The goal of the research was to identify the structural and functional characteristics of the rat's left ventricle under antiorthostatic suspension within 1, 3, 7 and 14 days, and subsequent 3 and 7-day reloading after a 14-day suspension. The transversal stiffness of the cardiomyocyte has been determined by the atomic force microscopy, cell respiration—by polarography and proteins content—by Western blotting. Stiffness of the cortical cytoskeleton increases as soon as one day after the suspension and increases up to the 14th day, and starts decreasing during reloading, reaching the control level after 7 days. The stiffness of the contractile apparatus and the intensity of cell respiration also increases. The content of non-muscle isoforms of actin in the cytoplasmic fraction of proteins does not change during the whole experiment, as does not the beta-actin content in the membrane fraction. The content of gamma-actin in the membrane fraction correlates with the change in the transversal stiffness of the cortical cytoskeleton. Increased content of alpha-actinin-1 and alpha-actinin-4 in the membrane fraction of proteins during the suspension is consistent with increased gamma-actin content there. The opposite direction of change of alpha-actinin-1 and alpha-actinin-4 content suggests their involvement into the signal pathways.
Exposure to microgravity causes various changes in the human cardiovascular system, particularly a cephalic fluid shift in the cranial direction [
In order to simulate most of the effects in the human body under weightlessness, the model of antiorthostatic suspension by the tail is used with rodents, such as rats. It has been demonstrated that the effects typical of the muscle and bone tissue in microgravity can be satisfactorily reproduced in ground experiments using antiorthostatic suspension [
However, the existing data about changes in the cardiovascular system are quite contradicting. Most researchers observe fluid shift and presence of hypovolemia in rats during the antiorthostatic suspension [
The effects of the early period cause increased volumetric load on the heart by activating cardiopulmonary receptors [
At the same time, there is data demonstrating a decreased contractility of the rat’s heart as a result of long-term suspension [
A number of authors observed decreased contractility of the myocard and decreased oxygen consumption during long-term suspension [
Thus, to sum up the existing literature, we can state that, at early stages of antiorthostatic suspension, the volumetric load on the heart is increased, and increased activity of mitochondrial ferments is observed. Later, during long-term antiorthostatic suspension, no changes in the heart weight and arterial pressure were observed in rats, even though the central venous pressure decreased. The contractile ability of the myocard decreased, including on the level of single cardiomyocytes. At the same time, no changes were identified in the intensity of cell respiration during long-term suspension, although oxygen consumption by the heart did decrease.
We suggested that at least the initial stages of suspension increase the mechanical load on the cardiomyocytes, unlike the cells of skeletal muscles, which have a lesser load under these conditions. Then, at early stages of reloading, the fluid shift is reversed and the mechanical tension of the cardiomyocytes should decrease. Meanwhile, changes in mechanical tension of the skeletal muscles are in the opposite direction. Our earlier data confirm that the stiffness of the cortical cytoskeleton of the skeletal muscle fibres is decreased, possibly due to the destruction of submembrane F-actin and dissociation of actin-binding proteins due to a change in the mechanical tension of the skeletal muscle cell [
The experiments have been performed with the tissue of the left ventricle of a Wistar rat (
All procedures with animals were approved by the biomedical ethics committee of the State Research Center of Russia at the Institute of Biomedical Problems of the Russian Academy of Sciences.
Cardiomyocytes were obtained from a part of the tissue of the rat’s left ventricle using the standard method [
On the day of the experiment, the samples were transferred to solution R where single glycerynized cardiomyocytes were singled out.
In order to obtain demembranized cardiomyocytes, single glycerynized cardiomyocytes in the R solution were incubated for 12 hours at +4°C with the Triton X-100 detergent with the final concentration of 2% v/v. Such concentration of the detergent used and the long incubation time enable completely removing the membranes of cardiomyocyte to analyze only the myofibrillar apparatus. After treatment with the detergent, the obtained demembranized cardiomyocytes were cleaned in the R solution.
In order to measure the transversal stiffness, the obtained cardiomyocytes were fixed on the bottom of the liquid cell of the atomic force microscope, attaching their tips with special Fluka shellac wax-free glue (Sigma). Depending on the series of experiments, the cell was filled either wih the relaxation solution R, or activation solution A (20 mM MOPS, 172 mM of potassium propionate, 2,38 mM of magnesium acetate, 5 mM CaEGTA, 2.5 mM of ATP), or rigor solution Rg (20 mM MOPS, 170 mM of potassium propionate, 2,5 mM of magnesium acetate, 5 mM of K2EGTA). All contractions of the cardiomyocyte were isometric since the tips of the cardiomyocytes were fixed.
All experiments were conducted at +16°C.
Atomic force microscopy was used in order to determine the transversal stiffness of various compartments of the cardiomyocyte. The method of obtaining images of the cardiomyocyte surfaces to perform local measurements of transversal stiffness has been described in detail earlier [
Measurements of transversal stiffness of both glycerynized and Triton X-100-treated cardiomyocytes were conducted using the Solver-P47-Pro platform (NT-MDT, Russia). The indentation depth was 150 nm. We tested at least 21 cardiomyocytes from each sample (
The results were processed in a special program in MatLab 6.5.
A part of the tissue of the left ventricle was immediately placed into cold solution A (2.77 mM of CaK2EGTA, 7.23 mM of K2EGTA, 6.56 mM of MgCl2
Oxygen adsorption rate was evaluated using the Saks polarography method [
The following parameters of respiration were measured:
In order to determine the protein content, a part of the rat’s left ventricle was frozen at the temperature of liquid nitrogen. The method described in Vitorino et al. [
In order to determine each protein, specific monoclonal primary antibodies based on mice immunoglobulines were used (Santa Cruz Biotechnology, Inc.) in the manufacturer-recommended dilutions: 1 : 200 for desmin, 1 : 300 for beta-actin, 1 : 100 for gamma-actin, 1 : 100 for alpha-actinin-1, 1 : 100 for alpha-actinin-4—1 : 100. For secondary antibodies, we used biotinylated goat antibodies against mice IgG (Santa Cruz Biotechnology, Inc.) diluted 1 : 5000.
In order to determine alpha-actinin-2 content, specific monoclonal primary antibodies based on rabbit immunoglobulines were used (Santa Cruz Biotechnology, Inc.) diluted 1 : 200 as recommended by the manufacturer. For secondary antibodies, we used biotinylated goat antibodies against rabbit IgG (Sigma, Germany) diluted 1 : 5000.
Afterwards, all membranes were treated with streptavidin conjugated with horseradish peroxidase (Sigma, Germany) diluted 1 : 5000. Protein lines were identified using 3,3′-diaminobenzidine (Merck, USA).
We tested
The results obtained during the experiments were statistically processed with ANOVA, using a post hoc
In the control group (Table
Transversal stiffness (pN/nm) of isolated rat’s Triton-treated left ventricular myocytes in liquid in relaxed, calcium activated (pCa = 4.2) and rigor states under gravitational unloading and subsequent reloading.
State | |||
---|---|---|---|
Group | Relaxed | Activated (pCa = 4.2) | Rigor |
Transversal stiffness of the half-sarcomere area |
|||
Control ( |
7.1 ± 0.4 | 11.0 ± 0.5@ | 13.2 ± 0.5@ |
1-HS ( |
6.8 ± 0.3 | 10.3 ± 0.3@ | 12.9 ± 0.3@ |
3-HS ( |
8.2 ± 0.3* | 13.8 ± 0.4*/@ | 14.7 ± 0.3*/@ |
7-HS ( |
8.9 ± 0.3* | 13.5 ± 0.3*/@ | 15.3 ± 0.3*/@ |
14-HS ( |
8.7 ± 0.4* | 13.6 ± 0.4*/@ | 15.6 ± 0.4*/@ |
14-HS + 3-R ( |
4.3 ± 0.3*/$ | 6.8 ± 0.3*/@/$ | 8.2 ± 0.5*/@/$ |
14-HS + 7-R ( |
7.7 ± 0.3 | 11.0 ± 0.9@ | 13.4 ± 0.6@ |
| |||
Transversal stiffness of the M-band area |
|||
Control | 9.9 ± 0.6 | 15.0 ± 0.6@ | 16.4 ± 0.5@ |
1-HS | 8.9 ± 0.5 | 14.1 ± 0.4@ | 15.8 ± 0.5@ |
3-HS | 9.3 ± 0.5 | 14.8 ± 0.6@ | 16.3 ± 0.5@ |
7-HS | 10.6 ± 0.4 | 14.9 ± 0.5@ | 16.5 ± 0.8@ |
14-HS | 9.8 ± 0.5 | 14.2 ± 0.6@ | 15.7 ± 0.3@ |
14-HS + 3-R | 6.2 ± 0.3*/$ | 12.6 ± 0.4*/@/$ | 13.1 ± 0.4*/@/$ |
14-HS + 7-R | 9.3 ± 0.4 | 14.3 ± 0.8@ | 17.1 ± 0.5@ |
| |||
Transversal stiffness of the Z-disk area |
|||
Control | 16.0 ± 1.3 | 22.5 ± 1.5@ | 24.5 ± 0.9@ |
1-HS | 15.4 ± 0.6 | 21.6 ± 0.3@ | 24.8 ± 0.6@ |
3-HS | 15.9 ± 0.6 | 23.4 ± 0.7@ | 26.1 ± 1.3@ |
7-HS | 15.9 ± 0.5 | 21.7 ± 0.3@ | 25.9 ± 0.6@ |
14-HS | 15.8 ± 0.8 | 21.9 ± 0.9@ | 22.9 ± 0.7@ |
14-HS + 3-R | 7.5 ± 0.4*/$ | 13.4 ± 0.5*/@/$ | 16.0 ± 0.4*/@/$ |
14-HS + 7-R | 15.4 ± 0.6 | 21.4 ± 0.5@ | 23.4 ± 1.4@ |
However, in three days, the stiffness of the contractile apparatus significantly increased near the semisarcomere both in the relaxed and in the activated/rigor states compared to similar states in the control group. This increase was even more prominent seven days after antiorthostatic suspension. 3-day reloading after 14-day suspension caused significant reduction in the transversal stiffness in all states relative to both the 14-HS group and the control group. However, after seven days of reloading, the transversal stiffness returned to the level of the control group.
Nevertheless, during the antiorthostatic suspension and subsequent reloading, when contraction was activated, the stiffness of the semisarcomere reliably decreased compared to the relaxed state.
At the M-line, the stiffness of the contractile apparatus of fibers of the left ventricular of rat was higher than the stiffness of the semisarcomere. During antiorthostatic suspension, the transversal stiffness of the M line remained unchanged, but it significantly decreased after three days of reloading that followed a 14-day suspension. After 7 days of reloading, the transversal stiffness of the M-line did not differ from the control group values.
The transversal stiffness of the Z-disk is significantly higher than in the semisarcomere and the M band. As described above, it also increased during activation and rigor. The changes in transversal stiffness of the Z-disk over time were similar to those of the M-line, except that it decreased more after 3 days of reloading.
The transversal stiffness of various segments of glycerynized fibers of the left ventricular of rat (Table
Transversal stiffness (pN/nm) of isolated rat’s permeabilized left ventricular myocytes in liquid in relaxed, calcium activated (pCa = 4.2) and rigor states under gravitational unloading and subsequent reloading.
State | |||
---|---|---|---|
Group | Relaxed | Activation (pCa = 4.2) | Rigor |
Transversal stiffness of sarcolemma between the M-band and Z-disk projections |
|||
Control ( |
4.03 ± 0.11 | 9.1 ± 0.4@ | 9.67 ± 0.24@ |
1-HS ( |
4.80 ± 0.22* | 9.5 ± 0.5@ | 10.1 ± 0.3@ |
3-HS ( |
6.22 ± 0.29* | 10.2 ± 0.3*/@ | 10.8 ± 0.4*/@ |
7-HS ( |
7.79 ± 0.12* | 10.5 ± 0.3*/@ | 13.3 ± 0.4*/@ |
14-HS ( |
12.3 ± 0.4* | 12.9 ± 0.4* | 13.3 ± 0.3*/@ |
14-HS + 3-R ( |
6.4 ± 0.5*/$ | 7.5 ± 0.3*/$ | 8.4 ± 0.6*/$/@ |
14-HS + 7-R ( |
4.3 ± 0.4 | 8.9 ± 0.5@ | 9.2 ± 0.3@ |
| |||
Transversal stiffness of sarcolemma at the M-band projection |
|||
Control | 2.85 ± 0.12 | 6.8 ± 0.5@ | 7.4 ± 0.6@ |
1-HS | 2.68 ± 0.17 | 6.6 ± 0.3@ | 7.0 ± 0.3@ |
3-HS | 3.77 ± 0.25* | 6.7 ± 0.4@ | 7.7 ± 0.5@ |
7-HS | 5.79 ± 0.19* | 6.9 ± 0.3@ | 7.9 ± 0.3@ |
14-HS | 7.55 ± 0.13* | 7.9 ± 0.6* | 9.4 ± 0.3*/@ |
14-HS + 3-R | 3.57 ± 0.20*/$ | 5.9 ± 0.4$/@ | 6.3 ± 0.3$/@ |
14-HS + 7-R | 3.1 ± 0.3 | 6.2 ± 0.4@ | 7.0 ± 0.6@ |
| |||
Transversal stiffness of sarcolemma at the Z-disk projection |
|||
Control | 10.0 ± 0.3 | 14.5 ± 1.2@ | 15.3 ± 0.5@ |
1-HS | 10.7 ± 0.8 | 15.4 ± 0.3@ | 16.3 ± 0.5@ |
3-HS | 10.6 ± 0.3 | 14.8 ± 0.3@ | 15.2 ± 0.7@ |
7-HS | 10.4 ± 0.3 | 11.9 ± 0.3@ | 16.5 ± 0.3@ |
14-HS | 17.2 ± 0.4* | 17.9 ± 0.4* | 18.2 ± 0.5*/@ |
14-HS + 3-R | 9.2 ± 0.3*/$ | 10.3 ± 0.6*/$ | 11.9 ± 0.5*/$/@ |
14-HS + 7-R | 9.9 ± 0.7 | 15.1 ± 0.8@ | 15.6 ± 0.4@ |
After one day of antiorthostatic suspension, the transversal stiffness of the sarcolemma between the Z-disk and M-band projections in the relaxed state significantly increased relative to the control values, but did not differ from the control values in the activated and rigor states. After three days of suspension, the transversal stiffness of the sarcolemma increased even more and continued to increase up to the 14th day, and it significantly differed from the control value in the relaxed, activated, and rigor states. Moreover, after 14 days of suspension, the transversal stiffness values of this area during activation and in rigor did not significantly differ from the values in the relaxed state. After 3 days of reloading, the transversal stiffness of the sarcolemma decreased compared to values in the 14-HS group, but only reached the levels of the control group by the 7th day of reloading.
The transversal stiffness of the sarcolemma in the M-band projection in the relaxed state significantly increased compared to the control levels by the 3rd day of suspension and subsequently increased up to the 14th day. The transversal stiffness values in this area in the activated and rigor states did not differ from the control level. After 3 days of reloading following a 14-day suspension, the transversal stiffness of the membrane in the M-line projection decreased, but reached the control group level only after 7 days of reloading.
The transversal stiffness of the sarcolemma in the Z-disk projection significantly increased only by the 14th day of suspension, significantly decreased after 3 days of reloading, and did not differ from the control group levels after 7 days.
Cell respiration of the rat’s left ventricular myocytes under gravitational unloading and subsequent reloading.
Parameter | ||||
---|---|---|---|---|
Group |
|
|
|
Respiration ratio |
·min-1·mg-1 | ·min−1·mg−1 | ·min−1·mg−1 | ||
Control ( |
14.8 ± 1.4 | 15.9 ± 0.9 | 22.7 ± 1.6 | 1.46 ± 0.11 |
1-HS ( |
21.2 ± 1.9* | 29.7 ± 2.8* | 49.0 ± 5.1* | 1.64 ± 0.07 |
3-HS ( |
16.3 ± 1.1& | 26.0 ± 1.5* | 47.5 ± 2.3* | 1.91 ± 0.21* |
7-HS ( |
16.9 ± 0.8& | 28.3 ± 1.9* | 44.9 ± 2.1* | 1.59 ± 0.13 |
14-HS ( |
16.4 ± 1.2& | 27.6 ± 1.5* | 44.5 ± 2.1* | 1.64 ± 0.14 |
14-HS + 3-R ( |
9.9 ± 0.6*/&/$ | 13.6 ± 0.7*/&/$ | 18.5 ± 1.9*/&/$ | 1.44 ± 0.12 |
14-HS + 7-R ( |
15.7 ± 1.7 | 17.2 ± 1.4 | 25.1 ± 1.6 | 1.58 ± 0.15 |
The basal respiration rate
The respiration rate on exogenous substrated
Changes in the maximum respiration rate determined by adding ADP
The estimated value reflecting the efficiency of coupling of oxidation and phosphorylation, known as respiratory control, increased after three days of antiorthostatic suspension by 31%
Relative alpfa-actinin-2 content in the total protein (Figure
Protein content in the rat’s left ventricular myocytes under gravitational unloading and subsequent reloading;
Desmin content (Figure
Beta-actin content (Figure
Beta-actin content in the rat’s left ventricular myocytes under gravitational unloading and subsequent reloading;
Gamma-actin content (Figure
Gamma-actin content in the rat’s left ventricular myocytes under gravitational unloading and subsequent reloading;
Alpha-actinin-1 content (Figure
Alpha-actinin-1 content in the rat’s left ventricular myocytes under gravitational unloading and subsequent reloading;
Alpha-actinin-4 content (Figure
Alpha-actinin-4 content in the rat’s left ventricular myocytes under gravitational unloading and subsequent reloading;
The results of this research suggest that antiorthostatic suspension causes a number of changes in the cardiomyocytes of rats.
The results of AFM measurements of the stiffness of the myofibrillar apparatus of rat’s left ventricle cardiomyocytes suggest that transversal stiffness of the Z-disk and M-line in the relaxed state was
In the course of antiorthostatic suspension, the transversal stiffness of the contractile apparatus of the rat’s left ventricle cardiomyocytes near the Z-disk and the M-line remained unchanged. However, after 3 days of reloading following a 14-day suspension, these parameters significantly decreased. Akiyama et al. [
The transversal stiffness of the contractile apparatus near the semisarcomere increased after 3 days and remained increased up to the 14th day of antiorthostatic suspension. The changes in the transversal stiffness as a mechanical parameter reflect the changes in the structure in question. Since the contractile apparatus is a significantly nonuniform structure, any changes in the transversal stiffness may be caused due to a number of physical and chemical factors (temperature, pH, and others), and the phosphorylation level of the light chains of myosin or interfilament spacing. However, in all of the experiments, we have conducted the physical and chemical parameters that remained stable. Changes in the level of phosphorylation of light myosin chains may cause higher probability of closing crossbridges [
On the other hand, the observed decrease of the maximum contractile force and calcium sensitivity of single cardiomyocytes of the rat during antiorthostatic suspension [
At the same time, desmin is one of the key proteins determining localization of mitochondria [
In view of the above, we decided to analyze the structure of cortical cytoskeleton of cardiomyocytes during antiorthostatic suspension and subsequent reloading. AFM results suggest that, in the relaxed state, the transversal stiffness of the membrane with adjacent cortical cytoskeleton increased near the middle of the semisarcomere, that is, between the projections of the Z-disk and M-line after 1 day, and continued to increase during antiorthostatic suspension. The stiffness of the membrane in the Z-disk and M-line projection also grew, but somewhat later, that is, after 14 and 3 days of suspension, respectively. Increased stiffness of all parts of the membrane during activation of contraction and rigor of fibre remained the same as in the control group, which was expected because desmin content remained unchanged, as desmin enables transfer of tension from the contractile apparatus to the membrane of the cardiomyocyte.
Similarly to the results of Costa et al. [
In order to verify this assumption (that increased transversal stiffness of the cortical cytoskeleton of cardiomyocytes may be linked to increased content of non-muscle actin isoforms), we have analyzed relative content of these proteins in the cytoplasmic and membrane fractions. Our results suggest that beta-actin content in the cytoplasmic and membrane fraction remained unchanged during antiorthostatic suspension and subsequent reloading. Gamma-actin content in the cytoplasmic fraction of proteins also remained the same as the control group level during suspension and reloading. However, gamma-actin content in the membrane fraction of proteins increased significantly on the first day of antiorthostatic suspension and continued to increase up to the 14th day, following the same trend as that of transversal stiffness. It should be noted that the increase of non-muscle F-actin (beta-actin) was observed in cat’s cardiomyocytes during stimulated hypertrophy [
At the same time, alpha-actinin-1 content in the cytoplasmic fraction of proteins decreased after 7 days of suspension, but increased in the membrane fraction. During 3-day reloading following 14 days of antiorthostatic suspension, alpha-actinin-1 content in the membrane fraction decreased, while it increased in the cytoplasmic fraction and did not differ from the control group’s level after 7 days of reloading in both fractions. At the same time, alpha-actinin-4 content in the membrane fraction of proteins grew on the first day of antiorthostatic suspension and continued to increase up to the 14th day, and, starting from the third day, its content in the cytoplasmic fraction exceeded the control group level. During the reloading period, alpha-actinin-4 content in the membrane fraction fell to the control group level, and also decreased in the cytoplasmic fraction although it did not reach the control group level.
It should be noted that hardly anything is known about the role of non-muscle forms of alpha-actinin in skeletal muscle cells and cardiomyocytes. Nevertheless, there is evidence that the increase of relative alpha-actinin-4 content in the cytoplasmic fraction is linked to the decrease of alpha-actinin-1 content, there and formation of a cancer pattern of fibroblasts [
In the conclusion, to sum up our experimental results and the above discussion, we can hypothesize the following sequence of events in cardiomyocytes at early stages of antiorthostatic suspension and subsequent reloading after the end of experimental exposure. Increased volumetric load on the heart at early stages of antiorthostatic suspension causes deformation (stretching) of the cardiomyocyte, involving coordinated stretching of the contractile apparatus and the cortical cytoskeleton. Stretching of the contractile apparatus may cause smaller inter-filament spacing and, consequently, the observed increase of myofibril stiffness, which can be the reason for reduced contractility of the cardiomyocyte. Furthermore, we can suggest a hypothesis (that has to be proven experimentally) that stretching of the cortical cytoskeleton will cause dissociation of alpha-actinin-1 from submembrane actin, and initiate overexpression of non-muscle actin and alpha-actinin-4, since the content of these two nonmuscle isoforms of alpha-actinin is interrelated. Increased content of non-muscle actin in the membrane fraction will require increased content of both isoforms of alpha-actinin in the membrane fraction as well to form the structure of the cortical cytoskeleton, which will be reflected in its increased stiffness that we observed in our experiments. At the same time, increased content of alpha-actinin-4 in the cytoplasmic fraction that we observed may cause higher cytochrome
Thus, in our study, we have determined the transversal stiffness of various parts of the contractile apparatus and the membrane with cortical cytoskeleton of rat’s cardiomyocytes in the course of antiorthostatic suspension and subsequent reloading, as well as the cell respiration rate and relative content of alpha-actinin-2, desmin, non-muscle actin (beta and gamma), alpha-actinin-1, and alpha-actinin-4 in the membrane and cytoplasmic fractions of proteins. The above parameters describe structural and functional state of rat’s cardiac muscle cells during antiorthostatic suspension and reloading, which led us to suggest a hypothetical mechanism of reaction of these cells to changes in external conditions, which requires further research to be experimentally confirmed.
The financial support of the Russian Foundation of Basic Research (RFBR Grant 10-04-00106-a) and program of fundamental research SSC RF-IBMP RAS greatly acknowledged. No conflict of interests are declared by the authors.