Keywords
ischemic preconditioning - hypoxic preconditioning - reperfusion injury
Introduction
Ischemic/reperfusion (I/R) injury is closely related to organ transplantation, stroke,
cardiovascular surgery, and disease. Myocardial I/R injury is the major reason of
death associated with heart desease.[1] Ischemic preconditioning (IPC), consisting of single or multiple episodes of sublethal
ischemia and reperfusion, markedly reduces myocardial infarct size and is the most
effective endogenous protective approach against I/R injury. However, clinical application
of IPC is limited for practical reasons and high risks.[2] New ways against I/R injury are urgently required.
Hypoxic preconditioning (HPC) is a phenomenon that sublethal hypoxia increases cellular
resistance against following lethal I/R injury. The clinical application of HPC may
be much easier and safer than that of IPC, for hypoxia was not accompanied by blood
flow cessation. HPC has been verified in various tissues. However, most studies have
been focused on prolonged HPC, which requires hours to weeks, or delayed HPC, in which
the protective effect occurs approximately 24 hours after the preconditioning stimulus.[3]
[4]
[5]
[6] Early cardioprotection, which occurs immediately after the preconditioning induced
by short duration HPC, would be of great value clinically. Only several studies demonstrated
that one-cycle short duration HPC (5-minute hypoxia followed by 10-minute reperfusion)
exerted early cardioprotection.[7]
[8]
[9] Cai et al reported that 30 minutes after preconditioning of mice with 5 cycles of
6-minute 6%O2 and 6-minute 21%O2, the hearts were not protected against I/R injury.[10] It has not been known that whether multiple-cycle short duration HPC could produce
early protective effects in isolated rat hearts. Therefore, we investigated the early
effects of various cycles and the number and hypoxic duration of each cycle on isolated
rat hearts subjected to I/R injury and compare the effects with cardioprotection by
IPC.
Materials and Methods
Animal and Rat Langendorff Model
Hearts from adult male Sprague-Dawley rats (220–300 g) anesthetized with pentobarbital
sodium (50 mg/kg intraperitoneally) were mounted in a Langendorff model (Radnoti,
Monrovia, California, United States) and perfused with Krebs–Henseleit buffer (in
mM: 118.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.0 NaHCO3, 11.1 glucose, and 0.125 EDTA-2Na, pH: 7.35 to 7.45) at 37°C and a constant pressure
of 100 cm H2O. Oxygenated KH buffer was continuously bubbled with 95%O2+5%CO2. Hypoxic Krebs Henseleit (KH) buffer was bubbled with 95%N2+5%CO2.
Experimental Protocol
After being stabilized with oxygenated KH buffer for 20 minutes, 49 isolated hearts
were randomly divided into 7 groups (n = 7 per group) and received 3 cycles of 3-minute global ischemia or hypoxic KH buffer
followed with 3-minute oxygenated KH buffer reperfusion (IPC33 or HPC33 group), 5
cycles of 3-minute global ischemia or hypoxic KH buffer followed with 3-minute oxygenated
KH buffer reperfusion (IPC53 group or HPC53 group), 3 cycles of 5-minute global ischemia
or hypoxic KH buffer followed with 5-minute oxygenated KH buffer reperfusion (IPC35
group or HPC35 group), or 30-minute perfusion with oxygenated KH buffer (I/R group),
respectively. Then all the hearts were subjected to 50-minute ischemia and 120-minute
oxygenated KH buffer reperfusion. Cardiac left ventricle (LV) function, infarct size,
and coronary flow rate (CFR) were evaluated. The experiment protocol was shown in
[Fig. 1].
Fig. 1 The experimental protocol. After 20-minute equilibration, rat hearts received 3 cycles
of 3-minute global ischemia or hypoxic KH buffer followed with 3-minute oxygenated
KH buffer reperfusion (IPC33 group and HPC33 group), 5 cycles of 3-minute global ischemia
or hypoxic KH buffer followed with 3-minute oxygenated KH buffer reperfusion (IPC53
group and HPC53 group), 3 cycles of 5-minute global ischemia or hypoxic KH buffer
followed with 5-minute oxygenated KH buffer reperfusion (IPC35 group and HPC35 group),
or 30-minute perfusion with oxygenated KH buffer (I/R group), respectively. Then all
the hearts were subjected to 50-minute ischemia and 120-minute oxygenated KH buffer
reperfusion.Abbreviations: HPC, hypoxic preconditioning (HPC); IPC, ischemic preconditioning;
IR, ischemic/reperfusion; KH, Krebs Henseleit.
Cardiac LV Function and CFR Measurement
A water-filled latex balloon connected to a pressure transducer (Model SP844; MEMSCAP,
Durham, North Carolina, United States) was inserted into the left ventricular cavity
via the left auricle to measure cardiac LV function. Left ventricular developed pressure
(LVDP, in mm Hg), its maximal and minimal rates of rise ([dp/dt]max and [dp/dt]min, in mm Hg/s), and heart rate (HR) were measured at the end of stabilization (baseline),
and at various times, during reperfusion. Coronary flow rate (mL/minute) was also
estimated at these time points. Recoveries of LVDP, (dp/dt)max, (dp/dt)min, HR, and CFR were, respectively, calculated as percentages of baseline values.
Infarct Size Assessment
At the end of 120 minutes of reperfusion, hearts were sliced into 2-mm transverse
sections from apex to base. The slices were incubated at 37°C with 1% 2,3,5-triphenyltetrazolium
chloride (TTC, Sigma–Aldrich, St. Louis, Missouri, United States) in phosphate buffer
(pH = 7.4) for 15 minutes and then fixed in a 4% paraformaldehyde phosphate buffer
for 24 hours. Subsequently, the slices were scanned (MRS-800P48U, MICROTEK, Shanghai,
China). The infarct (white) area was assessed by planimetry using Image Pro Plus 5.0
software (Media Cybernetics, Silver Spring, Maryland, United States) and expressed
as a percentage of total slice area.
Statistical Analysis
All values are expressed as means ± SE, the recovery of LVDP, (dp/dt)max, (dp/dt)min, HR, and CFR were analyzed by repeated-measures analysis of variance and using
least significant difference procedure for post hoc test. Infarct size was tested
with one-way analysis of variance followed by least significant difference procedure
for post hoc test. All p values less than 0.05 were considered statistically significant (version 16.0, SPSS,
Chicago, Illinois, United States).
Results
Recovery of Cardiac LV Function and CFR
During reperfusion, recovery of LVDP, (dp/dt)max, (dp/dt)min, and CFR in IPC35, HPC35,
and HPC53 groups were significantly higher than I/R group (p < 0.01). There was no significant difference in recovery of LVDP, (dp/dt)max, (dp/dt)min,
or CFR between IPC35 and HPC35 groups. Recovery of LVDP, (dp/dt)max, and CFR did not
differ significantly between HPC53 and HPC35 groups. Only recovery of (dp/dt) min
in HPC53 group was lower than that in HPC35 group (p < 0.05) ([Fig. 2]). There was no significant difference in HR among all the groups (data not shown;
[Fig. 2E]).
Fig. 2 Effects of IPC and HPC on left ventricle function of hearts subjected to ischemia/reperfusion
injury. (A–E) The recovery of LVDP, (dp/dt)max and (dp/dt)min, heart rate, and coronary flow rate during baseline and reperfusion. The zero
time point demonstrated the beginning of reperfusion. Data are expressed as mean ± SE,
n = 7. *P < 0.05, **P < 0.01 compared with I/R group.Abbreviations: HPC, hypoxic preconditioning; IPC,
ischemic preconditioning; LVDP, left ventricular developed pressure; (dp/dt) max and (dp/dt)min, LVDP's maximal and minimal rates of rise.
Infarct Size
Infarct size was significantly reduced in IPC35, HPC35, and HPC53 groups (14.15 ± 2.45%,
15.00 ± 3.17%, and 19.47 ± 3.54%, respectively) in comparison with I/R group (49.11 ± 5.99%,
p < 0.01). There was no significant difference in infarct size among IPC35, HPC35,
and HPC53 groups ([Fig. 3]).
Fig. 3 Effects of IPC and HPC on cardiac infarct size after prolonged ischemia and reperfusion.
(A) Histology picture. Sections of hearts were stained with triphenyltetrazolium chloride.
(B) Infarct sizes are expressed as a percentage of the total slice area. Data are
expressed as mean ± SE, n = 7. *P < 0.05, **P < 0.01 compared with I/R group.Abbreviations: HPC, hypoxic preconditioning; IPC,
ischemic preconditioning.
Discussion
In this study, our results demonstrated that multiple-cycle short duration HPC, which
was performed by 3 cycles of 5-minute hypoxia followed with 5-minute reperfusion or
5 cycles of 3-minute hypoxic followed with 3-minute reperfusion, exerted early cardioprotection
on isolated rat hearts subjected to I/R injury. The protection by multiple-cycle short
duration HPC was comparable with that of IPC. Our finding was evidenced by improved
recoveries of cardiac LV function and CFR and reduced myocardium infarct size.
Several studies have demonstrated that one-cycle IPC and HPC exerted similar cardioprotection.[7]
[8]
[9] However, it has been unknown whether multiple-cycle HPC and IPC elicited similar
cardioprotection. We demonstrated that early cardioprotection of multiple-cycle HPC
was as powerful as that of IPC. In a whole-body hypoxia model, 30-minute intermittent
hypoxia (repetitive cycles of 40-second hypoxia with 5 or 10% O2 followed by 20-second normoxia) did not induce delay cardioprotection in rat hearts.[11] However, we indicated that 30-minute intermittent HPC (3 cycles of 5-minute hypoxia
followed with 5-minute reperfusion or 5 cycles of 3-minute hypoxic followed with 3-minute
reperfusion) elicited early cardiac protection in isolated rat hears. Although Cai
et al reported that 1 hour whole-body HPC (5 cycles of 6 minute hypoxia and 6 minute
reoxygenation) could protect mice hearts against I/R injury 24 hours later but not
30 minutes later,[10] we demonstrated that short duration HPC induced immediate cardioprotection.
The present result demonstrated that HPC53 (5 cycles of 3 minute hypoxia followed
with 3 minute reperfusion) induced cardioprotection as robust as that of HPC35 (3
cycles of 5-minute hypoxia followed with 5-minute reperfusion). In our study, increasing
number of cycles and reducing hypoxic duration of each cycle, which made HPC safer,
were able to provide protective effects on I/R hearts. However, we found that IPC35
rather than IPC53 protected I/R hearts and there was little advantage of more than
3 cycles in IPC. Therefore, multiple-cycle HPC may have more attractive potential
for clinical application than IPC.
On one hand, the mechanisms of prolonged HPC and delayed cardioprotection by HPC include
upregulation and downregulation of various genes.[12] Recently, Pan et al reported that the expression of glucose-regulated protein 78
(GRP78) peaked at 24 hours after one-cycle HPC and the upregulation of GRP78 mediated
the delayed protection in cultured neonatal rat cardiomyocytes.[13] Another study demonstrated that the interaction of hypoxia inducible factor-1 with
myocardial inducible nitric oxide synthase gene played an important role in delayed
cardioprotection induced by intermittent hypoxia in rat.[14] On the other hand, there must be intrinsic mechanisms that could be rapidly triggered
involved in early cardioprotection by short duration HPC. A further understanding
of these mechanisms could contribute to minimizing early cardioprotection provided
by short duration HPC and developing new treatments against I/R injury in the future.
In conclusion, using an isolated rat heart model, our results demonstrated that multiple-cycle
short duration HPC exerted early cardioprotection, which was as powerful as that of
IPC. Further studies are needed to investigate the underlying mechanisms.
