AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 294: H2174-H2183, 2008. First published March 7, 2008; doi:10.1152/ajpheart.00058.2008
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/5/H2174    most recent
ajpheart.00058.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by O'Brien, J. D.
Right arrow Articles by Howlett, S. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by O'Brien, J. D.
Right arrow Articles by Howlett, S. E.

Effects of ischemia and reperfusion on isolated ventricular myocytes from young adult and aged Fischer 344 rat hearts

J. Darcy O'Brien,1 Jessica H. Ferguson,1 and Susan E. Howlett1,2

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

Submitted 16 January 2008 ; accepted in final form 3 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study examined the impact of age on contractile function, Ca2+ homeostasis, and cell viability in isolated myocytes exposed to simulated ischemia and reperfusion. Ventricular myocytes were isolated from anesthetized young adult (3 mo) and aged (24 mo) male Fischer 344 rats. Cells were field-stimulated at 4 Hz (37°C), exposed to simulated ischemia, and reperfused with Tyrode solution. Cell shortening and intracellular Ca2+ were measured simultaneously with an edge detector and fura-2. Cell viability was assessed by Trypan blue exclusion. Ischemia (20–45 min) depressed amplitudes of contraction equally in isolated myocytes from young adult and aged animals. The degree of postischemic contractile depression (stunning) was comparable in both groups. Ca2+ transient amplitudes were depressed in early reperfusion in young adult and aged cells and then recovered to preischemic levels in both groups. Cell viability also declined equally in reperfusion in both groups. In short, some cellular responses to simulated ischemia and reperfusion were similar in both groups. Even so, aged myocytes exhibited a much greater and more prolonged accumulation of diastolic Ca2+ in ischemia and in early reperfusion compared with myocytes from younger animals. In addition, the degree of mechanical alternans in ischemia increased significantly with age. The observation that there is an age-related increase in accumulation of diastolic Ca2+ in ischemia and early reperfusion may account for the increased sensitivity to ischemia and reperfusion injury in the aging heart. The occurrence of mechanical alternans in ischemia may contribute to contractile dysfunction in ischemia in the aging heart.

calcium transients; cell shortening; senescence

ADVANCED AGE IS BELIEVED to exacerbate deleterious effects of ischemia and reperfusion in the heart (25). Morbidity and mortality rates following myocardial ischemia are higher in older patients than in younger adults (37, 46, 48). Advanced age also increases the risk of death after clinical procedures that involve reperfusion injury, such as coronary angioplasty and coronary artery bypass graft surgery (5, 47). These data suggest that aging exacerbates harmful effects of ischemia and reperfusion in the human heart.

Studies in hearts from senescent animals also suggest that aging aggravates ischemia and reperfusion injury. Contractile function during ischemia is depressed to a greater extent in Langendorff-perfused hearts from aged animals than in hearts from younger animals (12). Furthermore, sustained contractile depression in reperfusion (known as stunning) is more prominent in perfused hearts from aged animals compared with younger hearts (2, 18, 28, 45, 49), although this has not been observed in all studies (27). Aged hearts have been shown to release greater amounts of creatine kinase and lactate dehydrogenase in response to myocardial ischemia than younger hearts (28). In addition, in vitro and in vivo studies have shown that infarct size after ischemia and reperfusion is greater in hearts from aged animals than in younger hearts (3, 38). Therefore, aging exacerbates ischemia and reperfusion injury in intact hearts from various animal models.

Elevated levels of intracellular free Ca2+ in individual cardiac myocytes are believed to play a central role in many of the harmful effects of myocardial ischemia and reperfusion (1, 13, 26, 39). It is possible that age-related alterations in intracellular Ca2+ homeostasis in individual cardiac myocytes may underlie the increased sensitivity to ischemia and reperfusion injury in the aging heart. However, whether aging alters Ca2+ homeostasis in isolated myocytes exposed to ischemia and reperfusion has not been investigated, and whether aging affects responses of isolated cardiac myocytes to ischemia and reperfusion injury is not known.

We have developed a model of simulated ischemia and reperfusion in isolated ventricular myocytes (6, 32, 35) that utilizes an "ischemic" Tyrode solution developed previously (9, 14). This model mimics many features of ischemia, such as hypoxia, acidosis, lactate accumulation, hyperkalemia, hypercapnia, and substrate deprivation. Individual myocytes exposed to simulated ischemia and reperfusion exhibit many characteristics of true ischemia such as depolarization, action potential abbreviation, abolition of contractions, and elevated intracellular Ca2+ levels (6, 32). Cells also exhibit abnormal electrical activity, stunning, and irreversible cell injury in reperfusion (6, 32, 35). The present study utilized this model to determine whether aging alters intracellular Ca2+ homeostasis in the setting of ischemia and reperfusion and to determine whether aging affects responses of individual cardiac myocytes to ischemia and reperfusion injury. In these experiments, we evaluated and compared differences in contractile function, intracellular Ca2+ levels, and cell viability throughout ischemia and reperfusion in ventricular myocytes isolated from the hearts of young adult (3 mo) and aged (24 mo) male Fisher 344 rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All procedures that involved the use of animals were approved by the Dalhousie University Committee on Animal Care, and experiments were performed according to the guidelines outlined in the books published by the Canadian Council on Animal Care (CCAC, Ottawa, Ontario; Vol. 1, 1980; Vol. 2, 1984). Aged male Fischer 344 rats (~24 mo) were obtained from the National Institute on Aging (Baltimore, MD). Young adult male Fisher 344 rats (~3 mo) were obtained from Charles River Laboratories (St. Constant, QC, Canada). In each facility, rats were housed behind specific pathogen-free barriers and monitored regularly for genetic purity and health status. A detailed health report accompanied each shipment of animals. Animals were housed in microisolator cages in the Carleton Animal Care Facility at Dalhousie University on a 12:12-h light-dark cycle with free access to food and water and were used within 2 wk of arrival.

Cardiac myocyte isolation. Ventricular myocytes were obtained by enzymatic dissociation as described previously (10). Briefly, rats were weighed and injected with heparin (3,000 U/kg ip; Pharmaceutical Partners of Canada, Richmond, ON, Canada) to inhibit blood coagulation. Thirty min later, animals were anesthetized with pentobarbital sodium (220 mg/kg ip; CDMV, Saint-Hyacinthe, QC, Canada). Hearts were cannulated in situ through the aorta and perfused retrogradely for 5 min (18–20 ml/min, 37°C, gassed with O2) with buffer solution containing (mM) 135 NaCl, 4 KCl, 10 HEPES, 1.2 MgSO4, 1.2 KH2PO4, and 12 glucose with 50 µM CaCl2 (pH 7.4 with NaOH). Next, hearts were enzymatically digested for 15–20 min by perfusion with the same buffer solution supplemented with protease dispase II (Roche Diagnostics, Laval, QC, Canada; 0.10 and 0.14 mg/ml for 3- and 24-mo hearts, respectively) and collagenase type 2 (240 U/mg, Worthington, Lakewood, NJ; 0.3 and 0.6 mg/ml for 3- and 24-mo hearts, respectively). After perfusion with the enzyme solution, the ventricles were separated from the atria and minced in high-potassium substrate-enriched buffer containing (mM) 80 KOH, 30 KCl, 3 MgSO4·7H2O, 30 KH2PO4, 50 L-glutamic acid, 20 taurine, 0.5 EGTA, 10 HEPES, and 10 glucose (pH 7.4 with KOH). The supernatant was then decanted and filtered through 225-µm polyethylene mesh to remove large particles. Only quiescent, rod-shaped myocytes with visible striations and no visible membrane damage were used experimentally. The relative yield of viable myocytes was 40–60% and 30–50% for 3- and 24-mo-old rat hearts, respectively, which is similar to results of previous studies (11, 29). In some experiments cell length, cell width, and cell area were measured and compared between the two groups as described below. No more than two myocytes per heart were included in any one data set.

Isolated myocytes from either young adult or aged animals were placed in a 0.75-ml chamber mounted on the stage of an inverted microscope (Nikon ECLIPSE TE200, Nikon Canada). These cells were allowed to settle for 15–20 min and were then superfused with Tyrode solution containing (mM) 129 NaCl, 20 NaHCO3, 0.9 NaH2PO4, 4 KCl, 0.5 MgSO4, 2.5 CaCl2, and 5.5 glucose (pH 7.2; gassed with 95% O2-5% CO2). All experiments were conducted at 37°C. Buffer solutions were heated to 37°C with a custom-made heat exchanger and a circulating water bath (Polystat Immersion Circulator; Cole Parmer Canada, Anjou, QC, Canada). Superfusion with buffer was controlled by gravity to maintain a flow rate of 6 ml/min.

Model of simulated ischemia and reperfusion. After 20 min of superfusion with normal Tyrode solution, isolated myocytes were superfused with a simulated ischemic solution containing (mM) 123 NaCl, 6 NaHCO3, 0.9 NaH2PO4, 8 KCl, 0.5 MgSO4, 2.5 CaCl2, and 20 sodium lactate (pH 6.8; gassed with 90% N2-10% CO2) (6). In most experiments, cells were exposed to simulated ischemia for 30 min. However, in some experiments the duration of the ischemic period was varied from 5 to 60 min, as described in RESULTS. During simulated ischemia, a 90% N2-10% CO2 gas phase was directed over the top of the chamber. After ischemia, the cells were reperfused with Tyrode solution for up to 30 min. The PO2 of normal and ischemic Tyrode solutions was measured with an i-STAT portable clinical analyzer (Abbott Diagnostics). The PO2 was 652.0 ± 14.5 mmHg (n = 3) in normal Tyrode solution; the PO2 declined to 71.7 ± 7.3 mmHg (n = 3) within 2 min of exposure to ischemic Tyrode solution. This represents a 90% decrease in PO2, which is greater than the 75–80% decrease reported in other cellular models of ischemia and reperfusion (33, 34, 36). In time control experiments, myocytes isolated from young adult and aged hearts were exposed to Tyrode solution for 80 min in the absence of ischemia. Fresh cells were placed in the chamber after each experiment, so that cells were exposed to simulated ischemia and reperfusion only once.

Contractions and Ca2+ transients. Contractions and Ca2+ transients were measured simultaneously in isolated myocytes incubated with the Ca2+-sensitive dye fura-2 AM (5 µM). Cells were incubated for 20 min in the dark at room temperature. Cells were then superfused with Tyrode solution as described above and field-stimulated at 4 Hz with 3-ms pulses delivered via a pair of platinum electrodes. Voltage and pulse duration were controlled with a Grass SD9 stimulator (Grass Technologies, West Warwick, RI) that was triggered by a Pulsar 6i digital stimulator (Frederick Haer, Bowdoinham, ME), which controlled the stimulation frequency. Unloaded cell shortening was measured with a video edge detector (model 105, Crescent Electronics, Sandy, UT) at 120 Hz. The edge detector was coupled to a video camera (Philips FTM800NH, Philips Canada, Markham, ON, Canada) mounted on the microscope. Cell length and fluorescence were simultaneously recorded from each cell by splitting the microscope light with a dichroic cube. The video edge detector received red light, and remaining light was sent to the photomultiplier tube for fluorescence measurements (described below). Axoscope 8.2 (Molecular Devices, Sunnyvale, CA) was used to collect contraction data. Ten-second recordings were taken every 5 min (with additional recordings at 1 and 2 min of reperfusion). Analog signals were converted to digital signals through a Digidata 1322A A/D board (Molecular Devices). Trains of 5–10 contractions were averaged and measured with Clampfit 8.2 (Molecular Devices). Contraction amplitude was defined as the difference between systolic and diastolic cell length.

Abnormal contractile activity, such as alternating large and small beats (mechanical alternans), that occurred during ischemia and reperfusion was also recorded. In some experiments, cell length and width were measured with the video edge detector to calculate cell area. Cell viability was evaluated by exposure of myocytes to Trypan blue dye, which has been used previously to assess cell survival in studies of ischemia and reperfusion injury in myocytes (see, e.g., Ref. 21). Cells that became spherical in shape, formed membrane blebs, and exhibited irreversible contracture and Ca2+ overload in reperfusion were considered not viable. These cells typically did not exclude Trypan blue dye.

Intracellular Ca2+ was measured by whole cell photometry (DeltaRam, Photon Technology International). The emission ratio at 510 nm, during alternate excitation at 340 nm and 380 nm was used to determine intracellular Ca2+ concentrations. Fluorescence emission was measured at 200 points/s for each of the excitation wavelengths. Background fluorescence values were determined at each excitation wavelength. These background values were subtracted from the recordings made at each wavelength during each experiment. Emission ratios were converted to intracellular Ca2+ concentrations with an in vitro calibration curve determined experimentally at pH 7.0 as reported previously (32). The Ca2+ concentration measured with this curve is expected to accurately approximate Ca2+ concentration within the pH range used in this study, since the effect of pH on fura-2 within the physiological range is negligible (17). Furthermore, calibration curves determined at pH 6.8, 7.0, or 7.2 in our laboratory were similar at Ca2+ concentrations between 100 nM and 1 µM. Felix32 software (Photon Technology International) was used to record data. Ca2+ transient amplitudes were the difference between systolic and diastolic Ca2+. Ca2+ transients were recorded for ~5 s, and trains of 5–10 Ca2+ transients were averaged and measured with Clampfit 8.2 (Molecular Devices). The occurrence of abnormal Ca2+ transients (alternating large and small transients called Ca2+ alternans) in ischemia and reperfusion also was recorded.

Data analysis. Statistical analyses were performed with SigmaStat 3.1 (Systat Software). Data other than cell viability are presented as means ± SE. Cell viability was plotted as a survival curve, which represents the probability of cell survival over time. Differences in survival curves between groups were assessed with a log rank test. All other differences between groups were assessed for significance with either a t-test or a two-way repeated-measures ANOVA. Differences within treatment groups were assessed with one-way repeated-measures ANOVA. Differences were considered significant if P < 0.05.

Chemicals. Fura-2 AM was obtained from Invitrogen (Burlington, ON, Canada), and all other chemicals were obtained from Sigma Aldrich (Oakville, ON, Canada). Fura-2 was prepared as a stock solution in anhydrous DMSO with a final concentration of 0.2% and stored at –20°C until use.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The aged rats used in this study were significantly older than young adult animals (24.2 ± 0.1 vs. 3.1 ± 0.1 mo, n = 47 animals/group; P < 0.05). Body weights were significantly greater in aged rats compared with younger animals (417.9 ± 4 vs. 300.3 ± 4.1 g, n = 47 animals/group; P < 0.05). Cell size was also compared in ventricular myocytes isolated from young adult and aged rat hearts (Table 1). Mean cell length was significantly greater in myocytes isolated from aged hearts than from younger hearts (Table 1). Cell width was not affected by age, but cell area was significantly greater in myocytes isolated from aged hearts compared with cells from younger hearts (Table 1). This characteristic age-related increase in cell size has been described previously in ventricular myocytes isolated from aged rat, mouse, and sheep hearts (7, 8, 11, 16, 29). To compensate for differences in cell size, all contraction data were normalized either to cell length or to control values before ischemia.


View this table:
[in this window]
[in a new window]

  		Table 1. Physical characteristics

 
Next, we conducted functional experiments in isolated myocytes superfused with Tyrode buffer at 37°C and field-stimulated at 4 Hz as described in MATERIALS AND METHODS. Effects of 30 min of ischemia followed by reperfusion on contraction amplitudes were compared in cells from young adult and aged rats; effects of other durations of ischemia are described below. Representative examples of contractions in young adult and aged cells at selected times during an experiment are shown in Fig. 1, A and B. Before ischemia, contraction amplitudes were similar in cells from young adult and aged rat hearts (3.0 ± 0.5% and 3.4 ± 0.4% cell length; n = 19 young adult and 18 aged cells), as reported in previous studies in rat and mouse myocytes (see, e.g., Refs. 11, 20). Ischemia caused a marked decrease in contraction amplitude in young adult and aged myocytes (Fig. 1, A and B). In the young adult cell, contractions exhibited a brief overshoot in early reperfusion but were depressed later in reperfusion (Fig. 1A). In the aged myocyte, contractions also were depressed later in reperfusion, but there was no overshoot in early reperfusion (Fig. 1B). Mean data are shown in Fig. 1, C and D. Contraction amplitudes were depressed in ischemia in young adult cells compared with time controls (Fig. 1C). Contractions showed a brief overshoot in early reperfusion but then were depressed compared with time controls for the remainder of reperfusion (Fig. 1C). This sustained depression of contraction in reperfusion is known as stunning. Contractions also were reduced in ischemia in aged cells compared with time controls (Fig. 1D). After a brief recovery to preischemic levels in early reperfusion, aged myocytes exhibited stunning throughout the remainder of reperfusion (Fig. 1D). The degree of contractile depression observed in ischemia and reperfusion was similar in young adult and aged cells (Fig. 1E).


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 1. A similar degree of stunning was seen in young adult and aged myocytes exposed to ischemia and reperfusion. A: in a young adult cell, contractions were diminished in ischemia and exhibited a brief overshoot relative to preischemic levels in early reperfusion. Contractions were depressed for the remainder of reperfusion. B: a similar degree of contractile depression in ischemia and in later reperfusion was observed in the aged myocyte. However, the aged cell did not exhibit an overshoot in contraction on reperfusion. C and D: contractions were significantly depressed in ischemia in young adult and aged cells compared with age-matched time controls. Young adult cells showed a significant overshoot in contraction on reperfusion that was not seen in aged cells. However, contractile depression (stunning) in reperfusion was similar in young adult and aged myocytes. E: there was little difference in the responses of young adult and aged cells to ischemia and reperfusion, except that aged cells did not exhibit an overshoot in contraction on reperfusion. Contraction amplitudes were normalized to control values before ischemia (t = 20 min, shown by dashed lines). n = 19 young adult and 18 aged cells (ischemia and reperfusion) and 14 young adult and 15 aged cells (time controls). *P < 0.05 vs. time controls.

 
We also evaluated effects of ischemia and reperfusion on Ca2+ concentrations in young adult and aged myocytes. Figure 2A shows recordings of Ca2+ transients in a young adult myocyte at selected times during an experiment. Ischemia caused an increase in diastolic Ca2+ (Fig. 2A). Diastolic Ca2+ levels decreased below preischemic values in early reperfusion but recovered later in reperfusion (Fig. 2A). In the aged myocyte, diastolic Ca2+ was markedly elevated throughout ischemia and recovered in early reperfusion (Fig. 2B). Ca2+ transient amplitudes appeared similar during ischemia and reperfusion in young adult and aged myocytes (Fig. 2, A and B). Mean measurements of diastolic and systolic Ca2+ plotted as a function of time are shown in Fig. 2, C and D. In young adult cells, diastolic Ca2+ was elevated early in ischemia compared with preischemic values (at t = 20 min) and then declined as ischemia progressed (Fig. 2C). Diastolic and systolic Ca2+ decreased significantly in early reperfusion and then recovered to preischemic values later in reperfusion (Fig. 2C). In aged myocytes, diastolic and systolic Ca2+ increased significantly in ischemia compared with preischemic values (Fig. 2D). In contrast to younger cells, diastolic Ca2+ remained elevated in early reperfusion and then declined to preischemic values as reperfusion progressed (Fig. 2D). Diastolic Ca2+ concentrations appeared lower in aged cells than in younger cells before ischemia, but this difference was not statistically significant. Systolic and diastolic Ca2+ concentrations increased slightly during the first 20 min of the experiment in aged cells, although Ca2+ concentrations remained stable after 20 min in young adult and aged cells used as time controls (data not shown).


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 2. Changes in systolic and diastolic Ca2+ during ischemia and reperfusion in young adult and aged myocytes. A: diastolic Ca2+ increased above control values in ischemia in a representative young adult myocyte. Diastolic Ca2+ declined relative to control in early reperfusion and recovered later in reperfusion. B: diastolic Ca2+ increased markedly in ischemia and returned to preischemic levels in early reperfusion in an aged cell. Arrows indicate preischemic levels of diastolic Ca2+. C: in young adult cells, diastolic Ca2+ increased relative to preischemic values early in ischemia but recovered later in ischemia. In early reperfusion, diastolic and systolic Ca2+ declined relative to preischemic values and then recovered. D: in aged myocytes, diastolic and systolic Ca2+ increased relative to preischemic values in ischemia. Diastolic Ca2+ remained elevated early in reperfusion but then recovered to preischemic levels. n = 19 young adult and 17 aged cells. Dashed lines represent control values before ischemia (at t = 20 min). *P < 0.05 vs. preischemic values at t = 20 min.

 
Figure 3 shows mean Ca2+ transient amplitudes recorded from young adult and aged myocytes plotted as a function of time. In young adult cells, Ca2+ transient amplitudes were similar to those in time controls before ischemia and declined slightly during ischemia. Ca2+ transients were depressed in early reperfusion and recovered later in reperfusion (Fig. 3A). In aged cells, Ca2+ transients were similar to time controls before and during ischemia but declined in early reperfusion and then partially recovered (Fig. 3B). Ca2+ transient amplitudes in ischemia and reperfusion were similar in young adult and aged cells (Fig. 3C). We also measured the time to 90% decay of the Ca2+ transient at selected times throughout these experiments. The time to 90% decay of the Ca2+ transient was similar in both young adult and aged myocytes before ischemia (160.8 ± 5.4 and 162.5 ± 4.6 ms at t = 20 min; n = 19 young adult and 16 aged cells). Ischemia caused a temporary prolongation of the Ca2+ transient in young adult and aged cells. After 5 min of ischemia, the Ca2+ transient was prolonged to a similar extent in young adult and aged myocytes (188.9 ± 5.4 and 193.5 ± 4.7 ms; n = 18 young adult and 16 aged cells; P < 0.05 compared with preischemic values at t = 20 min). However, this recovered to preischemic levels in reperfusion in both groups.


Figure 3
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 3. Changes in Ca2+ transient amplitudes throughout ischemia and reperfusion in myocytes from young adult and aged animals. A and B: Ca2+ transient amplitudes declined slightly in ischemia in young adult cells but not in aged cells. Ca2+ transient amplitudes were depressed in early reperfusion in both young adult and aged myocytes compared with time controls. C: amplitudes of Ca2+ transients were similar in young adult and aged cells. Data were normalized to Ca2+ transient amplitudes before ischemia (t = 20 min, dashed lines). n = 19 young adult and 17 aged cells (ischemia and reperfusion) and 11 young adult and 13 aged cells (time controls). *P < 0.05 vs. time controls.

 
Figure 4 shows mean diastolic Ca2+ concentrations plotted as a function of time in young adult and aged myocytes. In young adult cells, diastolic Ca2+ was significantly elevated on exposure to ischemia compared with time controls. However, diastolic Ca2+ levels declined throughout ischemia in younger cells (Fig. 4A). Diastolic Ca2+ was depressed relative to time controls in early reperfusion but recovered later in reperfusion (Fig. 4A). In contrast to young adult cells, diastolic Ca2+ concentrations were markedly elevated throughout ischemia in aged myocytes (Fig. 4B). Furthermore, diastolic Ca2+ levels were elevated in early reperfusion but returned to time control levels as reperfusion progressed (Fig. 4B). Figure 4C shows that the increase in diastolic Ca2+ was significantly greater in aged cells than in younger myocytes during the last 15 min of ischemia and in early reperfusion.


Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 4. Ischemia caused a more marked and prolonged increase in diastolic Ca2+ in aged cells than in younger cells. A: diastolic Ca2+ increased in early ischemia in young adult cells and recovered to time control levels in late ischemia. Diastolic Ca2+ was decreased in early reperfusion in young adult cells compared with time controls. B: diastolic Ca2+ was elevated for the duration of ischemia in aged myocytes exposed to ischemia and reperfusion compared with time controls. Diastolic Ca2+ remained elevated in early reperfusion but returned to levels similar to time controls later in reperfusion. C: the elevation in diastolic Ca2+ in ischemia and early reperfusion was more pronounced in aged myocytes than in young adult myocytes. Diastolic Ca2+ was normalized to preischemic values (t = 20 min, dashed lines). n = 19 young adult and 17 aged cells (ischemia and reperfusion) and 11 young adult and 13 aged cells (time controls). *P < 0.05 vs. time control (A and B) or young vs. aged cells (C).

 
In some experiments, contractions and Ca2+ transients exhibited a pattern of alternating small- and large-amplitude responses known as mechanical and Ca2+ alternans, respectively (Fig. 5A). This behavior was observed in ischemia but not in reperfusion. The mean mechanical alternans ratio was significantly elevated in aged myocytes compared with young adult cells (Fig. 5B). However, the Ca2+ alternans ratio was not significantly different between the two groups (Fig. 5C).


Figure 5
View larger version (6K):
[in this window]
[in a new window]

  		Fig. 5. Degree of mechanical alternans in ischemia increased significantly with age. To quantify the magnitude of alternans in young adult and aged myocytes, a ratio was calculated as 1 – S/L, where S is the amplitude of the smaller response and L is the amplitude of the larger response (51). Ratios near 1 represent a high degree of alternans, while ratios near 0 indicate the absence of this phenomenon. A: contractions and Ca2+ transients recorded from an aged myocyte in ischemia show Ca2+ (top) and mechanical alternans (bottom). B: mean mechanical alternans ratio was higher in aged myocytes than in younger cells. C: average Ca2+ alternans ratio was not significantly difference in young adult and aged myocytes. n = 19 young adult and 17 aged cells. *P < 0.05.

 
We also determined whether aging affected cell viability during exposure to ischemia and reperfusion by plotting survival curves, which show the probability of cell survival over time. Figure 6, A and B, show survival curves for young adult and aged myocytes exposed to ischemia and reperfusion compared with age-matched time controls. All young adult and aged cells that served as time controls remained viable for the duration of the experiments (Fig. 6, A and B). In contrast, cell survival was significantly reduced in both young adult and aged myocytes exposed to ischemia and reperfusion (Fig. 6, A and B). There was no significant difference in cell survival between young adult and aged myocytes exposed to ischemia and reperfusion (Fig. 6C).


Figure 6
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 6. Cell survival after ischemia and reperfusion was similar in young adult and aged cells. A and B: cell viability was significantly depressed in young adult (n = 34, A) and aged (n = 29, B) myocytes exposed to 30 min of ischemia and reperfusion compared with age-matched time controls (n = 13–15). C: cell viability was not significantly different between young adult and aged myocytes exposed to ischemia and reperfusion.

 
In some experiments, we investigated whether age-related differences in responses to ischemic stress could be observed when the ischemic period was varied from the 30-min period described above. When cells were exposed to very brief periods of ischemia (e.g., <10 min), there was no evidence of ischemic injury. Cells remained viable throughout reperfusion, and there was no evidence of stunning. When we exposed cells to 20 min of ischemia followed by reperfusion, diastolic Ca2+ levels at the end of ischemia were significantly greater in aged cells than in younger cells (117.9 ± 5.3% of preischemic levels in younger cells vs. 134.7 ± 8.2% of preischemic values in aged group; P < 0.05). However, the degree of stunning in reperfusion was similar in young adult and aged myocytes [contractions at 10 min of reperfusion were 59.6 ± 15.5% vs. 39.6 ± 11.8% of preischemic values in young adult and aged cells; not significant (NS)]. Cell survival also was similar in the two groups (45.5% of young adult cells and 33.3% of aged cells remained viable at the end of reperfusion, n = 10 or 11 cells/group; NS). When we increased the ischemic period from 30 to 45 min, diastolic Ca2+ levels at the end of ischemia were significantly higher in aged cells than in young adult cells (134.4 ± 6.3% of preischemic levels in younger cells vs. 169.0 ± 6.3% of preischemic values in aged group; P < 0.05). Still, a similar level of stunning was observed in young adult and aged myocytes (contractions at 10 min of reperfusion were 56.3 ± 9.7% vs. 42.0 ± 9.8% of preischemic values in young adult and aged cells; NS). In addition, cell survival was similar in the two groups (61.5% of young adult cells and 64.4% of aged cells remained viable at the end of reperfusion, n = 13 or 14 cells/group). When the ischemic period was increased to 60 min, cells rounded up, exhibited membrane blebs and hypercontracture, and did not exclude Trypan blue dye. These results demonstrate that exposure of myocytes to intermediate periods of ischemia (e.g., 20, 30, and 45 min) caused an increase in intracellular Ca2+ levels that was significantly greater in aged myocytes than in young adult cells. Exposure to these intermediate periods of ischemia also promoted stunning and reduced cell viability in reperfusion equally in both young adult and aged myocytes. Our results also show that prolonged ischemia (>60 min) produced irreversible cell injury, while very brief periods of ischemia (<10 min) produced little evidence of ischemic injury.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The objectives of this study were to determine whether aging alters intracellular Ca2+ homeostasis and affects responses of individual cardiac myocytes to ischemia and reperfusion injury. Our results showed that amplitudes of contractions were reduced equally by simulated ischemia in isolated myocytes from young adult and aged hearts. Furthermore, the degree of contractile dysfunction (stunning) in reperfusion was similar in young adult and aged myocytes. However, there was a marked overshoot in contraction amplitude in reperfusion in younger cells that was not observed in cells from aged hearts. Amplitudes of Ca2+ transients measured simultaneously with contractions decreased in early reperfusion and then recovered later in reperfusion in the two groups. Cell viability declined equally in reperfusion in young adult and aged myocytes. However, aged myocytes exhibited a much greater and more prolonged accumulation of diastolic Ca2+ in ischemia and early reperfusion than myocytes from younger animals. In addition, aged myocytes exhibited a higher degree of mechanical alternans during ischemia than cells from younger myocytes. Thus, although some cellular responses to simulated ischemia and reperfusion were similar in both groups, ischemia caused a marked increase in intracellular Ca2+ and augmented abnormal contractile activity in aged myocytes compared with younger cells.

To our knowledge, this is the first report of the effects of ischemia and reperfusion injury on intracellular Ca2+ homeostasis and functional responses of isolated ventricular myocytes from aged animals. An important finding in the present study was that exposure to simulated ischemia induced a much greater and more sustained increase in diastolic Ca2+ levels in aged individual myocytes compared with younger cells. This increase in diastolic Ca2+ persisted into early reperfusion in aged myocytes, while diastolic Ca2+ levels actually declined in early reperfusion in cells isolated from younger hearts. This observation is consistent with a previous finding that ischemia caused a greater increase in Ca2+ levels in intact hearts from aged animals compared with younger hearts (2). Our study extends this observation to demonstrate that intracellular Ca2+ levels increase in ischemia and in early reperfusion as a consequence of an increase in intracellular Ca2+ at the level of the cardiac myocyte. Elevated levels of free intracellular Ca2+ in cardiac myocytes are thought to contribute importantly to many detrimental effects of myocardial ischemia and reperfusion (1, 13, 26, 39). Thus our observation that individual aged myocytes accumulate more diastolic Ca2+ in ischemia and early reperfusion than cells from younger hearts may explain, at least in part, the increased sensitivity to ischemia and reperfusion injury in the aging heart (25).

It is possible that age-related changes in proteins involved in sequestration and removal of Ca2+ from the myocyte contribute to the elevation in diastolic Ca2+ in ischemia and early reperfusion. Indeed, a decrease in the activity and expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) has been reported in the aging heart (4, 41, 42, 44, 52). This would impair the sequestration of Ca2+ in the sarcoplasmic reticulum in aging myocytes, in particular under conditions such as ischemia when intracellular Ca2+ levels are high. Furthermore, the activity and expression of the Na+/Ca2+ exchanger also declines with age (19, 30), which would impair Ca2+ removal from aged myocytes. However, we found that the time to 90% decay of the Ca2+ transient was similar in young adult and aged cells during ischemia, which suggests that differences in SERCA activity in ischemia may not account for our observations. It is possible that the duration of the action potential may shorten more in young adult myocytes during ischemia than in aged myocytes. Abbreviation of action potential duration in ischemia could limit Ca2+ influx in ischemia and thereby minimize the rise in intracellular Ca2+ in young adult cells.

Our study also demonstrated that aged myocytes exhibited a greater degree of mechanical alternans in ischemia than cells isolated from younger animals. This increased abnormal activity in aged individual myocytes might be linked to the age-related decrease in activity and expression of SERCA (4, 41, 42, 44, 52). In support of this idea, previous studies have shown that inhibition of SERCA in young adult heart can give rise to various types of alternans (50). For example, reduced expression and regulation of SERCA in the failing heart (43) is thought to account for this phenomenon in heart failure (50). Alternatively, age-related differences in the incidence of mechanical alternans may reflect differences in action potential duration in ischemia between young adult and aged cells. If action potential duration shortens more in ischemia in young adult myocytes than in aged cells, this may contribute to the lower incidence of mechanical alternans in young adult cells compared with aged cells. To our knowledge, this is the first report of an increase in the occurrence of mechanical alternans during myocardial ischemia in aging. If this also occurs in vivo, it would be expected to contribute to contractile dysfunction in ischemia in the aging heart.

Results of this study also showed that some cellular responses to simulated ischemia and reperfusion were similar in young adult and aged isolated myocytes. Cell viability declined equally in reperfusion in young adult and aged myocytes. In addition, contractile responses to simulated ischemia and reperfusion were not dramatically different in myocytes isolated from young adult and aged rat hearts. Exposure to ischemia reduced the amplitudes of contraction equally in cells from young adult and aged rats, and the groups showed a similar degree of stunning in reperfusion. However, reperfusion initially induced an overshoot in contraction in young adult cells but not in aged cells. Previous studies in Langendorff-perfused hearts have shown that contractile dysfunction in ischemia and reperfusion is augmented in aging hearts. For example, ischemia depresses contractile function in aged hearts more than in younger hearts (12). In addition, aging promotes stunning in reperfusion in intact hearts (2, 18, 28, 45, 49). It is possible that these differences in results obtained in intact hearts and isolated myocytes arise because of the different experimental models used. Age-related changes in the vasculature may increase susceptibility to ischemia and reperfusion injury in buffer-perfused hearts (24) compared with individual myocytes. In addition, the severity of the ischemic insult in intact heart models may be greater than the 90% decrease in PO2 achieved during ischemia in the present study.

We measured contractions and Ca2+ transients simultaneously in ischemia and reperfusion to determine whether contractile changes were linked to changes in Ca2+ transients. We found that Ca2+ transient amplitudes changed very little in ischemia and reperfusion in myocytes isolated from young adult and aged animals. In early reperfusion, Ca2+ transients were reduced equally in young adult and aged cells. This reduction in Ca2+ transient amplitude might contribute to contractile dysfunction and stunning in early reperfusion. However, stunning persisted in late reperfusion in cells from both groups, despite recovery of Ca2+ transient amplitudes. This suggests that reduced myofilament Ca2+ sensitivity in reperfusion, which is believed to contribute importantly to the pathogenesis of stunning (15, 40), occurs equally in individual cells from young adult and aged rat hearts.

The aged rat ventricular myocytes utilized in the present study had many characteristics of aged myocytes reported in previous studies. Cells used in this study exhibited the age-related increase in cell size reported previously in ventricular myocytes from rat, mouse, and sheep hearts (7, 8, 11, 16, 29). We also found that the amplitudes of contractions, expressed as a percentage of diastolic cell length, were similar in young adult and aged cells under control conditions. Similar results have been reported in previous studies of rat and mouse myocytes subjected to field stimulation (see, e.g., Refs. 11, 20). We also found that Ca2+ transient durations were similar in young adult and aged cells, although ischemia caused a temporary prolongation of Ca2+ transients in both groups. In mouse myocytes investigated under conditions similar to ours, Ca2+ transients are prolonged in aging, at least at rapid pacing rates (>6 Hz; Ref. 31). We examined rat cells paced at 4 Hz, which may be too slow to observe prolongation of Ca2+ transients in aging. It is not clear why Ca2+ transients were prolonged in the initial ischemic period. However, acidosis prolongs action potential duration in rat myocytes (23), which may promote Ca2+ influx and account for the prolongation of Ca2+ transients observed in our study. Activation of ATP-sensitive potassium channels later in ischemia may counteract this effect (22).

There are some limitations to the data presented in this study. Our findings apply to field-stimulated rat ventricular myocytes paced near physiological frequency (e.g., 4 Hz) and investigated at physiological temperature. Whether similar observations would obtain in cells stimulated at lower frequencies and temperatures was not investigated here. In addition, our results apply to cells isolated from the hearts of male rats at 3 and 24 mo of age. However, age-related changes in cardiac function at the level of the individual myocyte have been shown to depend on the sex of the animal (16). Further experiments will be required to determine whether results can be extrapolated to cells isolated from the hearts of female animals at similar ages. An additional limitation of this study, as well as many other studies of isolated myocytes from aged hearts (see, e.g., Refs. 11, 29), is that the yield of viable myocytes is somewhat lower from aged hearts than from younger hearts. However, cells investigated in the present study had morphological and physiological characteristics similar to those previously described in aged ventricular myocytes (7, 8, 11, 16, 20, 29). Thus the cells utilized in this investigation are similar to those investigated in previous studies of the aging cardiac myocyte.

In summary, this study investigated the impact of aging on intracellular Ca2+ homeostasis and responses of individual myocytes to ischemia and reperfusion injury. The effect of ischemia and reperfusion on contractile function and Ca2+ transients was comparable in young adult and aged myocytes. Cell viability also declined equally in reperfusion in both groups. However, myocytes isolated from aged hearts exhibited a large, sustained increase in diastolic Ca2+ in ischemia and early reperfusion compared with cells from younger animals. Furthermore, the degree of mechanical alternans in ischemia increased significantly with age. If this occurs in vivo, it may contribute to contractile dysfunction in ischemia in the aging heart. The observation that aged myocytes accumulate more diastolic Ca2+ in ischemia and early reperfusion than cells from younger hearts may account for the increased sensitivity to ischemia and reperfusion injury in the aging heart.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study 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 express their appreciation for excellent technical assistance provided by Peter Nicholl and Cindy Mapplebeck.


   FOOTNOTES
 

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

  1. Allen DG, Orchard CH. Myocardial contractile function during ischemia and hypoxia. Circ Res 60: 153–168, 1987.[Abstract/Free Full Text]
  2. Ataka K, Chen D, Levitsky S, Jimenez E, Feinberg H. Effect of aging on intracellular Ca2+, pHi, and contractility during ischemia and reperfusion. Circulation 86: II371–II376, 1992.[Medline]
  3. Azhar G, Gao W, Liu L, Wei JY. Ischemia-reperfusion in the adult mouse heart influence of age. Exp Gerontol 34: 699–714, 1999.[CrossRef][Web of Science][Medline]
  4. Cain BS, Meldrum DR, Joo KS, Wang JF, Meng X, Cleveland JC Jr, Banerjee A, Harken AH. Human SERCA2a levels correlate inversely with age in senescent human myocardium. J Am Coll Cardiol 32: 458–467, 1998.[Abstract/Free Full Text]
  5. Christakis GT, Ivanov J, Weisel RD, Birnbaum PL, David TE, Salerno TA. The changing pattern of coronary artery bypass surgery. Circulation 80: I151–I161, 1989.[Medline]
  6. Cordeiro JM, Howlett SE, Ferrier GR. Simulated ischaemia and reperfusion in isolated guinea pig ventricular myocytes. Cardiovasc Res 28: 1794–1802, 1994.[Abstract/Free Full Text]
  7. Dibb KM, Rueckschloss U, Eisner DA, Isenberg G, Trafford AW. Mechanisms underlying enhanced cardiac excitation contraction coupling observed in the senescent sheep myocardium. J Mol Cell Cardiol 37: 1171–1181, 2004.[Web of Science][Medline]
  8. Dyachenko V, Rueckschloss U, Isenberg G. Aging aggravates heterogeneities in cell-size and stress-intolerance of cardiac ventricular myocytes. Exp Gerontol 41: 489–496, 2006.[CrossRef][Web of Science][Medline]
  9. Ferrier GR, Moffat MP, Lukas A. Possible mechanisms of ventricular arrhythmias elicited by ischemia followed by reperfusion. Studies on isolated canine ventricular tissues. Circ Res 56: 184–194, 1985.[Abstract/Free Full Text]
  10. Ferrier GR, Smith RH, Howlett SE. Calcium sparks in mouse ventricular myocytes at physiological temperature. Am J Physiol Heart Circ Physiol 285: H1495–H1505, 2003.[Abstract/Free Full Text]
  11. Fraticelli A, Josephson R, Danziger R, Lakatta E, Spurgeon H. Morphological and contractile characteristics of rat cardiac myocytes from maturation to senescence. Am J Physiol Heart Circ Physiol 257: H259–H265, 1989.[Abstract/Free Full Text]
  12. Frolkis VV, Frolkis RA, Mkhitarian LS, Fraifeld VE. Age-dependent effects of ischemia and reperfusion on cardiac function and Ca2+ transport in myocardium. Gerontology 37: 233–239, 1991.[Web of Science][Medline]
  13. Gao WD, Atar D, Liu Y, Perez NG, Murphy AM, Marban E. Role of troponin I proteolysis in the pathogenesis of stunned myocardium. Circ Res 80: 393–399, 1997.[Web of Science][Medline]
  14. Gilmour RF Jr, Zipes DP. Different electrophysiological responses of canine endocardium and epicardium to combined hyperkalemia, hypoxia, and acidosis. Circ Res 46: 814–825, 1980.[Free Full Text]
  15. Gowda RM, Khan IA, Vasavada BC, Sacchi TJ. Reversible myocardial dysfunction: basics and evaluation. Int J Cardiol 97: 349–353, 2004.[CrossRef][Web of Science][Medline]
  16. Grandy SA, Howlett SE. Cardiac excitation-contraction coupling is altered in myocytes from aged male mice, but not in cells from aged female mice. Am J Physiol Heart Circ Physiol 291: H2362–H2370, 2006.[Abstract/Free Full Text]
  17. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]
  18. Headrick JP, Willems L, Ashton KJ, Holmgren K, Peart J, Matherne GP. Ischaemic tolerance in aged mouse myocardium: the role of adenosine and effects of A1 adenosine receptor overexpression. J Physiol 549: 823–833, 2003.[Abstract/Free Full Text]
  19. Heyliger CE, Prakash AR, McNeill JH. Alterations in membrane Na+-Ca2+ exchange in the aging myocardium. Age 11: 1–6, 1988.[CrossRef][Web of Science]
  20. Huq F, Lebeche D, Iyer V, Liao R, Hajjar RJ. Gene transfer of parvalbumin improves diastolic dysfunction in senescent myocytes. Circulation 109: 2780–2785, 2004.[Abstract/Free Full Text]
  21. Ikonomidis JS, Tumiati LC, Weisel RD, Mickle DA, Li RK. Preconditioning human ventricular cardiomyocytes with brief periods of simulated ischaemia. Cardiovasc Res 28: 1285–1291, 1994.[Abstract/Free Full Text]
  22. Kane GC, Liu XK, Yamada S, Olson TM, Terzic A. Cardiac KATP channels in health and disease. J Mol Cell Cardiol 38: 937–943, 2005.[CrossRef][Web of Science][Medline]
  23. Komukai K, Brette F, Pascarel C, Orchard CH. Electrophysiological response of rat ventricular myocytes to acidosis. Am J Physiol Heart Circ Physiol 283: H412–H422, 2002.[Abstract/Free Full Text]
  24. Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises. I. Aging arteries: a "set up" for vascular disease. Circulation 107: 139–146, 2003.[Free Full Text]
  25. Lakatta EG, Schulman S. Age-associated cardiovascular changes are the substrate for poor prognosis with myocardial infarction. J Am Coll Cardiol 44: 35–37, 2004.[Free Full Text]
  26. Lee JA, Allen DG. Mechanisms of acute ischemic contractile failure of the heart. Role of intracellular calcium. J Clin Invest 88: 361–367, 1991.[Web of Science][Medline]
  27. Leichtweis S, Leeuwenburgh C, Bejma J, Ji L. Aged rat hearts are not more susceptible to ischemia-reperfusion injury in vivo: role of glutathione. Mech Ageing Dev 122: 503–518, 2001.[CrossRef][Web of Science][Medline]
  28. Lesnefsky EJ, Gallo DS, Ye J, Whittingham TS, Lust WD. Aging increases ischemia-reperfusion injury in the isolated, buffer-perfused heart. J Lab Clin Med 124: 843–851, 1994.[Web of Science][Medline]
  29. Lieber SC, Aubry N, Pain J, Diaz G, Kim SJ, Vatner SF. Aging increases stiffness of cardiac myocytes measured by atomic force microscopy nanoindentation. Am J Physiol Heart Circ Physiol 287: H645–H651, 2004.[Abstract/Free Full Text]
  30. Lim CC, Liao R, Varma N, Apstein CS. Impaired lusitropy-frequency in the aging mouse: role of Ca2+-handling proteins and effects of isoproterenol. Am J Physiol Heart Circ Physiol 277: H2083–H2090, 1999.[Abstract/Free Full Text]
  31. Lim CC, Apstein CS, Colucci WS, Liao R. Impaired cell shortening and relengthening with increased pacing frequency are intrinsic to the senescent mouse cardiomyocyte. J Mol Cell Cardiol 32: 2075–2082, 2000.[CrossRef][Web of Science][Medline]
  32. Louch WE, Ferrier GR, Howlett SE. Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning. Am J Physiol Heart Circ Physiol 283: H800–H810, 2002.[Abstract/Free Full Text]
  33. Lu J, Zang WJ, Yu XJ, Chen LN, Zhang CH, Jia B. Effects of ischaemia-mimetic factors on isolated rat ventricular myocytes. Exp Physiol 90: 497–505, 2005.[Abstract/Free Full Text]
  34. Lu J, Zang WJ, Yu XJ, Jia B, Chorvatova A, Sun L. Effects of postconditioning of adenosine and acetylcholine on the ischemic isolated rat ventricular myocytes. Eur J Pharmacol 549: 133–139, 2006.[CrossRef][Web of Science][Medline]
  35. Macdonald AC, Howlett SE. Differential effects of the sodium calcium exchange inhibitor, KB-R7943, on ischemia and reperfusion injury in isolated guinea pig ventricular myocytes. Eur J Pharmacol 580: 214–223, 2008.[CrossRef][Web of Science][Medline]
  36. Maddaford TG, Hurtado C, Sobrattee S, Czubryt MP, Pierce GN. A model of low-flow ischemia and reperfusion in single, beating adult cardiomyocytes. Am J Physiol Heart Circ Physiol 277: H788–H798, 1999.[Abstract/Free Full Text]
  37. Maggioni AP, Maseri A, Fresco C, Franzosi MG, Mauri F, Santoro E, Tognoni G. Age-related increase in mortality among patients with first myocardial infarctions treated with thrombolysis. The Investigators of the Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico (GISSI-2). N Engl J Med 329: 1442–1448, 1993.[Abstract/Free Full Text]
  38. McCully JD, Toyoda Y, Wakiyama H, Rousou AJ, Parker RA, Levitsky S. Age- and gender-related differences in ischemia/reperfusion injury and cardioprotection: effects of diazoxide. Ann Thorac Surg 82: 117–123, 2006.[Abstract/Free Full Text]
  39. Moens AL, Claeys MJ, Timmermans JP, Vrints CJ. Myocardial ischemia/reperfusion-injury, a clinical view on a complex pathophysiological process. Int J Cardiol 100: 179–190, 2005.[CrossRef][Web of Science][Medline]
  40. Murphy AM. Heart failure, myocardial stunning, and troponin: a key regulator of the cardiac myofilament. Congest Heart Fail 12: 32–38, 2006.[Medline]
  41. Narayanan N. Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium. Biochim Biophys Acta 678: 442–459, 1981.[Medline]
  42. Narayanan N. Comparison of ATP-dependent calcium transport and calcium-activated ATPase activities of cardiac sarcoplasmic reticulum and sarcolemma from rats of various ages. Mech Ageing Dev 38: 127–143, 1987.[CrossRef][Web of Science][Medline]
  43. Periasamy M, Kalyanasundaram A. SERCA pump isoforms: their role in calcium transport and disease. Muscle Nerve 35: 430–442, 2007.[CrossRef][Web of Science][Medline]
  44. Taffet GE, Tate CA. CaATPase content is lower in cardiac sarcoplasmic reticulum isolated from old rats. Am J Physiol Heart Circ Physiol 264: H1609–H1614, 1993.[Abstract/Free Full Text]
  45. Tani M, Suganuma Y, Hasegawa H, Shinmura K, Ebihara Y, Hayashi Y, Guo X, Takayama M. Decrease in ischemic tolerance with aging in isolated perfused Fischer 344 rat hearts: relation to increases in intracellular Na+ after ischemia. J Mol Cell Cardiol 29: 3081–3089, 1997.[CrossRef][Web of Science][Medline]
  46. Tofler GH, Muller JE, Stone PH, Willich SN, Davis VG, Poole WK, Braunwald E. Factors leading to shorter survival after acute myocardial infarction in patients ages 65 to 75 years compared with younger patients. Am J Cardiol 62: 860–867, 1988.[CrossRef][Web of Science][Medline]
  47. Wennberg DE, Makenka DJ, Sengupta A, Lucas FL, Vaitkus PT, Quinton H, O'Rourke D, Robb JF, Kellett MA Jr, Shubrooks SJ Jr, Bradley WA, Hearne MJ, Lee PV, O'Connor GT. Percutaneous transluminal coronary angioplasty in the elderly: epidemiology, clinical risk factors, and in-hospital outcomes. The Northern New England Cardiovascular Disease Study Group. Am Heart J 137: 639–645, 1999.[CrossRef][Web of Science][Medline]
  48. White HD, Barbash GI, Califf RM, Simes RJ, Granger CB, Weaver WD, Kleiman NS, Aylward PE, Gore JM, Vahanian A, Lee KL, Ross AM, Topol EJ. Age and outcome with contemporary thrombolytic therapy. Results from the GUSTO-I trial. Global Utilization of Streptokinase and TPA for Occluded coronary arteries trial. Circulation 94: 1826–1833, 1996.[Abstract/Free Full Text]
  49. Willems L, Zatta A, Holmgren K, Ashton KJ, Headrick JP. Age-related changes in ischemic tolerance in male and female mouse hearts. J Mol Cell Cardiol 38: 245–256, 2005.[CrossRef][Medline]
  50. Wilson LD, Wan X, Rosenbaum DS. Cellular alternans: a mechanism linking calcium cycling proteins to cardiac arrhythmogenesis. Ann NY Acad Sci 1080: 216–234, 2006.[CrossRef][Web of Science][Medline]
  51. Wu Y, Clusin WT. Calcium transient alternans in blood-perfused ischemic hearts: observations with fluorescent indicator fura red. Am J Physiol Heart Circ Physiol 273: H2161–H2169, 1997.[Abstract/Free Full Text]
  52. Xu A, Narayanan N. Effects of aging on sarcoplasmic reticulum Ca2+-cycling proteins and their phosphorylation in rat myocardium. Am J Physiol Heart Circ Physiol 275: H2087–H2094, 1998.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
K. Boengler, R. Schulz, and G. Heusch
Loss of cardioprotection with ageing
Cardiovasc Res, July 15, 2009; 83(2): 247 - 261.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Thorac. Surg.Home page
J. D. O'Brien, S. E. Howlett, H. J. Burton, S. B. O'Blenes, D. S. Litz, and C. L. H. Friesen
Pediatric cardioplegia strategy results in enhanced calcium metabolism and lower serum troponin T.
Ann. Thorac. Surg., May 1, 2009; 87(5): 1517 - 1523.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Kapur, J. A. Wasserstrom, J. E. Kelly, A. H. Kadish, and G. L. Aistrup
Acidosis and ischemia increase cellular Ca2+ transient alternans and repolarization alternans susceptibility in the intact rat heart
Am J Physiol Heart Circ Physiol, May 1, 2009; 296(5): H1491 - H1512.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. D. O'Brien and S. E. Howlett
Simulated ischemia-induced preconditioning of isolated ventricular myocytes from young adult and aged Fischer-344 rat hearts
Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H768 - H777.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/5/H2174    most recent
ajpheart.00058.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by O'Brien, J. D.
Right arrow Articles by Howlett, S. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by O'Brien, J. D.
Right arrow Articles by Howlett, S. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.