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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 7, 2005 as doi:10.1096/fj.04-2886fje. |
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* Medizinische Klinik I, RWTH Aachen, Aachen, Germany; and
Institut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Universität, Düsseldorf, Germany
1Correspondence: Medizinische Klinik I, Universitätsklinikum Aachen, Pauwelsstr. 30, Aachen 52057, Germany. E-mail: mmerx{at}ukaachen.de
SPECIFIC AIMS
Recent studies of myoglobin (Mb) knockout (myo–/–) mice have extended our understanding of Mb’s diverse functions and have demonstrated a complex array of compensatory mechanisms. The present study was aimed at detailed analysis of cardiac function and exercise endurance in myo–/– mice and at providing evidence for Mb’s functional relevance.
PRINCIPAL FINDINGS
1. Contractility and output efficiency is decreased in working myo–/– hearts
To assess the functional relevance of Mb without confounding systemic effects, isolated hearts from myo–/– and wild-type (WT) mice were compared using the working heart model. Stepwise increase in preload was performed to analyze the influence of filling pressure on the given mouse hearts. A marked decrease in contractility was detected in myo–/– hearts at all preloads studied. For example, dP/dtmax was 4688 ± 425 mmHg/s in myo–/– hearts vs. 5718 ± 435 mmHg/s in WT hearts at 5 mmHg preload (P<0.001, afterload fixed at 80 mmHg). Stepwise increases in afterload to simulate growing systemic resistance resulted in similar findings with lowered dP/dtmax in myo–/– hearts at all afterloads studied (P<0.05). Diminished contractility was also reflected by slower contraction and relaxation reflected as time to peak pressure (TPP) and relaxation half-time (RT1/2) in myo–/– hearts (P<0.001).
Under all loading conditions studied, coronary flow was significantly higher in myo–/– hearts than in their WT counterparts (e.g., 20.2±2.0 mL*min–1*g–1 vs. 13.0±1.9 mL*min–1*g–1; P<0.001; 5 mmHg preload; 80 mmHg afterload). Cardiac output (CO) being identical in both groups, aortic flow was significantly smaller in myo–/– hearts, reflecting the larger fraction of CO required to adequately perfuse the coronaries of myo–/– hearts. This implies reduced effective cardiac output (i.e., cardiac output available for perfusion of organs other than the heart).
2. Cardiac output is decreased in conscious myo–/– mice; this effect is partly attenuated by NO inhibition
To investigate whether the depressed contractility observed in the working heart model could be demonstrated in vivo, echocardiographic studies of conscious mice were performed. As the baseline measurements depicted in Fig. 1
demonstrate, HR and fractional shortening(SF) were significantly depressed in myo–/– mice. As a result, CO of myo–/– mice did not reach the level of their WT counterparts. To address the possibility of decreased contractility being mediated by higher levels of bioactive NO, NOS inhibition was achieved through continuous 2-ethyl-2-thiopseudourea (ETU) secretion by mini pumps implanted into the peritoneal cavity. SF increased by 7% in myo–/– mice, reaching the level of WT mice, which remained unaffected.
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Upon dobutamine stimulation, WT mice responded with a marked increase in CO by 27%, a 15% increase in SF and an 8% increase in HR as measured by stress echocardiography. In contrast, myo–/– mice showed only a slight increase of SF and HR (P=n.s.), with limited CO augmentation in comparison to WT mice. However, when NOS was inhibited by ETU myo–/– mice were capable of SF values equal to those of WT animals and demonstrated a marked increase of CO, albeit not to the level of WT mice, partially due to lower HR observed at baseline and during ß1 stimulation. To test whether the observed decrease in HR might be due to a shift in sympathetic/parasympathetic tone, ablation of parasympathetic tone was performed. Seven minutes after atropine application, a remarkable HR of above 800 bpm was measured by echocardiography in conscious myo–/– and WT animals. SF was decreased in both groups secondary to decreased diastolic filling, resulting in CO well below the values observed after dobutamine stimulation.
3. O2 consumption (
O2) and endurance are decreased whereas respiratory exchange ratio is increased in Mb knockout mice
At rest, O2 was decreased in myo–/– mice by 31% to 3082 ± 413 mL·kg·h (P<0.001). With the initiation of exercise, O2 in both groups rose sharply, reaching a similar level of maximum O2 (Fig. 2
). In contrast, for any given O2, RER at rest was higher in myo–/– than in WT mice. Exercising myo–/– mice showed a significantly larger increase in RER, rising to levels as high as 0.94 (P<0.001).
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In spiroergometric analysis, all mice were able to perform exercise for several minutes while subjected to increasing workloads. However, myo–/– mice had earlier onset of fatigue, mean endurance being reduced by an average of 6 min (31±5 vs. 37±7 min, myo–/– vs. WT, P<0.01). Distance run was calculated to be decreased by an average of 
120 min to 466 ± 113 min in myo–/– mice (WT 585±153 min, P<0.01).
CONCLUSIONS AND SIGNIFICANCE
The present study demonstrates Mb’s functional relevance on the whole animal level in that loss of Mb leads to impaired contractile function and exercise endurance. Despite unimpaired left ventricular developed pressure and CO measured in isolated working myo–/– hearts, dP/dt was lower in myo–/– hearts under all conditions studied. The decreased contractility with increased TPP and RT1/2 implies that contraction and relaxation span a longer period, thereby smoothing the peaks in energy and O2 demand. This decreased contractility, in itself, might be viewed as functional adaptation, as it counterbalances the loss of two major Mb functions: mass transport of O2 by facilitated diffusion and rapid deployment of O2 from Mb storage.
The dampened contractility observed in working myo–/– hearts in vitro is paralleled by the decreased SF seen in echocardiograms of conscious myo–/– mice. It is conceivable that decreased contractility is partly mediated by higher levels of bioactive NO due to the absence of Mb’s NO scavenging capacity in myo–/– mice. These higher bioactive NO levels possibly lead to direct mitochondrial respiratory chain inhibition by NO and thus to impaired ATP generation. In the present study, pharmacological NOS inhibition led to recovery of contractility in myo–/– mice in vivo. The dampened response to ß1-stimulation observed in myo–/– mice is partly reversible by NOS inhibition, suggesting NO as contributing factor. Recent studies have demonstrated decreased contractility mediated by NO in transplanted human cardiac tissue. In agreement with our findings, NO mediated contractility alterations in human hearts were characterized by an impaired response to ß1 stimulation. It is of note that low Mb levels and up-regulation of iNOS have been reported in heart failure. Thus, the loss of Mb and resulting lowered O2 supply in conjunction with the reduced NO scavenging capacity in failing hearts might contribute to the pathophysiology of this frequent disease state.
An important difference between our in vivo and in vitro findings is found in the HR obtained during echocardiography: whereas intrinsic HR did not differ in isolated hearts, it was significantly lower in intact myo–/– mice. Decreased HR would contribute to lowering O2 demand in myo–/– mice further cushioning reduced O2 availability. In line with this hypothesis, this study reports O2 of conscious animals at rest to be lower in myo–/– mice than their WT counterparts. The decrease in HR of myo–/– mice is achieved by a shift in sympathetic / parasympathetic tone toward the latter, as demonstrated by our atropine experiments. The application of ETU reveals that this shift in sympathetic/parasympathetic tone is not NO mediated. Nevertheless, the possible direct inhibition of the mitochondrial respiratory chain due to higher biologically active NO levels in myo–/– mice might also be viewed as an endogenous modulator of oxidative phosphorylation to reduce O2 consumption under conditions of diminished O2 supply.
In spite of these multiple functional and morphological compensating mechanisms, in vivo analysis of conscious animals reveals their limitations: in echocardiographic measurements myo–/– mice are incapable of achieving the cardiac output mustered by WT mice. In addition, myo–/– mice fall short in exercise endurance. This occurs despite their ability to increase O2 to the level of WT mice if challenged by the same degree of exercise stress. Moreover, the latter implies a larger absolute increase in O2 if one takes into account that O2 of the whole animal is 25% lower in resting myo–/– mice.
A possible cause for the decreased exercise endurance in spite of equal maximum O2 is found when analyzing the respiratory exchange ratio (RER). With RER defined as the (CO2) divided by O2 at any given time, it largely depends on substrate metabolism. However, under exercise conditions, it offers an indicator of lactic acid production as anaerobic metabolism supplements aerobic metabolism with rising exercise intensities. The lactic acid produced during exercise from anaerobic metabolism mainly in striated muscle promotes CO2 formation via the serum bicarbonate buffer in addition to the CO2 derived from aerobic metabolism, thus shifting the stoichiometric relation with respect to O2 uptake. Therefore, RER rises with increasing workload until exhaustion. In our studies, both myo–/– and WT mice displayed considerable increase of RER in the course of exertion. However, RER rose to significantly higher levels in myo–/– mice even though workload was lower and exercise duration shorter. Although a metabolic shift toward glucose metabolism is present in myo–/– mice, the accelerated RER rise, in conjunction with the diminished endurance, suggests that anaerobic metabolism contributes to meet the energetic demand in myo–/– mice after shorter periods of exertion and at lower levels of workload compared with WT mice.
In summary, the importance of oxygen supply and NO scavenging by Mb on the conscious animal level is clearly demonstrated. Cardiac function and exercise endurance of myo–/– mice are impaired. Lack of Mb results in a substrate utilization shift toward glucose metabolism. Decreased O2 in myo–/– mice is facilitated by higher parasympathetic tone. The cardiac dysfunction in myo–/– mice discovered here—namely, decreased contractility and fractional shortening as well as subdued response to ß1 stimulation—is partly attenuated by NOS inhibition.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2886fje; doi: 10.1096/fj.04-2886fje
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