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Intermittent Hypoxic Training Protects
Canine Myocardium from Infarction
PUZONG,*
,1
SRINATH SETTY,* WEI SUN,* RODOLFO MARTINEZ,* JOHNATHAN D. TUNE,*
IGOR V. E HRENBURG,ELENA N. TKATCHOUK,ROBERT T. MALLET,* AND H. FRED DOWNEY*
*Department of Integrative Physiology, University of North Texas Health Science Center, Fort Worth,
Texas 76107; and Clinical Research Laboratory of Hypoxia Medical Academy,
Moscow 123367, Russia
This investigation examined cardiac protective effects of
normobaric intermittent hypoxia training. Six dogs underwent
intermittent hypoxic training for 20 consecutive days in a
normobaric chamber ventilated intermittently with N
2
to reduce
fraction of inspired oxygen (FIO
2
) to 9.5%–10%. Hypoxic periods,
initially 5 mins and increasing to 10 mins, were followed by 4-
min normoxic periods. This hypoxia-normoxia protocol was
repeated, initially 5 times and increasing to 8 times. The dogs
showed no discomfort during intermittent hypoxic training. After
20 days of hypoxic training, the resistance of ventricular
myocardium to infarction was assessed in an acute experiment.
The left anterior descending (LAD) coronary artery was
occluded for 60 mins and then reperfused for 5 hrs. At 30 mins
of LAD occlusion, radioactive microspheres were injected
through a left atrial catheter to assess coronary collateral blood
flow into the ischemic region. After 5 hrs reperfusion, the heart
was dyed to delineate the area at risk (AAR) of infarction and
stained with triphenyl tetrazolium chloride to identify infarcted
myocardium. During LAD occlusion and reperfusion, systemic
hemodynamics and global left ventricular function were stable.
Infarction was not detected in 4 hearts and was 1.6% of AAR in
the other 2 hearts. In contrast, 6 dogs sham-trained in a chamber
ventilated with compressed air and 5 untrained dogs subjected
to the same LAD occlusion/reperfusion protocol had infarcts of
36.8% 65.8% and 35.2% 69.5% of the AAR, respectively. The
reduction in infarct size of four of the six hypoxia-trained dogs
could not be explained by enhanced collateral blood flow to the
AAR. Hypoxia-trained dogs had no ventricular tachycardia or
ventricular fibrillation. Three sham-trained dogs had ventricular
tachycardia and two had ventricular fibrillation. Three untrained
dogs had ventricular fibrillation. In conclusion, intermittent
hypoxic training protects canine myocardium from infarction
and life-threatening arrhythmias during coronary artery occlu-
sion and reperfusion. The mechanism responsible for this
potent cardioprotection merits further study. Exp Biol Med
229:806–812, 2004
Key words: cardiac protection; intermittent hypoxia; myocardial
infarction; collateral blood flow
Alower incidence of myocardial infarction and
mortality from coronary heart disease had been
observed in populations living in areas of high
altitude (1, 2). In 1966, Poupa et al.demonstrated
cardioprotective effect of hypobaric hypoxia against iso-
proterenol-induced myocardial necrosis in rats (3), and in
1973, Meerson et al. reported that exposure to simulated
high altitude for 5 hrs/day, 5 days/week, reduced the
mortality rate of rats with coronary artery ligation by 84%
and the size of myocardial infarction by 35% (4). Later,
Meerson et al. reported that ischemia/reperfusion–induced
ventricular arrhythmias were reduced and ventricular
contractile function was better preserved in rats exposed
to intermittent hypobaric hypoxia (5). In rats of widely
varying ages, McGrath et al. demonstrated that cardiac
resistance to anoxia was increased after exposure to
intermittent hypobaric hypoxia (6). More recently, other
studies have confirmed that intermittent hypobaric hypoxia
is cardioprotective in rats (7–11). Xi et al. (12) and Cai et al.
(13) examined ischemia/reperfusion injury in isolated
perfused hearts of mice sacrificed 24 hrs after normobaric
intermittent hypoxia. Both studies found that several cycles
of hypoxia reduced myocardial infarction by about 50%. To
date, however, no research has demonstrated cardioprotec-
tive effects of intermittent systemic hypoxia in a large
animal.
There is increasing interest in intermittent hypoxia
training (IHT) to improve exercise performance, enhance
acclimatization to high altitude, and prevent and treat
various illnesses (14–18). This training involves multiple
cycles of brief (;5 mins), moderate hypoxia interspersed
with normoxia, often on a daily basis for several weeks.
This study was supported by the National Institutes of Health Grants HL-64785 and
HL-71684 and by the Hypoxia Medical Academy, Moscow, Russia.
1
To whom correspondence should be addressed at Department of Integrative
Physiology, University of North Texas Health Science Center, 3500 Camp Bowie
Boulevard, Fort Worth, TX 76107-2699. E-mail: pzong@hsc.unt.edu
Received December 22, 2003.
Accepted May 21, 2004.
806
1535-3702/04/2298-0806$15.00
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Because neither sojourns to high altitude nor hypobaric
chambers are required for normobaric IHT, it can readily be
implemented in the clinic. Considering the demonstrated
cardioprotective effects of hypoxia in rodents, it seemed
conceivable that a clinically relevant IHT protocol would be
cardioprotective in dogs. Thus, the current investigation was
designed to test this hypothesis. We found that IHT was
remarkably effective in protecting canine hearts from
infarction and arrhythmias due to coronary artery occlusion
and reperfusion.
Materials and Methods
This investigation was approved by the institutional
animal care and use committee and was conducted in
accordance with the Guide for the Care and Use of
Laboratory Animals (NIH Publication No. 85-23, revised
1996). Seventeen adult mongrel dogs of either sex, free of
clinically evident disease, were used for this study. Six dogs
completed a 20-day IHT protocol and then were subjected to
acute experimentation to assess cardiac responses to
coronary artery occlusion and reperfusion. To provide
control data, this acute experimentation was also performed
on 6 dogs that had completed a 20-day sham IHT protocol
and also on five untrained dogs.
Intermittent Hypoxia Training Protocol. Dogs
were exposed to intermittent, normobaric hypoxia according
to the protocol described in Table 1. Dogs were subjected to
one session per day for 20 consecutive days. For this training,
the dogs were placed in a Plexiglas chamber (interior
dimensions: 114 333 371 cm), and N
2
was introduced into
the chamber to reduce fraction of inspired oxygen (FIO
2
)to
the prescribed level (Table 1). Chamber O
2
was monitored
with an Alpha Omega Instruments, Series 2000 O
2
analyzer
(Cumberland, RI). The dogs showed no distress during
hypoxic training. For sham IHT, the 20-day IHT protocol
was followed, except instead of N
2
, compressed air was
introduced into the chamber to keep the FIO
2
at 20%.
Assessment of Protection Against Myocardial
Infarction. Surgical Procedures. On the day following
completion of the hypoxia or sham training protocols, the
dogs were subjected to an acute myocardial ischemia/
reperfusion experiment. Untrained dogs were also subjected
to this acute experiment.
The dogs were fasted overnight and then anesthetized
with sodium pentobarbital (30 mg/kg, iv). The dogs were
intubated and mechanically ventilated with room air
containing supplemental O
2
. Arterial blood samples were
collected at frequent intervals and analyzed for PO
2
, PCO
2
,
and pH, which were kept within normal physiological limits
by adjusting supplemental O
2
, tidal volume, and respiratory
rate. Supplemental pentobarbital was administered as
needed to maintain stable anesthesia through a vinyl
catheter positioned in a femoral vein. A saline-filled vinyl
catheter was inserted into the thoracic aorta via a femoral
artery to measure aortic pressure. In the other femoral artery,
two Tygon catheters were placed to collect reference blood
samples required for measuring coronary collateral flow
with the radioactive microsphere technique (19). The heart
was exposed through a left thoracotomy in the fifth
intercostal space and suspended in a pericardial cradle.
The left anterior descending (LAD) coronary artery was
isolated near its origin, and a silk snare was passed around
it. A Millar catheter-tip pressure transducer (Millar Instru-
ments, Houston, TX) was inserted through the left atrium
and advanced to the left ventricle to measure left ventricular
pressure and dP/dt. Another vinyl catheter was positioned in
the left atrium for injecting microspheres. Limb lead II of
the electrocardiogram was recorded along with pressures
and dP/dt on a Grass polygraph (Grass Medical Instruments,
Quincy, MA). Body temperature was monitored with a
hypodermic needle probe and maintained at 36.58–37.58C
with a circulating H
2
O heating pad.
When surgical preparations were complete and the
animal stable, the LAD was occluded for 1 hr by tightening
the snare and then allowed to reperfuse for 5 hrs after
releasing the snare. Lidocaine (1.0 mg/kg, iv) was
administered 1 min before LAD occlusion and 1 min before
LAD reperfusion.
Hemodynamic and cardiac function variables were
measured before and at the midpoint of the LAD occlusion
Table 1. Intermittent Hypoxia Training Protocol
a
Session FIO
2
(%) Hypoxia (mins) Normoxia (mins) Replications RHypoxia (mins)
1105 4 5 25
2105 4 6 30
3105 4 7 35
4105 4 8 40
5105 4 8 40
6 9.5 6 4 7 42
7 9.5 6 4 8 48
8 9.5 6 4 8 48
9 9.5 7 4 7 49
10 9.5 8 4 7 56
11–20 9.5 10 4 7 70
a
Replications = number of cycles of hypoxia/normoxia per daily session. RHypoxia = total minutes of hypoxia per session. FIO
2
, fraction of
inspired oxygen.
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coincident with microsphere injection, at 60 mins of LAD
occlusion, and at 1, 3, and 5 hrs of LAD reperfusion. At 5
hrs of reperfusion, heparin (500 U/kg, iv) was administered
to facilitate coronary artery perfusion to demarcate the LAD
perfusion territory at risk of infarction (see below).
Coronary Collateral Blood Flow Measurement. Be-
cause the extent of myocardial infarction is highly depend-
ent on the amount of collateral flow, which varies among
dogs, radioactive microspheres were injected at the
midpoint of the LAD occlusion period to measure coronary
collateral blood flow into the LAD region and in the
normally perfused left circumflex region (19). The micro-
spheres were agitated on a vortex mixer and in an ultrasonic
bath for at least 15 mins before use. Microspheres (5
million; 15-lm diameter) labeled with
46
Sc,
85
Sr, or
141
Ce
were injected into the left atrium followed by a gentle 10-ml
saline flush. Beginning just before and continuing for 3 mins
after microsphere injection, duplicate reference arterial
blood samples were withdrawn from the thoracic aorta at
a constant rate of 3 ml/min. Adequacy of microsphere
mixing in the blood perfusate was verified by comparing
radioactivities in the duplicate reference blood samples.
After slicing the ventricle and determining the area at risk
(AAR) of infarction and the infarct size (see below),
ventricular samples were cut from the central ischemic region
and from the left circumflex region. Lateral border zones were
excluded to avoid errors associated with measuring blood
flow in samples of heterogeneous composition. The tissue
samples were divided into endocardial, mid-myocardial, and
epicardial thirds (;1 g each). Radioactivities of tissue and
blood reference samples were measured in a Packard gamma
counter (Packard Instrument Company, Meriden, CT). Blood
flow in these tissue samples (mlmin
1
g
1
) was calculated as
previously described (19, 20).
Collateral flow in the AAR was evaluated in two ways.
An average collateral flow was computed by averaging the
endocardial and mid-myocardial flows of all samples of the
AAR of each heart. This average collateral flow in the
central region of the AAR has previously been used to
evaluate cardioprotective interventions (21–24). A mini-
mum collateral flow was also computed by averaging the
endocardial and mid-myocardial flows in the slice of the
AAR with the lowest collateral flow.
Determination of Myocardial Infarct Size (IS). The size
of the AAR was determined with a dual-perfusion technique
applied in situ (21, 25). The descending aorta and the
brachiocephalic artery were ligated, and a large-bore
cannula was advanced into the root of the aorta through
the left subclavian artery. The LAD was cannulated at the
site of occlusion. Small-bore catheters within the aortic and
LAD cannulas were connected to pressure transducers, so
aortic root and LAD pressures could be monitored during
the dual-perfusion procedure. The aortic and LAD cannulas
were connected to pressurized reservoirs containing 2.5%
Evans blue dye and normal saline, respectively. The left and
right ventricles were vented to atmospheric pressure by
cannulas inserted through the apex of the heart. When these
preparations were complete, the left and right coronary
arteries were perfused from the aorta with saline containing
Evans blue dye, whereas the LAD was perfused with saline
alone. These solutions were infused simultaneously for 1–2
min at constant pressures of 85 mm Hg. This procedure
delineated the ischemic area of the LAD perfusion territory
at risk of infarction, as blue dye was excluded from this
region. The heart was excised for measurements of infarct
size and regional myocardial blood flow.
After excision of the atria and right ventricle, the left
ventricle (LV) was frozen and stored overnight before being
cut into four to six transverse slices approximately 1-cm
thick. The weight of the ventricular slices was measured
(LV), and then these slices were incubated in triphenyl
tetrazolium chloride (1% w/v) in phosphate buffer (0.1 mol/
l, pH 7.4) at 378C for 20 mins, which imparts a deep red
color to non-infarcted tissue (26). Undyed, infarcted tissue
was resected and weighed, and then the remaining red tissue
was cut away from the adjacent blue tissue and weighed.
The weight of the red tissue plus the weight of the infarcted
tissue equaled the AAR. IS/AAR and AAR/LV were
computed.
Statistical Analyses. Values are expressed as mean
6SE. Hemodynamic data were analyzed with a two-way,
repeated measures analysis of variance (ANOVA) to detect
effects of (i) treatment (i.e, IHT, sham training, no training)
and (ii) time period during the acute experimental protocol
(i.e., baseline, 30 mins ischemia, 60 mins ischemia, 1 hr
reperfusion, 3 hrs reperfusion, and 5 hrs reperfusion). Infarct
size/area at risk of infarction, AAR/LV, regional coronary
blood flow, and arterial hemoglobin and O
2
content were
analyzed with completely randomized ANOVA to detect
differences between IHT, sham training, and no training.
When significance (P,0.05) was detected by ANOVA, a
Student-Newman-Keuls multiple comparison test was
performed. Statistical procedures were performed with
GB-Stat statistical software, version 9.0 (Dynamic Micro-
systems, Silver Spring, MD).
Results
Hemodynamic variables are presented in Table 2. Mean
arterial pressure, heart rate, and global left ventricular
function were stable during LAD occlusion and reperfusion.
There were no significant differences in any hemodynamic
variable between IHT group and sham-trained or untrained
groups. Heart rate was elevated during the baseline
condition due to the vagolytic action of sodium pentobarbi-
tal anesthesia and remained elevated throughout the experi-
ment. Rate-pressure product, an index of myocardial oxygen
consumption, was also similar among the groups. At the
acute experiment following 20 days of hypoxic training,
arterial hemoglobin and arterial O
2
content in IHT dogs
were similar to those observed in sham-trained or untrained
dogs (Table 3).
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Left anterior descending coronary artery occlusion
produced clearly visible cyanosis in the myocardium distal
to the occlusion. Ventricular premature contractions were
observed in some dogs upon LAD occlusion and in all dogs
upon LAD reperfusion. No cases of ventricular tachycardia
or ventricular fibrillation (VF) occurred in the IHT dogs. In
comparison, 5 out of 6 sham-trained dogs and 3 out of 5
untrained dogs developed ventricular tachycardia or VF
during the same LAD occlusion/reperfusion protocol (Table
4). All cases of VF were successfully defibrillated, and the
protocol was completed.
Figure 1 illustrates the infarct size and AAR determined
after 5 hrs of reperfusion. In IHT dogs, 32% 62% of the
left ventricle was ischemic, and sham-trained and untrained
dogs had ischemic zones of 27% 63% and 30% 63%,
respectively. No infarcted myocardium was detected in four
IHT dogs. Two IHT dogs had infarcts weighing 0.5 g each,
which was 1.6% of the AAR. In sham-trained and untrained
dogs subjected to the same acute protocol, 36.8% 65.8%
and 35.2% 69.5% of the AAR infarcted, respectively.
Coronary blood flow in the normally perfused, left
circumflex (LC) region and in the AAR were computed
from radioactivity resulting from tissue trapping of radio-
active microspheres injected into the left atrium at 30 mins
of LAD occlusion. Left circumflex flow did not differ
significantly among the groups (Fig. 2). The average and
minimum collateral flows in the AAR of all 6 hypoxia-
trained dogs were 0.36 60.16 mlmin
1
g
1
and 0.20 6
0.09 mlmin
1
g
1
, respectively. These mean values were
affected by unusually high average collateral flows (.0.70
mlmin
1
g
1
) of 2 IHT dogs, so mean collateral flows of the
other 4 dogs were computed and are presented in Figure 2.
For these four dogs, coronary collateral flow to the ischemic
LAD region was similar to that observed in the ischemic
region of six sham-trained and five untrained dogs.
The extent of myocardial infarction in this canine
model of ischemia/reperfusion varies inversely with collat-
eral flow, such that infarct size may be small even in the
absence of a cardioprotective intervention (21–25). How-
Table 3. Arterial Blood Hemoglobin and O
2
Content
Measured During the Myocardial Ischemia/Reperfu-
sion Protocol
a
IHT
Sham
trained Untrained
(n=6) (n=6) (n=5)
Arterial hemoglobin
(g/100 ml blood) 12.6 60.4 13.8 60.5 13.3 60.7
Arterial O
2
content
(ml O
2
/100 ml blood) 16.6 60.8 18.5 60.6 17.6 61.0
a
Values are mean 6SE. IHT, intermittent hypoxic trained.
Table 2. Hemodynamic Data Measured During the Myocardial Ischemia/Reperfusion Protocol
a
30 mins 60 mins 1 hr 3 hrs 5 hrs ANOVA
Baseline ischemia ischemia reperfusion reperfusion reperfusion treatment
Mean aortic pressure (mm Hg) P=0.0797
IHT (n= 6) 119 68 119 69 119 67 100 68 102 689869
Sham (n= 6) 133 66 135 65 133 64 125 64 120 66 117 66
Untrained (n= 5) 119 67 106 69 114 65 112 66 112 64 117 66
Heart rate (bpm) P= 0.3222
IHT (n= 6) 143 615 145 614 149 616 161 65 171 68 164 65
Sham (n= 6) 162 66 169 66 165 65 165 64 175 63 177 63
Untrained (n= 5) 161 69 164 614 169 611 170 610 174 68 180 68
Left ventricular pressure (mm Hg) P= 0.1975
IHT (n= 6) 138 68 138 69 141 67 127 69 125 610 129 68
Sham (n= 6) 144 64 147 62 143 63 136 64 137 64 133 65
Untrained (n= 5) 126 68 119 69 126 66 123 63 129 66 134 65
Left ventricular dP/dt
max
(mm Hg/sec) P= 0.3473
IHT (n= 6) 1707 6221 1847 6244 1828 6257 1765 6271 1796 6258 1790 6332
Sham (n= 6) 2253 6216 2435 6193 2275 6166 2128 6116 2234 6137 2000 6127
Untrained (n= 5) 2216 6235 1858 6368 2020 6248 1804 6149 1826 692 1926 688
Rate pressure product (mm Hg 3bpm 310
3
)P= 0.4854
IHT (n= 6) 21.5 62.5 21.8 62.4 22.9 62.1 20.6 61.9 21.2 62.3 21.1 61.9
Sham (n= 6) 23.3 61.4 24.8 61.1 23.6 61.2 22.4 60.9 23.9 61.0 23.6 61.2
Untrained (n= 5) 20.3 61.8 19.7 62.6 21.3 61.9 21.0 61.3 22.5 61.7 24.1 61.4
a
Values are mean 6SE. ANOVA, analysis of variance; IHT, intermittent hypoxic trained; sham, sham trained.
Table 4. Ventricular Arrhythmias Recorded During
the Myocardial Ischemia/Reperfusion Protocol
a
PVC VT VF
IHT (n=6) 6 0 0
Sham trained (n=6) 6 3 2
Untrained (n=5) 5 0 3
a
PVC, premature ventricular contractions; VT, ventricular tachycar-
dia; VF, ventricular fibrillation; IHT, intermittent hypoxic trained.
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ever, 4 IHT dogs had low average (0.12 60.004
mlmin
1
g
1
) and minimum (0.075 60.018 mlmin
1
g
1
)
collateral flows, so absence of significant infarction in these
dogs cannot be explained by enhanced collateral flow. Figure
3 shows myocardial infarct size as a function of average
(Panel A) and minimum (Panel B) collateral flows to the
inner two-thirds of the ischemic myocardial wall, after
excluding animals with average collateral flow .0.20
mlmin
1
g
1
(21–25). Using this criteria, Figure 3 compares
data from four IHT dogs with those from five sham-trained
dogs and from four untrained dogs. It is clear from Figure 3
that the degree of infarction in these four IHT dogs was much
less than what would have been expected if intermittent
hypoxia had conferred no cardioprotective effect.
Discussion
The major findings of this investigation are that IHT
prevented significant myocardial infarction and lethal
ventricular tachyarrhythmias during canine myocardial ischemia and reperfusion. This is the first report of
cardioprotective effects of IHT in a large animal model.
Many investigations of interventions to protect ische-
mic myocardium have been stimulated by the observation in
1986 by Murry et al. that a brief period of acute ischemia
reduced the extent of myocardial infarction resulting from
subsequent, more prolonged ischemia (24, 27). In fact, the
cardioprotective effect of hypobaric hypoxia had been
reported many years earlier (3, 4, 28). Potentially beneficial
effects of hypoxia for cardiac protection have received much
less attention compared to ischemic preconditioning. This
seems somewhat surprising because hypoxic exposure
occurs normally at high altitude and can readily be
accomplished in the laboratory or clinic. However, recently
a hypobaric IHT protocol was employed to treat 46 patients
with coronary heart disease and dyslipidemia; 37 patients
were followed for 10 months, and none developed
myocardial infarction (18).
To date, experimental investigations of cardioprotective
Figure 1. Left ventricular (LV) infarct size (IS) expressed as
percentage of the area at risk (AAR), and AAR expressed as
percentage of total LV mass.
Figure 2. Coronary blood flow in the normally perfused left
circumflex (LC) region and collateral blood flow in the left anterior
descending (LAD) region. Data from two IHT dogs with high average
collateral flow (.0.70 ml/min/g) are not included.
Figure 3. Myocardial infarct size is plotted as a function of average
(Panel A) and minimum (Panel B) coronary collateral flow to the inner
2/3 of the ischemic myocardial wall. Each graph shows data from
dogs with average collateral flow ,0.20 ml/min/g.
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effects of simulated high altitude or intermittent normobaric
hypoxia have been performed only in small animals (mice,
rats, guinea pigs). Neckar et al. subjected rats to intermittent
hypobaric exposures simulating 5000–7000 m altitude for 8
hrs/day, 5 days a week. After 24–32 exposures, the rats were
anesthetized and subjected to 20–30 mins LAD occlusion
followed by 4 hrs reperfusion. They found that adaptation of
rats to intermittent hypobaric hypoxia decreased IS/AAR by
15%–25% (9, 10). It should be noted that in the current study,
20 days of intermittent normobaric hypoxic training produced
more substantial protection against myocardial infarction in
dogs than the protection observed in rodents adapted to more
severe intermittent hypobaric hypoxia. Furthermore, the
current study also indicates that IHT is effective in protecting
canine myocardium from infarction when the duration of
coronary artery occlusion has been extended to 60 mins
compared with the 20–30 mins regional myocardial ischemia
produced by Neckar et al. in rats (9, 10). However, it must be
acknowledged that dogs have greater native coronary
collateral flow than rats, and this factor could have contributed
to the smaller infarcts observed in the current study.
Xi et al. found that 4 hrs acute normobaric systemic
hypoxia (FIO
2
= 10%) protected isolated mice hearts from
infarction when the hearts were subjected to ischemia/
reperfusion 24 hrs after treatment (12). Similar findings
were reported by Cai et al., who found this cardioprotection
present at 24 hrs but not at 30 mins after hypoxia (13). We
did not test the resistance of myocardium to ischemia
immediately after IHT, but our results are consistent with
the myocardial protection observed by others 1 day after
IHT (9, 10, 12, 13). The results of Cai et al. (13) suggest
that the protective mechanism activated by IHT may differ
from that activated by ischemic preconditioning, because
ischemic preconditioning can induce both early and delayed
phases of resistance to ischemic injury (24, 27, 29, 30). The
minimum duration of IHT required to produce significant
protection against myocardial infarction and the duration of
this protection in the canine model of ischemia/reperfusion
remains to be determined.
It has been noted that adaptation to hypobaric hypoxia
protects the rat heart against ischemic ventricular tachyar-
rhythmias (5, 7, 8, 10). Meerson et al. reported that the
duration of extrasystole and VF induced by acute coronary
ligation in conscious rats adapted to hypobaric hypoxia was
decreased 2- to 3-fold compared to that of control rats (5). In
open-chest rats exposed to intermittent hypobaric hypoxia,
Neckar et al. observed no VF, compared with the 9.1%
incidence of VF in normoxic control rats (10). In the current
study, VF did not occur in any of the 6 IHT dogs subjected
to 60 mins LAD occlusion and 5 hrs reperfusion. In
contrast, two of six sham-trained dogs and three of five
untrained dogs developed VF during the same acute
experimental protocol. The apparent antiarrhythmic effect
of IHT cannot be attributed to the cardioprotective effect of
lidocaine (31), because the same dose of lidocaine was used
in all animals during the acute experiment.
Acute hypoxia-induced myocardial protection of the
canine heart has been reported by Shizukuda et al., who
perfused the LAD of anesthetized dogs with severely
hypoxic blood (,1mlO
2
/100 ml blood) for 5 mins in a
protocol to mimic ischemic preconditioning. After 10 mins
of normoxic perfusion, the LAD was then occluded for 1 hr
and reperfused for 5 hrs, as in the current study. Infarct size
in these hypoxic preconditioned hearts was 7.2% of the
AAR compared to 22.4% in untreated control hearts (21). In
the current study, 20 days of IHT was more cardioprotective
than acute hypoxic preconditioning. Furthermore, Shizuku-
da et al. found that acute hypoxic preconditioning provided
no protection against VF (21). Therefore, the protective
mechanism activated acutely by hypoxic preconditioning
may differ from that activated by more prolonged IHT.
As with ischemic preconditioning, there is currently no
definitive mechanism to explain intermittent hypoxia-
induced cardioprotection. Kolar reviewed putative mecha-
nisms of hypoxic adaptation of myocardium (32). These
mechanisms include altered (i) myocardial vascularity and
coronary blood flow, including collateral flow, (ii) blood
hematocrit and hemoglobin content, (iii) myocardial
myoglobin concentration, (iv) energy metabolism, (v)
neurohumoral factors, (vi) antioxidant enzymes, (vii) stress
proteins, (viii) prostaglandins, and (ix) adenosine release.
Recently, Asemu et al. (7), Neckar et al. (9), and Zhu et al.
(11) reported evidence that ATP-dependent potassium
channels are involved in hypoxia-mediated cardioprotec-
tion. Xi et al. demonstrated that the infarct-limiting effect of
acute systemic hypoxia is triggered and mediated by
inducible nitric oxide synthase but not by endothelial nitric
oxide synthase or cyclooxygenase-2 (12). Cai et al. found
that erythropoietin protected rodent hearts in a manner
similar to intermittent hypoxia, and that this protection was
critically dependent on activation of hypoxia-inducible
factor 1 (13). Thus, redundant mechanisms may be involved
in the cardioprotection conferred by IHT, and more research
is required to further clarify the contributions of these and
possibly other mechanisms.
Data from this study do permit comment on two
potential protective mechanisms. First, the hemoglobin and
arterial O
2
contents of IHT dogs were not different from
those of sham-trained and untrained dogs, so the amount of
O
2
transported in blood flowing through collateral vessels
was not enhanced by IHT. These results do not exclude a
role for erythropoietin but suggest that its effect would have
been independent of its stimulation of red blood cell
production. Second, augmented coronary collateral flow is
not required for IHT-induced cardioprotection, as essentially
no infarction occurred in four IHT dogs that had very low
collateral flow (Fig. 1). However, we did not measure
collateral flow prior to IHT, so we cannot exclude an effect
of IHT on collateral vessel development.
In summary, 20 consecutive days of IHT provided
remarkable protection against myocardial infarction and
ventricular tachyarrhythmias in a canine model of 60 mins
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coronary artery occlusion and 5 hrs reperfusion. This
cardioprotection did not result from increased arterial O
2
carrying capacity or increased coronary collateral blood
flow.
The expert technical assistance of Arthur G. Williams, Jr., is
gratefully acknowledged.
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