| BackgroundExposure to microgravity results in post-flight cardiovascular deconditioning,including orthostatic intolerance and decreased aerobic capacity,when astronauts return to earth(1 G environment).Postflight cardiovascular deconditioning has been considered as one of the major adverse effects when exposed to microgravity,which is associated with high risk to astronaut’s health and performance,and there are still no effective countermeasures.Uncovering the mechanisms underlying effects of microgravity on the cardiovascular system and finding effective countermeasures are the essential issues in aeromedical support.Hypovolemia,decreases in exercise tolerance and aerobic fitness,attenuated carotid baroreceptor-heart rate responsiveness,increased leg compliance,were proved to play roles in postflight cardiovascular deconditioning.However,the underlying mechanisms are still not fully clarified.Both human studies from spaceflights and head-down tilt bed tests and animal studies from the tail-suspended hindlimb-unweighting rat model revealed that region-specific vascular remodeling under microgravity exposure could be one of the fundamental causes in the postflight orthostatic intolerance.Simulated microgravity could induce hypertrophic remodeling of fore-body arteries like the cerebral arteries in rats.The functional and structural adaptation in cerebral arteries would be the most crucial component of region-specific vascular remodeling.It has been demonstrated that local renin-angiotensin system,oxidative stress injury,nitric oxide and nitric oxide synthase dysfunction in endothelial cells,ion channel remodeling and mitochondrial dysfunction in vascular smooth muscle cells(VSMCs),were involved in the process of microgravity or simulated microgravity induced remodeling of cerebral arteries.Nowadays,countermeasures like lower body negative pressure training,aerobic exercise training,supplement blood volume,taking Chinese medicine were applied in astronauts to fight against post-flight orthostatic intolerance.However,there is still no effective countermeasure.The application of artificial gravity would be the most effective fundamental countermeasure to eliminate the adverse effects of microgravity.Unfortunately,it is impossible to achieve continuous artificial gravity intervention at this stage,which makes intermittent artificial gravity intervention an alternate.Both human studies from head-down tilt bed tests and animal studies from tail-suspended hindlimbunweighting rats revealed that daily short-period gravitation can prevent functional and structural changes in cerebral arteries induced by simulated microgravity to fight against the occurrence of postflight cardiovascular deconditioning.However,the underlying mechanisms remain to be clarified.VSMC is the dominant cell type in blood vessels,which displays remarkable plasticity in different functional states or phenotypes and could undergo a rapid shift from a contractile phenotype to a synthetic phenotype in response to changes in local environmental cues including vascular injury,extracellular matrix,shear stress,mechanical stretches and growth factors,which is known as phenotype modulation.Contractile VSMC is characterized by the expression of contractile markers specific to smooth muscle,such as smooth muscle myosin heavy chain(SM-MHC),smooth muscle α-actin(SM-α-actin),smooth muscle protein 22-α(SM22α),and transgelin.Synthetic VSMCs have a more rounded and irregular morphology with a large number of organelles related to protein synthesis and secretion,such as endoplasmic reticulum and Golgi body,reduced and loose myofilaments.Osteopontin(OPN)is a type of adhesion glycoprotein expressed in the extracellular matrix and could be considered as a synthetic marker for almost nonexpression in the normal arterial wall.Phenotype modulation of VSMCs was considered as a crucial early step before alterations in cell proliferation,migration and apoptosis,and an initial and critical incident in series of cardiovascular physiologies and pathologies.Ca2+ sensitive transcription factors and mitochondrial dynamics are the crucial regulators in phenotype modulation of VSMC.Ca2+-dependent transcription factors,including serum response factor(SRF),c AMP response element-binding protein(CREB),NFAT and C-terminus of voltage-dependent L-type Ca2+ calcium channels are important regulators in phenotype modulation of VSMC.For example,SRF could interact with its co-factor Myocardin to form a protein complex,which activates the cis-acting element CAr G-box to initiate the expression of VSMC contractile-specific proteins.Recently,a relationship has been proposed between the VSMC phenotype modulation and mitochondrial dynamics.Emerging data suggest that synthetic VSMC from pulmonary artery of pulmonary arterial hypertension mice and proliferative vessels of debits patients manifest a fragmented mitochondrial phenotype,which indicated enhanced mitochondrial fission and suppressed mitochondrial fusion.Additional studies show that high glucose treatment in aortic cells leads to increased expression of mitochondrial fission protein and decrease expression of mitochondrial fusion proteins.Using a pharmacological inhibitor(Mdivi-1)of DRP1 to inhibit mitochondrial fission could prevent phenotype modulation of VSMC.However,the underlying mechanisms are still far from clear.The intracellular Ca2+ is an essential messenger in the phenotype modulation of VSMC.Studies revealed that the intracellular Ca2+ was closely related to its phenotype.Contractile VSMCs are characterized by low resting cytosolic Ca2+,whereas synthetic VSMCs are characterized by the sustained elevation of basal cytosolic Ca2+.The intracellular Ca2+ was tightly controlled both spatially and temporally by Ca2+ channels and transporters.One of the most important components of Ca2+ signaling in VSMC is the extracellular Ca2+ influx through voltage-dependent Ca2+ channels(VDCCs).The highvoltage activated L-type(large or long-lasting)and low-voltage activated T-type(tiny or transient)Ca2+ channels are the two main types of VDCCs in VSMC.Both L-type VDCC and T-type VDCC channels could be directly activated by increased blood pressure or mechanical stretch and lead to the Ca2+ influx into VSMCs.L-type VDCC is considered as the primary Ca2+ entry pathway in most vascular beds.T-type VDCCs played an important role in the sustained Ca2+ entry into VSMCs of small resistant arteries,such as the cerebral artery,renal artery,and mesenteric artery.It has been demonstrated that L-type VDCC could suppress the phenotype modulation of VSMC.Besides,studies revealed that T-type VDCCs were increased in VSMCs during phenotype modulation.However,the role of T-type VDCCs in modulating the dedifferentiation of VSMC has not been elucidated up to now.Recent studies have demonstrated that micro RNA and intracellular Ca2+ signals play important roles in phenotype modulation of VSMC induced by mechanical stimulation.mi RNAs are small non-coding RNAs that negatively regulate m RNA stability or protein translation by binding to the 3’UTR of the target m RNA,which leads to the degradation of target m RNA or inhibition of translation in most cases.It has been proved that mi RNAs were extensively involved in various cell biological processes including phenotype modulation of VSMCs,by regulating gene expressions at the post-transcriptional level.A series of specific mi RNAs,including mi R-143/145 and mi R-221/222,were proved to regulate phenotype modulation of VSMC.It has been demonstrated that mi R-143/145 were classic mi RNAs,which could sense the mechanic stimuli and promote VSMC to maintain contractile phenotype.It has also been reported that mi R-145 could control the expression of L-type Ca V1.2 channel and subsequently regulate the stretch-induced phenotype modulation in VSMCs.Interestingly,our research showed that simulated microgravity upregulated the expression of the T-type Ca V3.1 channel in a post-transcriptional way,indicating a potential role for mi RNAs in it.However,it is not clear whether there is a gravity-sensitive micro RNA as an upstream regulator of the T-type Ca V3.1 channel which can promote the phenotype modulation of VSMC from the contractile phenotype to synthetic phenotype.Our previous work demonstrated that 28-day simulated microgravity induced hypertrophic structural remodeling of cerebral arteries.However,it is not clear whether simulated microgravity could induce phenotype modulation of rat cerebral VSMCs from the contractile phenotype to synthetic phenotype.Compared with L-type VDCC,T-type VDCC was more sensitive to the change of transmural pressure,stretch stimuli and depolarization.However,it is unknown that whether T-type VDCC could regulate phenotype modulation,especially as a critical molecular in simulated microgravity induced phenotype modulation of rat cerebral VSMC and a target which intermittent artificial gravity act on.In this study,we aim to investigate the role of T-type VDCCs in phenotype modulation of VSMC under simulated microgravity and intermittent artificial gravity intervention and its underlying mechanisms.We sincerely wish our finding could provide a novel mechanism of microgravity-induced cerebrovascular adaptation and contributes to developing novel approaches as effective countermeasures against microgravity exposure.It also has implications for vascular physiologies and pathologies in general conditions.Purpose1.To confirm whether simulated microgravity induces phenotype modulation of rat cerebral VSMCs from the contractile phenotype to synthetic phenotype.2.To confirm whether T-type Ca V3.1 plays a crucial role in the regulation of simulated microgravity-induced phenotype modulation of rat cerebral VSMCs.3.To confirm whether there is a micro RNA target T-type Ca V3.1 as upstream signaling and whether T-type Ca V3.1 could regulate Ca2+-sensitive calcineurin/NFAT pathway and mitochondrial dynamics between mitochondrial fusion and fission in the regulation of simulated microgravity-induced phenotype modulation of rat cerebral VSMCs.4.To confirm whether intermittent artificial gravity intervention could prevent phenotype modulation of rat cerebral VSMCs induced by simulated microgravity by targeting Ttype Ca V3.1.Methods1.Animal models: Sprague Dawley rats were subjected to 28-day tail-suspension(SUS)to simulate the effects of simulated microgravity on the cardiovascular system.Daily standing(STD)for 1 h was used to provide short-period intermittent artificial gravity intervention as a countermeasure.2.In vitro cell culture model: A7r5 cells(rat thoracic aortic smooth muscle cell line)were applied in cell experiments.To induce phenotype modulation of VSMC in vitro,serum stimulation was given to A7r5 cells after 48 hours of serum starvation(cultured in serum-free medium)by incubated with 10% FBS.3.Isolation of cerebral VSMCs: Briefly,the cerebral arteries were carefully removed and placed in 4°C physiological salt solution(PSS).For electrophysiological using and mitochondria dyeing,cerebral arteries were digested for 18 minutes at 37°C with a solution containing 4 mg/m L papain,2 mg/m L dithioerythritol,1 mg/m L bovine serum albumin and 5 mmol/L taurine in PSS.Isolated VSMCs were stored at 4 °C for use within 8 hours.4.Transmission electron microscopy observation: Briefly,basilar arteries were fixed,dehydrated and embedded in epoxy resin.Ultrathin sections were obtained with a diamond knife using ultramicrotome and stained with lead citrate and uranyl acetate.After that,the sections were viewed and photographically recorded using a scanning transmission electron microscopy.For morphological assessments,individual cerebral VSMCs which reside in the tunica media of the basilar arterial were observed after being magnified 6,000 times.The mitochondrial size was observed after being magnified 16,500 times.Intracellular myofilaments,which support contractile apparatus,and cytoplasmic organelles including endoplasmic reticulum and mitochondria,which support synthetic apparatus,were observed after being magnified 20,000 times.5.Immunohistochemical staining: Basilar artery segments were dissected along with the partial brain,embedded and sectioned.Immunohistochemical staining was then performed following protocols ZSGB-bio recommended.Briefly,sections were incubated with the appropriate dilution of the primary antibodies,the secondary Ig G antibodies,and horseradish peroxidase(HRP)-conjugated streptavidin.Finally,3,3′-diaminobenzidine(DAB)was used as a chromogen to detect with an Olympus IX71 inverted microscope.Relative optical density(ROD)was calculated by normalizing integrated optical density(IOD)to vessel wall area.All image analyses were performed using Image-Pro Plus 6.0 software.6.Western blotting: Cerebral arteries and A7r5 cells lysis buffer were prepared in M-PER Mammalian Protein Extraction Reagent with freshly 1% protease inhibitor cocktail.After centrifugation,supernatants were denatured for Western blotting.Isolate and extract nuclear or cytoplasmic proteins was then performed following protocols Thermo recommended.Proteins were separated using Nu PAGE Bis-Tris gel and then transferred to polyvinylidene fluoride(PVDF)membranes.Membranes were blocked and subsequently incubated with appropriate primary antibodies at 4 °C overnight and HRP-conjugated secondary antibodies.Proteins were detected and visualized using the chemiluminescent HRP substrate.Image J software was applied for quantification.7.Real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR): Briefly,cerebral arteries and A7r5 cells were mixed with RNAiso and homogenized by grinding.After centrifugation,phase separation and precipitation,the resulting RNA pellet was dissolved in RNase-free water and stored at-80 °C until further analysis.After reverse transcription by using Mir-X mi RNA First-Strand Synthesis Kit,q RT-PCR was performed using a CFX96(BIO-RAD)instrument and SYBR Premix Ex Taq TM according to the manufacturer’s protocol.The data were analyzed via the ΔΔCt method.8.Mito Tracker Red Dyeing: Mito Tracker Red was used to observe mitochondrial morphology in isolated cerebral arteries VSMCs.Using a confocal laser scanning microscope,the cells were imaged with excitation at 579 nm and emission at 599 nm.9.Calcineurin phosphatase activity assay: The activity of calcineurin was determined using a calcineurin activity assay kit following the manufacturer’s protocol.Calcineurin activity was assessed by measuring the absorbance value at 660 nm using the following formula: calcineurin activity(U/mg prot)= [(testing tube OD value- control tube OD value)/standard tube OD value – standard blank tube OD value] × concentration of standard tube × dilution multiple of reaction system/sample protein concentration,the value in the control group was set to one.10.Oligonucleotides transient transfection: Oligonucleotides,including si RNA(target CACNA1 G sequence,Si Ca V3.1),si RNA control(Si NC),mi R-137 mimic/inhibitor,and corresponding controls(mi R-NC),were all purchased from Ribobio Corporation.According to the manufacturer’s instructions,A7r5 cells were transfected with Si Ca V3.1(100 nmol/L)or mi R-137 mimic/inhibitor(50 nmol/L/150 nmol/L),and their control using Lipofectamine 3000 reagent(Invitrogen)and harvested after 48 hours transfection.11.Dual-luciferase report assay: Psi CHECKTM-2 vector containing both Firefly and Renilla luciferase genes was used to introduce the wild/mutant 3’UTR sequences of CACNA1 G immediately downstream the stop codon of the Renilla luciferase gene to create a wild-type(WT)or mutant-type(MUT)CACNA1G 3’UTR plasmid.Psi CHECKTM-2 vector without inserted gene was used as negative control(NC)plasmid.Following the procedures provided by Ribobio and Promega,A7r5 cells were co-transfected with the Psi CHECKTM-2 vector(WT/MUT/NC)and oligonucleotides(mi R-137 mimic or mimic control)for 48 hours.The Firefly and Renilla luciferase activities(Fluc,Rluc)were sequentially measured using the Dual-Luciferase Reporter Assay System(Promega)as recommended.Relative luciferase activity was calculated by normalizing Rluc to Fluc,the value in NC plasmid plus mimic control-treated group was set to one.12.Ed U incorporation assay: Proliferation of A7r5 cells was determined by a 5-ethynyl-2-deoxyuridine(Ed U)incorporation assay.Ed U was added at 100 μmol/L,and the cells were cultured for an additional 2 hours.After the removal of the Ed U-containing medium,the cells were fixed,washed with glycine,treated with Trion X-100,reaction buffer,stained with Hoechst,and examined under a fluorescence microscope,according to the manufacturer’s instructions.13.CCK-8 assay: A7r5 cells were planted into 96-well plates at a density of 5,000 cells/well and added 10 μl CCK-8 reagent per well for the incubation at 37 °C.After that,viable cells were assessed by measuring the absorbance value at 450 nm with a Molecular Devices Spectra Max 190 Microplate Reader.14.Statistical analysis: Data are expressed as means ± SEM.All experiments were performed at least in triplicates.Statistical analysis using Student’s t test(two group means comparison),Chi-square test(two group rates comparison)or one-way ANOVA(multiple group comparison)was done with Graphpad Prism software.A P value < 0.05 was considered to be statistically significant.Results1)Simulated microgravity induced a phenotype modulation from the contractile phenotype to synthetic phenotype in rat cerebral VSMCs1.Ultrastructure of cerebral VSMCs in SUS rats showed a typical synthetic phenotype feature with less and loose myofilaments and more synthetic organelles including endoplasmic reticulum and mitochondria in the cytoplasm.2.The relative protein and m RNA expressions of contractile markers(including SMMHC,SM-α-actin,and SM22α)significantly reduced in cerebral arteries of SUS rats,as compared with that of CON rats.3.The relative protein and m RNA expressions of synthetic markers(including OPN and PCNA)markedly increased in cerebral arteries of SUS rats,as compared with that of CON rats.2)Activation of the T-type Ca V3.1 channel played a crucial role in phenotype modulation of rat cerebral VSMCs induced by simulated microgravity1.The protein and mRNA expressions of T-type Ca V3.1 channel significantly increased in cerebral arteries of SUS rats,as compared with that of CON rats.The expression of Ca V3.1 was subjected to post-transcriptional regulation.2.During the process of serum-induced phenotype modulation in cultured A7r5 cells,the m RNA expression of T-type Ca V3.1 was continuously increased.3.Whether silencing T-type Ca V3.1 channel by specific si RNA oligonucleotide or blocking it by mibefradil in cultured A7r5 cells,significantly inhibited VSMC phenotype modulation from contractile to synthetic phenotype and suppressed cell proliferation.3)T-type Ca V3.1,targeted by mi R-137,regulated simulated microgravity-induced phenotype modulation of rats cerebral VSMC by activating calcineurin/NFATc3 pathway and mitochondrial fission1.mi R-137 directly inhibited T-type Ca V3.1 as an upstream regulator1.1 We have screened eight candidate mi RNAs predicted to bind to 3’UTR of CACNA1G(m RNA of Ca V3.1)through the prediction of micro RNA target software programs.By RT-PCR analysis,mi R-137 was found to be the most significantly decreased mi RNA among candidate mi RNAs in cerebral arteries of SUS rats,as compared with that in CON rats.In addition,mi R-137 was confirmed to be significantly decreased in serum stimulation treated A7r5 cells,which is a model of VSMC phenotype modulation in vitro.1.2 Dual-luciferase reporter assay proved that mi R-137 negatively regulates the T-type Ca V3.1 channel by directly binding to CACNA1 G 3’UTR complementary sequences in VSMCs.1.3 The gain-and loss-of-function studies in A7r5 cells clearly showed that mi R-137 mimic significantly decreased the protein expression of T-type Ca V3.1,inhibited VSMC phenotype modulation and proliferation,whereas mi R-137 inhibitor significantly increased the protein expression of T-type Ca V3.1,promoted VSMC phenotype modulation and proliferation.1.4 The functional blockage of the T-type Ca V3.1 channel by mibefradil partially reversed the phenotype modulation and proliferation effects induced by mi R-137 inhibitor.2.Activation of the T-type Ca V3.1 channel activated calcineurin/NFATc3 pathway2.1 Calcineurin phosphatase activity was significantly increased in cerebral arteries of SUS rats,as compared with that in CON rats.The nuclear translocation of NFATc3 was markedly increased in cerebral arteries of SUS rats,as compared with that in CON rats.2.2 Silencing T-type Ca V3.1 channel by specific si RNA oligonucleotide or blocking it by mibefradil significantly inhibited calcineurin phosphatase activity and the nuclear translocation of NFATc3 in serum-induced synthetic A7r5 cells.3.Activation of T-type Ca V3.1 channel activated mitochondrial fission3.1 As compared with that in CON rats,the number of mitochondria was significantly increased,whereas the size of mitochondria was significantly decreased in cerebral VSMCs of SUS rats.In addition,the expressions of mitochondrial fusion proteins(including MFN1 and MFN2)were significantly reduced,whereas the expressions of mitochondrial fission proteins(including DRP1 and FIS1)were markedly increased in cerebral arteries of SUS rat,as compared with that of CON rats.3.2 The functional blockage of T-type Ca V3.1 channel by mibefradil in cultured A7r5 cells significantly increased percentage of filamentous mitochondria,decreased percentage of globular mitochondria,downregulated expressions of mitochondrial fission proteins(including DRP1 and FIS1),and upregulated expressions of mitochondrial fusion proteins(including MFN1,MFN2,and OPA1).3.3 Silencing T-type Ca V3.1 channel by specific si RNA oligonucleotide in the presence of mibefradil further inhibited FIS1 expression and promoted MFN1 and OPA1 expression,suggesting that T-type Ca V3.1 channel could promote mitochondrial fission in a non-canonical ion-conducting independent way.4)Intermittent artificial gravity prevented simulated microgravity-induced phenotype modulation of rat cerebral VSMCs by inhibiting T-type Ca V3.1 related signal pathways1.Daily short-period artificial gravity intervention for 1 h significantly inhibited phenotype modulation of cerebral VSMCs from the contractile phenotype to synthetic phenotype in SUS rats.2.Daily short-period artificial gravity intervention for 1 h significantly increased expression of mi R-137 and decreased expression of T-type Ca V3.1 in SUS rats.3.Daily short-period artificial gravity intervention for 1 h significantly inhibited the Ttype Ca V3.1-dependent calcineurin/NFATc3 pathway in SUS rats.4.Daily short-period artificial gravity intervention for 1 h significantly inhibited T-type Ca V3.1-dependent mitochondrial dynamics in SUS rats.ConclusionSimulated microgravity induced the phenotype modulation of rat cerebral VSMCs from the contractile phenotype to synthetic phenotype.The T-type Ca V3.1 channel played a crucial role in this process.Briefly,gravity-sensitive mi R-137 was downregulated in cerebral VSMCs under simulated microgravity conditions.As a direct target of mi R-137,Ca V3.1 was upregulated,and the T-type VDCC currents were enhanced.On the one hand,the Ca2+-sensitive calcineurin/NFATc3 pathway was activated due to enhanced Ca2+ entry through the Ca V3.1 channel.On the other hand,Ca2+-sensitive mitochondrial fission was promoted in an ion-conducting dependent and independent way.Both the activated calcineurin/NFATc3 pathway and enhanced mitochondrial fission promoted phenotype modulation of rat cerebral VSMCs.Intermittent artificial gravity intervention,as an effective countermeasure against post-flight cardiovascular deconditioning,prevented simulated microgravity-induced phenotype modulation of rat cerebral VSMCs by inhibiting expression of Ca V3.1. |
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