Protective effects of diltiazem and ryanodine against ischemia-reperfusion injury in neonatal rabbit hearts

Protective effects of diltiazem and ryanodine against ischemia-reperfusion injury in neonatal rabbit hearts The effects of diltiazem, a sarcolemmal Ca2+ channel blocker, and ryanodine, an inhibitor of sarcoplasmic reticulum function, were investigated in isolated newborn rabbit hearts (2 to 5 days old) subjected to ischemia and reperfusion. After cardioplegic arrest with St. Thomas' Hospital solution, global ischemia was induced at 37° C (normothermia) for 45 minutes or at 20° C (hypothermia) for 180 minutes. The hearts were then reperfused at 37° C for 30 minutes. Diltiazem or ryanodine, at concentrations that have minimal to moderately negative inotropic effects under nonischemic conditions, was added to the cardioplegic solution. After normothermic ischemia, reperfusion of untreated hearts resulted in recovery of left ventricular developed pressure to 52.9 % ± 2.5 % of the preischemic level. In hearts treated with diltiazem, recovery of left ventricular developed pressure was significantly improved (84.2% ± 2.9% at 3 X 10-8 mol/L; p < 0.01). Comparable improvement was achieved with ryanodine (90.5 % ± 4.1 % at 10- 9 mol/L; p < 0.01). Creatine kinase leakage and structural derangement of mitochondria were also reduced by both agents. With hypothermic ischemia, left ventricular developed pressure recovered in untreated hearts to 72.7% ± 3.3% of preischemic values. Treatment with diltiazem improved the recovery of left ventricular developed pressure to 96.9 % ± 3.5 % at 3 X 10- 8 mol/L and reduced creatine kinase leakage and mitochondrial damage. Ryanodine also improved the recovery of left ventricular developed pressure and attenuated ultrastructural damage. These findings suggest that Ca2+ handling by the sarcoplasmic reticulum, like transsarcolemmal Ca2+ influx, plays an important role in the pathogenesis of myocardial ischemia-reperfusion injury in the neonatal heart despite the morphologic and functional immaturity of the sarcoplasmic reticulum in the neonate. (J THORAC CARDIOVASC SURG 1993;106:55-66)

Toshiaki Akita, MD,a Toshio Abe, MD,a Satoru Kato, MD,a Itsuo Kodama, MD,b and Junji Toyama, MD,b Nagoya, Japan

HypothermiC, hyperkalemic cardioplegia is known to provide excellent myocardial protection during cardiac operations in adult patients. In pediatric patients, however, Bull, Cooper, and Stark' reported no beneficial effects From the Department of Thoracic Surgery, School of Medicine; and the Department of Circulation, Division of Regulation of Organ Function, Research Institute of Environmental Medicine," Nagoya University, Nagoya, Japan 466. This work was done in the Department of Circulation, Division of Regulation of Organ Function, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan. Received for publication Nov. 19, 1991. Accepted for publication July 27, 1992. Address for reprints: Toshiaki Akita, MD, The Department of Pediatrics, Emory University, School of Medicine, 2040 Ridgewood Drive NE, Atlanta, GA 30322. Copyright

\993 by Mosby-Year Book, Inc.

0022-5223/93 $1.00+ .\0

12/1/41338

of cardioplegia when compared with intermittent aortic perfusion. Since then, particular concerns for protection ofthe immature heart against ischemia-reperfusion injury have emerged and a number of experimental studies have been done to define age-related differences of cardioplegia."? With regard to the optimal cardioprotective protocols for immature hearts, the findings of these studies are controversial and much remains to be elucidated. Age-related differences exist in the interaction between the sarcolemma and the sarcoplasmic reticulum (SR) that affects the regulation of intracellular and myofibrillar Ca 2+.8- 16 It was reported that contraction of neonatal mammalian cardiac muscle depends primarily on transsarcolemmal Ca2+ influx,8-l3 and it has been suggested that Ca2+ releasefrom the SR is oflesser importance than in adult hearts.f 11, 14-16 Differences in Ca2+ dynamics would suggest a different pathogenesis for ischemia55

56

The Journal of Thoracic and Cardiovascular Surgery July 1993

Akita et al.

reperfusion injury in immature and mature mammalian hearts, because intracellular Ca2+ overload plays a major role in both ischemia and reperfusion-related injury.l?"" Sarcolemmal Ca2+ channel blockers and SR inhibitors would be expected to have different protective effects against ischemia-reperfusion injury in neonatal hearts than in adult hearts. 2o-24 The present study was designed to gain further insight into these issues. We investigated the efficacy of diltiazem, a sarcolemmal Ca2+ channel blocker, and ryanodine, an SR inhibitor, against the functional and morphologic derangement of neonatal rabbit hearts subjected to global ischemia under cardioplegic arrest and subsequent reperfusion. On the basis of the results obtained, we discussed possible protective procedures for the neonatal heart in terms of its unique calcium dynamics. Methods Preparation. Hearts were obtained from neonatal white Japanese rabbits, 2 to 5 days old. After intraperitoneal administration of heparin (200 U) and pentobarbital (50 mg/kg), hearts were rapidly excised and placed in cold perfusion solution. The aorta was cannulated for Langendorff perfusion at a constant pressure (80 em H20). The control perfusate was a modified Krebs-Henseleit bicarbonate buffer, which was composed of the following ingredients (rnmol/L): NaCI, 120; KCI, 4.7; NaHC0 3, 25; KH 2P0 4, 1.2; MgS0 4, 1.2; CaCI 2, 2.5; glucose, 11.1;and pyruvate, 2.0. The solution was equilibrated with 95% oxygen and 5% carbon dioxide to achieve a pH of7.4 at 37° C. A latex balloon (size 3 or 4, Hugo Sachs Electronik, March H ugstetten, Germany) was introduced into the left ventricle via the left atrium for measurement of isovolumic left ventricular developed pressure (LVDP). The balloon was filled with saline and adjusted to achieve a diastolic pressure of 5 mm Hg. Its volume was unchanged throughout the experiment. The first derivatives ( ± dp/dt) of LVDP were obtained by electronic differentiation. The pulmonary artery was incised to ensure adequate right ventricular venting. Heart temperature, which was monitored by a fine, soft-tipped thermistor in the right ventricle, was maintained at 37° ± 0.5° C. Protocols Effects of diltiazem and ryanodine on mechanical function. Hearts were constantly stimulated at a rate about 10% faster than their spontaneous rate through a pair of electrodes placed on the epicardial surface of the left atrium. Pulses were 3 msec in duration and twice the diastolic threshold in intensity. After stabilization periods of IS to 20 minutes, the perfusate was changed from the control to a solution including either diltiazem (10- 8 mol/L) or ryanodine (10- 10 mol/L), The concentration of each agent was increased incrementally to 10- 5 mol/L after each IS-minute equilibration period. Influence of diltiazem and ryanodine on ischemia-reperfusion injury. The effects of diltiazem and ryanodine on ischemiareperfusion injury were examined under normothermic (37° C) or hypothermic (20° C) conditions. Hearts were allowed to beat spontaneously (unstimulated) throughout the experiments. After a stabilization period of 15 minutes with control perfusate, preischemic baseline values for LVDP, ± dp/dt, heart rate, and

coronary flow were recorded. Hearts was then perfused for 2 minutes with St. Thomas' Hospital cardioplegic solution at either 37° Cor 20° C at a constant pressure (60 em H 20). The composition of the solution was as follows (rnrnol/L): NaC!, 110; KCI, 16; MgCI 2, 16; CaCI 2, 1.2; and NaHC0 3, 10.0; pH 7.8). Diltiazem or ryanodine was added to St. Thomas' Hospital solution at a specificconcentration. The cardioplegia infusion line was then clamped and global ischemia was produced for 45 minutes at normothermia (37° C) or for 180 minutes at hypothermia (20° C). Heart were bathed in the cardioplegic solution, which was surrounded by a water-filled jacket to keep the temperature at a constant level and to avoid oxygen diffusion from the surrounding air. 25 After ischemia, hearts were reperfused with the control perfusate at 37° C for 30 minutes. Hemodynamic measurements were repeated to document functional recovery. During the initial 15 minutes of reperfusion, coronary effluent was collected to allow for assessment of creatine kinase (CK) leakage from the heart. At the conclusion of each experiment, a 4 ° C 2.5%glutaraldehyde and 2% paraformaldehyde solution in phosphate buffer, 50 mrnol/L, was injected into the coronary arteries via the aortic cannula to fix the preparation. The whole wet weight of each heart was measured. Five small myocardial blocks were obtained from both the endocardial and epicardial layers of the left ventricle for examination with an electron microscope. Remaining heart muscle was reweighed and dried overnight at 110° C for assessment of dry weight. Statistical analysis and expression of results. All data were expressed as mean ± standard error. In the dose-response studies of diltiazem and ryanodine in nonischemic hearts, hemodynamic data were expressed as a percentage of pretreatmentcontrol values. In ischemia-reperfusion experiments, preischemic baseline function was expressed as an absolute value for each parameter. Postischemic recovery of cardiac function for each parameter was expressed as a percentage of its preischemic level. CK leakage into the coronary effluent was expressed as international units per IS minutes per gram dry weight. Morphologic damage to mitochondria was assessed with an electron microscope by means of a scoring system that graded the severity of structural derangement in mitochondria into fivecategories: 0, normal; I, separation of cristae; 2, further separation of cristae; 3, disruption of cristae; and 4, disruption of inner and outer membranes." Five hundred mitochondria in each heart were graded and scores were averaged for semiquantitative analysis. The morphologic features of other organelles and cellular components were only described qualitatively. Multiple comparisons of individual groups of ischemiareperfusion experiments were performed by analysis of variance. When significant F values were detected, Tukey's twotailed test of significance was carried out. Statistical significance was defined as p < 0.05. All animals received care in compliance with the "Principles of Laboratory Animal Care", formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). Results Effects of diltiazem and ryanodine on left ventricular contractility in nonischemic hearts. Cumulative

The Journal of Thoracic and Cardiovascular Surgery

Akita et al. 5 7

Volume 106, Number 1

"10 OF CONTROL

"10 OF CONTROL

100o-7'r-Q

100

~

mean±SEM (N=6)

80

:>

~

~

~ a.

lI) lI)

:>

a. 60

-0

>

--0-

a.

0 Qj

Q>

60

al

Q>

a.

-0

mean±SEM (N=6)

80

o

Qj

40

--0-

~

~

LVDP

~ 20

00

/

LVDP

40

20

/

-8

-7

-6

-5

100 80

80

_

~

60

~

+1

-c

+1

60

-c

40

----&- +dp/dl

40

----.-

-dp/dl

----&----.-

+dp/dl -dp/dt

20

20

/

L

-8

-7 Log

10

-5

[Diltiazem(Ml]

Fig. 1. Dose-response relationship of the effectofdiltiazemon left ventricular function under nonischemic conditions. Ordinates are left ventricular developed pressure (LVDP, upper panel) and its firstderivatives (± dpfdt, lower panel) presented as percentage of a controlvalue. Abscissas are drug concentration on a logarithmic scale. The values are mean ± standard error of the mean of six hearts. dose-response effects of diltiazem and ryanodine on mechanical function were assessed in six hearts for each agent under nonischemic conditions. The hearts were continuously stimulated through the left atrium at a rate between 222 and 286 beats/min. Average pretreatment control values for LVDP, --dp/dt, and -dp/dt in 12 hearts were 87.6 ± 5.1 mm Hg, +1881 ± 124 mm Hg/sec, and -1250 ± 44 mm Hg/sec, respectively. There were no significant differences in these baseline parameters between the two groups. Diltiazem, at doses above 3 X 10-8 mol/L, caused a significant decrease in LVDP and ±dp/dt in a dose-dependent manner as shown in Fig. 1. At 3 X 10-6 mol/L, LVDP was reduced by 77%. Comparative reductions in ±dp/dt were observed as well. Diltiazem, at a concen-

-10

-9

-8

-7

-6

-5

Log 10 [Ryanodine(Ml]

Fig. 2. Dose-response relationship ofthe effectof ryanodineon left ventricular function under nonischemic conditions. Ordinates and abscissas are the same as in Fig. l. tration over 10- 5 mol/L, often resulted in complete atrioventricular conduction block. Ryanodine, at concentrations above 10-9 mol/L, also suppressed LVDP and ±dp/dt in a dose-dependent manner as shown in Fig. 2. Unlike diltiazem, however, its negative inotropic effect reached a plateau at about 10- 7 mol/L, when LVDP and ±dp/dt were reduced to approximately half of control. Effects of diltiazem and ryanodine when administered with normothermic cardioplegic solution. Fiftyfour hearts subjected to normothermic cardioplegic arrest and ischemia were divided into nine groups of six hearts each. Four groups were treated with diltiazem in concentrations ranging from 10-8 to 3 X 10- 7 mol/L, and four were treated with ryanodine, in concentrations from 10- 10 to 3 X 10-9 mol/L. The remaining group comprised control hearts that received neither agent. Average of spontaneous heart rate, LVDP, ± dp/dt, and coronary effluent in the nine groups before ischemia is shown in

The Journal of Thoracic and Cardiovascular Surgery July 1993

58 Akita et at.

Table I. Baseline function of hearts before ischemia Treatment group Normothermic experiments Control Diltiazem 10- 8 mOI/L 3 x 10- 8 mol/L 10- 7 rnol/L 3 x 10- 7 mol/L Ryanodine 10- 10 mol/L 3 x 10- 10 mol/L 10- 9 mol/L 3 X 10-9 rncl/L Hypothermic experiments Control Diltiazem 3 X 10- 8 mol/L 10- 7 rnol/L 3 X 10- 7 mOI/L Ryanodine 10- 10 mol/L 3 X 10- 10 mol/L 10- 9 mol/L

LVDP (mmHg)

+dp/dt (mm Hgfsec}

-rdpldt (mm Hgfsec)

(mlfmin}

220 ± 7

86.0 ± 5.4

1954 ± 189

2080 ± 369

6.1 ± 0.4

227 246 230 216

± ± ± ±

16 5 9 9

73.5 80.3 69.5 86.6

± ± ± ±

6.1 8.0 6.9 6.5

2180 ± 1856 ± 1970 ± 2468 ±

164 259 66 129

1824±92 1408 ± 226 1398 ± 219 2080 ± 368

4.8 5.7 4.5 5.6

± ± ± ±

0.4 0.3 0.2 0.6

252 242 252 218

± ± ± ±

5 15 12 6

101.8 ± 92.2 ± 79.8 ± 96.4 ±

7.0 3.9 7.5 4.6

2514 ± 1908 ± 1780 ± 2244 ±

269 262 116 134

1567 ± 151 1178 ± 119 1140±127 1260 ± 74

7.8 7.3 4.6 6.9

± ± ± ±

0.8 0.5 0.4 0.3

HR (beats/min)

CF

211 ± 10

72.2 ± 3.7

2370 ± 242

1540 ± 222

5.4 ± 0.3

235 ± 88 254 ± 13 207 ± 9

73.4 ± 4.0 78.5 ± 4.9 78.9 ± 6.5

2540 ± 150 2240 ± 344 2224 ± 196

1930 ± 194 1728 ± 258 1832 ± 132

4.8 ± 0.3 5.0 ± 0.5 7.1 ± 0.6

207 ± 7 226 ± 6 225 ± 9

82.7 ± 5.8 75.3 ± 6.8 92.7 ± 7.5

2310 ± 343 1900 ± 182 2548 ± 278

1610 ± 250 1520 ± 197 2360 ± 348

4.3 ± 0.4 4.6 ± 0.4 5.9 ± 0.3

Values are mean ± standard error of the mean of six hearts in each group. H R, Heart rate; LVDP, left ventricular developed pressure; CF, coronary flow. There was no significant difference in these parameters among nine groups for normothermic experiments and among seven groups for hypothermic experiments.

Table II. Percent recovery of left ventricular function after 30 minutes of reperfusion Treatment group Normothermic experiments Control Diltiazem 10- 8 mol/L 3 X 10- 8 mol/L 10- 7 rnol/L 3 X 10-7 mol/L Ryanodine 10- 10 mOI/L 3 X 10- 10 mol/L 10-9 mol/L 3 X 10-9 mol/L Hypothermic experiments Control Diltiazem 10- 8 mol/L 3 X 10- 8 rnol/L 10- 7 mOI/L Ryanodine 10- 10 mol/L 3 X 10- 10 mol/L 10- 9 rnol/L

CF

HR (beats/min)

LVDP (mmHg)

+dp/dt (mm Hgfsec}

-dp/dt (mm Hgfsec}

(mltmin}

95.0 ± 2.9

52.9 ± 2.5

42.5 ± 4.5

45.8 ± 2.9

88.4 ± 2.3

94.4 95.9 94.3 91.5

± ± ± ±

3.4 2.5 2.9 1.8

76.8 84.2 79.0 72.1

± ± ± ±

2.8* 2.9* 5.8* 5.8t

75.4 84.3 72.9 65.7

7.5t 5.3* 4.7* 5.4t

79.6 91.2 78.1 69.8

± ± ± ±

5.9* 7.2* 4.1* 4.9t

95.7 ± 100.8 ± 100.3 ± 104.4 ±

4.5 8.2 4.2t 5.lt

95.8 96.1 96.4 94.2

± ± ± ±

2.0 2.8 3.0 1.4

86.6 90.0 90.5 76.1

± ± ± ±

2.9* 5.8* 4.5* 5.0t

84.9±8.1* 87.3 ± 6.6* 84.4 ± 6.9* 76.4 ± 5.9t

80.5 92.7 86.6 80.9

± ± ± ±

7.2* 4.0* 7.5* 6.6*

93.7 96.8 98.5 97.3

2.5 3.7 4.1 5.8

± ± ± ±

± ± ± ±

94.6 ± 1.7

72.7 ± 5.0

73.2 ± 3.1

74.9 ± 3.9

94.2 ± 6.1

93.6 ± 2.4 93.2 ± 2.1 91.7 ± 3.6

96.9 ± 3.3* 86.3 ± 2.0t 83.4 ± 4.5t

102.8 ± 4.5* 87.2 ± 5.4t 84.8 ± 3.7t

106.0 ± 3.2* 88.6 ± 1.9* 86.5 ± 6.5t

102.6 ± 5.1 110.3 ± 14.5 112.1 ± 9.9

92.8 ± 1.8 92.9 ± 1.4 92.9 ± 1.5

85.4 ± 5.0t 88.2 ± 3.lt 83.5 ± 2.8t

90.8 ± 6.2t 96.0 ± 4.7* 81.1 ± 5.3

92.5 ± 5.9t 88.3 ± 5.lt 76.5 ± 5.0

97.5 ± 8.5 98.0 ± 9.2 94.8 ± 7.3

Values are mean ± standard error of the mean of six hearts in each group. HR, Heart rate; LVDP, left ventricular developed pressure; CF, coronary flow. *p < 0.01 versus control. tp

< 0.05.

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 1

Akita et ai. 5 9

reperlusion

100mmHg

Control

DTZ

100mmHg

0.3x10 -7M

100mmHg

100mmHg

RY

10 ·9M

100mmHg

1 m in

Fig. 3. Sequential recordings of LVDP during normothermic ischemia and reperfusion. After cardioplegia with St. Thomas' Hospital solution (ST), normothermic (37° C) global ischemia was imposedfor 45 minutes. The hearts were then reperfused with Krebs-Henseleit solution (37° C) for 30 minutes. The St. Thomas' Hospital solution contained no drug (control), diltiazem (DTZ) at 0.3 X 10- 7 mol/L, 10- 7 mol/L, and 3 X 10- 7 mol/L, or ryanodine at 10- 9 mol/ L. The hearts were allowed to beat spontaneously throughout the experiments.

Table I. There were no significant differences in these parameters among the groups. Recovery of hemodynamic function. Representative recordings of LVDP in control, diltiazem-treated, and ryanodine-treated hearts are shown in Fig. 3. In the control group, diastolic pressure began to rise after 35 minutes of ischemia. In the hearts treated with diltiazem at concentrations between 3 X 10- 8 mol/L and 10- 7 mol/ L, there was no elevation in diastolic pressure during ischemia, and reperfusion was associated with a more rapid and greater recovery in LVDP than seen in control hearts. Increasing diltiazem concentration to 3 X 10- 7 mol/L diminished the rate and extent of LVDP recovery without affecting diastolic pressure during ischemia. In the ryanodine-treated groups, diastolic pressure was not elevated during ischemia, and reperfusion was associated with greater recovery of L VDP than in control hearts.

The percent recoveries of the hemodynamic parameters at the end of 30 minutes of reperfusion in the nine groups are listed in Table II and the recoveries of LVDP are illustrated in comparison with CK leakage in Fig. 4. The analysis of variance indicated that there were differences among the groups in the percent recovery ofLVDP, -i-dp/dt, -dp/dt, and coronary flow. In the control group, L VDP reached 52.9% ± 2.5% of its preischemic level, whereas -i-dp/dt and -dp/dt returned 42.5% ± 4.5% and 45.8% ± 2.9% of baseline, respectively. In the four groups treated with diltiazem, recovery of these three parameters was significantly better. At a diltiazem dosage of 3 X 10- 8 mol/L, L VDP reached 84.2% ± 2.9% of baseline, and --dp/dt and -dp/dt returned to 84.3% ± 5.3% and 91.2% ± 7.2%, respectively. Functional improvement relative to control values was most remarkable at this concentration and

The Journal of Thoracic and Cardiovascular Surgery July 1993

6 0 Akita et al.

A

A

(%j 100

••

•• •

** ••

4

*

80 c,

o > ...J

~

en

60

'0

.!l1

e

-0 c

Q>

>

0

o

Q>

ex:

3

oo

o s: o .9

40

'Q'?

••

~ 20

0 control

0.1 0.3

1

3

Diltiazem (10 ·7Mj

B

0.1 0.3

0

1

3 Ryanodine(10 ·9M j

80

control

0.1 0.3

1

3

Diltiazem(10 . 7M)

B

0.1 0.3

1

3

Ryanodine(10 ·9M)

4

.E 0>

~ 60

z"0

Q)

(;

~ c:

en

·E .,., 40

"0

0>

0

••

••

Q>

.!l!

'"c

·C

**

~ ~

3

o

••

20

2

s: o

.g

••

~

~

o

0 control

0.1 0.3

1

3

Diltiazem(10 ·7M)

0.1 0.3

1

3

Rya nodine(10 .9Mj

Fig. 4. Recovery of LVDP and CK leakage after normothermic (37 0 C) ischemia. A, LVDP recovery 30 minutes after reperfusionrelating to preischemic value. B,CK leakage during the initial 15 minutes of reperfusion (total enzyme amount per unit dry weightofthe heart). Valuesare mean ± standard error of the mean of six untreated (control) hearts and six hearts each treated with diltiazem at 0.1 to 3 X 10- 7 mol/L or ryanodineat 0.1 to 3 X 10- 9 mol/L. Symbols indicate significant difference from control at p < 0.05 (*) and p < 0.01 (**) . somewha t less at lower and higher concentrations ( 10-8, 10- 7, or 3 X 10- 7 mol jL). At a diltiazem concentration of 3 X 10- 7 mol /L, left ventricu lar functional recovery was significantly poorer than when 3 X i 0- 8 mol jL was administered. Similar improvements in recovery of left ventricular function were observed in the four groups treated with ryanodine (10- 10 to 3 X 10- 9 moljL). The maximal effect was obtained with concentrations of 3 X 10- 10 and 10-9 moljL, which resulted in L VDP recovery to 90 .0% ± 5.3% and 90.5 % ± 4.1% of baseline, respectively, in association with a substantial recovery of ± dp / dt. There wer e no significant differences between the

cont rol

0.3

3

Diltiazem (l 0. 7M)

0.1

0.3

Ryanodine (10· 9M)

Fig. 5. Mitochondrial score of ventricular muscles subjected to normothermic (A) or hypothermic (B)ischemia and reperfusion. Values are mean ± standard error of the mean of six untreated hearts (control) and six each treated with diltiazem at 0.1 to 3 X 10- 7 mol/L or ryanodine at 0.1 to 3 X 10- 9 moll L. Symbols are the same as in Fig. 4. maximal left ventricular functional recovery in diltiazema nd ryanodine-treated groups. The percent recovery of coronary flow in the control group at the end of 30 minutes of reperfusion was significantly less than the recoveries of diltiazem- and ryanodine-treated groups. T here were no significant differences in recovery of coronary flow among the eight drugtreated groups at the end of 30 minutes of reperfusion. However, during the initial 5 minutes of reperfusion, coronary flow in the diltiazem-treated groups ten ded to be higher than that in control and ryanodine-treated groups, a nd coronary flow increased as the concentration of diltiazem in the St. Thomas' Hospital solution increased. Fo r example, at the end of the initialS-minute reperfusion period, the per cent recovery of coronary flow in the

T he Journal of Th oracic and

Akita et al. 6 I

Card iova s cula r Surgery Volum e 10 6, Num ber 1

cont rol group was 101.7% ± 6.81 %, whereas perc ent recoveries in the diltiazem-treated groups were 108.4% ± 2.2% at 10- 8 mol/ L, 118.8% ± 5.5% at 3 X 10-8 mol/L, 120.5% ± 7.8% at 10- 7 moljL, an d 125.4% ± 5.1% a t 3 X 10- 7 mol/L, CK leaka ge. In the contro l gro up, CK leakage du ring the initial 15 minutes of reperfusion was 67.3 ± 6.0 IU per gram dry weight. In all th e groups treated with diItiazem or rya nodine, CK leakage was less than ha lf that of the control gro up (Fig, 4, B), U lt ras tructura l changes. Morphologic derangement in ventricular muscle indu ced by ischemia and repe rfusion was assessed by mitochondrial score in th e electron micrographs (Fig . 5, A). The average mitochondrial score of the nonischemic hearts (n = 6) was 0.23 ± 0.06. In the contro l group, subjected to nor mot hermic ischemia and reperfusio n, the average score was 2.35 ± 0.15, beca use of marked swelling of the mitochond ria and disruption of their internal cristae. In the hearts treated with diltiazem (10- 8 to 3 X 10- 7 rnol/L) or ryanodine (10- 10 to 3 X 10- 9 mol/L) during cardioplegia before ischemia, the structu re of th e intracellular organelles was relatively well preserved and contraction bands were rare. Average mitochondrial scores were, therefore, significantly lower than in the control group: 0.87 to 1.20 for the diltiazemtreated groups an d 0.75 to 1.14 for the ryanodine-treated groups (Fig . 5, A). Effects of diltiazem an d ryanodine a pplied during hypoth ermic cardioplegia Forty-two hearts, which were subjected to hypothermic cardioplegic arrest and ischemia, were divided into seven groups of six hearts each . Three groups were treated with diltiazern in concent ra tions rangi ng from 3 X 10-8 to 3 X 10-7 mol/ L, three were treated with ryanodine at 10- 10 to 10- 9 mol/L, and one group comprised an untreated control group. Average baseline values of heart rate, LV OP, ±dp/dt, and coronary flow in the seven gro ups before ischemia are presented in Table I. There was no significa nt difference in these parameters among the seven groups. Recover y of left ventr icular fun ction. T he percent recovery of the hemodynamic parameters at the end of 30 minutes of reperfusion a re listed in Table II an d the recoveries of LVOP are illustrated in comparison with CK leakage in Fig. 6. The analysis of variance ind icated that there were significant differences among the groups in the perce nt recovery of LVOP, -l-dp/dt, and -sdp/dt. In the control group, LVOP, --d p/ dt, a nd -xlp /dt had recovered to 72.7% ± 3.3%,71.1 % ± 3.4%, and 75.6% ± 4.3% of their preisc hemic values, respective ly (Fig. 6, A). In the three groups of hea rts treated with d iltiazem, the recovery of left ventricular function after 30 minutes of repe rfusion was significan tly better. The average

'"

••

(%) 100

60 o, 0

> ...J

60

'0

e-

'"o> a: '" 0

40

~

20

0 con trol Diltiazem{1 0.7 M)

B

Ryanodine (10 '9M)

60 E .~ 50 ~

e ii> c: '0

40

E

Ii)

~

'" ~

..>< I1l

30

20

.!!? ~

U

10

0 con trol Diltiazem (10.7 M)

Rya nod ine (10 ' 9 M)

Fig. 6. Recovery of LYOP and CK leakage following hypothermic (20 0 C) ischemia. A: LYOP recovery 30 minutes after reperfusion relating to pre-ischemic value. B: CK leakage during the initial 15 minutes of reperfusion (total enzyme amount per unit dry weight of the heart). Yaluesare mean ± SEM of six untreated hearts (control) and each six hearts treated with diltiazem at 0.3 to 3 X 10- 7 mol/L or ryanodine at 0.1 to I X 10- 9 mol/L. Symbols are the same as Fig. 4. recoveries of LVOP relative to baseline for hearts treated with different concentrations of diltiazem were 96.9% ± 3.3% (3 X 10-8 mol/ L), 86.3% ± 2.0% (10- 7 mol/ L), and 83.4% ± 4.5% (3 X 10- 7 mol/ L), Corresponding recovery of ± dp/ dt was also obtained. R ecovery of left ventricular function was a lso improved significantly by treatment with ryanodine; LVDP at 3 X 10- 10 mol/L reac hed 88.2% ± 3. 1% of baseline. T he extent of recovery with ryanodine was appreciably less than that with diltiazem. There were no significant differences in the percent recovery of coronary flow among the seven groups at the end of 30 minutes of reperfusion. However, like normo-

The Journal of Thoracic and Cardiovascular Surgery July 1993

6 2 Akita et al.

Fig. 7. Electron micrographs of ventricular muscles. Tissue was obtained from hearts subjected to hypothermic (20 0 C) ischemia and reperfusion after cardioplegia with St. Thomas' Hospital solution containing no drug (A, control), diltiazem at 3 X 10- 7 mol/L (B), or ryanodine at 10- 9 mol/L (C), and nonischemic hearts as a reference (D). Ventricular cells of the control hearts were characterized by marked swelling and disruption of mitochondrial internal cristae, as well as the outer membrane, and frequent development of contraction bands. Morphologic damage was appreciably less in the cells from diltiazem- or ryanodine-treated hearts (B, C).

thermic experiments, the percent recovery of coronary flow during the early reperfusion period was significantly greater in diltiazem-treated groups than that in control and ryanodine-treated groups. For example, at the end of the initial 5-minute reperfusion period, the percent recovery of coronary flowin the control group was 104.2% ± 8.8%, whereas recoveries in the diltiazemtreated groups were 155.8% ± 10.9% at 3 X 10- 8 moll L, 155.2% ± 18.7% at 10- 7 mol/L, and 152.1% ± 16.9% at 3 X 10- 7 mol/L, Such increases in coronary flow in diltiazem-treated groups gradually returned to preischemic value within 15 minutes of reperfusion. CK leakage. In the control group, CK leakage during the initial 15 minutesofreperfusion was 41.5 ± 13.7 IU per gm dry weight. In hearts treated with diltiazem in concentrations of 3 X 10-8 to 10-8 mol/L, CK leakage was one third that of the control group (Fig. 6, B). With a diltiazem concentration of 3 X 10- 7 mol/L, CK leak-

age was less than in the control group, but this difference was not statistically significant. In the three groups treated with ryanodine (10- 10 to 10- 9 mol/L), however, there was no significant reduction in CK leakage compared with the control group. Ultrastructural changes. Representative electron micrographs of ventricular muscles after hypothermic ischemia and reperfusion are shown in Fig. 7. The average mitochondrial score in the control group subjected to hypothermic ischemia was 1.73 ± 0.24. In all groups treated with diltiazem or ryanodine, mitochondrial scores were significantly less than scores of the control group (Fig. 5, B). Discussion In this study, the addition of diltiazem or ryanodine to St. Thomas' Hospital solution cardioplegic solution significantly enhanced recovery of left ventricular function

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 1

in neonatal rabbit hearts after normothermic or hypothermic global ischemia. CK leakage during the early reperfusion period was reduced and morphologic derangement of ventricular cells was attenuated by both treatments. To our knowledge, this is the first report to demonstrate that diltiazem or ryanodine, when added to cardioplegic solutions, protects against ischemia-reperfusion injury in newborn mammalian hearts at concentrations with minimal negative inotropic effects. The effects of diltiazem. Under the non ischemic condition, threshold and 50%-inhibitory concentrations of diltiazem for depression of L VDP and ± dP/dt were approximately 3 X 10- 8 mol/L and 10-6 mol/L, respectively. These values are comparable with those reported by Boucek and associates I 2 and Murashita, Hearse, and Avkiran/" for neonatal rabbit hearts, and they are appreciably less than those reported for adult hearts. The greater sensitivity of newborn hearts to the negative inotropic effects of organic calcium channel antagonists can be explained by developmental changes in excitationcontraction couplings and/or calcium channel density in the sarcolemma. II, 13 In mammalian hearts up to several days after birth, the SR is still underdeveloped II , 14 and the contraction is supposed to depend largely on transsarcolemmal Ca2+ influx through voltage-dependent highthreshold (L-type) Ca 2+ channels.f Coincident with the development ofthe SR with age, Ca2+ uptake and release by the SR is believed to play an increasingly important role in excitation-contraction coupling.v 14-16 resulting in a relatively high resistance to L-type channel blockers. Binding studies with [3H] nitrendipine have shown that sarcolemmal density of t.-type calcium channels is low during the fetal stage and increases rapidly during the first week after birth. I I, 15 Furthermore, Osaka and Joyner 28 have recently demonstrated, in whole-cell voltage clamp experiments on ventricular myocytes, that calcium current density through L-type channels in newborn rabbits (1 to 3 days old) is approximately half that of adults. This low-current density would potentiate the pharmacologic action of L-type Ca2+ channel blockers. Inhibition of transsarcolemmal Ca 2+ influx with Ca 2+ channel blockers has been studied extensively as a means of relieving Ca 2+ overload in the myocardium subjected to ischemia and reperfusion,29-32 These agents have been used alone or as additives to cardioplegic solutions in various experimental 20-23 and clinical U: 32 studies to investigate their poten tial cardioprotective action. However, conflicting results have been obtained. 21-23 In isolated adult rat hearts, Yamamoto and his collaborators" showed that diltiazem, when added to cardioplegic solutions under normothermic conditions, like verapamil and nifedipine,22 preserved myocardial function and reduced enzyme leakage after ischemia and reperfusion, and that

Akita et al. 6 3

such protective action was lost under hypothermic conditions. In contrast, the same laboratory recently reported that diltiazem had no protective effect when added to cardioplegic solutions in isolated neonatal and adult rabbit hearts under both normothermic and hypothermic conditions.i? In the present study in isolated neonatal rabbit hearts, the addition of diltiazem to cardioplegic solutions resulted in significant protection against functional and morphologic deterioration of cardiac muscle after normothermic as well as hypothermic ischemia and reperfusion. It should be stressed, however, that this protective effect was restricted to a specific range of diltiazem concentrations (10- 8 to 3 X 10-7 mol/L) that had a minimal negative inotropic effect under nonischemic conditions. Higher or lower concentrations of diltiazem offered no protective action (data not shown), These results may indicate that L-type Ca 2+ channel blockers must be administered in precise doses to be effective in protecting against the myocardial ischemia-reperfusion injury in neonatal hearts. The effects of ryanodine. Ryanodine, a plant alkaloid, is known to inhibit Ca2+ handling by the SR of striated and smooth muscle without affecting other functions of these cells. 33-35 In our experiments in nonischemic neonatal rabbits hearts, ryanodine caused a dose-dependent decrease in L VDP and ± dp/dt at concentrations above 3 X 10- 10 mol/L, but its negative inotropic effect reached a maximal plateau at around 10-7 mol/L, at which L VDP was reduced by approximately 50% from control. Because the action of ryanodine is specific to the Ca 2+releasing channels of the SR,34-36 these findings may indicate that the SR contributes substantially to the contraction of cardiac muscle in the newborn animal under physiologic conditions, despite its morphologic I 5.37 and functional's IO immaturity. Seguchi.P Chin,16 and their associates also reported that ryanodine had a negative inotropic effect in newborn rabbit hearts, although it was less potent than in adults. The negative inotropic effect of ryanodine observed in the present study is apparently greater than that reported by these previous investigators in terms of its concentration range and maximal inhibition of contraction. Seguchi, Harding, and J armakani 15 showed that ryanodine caused a significant decrease in contractility at concentrations above 3 X 10-8 mol/L, and it reached the maximal inhibition (20% to 42%) at 3 X 10- 7 mol /L, This difference from our results could be explained, at least in part, by different experimental conditions. We measured isovolumic left ventricular pressure in Langendorff-perfused hearts at 37° C, whereas Seguchi, Harding, and Jarmakani l5 monitored the isometric tension of ventricular muscle at Tl" c. In ventricular muscle isolated from adult

64

The Journal of Thoracic and Cardiovascular Surgery July 1993

Akita et al.

rabbits and rats, Shattock and Bers" demonstrated that the negative inotropic effect of ryanodine was largely attenuated by lowering the temperature from 370 to 23 0 C, probably because of a decrease in contribution of the SR to contraction. Varying stimulation frequency of the preparations may also modulate the action of ryanodine through an alteration in intracellular calcium dynamics.'? As an additive to normothermic cardioplegic solutions, ryanodine caused a significant improvement in functional recovery of neonatal rabbit hearts subjected to ischemia and reperfusion in our study. There is general agreement that metabolic depletion during ischemia leads to increases in the cytosolic calcium concentration, [Ca2+]i.40.41 This [Ca 2+]i elevation is believed to playa pivotal role in the functional and structural deterioration of cardiac muscle subjected to ischemia and reperfusion. A variety of mechanisms have been proposed to explain this [Ca 2+]i elevation, such as an alternation of coupled ion transportation (Na+/Ca H, Na+/H+ exchange) associated with acidosis and/or intracellular sodium loading,42.43 or an inhibition of adenosine triphosphatedependent Ca 2+ sequestration into the SR, or Ca H extrusion through the cell membrane.r' Most of these mechanisms are, however, unlikely to be influenced by ryanodine treatment. Recently, Feher, LeBolt, and Manson P assessed SR function during and after ischemia in terms of the calcium uptake rate of rat whole-heart homogenates in the presence of oxalate. They demonstrated that global ischemia of longer than 10 minutes caused a significant time-dependent decrease in the Ca H uptake rate, and this reduction was abolished when Ca 2+-release channels were blocked by very high concentrations of ryanodine (625 JImol/L) or ruthenium red. They suggested that [CaH]i elevation during ischemia is attributable, at least in part, to increased Ca 2+ efflux from SR into cytoplasm through ryanodine-sensitive Ca 2+-release channels. Ryanodine, at the low concentrations used in this study, unlike high concentration above micromolar range, enhances Ca H efflux from the SR through modulation of gating mode of the Ca 2+ release channel.l- 36, 46 When Ca H efflux occurs under nonischemic conditions, Ca H leaked into the cytoplasma would be quickly extruded to the extracellular space, primarily by Na+/Ca 2+ exchange. 47,48 Accordingly, it is reasonable to assume that in our experimental model, ryanodine may have caused a depletion of SR calcium during infusion of the cardioplegic solution and eventually prevented Ca H efflux from this organelle during the subsequent period of ischemia.'? In hearts treated with ryanodine, unlike untreated control hearts, left ventricular diastolic pressure did not rise throughout the ischemic period (Fig. 3). This observation adds credence to this assumption.

During hypothermia, the protective effect of ryanodine against ischemia-reperfusion injury in terms of left ventricular function was somewhat attenuated. CK leakage during the early reperfusion period was not significantly reduced by ryanodine. This is probably due to inhibition of active ion transport across the cell membrane under hypothermic conditions. Hypothermia has been shown to decrease N+, K+-adenotriphosphatase activity in mammalian cells,5o which results in intracellular Na+ accumulation and a decrease in the resting membrane potentiaI. 5!,52 Such conditions would limit Ca 2+ extrusion through the Na+ /Ca 2+ exchange. 47,48 Therefore, even if ryanodine causes a substantial leakage of Ca 2+ from the SR, it would not be extruded to the extracellular space as efficiently as normothermia. Bers and Christensen.t'' in their experiments using rapid cooling contracture to assess the Ca H content of SR, have demonstrated that the ability of ryanodine to deplete SR is largely inhibited under conditions that reduce the transsarcolemmal Na+ gradient. Nevertheless, we cannot rule out other possible mechanisms. For example, inasmuch as the negative inotropic effect of ryanodine is attenuated at lower temperatures.P' this might limit the energy preservation by this compound. Our data appear to suggest that conditions or procedures that deplete Ca H of the SR at the initiation of global ischemia may protect neonatal cardiac muscle from ischemia-reperfusion injury. In clinical practice, ryanodine cannot be used because of its undesirable side effects, including irreversible contracture of skeletal muscle.P Warm-induction blood cardioplegia could be used as an alternative protocol for depletion of SR Ca 2+, because long quiescence of the heartbeat and maintenance of Na+/Ca H exchange accelerates the efflux of intracellular Ca 2+, especially from the SR. 53 In fact, Williams and colleagues'" recently reported that warm-induction blood cardioplegia reduced the mortality of their infant cardiac operations. However, further studies will be required to substantiate the beneficial effect of this method.

REFERENCES I. Bull C, Cooper J, Stark J. Cardioplegic protection of the child's heart. J THORAC CARDIOVASC SURG 1984;88:28793. 2. Bove EL, Stammers AH. Recovery of left ventricular function after hypothermic global ischemia: age-related differences in the isolated rabbit heart. J THORAC CARDlOVASC SURG 1986;91:115-22. 3. Yano Y, Braimbridge MY, Hearse OJ. Protection of the pediatric myocardium: differential susceptibility to ischemic injury of the neonatal rat heart. J THORAC CARDIOVASC SURG 1987;94:887-96. 4. Watanabe H, Yokosawa T, Eguchi S, Imai S. Functional

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 1

and metabolic protection of the neonatal myocardium from ischemia. J THORAC CARDIOVASC SURG 1989;97:50-8. 5. Watanabe H, Yokosawa T, Eguchi S, Imai S. Difference in the mechanical response to a cardioplegic solution observed between the neonatal and adult guinea pig myocardium. J THORAC CARDIOVASC SURG 1989;97:50-8. 6. Baker JE, Boerboom LE, Olinger GN. Age-related changes in the ability of hypothermia and cardioplegia to protect ischemic rabbit myocardium. J THORAC CARDlOVASC SURG 1988;96:717-24. 7. Kempsford RD, Hearse DJ. Protection of the immature heart. J THORAC CARDIOVASC SURG 1990;99:269-79. 8. Fabiato A, Fabiato F. Calcium-induced release of calcium from the sarcoplasmic reticulum of skinned cells from adult human, dog, cat, rabbit, rat, and frog hearts and from newborn rat ventricles. Ann N Y Acad Sci 1978;307:491-522. 9. Maylie JG. Excitation-contraction coupling in neonate and adult myocardium of cat. Am J PhysioI1982;242:H834-43. 10. Nakanishi T, Jarmakani JM. Developmental changes in myocardial mechanical function and subcellular organelles. Am J PhysioI1984;246:H615-25. II. Wibo M, Brabo G, Godfraind T. Postnatal maturation of excitation-contraction coupling in rat ventricle in relation to the subcellular localization and surface density of 1,4 dihydropyridine and ryanodine receptor. Circ Res 1991 ;68:66273. 12. Boucek RD, Shelton M, Artman M, Mushlin PS, Starnes VA, Olson RD. Comparative effects of verapamil, nifedipine, and diltiazem on contractile function in the isolated immature and adult rabbit heart. Pediatr Res 1984;18:94852. 13. Boucek RD, Shelton M, Artman M. Myocellular calcium regulation by the sarcolemmal membrane in the adult and immature rabbit heart. Basic Res Cardiol 1985;80:316-25. 14. Nayer WG, Fassold E. Calcium accumulation and ATPase activity of cardiac sarcoplasmic reticulum before and after birth. Cardiovasc Res 1977;11:231-7. 15. Seguchi M, Harding JA, Jarmakani JM. Developmental change in the function of sarcoplasmic reticulum. J Mol Cell CardioI1986;18:189-95. 16. Chin TK, Friedman WF, Klitzner TS. Developmental changes in cardiac myocyte calcium regulation. Circ Res 1990;67:574-9. 17. Poole-Wilson PA, Harding DP, Bourdillion PDV, Tones MA. Calcium out of control. J Mol Cell Cardiol 1984; 16:175-87. 18. Cheung JY, Bonventre JV, Malis CD, Leaf A. Calcium and ischemic injury. N Engl J Med 1986;314: 1670-6. 19. Murphy JG, Marsh JD, Smith TW. The role of calcium in ischemic myocardial injury. Circulation 1987;75(Pt 2): VI5-24. 20. Van Gilst WH, Boonsta PW, Terpstra JA, et al. Improved recovery of cardiac function after 24 h of hypothermic arrest in the isolated rat heart: comparison of a prostacyclin analogue (ZK36 374) and a calcium entry blocker (diltiazem). J Cardiovasc Pharmacol 7:520,1985. 21. Yamamoto F, Manning AS, Braimbridge MV, Hearse DJ. Calcium antagonists and myocardiol protection: diltiazem

Akita et at. 6 5

22.

23.

24.

25. 26.

27.

28.

29.

30.

31.

32.

33. 34.

35.

36.

37.

38.

39.

during cardioplegic arrest. Thorac Cardiovasc Surg 1983; 31:369. Yamamoto F, Manning AS, Braimbridge MY, Hearse DJ. Cardioplegia and slow-channel blockers: studies with verapamil. J THORAC CARDlOVASC SURG 1983;86:252-61. Nayler WG. Protection of the myocardial against postischemic reperfusion damage. J THORAC CARDlOVASC SURG 1982;84:897-905. Thandroyen FT, MacCarthy J, Burton K, Opie LH. Ryanodine and caffeine prevent ventricular arrhythmias during acute myocardial ischemia and reperfusion in rat heart. Circ Res 1988;62:306-14. Gebhard MM, Bretschneider HJ. Myocardial protection. Curr Opinion Cardiol 1989;4:803-6. Kloner RA, Fishbein MC, Braunwald NC, Maroko P. Effect of propranolol on mitochondria morphology during acute myocardial ischemia. Am J Cardiol 1978;41 :880-6. Murashita T, Hearse DJ, Avkiran M. Effects of diltiazem as an additive to St. Thomas's Hospital cardioplegic solution in isolated neonatal and adult rabbit hearts. Cardiovasc Res 1991 ;25:496-502. Osaka T, Joyner RW. Developmental changes in calcium currents of rabbit ventricular cells. Circ Res 1991 ;68:78896. Nayler WG, Ferrari R, Williams A. Protective effect of pretreatment with verapamil, nifedipine, and propranolol on mitochondrial function in the ischemic and reperfused myocardium. Am J Cardiol 1980;46:242-8. Ferrari R, Albertini A, Ceconi CC, Raddino R, Visioli O. Myocardial recovery during post-ischemic reperfusion: effects of nifidipine, calcium, and magnesium. J Mol Cell Cardiol 1986; 18:487-98. Christakis GT, Fremes SE, Weisel RD, et al. Diltiazem cardioplegia: a balance of risk and benefit. J THORAC CARDIOVASC SURG 1986;91:647-61. Flameng W, De Meyere R, Daenen W, et al. Nifedipine as an adjunct to St. Thomas' Hospital cardioplegia. J THORAC CARDIOVASC SURG 1986;91 :723-31. Tenden DJ. The pharmacology of ryanodine. Pharmcol Rev 1969;21:1-25. Marban E, Wier WG. Ryanodine as a tool to determine the contributions of calcium transient and contraction of cardiac Purkinje fibers. Circ Res 1985;56: 133-8. Meisser G. Ryanodineactivation and inhibition ofthe Ca2+ release channel of sarcoplasmic reticulum. J BioI Chern 1986;261 :6300-6. Rousseau E, Smith JS, Meisser G. Ryanodine modifies conductance and gating behaviour of single Ca 2+ release channel. Am J Physiol 1987;253:C364-8. Page E, Buecker JL. Development of dyadic junction complexes between sarcoplasmic reticulumand plasmalemma in rabbit left ventricular myocardial cells: morphometric analysis. Circ Res 1981 ;48:519-22. Shattach M, Bers DM. Inotropic response to hypothermia and the temperature-dependence of ryanodine action in isolated rabbit and rat ventricular muscle: implication for excitation-contraction coupling. Circ Res 1987;61:761-71. Bouchard RA, Bose D. Analysis of the interval-force rela-

66

40.

41.

42.

43.

44.

45.

46.

Akita et ai.

tionship in rat and canine ventricular myocardium. Am J Physiol I 989;257(26):H2036-47 . Steenbergen C, Murphy E, Watts JA, London R. Correlation between cytosolic free calcium, contracture, ATP, and irreversible ischemic injury in perfused rat heart. Circ Res 1990;66:135-46. Koretsune Y, Marban E. Mechanism of ischemic contracture in ferret hearts: relative role of [Ca2+)i elevation and ATP depletion. Am J Physiol 1990;258:H9-26. Tani M, Neely J. Role of intracellular Na+ in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts: possible involvement of Na+-H+ and Na+-Ca2+ exchange. CircRes 1989;65:104556. Grinwald PM, Brosnahan C. Sodium inbalance as a cause of calcium overload in post-hypoxic reoxygenation injury. J Mol Cell Cardiol 1987;19:487-95. Krauss S, Hess ML. Characterization of cardiac sarcoplasmic reticulum dysfunction during short-term, normothermic global ischemia. Circ Res 1984;55:176-84. Feher JJ, LeBolt WR, Manson NH. Differential effects of global ischemia on the ryanodine sensitive and ryanodine insensitive uptake of cardiac sarcoplasmic reticulum. Circ Res 1989;65:1400-8. Lattanzio FA, Schlatterer RG, Nicar M, Campbell, Sutko JL. The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes. J Bioi Chem 1987;262:2711-8.

The Journal of Thoracic and Cardiovascular Surgery July 1993

47. Sutko JL, Bers DM, Reeves JP. Post-rest inotrophy in rabbit ventricle: N a+-Ca 2+exchange determined sarcoplasmic reticulum Ca content. Am J Physiol 1986;256:H654-61. 48. Bers DM, Christensen DM. Functional interconnection of rest decay and ryanodine effects in rabbit and rat ventricle depends on Na/Ca exchange. J Mol Cell Cardiol 1990; 22:715-23. 49. Hansfold RG, Lakatta EG. Ryanodine releases calcium from sarcoplasmic reticulum in calcium-tolerant rat cardiac myocytes. J Physiol (London) 1987;390:453-67. 50. Glitsch HG, Pusch H. On the temperature dependence of the Na pump in sheep Purkinje fibers. Pflugers Arch 1984; 402: 109-15. 51. Chapman RA. Sodium/calcium exchange and intracellularcalcium buffering in ferret myocardium: an ion-sensitive micro-electrode study. J PhysioI1986;373:163-79. 52. Navas J, Anderson W, Marsh JD. Hypothermia increases calcium content of hypoxic myocytes. Am J Physiol 1990; 259:H333-9. 53. Bers D. Ca influx and sarcoplasmic reticulum Ca release in cardiac muscles activation during postrest recovery. Am J Physiol 1985;248,H336-81. 54. Williams WG, Rebeyka 1M, Tibshirani RJ, et al. Warm induction blood cardioplegia in the infant. J THORAC CAR. DIOVASC SURG 1990;100:896-901.