ww.sciencedirect.com

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 8 ( 2 0 1 4 ) 3 8 1e3 8 6

Available online at w

ScienceDirect

journal homepage: www.JournalofSurgicalResearch.com

Improved cardioprotection using a novel stepwiseischemic preconditioning protocol in rabbit heart

Kentaro Yamakawa, MD,a,1 Wei Zhou, PhD, FAHA,a,1 Yoshihiro Ko, MD,a

Peyman Benharash, MD,b Mariko Takemoto, MD,a

and Aman Mahajan, MD, PhDa,*aDepartment of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CaliforniabDepartment of Cardiovascular Surgery, David Geffen School of Medicine, University of California, Los Angeles,

California

a r t i c l e i n f o

Article history:

Received 10 October 2013

Received in revised form

6 January 2014

Accepted 10 January 2014

Available online 16 January 2014

Keywords:

LV work efficiency

Myocardial ischemia

Preconditioning

Arrhythmia

* Corresponding author. 3302 Ronald ReaganE-mail address: amahajan@mednet.ucla.

1 Dr Yamakawa and Dr Zhou equally cont0022-4804/$ e see front matter ª 2014 Elsevhttp://dx.doi.org/10.1016/j.jss.2014.01.016

a b s t r a c t

Background: The current commonly used cardiac ischemic preconditioning (IPC) protocol,

involving three 5-min cycles of ischemiaereperfusion (I/R), may not be clinically beneficial

because of its acutely deleterious effects on hemodynamics. This study attempted to

assess the effects of a novel stepwise IPC scheme on cardiac function, infarct size, and

arrhythmogenesis in a rabbit model of prolonged I/R.

Methods: Anesthetized open-chest rabbits were subjected to 60-min occlusion of a proximal

branch of the left coronary artery followed by 180-min reperfusion. Animals were divided

into five groups (n ¼ 6 each): (1) control group (no IPC); (2) 2-min IPC group (three cycles of 2-

min IPC); (3) 5-min IPC group (three cycles of 5-min IPC); (4) 10-min IPC group (three cycles

of 10-min IPC); and (5) stepwise IPC group (2-, 5-, and 10-min I/R).

Results: Compared with control group, 2-, 5-, and 10-min IPC decreased arrhythmia score by

16%, 67%, and 33%, respectively. Remarkably, stepwise IPC resulted in a 78% reduction of

arrhythmias. Stepwise IPC also produced the least ventricular infarct size when compared

with 2-, 5-, and 10-min IPC groups (16.4% versus 39.3%, 28.1%, and 38.5%, P < 0.05).

Conclusions: These results suggest that stepwise IPC has better cardioprotective effects

against prolonged I/R injury and may serve as an acceptable approach to clinical revas-

cularization procedures on the heart, including catheter-based and surgical approaches.

ª 2014 Elsevier Inc. All rights reserved.

1. Introduction ventricular arrhythmias during ischemia [2,3]. In 1986, Murry

Yearly, more than a million patients undergo percutaneous

coronary interventions and more than 4,000,000 require sur-

gical revascularization procedures in the U.S. [1]. Ischemic

preconditioning (IPC) with brief episodes of nonlethal

ischemia and reperfusion (I/R), is known as one of the effec-

tive preventive strategies to reduce I/R injury, preventing pa-

tients from developing myocardial dysfunction and severe

Medical Center, UCLA, Cedu (A. Mahajan).ributed to this work.ier Inc. All rights reserved

et al. [4] first confirmed the beneficial effect of IPC in a canine

model with I/R injury. The cardioprotective effects of IPC have

been demonstrated experimentally by the reduction of

myocardial infarct size [5], prevention of the ventricular ar-

rhythmias [6], and improvement of work efficiency [7]. Clini-

cally, IPC has been shown to decrease ventricular arrhythmias

and the inotropic requirement after cardiopulmonary by-

pass [8e11]. In addition, IPC during percutaneous coronary

A 90095. Tel.: þ1 310 267 8680; fax: 310-267-3899.

.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 8 ( 2 0 1 4 ) 3 8 1e3 8 6382

interventions also demonstrated a decrease of in-hospital

adverse ischemic events and improved 1-y survival [12].

Despite the cardioprotective effects of IPC in animal and

clinical studies mentioned previously, an optimal and stan-

dard IPC protocol has not been established yet. The duration

and cycles of IPC likely affect its efficacy. For example, in a

swine model with prolonged I/R injury, Schulz et al. [13] have

compared the infarct-reducing effects of single 2-, 3-, and 10-

min period of IPC. They demonstrated that 10-min of IPC had

the most infarct-reducing effect, whereas 2-min IPC did not

affect the infarct size. However, others have shown that 10-

min of IPC may induce severe ventricular arrhythmias [14].

When comparing the cardioprotective effect between single

and a repeated IPC, previous studies have demonstrated that

multiple cycles of IPC produced a better cardioprotective ef-

fect [15e17]. Therefore, multiple cycles of IPC seem to exert

better cardioprotection in I/R injury.

We postulated that incrementally increasing the IPC

duration labeled “Stepwise IPC” may be optimal in preventing

against I/R injury. To evaluate this hypothesis, we assessed

hemodynamics along with infarct size, arrhythmia score, and

left ventricular (LV) work efficiency in a rabbit model of I/R

injury with different IPC protocols.

2. Methods

2.1. Animal model of myocardial ischemia andreperfusion

The animal study was approved by the Chancellor’s Animal

Research Committee at the University of California, Los

Angeles, and animals were treated in compliance with the

National Institutes of Health guidelines for the care and use

of laboratory animals. New Zealand white rabbits weighing

2.5e3.5 kg were anesthetized intramuscularly with keta-

mine (15 mg/kg) and xylazine (2 mg/kg). Intubation of the

trachea was performed for artificial ventilation. Subsequent

anesthesia was maintained with a-chloralose (50e60 mg/kg,

intravenously). Supplemental a-chloralose (5e10 mg/kg, i.v.)

Fig. 1 e The experimental protocols of IPC. The gray box is

was administrated to maintain an adequate depth of anes-

thesia as judged by stability of blood pressure (BP) and

respiration and lack of a withdrawal response to toe pinch.

The electrocardiogram (ECG) was monitored from limb

leads. One internal carotid artery and one jugular vein were

cannulated for BP measurement and drug infusion. The

animals underwent median sternotomy, and the pericar-

dium was incised carefully and sutured to the chest wall to

expose the heart and anterior coronary artery. A snare was

placed around the inferior vena cava for preload manipula-

tion. After a recovery of 30 min after the surgical operation, a

proximal branch of the left coronary artery (LAD) in the

rabbit was occluded with a 4-0 silk suture for 60 min fol-

lowed by 180-min reperfusion period. Evidence for a suc-

cessful intervention was a cyanotic-appearing left anterior

ventricular wall, elevated ST-segments, and peaked T-waves

on the ECG.

Rabbits were divided into five groups (n ¼ 6 each). All

rabbits except control groupwere subjected to one of four IPC

groups with three cycles of different time intervals of I/R as

follows: (1) control group (no IPC); (2) the 2-min IPC group

(2 min of LAD ligation followed by 2-min reperfusion); (3) the

5-min IPC group (5 min of LAD ligation followed by 5-min

reperfusion); (4) the 10-min IPC group (10 min of LAD liga-

tion followed by 10-min reperfusion); and (5) the Stepwise

IPC group (2, 5, and 10-min of incremental IPC; Fig. 1). In

addition, bolus administration of esmolol (0.1 mg/kg) fol-

lowed by continuous infusion of esmolol (0.05 mg/kg/min)

i.v. for 60 min was conducted during prolonged LAD ligation

after the 5-min IPC protocol in three animals. This protocol

was to determine if the classical 5-min IPC with beta

blockade has the same cardioprotective effect as seen in the

Stepwise IPC.

2.2. Pressure volume measurements by conductancecatheter

A Millar conductance catheter was used for continuous

measurement of LV pressure and volume. A 3 F 12-pole

multielectrode combination conductance-pressure catheter

ischemic period and white box is reperfusion period.

Table e Hemodynamic variables during the course of the experiment.

Variables Baseline 60-min ischemia 180-min reperfusion

Control 2-min 5-min 10-min Stepwise Control 2-min 5-min 10-min Stepwise Control 2-min 5-min 10-min Stepwise

HR (beats/

min)

204 � 12 203 � 15 200 � 15 209 � 15 201 � 15 181 � 7 180 � 6 185 � 15 168 � 13 176 � 10 174 � 6 166 � 9 177 � 18 170 � 3 140 � 14*

ESP (mm

Hg)

83.0 � 1.6 86.4 � 3.8 87.3 � 3.8 85.3 � 1.7 84.3 � 1.2 72.6 � 2.7* 77.2 � 1.5* 75.8 � 3.6 78.7 � 3.8 72.9 � 2.0 77.8 � 2.5* 74.5 � 2.3* 81.2 � 3.8 83.2 � 3.8 81.6 � 3.9

EDP (mm

Hg)

4.3 � 0.3 5.7 � 0.2 5.6 � 0.7 5.9 � 0.7 5.8 � 0.7 5.9 � 1.3 8.1 � 1.3* 5.4 � 1.2 10.6 � 3.7y 7.7 � 1.1 9.3 � 2.0* 8.8 � 1.4* 9.0 � 2.4 10.3 � 1.9 7.9 � 1.0

RPP (beats/

min$mm

Hg)

17.0 � 0.9 17.9 � 0.7 16.8 � 1.3 17.8 � 1.0 17.0 � 1.1 13.1 � 0.4* 14.0 � 0.8* 14.2 � 1.6 13.2 � 1.1* 13.0 � 1.1* 12.8 � 0.5* 11.2 � 0.5* 15.0 � 1.4y 13.7 � 0.3* 10.4 � 0.8*

SV (mL) 2.15 � 0.17 1.86 � 0.08 2.21 � 0.22 2.14 � 0.06 2.04 � 0.06 1.96 � 0.15 1.79 � 0.14 2.12 � 0.16 1.64 � 0.17* 2.23 � 0.09 1.95 � 0.18 1.93 � 0.06 2.28 � 0.19 2.19 � 0.03 2.60 � 0.07

SW (mm

Hg$mL)

178.3 � 9.9 168.2 � 9.6 196.5 � 9.5 182.7 � 9.1 166.1 � 6.7 134.6 � 4.3* 130.3 � 5.0* 152.4 � 4.6* 134.7 � 6.4* 160.4 � 9.9 146.3 � 7.0 127.0 � 7.8 183.0 � 6.0 171.7 � 2.4 189.0 � 4.7y

dP/dt max

(mm Hg)

2468 � 185 2309 � 271 2861 � 147 2548 � 254 2433 � 254 1901 � 165* 2499 � 236 2646 � 272 2173 � 375 2197 � 340 2225 � 173 2200 � 173 2551 � 234 2092 � 109 2532 � 216

dP/dt min

(mm Hg)

�3269 � 404 �3165 � 413 �2986 � 270 �2623 � 438 �2994 � 460 �1649 � 201* �2621 � 162* �2516 � 420 �2105 � 415 �2697 � 477 �2277 � 302* �2075 � 242* �2485 � 443 �2001 � 160 �2628 � 372

dP/dt max ¼ maximum rate of pressure change; dP/dt min ¼ minimum rate of pressure change; EDP ¼ end diastolic pressure; ESP ¼ end-systolic pressure; HR ¼ heart rate; SBP ¼ systolic blood

pressure; SV ¼ stroke volume; SW ¼ stroke work.

Values are mean � standard error of mean.*P < 0.05 versus baseline.y P < 0.05 versus control.

journalofsurgic

alresearch

188

(2014)381e386

383

Fig. 2 e Infarct size expressed as a percentage of the area-

at-risk in rabbit. *P < 0.05 versus the control group;xP < 0.05 versus 2-min group; zP < 0.05 versus 10-min

group; yP < 0.05 versus 5-min group.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 8 ( 2 0 1 4 ) 3 8 1e3 8 6384

(Millar Instruments, Inc, Houston, TX) was placed in the LV

via the LV apex wall and connected to a conductance pro-

cessor (MPVS Ultra; Millar Instruments, Inc, Houston, TX).

Proper electrode position was confirmed by the examination

of segmental volume signals. LV volume was calculated by

injecting a hypertonic saline (0.2 g NaCl/mL, 2 mL) into the

right atrium to obtain a constant offset volume (Vc). We

measured Vc before and at the end of each experiment and

confirmed that Vc remained stable. Hemodynamic indices

were obtained from steady-state pressureevolume loops

during sinus rhythm as previously reported [18]. Cardiac

performance was assessed by the measurement of heart rate,

stroke volume, end-systolic pressure, dP/dt max, dP/dt min,

and stroke work. Ventricular work efficiency was calculated

as the dimensionless ratio of stroke work-to-the rate-pres-

sure product (RPP) [19]. RPP is an index of myocardial oxygen

consumption, which was calculated as the product of systolic

BP and heart rate/1000 [20]. LV work efficiency was calculated

by stroke work divided by RPP.

2.3. Arrhythmia scoring system

Surface ECGs were recorded continuously in all animals with

the use of the GE Pruka Cardiolab Systems (GE Healthcare,

Waukesha, WI). Arrhythmias, such as premature atrial con-

tractions and ventricular arrhythmias including premature

ventricular contractions, ventricular tachycardia (VT), and

ventricular fibrillation, were observed for the first 20min after

reperfusion. Their severity was evaluated using a modified

arrhythmia scoring system [18], as follows: 0, no arrhythmia;

1 atrial arrhythmias; 2, <10 premature ventricular contrac-

tions; 3, VT (1e2 episodes); and 4, VT (3 episodes) or ventric-

ular fibrillation. This method of scoring has previously been

validated by other investigators [21] and was used because of

its simplicity.

2.4. Determination of ischemic risk zone and infarct size

Immediately after euthanasia, the coronary artery was reoc-

cluded, and the area-at-risk was determined by negative

staining. The Evans blue at a concentration of 0.25% was

infused into the heart via the jugular vein to determine the

perfused area. The atria, right ventricle, and connective tissue

were removed to isolate LV. The heart was frozen and cut into

5-mm slices. The slices were incubated in 1% 2,3,5-

triphenyltetrazolium chloride (TTC) buffer at pH 7.4 for

15 min at 37�C; then immersed in 10% formalin [22]. The

infarct (TTC negative) and area-at-risk (TTC stained) were

measured as percentages by comparing the weight-ratio of

these regions with the total LV mass [23]. Infarct severity was

determined by the ratio of infarct size-to-risk zone.

2.5. Statistical analysis

Data are presented as mean � standard error of mean. Ana-

lyses were performed using Sigma Stat (version 3.1, San Jose,

CA). The hemodynamic measurements and the LV work effi-

ciency were compared in the experimental stages by the use

of two-way repeated analysis of variance followed by a posthoc

Bonferroni correction. The infarct size and arrhythmia score

were analyzed by the c2 test. Statistical significance was

defined as P < 0.05.

3. Results

3.1. Heart rate, systolic, and diastolic function

The results shown in Table and demonstrate that all IPCs did

not significantly alter end-systolic pressure, dP/dt max, and

dP/dtmin. Stepwise IPC significantly reduced the heart rate by

70% after 180 min of reperfusion (P < 0.05). After 60-min

ischemia, the stroke work in all groups except for Stepwise

IPC group significantly decreased. After 180 min of reperfu-

sion, the strokework in Stepwise groupwasmaintained above

the baseline level and significantly higher than that in control

group (P < 0.05).

3.2. Infarct size and arrhythmia

The area-at-risk was similar and consistent amongst the

groups (41 � 0.4%). The Stepwise group demonstrated the

most reduction of infarct size compared with the control, 2-,

5-, and 10-min groups (16.4 � 2.7% versus 58.4 � 4.1%,

39.3 � 4.8%, 28.1 � 1.9%, and 38.5 � 2.4%, P < 0.05) (Fig. 2).

Furthermore, the Stepwise group demonstrated the lowest

arrhythmia score compared with all the other groups (Fig. 3).

In three animals, we observed the same infarct-reducing ef-

fect of 5-min IPC with beta blockade as seen in the Stepwise

IPC (14.9 � 2.3% versus 16.4 � 2.7%).

3.3. LV work efficiency

After 1 h of ischemia, the Stepwise, 5- and 10-min groups

maintained LV work efficiency similar to that at baseline

(P ¼ 0.59). After 180 min of reperfusion, the Stepwise group

showed a significant increase in LV work efficiency compared

with all other groups (P < 0.01; Fig. 4).

Fig. 3 e Effect of different IPC protocols on arrhythmia

score. *P < 0.05 versus the control group; xP < 0.05 versus

2-min group; zP < 0.05 versus 10-min group; yP < 0.05

versus 5-min group.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 8 ( 2 0 1 4 ) 3 8 1e3 8 6 385

4. Discussion

This study, to our knowledge, is the first to examine the car-

dioprotective effects of IPC using incrementally prolonged

time-steps on I/R injury. After coronary artery ligation and

reperfusion, this novel Stepwise approach yielded the lowest

infarct size, and arrhythmia incidence while significantly

improving LV work efficiency, compared with other IPC pro-

tocols described previously.

In the past three decades, the efficacy of IPC has been

explored in both experimental and clinical settings [24].

Despite the existence of many IPC protocols with various

times and frequencies, the optimal IPC parameters have not

been established [6,13e17]. The present study attempted to

bridge this knowledge gap and provide further evidence for

the physiological benefits of IPC.

Fig. 4 e LV work efficiency among different IPC groups. *P < 0.

versus baseline and ischemia.

Previous studies have demonstrated that repeated cycles

confer cardioprotection superior to a single period of IPC

[16,17]. Therefore, in this study we compared the effects of

stepwise IPC with the other multicycle IPC protocols on I/R

injury. Both Stepwise showed the most infarct-reducing and

antiarrhythmic effects compared with 2-, 5-, and 10-min IPCs.

The total occlusion time of IPC for each groupwas 6 (2-min IPC),

15 (5-min IPC), 17 (Stepwise IPC), and 30 min (10-min IPC). Our

data suggest that 15e20 min total occlusion duration during

IPC provides optimal cardioprotection. IPC of the myocardium

has been hypothesized towork throughmechanisms including

transient sympathetic activation, induction of protein kinase A

and C pathways, and likely involves bradykinin and adenosine.

Despite the existence ofmany IPC protocols with various times

and frequencies, the optimal IPC parameters have not been

established. In addition, the myocardium is better able to

tolerate longer ischemic episodes required for effective IPC if it

is first exposed to brief periods of ischemia.

Assessment of LV work efficiency is of importance in

myocardial ischemia because of the imbalance between oxy-

gen supply and demand. A ventricle with high work efficiency

is able to generate a higher cardiac output per amount of oxy-

gen consumed.We examined the LVwork efficiency among the

different IPC protocols by measuring LV pressure and volume.

The stepwise IPC demonstrated the highest value of LV work

efficiency after prolonged myocardial ischemia and at the

completion of reperfusion period. Stepwise IPC achieved this

through preservation of stroke work and reduction of the RPP.

The observed decrease in heart rate during Stepwise IPC may

be due to the inhibition of sympathetic nerve activity or

enhancement of vagal tone, both of which has been shown to

exert cardioprotection against myocardial ischemia reperfus-

ion injury [18]. In three animals, we added beta blockade to 5-

min IPC and observed the same infarct sizeereducing effect as

seen in the Stepwise IPC suggesting that the neural regulation

by Stepwise IPC plays a role in the cardioprotection.

Our study has several limitations. We did not examine

neurohumoral factors involved in cardioprotection. Some of

05 versus the control, 2-, 5-, and 10-min group. xP < 0.05

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 8 ( 2 0 1 4 ) 3 8 1e3 8 6386

these substances such as adenosine [23], bradykinin [25,26],

norepinephrine [27], and endogenous opioids [28] likely play

an important role in cardioprotection against I/R injury.

Further studies to evaluate the interaction between the Step-

wise IPC and humoral factors are warranted. As remote IPC is

increasingly being used in clinical studies, a comparison be-

tween remote and stepwise IPC in large animals deserve

further investigation.

5. Conclusions

In summary, our results suggest that Stepwise IPC with pro-

gressively longer period of transient ischemia confers better

cardioprotection against prolonged I/R injury. In particular,

reduction of arrhythmias and the relatively short time needed

for this IPC scheme, may allow our method to be widely used

during coronary interventions and beating heart coronary

surgical revascularization.

Acknowledgment

This study was supported by intramural funding from Uni-

versity of California, Los Angeles.

The authors declare that they have no conflict of

interest.

Author contributions: K.Y., W.Z., Y.K., and A.M. were

responsible for conception and design; K.Y., W.Z., Y.K., P.B.,

M.T., and A.M. participated in data analysis and interpreta-

tion; K.Y.,W.Z., Y.K., P.B., andM.T. collected the data; K.Y. and

W.Z. helped in writing the article; W.Z., P.B., and A.M. revised

the article. A.M. obtained funding.

r e f e r e n c e s

[1] Roger VL, Go AS, Lloyd-Jones DM, et al. Heart disease andstroke statisticse2012 update: a report from the AmericanHeart Association. Circulation 2012;125:e2.

[2] Haji Mohd Yasin NA, Herbison P, Saxena P, Praporski S,Konstantinov IE. The role of remote ischemicpreconditioning in organ protection after cardiac surgery: ameta-analysis. J Surg Res 2014;186:207.

[3] Mozaffari MS, Liu JY, Abebe W, Baban B. Mechanisms of loaddependency of myocardial ischemia reperfusion injury. Am JCardiovasc Dis 2013;3:180.

[4] Murry CE, Jennings RB, Reimer KA. Preconditioning withischemia: a delay of lethal cell injury in ischemicmyocardium. Circulation 1986;74:1124.

[5] Schott RJ, Rohmann S, Braun ER, Schaper W. Ischemicpreconditioning reduces infarct size in swine myocardium.Circ Res 1990;66:1133.

[6] Shiki K, Hearse DJ. Preconditioning of ischemic myocardium:reperfusion-inducedarrhythmias. Am J Physiol 1987;253:H1470.

[7] Sunderdiek U, Schmitz-Spanke S, Korbmacher B, Gams E,Schipke JD. Left ventricular dysfunction and disturbed O(2)-utilization in stunned myocardium: influence of ischemicpreconditioning. Eur J Cardiothorac Surg 2001;20:770.

[8] Walsh SR, Tang TY, Sadat U, Gaunt ME. Remote ischemicpreconditioning in major vascular surgery. J Vasc Surg 2009;49:240.

[9] Wu ZK, Iivainen T, Pehkonen E, Laurikka J, Tarkka MR.Ischemic preconditioning suppresses ventriculartachyarrhythmias after myocardial revascularization.Circulation 2002;106:3091.

[10] Wu ZK, Iivainen T, Pehkonen E, Laurikka J, Tarkka MR.Arrhythmias in off-pump coronary artery bypass graftingand the antiarrhythmic effect of regional ischemicpreconditioning. J Cardiothorac Vasc Anesth 2003;17:459.

[11] Wu ZK, Iivainen T, Pehkonen E, Laurikka J, Tarkka MR.Antiarrhythmic effect of ischemic preconditioning in recentunstable angina patients undergoing coronary artery bypassgrafting. World J Surg 2004;28:74.

[12] Laskey WK, Beach D. Frequency and clinical significance ofischemic preconditioning during percutaneous coronaryintervention. J Am Coll Cardiol 2003;42:998.

[13] Schulz R, Post H, Vahlhaus C, Heusch G. Ischemicpreconditioning in pigs: a graded phenomenon: its relationto adenosine and bradykinin. Circulation 1998;98:1022.

[14] Ovize M, Aupetit JF, Rioufol G, et al. Preconditioning reducesinfarct size but accelerates time to ventricular fibrillation inischemic pig heart. Am J Physiol 1995;269:H72.

[15] Eckle T, Grenz A, Kohler D, et al. Systematic evaluation of anovel model for cardiac ischemic preconditioning in mice.Am J Physiol Heart Circ Physiol 2006;291:H2533.

[16] Miura T, Goto M, Urabe K, Endoh A, Shimamoto K, Iimura O.Does myocardial stunning contribute to infarct size limitationby ischemic preconditioning? Circulation 1991;84:2504.

[17] Vegh A, Komori S, Szekeres L, Parratt JR. Antiarrhythmiceffects of preconditioning in anaesthetised dogs and rats.Cardiovasc Res 1992;26:487.

[18] Zhou W, Ko Y, Benharash P, et al. Cardioprotection ofelectroacupuncture against myocardial ischemia-reperfusion injury by modulation of cardiac norepinephrinerelease. Am J Physiol Heart Circ Physiol 2012;302:H1818.

[19] Baxley WA, Dodge HT, Rackley CE, Sandler H, Pugh D. Leftventricular mechanical efficiency in man with heart disease.Circulation 1977;55:564.

[20] Kitamura K, Jorgensen CR, Gobel FL, Taylor HL, Wang Y.Hemodynamic correlates of myocardial oxygen consumptionduring upright exercise. J Appl Physiol 1972;32:516.

[21] Curtis MJ, Walker MJ. Quantification of arrhythmias usingscoring systems: an examination of seven scores in anin vivo model of regional myocardial ischaemia. CardiovascRes 1988;22:656.

[22] Fryer RM, Wang Y, Hsu AK, Gross GJ. Essential activation ofPKC-delta in opioid-initiated cardioprotection. Am J PhysiolHeart Circ Physiol 2001;280:H1346.

[23] Ford WR, Clanachan AS, Hiley CR, Jugdutt BI. Angiotensin IIreduces infarct size and has no effect on post-ischaemiccontractile dysfunction in isolated rat hearts. Br J Pharmacol2001;134:38.

[24] Hausenloy DJ, Baxter G, Bell R, et al. Translating novelstrategies for cardioprotection: the Hatter WorkshopRecommendations. Basic Res Cardiol 2010;105:677.

[25] Iliodromitis EK, Kremastinos DT, Katritsis DG,Papadopoulos CC, Hearse DJ. Multiple cycles ofpreconditioning cause loss of protection in open-chestrabbits. J Mol Cell Cardiol 1997;29:915.

[26] Li GC, Vasquez JA, Gallagher KP, Lucchesi BR. Myocardialprotection with preconditioning. Circulation 1990;82:609.

[27] Takasaki Y, Adachi N, Dote K, Tsubota S, Yorozuya T, Arai T.Ischemic preconditioning suppresses the noradrenalineturnover in the rat heart. Cardiovasc Res 1998;39:373.

[28] Morris SD, Yellon DM. Angiotensin-converting enzymeinhibitors potentiate preconditioning through bradykinin B2receptor activation in human heart. J Am Coll Cardiol 1997;29:1599.