Gravity has a structural role for living systems. Tissue development, architecture, and organization are modified when the gravity vector is changed. In particular, microgravity induces a redistribution of blood volume and thus pressure in the astronaut body, abolishing an upright blood pressure gradient, inducing orthostatic hypotension. The present study was designed to investigate whether isolated vascular smooth muscle cells are directly sensitive to altered gravitational forces and, second, whether sustained blood pressure changes act on the same molecular target. Exposure to microgravity during 8 days in the International Space Station induced the decrease of ryanodine receptor subtype 1 expression in primary cultured myocytes from rat hepatic portal vein. Identical results were found in portal vein from mice exposed to microgravity during an 8-day shuttle spaceflight. To evaluate the functional consequences of this physiological adaptation, we have compared evoked calcium signals obtained in myocytes from hindlimb unloaded rats, in which the shift of blood pressure mimics the one produced by the microgravity, with those obtained in myocytes from rats injected with antisense oligonucleotide directed against ryanodine receptor subtype 1. In both conditions, calcium signals implicating calcium-induced calcium release were significantly decreased. In contrast, in spontaneous hypertensive rat, an increase in ryanodine receptor subtype 1 expression was observed as well as the calcium-induced calcium release mechanism. Taken together, our results shown that myocytes were directly sensitive to gravity level and that they adapt their calcium signaling pathways to pressure by the regulation of the ryanodine receptor subtype 1 expression.
- ryanodine receptor
- calcium signaling
- hindlimb suspension
during spaceflight, the microgravity (μG) induces blood volume redistribution and concomitant modification of the upright blood pressure gradient in the astronaut body from 70 mmHg in the head and 200 mmHg in the lowest resistance arteries to a constant pressure near 100 mmHg in the whole body (23). Thus a decrease in blood pressure below the heart seems to be one of the first consequences of μG, which can precede and trigger the adaptation of the vascular system (56). Many factors known to regulate blood pressure likely coexist in the vascular adaptation to μG: 1) the control of fluid volume and associated decrease in plasma volume (45), 2) the cardiac hypofunction and the alteration of baro- and cardiopulmonary reflexes (24), and 3) the adaptation of vascular responses at endothelial and vascular smooth muscle cell (VSMC) levels. Indeed, simulated μG, using the hindlimb unloaded (HU) rat model, can modify the vascular reactivity in infero-posterior vessels like abdominal aorta, mesenteric, femoral arteries, and portal vein (8, 20, 40, 47).
In the animal model of hypertension, a dysregulation of vasoreactivity encoded by Ca2+ signals has been reported. Indeed, the contraction induced by depolarization is greater in spontaneously hypertensive rats (SHR) than in control rats, and a change in Ca2+ signaling pathways implicating intracellular Ca2+ stores has been evidenced (1, 29, 30). More recently, a global adaptation of Ca2+ signaling pathways was suggested in this hypertensive model (51).
The vascular adaptation to changes in blood pressure is due to smooth muscle contractile activity and its regulation by endothelium, hormones, and neurotransmitters (13). In fact, the myogenic response can be considered as the equilibrium between contraction and relaxation pathways. Both pathways involve Ca2+ signaling. In VSMC, ryanodine receptors (RyRs) are reticulum Ca2+ channels likely implicated in both relaxation and contraction processes (5). Indeed, RyRs can be activated via the Ca2+-induced Ca2+ mechanism (CICR) that amplifies InsP3R and Ca2+ entry primary signals and thus participates to the cytoplasmic Ca2+ increase that can trigger contraction (22, 27). However, RyRs produce localized Ca2+ events (Ca2+ sparks), which activate Ca2+-activated K+ currents leading to membrane hyperpolarization and thus relaxation through inhibition of L-type channels (28). Three RyR genes have been described, and, although their expression pattern in VSMC varies between species and studies, the common expression of the three subtypes (RyR1–3) has been reported in rat aorta, superior and small mesenteric arteries, and hepatic portal vein (9, 44). Each RyR subtype had a specific function in Ca2+ signals. In smooth muscle, it has been shown that both RyR1 and RyR2 are able to generate Ca2+ sparks and Ca2+ waves (9). RyR expression is modulated in physiopathological conditions like muscular dystrophy and after blood redistribution following a chronic position change as hindlimb unloading (8, 10, 39, 40).
We designed the present study to investigate the effect of real μG on RyR expression in portal veins from mice and on rat primary cultured VSMC exposed to μG. To evaluate the functional effect of μG exposure, we have measured Ca2+ signals observed in HU rat model. Second, we have evaluated whether the regulation observed in HU (hypotension model) could be the opposite of those observed in SHR (hypertension model). In portal vein, the expression pattern of RyR1 was decreased by exposure to μG in both cultured VSMC and mouse portal vein as well as in portal vein from HU rat. We mimicked altered Ca2+ signals measured in portal vein from HU rats by in vivo injections of antisense oligonucleotide directed against RyR1. Finally, the increased pressure observed in SHR induced the opposite effect: the increase of RyR1 expression and the increase of Ca2+-induced Ca2+ release (CICR). Taken together, these findings suggest that VSMC adapt themselves to the forces that they experience, gravitational as hemodynamic, by modulating their expression of RyR1.
These investigations conform to the European Community and French guiding principles for the care and use of animals. Authorization to perform animal experiments (A-33-063-003) was obtained from the Prefecture de la Gironde (France). Wistar male rats as well as spontaneous hypertensive rats (SHR) and their control Wistar Kyoto rats (WKR) (120–150 g) were euthanized by cervical dislocation.
Hindlimb unloading rat.
Briefly, rats were hindlimb unloaded (HU; 8 days, angle 30–40°) by tail attachment as described previously (40) and in accordance with recommendations published by the National Aeronautics and Space Administration (NASA) (42). The hindlimb unloading time was chosen because 1) it was compatible to μG exposure during space flight missions (Soyuz TMA-8 transport mission and STS-118) and 2) we have previously shown that portal vein adaptation was significant after this HU duration. If there is no modification of the body weight, the HU effects calibrated on soleus wet weight are significant (52). Here, soleus wet weight from HU rats was 20% decreased compared with control rats. Moreover, vascular effects of μG such as orthostatic disorders can appear after only a few days of μG exposure (55).
Mice in STS118.
European Space Agency (ESA) gave us the opportunity to retrieve hepatic portal vein from mice that were exposed to μG during the STS-118 mission. The C57Bl6/J breeding was assumed by NASA. Animals were separated in three groups (n = 6 mice/group): conditioned in classical breeding environment baseline (BL); in cage used for the flight and stayed on Earth [ground control (GC)] and boarded on STS-118 (fly). Portal veins were removed, stored in 4% PFA for immunolabeling and RNA later solution (Qiagen) for RT-qPCR, and shipped to our laboratory for analysis.
Culture of VSMC from hepatic portal vein.
The dissociation of portal vein VSMC was detailed previously (41). Briefly, tissues were incubated twice in a cocktail of 0.8 mg/ml collagenase (EC 188.8.131.52) and 0.2 mg/ml pronase E (EC 184.108.40.206) in a low Ca2+ (40 μM) physiological solution (Hanks' balanced salt solution) at 37°C. VSMC were obtained by mechanical dispersion. VSMC used for Ca2+ measurements were seeded on glass slides and maintained in short-term primary culture in medium M199 containing 5% fetal calf serum (FCS), 20 U/ml penicillin, and 20 μg/ml streptomycin. Cells were kept in an incubator gassed with 95% air and 5% CO2 at 37°C and used during 24 h.
Packaging of VSMC to spaceflight.
Cells were packed in bags specific for cell culture in space flight. The packaging and the function of the hardware may be obtained from the CNES and ESA. Briefly, VSMC were in culture in appropriate medium, and the hardware contained programmed sequence to release angiotensin II (AII) solution (100 nM final) to verify their contractile phenotype and after formaldehyde fixative solution (3.7% final) to stop cell life after space exposure. The time profile was made to perform these experiments after either 24 or 200 h of μG exposure. Bags were placed into six MAMBA containers (Dutch-Space): three in μG conditions and three in the centrifuge to reproduce 1 G with space environment in the KUBIK incubator (COMAT). Finally, to measure eventual effects of travel or space environment, simultaneous and identical experiments were performed on cells kept on Earth. Cells were sent to the ISS with the Soyuz TMA-8 transport mission and returned with the Soyuz TMA-7 transport mission.
We used protocols that were previously described (12). Briefly, fixed VSMC were washed with PBS and permeabilized in PBS containing 2% bovine serum albumin and 1 mg/ml saponin. Anti-RyR antibody (34C, Santa Cruz Biotechnology, 1:1,000), anti-AT1 receptor antibody (SC-579, 1:500), or anti-smooth muscle actin antibody (SC-8432; 1:500) was added, and cells were incubated overnight at 4°C. Fixed portal veins from mice were treated as VSMC but were incubated with anti-RyR1 (AB9078, Millipore, 1:200), anti-RyR2 (C3.33, Millipore, 1:200), and anti-RyR3 (AB9082, Millipore, 1:200) antibodies. Samples were washed three times and incubated with the secondary Fluoprobe-488 antibody (1:250, Interchim) during 45 min at room temperature. After three washes in PBS, slides were mounted in Vectashied (Abcys). We have evaluated the non-specific fluorescence of secondary antibody by incubating cells only with the Fluoprobe-488 antibody (1:250), namely the primary antibody was omitted in the first steps. Microscope settings were defined with the non-specific preparation, and the acquisition parameters were kept constant to compare fluorescence emitted in each condition. The fluorescence counting was made only with non-saturated images and analyzed as described in Ref. 12.
Total RNA was extracted from cultured VSMC taken from hepatic portal vein using the RNA preparation kit from Epicentre following the instructions of the supplier. The reverse transcription reaction was performed on 50 ng of RNA using the Sensiscript-RT kit (Qiagen). Two different sets of sense and antisense primer pairs specific for RyR1, RyR2, and RyR3 were used as previously described (9). The first PCR protocol used with cutured portal vein cells was detailed in Ref. 12. In the second one, amplicons produced with CFX96 q-PCR machine (Biorad) were directly quantified by using CFX96 software. The normalization of RyR expression was realized with rat samples after verification of the stability of reference gene expression with GeNorm algorithms; the normalization was made with actin and GAPDH. The normalization used for the sample exposed to μG was made with Alien strategy (Agilent Technologies). It was consisting in addition to 1 pg of Alien RNA in RNA sample, and it was reverse-transcripted during PCR experiments with specific primer pair.
Antisense oligonucleotide treatment.
To mimic the decrease in RyR1 expression, we injected intraperitoneally a solution containing phosphorothioate antisense oligonucleotides (ASON, denoted with the prefix “as”) and JetPEI in vivo (Polyplus). Glucose solutions (5%), containing either asRyR1 associated to the JetPEI in vivo or only the vector, were injected following the recommendations of the supplier. After 4 days, a second injection was performed. Eight days after the first injection (D8), the rats were euthanized, and VSMC were isolated from hepatic portal vein as described above. To detect cells containing asRyR1, we used 5′-Cy5 indocarbocyanin-labeled ASON (Eurogentec). The fluorescence of Cy5 indocyanin was measured at 680 ± 32 nm (excitation 647 nm). The sequences of asRyR1 and the scrambled sequence of asRyR1 (asSCR) were previously described (9). The treated rats acted as control rats without visible troubles.
Contraction of portal vein strips.
Portal vein was dissected and opened to remove the endothelium. One muscle strip was taken in the middle of portal vein from rats, calibrated (mass 1 ± 0.2 mg), placed in an unloaded condition to minimize spontaneous contractions on a glass slide, and fully visualized with LSI macroscope setup (Leica Microsystems). The contraction was evaluated by the shortening of strip in both x and y directions.
Western blot analysis.
The protocols used were previously described (10). The muscle hepatic portal vein were homogenized in 50 μl of PBS 10% SDS solution. Supernatant was collected, and the protein content was measured according to the method of Bradford (6). Two different systems of revelations were used and described in supplementary methods. Briefly, in both cases, membranes were incubated (overnight, 4°C) with anti-RyR1 (AB9078), anti-RyR2 (C3.33), and anti-RyR3 (AB9082) primary antibodies (1:1,000). By using a stripping step, generally two different RyR subtypes have been probed per membrane. The secondary antibodies were coupled to a horseradish peroxydase and detected using an enhanced chemolumiscence kit (GE Healthcare) or coupled to Q-dot fluorophores (λem, 655 nm), and signal was detected using a ChemiGenius 2 system (Syngene).
Cytosolic Ca2+ measurements on VSMC from hepatic portal vein.
Physiological solution was composed as followed (in mM): 140 NaCl, 5.6 KCl, 1 MgCl2, 2 CaCl2, 11 glucose, 10 hepes, pH = 7.4 at 24°C with NaOH. Cells were loaded by incubation in a physiological solution containing 2 μM Fluo-4-AM or Fluo8L-AM, and cells were loaded for 20 min at 37°C. Probes were excited at 488 nm, and emitted fluorescence was filtered and measured at 540 ± 30 nm. Two different modes of image acquisition were used with the confocal system MRC1024ES (Bio-Rad) connected to a Nikon Diaphot microscope: image series were acquired at 1.2-s intervals and line-scan mode images were composed at a rate of 2 ms/scan. In this image, time increases from left to right. Image processing was performed by using Lasersharp 2000 software (Bio-Rad) and analysis by using IDL software as previously described (12). Background fluorescence was minimized by specific adjustment of MRC1024ES acquisition parameters. Caffeine, KCl, AII, and endothelin-1 (ET-1) were applied by pressure ejection from a glass pipette for the period indicated on the records, whereas other pharmacological agents were added to the perfused physiological solution. All experiments were carried out at 26 ± 1°C.
Cells from portal vein were loaded with the permeant form of InsP3 (cag-iso-2-145-InsP3, 2 μM in M199) during 30 min at 37°C before the Fluo8-AM loading. Photolysis produce by the xenon flash lamp (1-ms pulse) has been described previously (40). The flash intensity was adjusted from 150 to 250 V in these experiments.
Chemicals and drugs.
Collagenase was obtained from Worthington (Freehold, NJ). Fluo-4-AM, Fluo8L-AM, PBS, Sybr Green, TRITC-phalloidin, GeBa-Gels, and fluoprobe-488 secondary antibodies were from Interchim (Montluçon, France). Caffeine was from Merck (Darmstadt, Germany). Medium M199, streptomycin, and penicillin were from Invitrogen (Cergy Pontoise, France). As described in this section, antibodies were from Millipore (Saint-Quentin en Yvelines, France) and Santa-Cruz Biotechnologies (Tebu-Bio, Le Perray en Yvelines, France). Both used FCS were from PAA Laboratories (Linz, Austria). Cag-iso-2-145-InsP3 [d-2,3-O-isopropylidene-6-O-(2-nitro-4,5-dimethoxy)benzyl-myo-inositol 1,4,5-trisphosphate-hexakis(propionoxymethyl)ester] was synthesized by SiChem (Bremen, Germany). AII and ET-1 were synthesized by Genepep (Montpellier, France). All other chemicals were from Sigma (Lyon, France).
Data are expressed as means ± SE; n represents the number of tested cells. Significances between different conditions were tested with one-way ANOVA test with a Tukey's post test or with Student's t-test. P values of <0.05 were considered significant.
Orbital spaceflight decreased RyR expression in isolated vascular smooth muscle cells from hepatic portal vein.
To elucidate the effect of μG on RyR expression, VSMCs were isolated from rat hepatic portal vein on Earth and maintained in culture conditions designed to preserve their Ca2+ signals and phenotype integrity (Fig. 1). In space, vials containing a fixative solution were mechanically broken in the culture medium after 200 h of μG exposure. To test the contractile phenotype of the cultured smooth muscle cells, AII was released in specific plastic bags by breaking a first vial 15 min before fixation. After sample return, in AII-stimulated samples, the preservation of contractile phenotype was revealed by typical images of contracted cells after the labeling of actin filaments by TRITC-Phalloidin (Fig. 1). The proportion of contracted cells in AII-treated samples was ∼65 ± 10% in presence of AII vs. 10 ± 5% in CTL culture medium and was similar for both 1-G in-flight centrifuged control (1 G) and μG conditions.
RyR expression was investigated by RT-qPCR and immunostaining. No variations were found in actin expression between ground (Gr), 1-G, and μG conditions. Therefore, in each sample, level of actin expression as well as Alien expression were both used to normalize RyR expression. In culture, the expression of RyR2 subtype was progressively decreased. After 8 days in culture medium, the expression of RyR2 became undetectable by RT-qPCR in all experimental conditions (ground control, 1 G, and μG) with two different pairs of primers. Interestingly, the RyR1 expression was significantly decreased (67%) at the mRNA level in μG compared with Gr and 1-G in-flight control conditions, whereas the RyR3 expression was unchanged in the three different conditions (Fig. 2A). At the protein level, the limited number of samples allowed only an analysis based on the quantification of immunostaining with an antibody directed against a common RyR epitope (34C monoclonal antibody). First, we have verified that the nonspecific fluorescence was not modified in the three studied conditions. And then a significant decrease in RyR specific fluorescence (47%) was revealed in cells exposed to μG (Fig. 2, B and C). The μG is a unique environment because there is no force applied on the system. Therefore, by culturing VSMC in a μG environment, it allows one to study the influence of one particular variable, in this case, blood pressure.
We have also verified whether the transport of samples was deleterious for cell viability and RyR subtype expression by performing RT-qPCR on two different specific additive cassettes fixed exactly at the same time but stored in laboratory and on the launch area just before the launch. There was no difference in the level of expression of RyR subtypes between cell samples stored in laboratory and those that have traveled (RyR1/actin = 0.61 ± 0.09 vs. 0.48 ± 0.13; RyR2/actin = 0.20 ± 0.08 vs. 0.26 ± 0.07; RyR3/actin = 0.70 ± 0.05 vs. 0.74 ± 0.07 in laboratory vs. traveled samples, respectively).
RyR1 expression was also decreased in portal vein from mouse exposed to orbital spaceflight.
With a similar protocol, we have investigated the expression of RyR subtypes in portal vein from mice exposed to μG for the same duration during the STS-118 mission. The RT-qPCR experiments revealed that the expression of RyR1 was decreased (−61%), whereas RyR2 and RyR3 expressions were not changed (Fig. 3A). This result was also confirmed at the protein level by analysis of confocal microscopy stacks of RyR immunostaining because a Western blot analysis was not possible because the size of sample was too small. The immunolabeling using an anti-RyR1 antibody was decreased (−43%), whereas the immunostainnings of RyR2 and RyR3 revealed with specific antibodies against RyR2 and RyR3, respectively, were not changed (Fig. 3, B and C).
RyR1 expression is decreased by both HU and intraperitoneal injection of anti-RyR1 antisense oligonucleotides in portal vein.
The HU rat model has been widely used during the last two decades to mimic the fluid volume redistribution induced by μG. In portal vein, we previously demonstrated that the [3H]ryanodine binding was decreased by HU (40). To compare the effect of the decrease in gravitational force with the decrease in hemodynamic force on VSMC, we investigated the RyR expression pattern in hepatic portal vein from CTL and HU rats. The RT-qPCR revealed a significant decrease of RyR1 expression in HU rats compared with CTL rats, whereas the expression levels of RyR2 and RyR3 were identical in both conditions (Fig. 4A). To assess the RyR1 expression variation at the protein level, Western blots were made with protein extracted from portal vein from CTL and HU rats (Fig. 4B). The intensity of anti-RyR bands was normalized against an anti-actin antibody. The ratio of RyR1 to actin was significantly lower in portal vein taken from HU rats than from CTL, whereas the ratio of RyR2 or RyR3 to actin was not modified (Fig. 4C). RyR1 expression is thus decreased by the reduced infero-posterior blood pressure induced by HU.
To mimic the decrease of RyR1 expression, we developed an in vivo approach by antisense oligonucleotide (ASON) directed against RyR1 (asRyR1). Localization of the ASON in the VSMC was assessed by fluorescence microscopy using a Cy5-tagged asRyR1. Inhibition of the RyR1 expression was investigated by RT-qPCR following the same methodology as described earlier (Fig. 4A). In rats injected with the cationic vector only, the RyR1-to-actin ratio was similar to the ratio measured in CTL rat. Injection of asRyR1 causes similar reduction of the RyR1-to-actin ratio as observed in HU animals without effect on RyR2-to-actin and RyR3-to-actin ratios. Western analysis showed that ASON treatment reduced the density of the RyR1 protein band (Fig. 4D). The RyR1-to-actin ratio was −58.5 ± 18.7% less in ASON-treated than in vehicle-treated animals. The asRyR1 in vivo injection had no effect on RyR2 and RyR3 expression (Fig. 4E).
RyR1 has a functional role in portal vein VSMC adaptation to HU conditions.
To assess the role of RyR1 in hepatic portal vein adaptation to infero-posterior decreased blood pressure, different Ca2+ signals were compared between myocytes isolated from CTL, HU, and asRyR1-treated rats.
To measure the RyR-dependent Ca2+ release, caffeine (10 mM), known to activate RyRs by increasing their sensitivity to Ca2+ activation, was applied by puff on isolated VSMC from portal vein. Amplitudes and upstroke velocity of induced Ca2+ responses were identical between myocytes isolated from CTL and vehicle-treated rats and were thus pooled in the following experiments. Line-scan mode with a line acquired each 2 ms confirmed these differences and revealed a decreased upstroke velocity in HU and asRyR1-treated myocytes compared with control cells (Fig. 5A). Mean amplitude of caffeine-induced Ca2+ responses was decreased in HU and asRyR1-treated rats without any significant difference between these two last conditions (Fig. 5B). A SERCA inhibitor was used to check the effect of HU and asRyR1 injections on Ca2+ store refilling. The use of 1 μM thapsigargin and the reproducibility of the responses between CTL, HU, and asRyR1-treated rats suggested that the SR Ca2+ loading was unchanged (not shown).
In 1997, we showed a reduced cytosolic Ca2+ response evoked by AII in hepatic VSMC from HU rats (40). Then AII (100 nM) was first tested on VSMC from CTL, HU, and asRyR1-injected rats. As expected, HU decreased the amplitude of Ca2+ signal induced by application of AII compared with amplitude obtained in control condition (Fig. 6A, top). Interestingly, intraperitoneal injections of asRyR1 also reduced the AII-evoked Ca2+ response (Fig. 6A, bottom). In the presence of 10 μM ryanodine for 30 min, the amplitude of AII-evoked Ca2+ responses was decreased to a similar level in CTL, HU, and asRyR1-injected rats, suggesting that only changes in RyR function were responsible of the decreased AII-induced Ca2+ response in HU and asRyR1 conditions (Fig. 6B). We also evaluated the effect of asRyR1 and HU conditions on the AII-induced contraction (100 nM) measuring the shortening of portal vein strip placed on microscope setup used for Ca2+ measurement. The contraction of portal vein was decreased by HU from 22 ± 3% (n = 11) in CTL conditions to 12 ± 1% (n = 12) in HU conditions, and it was decreased from 17 ± 1% (n = 15) to 11 ± 2% (n = 20) in asRyR1-treated animals. This last result indicated that the decrease of Ca2+ signal possibly reduced the contraction induced by AII.
Since RyRs can be physiologically activated by a local increase in intracellular Ca2+, we tested the ability of the HU and asRyR1 treatment to modify this mechanism called Ca2+-induced Ca2+ release (CICR). The CICR mechanism can also be activated by membrane depolarization, leading to the opening of L-type Ca2+ channels and thus intracellular Ca2+ elevation. KCl (140 mM) was applied by puff on isolated VSMC (Fig. 7A); as a result, the induced Ca2+ responses were lower in HU and asRyR1 groups than in CTL group, whereas this difference was ablated by 10 μM ryanodine (Fig. 7, B and C).
We completed the investigation by the analysis of Ca2+ signal induced by ET1 (100 nM), known to activate InsP3 production (29). The amplitude of ET1-induced Ca2+ responses was decreased from 5.4 ± 0.3 (n = 39, CTL) to 4.2 ± 0.3 ratio units (n = 41, HU; P < 0.05, one-way ANOVA test) and to 3.9 ± 0.3 ratio units (n = 33, asRyR1-treated rats; P < 0.05, one-way ANOVA test). The mean amplitudes of the ET1-evoked Ca2+ response in HU and asRyR1-injected rats were similar. In the presence of 10 μM ryanodine, the mean amplitude of ET1-evoked Ca2+ responses was similar in all conditions [CTL: 1.6 ± 0.3 (n = 16); HU: 2.1 ± 0.4 (n = 25); asRyR1-injected rats: 1.8 ± 0.3 (n = 10)], suggesting that, in infero-posterior VSMCs, the InsP3 expression is not modified by HU condition. Taken together, these results clearly show the decrease in the RyR expression is sufficient to mimic the alteration of Ca2+ signaling induced by HU in VSMC from hepatic portal.
RyR1 was also regulated in portal vein VSMC from hypertensive model.
Finally, the SHR model could be used as the opposite model to μG and hindlimb suspension because if these last conditions induced a decrease of portal vein blood pressure (1), on the contrary, the blood pressure was increased in SHR model. Then, to complete our study, we wanted to analyze the RyR-dependent Ca2+ signaling in VSMC from SHR and WKR portal veins. First, the caffeine-induced Ca2+ release was significantly increased in VSMC from SHR portal vein compared with those obtained from WKR (Fig. 8A; ΔF/F0 = 4.48 ± 0.26, n = 51 in SHR vs. 2.94 ± 0.15, n = 23, in WKR; P < 0.05, t-test). Because the expression patterns of the ET-1 receptors were regulated in SHR model, the implication of RyR into the CICR induced by InsP3 pathway using ET-1 was not possible. Then, we used the photolysis of cag-iso-2-145-IP3 to activate IP3-dependent Ca2+ signals. In portal vein VSMC loaded with cag-iso-2–145-InsP3, the ultraviolet flash induced a Ca2+ signal whose amplitude was significantly increased in SHR compared with WKR (Fig. 8B; ΔF/F0 = 3.50 ± 0.13, n = 53 in SHR vs. 1.77 ± 0.03, n = 48, in WKR at 100-V intensity; and ΔF/F0 = 5.17 ± 0.47, n = 53, in SHR vs. 2.37 ± 0.20, n = 48, in WKR at 200-V intensity). As also illustrated in Fig. 8C, the KCl-induced Ca2+ response was significantly increased in SHR compared with WKR. The application of 10 μM ryanodine induced a more important decrease of KCl-induced Ca2+ response in SHR than in WKR (−68.3 ± 7.1% vs. −29.9 ± 4.2%, n = 6 different experiments), suggesting that the participation of CICR was more important in SHR than in WKR.
Finally, the expression of RyR subtype was investigated in SHR and WKR by RT-qPCR. Only the expression of RyR1 subtype was increased in SHR (Fig. 9), indicating an opposite effect to those observed in VSMC from HU rats or μG exposed mice and μG exposed VSMC.
The molecular basis of physiological effects of vascular adaptation to μG must be investigated at several levels. Indeed, vascular reactivity could be modified by changes in cell interactions, contractile proteins, and transduction pathways. The modifications could affect the expression level and also the functional state of proteins implicated in regulation of contraction. Vascular reactivity is due to the VSMC contraction/relaxation balance. The regulation of this balance is assumed by endothelial function principally via NO synthesis and by the regulation of Ca2+ signaling pathway of the VSMC. Here, we have investigated the regulation of Ca2+ signal encoding by μG and vessel pressure.
VSMC are directly sensitive to the real μG.
First, to delineate the relative contribution of vascular smooth muscle from the endothelial component in terms of the vascular adaptation to μG, experiments were performed in cultured VSMC. The expressions of RyR protein and RyR1 mRNA were decreased in cultured VSMC exposed to μG during TMA-8 spaceflight. These results suggested that Ca2+ signaling pathways were affected by μG. But one of the important challenges of this part of our work was to maintain the closest possible phenotype of the initial situation in the cultured VSMC. The presence of calf serum in culture medium is necessary to maintain the VSMC viability, but it also induced a dedifferentiation from a contractile to a proliferative phenotype, characterized by decreased SERCA2A and RyR expression and increased InsP3R, SOC, and CaV3 (26, 33, 34, 53). As we described previously, the use of calf serum decomplementation limits the VSMC proliferation (36), and the expression of RyR subtypes was not significantly modified during 4 days (9), but after 8 days only the RyR2 expression decreased to a level undetectable by RT-PCR. However, the RyR-dependent Ca2+ response and the contractile phenotype remained after 8 days, showing a preserved VSMC integrity. These culture conditions are usable to follow the effect of μG on Ca2+ signaling, despite a possible effect on RyR2 expression. There is no evidence in our work that μG induced drastic modifications of VSMC survival and proliferation, whereas RyR2 could be necessary to Ca2+ signals implicated in portal vein VSMC proliferation. In adherent cells, both real and simulated μG (clinorotation) decreased cell mitosis (37, 54). The differences between our work and these studies are due to the experimental procedures. Indeed, we defined particular conditions to maximally inhibit the cell proliferation. By indirect measurement, we have also shown that the contractile phenotype was preserved in μG because AII was still able to induce contraction of VSMC, and there was no modification in the proportion of contracted cells between 1-G and μG samples. Finally, at this stage of investigation, we cannot exclude that the decrease in RyR1 expression in μG can also be due, at least partly, to a decreased VSMC metabolism as observed in cultured cells in clinorotation (7). In the vascular physiology, it will be important to use the μG environment to characterized the pressure sensor and the level of gravity change that is perceptible by the VSMC as performed in vegetal root cells (16).
In μG conditions, the VSMCs have been packaged in Earth atmosphere, and only the gravity was not applied on cells in the ISS environment; blood and adjacent tissue pressures were then absent. The use of animals living in space environment has been necessary to verify whether the effect of μG observed in cultured VSMC made sense in vascular physiology.
In portal vein from mice exposed to μG during the STS-118 Endeavour mission, the expression of RyR1 was decreased as observed in cultured VSMC, indicating a molecular adaptation to real μG and potentially to changes in applied forces.
In a specific situation, the forces applied on cells change and modify the gene expression pattern as observed in myometrium near parturition (10, 25), in portal hypertension (48), or in renal hypertonic stress (31). Taken together, these results suggest that VSMC can rapidly (8 days) adapt their Ca2+ signaling pathways to μG during spaceflight and probably to blood pressure as observed in hypertensive situation.
Our study is the first direct demonstration that shows the absence of force obtained by μG exposure during spaceflight induces a modulation of VSMC and that reinforces the “pressure effect theory,” indicating that the intravascular pressure is a key regulator of vascular reactivity.
The asRyR1 in vivo treatment induces the decrease in vascular reactivity.
To investigate the significance of μG-induced RyR1 decrease in Ca2+ signaling, we have induced the decrease of RyR1 expression in rat by intraperitoneal injection of asRyR1. The specific decrease in RyR1 expression was followed at mRNA and protein levels and was responsible for decreases in Ca2+ responses induced by AII, ET1, and depolarization. This result indicates diverse Ca2+ signals are modified to likely affect many cell functions such as contraction and proliferation. First, the decrease in contraction was suggested by the decrease in Ca2+ signals evoked by vasoconstrictors such as depolarization, ET1, AII, and NE in our laboratory's previous work (40). In the same way, we have verified in confocal microscopy that AII-induced contraction of portal vein strips significantly decreased asRyR1 treatments. These experiments showed that the level of RyR1 expression was determinant to encode the magnitude and upstroke velocity of CICR in VSMC, confirming other studies using RyR1 KO (19, 32). In a previous work, our laboratory determined that the 80% inhibition of RyR1 expression by asRyR1 in cultured VSMC from the same model decreased the amplitude and the upstroke velocity of caffeine-activated Ca2+ response in the range of 60% and 80%, respectively (9). Here, the effects of asRyR1 and HU were similar, probably because the effects of the used concentration of asRyR1 inhibits RyR1 expression in the same range to those observed with HU.
But at this stage of the discussion, we cannot link together the decrease of RyR1 expression level as an adaptation to the decrease of pressure. To have some arguments to discuss this point, we have compared results obtained on asRyR1-treated animals and HU rats. This last model was admitted to mimic vascular adaptation to μG because HU reproduces the hyporeactivity of infero-posterior vessels.
The HU decreases the RyR1 expression.
The observed decrease of AII-induced contraction of portal vein strips in HU could be considered as a confirmation to the decrease in vasoconstriction that was also found in other peripheral vessels from HU rats, including abdominal aorta and femoral, pulmonary, and mesenteric arteries (4, 8, 15, 46, 47). In infero-posterior veins, the effect of HU is less clear than in arteries. HU also decreased, in a few days, the NE-induced contraction in vena cava, but it was due to the modifications of transduction pathways via α1-adrenoceptor desensitization (50). The decrease of contraction in mesenteric veins from Sprague-Dawley rats was also observed (4, 17), but on the contrary Purdy and coworkers concluded that jugular and femoral veins were globally not sensitive to HU (47). In these last models, the effects also could be analyzed by focusing on the nature of vasoconstrictors: NE-induced contraction was unchanged, whereas KCl-induced contraction was inhibited in HU rats. This last point would be consistent with our study, indicating the evidence that InsP3R Ca2+ release is not affected by HU (NE pathway), whereas CICR is affected (CICR between VDCC-induced Ca2+ influx and RyR).
However, the molecular basis of vascular adaptation to HU could be due to GPCR-activated pathway as suggested (3, 50), but it was not sufficient to explain the observed decrease in depolarization-induced and caffeine-induced contraction even if the activation of GPCR by depolarization was possible (35). In 1997, our laboratory showed for the first time that RyR expression is modified by HU conditions and may explain the decrease in vasoreactivity of portal veins (40). In 2008, Colleran and coworkers associated a decrease in RyR2 expression in HU rats with diminished vasoconstrictor responses to KCl, NE, and caffeine in mesenteric resistance arteries (8). The apparent discordance of their results with ours might be explained in that they only detected RyR2 and RyR3 mRNA expression. We have shown that RyR1 and RyR2 functions seemed to be interchangeable in VSMC (9). So it is not surprising to observe a HU-induced decrease in RyR2 expression in the absence of RyR1 protein. The origin of this different RyR subtype expression pattern could lie in the tissue used (10, 11, 38).
Finally, the present study might explain the medical observation during bed-rest experiments indicating a significant splanchnic blood stagnation. Indeed, a decreased contractility of the portal vein appears to be linked to a decreased RyR1 function (2). With mesenteric vessels, portal vein is the most important blood reserve; a loss of VSMC contractile ability in μG can induce the decrease in portal vein ability to transport the blood and then becomes essential in spaceflight-consecutive orthostatic intolerance. The second consequence of the decrease in Ca2+ signals can be the remodeling of the vascular wall. In fact, HU-induced decreased contraction was associated with vascular hypotrophy in aorta and skeletal muscle arteries (14, 21), but this point must be studied more precisely by following the proliferative pathway at the molecular level.
Regulation of RyR1 expression was also observed in SHR hypertension model.
The decrease in RyR1 expression observed in HU model or after μG exposure is possibly due to the drop of pressure applied on cells or blood pressure in vessels. Our results obtained from SHR showed that the chronic increase in blood pressure produces the opposite effect. Our results could also explain several effects observed in SHR model. In fact, the increase of RyR1 expression and the associated increase of Ca2+ signaling could explain the increase of the contraction induced by depolarization, caffeine, and agonists (29, 30, 43, 49) but also the effect of the inhibitor of sarcoplasmic reticulum Ca2+ stores (18).
In conclusion, our study shows that, during spaceflight, μG directly affects cultured VSMC by modulating the same molecular target as hemodynamic changes. Thus μG-induced blood pressure changes should induce the regulation of RyR1 expression in the same way. Nevertheless, it is necessary to investigate as soon as possible the sensor of gravity and/or blood pressure variation and pathways activated to regulate gene expression to adapt the Ca2+ pathways to understand the basis of vascular adaptation to spaceflight and also to give a new target to control and regulate blood pressure in hypertensive and hypotensive situations.
This work was supported by grants from CNES (1999–2011), Agence Nationale pour la Recherche (AdapHyG, no. ANR-09-BLAN-0148), and a postdoctoral fellowship from CNES to F. Dabertrand. Equipment was financed by Centre National de la Recherche Scientifique (CNRS) and Conseil régional d'Aquitaine.
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: F.D., N.M., and J.-L.M. conception and design of research; F.D., Y.P., and J.-L.M. performed experiments; F.D., Y.P., and J.-L.M. analyzed data; F.D. and J.-L.M. interpreted results of experiments; F.D. and J.-L.M. prepared figures; F.D. and J.-L.M. drafted the manuscript; F.D. and J.-L.M. edited and revised the manuscript; F.D., Y.P., N.M., and J.-L.M. approved the final version of the manuscript.
The authors thank the people with whom μG experiments have been prepared: G. Gauquelin-Koch (CNES); G. Gasset (GSBMS); L. Stodieck and V. Ferguson (BioServe Space Technologies, University of Colorado); D. Chaput (CADMOS, CNES); J. Hatton (ESA); all astronauts of the TMA-8 and TMA-7 soyouz missions and STS-118 mission; J. Mironneau for the helpful discussion; and A. Prevot for manuscript editing.
Present address of F. Dabertrand: Department of Pharmacology, University of Vermont, UVM College of Medicine, Burlington, VT 05405.
- Copyright © 2012 the American Physiological Society