spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online March 12, 2009
Journal of Experimental Biology 212, 954-960 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.025171
This Article
Right arrow Summary Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Misfeldt, M.
Right arrow Articles by Gesser, H.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Misfeldt, M.
Right arrow Articles by Gesser, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Nitric oxide increases myocardial efficiency in the hypoxia-tolerant turtle Trachemys scripta

Mikkel Misfeldt, Angela Fago and Hans Gesser*

Department of Biological Sciences, Building 1131, University of Aarhus, DK-8000, Aarhus C, Denmark

* Author for correspondence (e-mail: hans.gesser{at}biology.au.dk)

Accepted 13 January 2009


   Summary
TOP
 Summary
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Nitric oxide (NO) may influence cardiac mechanical performance relative to O2 consumption by depressing respiration rate and by affecting the excitation–contraction coupling. Such effects of NO should be particularly important during hypoxia in species such as the hypoxia-tolerant turtle Trachemys scripta. In heart ventricle preparations from this species, the ratio of twitch force to O2 consumption increased by approximately 15% during full oxygenation and by approximately 60% during hypoxia in the presence of added L-arginine [the substrate for nitric oxide synthase (NOS)]. This effect was primarily due to a decrease in O2 consumption and may represent an increase in the twitch force obtained per ATP and/or in the ATP obtained per O2. Lactate production during hypoxia did not differ between preparations treated with either L-arginine or asymmetric dimethylarginine (ADMA), an inhibitor of NOS, suggesting that NO does not elicit a compensatory increase in anaerobic metabolism. ADMA did not reverse the effects of L-arginine on O2 consumption significantly, although pre-treatment with ADMA abolished the effect of L-arginine, consistent with the competitive binding of L-arginine and ADMA to NOS. Histochemical studies using the fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) revealed NO production in the presence of added L-arginine. In conclusion, NO may augment heart contractility obtained per O2 by deceasing O2 consumption without affecting either lactate production or developed force. This effect was particularly pronounced under O2 deficiency and may therefore contribute towards preserving cardiac function and to the overall excellent hypoxic tolerance of the turtle.

Key words: nitric oxide, heart muscle, freshwater turtle, contractile force, oxygen consumption, hypoxia


   INTRODUCTION
TOP
 Summary
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Nitric oxide (NO) has been recognized as a signal molecule, having autocrine and paracrine actions, and as a key regulator of a number of cellular processes in vertebrates, including ectotherms (Skovgaard et al., 2005Go; Tota et al., 2005Go; Imbrogno et al., 2006Go). Different isoforms of nitric oxide synthase (NOS), which catalyzes the formation of NO from L-arginine (L-Arg), are expressed in the mammalian heart muscle. The extent of this expression varies between and within cells as well as with animal living conditions; however, typically, endothelial NOS is quantitatively the predominant isoform expressed. Although endothelial NOS isoforms from non-mammalian species have not yet been isolated, recent evidence indicates the existence of functional NOS isoforms in the heart and vasculature of dipnoi, teleosts, reptiles and amphibians (e.g. Tota et al., 2005Go; Amelio et al., 2006Go; Amelio et al., 2008Go; Jennings and Donald, 2008Go). As an important general action, NO downregulates the O2 consumption rate, primarily by inhibiting the binding of O2 to mitochondrial cytochrome c oxidase both competitively and uncompetitively (Mason et al., 2006Go; Erusalimsky and Moncada, 2007Go; Cooper et al., 2008Go). Because of its competition with O2, NO inhibits cellular respiration to a larger extent at low O2 tensions and may thus protect cellular functions under hypoxia by extending O2 availability (Hagen et al., 2003Go). Such effects may also become evident during normal oxygenation. NO has been found to increase efficiency in oxygenated heart muscle from guinea pig, by downregulating cellular respiration relative to both mechanical performance and ATP synthesis rate (Shen et al., 2001Go). NO has also been found to increase contractile performance relative to O2 consumption in dog cardiac muscle (Setty et al., 2002Go). A number of NO-dependent mechanisms may be important for myocardial hypoxia. NO may open mitochondrial ATP-sensitive K+ channels (KATP) (Ljubkovic et al., 2007Go; Jennings and Donald, 2008Go), which appear to be an element in pre-conditioning (Lebuffe et al., 2003Go), or may influence the fatty acids to carbohydrates substrate preference in the mammalian heart (Canty 2000Go; Williams et al., 2008Go) and consequently change the amount of ATP formed per O2 consumed. Apart from influencing energy metabolism, NO produced by NOS also influences Ca2+ regulation in cells and enhances the release of Ca2+ from the sarcoplasmatic reticulum (SR) in the mammalian myocardial cell (Xu et al., 1999Go; Viner et al., 2000Go; Petroff et al., 2001Go).

With this background, NO may be implicated in the remarkable hypoxic tolerance of certain ectothermic vertebrates such as freshwater turtles. Some turtle species experience prolonged periods of severe hypoxia, e.g. being submerged in water without access to air during winter hibernation and during dives in the summer (e.g. Shi et al., 1999Go; Jackson, 2002Go). Accordingly, heart muscle from turtles (Trachemys scripta and Chrysemys picta) shows an excellent maintenance of contractility and energy state when subjected to severe hypoxia (Overgaard and Gesser, 2004Go; Overgaard et al., 2007Go). The finding that hypoxia, compared with full oxygenation, increased twitch force development relative to the energy liberated, as assessed by O2 consumption and lactate production (Overgaard and Gesser, 2004Go), is of particular interest in the present study as NO may contribute to this process. For these reasons, we examined the influence of NO on mechanical performance, recorded as twitch force and resting tension, and also on cellular energy liberation, in terms of O2 consumption and lactate production, in turtle ventricular muscle under exposure to full oxygenation and hypoxia.


   MATERIALS AND METHODS
TOP
 Summary
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Experimental animals and myocardial preparations
Freshwater turtles (Trachemys scripta Schoepff) were obtained from Lemberger Reptiles (Oshkosh, WI, USA) and kept in water tanks at 20°C in a 12 h:12 h light:dark cycle. They had free access to basking platforms and were fed regularly.

The turtles were captured with a net and decapitated. The plastron was opened with a clinical bone saw, and the heart was quickly transferred to an ice-cold physiological solution containing (mmol l–1): NaCl (95), KCl (2.5), MgSO4 (0.94), NaHCO3 (25), NaH2PO4.H2O (1), CaCl2 (1) and glucose (5), equilibrated with 48% O2, 2% CO2 and 50% N2, which resulted in pH 7.7.

Ring-shaped preparations were made from the ventricle. The rings weighed between 18 and 50 mg.

Experimental setup
The experimental setup has been described previously (Kalinin and Gesser, 2002Go; Overgaard and Gesser, 2004Go). Briefly, a single ring-shaped preparation was mounted on two hooks. The lower end of the myocardial preparation was attached to one hook fixed in the measuring chamber whereas the upper end of the preparation was carried by a second hook connected to the force transducer (Fort 10; World Precision Instruments, Sarasota, FL, USA) through a hole in the lid of the measuring chamber. A micrometer screw allowed the force transducer to be moved vertically and the length of the myocardial preparation to be adjusted. The micrometer screw also provided the distance between the two hooks and thus an estimate of the preparation length, which was in the range of 4–10 mm.

Two silver electrodes, coated with silver chloride, were placed on opposite sides of the myocardial preparation and connected to a stimulator (Grass SD 9, Quincy, MA, USA) through two separate holes in the lid of the measuring chamber. The stimulator provided square pulses at a rate of 0.25 Hz and with a polarity that was altered between stimulations. Each stimulation had a duration of 5 ms and a voltage 1.5 times that which was necessary to elicit full twitch force development (e.g. Kalinin and Gesser, 2002Go).

The physiological solution was recirculated between the measuring chamber (2.56 ml) and the reservoir (20 ml). The solution contained in the reservoir was continuously bubbled with a gas mixture delivered by a precision gas-mixing pump (Wösthoff, Bochum, Germany). The chamber with the O2 electrode and the reservoir were thermostatically controlled to 20°C, and the solution in the chamber was continuously stirred using a glass-covered magnetic stir bar. During measurements of O2 consumption and of the associated twitch force, the recirculation of the solution was stopped and the decrease in O2 tension in the chamber was recorded over time using an O2 electrode (Radiometer E5046, Copenhagen, Denmark).

Values of O2 tension and force were sampled at a rate of 100 s–1 using a Biopack MP100 data acquisition system (Biopack Systems, Goleta, CA, USA) connected to a computer using the program Acqknowledge 3.7.0 (Biopack Systems). The rate of O2 tension change was obtained by linear regression analysis, and the twitch force was determined as the difference between the minimal (resting tension) and maximal force values.

The myocardial preparations were stretched to the peak of the force–length relationship. Force development was allowed to stabilize for at least 60 min before experiments began.

Equipment calibration
The O2 electrode was calibrated each day. A zero value was obtained using a solution of 1.6 mmol l–1 NaSO3 in 10 mmol l–1 Na2B4O7 Borax (sodium tetraborate), and the value of atmospheric O2 was obtained by gassing the solution with air. The physiological solution was equilibrated with 48% O2, 50% N2 and 2% CO2. A 20 min test was performed to assess any background O2 changes without tissue in the chamber and with stimulation switched on. Values below 0.2 µmol O2 min–1 were accepted and subtracted from the O2 consumption rate in the presence of myocardial tissue.

Experimental protocol
The effects of 0.1 mmol l–1 L-Arg on O2 consumption and twitch force were examined in an experimental series in which O2 was either high (48% O2, 50% N2, 2% CO2) or low (8% O2, 90% N2, 2% CO2). This concentration of L-Arg was chosen to maximize NO production by saturating NOS with L-Arg, as Michaelis constant, Km, for L-Arg has been assessed to be 0.6 µmol l–1 (Matsuoka et al., 1994Go). Experiments also included the application of 1 mmol l–1 asymmetric dimethylarginine (ADMA), which inhibits cellular NOS activity (Kodja and Kottenberg, 1999Go; Böger et al., 2003Go). Preliminary experiments using L-NAME, a commonly used NOS inhibitor, have shown no significant effects on the parameters examined in the present study (data not shown), which agrees with a previous report of no effect of L-NAME on the in vivo heart function in this species (Crossley et al., 2000Go). After the initial stabilization, the recirculation of solution between the measuring chamber and the reservoir solution was stopped for 40 min, during which O2 consumption and twitch force were recorded.

At the end of this period, the substance to be tested (either L-Arg or ADMA) was added to the reservoir solution, whereby it reached the myocardial preparation when circulation was resumed. After 40 min, circulation was again stopped and O2 consumption and twitch force were recorded as before in the presence of the substance added. The sequence with open and closed circulation could be repeated, allowing the effects of several substances to be tested in each experiment (Fig. 1).


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 1. Representative traces showing three consecutive 40 min recordings of twitch force (upper panel) and O2 consumption (lower panel). (1 mmHg= 0.133 kPa.)

 
To examine spontaneous changes in O2 consumption and twitch force development during the experiments, myocardial preparations were exposed to two or three cycles of recordings without any additions.

Hypoxic lactate production
Tissue lactate production and twitch force development were recorded in a separate experimental series. Two rings were prepared from each heart and mounted around two hooks, in a setup identical to that described above, and immersed in 15 ml of thermostatted physiological solution.

Myocardial ring-shaped preparations were stimulated and stretched to produce maximal twitch force. After stabilization in 48% O2, the physiological solution was exchanged for a new solution equilibrated with 8% O2 (hypoxia). After 30 min under hypoxia, the bath solution for each myocardial preparation was replaced with a new solution, which contained 0.1 mmol l–1 L-Arg for one of the two preparations and 1 mmol l–1 ADMA for the other preparation to inhibit endogenous NO production (i.e. in the absence of added L-Arg). After a further 60 min, the myocardial preparations were rapidly frozen in liquid N2 and stored at –80°C together with samples of the physiological bath solution. The lactate contained in both the tissue and the bath solution was measured so as to estimate the anaerobic glycolytic rate of the heart preparations treated with either L-Arg or ADMA. To assess tissue lactate production, the frozen heart tissue preparation was homogenized (Ultra-Turrax T25, Jancke and Kunkel, Steufen, Germany) in 3 mol l–1 perchloric acid and centrifuged. The lactate contained in the supernatant and in the physiological bath solution was determined enzymatically (Lowry and Passonneau, 1972Go).

Confocal imaging of tissue NO
Four ring-shaped preparations from each ventricle were stretched and maintained at 48% O2, 50% N2 and 2% CO2 for 40 min. The gas was then switched to 8% O2, 90% N2 and 2% CO2 and, after 30 min, L-Arg and ADMA were added. After 40 min, the presence of NO in the tissue was examined according to the protocol provided by Rodriguez and colleagues (Rodriguez et al., 2005Go). The cell-permeable fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA, Alexis, Lausanne, Switzerland) was dissolved in dimethylsulphoxide (DMSO) and immediately added to a final concentration of 0.001 mmol l–1 in the bath solution. After 60 min, the bath solution was replaced by a solution containing 1 mmol l–1 ADMA to stop NO production.

Heart tissue preparations were then mounted on a coverslip to detect fluorescence of DAF-2T, the product of the reaction between NO and DAF-2 DA, using a confocal microscope (Zeiss LSM 5 pascal, Zeiss, Germany) with 488 nm as excitation and 515 nm as emission wavelengths, respectively (Rodriguez et al., 2005Go).

Calculations and statistics
During the 40 min with a closed chamber, the mean value of the twitch force tension, sampled every 5 min, was taken to measure the twitch force production associated with O2 consumption.

Myocardial O2 consumption per gram wet mass was calculated according to the following equation:

Formula
Here, VO2 is the O2 consumption rate (µmol O2 min–1 g–1), k is {Delta}PO2/min, {alpha}O2 is the O2 solubility coefficient (12.93 µmol l–1 kPa–1), v is the volume of the chamber (2.56 ml) and m is the tissue preparation mass (g).

All data are expressed as means ± s.e.m. Fractional changes were tested for significance with Student's t-test. A P-value of <0.05 was considered to indicate significance.

In order to use isometric twitch force as an indicator of mechanical function, the thickness and cross-sectional area of the myocardial preparations were kept as constant as possible so that the twitch force given in mN could be assumed to be proportional to the twitch force related to the cross-sectional area. The relationship between mechanical performance and aerobic metabolism was assessed by the ratio of developed twitch force to O2 consumption rate (mN)/(µmol O2 min–1 g–1). Changes in this ratio were analyzed by normalizing it to the ratio obtained under control situations.


   RESULTS
TOP
 Summary
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Full oxygenation
The effects of 0.1 mmol l–1 L-Arg during full oxygenation (48% O2, 50% N2 and 2% CO2) were examined in the first series of experiments. In the first 40 min run, twitch force and O2 consumption were recorded in the absence of L-Arg and in the second run with 0.1 mmol l–1 L-Arg in the bath solution. The addition of L-Arg caused a 12±2% decrease in O2 consumption (Fig. 2A) whereas twitch force did not change significantly (Fig. 2B). The decrease in O2 consumption together with an unchanged twitch force resulted in an increased efficiency (i.e. twitch force to O2 consumption ratio) by 15±3% (Fig. 2C).


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 2. (A) O2 consumption (µmol O2 min–1 g–1). (B) Developed twitch force (mN). (C) Ratio of twitch force to O2 consumption recorded before (black bars) and after (gray bars) the addition of 0.1 mmol l–1 L-arginine (L-Arg) in full oxygenation (N=6). Data are means ± s.e.m. *Significant change from the initial value (P<0.05).

 
The addition of ADMA during full oxygenation did not change the parameters significantly (data not shown). Three preparations subjected to the same experimental time course but without the addition of L-Arg displayed no significant spontaneous changes in the parameters recorded (data not shown).


Figure 3
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 3. (A) O2 consumption (µmol O2min–1 g–1). (B) Developed twitch force (mN). (C) Ratio of twitch force to O2 consumption recorded for preparations sequentially subjected to no addition, 0.1 mmol l–1 L-arginine (L-Arg) and L-Arg+1 mmol l–1 asymmetric dimethylarginine (ADMA) (N=6), as indicated. As control (D–F) the three parameters were also recorded according to the same time schedule in preparations receiving neither L-Arg nor ADMA (N=5). Data are means ± s.e.m. *Significant change from the initial value (P<0.05).

 
Low O2
After lowering O2 from 48% to 8% (8% O2, 90% N2 and 2% CO2), O2 consumption and twitch force were recorded in three subsequent runs; the first one without any additions, the second run in the presence of 0.1 mmol l–1 L-Arg and the third run with L-Arg and 1 mmol l–1 ADMA. O2 consumption decreased significantly by 30±4%, following the addition of 0.1 mmol l–1 L-Arg, which is significantly larger than the decrease observed at full oxygenation. O2 consumption also remained significantly lower after the addition of ADMA (Fig. 3A).

Neither L-Arg nor ADMA influenced twitch force significantly (Fig. 3B) whereas L-Arg entailed an increase in the ratio of twitch force to O2 consumption by 60±18% (Fig. 3C), which was significantly higher than that of 15±3% recorded at full oxygenation, i.e. with 48% O2 in the gas mixture. ADMA tended to reverse the effects of L-Arg, although not significantly (Fig. 3A,C). The control experiment did not indicate any significant spontaneous changes over the course of the experiment (Fig. 3D–F). In accordance with the result observed at 48% O2, ADMA seemed to be unable to efficiently displace L-Arg from the enzyme active site under the conditions applied. At a reversed order of additions, i.e. when ADMA was added before L-Arg, we did not observe any significant changes in either O2 consumption or twitch force (Fig. 4), indicating that ADMA is an efficient NOS inhibitor in this species.


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. (A) O2 consumption (µmol O2 min–1 g–1). (B) Developed twitch force (mN) recorded for preparations sequentially subjected to no addition, 1 mmol l–1 asymmetric dimethylarginine (ADMA) and 1 mmol l–1 ADMA+0.1 mmol l–1 L-arginine (L-Arg), as indicated (N=5).

 
Confocal NO detection
In order to verify whether the effects seen with added L-Arg could be ascribed to an increased NOS activity, we examined NO production in hypoxic heart tissue using the fluorescence NO probe DAF-2DA. The L-Arg-dependent production of NO was examined using the procedure described in Table 1. We observed an increase in fluorescence in the presence of L-Arg, indicating a stimulation of NO production, which tended to be only slightly reversed by ADMA (Fig. 5). The basal production of NO in the absence of externally added L-Arg was too low to be detected by this technique.


View this table:
[in this window]
[in a new window]

  		Table 1. Experimental conditions for the samples depicted in Fig. 5

 

Figure 5
View larger version (45K):
[in this window]
[in a new window]

  		Fig. 5. Results for four ventricular samples treated with DAF-2 DA as described in Table 1. (A, sample 1) Tissue autofluoroscence in the presence of L-arginine (L-Arg) (0.1 mmol l–1) and absence of 4,5-diaminofluorescein diacetate (DAF-2 DA). (B, sample 2) Basal nitric oxide (NO) production without L-Arg added. (C, sample 3) NO production with 0.1 mmol l–1 L-Arg. (D, sample 4) Effect of asymmetric dimethylarginine (ADMA) (1 mmol l–1) in the presence of 0.1 mmol l–1 L-Arg.

 

Lactate
Anaerobic glycolysis and lactate production are typically enhanced at low O2. Lactate might also have contributed to the higher ratio of twitch force to O2 consumption observed in the presence of L-Arg (Fig. 2C, Fig. 3C). This possibility was, however, not supported as a separate experimental series did not reveal any significant difference in lactate production for hypoxic heart preparations exposed to either L-Arg or ADMA, the values being 0.17±0.04 and 0.19±0.05 µmol min–1 g–1 lactate, respectively (N=7).


   DISCUSSION
TOP
 Summary
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
A main finding of the present study was that the O2 consumption rate in turtle ventricular preparations stimulated to isometric force development was diminished in the presence of L-Arg and particularly during hypoxia. The fact that the myocardial preparations were stretched to maximize twitch force should stimulate NOS activity according to previous studies, showing that stretch is an important regulator of this activity (e.g. Tota et al., 2005Go). In our confocal studies, we could not detect a basal NO production from endogenous L-Arg under control conditions, whereas NO production was clearly observed in the presence of L-Arg added as a substrate. Hence, the turtle myocardium seems to differ from the mammalian myocardium, in which a basal production of NO was evident in the absence of added L-Arg (Shen et al., 2001Go; Eu et al., 2003Go; Palacios-Callander et al., 2004Go). Although the in vivo levels of L-Arg in ectotherms are unknown, it should be noted that the L-Arg concentration (0.1 mmol l–1) used in the present study to stimulate NO production is approximately the same as the plasma concentrations recorded in mammals [~0.1 mmol l–1 (Morris, 2000Go)]. It should also be recalled that Km for L-Arg in mammalian NOS is about 100-fold lower [~0.6 µmol l–1 (Matsuoka et al., 1994Go)].

Despite its importance in the control of cardiovascular function in mammals, endothelial NOS isoforms have not been cloned in non-mammalian animals. The results shown in the current study, and previously by other groups (Tota et al., 2005Go; Amelio et al., 2006Go; Amelio et al., 2008Go; Jennings and Donald, 2008Go), strongly suggest that functional (possibly non-endothelial) NOS isoforms are expressed in several ectotherm species, belonging to dipnoi, teleosts, amphibians and reptiles, and that NO as a cardiovascular signaling molecule has been conserved in the evolution of vertebrates.

The decrease in O2 consumption rate of the turtle myocardium observed in the present study in the presence of L-Arg may be explained by an inhibition by NO of mitochondrial respiration due largely, but not exclusively, to a competitive inhibition of the binding of O2 to cytochrome c oxidase, i.e. the final step in the respiratory chain (Erusalimsky and Moncada, 2007Go; Cooper et al., 2008Go). Accordingly, the relative decrease in O2 consumption was significantly more pronounced during hypoxia than during full oxygenation. As a further contribution to this difference, cytochrome c oxidase seems to remove NO more efficiently at a high oxidation level (Palacios-Callander et al., 2007Go). Consistent with this, Kojic and colleagues found no effect of NO on O2 consumption in the mouse heart in vivo; a result suggested to be due to high tissue oxygenation in combination with a high concentration of the NO scavenger, myoglobin (Kojic et al., 2003Go). It should be noted that NO may not only act on the respiratory chain but it may also inhibit O2 consumption by inhibiting creatine kinase and, in turn, the sensitivity of mitochondrial respiration to cytosolic ADP (Kaasik et al., 1999Go).

Besides cytochrome c oxidase of mitochondria, another biological target of NO is soluble guanylate cyclase, whose activation by NO generates cGMP, which induces muscle relaxation in the vasculature and influences heart performance (Balligand et al., 2000Go). Under the conditions used in our present study, an increase in L-Arg levels only affected O2 consumption and not contractility. However, our data do not exclude a physiological role of the NO–cGMP pathway in the regulation of turtle heart contractile function.

In the myocardial tissue of many species, NO affects contractility either positively, as in the icefish (Pellegrino et al., 2003Go), or negatively, as in the eel (Imbrogno et al., 2001Go). However, in guinea pig myocardial tissue, Shen and colleagues found that NOS-derived NO depressed O2 consumption, while leaving contractility unchanged (Shen et al., 2001Go). We obtained similar results with turtle myocardium as L-Arg augmented the ratio of twitch force to O2 consumption by diminishing O2 consumption without affecting twitch force development. This effect was particularly evident under hypoxia but also occurred under full oxygenation. The drop in O2 consumption appeared not to involve any significant compensatory enhancement of anaerobic metabolism in the heart recorded as lactate production. Hence, NO seems to augment either contractility relative to the rate of ATP consumption or alternatively to the ATP produced aerobically relative to O2 consumed. A number of possible mechanisms can be envisaged. In mammals, the inhibition of the cytochrome c oxidase activity by NO and a consequent increase in reactive oxygen species (ROS) formation at least in some cell types activates AMP kinase and, in turn, glycolysis (Erusalimski and Moncada, 2007) whereas in ectotherms activation of AMP kinase may inhibit glycolysis (Bickler and Buck, 2007Go). However, and of relevance to our results with turtle heart, the activity of AMP kinase also entails a decrease in biosynthetic activity (Erusalimsky and Moncada, 2007Go), which may potentially increase the fraction of ATP available for contractility. Furthermore, during hypoxia the aerobic ATP production may possibly decrease less than the O2 consumption, because of a partial inhibition of the respiratory chain that may reduce the proton gradient and the proton leak across the inner mitochondrial membrane and tighten the coupling between transmembrane proton flux and ATP synthetase; a coupling that has proven to be rather variable (Gnaiger et al., 2000Go; Brand, 2005Go). This scenario is compatible with the study of Shen and colleagues (Shen et al., 2001Go), providing evidence that NO may entail a lowering of myocardial O2 consumption relative to twitch force development without influencing either ATP synthesis rate or the concentrations of phosphocreatine and inorganic phosphate. Alternatively, an increase in ATP production relative to O2 consumption may be due to a shift from fatty acids to carbohydrates (Canty 2000Go; Williams et al., 2008Go). In opposition to this, another study has shown an increased O2 consumption relative to mechanical performance upon inhibition of NOS in dog ventricular muscle exposed to noradrenaline (Setty et al., 2002Go), although NOS inhibition seems to shift substrate selection from fatty acids to glucose in dog heart (Recchia et al., 1999Go).

In conclusion, the present study suggests that NO generated from L-Arg contributes to the superior hypoxic tolerance of the freshwater turtle and its heart muscle by reducing the O2 consumption needed for the maintenance of a given contractility. This effect may represent an increase in the force obtained per ATP and/or in the number of ATP obtained per O2.


   Footnotes
 
This work was supported by the Danish Natural Science Research Council and the Novo Nordisk Foundation. Thanks are also due to Dr Sebastian Frische for help with confocal microscopy and technician Dorte Olsson for skillful assistance.


   References
TOP
 Summary
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

Amelio, D., Garofalo, F., Pellegrino, D., Giordano, F., Tota, B. and Cerra, M. C. (2006). Cardiac expression and distribution of nitric oxide synthases in the ventricle of the cold-adapted Antarctic teleosts, the hemoglobinless Chionodraco hamatus and the red-blooded Trematomus bernacchii. Nitric Oxide 15,190 -198.[CrossRef][Medline]

Amelio, D., Garofalo, F., Brunelli, E., Loong, A. M., Wong, W. P., Ip, Y. K., Tota, B. and Cerra, M. C. (2008). Differential NOS expression in freshwater and aestivating Protopterus dolloi (lungfish): heart vs kidney readjustments. Nitric Oxide 18,1 -10.[CrossRef][Medline]

Balligand, J. L., Feron, O. and Kelly, R. A. (2000). Role of nitric oxide in myocardial function. In Nitric Oxide (ed. l. J. Ignarro), pp.585 -607. San Diego, CA: Academic Press.

Bickler, P. E. and Buck, L. T. (2007). Hypoxia tolerance in reptiles, amphibians, and fishes. Annu. Rev. Physiol. 69,145 -170.[CrossRef][Medline]

Böger, R., Vallance, P. and Cooke, J. P. (2003). Asymmetric dimethylarginine (ADMA): a key regulator of nitric oxide synthase. Altherosclerosis Suppl. 4, 1-3.

Brand, M. D. (2005). The efficiency and plasticity of mitochondrial energy transduction. Biochem. Soc. Trans. 33,897 -904.[CrossRef][Medline]

Canty, J. M., Jr (2000). Nitric oxide and short-term hibernation: friend or foe? Circ Res. 87, 85-87.[Free Full Text]

Cooper, C. E., Mason, M. G. and Nicholls, P. (2008). A dynamic model of nitric oxide inhibition of mitochondrial cytochrome c oxidase. Biochim. Biophys. Acta 1777,867 -876.

Crossley, D. A., Wang, T. and Altimiras, J. (2000). Role of nitric oxide in the systemic and pulmonary circulation of anesthetized turtles (Trachemys scripta). J. Exp. Zool. 271,683 -689.

Erusalimsky, J. D. and Moncada, S. (2007). Nitric oxide and mitochondrial signaling: from physiology to pathophysiology. Arterioscler. Thromb. Vasc. Biol. 27,2524 -2531.[Abstract/Free Full Text]

Eu, J. P., Hare, J. M., Hess, D. T., Skaf, M., Sun, J., Navina-Cardenas, I., Sun, Q. A., Dewhirst, M., Meissner, G. and Stamler, J. S. (2003). Concerted regulation of skeletal muscle contractility by oxygen tension and endogenous nitric oxide. Proc. Natl. Acad. Sci. USA 100,15229 -15234.[Abstract/Free Full Text]

Gnaiger, E., Mendez, G. and Hand, S. C. (2000). High phosporylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc. Natl. Acad. Sci. USA 97,11080 -11085.[Abstract/Free Full Text]

Hagen, T., Taylor, C. T., Lam, F. and Moncada, S. (2003). Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF1alpha. Science 302,1975 -1978.[Abstract/Free Full Text]

Imbrogno, S., De Iuri, L., Mazza, R. and Tota, B. (2001). Nitric oxide modulates cardiac performance in the heart of Anguilla anguilla. J. Exp. Biol. 204,1719 -1727.[Abstract]

Imbrogno, S., Angelone, T., Adamo, C., Pulerà, E., Tota, B. and Cerra, M. C. (2006). Beta3-adrenoceptor in the eel (Anguilla anguilla) heart: negative inotropy and NO-cGMP-dependent mechanism. J. Exp. Biol. 209,4966 -4973.[Abstract/Free Full Text]

Jackson, D. C. (2002). Hibernating without oxygen: physiological adaptations of the painted turtle. J. Physiol. 543,731 -737.[Abstract/Free Full Text]

Jennings, B. L. and Donald, J. A. (2008). Neurally-derived nitric oxide regulates vascular tone in pulmonary and cutaneous arteries of the toad, Bufo marinus. Am. J. Physiol. Regul. Integr. Comp. Physiol. 295,R1640 -R1646.[Abstract/Free Full Text]

Kaasik, A., Minajeva, A., De Sousa, E., Ventura-Clapier, R. and Veksler, V. (1999). Nitric oxide inhibits cardiac energy production via inhibition of mitochondrial creatine kinase. FEBS Lett. 444,75 -77.[CrossRef][Medline]

Kalinin, A. and Gesser, H. (2002). Oxygen consumption and force development in turtle and trout cardiac muscle during acidosis and high extracellular potassium. J. Comp. Physiol. B 172,145 -151.[CrossRef][Medline]

Kodja, G. and Kottenberg, K. (1999). Regulation of basal myocardial function by NO. Cardiovasc. Res. 41,514 -523.[Free Full Text]

Kojic, Z. Z., Flögel, U., Schrader, J. and Decking, U. K. M. (2003). Endothelial NO formation does not control myocardial O2 consumption in mouse heart. Am. J. Physiol. Heart Circ. Physiol. 285,392 -397.

Lebuffe, G., Schumacker, P. T., Shao, Z. H., Anderson, T., Iwase, H. and Vanden Hoek, T. L. (2003). ROS and NO trigger early preconditioning: relationship to mitochondrial KATP channel. Am. J. Physiol. Heart Circ. Physiol. 284,H299 -H308.[Abstract/Free Full Text]

Ljubkovic, M., Shi, Y., Cheng, Q., Bosnjak, Z. and Jiang, M. T. (2007). Cardiac mitochondrial ATP-sensitive potassium channel is activated by nitric oxide in vitro. FEBS Lett. 581,4255 -4259.[CrossRef][Medline]

Lowry, O. H. and Passonneau, J. V. (1972). A Flexible System of Enzymatic Analysis. London: Academic Press.

Mason, M. G., Nicholls, P., Wilson, M. T. and Cooper, C. E. (2006). Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 103,708 -713.[Abstract/Free Full Text]

Matsuoka, A., Stuehr, D. J., Olson, J. S., Clark, P. and Ikeda-Saito, M. (1994). L-arginine and calmodulin regulation of the heme iron reactivity in neuronal nitric oxide synthase. J. Biol. Chem. 269,20335 -20339.[Abstract/Free Full Text]

Morris, S., Jr (2000). Regulation of arginine availability and its impact on NO synthesis. In Nitric Oxide (ed. L. J. Ignarro), pp. 187-197. San Diegeo, CA: Academic Press.

Overgaard, J. and Gesser, H. (2004). Force development, energy state and ATP production of cardiac muscle from turtles and trout during normoxia and severe hypoxia. J. Exp. Biol. 207,1915 -1924.[Abstract/Free Full Text]

Overgaard, J., Gesser, H. and Wang, T. (2007). Tribute to P. L. Lutz: cardiac performance and cardiovascular regulation during anoxia/hypoxia in freshwater turtles. J. Exp. Biol. 10,1687 -1699.

Palacios-Callander, M., Quintero, M., Hollis, S. V., Springett, R. J. and Moncada, S. (2004). Endogenous NO regulates superoxide production at low oxygen concentrations by modifying the redox state of cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 101,7630 -7635.[Abstract/Free Full Text]

Palacios-Callander, M., Hollis, V., Mitchison, M., Frakich, N., Unitt, D. and Moncada, S. (2007). Cytochrome c oxidase regulates endogenous nitric oxide availability in respiring cells: a possible explanation for hypoxic vasodilation. Proc. Natl. Acad. Sci. USA 104,18508 -18513.[Abstract/Free Full Text]

Pellegrino, D., Acierno, R. and Tota, B. (2003). Control of cardiovascular function in the icefish Chionodraco hamatus: involvement of serotonin and nitric oxide. Comp. Biochem. Physiol. A 134,471 -480.[CrossRef][Medline]

Petroff, M. G., Kim, S. H., Pepe, S., Dessy, C., Marbán, E., Balligand, J. L. and Sollott, S. J. (2001). Endogenous nitric oxide mechanisms mediate the stretch dependence of Ca2+ release in cardiomyocytes. Nat. Cell Biol. 10,867 -873.

Recchia, F. A., McConnell, P. I., Loke, K. E., Xu, X., Ochoa, M. and Hintze, T. H. (1999). Nitric oxide controls cardiac substrate utilization in the conscious dog. Cardiovasc. Res. 44,325 -332.[Abstract/Free Full Text]

Rodriguez, J., Specian, V., Malony, R., Jourd'heuil, D. and Feelisch, M. (2005). Performance of diamino flourophores for the localization of sources and targets of nitric oxide. Free Radic. Biol. Med. 38,356 -368.[CrossRef][Medline]

Setty, S., Tune, J. D. and Downey, H. F. (2002). Nitric oxide modulates right ventricular flow and oxygen consumption during norepinephrine infusion. Am. J. Physiol. Heart Circ. Physiol. 282,H696 -H703.[Abstract/Free Full Text]

Shen, W., Tian, R., Saupe, W. K., Spindler, M. and Ingwall, S. J. (2001). Endogenous nitric oxide enhances coupling between O2 consumption and ATP synthesis in guinea pig hearts. Am. J. Physiol. Heart Circ. Physiol. 281,838 -846.

Shi, H., Hamm, P. H., Lawler, R. G. and Jackson, D. C. (1999). Different effects of simple anoxic lactic acidosis and simulated in vivo anoxic acidosis on turtle heart. Comp. Biochem. Physiol. 122,173 -180.[CrossRef][Medline]

Skovgaard, N., Galli, G., Abe, A., Taylor, E. W. and Wang, T. (2005). The role of nitric oxide in regulation of the cardiovascular system in reptiles. Comp. Biochem. Physiol. A 142,205 -214.[CrossRef][Medline]

Tota, B., Amelio, D., Pellegrino, D., Ip, Y. K. and Cerra, M. C. (2005). NO modulation of myocardial performance in fish hearts. Comp. Biochem. Physiol. A 142,164 -177.[CrossRef][Medline]

Viner, R. I., Williams, T. D. and Scöneich, C. (2000). Nitric oxide-dependent modification of the sarcoplasmic reticulum Ca-ATPASE: localization of cystine target sites. Free Radic. Biol. Med. 29,489 -496.[CrossRef][Medline]

Williams, J. G., Ojaimi, C., Qanud, K., Zhang, S., Xu, X., Recchia, F. A. and Hintze, T. H. (2008). Coronary nitric oxide production controls cardiac substrate metabolism during pregnancy in the dog. Am. J. Physiol. Heart Circ. Physiol. 294,H2516 -H2523.[Abstract/Free Full Text]

Xu, K. Y., Huso, D. L., Dawson, T. M., Bredt, D. S. and Becker, L. C. (1999). Nitric oxide synthase in cardiac sarcoplasmic reticulum. Proc. Natl. Acad. Sci. USA 96,657 -662.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



This Article
Right arrow Summary Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Misfeldt, M.
Right arrow Articles by Gesser, H.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Misfeldt, M.
Right arrow Articles by Gesser, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?