Skip Navigation

Cardiovascular Research 1997 34(2):313-322; doi:10.1016/S0008-6363(97)00021-7
© 1997 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
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 Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Kasamaki, Y.
Right arrow Articles by McDonald, T. F
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kasamaki, Y.
Right arrow Articles by McDonald, T. F
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1997, European Society of Cardiology

Protection by hypoxic preconditioning against hypoxia-reoxygenation injury in guinea-pig papillary muscles

Yuji Kasamaki, An Chi Guo and Terence F McDonald*

Department of Physiology and Biophysics, Dalhousie University, Halifax, NS B3H 4H7, Canada

* Corresponding author. Tel. +1 902 494-3392; Fax +1 902 494-1685.

Received 4 April 1996; accepted 13 December 1996


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Developed tension in guinea-pig papillary muscles is depressed by prolonged hypoxia; subsequent reoxygenation leads to a partial recovery that stabilizes after an early period of arrhythmia. We have investigated whether hypoxic preconditioning in these muscles (1) improves the recovery of developed tension, (2) protects against reoxygenation arrhythmia, and (3) causes other significant electromechanical changes. Methods: Papillary muscles stimulated at 1 Hz were superfused with oxygenated Krebs solution for 60 min and either preconditioned (5 min of 3 Hz pacing under substrate-free hypoxic conditions, 10 min of normoxic recovery) or equilibrated for an extra 15 min. Muscles were subsequently challenged with substrate-free hypoxia (1 Hz), and reoxygenated (1 Hz) for 60 min. Contractile performance, action potential parameters, and indicators of arrhythmic activity were measured in 10 preconditioned and 10 non-preconditioned muscles. Results: Developed tension in preconditioned muscles declined to the same level (10–15% control) as in non-preconditioned muscles after 60 min hypoxia. A notable difference was that developed tension in the preconditioned muscles failed to rebound during mid-hypoxia, a hallmark feature in non-preconditioned muscles. The action potential duration and overshoot collapsed at a significantly faster rate in hypoxic preconditioned muscles. Action potential recovery during reoxygenation was similar in the two groups of muscles, but recovery of developed tension was significantly stronger in preconditioned (76.7±5.4%) than in non-preconditioned (42.9±1.7%) muscles (P<0.001). Reoxygenation provoked arrhythmic activity in all muscles, but the summed average duration was shorter (5.5±1.0 vs. 9.4±1.5 min) (P<0.05) in the preconditioned muscles. Conclusions: Hypoxic preconditioning can significantly enhance post-hypoxia recovery of developed tension, and significantly attenuate arrhythmic activity, in guinea-pig papillary muscles. © 1997 Elsevier Science B.V.

KEYWORDS Hypoxia; Preconditioning; Contraction; Contracture; Membrane potential; Reperfusion; Arrhythmias; Guinea pig, ventricular muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Ischemic preconditioning was first described by Murry et al. [1]as the cardioprotective mechanisms that are activated by brief ischemic stress; these mechanisms can reduce the rate of cardiac cell necrosis during a subsequent episode of prolonged ischemia. Depending on the species, the forward protection afforded by the brief (2–5 min) preconditioning ischemia lasts for several hours [2]. It is now evident that this endogenous myocardial protective mechanism can be activated by either coronary occlusion or hypoxia [3, 4], and that protection can be gauged from measurement of a number of functional indices that include post-ischemic recovery of contractile performance [5–7]and post-ischemic occurrence of arrhythmia [8–10].

Cardioprotection afforded by ischemic/hypoxic preconditioning has primarily been investigated in open-chest or Langendorff whole-heart preparations. However, it has recently been detected and studied in isolated tissue strands. Tan et al. [11]investigated no-flow ischemia in isolated vascular-perfused rabbit papillary muscles and reported that preconditioning delayed the onset of electrical uncoupling during the ischemia challenge. Walker et al. [12]found that 3 min of hypoxia preconditioning prior to prolonged hypoxia challenge improved the post-hypoxia recovery of developed force in rabbit papillary muscles; the same group have recently reported similar results on human atrial trabeculae [13, 14].

Our objective was to determine whether hypoxic preconditioning offers protection against hypoxia-induced dysfunction in superfused guinea-pig papillary muscles. Although the primary index of protection was the recovery of contractile function after the hypoxia challenge, we also monitored action potential configuration, and examined whether the incidence and duration of arrhythmias induced by reoxygenation of these tissues (cf. [15]) were modified by preconditioning. In addition, we report on experimental conditions that modify the development of a mid-hypoxia transitory rebound in developed tension that is strongly attenuated by preconditioning.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Experimental preparations and solutions
The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Male guinea-pigs (250–350 g) were killed by cervical dislocation, and the hearts quickly removed and immersed in cold oxygenated (95% O2/5% CO2) bicarbonate-buffered Krebs solution. A suitable papillary muscle (length >2 mm, diameter <0.8 mm) was dissected from the right ventricle, along with an attached small portion of the interventricular septum and valve leaflet. Muscles were mounted horizontally in a 0.25 ml perfusion chamber, and continuously superfused with oxygenated Krebs solution at 6 ml/min. The Krebs solution (36±0.5°C) contained (in mM): 113.1 NaCl, 4.6 KCl, 21.9 NaHCO3, 3.58 NaH2PO4, 1.15 MgCl2, 2.45 CaCl2 and 10 glucose (pH 7.4). Five millimolar glucose solution was made by reducing glucose concentration, and substrate-free solution by omission of glucose.

2.2 Measurement of contractile and electrical activity
The clamped mural end of the muscle was electrically stimulated through a bipolar Ag–AgCl electrode. Stimulating pulses (1–2 ms duration, 1.2 times threshold intensity) were applied at 1 Hz unless otherwise stated. The valve leaflet was attached to an isometric force transducer (model UC2, Gould Statham, Oxnard, CA, USA), and the muscle stretched to a length that produced 80–90% maximal developed tension. Action potentials were recorded with conventional 3 M KCl-filled microelectrodes (resistance 8–12 M{Omega}) connected to a high-input impedance amplifier (model 750, WP Instruments, New Haven, CT, USA) via an Ag–AgCl half-cell. A flowing 3 M KCl/Ag–AgCl unit with frittered glass junction was used as the reference electrode. The maximum rate of rise of the action potential upstroke (Vmax) was obtained by electronic differentiation of the action potential. The electrical signals and tension were displayed on a storage oscilloscope (model 5113, Tektronix, Beaverton, OR, USA) and recorded on film, tape, and/or chart recorder (model 2400, Gould, Cleveland, OH, USA) as required.

The contractile parameters that were measured included developed tension (DT), resting tension (RT), rate of tension development (+dT/dt), rate of relaxation (–dT/dt), time to half-relaxation (RT1/2) and time to peak tension (TTP). The action potential parameters that were measured included Vmax, action potential duration at 90% repolarization (APD90), action potential duration at 0 mV (APD0 mV), action potential amplitude, overshoot amplitude (action potential amplitude above 0 mV), and resting membrane potential. Arrhythmic activity provoked by reoxygenation was assessed from recordings of electrical and contractile activity by (1) scoring the incidence of arrhythmic episodes in given time periods, (2) measuring the summed duration of such episodes within given time periods, and (3) measuring the summed duration of the activity over the entire 60-min reoxygenation period. The incidence of automaticity was scored by scrutinizing whether an extrasystolic action potential occurred during the initial 60–90 s period after reoxygenation, and during subsequent 30-s periods spaced 1 min apart.

2.3 Hypoxic preconditioning, hypoxia challenge, and reoxygenation protocols
All muscles were equilibrated for 60 min in oxygenated Krebs solution prior to an experimental procedure. Hypoxic conditions were achieved by gassing glucose-free Krebs solution with 95% N2/5% CO2; this resulted in PO2 <5 kPa at the bath inflow port as analyzed by an automated blood gas analyzer (model 158, CIBA Corning, Markham, Ont., Canada). Reoxygenation was achieved by switching from hypoxic superfusate to oxygenated (10 mM glucose) Krebs solution. A preconditioning treatment included a brief period (e.g., 5 min) of superfusion with substrate-free hypoxic solution, and a recovery period (e.g., 10 min) with oxygenated Krebs solution. Preconditioning was followed by a prolonged (30–120 min) challenge with substrate-free hypoxic solution, and eventual reoxygenation with normal Krebs solution. The stimulation rate was kept at 1 Hz except for the brief hypoxic preconditioning period when it was 1, 3 or 5 Hz depending on the protocol being examined. Results were compared with those from non-preconditioned muscles that were treated in the same way except that a normoxic dummy treatment (1 Hz stimulation) was substituted for the preconditioning (hypoxia, short recovery) treatment. An additional smaller group of control muscles was paced at 3 Hz for the first 5 min of the normoxic dummy treatment. Further details are provided in Section 3. All of the superfusates were warmed to 36±0.5°C.

2.4 Drugs
Ryanodine was obtained from Calbiochem. A 1 mM stock solution of ryanodine was made in dimethyl sulfoxide (DMSO) and then diluted in the buffer (final DMSO after dilution in buffer: 0.01%).

2.5 Statistics
Data are expressed as means±s.e.m. Differences between means in two groups at chosen time points were evaluated using analysis of variance (ANOVA) for repeated measures followed by a Student's unpaired t-test with Bonferroni's correction for multiple comparisons. For comparison between two mean values, a Student's unpaired t-test was performed. P<0.05 was considered to be statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Preconditioning protocol and representative results
Effective preconditioning is dependent on at least two experimental variables—the brief preconditioning protocol and the duration/severity of the subsequent challenge [36, 37]. Since we were unaware of any published studies on hypoxic preconditioning of guinea-pig papillary muscles, we examined the outcomes of a number of different experimental protocols applied to small groups (n=3–5) of papillary muscles. In this screening, we varied the severity (1–5 Hz pacing) and duration (3–10 min) of the hypoxic preconditioning, as well as the severity (0–10 mM glucose) and duration (30–120 min) of the hypoxia challenge, and measured the recovery of developed tension during subsequent 60 min reoxygenation. For example, muscles were preconditioned with 10 min substrate-free hypoxia at 1 Hz, recovered under normoxic conditions for 10 min, challenged with 30 min substrate-free hypoxia, and reoxygenated. Developed tension at the end of the reoxygenation period in preconditioned muscles (73.5±5.8%, n=5) was not significantly better than in non-preconditioned muscles (66.0±8.9%, n=5), and a similar result was obtained when the hypoxic preconditioning was reduced from 10 to 5 min (n=3). However, 5 min substrate-free hypoxia (1 Hz) combined with a longer substrate-free challenge (60 min) produced more encouraging results: i.e., developed tension recovered to 62.5±9.1% (n=5) after preconditioning versus 44.4±2.0% (n=5) without preconditioning (P<0.09). Longer hypoxia challenge (120 min) reduced the recovery of both test and control muscles.

Based on the findings of Walker et al. [12], we examined whether the inclusion of rapid pacing during hypoxic preconditioning improved the protocol. Preconditioning at 5 Hz for 10 min was unsatisfactory because muscles did not always return to within 10% of pre-preconditioning contractile force after 10 min normoxic recovery. However, the latter was less of a problem when the rate was reduced to 3 Hz for 5 min, and initial trials on contractile recovery were promising. On this basis, we selected this experimental protocol to study the effects of preconditioning on contractile activity, electrical activity, and reoxygenation arrhythmia. As depicted in Fig. 1, muscles were preconditioned for 5 min by 3 Hz pacing under substrate-free hypoxic conditions. This was followed by a 10 min normoxic (1 Hz) recovery period, 60 min substrate-free 1 Hz hypoxia challenge, and 60 min normoxic (1 Hz) reoxygenation. Control non-preconditioned muscles were equilibrated for an extra 15 min prior to challenge and reoxygenation. The baseline characteristics of the control and test groups of muscles (n=10 each) investigated with this protocol are provided in Table 1.


Figure 1
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Experimental protocols.

 

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

  		Table 1 Baseline characteristics of papillary muscles

 
The slow speed records of typical contractile responses of non-preconditioned and preconditioned muscles shown in Fig. 2A illustrate that there were substantial declines in developed tension and increases in resting tension during hypoxia challenges. Both parameters recovered to a variable degree during arrhythmia-provoking reoxygenation.


Figure 2
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Example responses of non-preconditioned and preconditioned guinea pig papillary muscles. (A) Force development in representative non-preconditioned (top) and preconditioned (bottom) muscles. The spike at –10 min in the record from the preconditioned muscle was due to a potentiated contraction when the 3 Hz stimulation during hypoxic preconditioning was reduced to 1 Hz at the start of normoxic recovery. (B) Action potentials recorded from representative muscles.

 
Fig. 2B (top) shows representative action potentials recorded from a non-preconditioned papillary muscle. The 60-min hypoxia challenge induced a progressive slight depolarization, a 30% reduction in action potential overshoot, and a pronounced shortening of the action potential duration. Similar changes in action potential configuration were recorded from the example preconditioned muscle, but they occurred more quickly and to a greater degree (Fig. 2B, bottom).

3.2 Effects of preconditioning on contractile performance
A summary of the changes in developed tension recorded from the 10 preconditioned and 10 non-preconditioned muscles is provided in Fig. 3A. The preconditioning treatment caused an increase in developed tension to 213±15% of control after 2.5 min of the 3 Hz hypoxia (filled circles). However, unlike the stimulation during 3 Hz normoxia trials (open triangles), the positive inotropy was not sustained for the full 5 min of hypoxia, and force declined to 79.1±6.6% at its termination. A further decline to 66.7±6.1% after 5 min normoxic recovery was primarily due to the change in stimulation rate from 3 to 1 Hz. Thereafter, there was a quick improvement to 90.1±4.8% by the end of the normoxia recovery period.


Figure 3
View larger version (33K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Force development in non-preconditioned and preconditioned muscles. (A) Developed tension expressed as percent of 60-min equilibration values. Means±s.e., n=10 muscles in each group. The open triangles at –10, 10, 20, 35, 60, 70, 90 and 120 min are data from a second group of control muscles (n=5) that (unlike the standard control muscles) were subjected to 3-Hz stimulation for the first 5 min of the 15-min dummy preconditioning. (B) Resting tension measured from the two principal groups of muscles in panel A. Time to onset of contracture was measured as the duration of hypoxia that caused a 10% rise in the pre-hypoxia resting tension. *P<0.05, **P<0.01, {dagger}P<0.001 (Ctl vs. PC).

 
During the first 15 min of the hypoxia challenge, developed tension fell to about 33% of control in both preconditioned and non-preconditioned muscles. However, during the next 15 min of hypoxia there was a strong rebound to 71.5±9.5% in the non-preconditioned muscles, in marked contrast to the more or less stable situation (37.0±5.4%) in preconditioned muscles (P<0.07). The rebound in the non-preconditioned muscles was transitory, and by the end of the 60-min hypoxia the developed tension had declined to 15.4±1.7%, a value similar to that (11.9±1.7%) measured in preconditioned muscles.

Evaluation of the recovery of developed tension during the first 20 min of reoxygenation was complicated by the inotropic influence of episodes of arrhythmic activity. As assessed at times of non-arrhythmogenicity, developed tension recovered more quickly in preconditioned muscles (Fig. 3A). More importantly, developed tension during the arrhythmia-free 30–60 min reoxygenation period (see below) was much stronger in the preconditioned muscles (e.g., 76.7±5.4% after 60 min) than in the non-preconditioned muscles (42.9±1.7%) (P<0.001).

The hypoxia challenge caused a delayed rise in resting tension that levelled off at later times; this increase was largely but not completely reversed during subsequent reoxygenation (Fig. 3B). The time to onset of contracture was significantly shorter in preconditioned (17.4±1.6 min) than in non-preconditioned (23.3±2.1 min) muscles (P<0.05). However, it peaked at the same time (50.8±1.4 vs. 50.9±1.5 min), had a similar magnitude (606±90 vs. 706±89%), and declined to the same level after reoxygenation (191±16 vs. 222±25%) as in non-preconditioned muscles.

Since the preconditioning involved rapid pacing in addition to hypoxia, we also assessed the performance of non-preconditioned muscles that were stimulated at 3 Hz for 5 min under normoxic conditions. The open triangles at selected times in Fig. 3A indicate that, aside from the dummy preconditioning period, developed tension in these muscles was similar to that in the other non-preconditioned muscles. In particular, there was a rebound during mid-hypoxia, and weak recovery after 60 min reoxygenation (38.1±4.6%, n=5) (P<0.001 vs. preconditioned muscles).

An analysis of the changes in the twitch parameters caused by preconditioning is shown in Fig. 4. Peak +dT/dt and –dT/dt closely followed the pattern recorded for developed tension. The rates declined to a lower level with smaller rebound during the hypoxia challenge, and subsequently recovered to higher levels than in the non-preconditioned muscles (e.g., recovery to 87.3±8.2% versus 52.3±5.4% for peak +dT/dt after 60 min reoxygenation) (P<0.025) (Fig. 4A,B). The time to peak tension (TTP) in preconditioned muscles overshot control by 25–35% during the 10 min recovery period (a typical response after short-term hypoxia: 20) (Fig. 4C). It declined rapidly during the hypoxia challenge and recovered to the same degree (~80% control) as in non-preconditioned muscles. The halftime for twitch relaxation (RT1/2) was near control after preconditioning, declined to lower levels during challenge than in non-preconditioned muscles, and recovered to an insignificantly lower 89.0±3.8% (vs 99.1±7.6%) after 60 min reoxygenation (Fig. 4D).


Figure 4
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Contraction parameters measured from non-preconditioned and preconditioned muscles. All parameters are expressed as percent of 60 min equilibration values. Means±s.e., n=10 for each group; same muscles as in Fig. 3. *P<0.05, {dagger}P<0.001.

 
3.3 Preconditioning and action potential configuration
A summary of the changes in APD90 measured in the two groups of muscles is shown in Fig. 5A. The 5 min/3 Hz hypoxia preconditioning shortened the APD90, but it quickly returned to control level during the 10 min recovery period. Subsequent hypoxia challenge caused a faster shortening than in non-preconditioned muscles; for example, values at 20 and 30 min (51.2±1.6 and 39.4±3.4%) were significantly (P<0.05) shorter than in non-preconditioned muscles (72.3±3.7 and 59.8±3.1%). After 60 min hypoxia, the APD90 was not significantly different (P<0.1) in preconditioned muscles (7.4±0.9% control) than in non-preconditioned tissues (12.8±1.5%). Reoxygenation produced a rapid lengthening of the APD90 in all muscles.


Figure 5
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effects of preconditioning on action potential parameters in papillary muscles. All values are expressed as percent of 60-min equilibration values. Same muscles as in Fig. 3A; the open triangles at 40–60 min hypoxia indicate that 3-Hz pacing of non-preconditioned muscles before hypoxia had no effect on action potential parameters during subsequent hypoxia (see legend, Fig. 3). (A) Action potential duration at 90% repolarization. (B) Action potential duration at 0 mV. (C) Overshoot amplitude. Means±s.e., n=10 in the two principal groups. *P<0.05, **P<0.01, {dagger}P<0.001. Means±s.e., n=5 for second control (open triangles).

 
The action potential duration at 0 mV (APD0 mV) and the action potential overshoot were sensitive discriminators of the effects of preconditioning on action potential configuration during hypoxia challenge. The APD0 mV declined more quickly in preconditioned than in non-preconditioned muscles; after 20 min hypoxia it was 43.8±3.0 versus 74.4±1.7% in non-preconditioned muscles (P<0.001), and after 60 min it was 2.4±0.3 versus 6.8±1.3% (P<0.05) (Fig. 5B). Overshoot amplitude also declined at a faster rate; after 60 min hypoxia it was 9.5±1.2 mV in preconditioned muscles versus 19.6±0.8 mV in non-preconditioned muscles (P<0.01) (Fig. 5C). Vmax after 60 min hypoxia was slightly lower in the preconditioned muscles (80.5±2.1 vs. 87.2±2.1%) (not shown).

Action potential parameters recorded from the 3 Hz non-preconditioned muscles after 40–60 min hypoxia were very similar to those in the main control group of muscles (see open triangles in Fig. 5A–C).

3.4 Effects of preconditioning on reoxygenation arrhythmia
Records of electrical and mechanical events from a non-preconditioned muscle illustrate that long-lasting periods of automaticity during early reoxygenation gradually gave way to sporadic, single-extrasystole-type activity at later times (Fig. 6A). The same general features (pronounced activity at early times, reduced activity at later times) were observed in preconditioned muscles. The incidence of automaticity in non-preconditioned muscles climbed from 60% (6 of 10 muscles) at 1–1.5 min reoxygenation to 100% after 3–4 min, and then slowly fell to ≤20% after 20 min. The incidence in preconditioned muscles was lower at early times (e.g., 10% at 1–1.5 min), similar at intermediate times, and lower again at later times (not shown). The summed duration of arrhythmic activity during the 0–5 min reoxygenation period was significantly shorter in preconditioned muscles (2.13±0.32 min) than in the non-preconditioned muscles (3.66±0.40 min) (P<0.01), similar (2.99±0.59 vs. 3.19±0.52 min) during the 5–10 min period, and shorter (0.40±0.21 vs. 2.15±0.69 min) (P<0.025) during the 10–20 min period (Fig. 6B). The average total duration of activity during 60 min reoxygenation was 5.5±1.0 min in preconditioned muscles, and 9.4±1.5 min in non-preconditioned muscles (P<0.05) (Fig. 6C).


Figure 6
View larger version (35K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Arrhythmic activity in non-preconditioned and preconditioned muscles. Same muscles as in Fig. 3; n=10 in each group. (A) Examples of arrhythmic activity in a non-preconditioned muscle after 3 min (a) and 15 min (b) reoxygenation. The arrowheads indicate the stimulated (1 Hz) events. (B) Average summed duration of arrhythmic activity per observation period. The sums were compiled for each muscle from 1-s arrhythmic periods (see panel A). Means±s.e., *P<0.05, **P<0.01. (C) Comparison of average total duration of arrhythmic activity in non-preconditioned and preconditioned muscles during 60 min of reoxygenation. Means±s.e., *P<0.05.

 
3.5 Effects of modified hypoxia on the mid-hypoxia rebound in contractile force
It is apparent from Fig. 2A and 3A that preconditioning depressed the strong transient rebound in contractility that normally occurred after 20–30 min substrate-free hypoxia. Since this response may turn out to be of practical value in the investigation of preconditioning mechanisms, we examined whether modifications of the hypoxia conditions affected the rebound in non-preconditioned muscles. The first modification was a reduction in the severity of the challenge by including either 10 or 5 mM glucose in the superfusate. Compared to ‘control’ hypoxia (zero glucose), the 10 mM glucose superfusate greatly slowed the rate of decline of developed tension during the first 15 min (Fig. 7, open squares). Thereafter, tension steadily declined, and there was no sign of a rebound; at the end of the 60-min challenge, force was 15.3±4.6% of pre-hypoxia control (n=5) and not significantly different from that in muscles under substrate-free conditions. However, recovery at 60-min reoxygenation was significantly (P<0.001) better (70.9±5.1%) than after zero glucose hypoxia (42.9±1.7%).


Figure 7
View larger version (40K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Effects of modified hypoxia on the development of mid-hypoxia rebound in developed tension. The ‘control’ data (substrate-free hypoxia (0 G); open circles, n=10) are from Fig. 3A. There was no rebound when hypoxic superfusate was supplemented with glucose (10 mM (10 G), open squares, n=5; 5 mM (5 G), open triangles, n=6). There was also no rebound when the Ca2+ concentration of substrate-free hypoxic solution was lowered from standard 2.45 to 0.25 mM (filled circles, n=5), whereas a small rebound occurred when standard substrate-free hypoxic solution contained 0.1 µM ryanodine. Basal DT values were 88±18 mg (10 G), 96±13 mg (5 G), 103±18 mg (0 G), 94±17 mg (0 G, low Ca2+), and 90±8 mg (ryanodine). Means±s.e., {dagger}P<0.001 vs. 0 G.

 
The initial rate of decline of developed tension was faster during hypoxia with 5 mM glucose than with 10 mM, but not as fast as with zero glucose (Fig. 7, open triangles); after 15 min, the tension was 58.1±5.1% (n=6) versus 35.4±4.0% with zero glucose. Developed tension remained near that level for the next 15 min, before declining to 17.2±3.0% at 60 min. After 60-min reoxygenation, developed tension (63.5±4.3%) was only slightly smaller than under the 10 mM glucose condition.

To assess whether a release of Ca2+ from intracellular stores might be responsible for the mid-hypoxia rebound, 0.1 µM ryanodine was added to the substrate-free hypoxic solution. Under these conditions, developed tension declined more quickly than in the absence of the drug (Fig. 7, filled inverted triangles), and there was a rebound from 18.2±2.6% (n=5) at 20 min to 32.9±8.0% at 35 min. Thereafter, the developed tension declined to 7.1±2.0%, before recovering to a weak 32.9±5.4% after 60-min reoxygenation. The latter may not be a useful estimator of functional recovery due to poor reversibility from the drug treatment alone.

The final modification tested was a reduction of the Ca2+ concentration of the substrate-free hypoxic superfusate from 2.45 to 0.25 mM. This solution provoked a rapid decline in developed tension to 7.8±2.5% (n=5) of pre-hypoxia control after 15 min (Fig. 7, filled circles). There was no mid-hypoxia rebound, and developed tension at the end of hypoxia was 3.5±0.6%. However, 60-min reoxygenation under standard conditions (2.45 mM Ca2+) resulted in a recovery to 74.4±3.6% of pre-hypoxia control, a value significantly (P<0.001) larger than that following substrate-free hypoxia with 2.45 mM Ca2+ solution. Although not shown in the figure, we found that all three modifications (glucose, Ca2+, ryanodine) reduced the incidence of reoxygenation arrhythmia.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Preconditioning and contractile function
The extent of residual contractile function, a primary determinant of mortality following acute myocardial infarction [16], is a commonly used index of myocardial injury and cardioprotection. Ischemic preconditioning has been shown to enhance post-ischemic recovery of contractile function in isolated rabbit [17, 18]and rat hearts [5, 19, 20], and hypoxic preconditioning was as effective as ischemic preconditioning in preserving post-ischemic contractile function in isolated dog [3]and rat [4]heart. A significant fraction of the hypoxia-preconditioning effect originates at the myocardial cell level since protection of post-hypoxia contractile function has been demonstrated in superfused rabbit papillary muscles [12], human atrial trabeculae [13, 14], and cultured rat cardiomyocytes [21].

A primary conclusion from the present study is that hypoxic preconditioning can significantly improve the recovery of contractile function in reoxygenated guinea-pig papillary muscles. The improvement may have been due to enhanced recovery of ‘stunned’ muscle cells and/or to a reduction in the fraction of cells irreversibly injured during the hypoxia challenge. Improved contractile recovery following global ischemia in isolated preconditioned rat and rabbit hearts was associated with a reduction of enzyme leakage, and therefore attributed to a reduction of cell necrosis [5–7]. Cohen et al. [6]have reported that ischemic preconditioning in rabbits not only reduced myocardial infarct size but also improved the recovery of contractile function in the myocardial ischemic area at risk. However, Ovize et al. [22]failed to detect improved recovery of contractile function in the myocardial ischemic area at risk in dogs. A recent study on ischemic preconditioning in isolated rabbit heart by Jenkins et al. [17]provided strong evidence that improved recovery of contractile force development is closely correlated with improved salvage of muscle cells.

In the present study, hypoxic preconditioning markedly enhanced the recovery of contractile function when guinea-pig papillary muscles were challenged with 60 min substrate-free hypoxia, but only slightly enhanced recovery when they were challenged with 30 min substrate-free hypoxia. This result, and similar ones on time-dependent protection of superfused rabbit and human myocardial bundles [12, 14], suggest that the beneficial effects of preconditioning in these tissues is primarily due to improved salvage of muscle cells rather than to improved performance of stunned cells (cf. [14]).

4.2 Preconditioning and arrhythmogenic activity
Shiki and Hearse [9]were the first to demonstrate that ischemic preconditioning in rats can reduce the severity of reperfusion-induced arrhythmias. Hager et al. [8]confirmed this result, and a reduced incidence of reperfusion-induced ventricular arrhythmias after ischemic preconditioning has also been demonstrated in dogs [23–25]. However, ischemic preconditioning did not limit the incidence of ventricular fibrillation during reperfusion of pig heart [18]. The mechanism by which ischemic preconditioning affords protection against reperfusion-induced arrhythmias is not known. Bradykinin-mediated release of nitric oxide and prostanoid release may play a role in dogs [23, 25], but may not be of importance in rats [26].

The foregoing studies on protection against reperfusion-arrhythmia were conducted on in-situ or perfused whole hearts. In the present study on superfused guinea-pig papillary muscles, the arrhythmic activity provoked by reoxygenation was significantly less severe in preconditioned muscles than in non-preconditioned muscles. The mechanisms responsible for this protection are unknown, but a number of possibilities are explored below in the context of the overall electromechanical responses to hypoxia and reoxygenation.

4.3 Analysis of the electromechanical findings
The primary index of protection in guinea-pig papillary muscles in this study was the recovery of developed tension. If, as seems likely, improved recovery in preconditioned muscles was due to improved salvage of cells, it remains unclear whether this protection was exerted during the hypoxia challenge or at a later time (e.g., during early reoxygenation). However, there may well be indications of the protection in the electromechanical activity recorded during the hypoxia. In the preconditioned muscles, there was a faster decline of developed tension, relaxation of twitch tension, onset of contracture, action potential shortening, and overshoot depression. In these tissues, the rate of action potential shortening during hypoxia is related to cell energy metabolism [27], and the faster collapse of the action potential in preconditioned muscles suggests that there was a more rapid decline of ATP in these tissues. As a result, there may have been a faster activation of the ATP-regulated K+ current (IK,ATP) that is thought to be the dominant factor in the shortening of the action potential in metabolically-stressed cardiac muscle [28, 29](although additional factors such as depressed inward Na+ and Ca2+ currents and/or augmented outward delayed-rectifier K+ current may also be involved in the faster collapse).

The faster collapse of the action potential may also have been the consequence of events set in motion during the preceding preconditioning. For example, there is strong evidence that (1) ischemic preconditioning in rabbits triggers a release of adenosine that, for unknown reasons, results in protection [30–33], and (2) adenosine activates IK,ATP in cardiac myocytes [34–36]. An adenosine-mediated, faster collapse of the action potential could explain the faster decline of developed tension by more quickly restricting Ca2+ inflow during the action potential plateau (cf. [37]). Such an action might conserve ATP and enhance cell survival. The major argument against this being the sole explanation is that resting tension, an apparent indicator of relative metabolic state [27], rose at an earlier time in preconditioned (17.4±1.6 min) than in non-preconditioned (23.3±2.1 min) muscles (Fig. 3Ba).

The rebound in developed tension after 20–40 min substrate-free hypoxia was far stronger in control non-preconditioned muscles than in preconditioned muscles (Fig. 3A). To acquire insight into this finding, we examined the effects of modified hypoxia on the development of the rebound in non-preconditioned muscles. The rebound was absent when the hypoxic solution contained 10 mM glucose, and quite minor when the glucose was lowered to 5 mM (Fig. 7): i.e., the rebound appears to be directly related to the severity of the metabolic disruption. Since the rebound was not prominent in hypoxic muscles treated with ryanodine, and absent in muscles superfused with low Ca2+ solution, it appears to be related to an influx of Ca2+ and/or release of Ca2+ from the sarcoplasmic reticulum. In that regard, we note that large increases in Ca2+-related fluorescence signals from rat ventricular myocytes occur not during early hypoxia but shortly after hypoxic contracture [38, 39]. In the present study, the rebound in developed tension occurred just after the initial small rise in resting tension (e.g., Fig. 2A).

In summary, a rise in intracellular Ca2+ that begins approximately 20–25 min after the onset of substrate-free hypoxia appears to cause the rebound in developed tension. The ‘compensatory’ inotropy lasts for approximately 20 min, and must extol a price in terms of energetic requirements. Dissipation of ATP and possibly a further rise in intracellular Ca2+ during early reoxygenation are expected to aggravate cell damage and promote Ca2+-overload-triggered arrhythmic activity (cf. [15]). Compared to the poor recovery of developed tension after reoxygenation of control muscles exposed to substrate-free hypoxia, recovery was much improved in muscles reoxygenated after hypoxia with 5–10 mM glucose or low Ca2+. In fact, the enhanced recovery was similar to that achieved by preconditioned muscles reoxygenated after substrate-free hypoxia. Although it is premature to invoke a common denominator in explaining these findings, further investigation of electromechanical events during hypoxia may prove to be helpful in unravelling particular features of the protective mechanisms initiated by preconditioning.

Time for primary review 29 days.


   Acknowledgements
 
We thank Jean Crozsman for technical assistance. This work was supported by the Heart and Stroke Foundation of Nova Scotia.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–1136.
  2. Lawson CS, Downey JM. Preconditioning: state of the art myocardial protection. Cardiovasc Res 1993;27:542–550.
  3. Shizukuda Y, Mallet RT, Lee S, Downey HF. Hypoxic preconditioning of ischaemic canine myocardium. Cardiovasc Res 1992;26:534–542.
  4. Zhai X, Lawson CS, Cave AC, Hearse DJ. Preconditioning and post-ischemic contractile dysfunction: The role of impaired oxygen delivery versus extracellular metabolite accumulation. J Mol Cell Cardiol 1993;25:847–857.
  5. Cave AC, Collis CS, Downey JM, Hearse DJ. Improved functional recovery by ischaemic preconditioning is not mediated by adenosine in the globally ischaemic rat heart. Cardiovasc Res 1993;27:663–668.
  6. Cohen MV, Liu GS, Downey JM. Preconditioning causes improved wall motion as well as smaller infarcts after transient coronary occlusion in rabbits. Circulation 1991;84:341–349.
  7. Omar BA, Hanson AK, Bose SK, McCord JM. Reperfusion with pyruvate eliminates ischemic preconditioning in the isolated rabbit heart: an apparent role for enhanced glycolysis. Coron Artery Dis 1991;2:799–804.
  8. Hagar JM, Hale SL, Kloner RA. Effects of preconditioning ischemia on reperfusion arrhythmias after coronary artery occlusion and reperfusion in the rat. Circ Res 1991;68:61–68.
  9. Shiki K, Hearse DJ. Preconditioning of ischemic myocardium: reperfusion-induced arrhythmias. Am J Physiol 1987;253:H1470–H1476.
  10. Vegh A, Szekeres LS, Parratt JR. Protective effects of preconditioning of the ischaemic myocardium involve cyclo-oxygenase products. Cardiovasc Res 1990;24:1020–1023.
  11. Tan HL, Mazón P, Verberne HJ, et al. Ischaemic preconditioning delays ischaemia induced cellular electrical uncoupling in rabbit myocardium by activation of ATP sensitive potassium channels. Cardiovasc Res 1993;27:644–651.
  12. Walker DM, Marber MS, Walker JM, Yellon DM. Preconditioning in isolated superfused rabbit papillary muscles. Am J Physiol 1994;266:H1534–H1540.
  13. Speechly-Dick ME, Grover GJ, Yellon DM. Does ischemic preconditioning in the human involve protein kinase C and the ATP-dependent K+ channel? Studies of contractile function after simulated ischemia in an atrial in vitro model. Circ Res 1995;77:1030–1035.
  14. Walker DM, Walker JM, Pugsley WB, Pattison CW, Yellon DM. Preconditioning in isolated superfused human muscle. J Mol Cell Cardiol 1995;27:1349–1357.
  15. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature 1983;305:147–148.
  16. Pasternak R, Braunwald E, Sobel B, Braunwald E, eds. Acute myocardial infarction. In: A Textbook of Cardiovascular Medicine, 3rd edn. Philadelphia: Saunders Company, 1988:1222–1313.
  17. Jenkins DP, Pugsley WB, Yellon DM. Ischaemic preconditioning in a model of global ischaemia: infarct size limitation, but no reduction of stunning. J Mol Cell Cardiol 1995;27:1623–1632.
  18. Ovize M, Aupetit J, Rioufol G, et al. Preconditioning reduces infarct size but accelerates time to ventricular fibrillation in ischemic pig heart. Am J Physiol 1995;269:H72–H79.
  19. Guo AC, Diacono J, Feuvray D. Comparison of effects of aprikalim and of hypoxic and ischaemic preconditioning on extracellular potassium accumulation, metabolism, and functional recovery of the globally ischaemic rat heart. Cardiovasc Res 1994;28:864–871.
  20. Steenbergen C, Perlman ME, London RE, Murphy E. Mechanism of preconditioning. Ionic alterations. Circ Res 1993;72:112–125.
  21. Webster KA, Discher DJ, Bishopric NH. Cardioprotection in an in vitro model of hypoxic preconditioning. J Mol Cell Cardiol 1995;27:453–458.
  22. Ovize M, Przyklenk K, Hale SL, Kloner RA. Preconditioning does not attenuate myocardial stunning. Circulation 1992;85:2247–2254.
  23. Vegh A, Papp JG, Parratt JR. Attenuation of the antiarrhythmic effects of ischaemic preconditioning by blockade of bradykinin B2 receptors. Br J Pharmacol 1994;113:1167–1172.
  24. Vegh A, Papp JG, Szekeres LS, Parratt JR. Are ATP sensitive potassium channels involved in the pronounced antiarrhythmic effects of preconditioning? Cardiovasc Res 1993;27:638–643.
  25. Vegh A, Szekeres LS, Parratt JR. Preconditioning of the ischaemic myocardium; involvement of the L-arginine nitric oxide pathway. Br J Pharmacol 1992;107:648–652.
  26. Li Y, Whittaker P, Kloner RA. The transient nature of the effect of ischemic preconditioning on myocardial infarct size and ventricular arrhythmia. Am Heart J 1992;123:346–353.
  27. Hayashi H, Watanabe T, McDonald TF. Action potential duration in ventricular muscle during selective metabolic block. Am J Physiol 1987;253:H373–H379.
  28. Cole WC, McPherson CD, Sontag D. ATP-regulated K+ channels protect the myocardium against ischemia/reperfusion damage. Circ Res 1991;69:71–581.
  29. Hayashi H, Ponnambalam C, McDonald TF. Arrhythmic activity in reoxygenated guinea pig papillary muscles and ventricular cells. Circ Res 1987;61:24–133.
  30. Liu GS, Thornton JD, Van Winkle DM, Stanley AWH, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation 1991;84:350–356.
  31. Parratt JR. Possibilities for the pharmacological exploitation of ischaemic preconditioning. J Mol Cell Cardiol 1995;27:991–1000.
  32. Thornton JD, Liu GS, Olsson RA, Downey JM. Intravenous pretreatment with A1-selective adenosine analogues protects the heart against infarction. Circulation 1992;85:659–665.
  33. Tsuchida A, Miura T, Miki T, Shimamoto K, Iimura O. Role of adenosine receptor activation in myocardial infarct size limitation by ischaemic preconditioning. Cardiovasc Res 1992;26:456–461.
  34. Kirsch GE, Codina J, Birnbaumer L, Brown AM. Coupling of ATP-sensitive K+ channels to A1-receptors by G proteins in rat ventricular myocytes. Am J Physiol 1990;259:H820–H826.
  35. Li GR, Feng J, Shrier A, Nattel S. Contribution of ATP-sensitive potassium channels to the electrophysiological effects of adenosine in guinea-pig atrial cells. J Physiol 1995;484.3:629–642.
  36. Terzic A, Tung RT, Inanobe A, Katada T, Kurachi Y. G proteins activate ATP-sensitive K+ channels by antagonizing ATP-dependent gating. Neuron 1994;12:885–893.
  37. Shuba LM, McDonald TF, Excitation–contraction coupling: Relationship of calcium currents to contraction. In: Physiology and Pathophysiology of the Heart. Boston: Kluwer Academic Publishers, 1995:269–287.
  38. Allshire A, Piper HM, Cuthbertson KSR, Cobbold PH. Cytosolic free Ca2+ in single rat heart cells during anoxia and reoxygenation. Biochem J 1987;244:381–385.
  39. Siegmund B, Ladilov YV, Piper HM. Importance of sodium for recovery of calcium control in reoxygenated cardiomyocytes. Am J Physiol 1994;267:H506–H513.

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
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 Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Kasamaki, Y.
Right arrow Articles by McDonald, T. F
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kasamaki, Y.
Right arrow Articles by McDonald, T. F
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?