. 5.59 ± 1.29 sparks·100 µm–1·s–1 in 19 young adult and 45 aged myocytes, respectively).


Figure 4
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  		Fig. 4. Incidence and frequency of Ca2+ sparks are significantly higher in aged myocytes than in cells from younger hearts. A: representative line scan diagram shows a single Ca2+ spark recorded from a young adult myocyte. B: line scan diagram shows that multiple Ca2+ sparks were recorded in an aged ventricular myocyte. C: percentage of cells in which Ca2+ sparks occurred during a 6-s line scan recording was significantly greater in aged myocytes ({chi}2-test). D: mean frequency of Ca2+ sparks, expressed as number of sparks per 100 µm per second, was significantly higher in aged myocytes than in young adult cells. Data are from line scans recorded from 60 young adult and 72 aged myocytes. *P < 0.05.

 
The increase in incidence and frequency of spontaneous Ca2+ sparks in aging myocytes could reflect an increase in SR Ca2+ load in aging heart. To determine whether SR Ca2+ load is altered in aged myocytes, we compared SR Ca2+ content in young adult and aged myocytes. SR content was assessed at 37°C by rapid application of 10 mM caffeine in cells loaded with fura-2. Cells were impaled with high-resistance electrodes to stabilize the myocytes during rapid solution switches and then voltage clamped at a holding potential of –80 mV. Caffeine was rapidly applied for 1 s after a series of conditioning pulses delivered at a frequency of 2 Hz, as described in METHODS. Caffeine was prepared in a solution with 0 mM Na+ and 0 mM Ca2+ to minimize loss of released Ca2+ through the Na+-Ca2+ exchanger. Figure 5, A and B, shows recordings of caffeine-induced Ca2+ transients in representative young adult and aged myocytes, respectively. The peak amplitudes of caffeine-induced Ca2+ transients appeared similar in young adult and aged myocytes. Figure 5C shows that the mean caffeine-induced increase in peak Ca2+ concentration was similar in young adult and aged myocytes. Thus SR Ca2+ content was similar in young adult and aging ventricular myocytes. We also measured resting cytosolic Ca2+ concentrations just before application of caffeine in young adult and aged myocytes. Mean cytosolic Ca2+ concentrations at rest were similar in cells from young and aged hearts (Fig. 5D). We also measured caffeine-induced Ca2+ transients in cells stimulated at a faster rate (e.g., 6 Hz) before application of caffeine. The mean increase in peak Ca2+ concentration was similar in young adult and aged cells stimulated at 6 Hz (values were 437.7 ± 47.2 vs. 367.5 ± 25.5 nM in 13 young adult cells and 19 aged cells, respectively). However, the amplitudes of fura-2 Ca2+ transients in cells stimulated at 6 Hz were significantly smaller in aged myocytes than in young adult cells (values were 146.7 ± 21.3 vs. 88.3 ± 13.2 nM for young adult and aged cells, respectively; P < 0.05).


Figure 5
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  		Fig. 5. SR Ca2+ content was similar in young adult and aged mouse ventricular myocytes. A: representative example of a caffeine-induced Ca2+ transient recorded from a young adult myocyte. Caffeine (10 mM) was applied for 1 s with a rapid solution switcher. B: caffeine-induced Ca2+ transient recorded from an aged cardiac myocyte. C: mean caffeine-induced increase in peak Ca2+ transient was similar in cells from young adult and aged mice. D: mean resting free Ca2+ concentrations were similar in young adult and aged myocytes. n = 19 young and 27 aged cells.

 
These observations suggest that the increased frequency and incidence of Ca2+ sparks in aging myocytes cannot be attributed to increased SR Ca2+ load or increased cytosolic Ca2+. This suggests that there might be age-related changes in the properties of fundamental SR Ca2+ release units. Therefore, we next determined and compared the characteristics of Ca2+ sparks recorded from young adult and aged myocytes. Fig. 6, A and B, shows line scan diagrams of representative Ca2+ sparks recorded from young adult and aged cells, respectively. The example recorded from the aged myocyte appears to have a shorter duration than the example recorded from the younger cell. We examined the time course of Ca2+ sparks by recording the fluorescence intensity of three contiguous pixels corresponding to the brightest part of the spark. Figure 6, C and D, shows the time courses of Ca2+ release during these two sparks. The fluorescence intensity is presented as the ratio of the fluorescence intensity at any given point (F) to the background intensity preceding the spark (F0). Spark duration appeared to be shorter in the aged myocyte. Next, we examined spark width by recording the intensity of three contiguous pixels through the peak of the spark in the distance axis. Figure 6, E and F, shows spark width profiles for the representative sparks shown in Fig. 6, A and B. Spark width appeared to be similar in the young adult and aged cells.


Figure 6
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  		Fig. 6. Representative Ca2+ sparks recorded from young adult and aged myocytes. A and B: representative line scan diagrams illustrate representative Ca2+ sparks recorded from young adult and aged cells. C and D: intensity-time profiles for representative sparks in A and B. The spark recorded in the aged cell appeared to have a shorter duration than the spark in the young cell. E and F: intensity-distance profiles for representative sparks recorded from young and aged myocytes. Spark width appeared similar in the young adult and aged cells.

 
We next compared the mean amplitudes, spatial widths, and time courses of individual spontaneous Ca2+ sparks in cells isolated from young adult and aged hearts. Frequency distributions of spark characteristics in 100 young adult myocytes and 105 aged myocytes are plotted in Fig. 7. Figure 7A shows that the distributions of spark amplitudes were similar in young adult and aged myocytes. The frequency distributions for FWHM also were similar in both groups (Fig. 7B). Figure 7C shows that there were more events with half-rise times of 4 ms or less, and few events longer than 7 ms, in the aged group than in the young adult group. Figure 7D shows distributions of the half-decay times for young adult and aged cells. The distribution of half-decay times was shifted to the left in the aged cells when compared with cells from younger hearts.


Figure 7
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  		Fig. 7. Frequency distributions for Ca2+ spark characteristics recorded from young adult and aged myocytes. A: frequency distributions for amplitudes of Ca2+ sparks (F/F0), placed in bins of 0.30 units, were similar in young adult and aged cells. B: frequency distributions for full width half-maximum amplitude (FWHM), in bins of 0.25 µm, were similar in both groups. C: frequency distribution of half-rise times in 1.0-ms bins. Half-rise times included more events with brief durations for Ca2+ sparks measured in aged myocytes compared with young adult cells. D: frequency distribution of half-decay times in bins of 3.0 ms. Half-decay time with the largest number of events was shorter in the aged group.

 
Mean values for these spark parameters are shown in Fig. 8. Figure 8A shows that mean spark amplitudes, expressed as F/F0, were similar in young adult and aged cells. Similarly, spark width, measured as FWHM, was not significantly different between the two groups (Fig. 8B). However, the half-rise times and half-decay times were significantly shorter for sparks recorded from aged myocytes than for sparks recorded from young adult myocytes (Fig. 8, C and D). Thus Ca2+ spark duration was abbreviated in ventricular myocytes isolated from aging hearts.


Figure 8
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  		Fig. 8. Ca2+ sparks recorded from aged myocytes have faster rise times and faster half-decay times when compared with sparks recorded from young adult cells. A: mean spark amplitudes (F/F0) were similar for sparks from young adult and aged cells. B: mean widths (FWHM) of sparks were not significantly different between young and aged cells. C: half-rise time was significantly shorter for sparks recorded from aged myocytes compared with young adult myocytes. D: half-decay time of Ca2+ sparks was significantly shorter in aged cells than in young adult cells. n = 100 sparks from young adult myocytes and 105 sparks from aged myocytes. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we determined whether there were age-related changes in Ca2+ transients both at the whole cell level and at the level of unitary Ca2+ release. Our results demonstrated that the amplitudes and time courses of Ca2+ transients were similar in young adult and aged myocytes stimulated at 2 Hz. However, Ca2+ transient amplitudes were reduced and times to peak Ca2+ transient were shorter in aged cells paced at a frequency of 8 Hz than in young adult cells. We also compared the frequency, incidence, and properties of spontaneous Ca2+ sparks in young adult and aging cells. Our results showed that the frequency and incidence of spontaneous Ca2+ sparks increased markedly in aged myocytes. There was no difference in spark amplitudes or spatial widths between young and aged cells. However, spark half-rise times and half-decay times were significantly abbreviated in aged cells. These results show that there are alterations in the frequency and characteristics of fundamental SR Ca2+ release units in aging ventricular myocytes. Thus there are significant age-related changes in Ca2+ transients at the whole cell level and at the Ca2+ spark level.

We found that whole cell Ca2+ transients had F/F0 ratios near 2 in young adult and aged cells paced at 2 Hz; similar values of F/F0 have been reported previously in field-stimulated murine ventricular myocytes (2, 6, 13, 28). However, we also found that Ca2+ transient amplitudes were smaller in aged cells than in younger myocytes when cells were stimulated at 8 Hz. These observations are in general agreement with results of previous studies in aging mouse ventricular myocytes. Lim et al. (24) and Isenberg et al. (19) found that amplitudes of Ca2+ transients were similar in young and aged myocytes stimulated at 2 Hz but that Ca2+ transients were smaller in aged cells when the stimulation rate was increased to 8 or 9 Hz. However, unlike these previous studies (19, 24), we found that the amplitudes of Ca2+ transients declined as stimulation frequency was increased in both young adult and aged myocytes. It is not clear why we observed a negative effect of increased stimulation frequency in mouse myocytes while Lim et al. (24) and Isenberg et al. (19) did not. However, many previous studies (5, 11) also have reported negative "force-frequency" effects in mouse and rat ventricular myocytes. These negative force-frequency relations may be due, at least in part, to the relatively high SR Ca2+ content in mouse and rat cells, even at low stimulation rates (5).

We also found that Ca2+ transient decay times were similar in young and aged myocytes and that times to peak Ca2+ transient were prolonged in cells from young adult hearts paced at 8 Hz but not in cells from aged animals. In contrast, previous studies (19, 24) have reported that Ca2+ transients are prolonged in aging mouse myocytes when compared with cells from younger animals. One explanation for the discrepancy between studies is differences in specific experimental conditions between studies. For example, the present study used 1 mM extracellular Ca2+, whereas higher concentrations of Ca2+ were used in previous studies (19, 24). Furthermore, previous studies used indo-1 (19) or fura-2 (24) rather than fluo-4, which was used in the present study. Differences in extracellular Ca2+ concentrations and/or the rate at which Ca2+ dissociates from different Ca2+-sensitive dyes could affect the rate of decay of the Ca2+ transient measured in different studies. Furthermore, contractions were activated by voltage-clamp pulses of fixed duration in one previous study (19), whereas the present study used field stimulation to activate contractions with action potentials. Differences in the duration of depolarization could affect the rate of decay of the Ca2+ transient observed in each study.

Our results also showed that the incidence and frequency of spontaneous Ca2+ sparks were increased markedly in ventricular myocytes from aged mice when compared with young adult cells. Interestingly, an increase in the frequency of Ca2+ sparks in rat aging ventricular myocytes was recently reported in studies conducted at room temperature (42). The results of the present study demonstrate that the increase in the frequency of Ca2+ sparks in aging also occurs at physiological temperature, where Ca2+ sparks occur much less frequently than at room temperature (13). Indeed, in young adult mouse myocytes investigated at 37°C, spontaneous Ca2+ sparks occurred at a low frequency as reported in our previous study (13). These spontaneous Ca2+ sparks are believed to provide a pathway for SR Ca2+ leak that limits SR Ca2+ content in cardiac myocytes (4, 27). Therefore, the increase in Ca2+ spark frequency in aging myocytes might be expected to increase Ca2+ leak from the SR. However, we found that SR Ca2+ stores were similar in myocytes from young adult and aging mouse hearts, as reported previously for young and aging sheep myocytes (12). Thus the increase in Ca2+ spark frequency in aging cells does not appear to deplete SR Ca2+ stores. Furthermore, the reduction in Ca2+ transient amplitude in aging cells is not due to a decrease in SR Ca2+ stores.

There are several possible mechanisms that could account for the increased occurrence of Ca2+ sparks in aging myocytes. Spark frequency has been reported to increase when cytosolic Ca2+ levels rise (5). Thus one might propose that the increase in Ca2+ sparks in aging cells occurs as a result of elevated cytosolic Ca2+. However, this is unlikely because we found that resting Ca2+ concentrations were similar in cells from young adult and aged hearts. Spark frequency also increases when SR Ca2+ stores are increased (29, 32, 33). Therefore, if SR Ca2+ content increases in aging cells, this could account for the increase in spark occurrence in aging. However, we found that SR Ca2+ stores were similar in young adult and aged myocytes, as reported previously (12). It also is possible that depolarization of aged myocytes might activate Ca2+ sparks, although previous studies have shown that the resting membrane potential does not change in aging (37, 38). Thus changes in SR Ca2+ content, resting cytosolic Ca2+ levels, or resting membrane potential are not likely to account for increased spark activity in aging myocytes. The frequency of Ca2+ sparks also is modulated by factors such as ATP, H+, calmodulin, cADP ribose, and protein phosphatases that influence gating properties of cardiac SR Ca2+ release channels (3, 10, 28, 36, 41). It is possible that alterations in one or more of these factors could account for the increased occurrence of Ca2+ sparks in the aging heart, in the absence of changes in SR Ca2+ and resting Ca2+.

We also found that spark widths and spark amplitudes were similar in young and aged cells. Our observation that spark widths did not differ between young and aged cells suggests that the number of ryanodine receptors in each spark unit does not change with age. A recent model suggests that the number of ryanodine receptors in a cluster is a determinant of spark amplitude (34). Our observation that spark amplitudes did not change in aging cells also suggests that the number of ryanodine receptors per spark unit is similar in young adult and aged cells. However, we found that spark duration was abbreviated significantly in aged cells. Spark duration can be modulated by the degree of coupling between ryanodine receptors in a cluster (14, 34). Application of FK506, a compound that binds to FKBP12.6 proteins and disrupts ryanodine receptor coupling, causes prolongation of Ca2+ sparks (14, 34), whereas overexpression of FKBP12.6 abbreviates spark duration (14). Because abnormalities in ryanodine receptors have been reported in the aging heart (40), it is possible that disruptions in ryanodine receptor coupling might contribute to the abbreviation of spark duration in aging myocytes. It also is possible that the mechanism responsible for termination of Ca2+ sparks may be altered in the aging heart. At present, the mechanism by which Ca2+ sparks terminate is quite controversial (35). Therefore, additional studies will be required to determine the means by which spark duration is abbreviated in aging.

The results of this study showed that the time to peak Ca2+ transient was prolonged when the stimulation rate was increased in young adult myocytes but not in aging myocytes. It is possible that the time to peak Ca2+ transient might be shorter in aged myocytes paced at 8 Hz because spark duration is shorter in aging cells than in young adult cells. However, other factors, such as the synchrony of spark recruitment and the duration of the latent period between depolarization and initiation of sparks, also are important determinants of time to peak transient. For example, isoproterenol has been shown to shorten rise times for Ca2+ transients through synchronization of Ca2+ sparks and abbreviation of the latent period (25). It is possible that increased stimulation frequency disrupts the synchronization of sparks and/or prolongs the latent period in young adult myocytes but not in aged myocytes, although this remains to be demonstrated experimentally. The present study also demonstrates that the reduction in Ca2+ transient amplitudes in aged cells was not reflected in a reduction in the amplitude of spontaneous Ca2+ sparks. Furthermore, the reduction in amplitudes of Ca2+ transients in aged myocytes is not due to a reduction in SR Ca2+ stores in the aging heart. Thus the mechanism for the reduction in Ca2+ transient amplitude is not clear. However, Ca2+ transients might be smaller if fewer spark units are available at higher stimulation rates in aged myocytes.

The present study demonstrates that there are significant age-associated changes in whole cell Ca2+ transients and in spontaneous Ca2+ sparks. Ca2+ transient amplitudes are smaller in aging myocytes paced at a frequency of 8 Hz. In addition, the incidence, frequency, and characteristics of spontaneous Ca2+ sparks are markedly altered in cardiac myocytes isolated from aging hearts. Because Ca2+ sparks represent the activation of clusters of ryanodine receptors, our findings suggest that alterations in ryanodine receptor function occur in the aging heart. Thus alterations in SR Ca2+ release in the aging heart may reflect disruption of the function of individual SR Ca2+ release units rather than changes in SR Ca2+ stores and diastolic Ca2+ levels.


   GRANTS
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 ABSTRACT
 METHODS
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 DISCUSSION
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This work was supported in part by grants from Canadian Institutes of Health Research and from Heart and Stroke Foundation of Nova Scotia. S. Grandy was supported by a graduate studentship from Canadian Institutes of Health Research and Heart and Stroke Foundation of Canada.


   ACKNOWLEDGMENTS
 
We thank Peter Nicholl, Dr. Jiequan Zhu, and Steve Foster for excellent laboratory technical support and for assistance in preparation of illustrations. We also thank Steven Whitefield for outstanding technical support for confocal microscopy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. E. Howlett, Dept. of Pharmacology, Sir Charles Tupper Medical Bldg., Dalhousie Univ., Halifax, Nova Scotia, Canada B3H 1X5 (e-mail: Susan.Howlett{at}Dal.ca)

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.

{dagger} Deceased 30 August 2005. Back


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