© 1998 American Heart Association, Inc.
Original Contributions |
Inhibition of Rat Cardiac Muscle Contraction and Mitochondrial Respiration by Endogenous Peroxynitrite Formation During Posthypoxic Reoxygenation
From the Department of Physiology, New York Medical College, Valhalla.
Correspondence to Michael S. Wolin, PhD, Professor, Department of Physiology, New York Medical College, Valhalla, NY 10595.
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Abstract—This study was designed to investigate the potential role of endogenous peroxynitrite (ONOO-) formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation. Isometric contraction of isolated rat left ventricular posterior papillary muscle was virtually eliminated at the end of an exposure to 15 minutes of hypoxia and remained 40±5% depressed an hour after the reintroduction of O2. O2 uptake by rat left ventricular cardiac muscle, measured by a Clark-type O2 electrode, was also inhibited by 24±2% at 10 minutes after reoxygenation. The inhibition of contractility and respiration during posthypoxic reoxygenation was markedly attenuated by the NO synthase inhibitor nitro-L-arginine, exogenous superoxide dismutase, and the ONOO- scavenger urate but not by the hydroxyl radical scavenger mannitol. Generation of ONOO- with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) plus the superoxide-releasing agent pyrogallol caused an irreversible inhibition of cardiac contractile and respiratory function. Unlike ONOO-, exogenous (SNAP) and endogenous (bradykinin) sources of NO inhibited contractility in a reversible manner. Under conditions of comparable amounts of respiratory inhibition in unstimulated incubated muscle, the NO-dependent agents and the mitochondrial antagonist NaCN produced a smaller degree of suppression of contractility compared with ONOO- and posthypoxic reoxygenation. These results are consistent with a contributing role for endogenous ONOO- formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation.
Key Words: cardiac contractility • mitochondrial respiration • hypoxia/reoxygenation • nitric oxide • superoxide
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Introduction |
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Mechanisms leading to tissue injury and organ dysfunction after ischemia/reperfusion or hypoxia/reoxygenation are generally rather poorly understood. Aspects of cardiac contractile performance are markedly depressed during postischemic reperfusion or posthypoxic reoxygenation.1 2 3 4 5 6 After exposure of the heart to ischemia/reperfusion or hypoxia/reoxygenation, contractile dysfunction is observed together with an elevated production of reactive O2 and N2 species4 5 6 and impaired mitochondrial function.7 8 9 Endogenous peroxynitrite (ONOO-) formation derived from NO and superoxide (O2-) has been suggested to be responsible for the cardiac contractile dysfunction during postischemic reperfusion.5 6 Infusion of ONOO- into isolated rat hearts has been found to impair cardiac contractile performance by decreasing cardiac efficiency.10 However, the exact mechanisms involved in the mediation of contractile dysfunction remain an open question. The activities of components of the mitochondrial respiratory chain have been reported to be largely reduced after exposure to hypoxia/reoxygenation or ischemia/reperfusion.7 8 9 Furthermore, we have previously shown that endogenous ONOO- formation during posthypoxic reoxygenation contributes to a prolonged suppression of mitochondrial respiration in intact cardiac muscle.11 Because aerobic metabolism is essential in maintaining cardiac contractile performance, impaired mitochondrial function may contribute to the contractile dysfunction observed during postischemic reperfusion or posthypoxic reoxygenation. Therefore, the present study was focused on examining whether endogenous ONOO- formation contributes to cardiac contractile dysfunction and respiratory inhibition after exposure to a short-term model of hypoxia and reoxygenation.
Evidence exists that NO inhibits cardiac myocyte contractility and that this mechanism could be an important process in the control of cardiac function.12 13 14 We have previously shown that NO and ONOO- suppress respiration in bovine cardiac muscle through reversible and irreversible mechanisms, respectively.11 Therefore, the present study was also designed to investigate the relationship between the effects of NO and ONOO- on contractile performance and mitochondrial respiration in isolated rat cardiac muscle.
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Materials and Methods |
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Materials
BK, 8-bromo-cGMP, catalase, mannitol, uric acid, SOD from bovine erythrocytes, HEPES, and lucigenin were purchased from Sigma Chemical Co. L-NA was obtained from Aldrich Chemical Co, and sodium cyanide and pyrogallol were from JT Baker Chemical Co. SNAP was synthesized as described.15 All materials were dissolved in Krebs' solution containing (mmol/L) NaCl 118, KCl 4.7, CaCl2 1.5, NaHCO3 25, KH2PO4 1.2, MgSO4 1.1, and glucose 5.6, pH 7.4.
Cardiac muscle was obtained from male Wistar rats at 3 months of age that were anesthetized with pentobarbital sodium (50 mg/kg body wt IP). Hearts were immediately excised and rinsed at least twice in ice-cold Krebs' solution.
Respiration Measurement
Cardiac muscle was isolated from the left ventricle (free
wall only) of these rat hearts and then cut into small segments that
were 20 to 30 mg in weight and 1 to 2 mm in thickness. Before
studies of tissue respiration, these muscle segments were bathed at
37°C for 2 hours in Krebs' solution, in which 21%
O2/5% CO2/balance
N2 was continuously bubbled.
O2 uptake by cardiac muscle slices was measured
polarographically in 3 mL of air-saturated Krebs' solution buffered
with 10 mmol/L HEPES-NaOH (pH 7.4) at 37°C by a Clark-type
O2 electrode (Yellow Springs Instrument Co), as
previously described.11 16 17 Tissue respiration
was calculated as the rate of decrease of O2
concentration after the addition of muscle segments, assuming an
initial O2 concentration of 224 nmol/mL, and was
expressed as nanomoles consumed per minute per gram tissue (wet
weight). O2 uptake by muscle slices was monitored
only before one third of the O2 was consumed, and
any spontaneous changes in the electrode recording in the
absence of tissue were subtracted from each measured rate of tissue
O2 consumption. The mitochondrial
inhibitor NaCN, the NO donor SNAP, the
endogenous NO stimulus BK, the cell-permeable cGMP analogue
8-bromo-cGMP, and ONOO- generation with
0.01 mmol/L SNAP plus 0.01 mmol/L pyrogallol were examined
for their acute effects on cardiac muscle respiration. The presence of
the ONOO--generating system directly caused a
small amount of O2 consumption, and this was
subtracted from the amount of respiration observed in the presence of
cardiac muscle. Respiration was also examined 15 minutes after the
ONOO--generating system was washed out of the
tissue. Effects of a short-term model of hypoxia (15 minutes)
and reoxygenation (10 minutes) on
O2 consumption by cardiac muscle were also
studied in the absence or presence of the NO synthase
inhibitor L-NA, the
O2- scavenger SOD, the
ONOO- scavenger uric acid, and the hydroxyl
radical scavenger mannitol. The typical observation time for
respiration measurements was 6 to 8 minutes, and new muscle segments
were used for each drug examined. At the end of each measurement,
1 mmol/L of NaCN was added to confirm that changes in
O2 consumption were from mitochondrial sources.
Data were analyzed as the percent change of the rate of
respiration for each muscle slice studied.
Tension Measurement
Preparation of left ventricular papillary muscle
from male Wistar rats was similar to previous
reports.18 19 The posterior papillary muscle (0.5
to 1 mm in diameter, 5 to 7 mm in length, and 
30 mg in
weight) was carefully and rapidly removed. The nontendinous end of the
papillary muscle was tied to a fixed steel segment with Ethicon 6-0
braided silk, and the tendinous end of the papillary muscle was tied to
a steel wire hook that was connected to a force transducer for
measurement of changes in isometric contraction with a Grass polygraph
recorder (model 79). The papillary muscle was placed vertically in
3 mL of Krebs' buffer and gassed continuously with
21%O2/5% CO2/balance
N2 in a cuvette mounted in a cell holder
thermostated at 37°C. The papillary muscle was maintained at a basal
tension of 5 to 20 mN/mm2 to attain maximal
developed tension, and the papillary muscle was stimulated at 0.1 Hz by
rectangular depolarizing pulses 5 milliseconds in duration and twice
diastolic threshold in intensity (30 to 50 V) with a Grass
S44 stimulator. After an equilibration of 60 to 90 minutes, changes in
developed and diastolic tension of the papillary muscle
that were caused by addition of probes or exposure to a short-term
hypoxia (15 minutes) and reoxygenation were
recorded on-line with a simultaneous measurement of
O2- production in the
presence of 0.25 mmol/L lucigenin. NaCN, SNAP, BK, 8-bromo-cGMP,
and the ONOO--generating system (0.01
mmol/L SNAP+0.01 mmol/L pyrogallol) were examined for their acute
effects on the contractile performance of rat papillary muscle,
and the effects of SNAP and BK were examined 15 minutes after these
agents were washed out of the tissue. During the 15 minutes of
hypoxia, L-NA, uric acid, and SOD were added to examine their
effects on papillary muscle contractile function during posthypoxic
reoxygenation. This protocol also minimized
complications associated with SOD causing a slowly developing foaming
effect. Since mannitol was observed to increase force generation by
28±4.2%, it was added before hypoxia, and responses were
compared with the level of force generated in the presence of mannitol
under 21% O2. The recorded contractile
amplitude was measured in gram units, and data were transformed into
millinewtons and then divided by the cross-sectional area of the
papillary muscle for expression of force generation in units of
millinewtons per millimeter squared.
Detection of Superoxide Anion
Superoxide anion was detected primarily to help identify
conditions in which cardiac muscle superoxide production or
scavenging was altered by the treatments used. Under the same
conditions as the contractility study, lucigenin
chemiluminescence was used to detect
O2- production by rat
posterior papillary muscle in a lighttight box with a photon-counting
apparatus (Thorn EMI electron tubes) that we have
previously described.20 This photon-counting
apparatus includes a cooled Thorn EMI photomultiplier tube
(model 9235B), a Thorn EMI amplifier-discriminator (model C604), and a
photon counter (model C660). The photon counts were integrated over
5-second periods and shown on a computer monitor; meanwhile, an analog
signal of the integrated photon counts was continuously recorded on
the same polygraph recorder as was the contractile
performance. The photon counts are expressed in the text as
counts per minute per gram after subtraction of the background dark
current of the photomultiplier. Our previous
work20 21 examining endogenously
produced O2- in vascular and
cardiac myocyte preparations by lucigenin chemiluminescence is
consistent with lucigenin detecting intracellular
O2- and its scavenging by NO.
In the absence of tissue, interventions such as hypoxia and
reoxygenation did not cause changes in lucigenin
chemiluminescence. In addition, the absolute magnitudes of
modifications in cardiac muscle–derived chemiluminescence during
hypoxia and reoxygenation need to be
interpreted with caution, since they could be affected by processes
that potentially alter the sensitivity of the lucigenin method, such as
changes in the redox state of lucigenin, pH, and alterations in lipid
metabolism (eg, see References 2222 to 24).
Statistical Analysis
All data in the text are reported as mean±SE, and n in all
instances represents the number of different animals studied.
Differences in the mean values were analyzed using unpaired
and, where appropriate, paired Student t tests. A Bonferroni
correction was applied when multiple groups were compared. Statistical
significance was assumed at P<0.05.
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Results |
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Effects of Exogenous NO on Cardiac Muscle Contractile and Respiratory Function
The NO donor SNAP reduced the developed tension of isolated rat posterior papillary muscle by up to 23±6.5% (n=6) at cumulative doses of 10-7 to 10-4 mol/L (Figure 1
), with statistical significance
starting at 10-5 mol/L. Under these conditions,
O2- generation detected by
lucigenin chemiluminescence (2.7±1.3x104 cpm/g,
n=6) was significantly decreased by 23±8% and 32±13% at the two
larger concentrations of SNAP, respectively. Similar doses of SNAP
significantly suppressed tissue respiration of rat cardiac muscle by up
to 35±4% (n=5, Figure 1), which is comparable to its previously
reported effects on O2 consumption by canine and
bovine myocardial muscle.11 16 The inhibition of
respiration at each dose of SNAP below 10-4
mol/L was significantly greater than the attenuation of developed
tension. The average O2 consumption rate of rat
cardiac muscle was 299±22 nmol ·
min-1 · g-1
(n=10). The effects of SNAP on the attenuation of developed tension
were reversed after its removal by washout (2.6±1.0% inhibition).
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Effects of Endogenous NO on Contraction and
Respiration
The stimulus of endogenous NO biosynthesis, BK,
significantly depressed the developed tension of papillary muscle by up
to 23±4.4% (n=7) at cumulative doses of 10-7
to 10-4 mol/L (Figure 2). Meanwhile, none of these doses of BK
significantly altered O2-
generation detected by lucigenin chemiluminescence
(8.4±1.9x104 cpm/g, n=7). Similar
concentrations of BK significantly suppressed tissue respiration of rat
cardiac muscle by up to 34±1.4% (n=5, Figure 2), which is comparable
to its previously reported effects on O2
consumption by canine and bovine myocardial
muscle.11 16 The inhibition of respiration at
each dose of BK was significantly greater than the attenuation of
developed tension. The NO synthase inhibitor L-NA (0.1
mmol/L) did not significantly affect the contractile amplitude of
papillary muscle or detection of
O2- production (not
shown), but it did eliminate the suppression of developed tension
caused by BK (n=4, Figure 2). Similarly, L-NA markedly attenuated the
inhibition of tissue respiration of cardiac muscle caused by BK (n=5,
Figure 2). The effects of BK on the attenuation of developed tension
were reversed after its removal by washout (5.7±6.0% inhibition).
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Effects of a cGMP Analogue on Contraction and Respiration
The cell-permeable and stable cGMP analogue 8-bromo-cGMP reduced
the developed tension of papillary muscle by up to 18±4% (n=7) at
cumulative doses of 10-7 to
10-4 mol/L (Figure 3), with statistical significance
starting at 10-5 mol/L. None of these
concentrations of 8-bromo-cGMP significantly affected
O2- generation detected by
lucigenin chemiluminescence (11±2.4x104 cpm/g,
n=7). As previously observed with canine myocardial
muscle,16 similar doses of 8-bromo-cGMP
suppressed tissue respiration of rat cardiac muscle by up to 18±3%
(n=4, Figure 3), with statistical significance starting at
10-6 mol/L. The inhibition of respiration was
not significantly greater than the attenuation of developed tension at
any dose of 8-bromo-cGMP.
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Effects of NaCN on Contraction and Respiration
The mitochondrial cytochrome oxidase inhibitor
NaCN significantly reduced the developed tension of papillary muscle by
up to 76±6.7% (n=9) at increasing doses of
10-6 to 10-3 mol/L
(Figure 4). Meanwhile,
O2- generation measured by
lucigenin chemiluminescence (2.1±0.4x104 cpm/g,
n=9) was significantly increased by 21±12% and 142±22% at the two
larger concentrations of NaCN, respectively. Similar doses of NaCN
significantly suppressed tissue respiration of the cardiac muscle by up
to 73±2.5% (Figure 4). The inhibition of respiration at each dose of
NaCN below 10-3 mol/L was significantly greater
than the attenuation of developed tension.
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Effects of ONOO- Generation on Contraction and
Respiration
The developed tension of papillary muscle was markedly suppressed
by 36±5.3% (n=5, Figure 5) by exposure
to an ONOO--generating system using 0.01
mmol/L SNAP plus 0.01 mmol/L pyrogallol.11
This depression of force generation by ONOO-
remained stable for at least 1 hour. The degree of inhibition of force
generation by this ONOO--generating system was
significantly greater than the inhibition of 9.9±3.2% caused by
0.01 mmol/L SNAP (in the absence of pyrogallol), and treatment
with 0.01 mmol/L pyrogallol alone did not detectably alter force
generation. As detected by lucigenin chemiluminescence,
O2- production by the
papillary muscle was not significantly different after exposure to the
ONOO--generating system (7.3±7.4%, n=5).
Similar to observations made in bovine myocardial
muscle,11 the generation of
ONOO- acutely inhibited the tissue respiration
of rat cardiac muscle by 30±1.1% (n=4, Figure 5); cardiac muscle
respiration remained suppressed by 29±3.3% (n=4) when it was
reexamined 15 minutes after washout of the
ONOO--generating system.
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Effects of Hypoxia/Reoxygenation on Cardiac
Muscle Contractile and Respiratory Function
The developed tension of rat papillary muscle was severely
depressed 81±4% (n=6) at the end of a 15-minute period of exposure to
severe hypoxia (Figure 6).
Reintroduction of O2 resulted in some recovery of
contractile function, but the developed force was still depressed by
40±5.2% for up to 1 hour after reoxygenation (Figure 6). Interestingly, in the presence of 0.1 mmol/L L-NA, the
depression of contractile performance during
reoxygenation was only 11±1.8% (n=6, Figure 6). The
depression of the developed force during hypoxia was not
altered by L-NA (Figure 6). Similar to L-NA, uric acid (0.1
mmol/L), an intracellular scavenger of
ONOO-,11 markedly
attenuated the depression of the developed tension of papillary muscle
during reoxygenation to 14±2.8% inhibition, without
affecting the depression of contractile function by hypoxia
(n=7, Figure 6). The addition of SOD (3 µmol/L) 5 minutes before
reoxygenation also largely decreased the suppression of
the contractile amplitude of papillary muscle during
reoxygenation to 18±7%, without altering the effects
of hypoxia (n=6, Figure 6). However, mannitol (100 mmol/L,
added before hypoxia), a scavenger of the hydroxyl radical, did
not alter the effects of reoxygenation. These results
with L-NA, uric acid, and SOD provide evidence for an involvement of
endogenous ONOO- formation from NO
and O2- in the depression of
cardiac contractile function during posthypoxic
reoxygenation.
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The simultaneous detection of
O2- with lucigenin
chemiluminescence revealed a 91±2% decrease during hypoxia
(Figure 7), followed by a transient
overshoot (130±27% increase, Figure 7) within the first 2 to 3
minutes of reoxygenation. The steady-state level of
detectable O2- after the
transient increase during reoxygenation was not
significantly different from control (n=6). During
reoxygenation, the overshoot of
O2- levels detected by
lucigenin chemiluminescence was significantly attenuated by exogenous
SOD (Figure 7). However, SOD did not alter the detection of superoxide
anion when its production returned to the steady-state level
seen before exposure to hypoxia. An observation for which we
currently do not have an explanation is that the markedly depressed
level of lucigenin chemiluminescence observed under hypoxia was
slightly greater in the presence of SOD. On the basis of the effects of
the addition of SOD on the detection of
O2- and the protective effects
of SOD on contractile function, superoxide anion seems to be released
into the extracellular environment only during the first 2 to 3 minutes
of reoxygenation.
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Tissue respiration of rat cardiac muscle after the 15-minute
exposure to hypoxia and 10-minute period of
reoxygenation was also significantly depressed by
24±2.4% (n=10, Figure 8), which is
similar to the degree of inhibition of O2
consumption produced by a comparable treatment of bovine cardiac
papillary muscle.11 The inhibition of respiration
by hypoxia/reoxygenation was significantly less
than the attenuation of developed tension. This suppression of tissue
respiration by posthypoxic reoxygenation was markedly
attenuated (see Figure 8) by the addition of 0.1 mmol/L L-NA
(6.7±2.9%, n=6), 0.1 mmol/L uric acid (7.1±3.2%, n=6), and
3 µmol/L SOD (8.8±2.9%, n=5) but not by 100 mmol/L
mannitol (27±3.6%, n=5).
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Discussion |
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The data reported in the present study are consistent with a key role for ONOO- in the prolonged inhibition of contractility and respiration of rat cardiac muscle observed during posthypoxic reoxygenation. Since L-NA and uric acid did not alter the marked suppression of contractile performance of rat cardiac papillary muscle caused by hypoxia, NO and ONOO- may not be involved in this response. The reduction in contractile performance during ischemia has been previously suggested to be due to metabolic changes associated with the loss of creatine phosphate, increased intracellular acidosis, and the accumulation of inorganic phosphate and lactate.25 26 27 28 During reoxygenation after the 15-minute period of hypoxia, the contractile performance of electrically stimulated rat papillary muscle was markedly depressed by 40% for up to 60 minutes, consistent with the findings of previous studies with ischemia/reperfusion1 2 and hypoxia/reoxygenation.3 Since L-NA, SOD, and uric acid markedly suppressed the observed depression of contractile function during posthypoxic reoxygenation, NO-derived endogenous ONOO- formation is mainly responsible for the suppression of force generation during reoxygenation. The absence of an effect of mannitol suggests that hydroxyl radicals do not detectably contribute to this effect of reoxygenation. The transient increase in O2- detected by lucigenin on reoxygenation is also consistent with the formation of ONOO- during this period. Similar to studies with bovine cardiac muscle,11 endogenous ONOO- appears to form in rat cardiac muscle during posthypoxic reoxygenation in amounts sufficient to inhibit irreversibly tissue respiration. Thus, endogenous ONOO- formation during posthypoxic reoxygenation is likely to contribute to the observed depression of cardiac contractility and tissue respiration.
In rat cardiac muscle, ONOO- formation appears to increase the potency and duration of NO as an inhibitor of cardiac muscle contractile performance. Both exogenous and endogenous sources of NO from SNAP and BK, respectively, significantly depressed the contractile amplitude of rat left ventricular papillary muscle. Increased cGMP formation is likely to contribute to these NO-elicited responses, because the cGMP analogue 8-bromo-cGMP also caused an inhibition of contraction. Previous studies indicate that NO has an inhibitory effect on the contractile amplitude of cardiac muscle.12 13 14 Elevated cGMP levels12 13 and inhibited Ca2+ transients, potentially originating from modulation of sarcoplasmic L-type Ca2+ channels,29 have been previously suggested to contribute to the effect of NO on contractile function.14 The generation of O2- potentiated the inhibitory effect of NO on rat cardiac muscle contractile function, suggesting that the potency of NO was enhanced by conditions that promote ONOO- formation. Studies on the actions of ONOO- produced endogenously during posthypoxic reoxygenation indicate that its effects on rat papillary muscle contractility and respiration in rat and bovine11 cardiac muscle seem to be irreversible. The inhibition of contraction and respiration elicited by BK and SNAP did not appear to involve an interaction of NO with O2- and the formation of ONOO-, since the effects of these agents were reversible. Although BK did not alter the detection of O2- by lucigenin chemiluminescence, the two larger doses of SNAP significantly depressed the detection of endogenous O2-. Thus, if ONOO- is being produced by these doses of SNAP, the amounts formed do not have appreciable irreversible effects. The inactivation of systems that contribute to energy metabolism (mitochondrial proteins,30 glycolytic enzymes,31 and possibly creatine kinase32 ), calcium homeostasis (a sarcoplasmic reticulum Ca2+-ATPase33 ), and perhaps other processes could potentially contribute to the observed increased potency of ONOO- over NO as an inhibitor of cardiac muscle contractility. In addition, many of the systems potentially altered by ONOO-, including mitochondrial function,7 8 9 glycolytic enzymes,34 creatine kinase,35 36 and sarcoplasmic reticulum Ca2+ handling,3 also seem to be impaired by hypoxia/reoxygenation or ischemia/reperfusion.
The extent to which mitochondrial inhibition contributes to the
depression of contractile function after exposure to NO,
ONOO-, or posthypoxic
reoxygenation is difficult to assess because of the
potential complexity of the mechanisms involved and limitations of the
present study originating from measuring respiration and
contraction in different segments of cardiac muscle under basal and
stimulated levels of energy consumption, respectively. The ability of
the mitochondrial cytochrome oxidase inhibitor NaCN to
depress cardiac contraction and respiration is consistent with
the well-established concept that suppression of mitochondrial energy
metabolism can lead to depression of cardiac contractile
function. However, the effects of the higher doses of NaCN need to be
cautiously interpreted, because additional mechanisms are potentially
activated by processes, such as an increase in
O2-, resulting from the
inhibition of SOD by NaCN. NO is known to be similar to NaCN as a
potent reversible inhibitor of cytochrome
oxidase.37 38 39 It is difficult to determine the
cause-effect relationship between actions of NO on respiration and
contractile function, because NO appears to activate mechanisms
that alter both of these processes and because force generation and
mitochondrial energy metabolism are likely to be dependent
on each other in actively contracting cardiac muscle. For example, our
previous work involving incubated slices of canine cardiac and skeletal
muscle detected evidence that NO suppressed respiration by both a
cGMP-derived effect on energy consumption and through a direct
inhibitory effect of NO on mitochondrial
respiration.16 17 The inhibitory
effects of NO on O2 consumption are also seen in
the presence of mitochondrial respiration, which is stimulated in vitro
by an uncoupler of electron transport
(dinitrophenol16 ) and in vivo during increased
levels of cardiac work,40 suggesting that an
interaction of NO with cytochrome oxidase is likely to be occurring in
actively contracting cardiac muscle. Although the design of the
present study does not permit us to evaluate whether the
attenuation of contractile function by NO originated from a signaling
effect that reduces force generation or from a suppression of
respiration, the modest effect observed at large doses of NO suggests
that it could have only a minor inhibitory effect on
respiration. Several processes may limit the inhibitory
effect of large concentrations of NO on mitochondrial function. The
inhibition of cytochrome oxidase by NO may reach an equilibrium with a
reactivation process, through its ability either to metabolize
NO41 42 43 or to cause a rapid dissociation of NO
from this enzyme.44 This may explain why NO
mechanisms produce a maximum of 40% inhibition of tissue
respiration at the very high levels of NO that were examined in the
present study and in previous studies with
canine16 and bovine11
cardiac muscle. In addition, the cardiac muscle mitochondrial
metabolic state may control the coupling between electron
transport and proton pumping by cytochrome oxidase and
thermogenesis.45 Interestingly, the inhibition of
NO biosynthesis in resting conscious dogs increases whole-body
O2 consumption associated with an elevation of
body temperature.46 Thus, an action of NO on
cytochrome oxidase may improve the efficiency of
O2 utilization for energy metabolism.
In the present study, under conditions of equivalent amounts of
respiratory inhibition in unstimulated muscle,
ONOO- and posthypoxic
reoxygenation elicited a greater suppression of
contractile function than did the NO-generating agents or the
mitochondrial antagonist NaCN. These observations seem to
be consistent with the previously reported loss of cardiac
efficiency associated with exposure of isolated rat hearts to an
infusion of 40 µmol/L
ONOO-.10 Thus, limitations
of the present study, together with the apparent complexity of the
interactions that NO and its metabolites have with contractile and
respiratory systems, prevent interpretation of the influence of
mitochondrial respiration on contractile function.
In summary, an interaction of O2- with NO, associated with the formation of ONOO-, appears to be an important process that contributes to the suppression of rat cardiac muscle contractile performance and mitochondrial respiration caused by hypoxia and reoxygenation. The ability of the extracellular scavenger SOD to alleviate contractile dysfunction during posthypoxic reoxygenation observed in the present study or during postischemic recovery in isolated rat hearts47 is consistent with a potential role for O2- production by nearby cells, such as endothelium,48 in the depression of cardiac contractile and respiratory function. Development of therapeutic approaches for situations in which cardiac muscle is exposed to hypoxia/reoxygenation, such as coronary bypass surgery, heart transplantation, or thrombolytic therapy, may benefit from considering factors that control the generation and scavenging of ONOO- and the impact of its formation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was funded by US Public Health Service grants HL-43023 and HL-31069. The authors thank Dr Gabor Kaley for providing sources of Wistar rat hearts and Dr Francis L. Belloni for providing the Grass S44 stimulator.
Received December 5, 1997; accepted February 12, 1998.
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