Download Reduced reactive O2 species formation and preserved

Cardiovascular Research 61 (2004) 580 – 590
www.elsevier.com/locate/cardiores
Reduced reactive O2 species formation and preserved
mitochondrial NADH and [Ca2+] levels during short-term
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17 jC ischemia in intact hearts
Matthias L. Riess a,b,c, Amadou K.S. Camara a, Leo G. Kevin a,
Jianzhong An a, David F. Stowe a,b,d,e,f,*
a
Anesthesiology Research Laboratory, Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
b
Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
c
Department of Anesthesiology and Intensive Care Medicine, University Hospital Münster, 48129 Münster, Germany
d
Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
e
Research Service, Veterans Affairs Medical Center, Milwaukee, WI 53295, USA
f
Department of Biomedical Engineering, Marquette University, Milwaukee, WI 53233, USA
Received 1 June 2003; received in revised form 12 August 2003; accepted 15 September 2003
Time for primary review 22 days
Abstract
Objective: Different cardioprotective strategies such as ischemic or pharmacologic preconditioning lead to attenuated ischemia/
reperfusion (I/R) injury with less mechanical dysfunction and reduced infarct size on reperfusion. Improved mitochondrial function during
ischemia as well as on reperfusion is a key feature of cardioprotection. The best reversible cardioprotective strategy is hypothermia. We
investigated mitochondrial protection before, during, and after hypothermic ischemia by measuring mitochondrial (m)Ca2 +, NADH, and
reactive oxygen species (ROS) by online spectrophotofluorometry in intact hearts. Methods: A fiberoptic cable was placed against the left
ventricle of Langendorff-prepared guinea pig hearts to excite and record transmyocardial fluorescence at the appropriate wavelengths during
37 and 17 jC perfusion and during 30 min ischemia at 37 and 17 jC before 120 min reperfusion/rewarming. Results: Cold perfusion caused
significant reversible increases in m[Ca2 +], NADH, and ROS. Hypothermia prevented a further increase in m[Ca2 +], excess ROS formation
and NADH oxidation/reduction imbalance during ischemia, led to a rapid return to preischemic values on warm reperfusion, and preserved
cardiac function and tissue viability on reperfusion. Conclusions: Hypothermic perfusion at 17 jC caused moderate and reversible changes
in mitochondrial function. However, hypothermia protects during ischemia, as shown by preservation of mitochondrial NADH energy
balance and prevention of deleterious increases in m[Ca2 +] and ROS formation. The close temporal relations of these factors during cooling
and during ischemia suggest a causal link between mCa2 +, mitochondrial energy balance, and ROS production.
D 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Calcium; Free radicals; Hypothermia; Ischemia; Mitochondria
1. Introduction
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Portions of this work have been published in abstract form and
presented at the 2002 ASA annual meeting in Orlando, FL, USA, October
12 – 16, 2002 (Anesthesiology 2002; 97: A608) and at the IARS 77th
Congress, New Orleans, LA, USA, March 21 – 25, 2003 (Anesth Analg
2003; 96 (2S): S18).
* Corresponding author. M4280, 8701 Watertown Plank Road, Medical
College of Wisconsin, Milwaukee, WI 53226, USA. Tel.: +1-414-4565722; fax: +1-414-456-6507.
E-mail address: [email protected] (D.F. Stowe).
Cardiac ischemia/reperfusion (I/R) injury is frequently
induced or may occur during coronary angioplasty, cardiac
valve replacement, and coronary artery bypass grafting; it
is a necessary consequence for transplantation. Major
surgery and anesthesia create a cardiovascular risk, particularly in patients with cardiovascular disease. Many approaches and therapies have been tested to reduce the
deleterious effects of I/R. Hypothermia affords the best
protection against an expected ischemic insult in patients
undergoing cardiac surgery for coronary bypass grafting or
valve repair (25 – 27 jC), ascending aortic arch repair with
circulatory arrest (15 – 18 jC), and cardiac transplantation
0008-6363/$ - see front matter D 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2003.09.016
M.L. Riess et al. / Cardiovascular Research 61 (2004) 580–590
(3– 7 jC). The greater the cooling, the longer hearts can
remain non-perfused without damage on normothermic
reperfusion. Protection by hypothermia during ischemia
includes better tissue perfusion, improved metabolic and
mechanical function, fewer dysrhythmias and reduced
infarct size on reperfusion [1,2]. Although it is generally
agreed that hypothermia decreases energy utilization during ischemia and preserves essential mechanisms to rapidly
regenerate ATP on reperfusion, little is known about the
impact of hypothermia on mitochondrial function during
cold perfusion, cold ischemia, and reperfusion/rewarming,
nor the implications of altered mitochondrial bioenergetics
for cardioprotection.
Cytosolic, and subsequently, mitochondrial (m)Ca2 +
overload, impaired mitochondrial bioenergetics, i.e., a mismatch of mitochondrial dinucleotide reduction and oxidation (NADH/NAD, FADH2/FAD), and formation of reactive
oxygen species (ROS) by impaired mitochondrial electron
transport are key features of normothermic I/R injury [3– 5].
Consequently, different cardioprotective strategies such as
ischemic and pharmacologic preconditioning have been
shown not only to improve cardiac function and reduce
infarct size on reperfusion, but also to attenuate mitochondrial dysfunction as evidenced by less mCa2 + overload [3],
a better NADH balance [4,6] and reduced ROS formation
[5] during ischemia as well as on reperfusion. Although
hypothermia is cardioprotective, it can also exert deleterious
effects itself, such as hypercontracture due to elevated
cytosolic Ca2 + [7] that may jeopardize a full return of
function on rewarming.
To better understand the relationship of those mechanisms that lead to I/R injury, and those that prevent it during
hypothermia, the aim of the present study was to investigate
how hypothermia alters mitochondrial function before,
during, and after ischemia. To do so we measured mitochondrial [Ca2 +], NADH, and ROS formation by near
continuous online fluorescent measurements in guinea pig
isolated hearts during cold vs. warm perfusion as well as
during cold vs. warm ischemia.
2. Materials and methods
2.1. Langendorff heart preparation
The investigation conforms to the Guide for the Care and
Use of Laboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85-23, revised
1996) and was approved by the institutional animal studies
committee. Our methods have been previously described
[1– 7]. Thirty milligrams of ketamine and 1000 units (U) of
heparin were injected intraperitoneally into 96 albino English short-haired guinea pigs (weight 250– 300 g). Animals
were decapitated when unresponsive to noxious stimulation.
After thoracotomy, the aorta was cannulated distal to the
aortic valve, and the heart was immediately perfused retro-
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grade with 4 jC cold oxygenated Krebs – Ringer (KR)
solution. The inferior and superior venae cavae were ligated,
and the heart was rapidly excised. After cannulation of the
pulmonary artery to collect the coronary effluent, the heart
was placed in the support system, immersed in a waterbath
containing KR at 37 jC and perfused at 55 mm Hg at 37 jC.
The KR perfusate was equilibrated with f 95% O2 and
f 5% CO2 to maintain a constant pH of 7.4 F 0.01 at 37 jC.
The KR perfusate was filtered (5 Am pore size) in-line and
had the following calculated composition in mM: 138 Na+,
4.5 K+, 1.2 Mg2 +, 2.5 Ca2 +, 134 Cl, 14.5 HCO3, 1.2
H2PO4, 11.5 glucose, 2 pyruvate, 16 mannitol, 0.1 probenecid, 0.05 EDTA, and 5 U/l insulin.
Left ventricular pressure (LVP) was measured isovolumetrically with a saline-filled latex balloon inserted into the
left ventricle through the left atrium. At the beginning of
each experiment, the balloon volume was adjusted to
achieve a diastolic LVP of zero mm Hg, so that any
subsequent increase in diastolic LVP reflected left ventricular diastolic contracture. Spontaneous heart rate was monitored with bipolar electrodes placed in the right atrial and
ventricular walls. Coronary flow (CF) was measured by an
ultrasonic flowmeter (Transonic T106X, Ithaca, NY) placed
directly into the aortic inflow line. Coronary inflow (a) and
coronary venous (v) Na+, K+, Ca2 +, oxygen partial pressure
( pO2), pH, and carbon dioxide partial pressure ( pCO2) were
measured off-line with an intermittently self-calibrating
analyzer system (Radiometer Copenhagen ABL 505,
Copenhagen, Denmark). pvO2 tension was also measured
continuously online with an O2 Clark type electrode (model
203B; Instech, Plymouth Meeting, PA). Myocardial oxygen
consumption (MVO2) was calculated as CFheart wet
weight 1[ paO2 pvO2]24 Al O2/ml at 760 mm Hg and
37 jC; temperature-dependent changes in O2 solubility were
taken into account.
NADH, mCa2 +, and ROS were measured online by
spectrophotofluorometry. Experiments were conducted in a
light-proofed Faraday cage to block incident room light. The
distal end of a bifurcated fiberoptic cable (6.8 mm2 per
bundle) was placed gently against the left ventricular anterior
wall. The two proximal ends of the fiberoptic cable were
connected to a modified spectrophotofluorometer (Photon
Technology International, London, Canada). Fluorescence
( F) was excited with light at the appropriate wavelength (k)
from a xenon arc lamp at 75 W filtered through a monochromator (Delta RAM; Photon Technology International). The
beam was focused onto the in-going fibers of the optic
bundle. The arc lamp shutter was opened for 2.5-s recording
intervals to prevent photobleaching; there were 22 recordings
for a total exposure of 55 s over the course of each experiment. Light at the wavelengths used in our study penetrates
transmurally, i.e., all cells from the epicardium to the endocardium contribute to the measured fluorescence signal [8]. F
emissions were collected by fibers of the second limb of the
cable. This light was separated by a dichroic beamsplitter at
430 nm and filtered by interference filters (Chroma Technol-
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M.L. Riess et al. / Cardiovascular Research 61 (2004) 580–590
ogy, Brattleboro, VT) at the appropriate k. F intensity was
measured by photomultipliers (Photomultiplier Detection
System 814; Photon Technology International).
Ca2 +. In previous experiments, S460 was calculated as 2.29,
Rmin as 0.57, and Rmax as 6.22 at the chosen photomultiplier
settings [3]. Kd was calculated as 249 nM at 37 jC [10], and
was corrected for changes in temperature [7].
2.2. Online assessment of NADH
2.4. Online assessment of ROS
Tissue auto F at kem 460 nm (kex 350 nm) was used to
measure changes in mitochondrial NADH as previously
described [4,6,9,10]. Although F may also arise from
unknown intracellular constituents or cytosolic NADH,
the majority is derived from mitochondrial NADH [11].
Motion artifacts are diminished by using kem 405 nm as a
second reference that is less sensitive to changes in
NADH; thus, the ratio of F at kem 460 and at kem 405
nm, F460/F405, is interpreted as a measure of NADH [12].
The use of these two wavelengths also accounts for
possible alterations in myoglobin light absorption, e.g.,
by hypoxia [12]. Experiments in cell free solutions
showed that NADH fluorescence per se is independent
of temperature changes from 37 to 17 jC. NADH is
given in arbitrary fluorescence units (afu).
2.3. Online measurement of mCa2+ concentration
Averaged mitochondrial Ca2 + was measured with the
probe indo 1 using a method adapted for use in the intact
heart [3,10,13]. After equilibration, background F at kem 405
and 460 nm at kex 350 nm was determined for each heart. Indo
1-AM (6 AM) (Molecular Probes, Eugene, OR) was prepared
and perfused for 30 min; this increased each F signal by
approximately 10-fold. After washout of residual interstitial
indo 1-AM for 20 min, cytosolic Ca2 + was quenched from
cytosolic bound indo 1 by perfusing with 100 AM MnCl2 for
15 min. The remaining F originates predominantly from
mitochondrial sources. Although mitochondrial Ca2 + transients have been described in stimulated myocytes [14], F
measured by our technique in isolated hearts lacks the phasic
character of cytosolic Ca2 + during the contractile cycle
[3,10]. While both F405 and F460 declined over time, the
F405/F460 ratio remained stable during the course of our
studies. Since background F values obtained before indo 1
loading represent measures of NADH [9] all F signals were
corrected for the corresponding temperature- and I/R-induced
changes in auto F (NADH) obtained in the previous group of
experiments. The indo 1 transient is nonlinearly proportional
to m[Ca2 +], which was calculated according to the following
equation [3,10,13]:
m½Ca2þ ¼ S460 Kd ½ðRm Rmin Þ=ðRmax Rm Þ;
where S460 is the ratio of F intensities at 460 nm at zero and
saturated Ca2 +, Kd is the dissociation constant of indo 1, Rm is
the actually measured F405/F460 ratio, Rmin is the F405/F460
ratio at zero Ca2 +, and Rmax is the F405/F460 ratio at saturated
We used the oxidation of the fluorescent dye dihydroethidium (DHE; Molecular Probes) to measure ROS
formation, most likely superoxide (O2 ) [15 –17], which
converts DHE to ethidium that intercalates with DNA to
cause a red shift in fluorescence. DHE and ethidium
remain within cells with minimal leakage. Tissue was
excited at kex 540 nm and F obtained at kem 590 nm. In
preliminary experiments, background auto F at these
wavelengths was determined for each experimental protocol. All subsequent DHE F recordings were adjusted
for these minimal changes in background auto F with
changes in temperature and I/R over time. DHE (10 AM)
was prepared and perfused for 30 min; this was followed
by washout of residual DHE for 20 min [5]. DHE loading
increased F from 0.3 F 0.0 afu before loading to 2.4 F0.2
afu after washout.
All analog signals, including F signals, were digitized
(PowerLab/16 SP; ADInstruments, Castle Hills, Australia)
and recorded at 200 Hz (Chart and Scope v3.6.3; ADInstruments) on Power MacintoshR Computer G4 (Apple,
Cupertino, CA) for later analysis using MATLABR (The
MathWorks, Natick, MA) and Microsoft ExcelR (Microsoft, Redmond, WA) software. All variables were averaged over the sampling period of 2.5 s, which is about 10
cycle lengths at 37 jC and two cycle lengths at 17 jC.
2.5. Protocol
Hearts were randomly assigned to one of four experimental groups, each with a subset to independently measure either
NADH, mCa2 +, or ROS. Each of the four groups consisted of
eight hearts for each of the three mitochondrial measures.
After a 30-min equilibration period: (a) normothermic, nonischemic control hearts (37 jC Time Con) were perfused at 37
jC for 180 min; (b) normothermic, ischemic hearts (37 jC
ISC) were perfused at 37 jC for 30 min, before 30 min of
global no-flow ischemia and 120 min reperfusion at 37 jC;
(c) hypothermic, non-ischemic control hearts (17 jC Time
Con) were perfused at 37 jC for 10 min and then subject to
cold perfusion at 17 jC for 50 min before rewarming to 37 jC
for 120 min; and (d) hypothermic, ischemic hearts (17 jC
ISC) were perfused at 37 jC for 10 min, and cooled to 17 jC
for 20 min before 30 min of global no-flow ischemia at 17 jC
and 120 min reperfusion at 37 jC. At the end of each
experiment, hearts were removed and ventricles cut into
transverse sections of 3-mm thickness. These were stained
with 0.1% 2,3,5-triphenyltetrazolium chloride and digitally
imaged. Ventricular infarct size was measured in % by
cumulative planimetry as described previously [6]. Repro-
M.L. Riess et al. / Cardiovascular Research 61 (2004) 580–590
ducibility of this method is approximately F 5% based on
studies of fresh ex vivo, but non-perfused Langendorff
prepared hearts (ML Riess, laboratory observations).
All data are expressed as mean F S.E.M. Among-group
data were compared by analysis of variance to determine
significance (Super ANOVA 1.11R software for MacintoshR; Abacus Concepts, Berkeley, CA) at the following
selected time points: before (at 0 and 10 min) and after
cooling (at 30 min), during ischemia (at 31 and 60 min); and
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during reperfusion/rewarming (at 61, 120, and 180 min). If
F values ( P < 0.05) were significant, post hoc comparisons
of means tests (Student– Newman – Keuls) were used to
compare the four groups within each subset. Differences
among means were considered statistically significant when
P < 0.05 (two-tailed). Statistical symbols used were: *17 jC
Time Con vs. 37 jC Time Con; y17 jC ISC vs. 37 jC ISC;
z
17 jC Time Con vs. 17 jC ISC; and §37 jC Time Con vs.
37 jC ISC.
Fig. 1. Averaged mitochondrial Ca2 + fluorescence (A), NADH fluorescence (B), and DHE fluorescence (C) for each of the four groups (n = 8 each) before and
during cooling, before and during ischemia, and on reperfusion/rewarming. P < 0.05 for *17 jC Time Con vs. 37 jC Time Con; y17 jC ISC vs. 37 jC ISC;
z
17 jC Time Con vs. 17 jC ISC; and §37 jC Time Con vs. 37 jC ISC.
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M.L. Riess et al. / Cardiovascular Research 61 (2004) 580–590
3. Results
not different from the non-ischemic control level throughout reperfusion.
3.1. Changes in mCa2+ fluorescence
3.2. Changes in NADH fluorescence
Fig. 1A shows changes in m[Ca2 +] in each of the two
warm and two cold groups before and during cooling, before
and during ischemia, and on reperfusion/rewarming.
m[Ca2 +] remained stable for 180 min in the warm, nonischemic control group. Cooling to 17 jC increased m[Ca2 +]
in both hypothermic groups. Rewarming to 37 jC after
continued cold perfusion without ischemia for 30 min
(17 jC Time Con) led to a rapid return of m[Ca2 +] to
the baseline level. During normothermic ischemia (37 jC
ISC), m[Ca2 +] increased continuously and peaked at nearly
four times the baseline value. On reperfusion, this was
followed by a slow continuous decline toward preischemic
values. Hypothermic ischemia (17 jC ISC) did not lead to
a further increase in m[Ca2 +]. On reperfusion/rewarming,
m[Ca2 +] rapidly decreased to the baseline level and was
Fig. 1B shows NADH fluorescence in each of the two
warm and two cold groups before and during cooling,
before and during ischemia, and on reperfusion/rewarming.
NADH remained stable for 120 min in the warm, nonischemic control group and then slowly decreased by about
5% at 180 min (non-significant). Cooling to 17 jC increased
NADH in both hypothermic groups. Rewarming to 37 jC
after continued cold perfusion without ischemia for 30 min
(17 jC Time Con) led to a rapid return of NADH to the
baseline level. During warm ischemia (37 jC ISC) NADH
increased initially, but this was followed by a gradual
decline below the preischemic level. Reperfusion led to a
further decrease in NADH to a level significantly below the
baseline level. Cold ischemia (17 jC ISC) also led to an
Fig. 2. Ventricular heart rate (A), systolic and diastolic left ventricular pressure (B) for each of the four groups (n = 8 each) before and during cooling, before
and during ischemia and on reperfusion/rewarming. Data were obtained from the NADH subgroups. Note that identical systolic and diastolic pressure during
ischemia reflects the absence of developed left ventricular pressure (systolic – diastolic pressure). P < 0.05 for *17 jC Time Con vs. 37 jC Time Con; y17 jC
ISC vs. 37 jC ISC; z17 jC Time Con vs. 17 jC ISC; and §37 jC Time Con vs. 37 jC ISC.
M.L. Riess et al. / Cardiovascular Research 61 (2004) 580–590
initial increase in NADH to a level similar to that during
normothermic ischemia. However, during continued cold
ischemia, NADH remained elevated. On reperfusion/
rewarming, NADH rapidly returned to the baseline level
and was not different from the non-ischemic control level
throughout reperfusion.
3.3. Changes in DHE fluorescence
Fig. 1C shows changes in DHE F as a measure of ROS in
each of the two warm and two cold groups before and
during cooling, before and during ischemia, and on reperfusion/rewarming. DHE F remained stable for 180 min in
the warm, non-ischemic control group. Cooling to 17 jC
caused a reversible increase in DHE F in both hypothermic
groups. During continued perfusion at 17 jC (17 jC Time
Con) DHE F remained elevated; this was followed by a
rapid return to the baseline value on rewarming to 37 jC.
During warm ischemia (37 jC ISC) DHE F initially
increased to a level higher than during cold perfusion and
585
remained stable for approximately 15 min. DHE F then
began to increase to approximately twice its baseline value.
Reperfusion caused DHE F to decrease initially and then to
increase slightly; DHE F remained above the non-ischemic
control level throughout reperfusion. Cold ischemia (17 jC
ISC) also led to an increase in DHE F, but to a lesser extent
than during warm ischemia so that DHE F was not different
between warm and cold ischemic hearts for up to 25 min of
ischemia. However, cold ischemic hearts did not exhibit the
marked peak in DHE F during the last 5 min of ischemia as
did warm ischemic hearts. Reperfusion/rewarming caused a
rapid return of DHE F to the baseline level, and this was not
different from the non-ischemic control level throughout
reperfusion.
3.4. Changes in mechanical function
There were no significant differences in heart rate and
LVP among the three subgroups within each of the four
experimental groups. All data shown are from the four
Fig. 3. Changes in coronary flow (A) and myocardial oxygen consumption (B) for each of the four groups (n = 8 each) before and during cooling, before and
during ischemia and on reperfusion/rewarming. Data were obtained from the NADH subgroups. P < 0.05 for *17 jC Time Con vs. 37 jC Time Con; y17 jC
ISC vs. 37 jC ISC; z17 jC Time Con vs. 17 jC ISC; and §37 jC Time Con vs. 37 jC ISC.
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M.L. Riess et al. / Cardiovascular Research 61 (2004) 580–590
NADH groups. Cooling to 17 jC reversibly lowered heart
rate to approximately one-third (Fig. 2A). Cold ischemia
decreased heart rate even more, but the decrease toward
cessation of cardiac activity (at approximately 20 min) was
slower than in the 37 jC ischemic group, resulting in a
longer period of measurable developed LVP. On reperfusion/rewarming, heart rate recovered fully in both ischemic
groups. Cold perfusion caused systolic LVP to increase
transiently (Fig. 2B). Systolic LVP was sustained during
cold perfusion and was not different from the value obtained
during warm perfusion. Although warm ischemia led to a
rapid initial decrease in systolic LVP, this decrease was
delayed during initial cold ischemia. Also, normothermic
ischemic hearts exhibited a marked diastolic contracture
during the last 5 min of ischemia that was completely absent
during cold ischemia. On reperfusion, warm ischemic hearts
showed a lower return of systolic LVP and a markedly
elevated diastolic LVP throughout reperfusion, while systolic LVP rapidly returned to the control level in the cold
ischemic group.
3.5. Changes in coronary function, myocardial oxygen
consumption, and tissue viability
Fig. 3A shows changes in coronary flow for each of the
two warm and two cold groups. Coronary flow decreased
approximately 25% with cooling; this decrease was completely reversed on rewarming. Reperfusion/rewarming after
cold ischemia decreased coronary flow about 10% compared to the non-ischemic control group, whereas reperfusion after warm ischemia led to almost a 40% decrease in
coronary flow throughout reperfusion.
MVO2 is displayed in Fig. 3B. Cooling from 37 to 17 jC
decreased MVO2 by about 70%; this was completely
reversed on rewarming. Reperfusion/rewarming after cold
ischemia decreased MVO2 moderately over the time course
Fig. 4. Infarct size as a percentage of total ventricular weight measured at
the end of each experiment for each of the four groups (n = 8 each). Data
were obtained from the NADH subgroups. P < 0.05 for *17 jC Time Con
vs. 37 jC Time Con; y17 jC ISC vs. 37 jC ISC; z17 jC Time Con vs. 17
jC ISC; and §37 jC Time Con vs. 37 jC ISC.
of reperfusion. Reperfusion after warm ischemia resulted in
a 30% lower MVO2 compared to non-ischemic hearts.
Fig. 4 shows that 17 jC hypothermia protected hearts
against infarction measured at 120 min reperfusion following 30 min of ischemia. Infarct size in cold ischemic hearts
was not different from that in the normothermic or hypothermic time control hearts.
4. Discussion
The present study first demonstrates in the intact beating
heart that cardiac perfusion at 17 jC causes a moderate and
steady-state increase in m[Ca2 +], a more reduced mitochondrial redox state, as evidenced by an increase in NADH, and
moderate production of ROS. Moreover, compared to 37 jC
30 min ischemia, 17 jC 30 min ischemia resulted in no
additional increase in m[Ca2 +], no delayed decrease in
NADH, and no delayed increase in ROS. Each of these
mitochondrial changes was completely reversed on rewarming/reperfusion after 17 jC ischemia, but not 37 jC ischemia, and caused no remaining dysfunction or damage. Our
results, obtained in a nearly physiologic intact heart model,
suggest that although hypothermia, per se, causes a moderate derangement in mitochondrial bioenergetics, mitochondrial function is wholly preserved during and after shortterm ischemia. The close temporal association of these
changes in mitochondrial function in both the normothermic
and hypothermic hearts indicates a tight causal link between
mCa2 +, energy metabolism, and ROS production before,
during and after ischemia.
4.1. Mitochondrial bioenergetics
ATP is primarily made in the presence of O2 by oxidative
phosphorylation within mitochondria through a chemiosmotic mechanism [18]. The chemiosmotic theory suggests
that the rate of respiration is a function of a thermodynamic
driving force. In this way, a decrease in the electrochemical
proton gradient, DAH, stimulates a faster rate of proton
outward pumping by the electron transport chain (ETC) and
hence an increase in the electrical potential established
across the inner mitochondrial membrane (Dcm) which
stimulates ATP production.
It is unclear how this is regulated. For example, it is
unresolved if an increase in NADH/NAD+ drives oxidative
phosphorylation, or if changes in this ratio are secondary to
changes in respiratory chain activity [19 – 22]. NADH and
FADH2 are products of the Krebs cycle and h-oxidation of
fatty acids. NADH is also generated by glycolysis. NADH
feeds reducing equivalents, i.e., electrons, into the ETC
where they are transferred from cytochrome aa3 to 1/2 O2.
Thus, the steady-state NADH level is established by the
balance between its consumption by electron transport
(oxidation) and its generation by mitochondrial dehydrogenases (reduction). DAH is mainly generated by NADH,
M.L. Riess et al. / Cardiovascular Research 61 (2004) 580–590
and Dcm is largely dependent on DAH, so mitochondrial
NADH is a useful measure of the mitochondrial energy
state.
There is evidence that mitochondria transport Ca2 + in
order to regulate m[Ca2 +], which thereby controls activation
of Ca2 +-sensitive dehydrogenases, and in turn, oxidative
phosphorylation [23]. Ca2 + is a ubiquitous second messenger; it is the only one known to interact directly with
mitochondria [24]. mCa2 + controls critical elements of the
Krebs cycle, including several non-mitochondrial dehydrogenases, and may modulate mitochondrial membrane permeability of H+. Thus, it is plausible that a small increase in
m[Ca2 +], as with increased contractile [Ca2 +], stimulates
respiration to match energy demand with supply.
Mitochondria continuously generate O2 at about 3 – 5%
of total O2 consumption due to electron leak by mitochondrial oxidoreductases, especially complexes I and III [25,26].
Efficiency of mitochondrial respiration is related to the
activities of these complexes. Slower activity is proportional
to electron leak and ROS formation [27].
4.2. Cardiac hypothermic perfusion
Hypothermic perfusion, i.e., in the absence of ischemia or
cardioplegia, is known to increase cytosolic [Ca2 +] which
impairs relaxation; the mechanism for Ca2 + loading is not
clear but it is likely multi-factorial [7]. The positive inotropic
effect of cold perfusion at temperatures between 37 and 15 jC
depends in part on an increase in maximal Ca2 + activated
force [28]. Slowing of sarcoplasmatic reticulum function is
also a factor [29], but likely not the primary mediator. Altered
myofibrillar Ca2 + sensitivity [7] may also contribute to the
increased strength of contraction. However, it is widely
believed that cold perfusion increases cytosolic [Ca2 +] and
contractile force primarily by slowing Na+/K+-ATPase and
Ca2 +-ATPase activities. Ion pumps and co-transporters are
very sensitive to temperature changes so that an imbalance
between ion pump activity and ion leaks can cause cytosolic
Ca2 + loading [30 – 32]. For example, 10 jC hypothermia
reduced Na+/K+-ATPase activity to 15% of that at 37 jC and
increased total cell Na+ and Ca2 + content [33].
Mitochondria take up Ca2 + in an energy-requiring process driven by Dcm, H+ extrusion, and extramitochondrial
(cytosolic) Ca2 + [34]. Ca2 + enters mitochondria primarily
by the Ca2 + uniporter and exits primarily by the mitochondrial Na+/Ca2 + exchanger (NCE). But little is known about
mCa2 + loading during hypothermic perfusion and cold I/R.
We suggest that an alteration in Dcm, as supported by the
concomitant increases in NADH and ROS, is associated
with a net influx of Ca2 + from the cytosolic to the mitochondrial compartment. However, it is unclear if mCa2 +
loading results in mitochondrial dysfunction or if mitochondrial dysfunction contributes to mCa2 + loading.
We observed that NADH increased during cold perfusion.
This could arise either as a result of increased production or
decreased consumption of NADH; thus either effect could
587
change Dcm and alter respiratory efficiency to allow electron
leak. Since m[Ca2 +] was elevated and because mCa2 + contributes to regulation of Ca2 + sensitive dehydrogenase activity, the increase in m[Ca2 +] may cause an increase in NADH
production. On the other hand, if ATP synthesis at complex V
is reduced because of decreased metabolic demand, NADH
would also increase, i.e., decreased oxidation.
We have explored the source, kind, and role of ROS
generation during 17 jC cardiac perfusion in preliminary
experiments [35]. Since complexes I and III are sources of
electron leak with formation of ROS in the presence of O2,
it is possible that available excess reducing equivalents,
coupled with slowed mitochondrial oxidoreductase and
ROS scavenger enzyme activities, allow electron leak
and formation of O2 and other reactive species and
reactants. The reversible increases in NADH and ROS
during hypothermia suggest an attenuation of ETC primarily at complex I and/or III.
It may appear paradoxical that ROS induced by cold
perfusion for nearly 1 h does not cause mitochondrial or
cellular dysfunction. But hypothermia itself does not cause
cardiac ischemia; in contrast severe cold induces reductions
in metabolism and contractility to confer cardioprotection,
although it is suboptimal. Rauen et al. [36] found that
cultured rat hepatocytes as well as endothelial cells incubated in 4 jC Krebs buffer were injured under normoxic
conditions and protected under hypoxic conditions; this
injury was largely decreased when cells were incubated
with one of several ROS scavengers. Their study and our
results imply that ROS generation may be deleterious
consequence of longer and more severe cold perfusion.
Moreover, the changes in NADH, ROS and mCa2 + during
cold perfusion in our study indicate that these measures of
mitochondrial function are indeed tightly interrelated.
4.3. Cold cardiac ischemia
It is well known that the lower the temperature, the longer
the heart can be protected against I/R injury. We have shown
previously that hypothermia, with and without cardioplegia,
provides protection against increasingly longer ischemia as
temperature is reduced [1]. Nevertheless, hypothermia, even
at increasingly lower temperatures, does not provide complete protection as the duration of ischemia is lengthened. In
the present study it was not our intention to examine the
protective benefits of hypothermia with long-term ischemia,
but rather to better understand how hypothermia protects
mitochondrial function during the early phase of ischemia
and to assess which mitochondrial factors are important for
eliciting overall myocardial protection.
During ischemia, the normal flow of electrons via the ETC
is reduced because of the paucity of O2 to accept electrons at
complex IV. This results in NADH accumulation, electron
leak with escape of ROS outside the ETC, decreased Dcm,
and reduced ATP synthesis. Although both warm and cold
ischemia initially caused NADH to increase in our study,
588
M.L. Riess et al. / Cardiovascular Research 61 (2004) 580–590
NADH remained elevated during cold ischemia, whereas
during warm ischemia NADH decreased after the initial
increase. This suggests, along with our previous observations
in preconditioned hearts [4,6], that it is not the decreased
oxidation of NADH during early ischemia, but rather its
increase during continued ischemia, i.e., an imbalance in
mitochondrial bioenergetics, that is deleterious during ischemia and associated with I/R injury. In this way, it appears that
hypothermia protects the myocardium during ischemia by
preserving mitochondrial bioenergetics. This is also clearly
reflected in the prevention of excess ROS formation during
cold ischemia compared to warm ischemia. That ROS are
produced during warm ischemia before reperfusion is in
agreement with other studies [5,16,37].
Hypothermia and warm ischemia both alter Ca2 + balance
across cells, but by different mechanisms. Ferrari et al. [38]
observed a lesser increase in tissue and mCa2 + content and a
maintained rate of ATP production in isolated rabbit hearts
after hypothermic vs. normothermic ischemia; they suggested that the protective effect of hypothermia on reperfusion function was related more to reduced Ca2 + content than
to a slowed rate of energy utilization. Cytosolic Ca2 +
overload and a subsequent mCa2 + overload have been
causally implicated in normothermic I/R injury [3,10,39].
Excess mCa2 + can compromise oxidative phosphorylation
[18]. The rise in m[Ca2 +] during ischemia is due in large part
to the rise in cytosolic [Ca2 +] that occurs with slowed Na+
and Ca2 + pump activity and enhanced NCE that leads to
increased m[Ca2 +] entry through its uniporter. Under normal
conditions, a small increase in m[Ca2 +] during increased
workload stimulates the Krebs cycle and NADH production
via Ca2 +-dependent mitochondrial dehydrogenases to accelerate ATP synthesis [22]. This suggests preserved handling
of mCa2 +, possibly by better maintenance of Dcm with less
mitochondrial damage and reduced leakage of ions through
mitochondrial membranes, or because cytosolic [Ca2 +] is
less elevated [1,2]. The present study is the first to show that
hypothermic ischemia protects the intact myocardium by
preventing further mCa2 + loading during ischemia.
4.4. Reperfusion after cardiac ischemia
Reperfusion/reoxygenation after short-term normothermic ischemia/hypoxia can result in severe m[Ca2 +] loading
[3,40,41] and decrease mitochondrial potentials, DAH and
Dcm, necessary for ATP synthesis [42]. The mechanism of
mCa2 + overload with warm I/R is believed to involve increased cytosolic Ca2 + overload leading to mCa2 + overload
which then inhibits ETC; reduced ATP synthesis in turn
reduces ATP-dependent Na+ and Ca2 + pumps and causes a
vicious cycle of mCa2 + loading [43]. We have already shown
that warm ischemia depletes the reducing equivalents necessary to drive respiration on reperfusion [4,6].
The role of ROS in effecting cardiac injury on aerobic
reperfusion after normothermic ischemia is now well known
[44,45]. Sources of ROS are primarily complexes I and III;
other sources are NAD(P)/NADH oxidases, xanthine oxidase, cyclooxygenase/lipoxygenase, cytochrome P450 [46 –
49]. Increasing redox potential at a given [O2] increases ROS
generation [50].
Excess ROS cause cell damage by oxidizing DNA,
proteins, carbohydrates and membrane phospholipids.
ROS damage the Na+ pump [51] that may cause a positive
feedback generation of ROS. ROS are in part responsible for
increasing myocyte Ca2 + [52], in part by slowing Ca2 +
uptake and augmenting reverse mode NCE [53]. Exposure
to ROS generators decreases myofilament Ca2 + sensitivity
[54], while ROS scavengers given after ischemia improve
Ca2 + sensitivity [55]. Thus, in addition to ROS, elevated
cytosolic and mCa2 + are thought to be major factors in I/R
injury. As during ischemia, the exact relationship between
Ca2 + and ROS in warm reperfusion injury, however, is
controversial [48,56]. mCa2 + overload could occur first to
inhibit mitochondrial function so that ROS are produced.
Alternatively, ROS could precede Ca2 + loading and make
way for Ca2 + influx into the mitochondria. ROS formation
may also impair other cellular metabolic pathways and
indirectly lead to decreased ATP synthesis [53]. We showed
that ROS are formed during hypothermia and that, despite a
further increase in ROS at the beginning of cold ischemia,
hypothermia prevents the excess formation of ROS during
prolonged ischemia and the elevated ROS levels throughout
reperfusion observed in normothermic hearts. Our results
contribute to an understanding of these interrelationships by
examining how cooling alters changes in mitochondrial
function.
In summary, we have shown that hypothermic perfusion
increases mCa2 +, attenuates electron transport, and causes
moderate formation of ROS. During short-term ischemia,
hypothermia preserves mitochondrial energy balance, decreases ROS formation, and prevents Ca2 + overload. This
is associated with cardioprotection during reperfusion. Although it is not clear at this point which of these phenomena
are causes and which are effects, their temporal association
underlines the tight relationship of mCa2 +, redox state and
ROS formation. Also, moderate increases in m[Ca2 +] levels,
ROS formation or a NADH reduction do not lead to damage,
per se. Moreover, it is the level of m[Ca2 +] and ROS
formation and the rate of NADH oxidation during continued
ischemia that determines the degree of I/R injury. A potential
clinical benefit of this study is the knowledge that protection
by hypothermia occurs early during the phase of ischemia and
not just on reperfusion. Future studies will be directed toward
improving techniques to ultimately better preserve mitochondrial bioenergetics during hypothermic ischemia.
Acknowledgements
The authors wish to thank Dr. Janis Eells, Dr. Samhita
Rhodes, Dr. Ming Tao Jiang, Michelle Henry, and James
Heisner for their valuable contributions to this study.
M.L. Riess et al. / Cardiovascular Research 61 (2004) 580–590
This work was supported in part by grants No. HL58691
(to Drs. Stowe and Camara) from the National Institutes of
Health, Bethesda, MD, and by grant No. Ri 610005 (to Dr.
Riess) from the Medical Faculty of the WestfälischeWilhelms-Universität, Münster, Germany.
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