International Scientific Training Center for Information Technologies and Systems Nathional Academy of Sciences of Ukraine,
prosp. Acad.Glushkova, 40, Kiev, 03680, Ukraine
While adapting to environmental changes, cells tend to eliminate en- ergy imbalance. Being at the core of cellular energetic process, mitochondria monitor O2 supply and play central role in cell resistance to hypoxia. Appar- ently, a cell, and even its mitochondria, has the ability to sense O2 and to acti- vate adaptive processes that will enhance the survival in anticipation that oxygen availability might become limiting. Mitochondria move within the cell, their fission, fusion, motility, proliferation, and redistribution may change their oxidative power qO2 and affect the cell oxygen regimen [1,2].
Our goal is to examine the enhancement of oxidative power and redistribu- tion of mitochondria as a regulator of cell oxygen regimen under hypoxia.
Methods
To test some of mitochondria abilities to meet the oxygen lack chal- lenge we used mathematical modeling of transport and utilization of O2 in skeletal muscles under hypoxia [3]. The model describes steady state of capil- lary blood flow and three-dimensional oxygen diffusion - reaction in the myo- cyte. Solving the equation system, we get the distribution of pO2 mm Hg and VO2 (ml/min/100g tissue) in muscle fiber and mean values of tissue pO2 (PtO2), pO2 in capillary (PaO2, PvO2) and mean value of tissue VO2. Muscle blood flow F (ml/min/100g), oxygen concentration in arterial blood CaO2 (ml O2/l), and mitochondria oxidative power qO2 (ml/min/100g tissue) serve as input vari- ables. Oxidative power qO2 may be defined as mitochondrion number per unit of cell volume multiplied by their oxidative potential. The calculated values of the ratio S(x,y,z) = VO2(x,y,z)/qO2 describe the degree of hypoxia at (x,y,z).
They set up a field of hypoxia where pO2<pO2crit. We refer to a hypoxia if VO2< 0.9 qO2. The variation of S from 1 to 0 shows gradual decline of cellular VO2. The zone where VO2/qO2 < 1 represents the picture of hypoxia of vary- ing severity. The computational experiments take into account hypoxic influ- ence - various values of CaO2 and regulatory impact – values of oxidative power of mitochondria qO2, with which mean value of VO2 could stay ap- proximately constant. CaO2 varied within the range of 100-195mlO2/l, the F varied within the range of 10 - 70 ml / min/100g of tissue, at the selected minimum value qO2=3 ml/min/100g tissue. In the first series of calculations, we considered homogeneous distribution of qO2; in the second series qO2 was assumed to be proportional to values of VO2 obtained in the first series. We supposed that certain mitochondria may move from areas of poor oxygen sup- ply to the region of better oxygenation where oxygen demand would be satis- fied. Parameters of both oxygen regimens were compared.
Results
It is obvious that inadequate O2 delivery results in lower muscle oxy- gen consumption rate VO2, but carrying out the work with required intensity necessitates keeping VO2 at a required level. According to our calculations, if oxidative power of mitochondria qO2 increases, VO2 can remain unchanged despite increasing hypoxemia (Fig.1). We can see that maintain a constant level of VO2 is possible only within definite range of qO2. At a certain value of CaO2, the curve VO2 (qO2) goes abruptly upwards. Further increase of qO2
becomes ineffective because O2 cannot be extracted from blood. To keep a constant VO2 at hypoxia by increasing qO2 it is necessary to enhance O2 de- livery. Thus regulatory resource of increasing qO2 can be limited by the level of blood flow F (Fig. 1).
Calculated values oxygen mode parameters in muscle under arterial hypoxemia and increased oxidative power are listed in Table 1. It is shown that with decreasing of CaO2 qO2 have to be increased to get VO2=const on a background of certain Fi. With further decrease in CaO2, F must be increased in order to regain the possibility of regulating VO2 by varying qO2. We repre- sented the limiting values of F and qO2 at CaO2 = 130 and 110ml/l in Table 1 Maximum value of qO2 6ml/min/100g.
As can be seen, hypoxia area in the myocite grows ( S decline to 0.61) , but VO2 stays unchanged provided F is at an appropriate level.
Table 1 Oxygen mode parameters in the cell under arterial hypoxemia: homogene- ous distributed qO2 in the cell is increased. Pmin is pO2 in the dead corner
CaO2 195 160 150 130 110
F 30 30 30 40 60
qO2 4 4.7 6 5.7 6
PaO2 105 52 47 38 31
PvO2 22 14 11 14 16
PtO2 20 8 4.3 4 3
Pmin 2.8 0.3 0 0.2 0.2
S 0.92 0.78 0.62 0.65 0.61
AVD 123 121 123 92 61
VO2 3.7 3.7 3.7 3.7 3.7
Fig. 1. Cell VO2 remains constant under arte- rial hypoxemia due to the increase of qO2.
Based on the results of mathematical modeling and literature data [1, 2, 4, 5], we assume that mitochondrial oxidative power may be redistrib- uted inside a cell by increasing individual oxidative power of each mitochon- drion and by their migration inside a cell. Using previously obtained values of VO2, we redistribute qO2 according to cell gradient of VO2 and calculate parameters of a new oxygen regimen (Table 2).
Table 2 Oxygen mode parameters in the cell under hypoxic hypoxia: qO2 is in- creased and redistributed to avoid hypoxia
Ca 2 195 160 150 130 110
F 30 30 30 40 60
qO2 3.7 4.7 6 5.7 6
PaO2 105 52 47 38 31
PvO2 23 16 13 15 17
PtO2 20 11 7.3 7 6.2
Pmin 5.8 4 3.4 3.6 3.7
S 0.95 0.94 0.95 0.94 0.93
AVD 117 113 117 86 57
VO2 3.5 3.5 3.5 3.5 3.5
Migration and increase of oxidative capacity create such distribution of mitochondria power inside a cell that hypoxia is reduced or eliminated (S 93%, Pmin> Pcrit) and mean value of cell VO2 achieves the required level (Table 2).
Thus for convenience we may assume: that the first step of tissue ad- aptation process is an increase of mitochondrial oxidative power. This step permits to maintain VO2=const. Perhaps it will not be enough and the cell will have to send signals about O2 deficit and “to ask for” a change of blood flow level. Next step in adaptation is redistribution of mitochondria according to VO2 gradient. This will require a certain reconstruction of mitochondrial system, which implies not just increase of oxidative power but also parallel relocation of mitochondria inside the cell and increasing their number. Ac- cording to [4], mitochondrial clusters eliminate cell hypoxia. Our results show that inhomogeneous distribution of mitochondria can improve oxygen regimen even without clusters. As a result, hypoxia is eliminated, and new energetic level is attained.
Conclusion
Augmentation of oxidative power of mitochondria in a cell enables to keep constant mean value of oxygen consumption rate under hypoxia. Redis- tribution of oxidative power of mitochondria may improve tissue oxygen regimen by decreasing or elimination tissue hypoxia.
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MODIFICATION OF FUNCTIONAL STATE OF MUSCLE TISSUE UPON INFLUENCE EXTREMELY LOW FREQUENCY
OF ELECTROMAGNETIC FIELD
V.S. Martynyuk, Yu.V. Tseyslyer, O.V. Tsymbalyuk, O.V. Shelyuk, N.E. Nurishenko
Taras Shevchenko National University of Kyiv, 64, Volodymyrs'ka St., Kiev, 01061, Ukraine;
E-mail: mavis@science-center.net, yuliya.tseysler@gmail.com Search of mechanisms of the effect of weak electromagnetic fields of extremely low frequency (EMF ELF) on biological objects is one of the in- triguing problems of modern biophysics. The action of ELF EMF on biologi- cal objects is realized though different primary and system mechanisms and depended on frequency and amplitude of EMF, exposure, biological nature of experimental objects and its initial physiological state [1], as well as con- comitant physical factors and chemical agents [2]. It is known that different functional systems of humans and animals demonstrate unequal sensitivity to the influence of EMF ELF. Analysis of published data shows that the muscle tissue and its components are regarded as one of the targets of EMF [3], but this question remains poorly understood. In this regard it should be noted that the greatest number of papers devoted to studying the effect of EMF ELF on the activity of the myocardium and functional status of the cardiovascular system [4]. At the same time, there are small amount data that testified on direct effects of EMF on smooth muscle tissue and structural-functional properties of contractile proteins.
Smooth muscles are part of shells of internal organs: the intestines, blood vessels, respiratory tract, excretory and sexual organs, and many glands. Smooth muscles play an important role in regulating the functional activity of these organs.
The actomyosin complex is basic contractile element of muscle proteins.
Its ATPase activity is the key property of the muscle elements to carry out their contractile function. The basis of functional disorders of muscle contrac-
tility can be changes of activity of the myosin ATPase that is the main mech- ano-chemical energy converter. Violation of actin-myosin interaction can lead to changes in ATP utilization in muscle tissue and, as a consequence, to changes in the rate of contraction of myofibrils. Physical factors of different nature are able to influence on the ATPase activity of actomyosin. Among these factors the EMF ELF are considered, but its effects are poorly under- stood on the level of muscle tissue and its components.
Therefore, the aim of our study was to investigate the effect of EMF ELF on some functional parameters of smooth muscle and ATP-ase activity of skeletal muscle actomyosin. In particular, the effects of ELF EMF on spontaneous and K +-induced and also acetylcholine-induced contraction of smooth muscle strips of rat caecum were studied. At the same time the influ- ence of the EMF ELF on the ATPase activity of actomyosin in vitro that iso- lated from rabbit skeletal muscle was estimated.
Materials and methods. The smooth muscle contractile activity of cir- cular smooth muscles of the rat’s caecum was evaluated in this study. Strips of smooth muscles were placed in the chamber 2 ml with flow Krebs’ solu- tion (the flow rate is 4 ml/min) at 37°C. Contractions was measured by using an electric potentiometer 339. Contractions were induced by application of acetylcholine (ACh, 10 M) in Krebs’ solution, as well as hyper-potassium solution (K+, 80 mM). The contractile responses of smooth muscle strips were analyzed by the method of kinetic analysis proposed by T.V. Burdyga and S.O. Kosterin [5].
The actomyosin of skeletal muscle from rabbit was isolated by the Perry’s method modified by A.D. Tartakovsky [6] and us. Actomyosin was purified additionally by centrifugation at 20 000g for an hour. The Mg+2/Ca+2 - and K+-ATPase activity of actomyosin evaluated by the method described in [7]. The exposure of actomyosin solutions (2 mg/ml) in EMF ELF was 1, 2 and 3 hours at 37oC.
EMF (meander wave) with frequency 8 Hz and 25 T was created by us- ing the special generator G6-28 and Helmholtz coils.
Student’s test was used for estimation of statistical significance of differ- ences between independent statistical samplings.
Results. In the first part of this research the study of the spontaneous ac- tivity of smooth muscle strips showed that the effect of EMF ELF exposure caused a statistically significant (p<0.05) increase of frequency of contrac- tions and tends to increase the amplitude of spontaneous contractions. Simul- taneously, the electromagnetic treatment organizes a rhythmic activity. One of the causes of such changes can be synchronizing effect of EMF on the pacemaker activity of Cajal cells that play key role in the spontaneous activ- ity of smooth muscles in different organs.
EMF ELF caused inhibition of K+-induced contractions (23%, p<0.001) and also significant increase in the duration of the contraction and slowdown of relaxation (32.5%, p<0.01) in smooth muscle strips (fig. 1).
Fig. 1. Examples of recordings of K+-induced (80 M) (A) and acetylcholine- induced (10 µM) (B) contraction of smooth muscle strips of rat’s caecum upon in- fluence of EMF 8 Hz 25 µT.
Acetylcholine-induced contraction was less sensitive to the EMF- influence, however, the statistically significant decrease of the maximum power of contraction on average 11% was revealed (fig. 1). The other me- chanic-kinetic parameters of acetylcholine-induced contractions remained at control values. EMF-induced changes in acetylcholine contractions were sig- nificantly weaker compared with the responses to K+-depolarization, and no changes of ratio phase and tonic components was revealed in this case. The appearance of ordered cyclic tonic components was the feature of the EMF influence on the acetylcholine-induced contraction.
The effect of EMF ELF on the protein contractile elements of skeletal muscle, in particularly on ATPaase activity of actomyosin, was evaluated in the second part of the study. The analysis of the effect of EMF ELF on the ATPase activity of actomyosin showed that this process is characterized by certain dynamics. During first two hours of exposure of the protein solutions the EMF ELF inhibited the ATPase activity in comparison with control sam- ples that are not exposed to the EMF for all other equal conditions. This fact confirms data previously obtained by Lednev V. et al. [3]. However, the sig- nificant (p<0.05) increase of ATPase activity of actomyosin on the third hour of EMF-exposure was revealed but unexpected. Moreover, it is necessary to note that a similar pattern of change of enzyme activity was universal both for media with ions Mg+2 and Ca+2, and for the absence of these ions in the buffer (fig. 2). So, Mg+2/Ca+2- and K+-ATPase activity for one hour exposure to EMF were almost identical and were decreased by 16.5% and 16.3% rela- tive to control samples. Two hours later, Mg+2/Ca+2-ATPase activity de- creased by 19.5%, and K+-ATPase by 15.5%. After three hours of EMF ex- posure stimulate the Mg+2/Ca+2-ATPase activity by 33.6%, whereas the K+- ATPase activity by 50.3% (see Fig. 2). In our opinion, such effect of the EMF ELF exposure evidences on dynamic changes in the structural- functional properties of actomyosin in solution during long time that is fea- ture of nonequilibrium system.
Fig. 2. Change the Mg2 +/Ca2 +- and K+-ATPase activity (%) upon the influence of EMF (n=30) during 3 hour exposure.
Conclusions. Thus, our findings suggest that extremely low frequency EMF can alter both the contractile activity of smooth and skeletal muscle tissues. The direction and magnitude of the EMF-induced changes depends on the chemical agents that induce contraction and also on time of exposure.
The Ca+2, Mg+2-independent effects of the EMF ELF are intriguing and opens the prospect of learning a new primary mechanisms of action of this physical factor that based on sensitivity of magnetic moments of the nuclear spins of the hydrogen atom and diamagnetic currents of electrons in molecules [8].
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ISOFORM-SPECIFIC EFFECTS OF HINDLIMB UNLOADING ON Na,K-ATPASE IN THE RAT SOLEUS MUSCLE
V. Matchkov2, I.A. Razgovorova1, V.V. Kravtsova1, B.S. Shenkman3, I.I. Krivoi1
1St. Petersburg State University, St. Petersburg, Russia;
2Aarhus University, Aarhus, Denmark;
3RAS Institute for Biomedical Problems, Moscow, Russia
The Na,K-ATPase is a p-type ATPase which catalyzes the active trans- port of K+ into and Na+ out of the cell, thereby maintaining the steep Na+ and K+ gradients that provide electrical excitability and the driving force for many other transport processes. The Na,K-ATPase composed of alpha-catalytic and beta-glycoprotein subunits. Four isoforms of the alpha subunit are known to exist in tissues of vertebrates. It is generally accepted that the ubiquitous alpha1 isoform plays the main "house-keeping" role while the other isoforms express- ing in a cell- and tissue-specific manner possess additional regulatory functions that are still poorly understood. The largest pool of Na,K-ATPase in a verte- brate's body is contained in the skeletal muscles where the alpha1 and alpha2 isoforms of alpha-subunit are expressed [Orlowski, Lingrel, 1988]. The Na,K- ATPase is critically important for excitability, electrogenesis and contractility of skeletal muscle. Although the alpha2 isoform is expressed in high abundance in skeletal muscle, the functional role and mechanisms of regulation of this isoform remain unclear and are now being intensively investigated. It is well known that content of Na,K-ATPase strong depends on skeletal muscle activ- ity: muscle inactivity decreases Na,K-pump concentration while activity in- creases it [Clausen, 2008]. Some data indicates that increased skeletal muscle activity differently regulates alpha1 and alpha2 isoforms of the Na,K-ATPase [Kristensen et al., 2008]. Earlier it was shown that disuse induced by hindlimb unloading (HU) leads to progressive atrophy of postural skeletal muscle; the muscle undergoes a number of dramatic remodeling events. The isoform- specifity of HU effects on the Na,K-ATPase are not studied. In the present study, the effect of HU on the resting membrane potential (RMP), the electro- genic activity of the Na,K-ATPase 1 and 2 isoforms as well as their expres- sion in the rat soleus muscle were investigated.
Experiments were performed on male Wistar rats (210–230 g). To in- duce muscle unloading, the animals were tail suspended individually in spe- cial cages for 3 days. Control animals were not suspended. At the end of the period of unloading, soleus muscles were removed from the animals under deep anesthesia. Isolated muscles were perfused in a chamber with normal physiological solution aerated with a mixture of 95% O2 and 5% CO2 at 28oC (pH=7.4). The RMPs were recorded intracellularly in nonsynaptic region of muscle fibers using standard microelectrode techniques. Since in rodents the alpha1 isoform of the Na,K-ATPase is ouabain-resistant whereas the alpha2 isoform is highly ouabain-sensitive, we used ouabain at different concentra- tions to separate electrogenic contributions of these isoforms. We estimated the electrogenic contribution of the alpha2 isoform to RMP by administration
of 1 µM ouabain, a concentration selectively blocking alpha2 isoform in ro- dents without effect on ouabain-resistant alpha1 isoform [Heiny et al., 2010].
The electrogenic contribution of the alpha2 isoform was estimated as a differ- ence between RMP before and during 15–30 min of 1 µM ouabain action.
Then, for complete block of the alpha1 isoform an ouabain concentration of 500 µM was used. The electrogenic contribution of the alpha1 isoform was estimated as a difference between RMP during 15–30 min of 1 µM ouabain action and during 15–30 min after 500 µM ouabain addition.
Western blots were performed using lysates of frozen muscles. The al- pha1 and alpha2 isoform expressions were identified using specific antibod- ies. Membranes were then stripped for antibodies and stained for pan-actin which served as a house-keeping protein. Detected protein was quantified as a ratio to pan-actin. The mRNA expression was estimated with quantitative PCR using Taqman probe (FAM) technology. Gene expression was normal- ized to two house-keeping genes, GAPDH and transferrin receptor.
After 3 days of HU RMP value in soleus muscles (–66.8 0.7 mV, 187 fibers) was significantly more positive (by 4.2 0.9 mV; p<0.01) than in con- trol rats (–71.0 0.5 mV, 222 fibers). RMP distributions were close to normal;
in experimental rats the distribution was shifted to more depolarized RMPs as compared with control rats. In control rats, electrogenic activities of the al- pha1 and the alpha2 isoforms were 4.6 0.6 mV and 6.2 0.6 mV, respec- tively. The total Na,K-ATPase electrogenic contribution in control rats was 10.8 0.6 mV. After 3 days of HU, electrogenic activity of the alpha1 isoform decreased to 2.6 0.6 mV (p<0.01) compare to the control; the activity of the alpha2 isoform was not detectable (0.5 0.8 mV). The total electrogenic Na,K-ATPase contribution after HU was 3.1 0.9 mV (p<0.01 vs. control).
3 days of HU did not significantly changed the whole-muscle protein con- tent of Na,K-ATPase alpha1 subunit, while alpha2 subunit protein content de- creased to 48 7% (p < 0.01) of the control. At the same time, there was not seen any changes in Na,K-ATPase alpha1 and alpha2 subunits mRNA expression.
It was previously been shown that HU leads to accumulation of Ca2+
in myoplasm as well as increases the level of -calpains, Ca2+-dependent pro- tease [see for review: Kachaeva, Shenkman, 2012]. Our results suggest that the decreased alpha2 subunit content in muscle after HU might be due to in- creased protein degradation rather than alteration in the expression of Na,K- ATPase alpha subunits. The translocation mechanism by which Na,K- ATPase subunits are recruited to plasma membrane from intracellular com- partments [Benziane, Chibalin, 2008] could also be involved in HU responses of skeletal muscle. Our data provide further evidence that in the rat skeletal muscle the alpha1 isoform of Na,K-ATPase serves to maintain basal electro- genesis and contractility while the alpha2 isoform seems to provide some regulatory function(s) and more adjustable under functional unloading.
Supported by RFBR #10-04-00970; Saint-Petersburg State University research grant #1.37.118.2011; the Danish Research Council and the Novo Nordisk Foundation.