Author + information
- Received October 29, 2007
- Revision received April 29, 2008
- Accepted June 3, 2008
- Published online August 26, 2008.
- Tetsuya Watanabe, MD, PhD⁎,
- Toshihiro Takeda, MD, PhD⁎,
- Shigemiki Omiya, MD⁎,
- Shungo Hikoso, MD, PhD⁎,
- Osamu Yamaguchi, MD, PhD⁎,
- Yuko Nakano, DDS†,
- Yoshiharu Higuchi, MD, PhD⁎,
- Atsuko Nakai, PhD⁎,
- Yusuke Abe, MS‡,
- Yayoi Aki-Jin, PhD§,
- Masayuki Taniike, MD, PhD⁎,
- Isamu Mizote, MD⁎,
- Yasushi Matsumura, MD, PhD∥,
- Takahiko Shimizu, PhD‡,
- Kazuhiko Nishida, MD, PhD⁎,
- Kiyohiro Imai, PhD§,
- Masatsugu Hori, MD, PhD, FACC⁎,
- Takuji Shirasawa, MD, PhD‡ and
- Kinya Otsu, MD, PhD⁎,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Kinya Otsu, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Objectives This study examined whether a reduction in hemoglobin–oxygen affinity improves exercise capacity in mice with heart failure.
Background Exercise intolerance is a major determinant of quality of life in patients with chronic heart failure. One of the major goals of the treatment for chronic heart failure is to improve quality of life.
Methods Four weeks after left coronary ligation, we transplanted bone marrow cells isolated from the transgenic mice expressing a hemoglobin variant with low oxygen affinity, Presbyterian, into the lethally irradiated mice with heart failure or administered a synthetic allosteric modifier of hemoglobin. The mice were then exercised on a treadmill.
Results Four weeks after the left coronary artery ligation, mice showed cardiac dysfunction and chamber dilation, which were characteristics of heart failure. The transplantation led to a reduction in hemoglobin–oxygen affinity and an increase in oxygen supply for skeletal muscle without changes in muscle properties. The transplanted mice showed improved running performance on a treadmill despite impaired cardiac contractility. Furthermore, administration of the synthetic allosteric modifier of hemoglobin showed a similar effect.
Conclusions Allosteric modification of hemoglobin represents a therapeutic option for improving exercise capacity in patients with chronic heart failure. One mechanism of improvement in exercise capacity is enhanced oxygen delivery in the skeletal muscle.
The 3 major goals of treatment for chronic heart failure (CHF) are to reduce symptoms, prolong survival, and improve quality of life (1). A nearly universal manifestation of CHF is a reduction in exercise capacity, which diminishes quality of life. The pathophysiology of exercise intolerance in this syndrome is incompletely understood, but it is believed to be multifactorial.
The oxygen (O2) affinity of hemoglobin (Hb), a tetrameric protein consisting of α- and β-globin subunits, is critical for gas exchange in the lung and O2delivery in the peripheral tissues. A variety of mutations in the genes of α- and β-globin have been reported, one of which, hemoglobin Presbyterian (HbPres), carries lysine at asparagine-108 of β-globin and shows a low Hb–O2affinity in vitro (2). We previously reported that mutant mice, carrying the Presbyterian mutation generated with a targeted knock-in strategy, also showed a low Hb–O2affinity (2). The mutant mice showed various phenotypes, including mild anemia, respiratory acidosis, enhanced tissue oxygenation, increased O2consumption, a higher ratio of type IIA/IIB muscle fibers, and increased spontaneous physical activity.
The 2-[4-[(3,5-dimethylanilino carbonyl)-methyl]phenoxy]-2-methylpropionic acid, known as RSR13, is a synthetic allosteric modifier of Hb that acts to increase the release of O2from Hb to the surrounding tissues (3,4). This agent markedly shifts the Hb–O2curve rightward in both in vitro and in vivo rodent models through the stabilization of deoxyhemoglobin in a manner similar to the natural allosteric effecter of Hb, 2, 3-diphosphoglycerate.
In the present study, we investigated whether a reduction in Hb–O2affinity could improve exercise capacity of CHF mice by transplanting bone marrow cells (BMCs) obtained from the HbPresmice or by the administration of RSR13. We showed that a reduction in Hb–O2affinity leads to improved running performance on a treadmill despite impaired cardiac function.
This study was carried out under the supervision of the Animal Research Committee in accordance with the Guidelines on Animal Experiments of Osaka University and the Japanese Government Animal Protection and Management Law (no. 105).
The BMCs were obtained by flushing the bone marrow cavity of femurs of 6-week-old heterozygous Presbyterian mice with a C57Bl6/J background (2) and C57Bl6/J (wild-type [WT]) mice. The 5 × 106cells were intravenously injected into WT mice irradiated with a single whole-body dose of 10 Gy (5).
Detection and quantification of βPres-globin and red blood cell oxygen dissociation curve
Erythrocytes lysate sample containing 50 μg Hb was subjected to reversed-phase high-performance liquid chromatography, as previously described (6). Oxygen dissociation curve measurements were performed at pH 6.9 and 7.4 at 37°C as we previously reported (7).
Surgical procedure and in vivo assessment of cardiac functions
Eight-week-old male WT mice were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and silk thread (7-0 type) was passed around the left coronary artery (LCA) about 1 mm distal to the LCA origin (8). Echocardiography and hemodynamic measurements were performed on mice as we previously described (8,9).
Training and exercise protocol
Exercise was performed on a treadmill (10). Four weeks after bone marrow cell transplantation (BMT), mice were initially trained 3 times daily for 10 min for 3 days. The velocity of the treadmill was 10 m/min. Then, the exercise test was performed. The mice underwent 1 exercise session, which consisted of running on a treadmill at 10 m/min at a gradient of 0° for 30 min. The running of mice was video recorded for measuring the periods for running and resting.
Analysis of tissue O2and characterization of muscle fiber
Four weeks after BMT, an O2electrode with a thermocouple (Needle-type electrode, Laboratory and Medical Supplies, Tokyo, Japan) was inserted into the left gastrocnemius muscle to measure tissue O2in a double-chamber plethysmograph (2).
Muscle fiber type classification and succinate dehydrogenase (SDH) activity were examined as previously reported (2). Total SDH activity was measured as previously described (11). Capillary density from tibialis anterior muscle was measured using von Willebrand factor antibody.
Physical activity after RSR13 intravenous injection
Twelve-week-old male WT mice were initially trained 3 times per day for 10 min. The velocity of the treadmill was 15 m/min. The mice were then exercised 5 times per week for 30 min at 15 m/min for 3 weeks. The RSR13 was synthesized (3), dissolved in dimethyl sulfoxide and 2-amino-2-hydroxymethyl-1,3-propanediol/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), and injected intravenously (150 mg/kg) (12). Thirty minutes after the injection, the exercise test was performed. The exercise test consisted of running at 15 m/min for 30 min on a level surface, during the next 30 min on a 5° incline, and for the last 30 min on a 10° incline. The run was calculated every 30 min. To examine the effect of RSR13 on exercise capacity in myocardial infarct (MI) mice, male mice were used 4 weeks after LCA ligation. The mice were initially trained 3 times daily for 10 min for 3 days at a treadmill velocity of 10 m/min, followed by the exercise test. The graded exercise test was performed for 90 min at a constant speed of 12.5 m/min for first 30 min, followed by 30 min at an incline of 5°, and another 30 min at an angle of 10°.
All experiments were performed in a blinded fashion. Results are shown as mean ± SEM. Paired data were evaluated by the Student ttest. A one-way analysis of variance with the Bonferroni post hoc test was used for multiple comparisons. For the analysis of exercise capacity, data were evaluated by nonparametric methods. A value of p < 0.05 was considered statistically significant.
Analysis of peripheral blood cells in the mice transplanted with Presbyterian BMCs
The BMC obtained from femurs of heterozygous Presbyterian or WT mice were transplanted into WT mice. Reversed-phase high-performance liquid chromatography profiles of purified Hb prepared from Presbyterian BMT mice showed double peaks for the β-chain (Fig. 1A),which enabled the estimation that 32.2 ± 5.8% (n = 5) of the Hb in the peripheral blood consisted of HbPres. Genotyping of BMC revealed that 43.2% of BMC obtained from Presbyterian BMT mice had the Presbyterian allele. In O2dissociation plots, red blood cells prepared from Presbyterian BMT mice showed a rightward shift in comparison with control subjects (Fig. 1B). At pH 6.9, Presbyterian BMT mice showed a more extensive rightward shift than at pH 7.4 (P50= 99 mm Hg for Presbyterian BMT and 86 mm Hg for WT BMT at pH 6.9; P50= 54 mm Hg for Presbyterian BMT and 48 mm Hg for WT BMT at pH 7.4).
Effect of Presbyterian BMT on exercise capacity of CHF mice
The 8-week-old WT mice were subjected to LCA ligation (Fig. 2A).Four weeks later, echocardiographic analysis indicated that the left ventricular end-diastolic and end-systolic diameters showed comparatively significant increases (Table 1).Cardiac contractility as assessed by fractional shortening was significantly reduced. Infarcts causing this degree of CHF did not result in early mortality in our mouse model (8). The BMC isolated from the transgenic mice expressing Presbyterian Hb or from WT mice were then transplanted into the lethally irradiated sham-operated or MI WT mice. No mice died during 4 weeks after BMT. Echocardiography showed no significant differences in chamber size or cardiac function between sham-operated WT BMC-transplanted (Sham-WT BMT) and sham-operated Presbyterian BMC-transplanted (Sham-Pres BMT) groups, nor between MI WT BMC-transplanted (MI-WT BMT) and MI Presbyterian BMC-transplanted (MI-Pres BMT) groups either before or 4 weeks after BMT (Table 1, Fig. 2B). In addition, the chamber size and cardiac function of any group showed no changes between before and 4 weeks after BMT. These findings suggest that either WT or mutant BMT had no effect on cardiac remodeling. Catheterization of the left ventricle revealed a similar extent of impaired cardiac function in both MI groups (Fig. 2C). The heart weight to body weight ratio and cross-sectional area of cardiomyocytes at remote area in the MI-WT BMT or MI-Pres BMT groups were increased compared with sham-operated control subjects, but there were no significant differences between the MI-WT BMT and MI-Pres BMT groups (Figs. 2D and 2E). The lung weight to body weight ratio was not different among groups. We observed enhanced cardiac fibrosis at the border area of MI-WT BMT and MI-Pres BMT hearts to a similar degree (Fig. 2F). The peripheral blood cell analysis performed 4 weeks after BMT indicated that Sham-WT BMT, MI-WT BMT, Sham-Pres BMT, and MI-Pres BMT mice did not show anemia, and the number of red blood cells, Hb level, and serum erythropoietin level did not significantly differ among all groups (data not shown). Furthermore, there were no significant differences in the pH, Paco2, and Pao2level of arterial blood among all groups (data not shown).
Four weeks after BMT, the mice were exercised on a treadmill at 10 m/min and at a 0° gradient for 30 min (Fig. 3A).The MI-WT BMT mice used a longer total resting time during the exercise test than sham-WT BMT mice, whereas Presbyterian BMT enabled MI mice to run for a longer time (Fig. 3A). The Presbyterian BMT resulted in decreased total resting time during the exercise test, which was not significantly different from that for Sham-WT BMT or Sham-Pres BMT mice. As a result, MI-Pres BMT mice could run 292.6 ± 4.2 m, but MI-WT BMT mice could only run 189.3 ± 37.7 m (Fig. 3B).
Capacity of O2delivery in skeletal muscle tissues
The mice were exposed to 15% O2for 4 min, and tissue O2was measured with an O2electrode. During the exposure, the tissue O2of MI-Pres BMT mice was retained and sustained a higher level than MI-WT BMT mice (Fig. 3C). These results suggest that more O2is delivered to the tissues of MI-Pres BMT mice than of MI-WT BMT mice over a certain range of hypoxic conditions. Serum lactate level after exercise was significantly higher in MI-WT BMT mice than in MI-Pres BMT mice (Fig. 3D).
Characterization of skeletal muscle in Presbyterian BMT mice
We characterized the tibialis anterior muscle of Presbyterian BMT mice 4 weeks after BMT. There were no significant differences in cross-sectional area between the WT BMT and Presbyterian BMT groups (data not shown). Histochemical staining for adenosine triphosphatase activity showed a similar percentage of type IIA and IIB fibers for both groups of mice (Figs. 4Aand 4B). The fiber type distributions were not affected by Presbyterian BMT as long as 24 weeks after transplantation (data not shown). There were no significant differences in mitochondrial SDH activity in either type IIA or IIB fibers between the WT and Presbyterian BMT groups (Figs. 4A and 4C). Total SDH activity measured by biochemical methods was not different in each group (Fig. 4D). These results indicate that Presbyterian BMT did not affect oxidative energy metabolism. Furthermore, capillary density of the muscle was not altered in the Presbyterian BMT groups (Figs. 4E and 4F).
Effect of RSR13 on exercise capacity
We examined the effect of RSR13 (Fig. 5A)on exercise capacity of WT mice. Because both RSR13- and vehicle-treated mice could complete a run for 90 min on a treadmill under the conditions used for the BMT treatment, the exercise test was started 30 min after RSR13 injection at 15 m/min for 30 min on a level treadmill, after which the gradient was increased by 5° every 30 min. Control and RSR13-treated mice ran for a similar distance during the first 30 min, but RSR13 treatment enabled the mice to run for a longer distance than the control mice thereafter (Fig. 5B). Next, we examined the effect of RSR13 on the exercise capacity of MI mice. Four weeks after LCA ligation, MI mice were injected with RSR13 or a vehicle. The RSR13 had no effect on heart rate, blood pressure, and echocardiographic parameters (data not shown). The exercise test was then performed as described for RSR-treated normal mice, except that the running speed was 12.5 m/min because of the lower exercise capacity of CHF mice. Three days later, the exercise test was performed again, but each group received the opposite treatment from the one received the previous session. The test was repeated 4 times. Up to 60 min, RSR13- and vehicle-injected mice registered a similar total resting time and running distance (Figs. 5C and 5D). After 60 min, however, the RSR13-treated mice showed a significantly shorter total resting time and longer running distance than the control mice (running distance for 30 min, RSR-treated: 344.7 ± 19.7 m; control: 267.6 ± 33.9 m).
The main finding of the present study is that a reduction in Hb–O2affinity resulted in improvement of the exercise capacity of CHF mice independent of cardiac function. There still will be a risk that the short-term benefit in terms of enhanced exercise capacity may shorten long-term survival or that the increased physical activity itself may lead to sudden death during exercise. A theoretical drawback is that increasing oxygen delivery to tissues may lead to the generation of oxygen free radicals, which can be harmful to the cardiovascular system. In our experimental setting, however, no mice died during the follow-up period of 24 weeks after BMT or during the exercise tests, and Presbyterian BMT did not affect blood gas levels or cardiac function. Thus, the treatments described in this study will provide a novel therapeutic strategy that improves exercise tolerance without increasing mortality.
In this study, we examined the exercise capacity of MI WT mice transplanted with Presbyterian BMC rather than MI Presbyterian transgenic mice because the latter showed anemia, acidosis, alteration in muscle properties, and increased spontaneous physical activity (2), which may directly or indirectly affect exercise capacity. The Presbyterian BMT and WT BMT mice did not show any differences in such phenotypic parameters. We used the different exercise protocols for the different therapeutic treatments to detect significant effects of reduction in Hb–O2affinity by BMT or RSR13 treatment on exercise capacity. Our results indicate that the BMT procedure including radiation alone substantially reduced exercise tolerance. Although we cannot exclude a possibility that the difference in sensitivity to the BMT procedure may influence exercise capacity, quite different procedures such as RSR13 treatment gave similar results in terms of exercise capacity of mice with CHF, and it is likely that Hb–O2affinity is a major determinant in exercise capacity.
The most common causes of exercise intolerance associated with CHF are the development of dyspnea caused by pulmonary congestion and the failure of the heart to provide sufficient blood flow to exercising muscles (1). A number of other peripheral factors may explain exercise intolerance, including abnormalities of vasodilatory capacity, endothelial function, ergoreflex activation, muscular metabolism, and distribution of cardiac output (13). The impairment of vasodilatory capacity has been attributed to excessive sympathetic stimulation, activation of the plasma renin–angiotensin system, higher levels of endothelin, and impaired release of nitric oxide. Anaerobic metabolism occurs early during exercise in CHF and is likely an important cause of exercise intolerance (14). Patients with CHF have shown a decrease in oxidative type I fibers and an increase in glycolytic type IIB in the skeletal muscles (14). Levels of oxidative enzymes of mitochondrial enzymes, including SDH, are decreased in CHF (15). In this study, the improved exercise performance of Presbyterian BMT mice was not accompanied with changes in cardiac function, properties of skeletal muscle, or sympathetic nerve activity (data not shown). In addition, growth factors or endothelial stem/progenitor cells contained in BMC solution may function differently between WT and Presbyterian BMC. The fact that the acute administration of RSR13 showed a similar effect with Presbyterian BMT, however, excludes such possibilities. Furthermore, it is unlikely that slow processes such as structural alterations or metabolic changes containing protein synthesis are involved in the enhanced exercise capacity by Presbyterian BMT. Thus, the mechanism producing enhanced exercise capacity in response to the therapies seems to be derived from enhanced O2delivery in the skeletal muscle. However, it is still possible that reducing Hb–O2affinity may directly or indirectly affect a number of central or peripheral factors contributing to exercise intolerance in CHF. Although the exact underlying molecular mechanism for the improved exercise capacity remains to be elucidated, this study clearly indicates that a synthetic allosteric modifier of Hb, such as an RSR13, will be useful for the treatment of patients with CHF to improve their daily physical activity.
Because RSR13 is a relatively short-acting drug and can be administered only intravenously, modification of the drug is necessary for its clinical application. Further investigations using larger animals over a longer period of time will be necessary to determine whether the reduction in Hb–O2affinity and increased physical activity actually benefit the lives of patients with CHF.
The authors thank Naoko Watanabe and Sunao Tanaka for their technical assistance.
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a research grant from Nakatomi Foundation to Dr. Otsu. Drs. Watanabe, Takeda, and Omiya contributed equally to this work.
- Abbreviations and Acronyms
- bone marrow cell
- bone marrow cell transplantation
- chronic heart failure
- hemoglobin Presbyterian
- left coronary artery
- myocardial infarct/infarction
- succinate dehydrogenase
- wild type
- Received October 29, 2007.
- Revision received April 29, 2008.
- Accepted June 3, 2008.
- American College of Cardiology Foundation
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