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Am J Physiol Heart Circ Physiol 295: H768-H777, 2008. First published June 20, 2008; doi:10.1152/ajpheart.00432.2008
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Simulated ischemia-induced preconditioning of isolated ventricular myocytes from young adult and aged Fischer-344 rat hearts

J. Darcy O'Brien1 and Susan E. Howlett1,2

1Department of Pharmacology and 2Division of Geriatric Medicine, Dalhousie University, Halifax, Nova Scotia, Canada

Submitted 24 April 2008 ; accepted in final form 12 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The impact of ischemic preconditioning (IPC) on contraction, Ca2+ homeostasis, and cell survival was compared in isolated ventricular myocytes from young adult (~3 mo) and aged (~24 mo) male Fischer-344 rats. Myocytes were field stimulated at 4 Hz (37°C). Contraction (edge detector) and intracellular Ca2+ (fura-2) were measured simultaneously. Viability was assessed with trypan blue. All cells were exposed to 30 min of simulated ischemia followed by reperfusion. Some cells were preconditioned by exposure to 5 min of simulated ischemia before prolonged ischemia. Pretreatment with IPC abolished postischemic contractile depression, inhibited diastolic contracture, and increased Ca2+ transient amplitudes in reperfusion in young adult and aged cells. IPC did not affect the modest rise in diastolic Ca2+ in ischemia in young adult myocytes. However, IPC abolished the marked rise in diastolic Ca2+ observed in ischemia and early reperfusion in aged myocytes. IPC also suppressed mechanical alternans in ischemia in aged cells, but younger myocytes showed little evidence of mechanical alternans whether or not cells were preconditioned. IPC markedly improved cell viability in reperfusion in young adult but not aged cells. These results suggest that IPC augments the recovery of contractile function in reperfusion by increasing Ca2+ transient amplitudes in ventricular myocytes from young adult and aged rats. IPC reduced diastolic Ca2+ accumulation in ischemia in aged myocytes, which may diminish the severity of mechanical alternans in aged cells. Nonetheless, the efficacy of IPC is compromised in aging, as IPC did not improve survival of aged myocytes exposed to ischemia and reperfusion.

cardioprotection; reperfusion; mechanical alternans; senescence

IT IS WELL ESTABLISHED that aged hearts are more sensitive to ischemia and reperfusion injury than younger adult hearts (14, 22, 27). Studies in humans have shown that morbidity and mortality rates after acute myocardial infarction are much higher in older adults than in younger patients (37, 50, 54). Also, the risk of death following procedures that cause reperfusion increases with age (9, 53). Similar results have been reported in animal models of aging. Studies of ischemia and reperfusion injury in Langendorff-perfused hearts have shown that infarct size is greater in hearts from aged animals than in younger hearts, while recovery of contractile function in reperfusion declines with age (3, 16, 19, 31, 39, 48, 55). Many of these detrimental effects of myocardial ischemia and reperfusion are thought to be linked to a rise in intracellular free Ca2+ levels in cardiac myocytes (30, 40). Indeed, individual ventricular myocytes isolated from aged rats accumulate more diastolic Ca2+ in ischemia and early reperfusion than cells from younger hearts (43). These findings indicate that aging hearts are more sensitive to ischemia and reperfusion injury than younger hearts and suggest that alterations at the level of the cardiac myocyte may increase the susceptibility to ischemia and reperfusion injury with age.

It is possible that the increase in sensitivity to ischemia and reperfusion injury may arise, at least in part, from the loss of endogenous mechanisms for cardioprotection in the aging heart. Ischemic preconditioning (IPC) is a cardioprotective phenomenon triggered by exposure to brief periods of ischemia that protects the heart from detrimental effects of subsequent prolonged ischemia and reperfusion (17, 23, 24, 57). Most studies of IPC have investigated this phenomenon in young adult animals. It is well established that IPC markedly reduces infarct size and attenuates postischemic contractile depression, known as stunning, in young adult hearts (17, 23, 24, 57). IPC also has been shown to improve cell survival in isolated cardiac myocyte models when cells from young adult hearts are investigated (12).

Although IPC is a potent cardioprotective mechanism in young adult hearts, the aging process appears to reduce the efficacy of IPC (14, 20, 22). The marked reduction in infarct size caused by IPC in hearts from young adult animals is not observed in hearts from aged animals (see Refs. 6, 13, 38, and 46 but cf. Ref. 7). In addition, although IPC attenuates stunning in intact hearts from young adult animals, IPC does not improve the recovery of contractile function in reperfusion in hearts from aged animals (1, 13, 25, 38, 49). The cardioprotective effects of IPC also are reduced in aging humans, as shown in a clinical study (2), as well as in isolated cardiac muscle preparations (see Ref. 4 but cf. Ref. 33). The decrease in efficacy of IPC in the aging heart has recently been linked to age-related changes in proteins that are implicated in preconditioning-induced cardioprotection (6, 25).

Previous studies of IPC in the aging heart have been conducted in vivo or have used intact hearts or multicellular preparations subjected to ischemia and reperfusion. In the intact heart, age-related changes in endothelial cells and vascular smooth muscle cells are well documented (26). These vascular changes may contribute to the decrease in the efficacy of IPC in aging. Still, the observation that IPC is a powerful protective mechanism in individual cardiac myocytes suggests that beneficial effects of IPC also reside within the myocytes themselves (12). The overall goal of this study was to determine whether a decrease in the effectiveness of IPC at the level of the individual cardiac myocyte contributes to the decline of this important cardioprotective mechanism in aging.

The specific objectives of this study were 1) to determine whether IPC can improve contractile function, intracellular Ca2+ homeostasis, and cell viability in isolated ventricular myocytes exposed to simulated ischemia and reperfusion; and 2) to determine whether these beneficial effects of IPC are attenuated or abolished in myocytes from aged animals. The present study used a cellular model of simulated ischemia and reperfusion developed in this laboratory and recently adapted for use in rat myocytes (43). Here, we evaluated and compared differences in contractile function, intracellular Ca2+ homeostasis, and cell viability throughout ischemia and reperfusion in ventricular myocytes isolated from the hearts of young adult (~3 mo) and aged (~24 mo) male Fischer-344 rats in the absence and presence of IPC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation. All experiments in this study were performed in conformity with the guidelines published by the Canadian Council on Animal Care (Ottawa, ON, Canada; vol. 1, 2nd ed., 1993; and vol. 2, 1984). The Dalhousie University Committee on Laboratory Animals approved all animal protocols. Male Fischer-344 rats (24.7 ± 0.1 vs. 3.2 ± 0.1 mo, n = 28 aged rats and 23 young rats, P < 0.05) were obtained either from Charles River Laboratories (St. Constant, QC, Canada) or from the National Institute on Aging (Baltimore, MD). In each of these facilities, rats were housed behind pathogen-free barriers and monitored on a regular basis for genetic purity and health status. Animals were housed in microisolator cages in the Animal Care Facility at Dalhousie University and used within 2 wk of arrival. Rats were maintained on a 12:12-h light-dark cycle, and animals had free access to food and water.

The isolation techniques for enzymatic dissociation of ventricular myocytes from young adult and aged rat hearts have been previously described (43). Animals were transported from the animal care facility to the laboratory, where they were weighed. Aged rats were significantly heavier than young adult rats (414.4 ± 8.7 vs. 293.0 ± 4.3 g, n = 28 aged and 23 young rats, P < 0.05). Rats were then injected with heparin (3,000 U/kg ip) 30 min before they were anesthetized with pentobarbital sodium (220 mg/kg ip). The aorta was cannulated, and hearts were perfused at a rate of 18–20 ml/min with a perfusion buffer that contained (in mM) 135.5 NaCl, 4 KCl, 10 HEPES, 1.2 MgSO4, 1.2 KH2PO4, and 12 glucose with 200 µM CaCl2 (pH 7.4, gassed with 100% O2, 37°C). After 5 min, the heart was perfused for an additional 5 min with the same buffer without Ca2+. Hearts were then perfused with the perfusion buffer supplemented with protease dispase II (Roche Diagnostics, Laval, QC, Canada; 0.10 and 0.14 mg/ml for 3- and 24-mo-old hearts), collagenase type 2 (240 U/mg, Worthington, Lakewood, NJ; 0.3 and 0.6 mg/ml for 3- and 24-mo-old hearts), and 50 µM Ca2+. After 15–20 min of perfusion with the enzyme solution, ventricles were removed and minced in a high-potassium buffer that contained (in mM) 80 KOH, 30 KCl, 3 MgSO4, 30 KH2PO4, 50 L-glutamic acid, 20 taurine, 0.5 EGTA, 10 HEPES, and 10 glucose (pH 7.4 with KOH). The supernatant was filtered through polyethylene mesh (pore size: 225 µm). Quiescent, rod-shaped myocytes with clear striations and no visible membrane damage were used in our experiments. The yield of viable myocytes with these characteristics was between 40% and 60% for young adult hearts and 30% and 50% for aged hearts in our study. A decrease in myocyte yield with age has been reported previously in studies of aged rat ventricular myocytes (15, 32).

Experimental protocols. Cell shortening and intracellular Ca2+ were measured simultaneously in each myocyte. Ventricular myocytes were loaded with the Ca2+-sensitive dye fura-2 AM (5 µM) for 20 min in the dark at room temperature. Cells were then transferred to a 0.75-ml chamber with an optical grade glass bottom. The chamber was mounted on the stage of an inverted microscope (Nikon ECLIPSE TE200, Nikon Canada). Next, cells were superfused with "normal" Tyrode buffer, which contained (in mM) 129 NaCl, 20 NaHCO3, 0.9 NaH2PO4, 4 KCl, 0.5 MgSO4, 1.8 CaCl2, and 5.5 glucose (pH 7.2, gassed with 95% O2-5% CO2, 37°C). All buffer solutions were heated to 37°C, and all experiments were conducted at this temperature. Cells were superfused at a rate of 6 ml/min.

In all experiments, myocytes were initially equilibrated in normal Tyrode buffer solution. Simulated ischemia and IPC were achieved by exposing myocytes to an "ischemic" Tyrode solution as previously described (43). The ischemic Tyrode buffer contained (in mM) 123 NaCl, 6 NaHCO3, 0.9 NaH2PO4, 8 KCl, 0.5 MgSO4, 1.8 CaCl2, and 20 sodium lactate (pH 6.8, gassed with 90% N2-10% CO2, 37°C). This solution mimics the hypoxia, hyperkalemia, hypercapnia, acidosis, and substrate deprivation associated with myocardial ischemia. During simulated ischemia and IPC, a 90% N2-10% CO2 gas phase was directed over the top of the chamber. For ischemia and reperfusion experiments, myocytes were first superfused with Tyrode solution for 20 min, followed by 30 min of simulated ischemia and up to 30 min of reperfusion. To simulate IPC, separate groups of young adult and aged myocytes were exposed to 5 min of simulated ischemia, 10 min before prolonged ischemia and reperfusion. The PO2 value was ~650 mmHg in normal Tyrode buffer, and this rapidly declined by ~90% (to 70 mmHg) in ischemic Tyrode buffer, as previously described (43). This 90% decrease in PO2 values is greater than the 75–80% reduction in PO2 reported in other cellular models of simulated ischemia and reperfusion (35, 36). A PO2 value of 70 mmHg represents hypoxia (47). In time control experiments, myocytes were preconditioned with a 5-min exposure to ischemia and then exposed to normal Tyrode solution only for the duration of the experiment. In all experiments, cells were exposed to only one cycle of ischemia and reperfusion or to IPC followed by ischemia and reperfusion.

Cell shortening. Myocytes were stimulated continuously at 4 Hz with 3-ms pulses delivered though a pair of platinum electrodes. Voltage and pulse duration were controlled with a Grass stimulator (SD9, Grass Technologies, West Warwick, RI). The electrical stimulus was set at a voltage ~50% greater than the threshold to induce cell shortening, and this was maintained throughout the experiment. A pulse generator was used to trigger the stimulator and control stimulation frequency (Pulsar 6i, Frederick Haer & Co., Bowdoinham, ME). Unloaded cell shortening was measured at 120 Hz with a video edge detector (model no. 105, Crescent Electronics, Sandy, UT) coupled to a video camera (Philips FTM800NH, Philips Canada, Markham, ON, Canada). The microscope light was split with a dichroic cube to allow simultaneous recording of cell length and whole cell fluorescence. Contraction data were collected with Axoscope 8.2 (Molecular Devices, Sunnyvale, CA). Recordings (10 s) were made at 5-min intervals throughout each experiment with additional recordings at 1 and 2 min of reperfusion. Analog signals were digitized with a Digidata 1322A analog-to-digital board (Molecular Devices). Contractions recorded at each time point were averaged and measured with Clampfit 8.2 (Molecular Devices). Contraction amplitude was defined as the difference between systolic and diastolic cell lengths.

Intracellular Ca2+ concentrations. Intracellular Ca2+ concentrations were measured in all cells with whole cell photometry (DeltaRam, Photon Technology, Birmingham, NJ) as previously described (43). In brief, cells were alternately excited at 340 and 380 nm. The ratio of fluorescence emission at 510 nm was used to determine intracellular Ca2+ concentrations (Nikon Super Fluor oil-immersion objective, x40/1.3 numerical aperture). In each experiment, background fluorescence values were recorded and used to correct the data. Emission ratios were converted to Ca2+ concentrations with an in vitro calibration curve. The calibration curve used to convert all fluorescence data to Ca2+ concentration was determined at pH 7.0. We found that calibration curves determined at pH 6.8, 7.0, or 7.2 were similar for Ca2+ concentrations between 100 nM and 1 µM, as previously described (43). Data were recorded with Felix32 software (Photon Technology). Ca2+ transient amplitudes were measured as the difference between systolic and diastolic Ca2+. Ca2+ transients were recorded at each time point for approximately 5 s. Trains of transients were averaged and measured with Clampfit 8.2 (Molecular Devices).

Abnormal contractile activity was observed in ischemia in some cells. This activity consisted of alternating patterns of large and small amplitude contractions, known as mechanical alternans. This was frequently accompanied by alternating Ca2+ transients (Ca2+ alternans). To quantify this, an alternans ratio was calculated for each cell. The alternans ratio was as follows: 1 – S/L, where S is the magnitude of the smaller response and L is the amplitude of the larger response (56). Cell viability was assessed with trypan blue exclusion as in a previous study of ischemia and reperfusion injury in myocytes (12). Cells that took up trypan blue dye also became spherical in shape, formed membrane blebs, and exhibited irreversible contracture and Ca2+ overload. Cells that excluded trypan blue dye were considered viable at the end of the experiment.

Data analysis. All statistical analyses were performed with SigmaStat 3.1, and graphs were constructed with SigmaPlot 8.0 (Systat Software, Point Richmond, CA). All data except cell viability data and incidence are expressed as means ± SE. Cell viability is shown in a survival curve that depicts the probability of cell survival over time. Differences in survival curves were determined with a log rank test. Differences in the incidence of alternans between groups were evaluated with a {chi}2-test. All other differences between groups were evaluated with two-way ANOVA or two-way ANOVA with repeated measures. No more than two myocytes per heart were included in any one data set. Differences were considered significant when P < 0.05.

Chemicals and reagents. Fura-2 AM was purchased from Invitrogen (Burlington, ON, Canada). A stock solution of fura-2 was prepared in anhydrous DMSO (final concentration of DMSO in the cell suspension was 0.2%), and aliquots of this solution were stored at –20°C until use. All other chemicals were obtained from Sigma-Aldrich Canada (Oakville, ON, Canada).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The aged myocytes used in this study exhibited the characteristic increase in cell length previously described for ventricular myocytes isolated from aged rat hearts (15, 32, 43). In our experiments, mean values for cell length increased significantly with age [values were 131.4 ± 2.4 µm for young adult cells (n = 33) and 142.5 ± 2.6 µm for aged cells (n = 30), P < 0.05]. To account for differences in cell size between groups, contraction data were normalized to either cell length or the magnitude of contraction recorded before prolonged ischemia. When contractions were normalized to diastolic cell length under control conditions (before ischemia), amplitudes of contractions were similar in young adult and aged cells (2.5 ± 0.3% and 3.0 ± 0.3% diastolic cell length, n = 33 young adult and 30 aged cells). These findings agreed with results of previous studies of contractile function in field-stimulated young adult and aged rat ventricular myocytes (15, 43). In the experiments presented in this study, data were normalized to control values at time (t) = 20 min, just before prolonged ischemia. However, similar results were obtained when data were normalized to control values at t = 0 min, before IPC (data not shown).

All experiments were conducted in cells loaded with fura-2, superfused with normal Tyrode buffer at 37°C, and field stimulated at 4 Hz, as described in MATERIALS AND METHODS. The effects of simulated ischemia and reperfusion on Ca2+ transients and contractions were compared in cells from young adult and aged animals in the absence or presence of IPC. Figure 1A shows representative examples of Ca2+ transients (top) and contractions (bottom) recorded from a young adult myocyte in the absence of IPC. Ischemia reduced the size of contractions and increased diastolic Ca2+ levels (Fig. 1A). Diastolic Ca2+ recovered in reperfusion, but contractions were depressed, and the cell exhibited a sustained diastolic contracture (Fig. 1A). IPC did not affect cellular responses during prolonged ischemia in the young adult cell (Fig. 1B). However, IPC increased Ca2+ transient amplitudes, prevented postischemic contractile depression, and attenuated diastolic contracture in reperfusion in the young adult cell (Fig. 1B). Figure 1C shows representative recordings from an aged myocyte exposed to ischemia and reperfusion. Ischemia attenuated contractions in the aged cell. Additionally, ischemia caused a large increase in diastolic Ca2+ in the aged cell (Fig. 1C). Diastolic Ca2+ recovered in reperfusion, but the aged cell showed postischemic contractile depression and diastolic contracture (Fig. 1C). IPC reduced the marked rise in diastolic Ca2+ in ischemia in the aged cell (Fig. 1D). IPC also inhibited postischemic contractile depression, increased Ca2+ transient amplitudes, and reduced diastolic contracture in reperfusion in the aged cell (Fig. 1D).


Figure 1
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  		Fig. 1. Representative recordings of contractions and Ca2+ transients at selected time points during experiments. In A–D, Ca2+ transients are shown at the top and contractions are shown at the bottom. Cell shortening is shown as downward deflections in the recordings. A: in a young adult cell in the absence of ischemic preconditioning (IPC), contractions were suppressed by ischemia. Contractions remained depressed in reperfusion, and the cell exhibited sustained contracture. Diastolic Ca2+ levels increased in ischemia but recovered in reperfusion. B: IPC eliminated postischemic contractile depression and diastolic contracture and markedly increased Ca2+ transient amplitudes in reperfusion in a young adult cell. C: in the absence of IPC, contractile responses to ischemia and reperfusion in an aged cell were similar to responses in the younger cell. However, the aged cell exhibited a greater increase in diastolic Ca2+ in ischemia compared with the younger cell. D: IPC reduced the increase in diastolic Ca2+ observed in ischemia in the aged cell. IPC also abolished postischemic contractile depression, reduced diastolic contracture, and augmented Ca2+ transients in reperfusion in the aged myocyte. Control recordings were made at the beginning of the experiment [time (t) = 0 min]; recordings in ischemia were made 5 min after exposure to prolonged ischemia (t = 25 min); recordings in reperfusion were made after 15 min of reperfusion (t = 65 mins).

 
Mean amplitudes of contractions in the absence or presence of IPC are shown in Fig. 2. A 5-min exposure to IPC attenuated contractions in both young adult and aged cells, but this quickly recovered (Fig. 2, A and B). Prolonged ischemia inhibited contractions in young adult and aged groups regardless of whether cells were preconditioned (Fig. 2, A and B). However, in the absence of IPC, young adult and aged myocytes showed postischemic contractile depression throughout most of reperfusion (Fig. 2, A and B). Interestingly, pretreatment with IPC abolished postischemic contractile depression in both groups (Fig. 2, A and B). Figure 2C directly compares mean amplitudes of contractions in young adult and aged cells that were pretreated with IPC. Responses were similar in the two groups except that contractions recorded in young adult cells showed an overshoot in early reperfusion, which did not occur in the aged group (Fig. 2C). We also measured contractions over the course of an experiment in cells that served as time controls, as described in MATERIALS AND METHODS. Mean amplitudes of contractions changed by <5% over the course of the experiments in both young adult (n = 9) and aged (n = 11) groups, and this effect was not statistically significant (not shown).


Figure 2
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  		Fig. 2. IPC abolished postischemic contractile depression in reperfusion in young adult and aged myocytes. A: in young adult myocytes, IPC caused a transient decrease in contraction amplitude, which recovered before prolonged ischemia. Still, IPC abolished postischemic contractile depression in reperfusion in young cells. B: IPC also caused a transient decrease in contraction amplitudes and abolished postischemic contractile depression in aged cells. C: responses of preconditioned young adult and aged cells to ischemia and reperfusion were similar except that aged cells did not exhibit a brief overshoot in contraction upon reperfusion. Contraction amplitudes were normalized to control values before prolonged ischemia (at t = 20 min; dashed lines). n = 18 young adult and 16 aged cells in the absence of IPC and n = 15 young adult and 13 aged cells pretreated with IPC. *Significant difference from the appropriate comparison group (P < 0.05).

 
Figure 3 shows diastolic cell lengths in the absence and presence of IPC in myocytes isolated from young adult and aged rat hearts. In the absence of IPC, cell lengths remained relatively constant until reperfusion in both age groups (Fig. 3, A and B). However, young adult and aged cells developed diastolic contracture upon reperfusion in the absence of IPC (Fig. 3, A and B). Pretreatment with IPC significantly inhibited diastolic contracture in reperfusion in both age groups (Fig. 3, A and B). We also directly compared mean diastolic cell lengths in young adult and aged cells exposed to IPC. Mean diastolic cell lengths in cells exposed to IPC were virtually identical in the two groups (not shown). Diastolic cell lengths did not change significantly with time in cells used as time controls in either age group (n = 9 young adult and 11 aged cells; not shown).


Figure 3
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  		Fig. 3. IPC attenuated diastolic contracture in reperfusion in young adult and aged myocytes. A and B: IPC significantly attenuated the diastolic contracture that accompanied reperfusion in both young adult (A) and aged (B) myocytes. Diastolic cell length was normalized to control values before prolonged ischemia (at t = 20 min; dashed lines). n = 19 young adult and 18 aged cells in the absence of IPC and n = 15 young adult and 13 aged cells pretreated with IPC. *Significant difference from the appropriate comparison group (P < 0.05).

 
Figure 4 shows mean amplitudes of Ca2+ transients from young adult and aged myocytes in the absence and presence of IPC. In young adult cells, Ca2+ transient amplitudes were increased by IPC in late ischemia and throughout reperfusion compared with cells that were not preconditioned (Fig. 4A). IPC also increased the amplitudes of Ca2+ transients recorded from aged cells in reperfusion (Fig. 4B). We directly compared mean Ca2+ transient amplitudes in young adult and aged cells in the presence of IPC. Ca2+ transient amplitudes were similar throughout ischemia and reperfusion in young adult and aged cells exposed to IPC (not shown). In addition, we measured the time to 90% decay of the Ca2+ transient at selected times during each experiment in the absence and presence of IPC (Fig. 5). Representative examples of averaged Ca2+ transients recorded from young adult and aged cells are shown in Fig. 5, A and B. In young adult and aged cells, ischemia caused a temporary prolongation of the Ca2+ transient, which recovered in reperfusion (Fig. 5, C and D). Pretreatment with IPC had no effect on the prolongation of the Ca2+ transient induced by ischemia in either young adult or aged myocytes (Fig. 5, C and D). There were no significant differences in the duration of the Ca2+ transient between young adult and aged myocytes regardless of whether or not cells were preconditioned. Mean amplitudes of Ca2+ transients changed by <10% in time control experiments in both age groups (n = 9 young adult and 11 aged cells); this effect was not statistically significant (not shown).


Figure 4
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  		Fig. 4. IPC augmented the amplitudes of Ca2+ transients in reperfusion in both young adult and aged myocytes. A: IPC increased the size of Ca2+ transients in late ischemia and throughout reperfusion in young adult myocytes. B: Ca2+ transient amplitudes also were increased in reperfusion in aged cells pretreated with IPC. Ca2+ transient amplitudes were normalized to control values before prolonged ischemia (at t = 20 min; dashed lines). n = 18 young adult and 15 aged cells in the absence of IPC and n = 13 young adult and 13 aged cells pretreated with IPC. *Significant difference from the appropriate comparison group (P < 0.05).

 

Figure 5
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  		Fig. 5. IPC did not inhibit the prolongation of Ca2+ transient duration caused by ischemia in young adult and aged myocytes. A and B: representative recordings of Ca2+ transients recorded at selected times during an experiment to illustrate prolongation of the transient by ischemia in young adult (A) and aged (B) cells in the absence of IPC. Each recording represents the average of a 5-s train of Ca2+ transients. C and D: the duration of the Ca2+ transient at 90% decay was prolonged by ischemia in young adult (C) and aged cells (D), even when cells were pretreated with IPC. There was no significant difference in the effect of IPC on Ca2+ transient duration between young adult and aged cells. Control recordings were made 20 min after the start of the experiment, just before prolonged ischemia (t = 20 min); recordings in ischemia were made 5 min after the start of ischemia (t = 25 min); recordings in reperfusion were made 15 min after the start of reperfusion (t = 65 mins). Ca2+ transient durations were normalized to control values before prolonged ischemia (t = 20 min). n = 12 young adult and 15 aged cells in the absence of IPC and n = 14 young adult and 15 aged cells pretreated with IPC. *Significant difference from the control group (P < 0.05).

 
Figure 6 shows mean diastolic Ca2+ concentrations in young adult and aged myocytes in the absence and presence of IPC. IPC caused a transient increase in diastolic Ca2+ levels in young adult cells but had no impact on the rise in diastolic Ca2+ induced by prolonged ischemia in this group (Fig. 6A). IPC also caused a brief rise in diastolic Ca2+ levels in aged cells (Fig. 6B). However, in contrast to young adult cells, IPC inhibited the marked rise in diastolic Ca2+ that occurred throughout ischemia and early reperfusion in aged cells (Fig. 6B). We also directly compared diastolic Ca2+ concentrations throughout an experiment in young adult and aged cells exposed to IPC. Diastolic Ca2+ levels throughout these experiments were virtually identical in both age groups when cells were preconditioned (not shown). Note that although diastolic Ca2+ was elevated in ischemia in all groups, this was not accompanied by a change in cell length (Fig. 3, A and B). Time control experiments showed that diastolic Ca2+ levels did not change with time in either age group (n = 9 young adult and 11 aged cells; not shown).


Figure 6
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  		Fig. 6. IPC abolished the marked increase in diastolic Ca2+ in ischemia and early reperfusion in aged cells but had no impact on diastolic Ca2+ in younger cells. A: IPC itself increased diastolic Ca2+ in ischemia but had no effect on the increase in diastolic Ca2+ induced by ischemia in young adult cells. B: IPC inhibited the increase in diastolic Ca2+ caused by prolonged ischemia in aged myocytes. Diastolic Ca2+ levels were normalized to control values before prolonged ischemia (at t = 20 mins; dashed lines). n = 18 young adult and 15 aged cells in the absence of IPC and n = 13 young adult and 13 aged cells pretreated with IPC. *Significant difference from the appropriate comparison group (P < 0.05).

 
Ischemia induced an alternating pattern of small and large amplitude contractions and/or Ca2+ transients in some experiments. Representative examples of these responses, called Ca2+ (top) and mechanical (bottom) alternans, are shown in Fig. 7A. The overall incidence of Ca2+ and mechanical alternans was calculated for all groups. In addition, the mean mechanical alternans and Ca2+ alternans ratios in the absence and presence of IPC were calculated as described in MATERIALS AND METHODS. IPC had no significant effect on the incidence of mechanical alternans in young adult cells (Fig. 7B). In contrast, IPC markedly reduced the incidence of mechanical alternans in the aged group (Fig. 7C). IPC also had no effect on the mechanical alternans ratio in younger cells (Fig. 7D) but reduced the mechanical alternans ratio in aged cells (Fig. 7E). Ca2+ alternans occurred rarely in all groups, and IPC had no effect on the incidence of Ca2+ alternans in our study (values were 5.3% vs. 7.7% of young adult cells in the absence and presence of IPC and 11.8% vs. 0% of aged cells in the absence and presence of IPC). IPC also had no impact on the Ca2+ alternans ratios in young adult or aged cells (Fig. 7, F and G).


Figure 7
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  		Fig. 7. IPC suppressed the occurrence of mechanical alternans in ischemia in aged cells but not in young adult cells. A: representative recordings of Ca2+ alternans (top) and mechanical alternans (bottom) in ischemia. Cell shortening is shown as downward deflections in the recordings. The stimulus pulse (4 Hz) is shown below. Data were recorded from an aged myocyte. B: IPC had no significant effect on the incidence of IPC in young adult cells. C: IPC reduced the incidence of mechanical alternans in aged cells. D and E: preconditioning had no effect on the mechanical alternans ratio in younger cells (D) but significantly reduced the mechanical alternans ratio in aged myocytes (E). F and G: average Ca2+ alternans ratios in young adult (F) and aged (G) myocytes were not affected by IPC. n = 18 young adult and 16 aged cells in the absence of IPC and n = 13 young adult and 15 aged cells pretreated with IPC. *Significant effect of IPC (P < 0.05).

 
Cell viability during exposure to ischemia and reperfusion was evaluated by plotting survival curves, which show the probability of cell survival as a function of time. Figure 8A shows survival curves for young adult myocytes exposed to ischemia and reperfusion in the absence and presence of IPC. Figure 8A also shows survival curves for young adult myocytes that served as time controls. Time controls were exposed to 5 min of IPC but not to subsequent prolonged ischemia, as described in MATERIALS AND METHODS. Cell survival declined in reperfusion in the absence of preconditioning, but IPC markedly improved cell survival in the young adult group (Fig. 8A). When young adult cells were preconditioned, cell survival was virtually identical to survival in the time control group (Fig. 8A). In contrast to results in young adult cells, IPC had no significant effect on cell survival in the aged group (Fig. 8B). Furthermore, preconditioning did not improve the survival of aged cells compared with the time control group (Fig. 8B).


Figure 8
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  		Fig. 8. IPC improved cell viability in reperfusion in young adult cells but not in aged cells. Cells were field stimulated at 4 Hz throughout the protocol. A: in young adult myocytes in the absence of IPC, cell viability declined after ischemia and reperfusion (n = 33). However, IPC abolished the decline in viability in reperfusion in young cells, and this effect was statistically significant (P < 0.05). There were no significant differences in cell survival between young adult cells that were preconditioned (n = 15) and young adult cells that served as time controls (n = 10). B: cell viability also declined after ischemia and reperfusion in aged myocytes (n = 28). In contrast to the young adult group, IPC did not improve cell viability in the aged group (n = 13). There was a significant difference in cell survival between aged cells that were preconditioned compared with time controls (n = 11, P < 0.05). NS, not significant.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
This study evaluated the beneficial effects of IPC on cell function and viability in isolated myocytes exposed to ischemia and reperfusion and determined whether these effects were compromised in cells from aged hearts. The results showed that IPC inhibited diastolic contracture, abolished postischemic contractile depression, and augmented Ca2+ transients in reperfusion in young adult and aged cells. Thus, some cellular responses to IPC were similar in young adult and aged cells. Despite these similarities, there were age-related differences in responses to IPC. Preconditioning had no effect on the rise in diastolic Ca2+ in ischemia in younger cells but inhibited the marked rise in diastolic Ca2+ in ischemia and early reperfusion in aged myocytes. Pretreatment with IPC also suppressed mechanical alternans in ischemia in cells from aged hearts, whereas cells from younger hearts showed little mechanical alternans in the absence or presence of IPC. Preconditioning also significantly increased the survival of young adult cells in reperfusion but had no significant effect on cell survival in the aged group. These results demonstrate that IPC has significant cardioprotective effects in isolated myocytes from young adult hearts, and many of these protective effects are preserved in cells from aged rats. Nonetheless, as IPC did not improve the survival of aged cells in reperfusion, our results also indicate that the efficacy of IPC is reduced in the aging heart.

To our knowledge, this is the first study to investigate the impact of IPC on Ca2+ homeostasis and functional responses of young adult and aged myocytes throughout simulated ischemia and reperfusion. An important finding in this study was the observation that IPC had beneficial effects on the recovery of contractile function in reperfusion in young adult myocytes. We found that IPC attenuated diastolic contracture in reperfusion and dramatically improved postischemic contractile function in individual ventricular myocytes. This finding agrees with previous studies in intact hearts, which report that IPC improves postischemic contractile function in a variety of different young adult animals (see Refs. 8, 11, 28, 41, 44, and 51 but cf. Ref. 21). However, we also showed that the improvement in contractile function in reperfusion was accompanied by an increase in the size of Ca2+ transients recorded together with contractions. Thus, our observations suggest a mechanism that may contribute to the beneficial effects of IPC on recovery of contractile function in the young adult heart. The increased availability of intracellular Ca2+ in preconditioned cells would provide more Ca2+ to activate contraction (5). This could help to counteract the decrease in myofilament Ca2+ sensitivity that contributes importantly to the pathogenesis of postischemic contractile depression and stunning in reperfusion (42).

The present study also showed that IPC improved postischemic contractile function and increased the size of Ca2+ transients in reperfusion in myocytes from aged rats. The improvement in contractile function was almost as great as that seen in younger cells. This finding was unexpected, as previous studies have suggested that IPC does not improve postischemic contractile function in intact hearts from aged animals (1, 13, 38, 49). There are several possible explanations for these findings. One major difference between the present study and previous investigations is that earlier studies used intact heart models, whereas the present study used isolated myocytes. In the intact heart, age-related changes in the structure and function of the vasculature (26) may contribute to the decrease in efficacy of IPC observed in aging. It also is possible that vascular changes associated with aging might increase the severity of the ischemic insult in intact hearts from aged animals compared with younger hearts. However, the present study clearly shows that IPC can have beneficial effects on contractile function in individual ventricular myocytes from aged hearts and suggest that this occurs through effects on intracellular Ca2+ homeostasis.

We found that diastolic Ca2+ levels were elevated in ischemia, but this was not reflected in cell length, which actually increased slightly in ischemia in all groups. This dissociation between Ca2+ levels and cell length in ischemia is thought to be due to a decrease in myofilament sensitivity to Ca2+, which arises as a consequence of acidosis and inorganic phosphate accumulation in ischemia (5, 30). The decrease in myofilament Ca2+ sensitivity in ischemia is even more dramatically illustrated by the marked depression of contractions, despite the presence of normal Ca2+ transients. We also found that contractile depression during ischemia showed some recovery in both age groups in the absence and presence of IPC. This improvement in contractile function throughout ischemia appears to be characteristic of rat ventricular myocytes, as it was not observed in guinea pig ventricular myocytes exposed to the same ischemic solution (34). Previous studies have shown that contractions in rat ventricular myocytes do recover gradually during exposure to acidosis and have suggested that this may be linked to Na+ loading via the Na+/H+ exchanger in response to the decrease in intracellular pH (e.g., Ref. 18). A similar mechanism may explain the recovery of contractions in ischemia in rat ventricular myocytes observed in this study.

We examined the impact of IPC on the increase in diastolic Ca2+ levels in ischemia and found that IPC had no effect on the modest rise in diastolic Ca2+ observed in ischemia in younger cells. In contrast, preconditioning inhibited the large, sustained increase in diastolic Ca2+ observed throughout ischemia and in early reperfusion in aged myocytes. Elevated levels of diastolic Ca2+ and prolonged Ca2+ transients are believed to play an important role in the development of alternans in the heart (10, 29, 52). Interestingly, the incidence and severity of mechanical alternans were highest in aged myocytes, where ischemia also caused a large, sustained increase in diastolic Ca2+. IPC attenuated this rise in diastolic Ca2+ in aged myocytes, and IPC also reduced the incidence and severity of mechanical alternans in aged cells. In contrast, IPC had no effect on either diastolic Ca2+ levels or the occurrence of alternans in young adult cells. IPC also had no effect on the prolongation of Ca2+ transients caused by ischemia in young adult or aged cells. Taken together, these observations suggest that the decrease in diastolic Ca2+ levels caused by IPC in aged cells may contribute to the decline in severity of mechanical alternans in these cells. To our knowledge, this is the first report to evaluate the impact of IPC on mechanical alternans in cardiac myocytes. Our results suggest that beneficial effects of IPC in the heart may include a reduction in the severity of alternans under conditions, such as myocardial ischemia, where this activity occurs.

The mechanism by which IPC reduces the occurrence of mechanical alternans in aged cells in ischemia is not yet clear. However, one possibility involves the effects of IPC on action potential duration. Action potential duration is prolonged in aged myocytes compared with younger cells (27), and this prolongation may persist in aged myocytes exposed to ischemia. IPC is thought to abbreviate action potential duration, which may contribute to cardioprotective effects of IPC by limiting Ca2+ influx (14). If IPC abbreviates prolonged action potentials in aged myocytes in ischemia, this may reduce diastolic Ca2+ levels and decrease the occurrence of alternans in these cells.

This study also showed that IPC significantly improved the viability of young adult myocytes in reperfusion but had no effect on survival of aged myocytes in reperfusion. These observations are compatible with many previous studies in intact animal hearts, where the marked reduction in infarct size caused by IPC in young adult hearts was not observed in hearts from aged animals (see Refs. 6, 13, 38, and 46 but cf. Ref. 7). Therefore, despite the cardioprotective effects of IPC on Ca2+ homeostasis and contractile function in aged myocytes reported here, our results do suggest the efficacy of IPC is reduced in myocytes isolated from the aging heart. The cardioprotective effects of IPC on Ca2+ homeostasis and contractile function appear to be independent of the impact of IPC on cell viability, at least in cells from aged hearts. Our findings show that although IPC reduces diastolic Ca2+ in ischemia and early reperfusion in aged myocytes, this does not translate into an improvement in cell viability in reperfusion. Conversely, IPC has no effect on diastolic Ca2+ levels, but IPC improves cell survival in reperfusion in the younger group. These results suggest that the beneficial effects of IPC on cell viability are not linked to modulation of diastolic Ca2+ levels. However, the aging process is thought to modify one or more effector mechanisms that are implicated in IPC (6, 25), and this could reduce the efficacy of IPC in the aging heart.

The cellular model used here allowed us to investigate cardioprotective effects of IPC in individual ventricular myocytes without the potential confounding effects of changes in the vasculature that occur with age (26). Still, the absence of cell types other than cardiac myocytes in our IPC protocol may not allow full activation of the pathways that are important in IPC in the intact heart. A recent study (45) in young adult rat hearts demonstrated that ischemic preconditioning of Langendorff-perfused hearts conferred cardioprotection on individual myocytes that were subsequently isolated from the preconditioned hearts. These preconditioned myocytes had better contractile function, less hypercontracture, less diastolic Ca2+ accumulation, and improved survival in reoxygenation compared with cells that were not preconditioned (45). Interestingly, many of the cardioprotective effects observed in myocytes that were preconditioned in the intact heart (45) also occurred in the myocytes investigated in our study, where cells were preconditioned after isolation. These observations suggest that many beneficial effects of IPC reside within the myocytes themselves, as previously suggested (12).

There are some limitations to the present study. The 90% decrease in PO2 achieved in our model represents hypoxia, and the degree of ischemia is likely to be less severe than the ischemic insult achieved in the intact heart (47). Our model also does not specifically incorporate factors such as opiates, catecholamines, or adenosine that are released by ischemia in intact tissues. One additional limitation of this study, as in other studies of isolated myocytes from aging hearts, is that the yield of viable myocytes declines slightly with age. However, the aged cells used in our investigations exhibited the characteristic increase in cell size observed in aged ventricular myocytes (15, 32). Therefore, the myocytes used in our study are similar to those used in previous studies of aging cardiac myocytes.

In summary, this study demonstrated that IPC has significant cardioprotective effects on isolated ventricular myocytes from both young adult and aged rat hearts. IPC inhibited diastolic contracture, improved the recovery of contractile function, and increased Ca2+ transient amplitudes in reperfusion cells from animals of both ages. IPC also inhibited the increase in diastolic Ca2+ and suppressed mechanical alternans in ischemia in cells from aged hearts but had no effect on these parameters in cells from younger animals. In contrast, IPC improved survival of younger cells in reperfusion but had no effect on the survival of aged cells. Thus, although IPC has protective effects on ventricular myocytes from young adult and aged rats, the efficacy of IPC is reduced in cells from aged hearts.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by grants from the Canadian Institutes for Health Research and the Heart and Stroke Foundation of Nova Scotia.


   ACKNOWLEDGMENTS
 
The authors thank Peter Nicholl and Cindy Mapplebeck for excellent technical assistance. The authors are grateful to Spring Farrell for helpful comments on this manuscript and to Jenna Ross for assistance with PO2 measurements.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. E. Howlett, Dept. of Pharmacology, Dalhousie Univ., 5850 College St., Sir Charles Tupper Medical Bldg., Halifax, NS, 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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