Mitochondria Dysfuction Is Involved In The Genesis Of Low Vasoreactivity In Severe Shock

Vascular hyporeactivity may lead to persistent or refractory hypotension with low perfusion which is poorly responsive to routine therapies,including blood transfusion,fluid replacement,and vasopressor treatment.Therefore,it is one of the major causes of mortality following severe shock.Our previous data showed membrane hyperpolarization of arteriolar smooth muscle cells(ASMCs) caused by ATP-sensitive potassium channels(K_ATP) activation contributes to vascular hyporeactivity in shock.Despite supplication of oxygen and nutrients,vascular hyporeactivity to vasoconstrictor agents still remains,which may result from low ATP level.Currently,insufficient delivery of nutrients and oxygen and mitochondria dysfunction seemed to be responsible for the diminished ATP level.Increasing scientific interests are focused on the study how to protect mitochondria from dysfunction since it might be a potential therapeutic target in shock therapy,however, the mechanism still remain poorly understood.In this study,we investigated the role of mitochondrial dysfunction in the genesis of low vasoreactivity during severe shock in hemorrhagic shock rat model.By using transmission electron microscope, fluorescent probe technique,bioluminescence and patch clamp,four levels of mitochondria,ASMCs,vascular reactivity and overall anti-shock were determined.. This study includes three parts:Part one:morphological and physiological changes of ASMC mitochondria in severe shock1.ASMC mitochondria ultrastructure changes.ASMCs mitochondria from rats in time-matched sham group were sausage-shaped with more cristae projected into the internal cavity of the mitochondrion.In comparison to the normal ultrastructure of mitochondria in the sham group,that of ASMCs mitochondrion at 60min post reinfusion from hemorrhagic shock group appeared to be more spherical or irregular shaped,apparently swollen with increased size and with electron-lucent matrix,and extensive disruption of membrane integrity with poorly defined cristae,which indicated that significant ultrastructural mitochondrial injury of ASMCs took place in severe shock.By contrast,CsA(6mg/kg),a mitochondria protector by inhibition of MPTP,normalized mitochondrial ultrastructural injury,and while cotreatment of ATR(5mg/kg),a potent MPTP inducer,abolished the CsA protection function. Morphological assessments of ASMCs mitochondria shows following hemorrhagic shock,expansion of the mitochondrial matrix space(swelling) was reflected by having greater area(μm²) and parameter(μm),and a decrease in the area ratio and roundness measurements.Pretreatment with CsA ameliorated shock-induced changes in mitochondrial morphology,which was abolished by ATR.The mitochondrial permeability transition(MPT) is a sudden and sustained increase of the permeability of the inner mitochondrial membrane for solutes smaller than 1.5 kD mainly through mitochondria permeability transition pore(MPTP).This has catastrophic consequences for this organelle,including mitochondria swelling.So the results indicated the mitochondria ultrastructural changes observed in severe shock may be connected with MPTP alterations,and inhibition of MPT could protect mitochondria.2.ASMC mitochondria membrane potential changes.To confirm mitochondrial damage in hemorrhagic shock,mitochondrial membrane potential(Δψm) in ASMCs was determined by flow cytometry with the potential-sensitive fluorescent dye JC-1. Mitochondrial depolarization is indicated by an increase in green fluorescence intensity.Compared with time-matched intensity in the sham group,the percentage of cells with lowΔψm(green fluorescent intensity of JC-1 monomer) was much higher in the shock group(P=0.000).In response to the administration of CsA in shock+CsA group,the percentage of gated cells in the lower quadrants decreased to 38.7±4.0%, which was much lower than 61.1±8.3%in shock group(P=0.000).Meanwhile the percentage of cells with high green fluorescence increased to 59.4±7.0%by cotreatment of ATR with CsA(P=0.972 vs shock group).It was indicated that over this period of severe shock increase in MPT might contribute to mitochondrial membrane potential depolarization,following mitochondrial structure injury and dysfunction.3.Changes of ASMC intracellular ATP level.In order to evaluate the function of ASMCs mitochondria in severe shock,intracellular ATP level was determined by the CellTiter-Glo(?) luciferase bioluminescence method.It was shown that the ATP level was only 17.9±6.4%of sham control group in hemorrhagic shock group(P=0.000), which indicated presence of ASMCs mitochondrial dysfunction in the severe shock model.After pretreatment with mitochondrial protector CsA,ASMCs ATP level could enhance to 40.1±9.9%,which was much higher than that in shock group(P=0.003). And cotreatment with ATR could abolish the CsA protection effect.The ATP level in shock+ATR+CsA group reduced to 21.6±8.9%of sham control group,which implied the significance of protecting mitochondrial function in severe shock.4.Changes of ASMC membrane potential and K_ATP channels.K_ATP channels of freshly isolated ASMC were recorded by whole cell patch-clamp technique.K_ATP currents densities of ASMC at holding potential -40mV were increased from -1.31±1.75 pA/pF in sham group to -8.94±4.40 pA/pF in shock group(F=30.823,P =0.000),which indicated the activation of ASMC K_ATP channels in severe shock.The administration of CsA reduced ASMC K_ATP currents densities to -3.71±2.64 pA/pF in shock+CsA group,which was much lower than that in shock group(F=30.823,P =0.002);cotreatment of ATR and CsA could abolish the CsA effect with -12.21±5.46 pA/pF of the K_ATP currents densities in shock+ATR+CsA group.ASMCs membrane potential increased from 32.1±5.1 mV in sham control group to -48.5±8.2 mV in shock group(P=0.000 vs sham group),which indicated existence of membrane hyperpolarization in severe shock.However,the ASMC hyperpolarization was partially recovered after pretreatment of mitochondrial protector CsA,since the negative value of ASMC membrane potential was reduced to -37.8±7.7 mV in shock+CsA group,which was much lower than that in shock group(P=0.011).The membrane potential level was -50.6±11.1 mV in shock+ATR+CsA group,which indicated that cotreatment of ATR could abolish the CsA protection effect.These results indicated inhibition of MPT and protecting mitochondrial function contributed to preventing from membrane hyperpolization of ASMCs.Part two:Establishment of ASMC mitochondrial injury model and effects of what on the contractile responseIn order to confirm mitochondrial damage can lead to decreased contractile response of smooth muscle,ASMC model of mitochondrial injury was established by using ATR,MPTP opener,and the following experiment was conducted.1.Effects of ATR on mitochondrial membrane potential(Δψm) of normal and shock ASMCs.ATR in different concentration(0,2.5,5,7.5μM) caused increase in the percentage of normal and shock cells with lowΔψm.In addition the percentage of shock cells(48.52%) with lowΔψm was significant higher than that of normal cells (23.13%)(F=204.676,P=0.000).There were significantly different effects among ATR of different concentration(F=101.780,P=0.000).The higher concentration of ATR,the higher percentage of normal cells with lowΔψm.However,there was no significant difference between 5μM and 7.5μM ATR in shock cells.Besides,at the time of 10min of administration of ATR,changes ofΔψm were similar to those at the time of 30min(F=1.055,P=0.309).It was indicated that ATR depolarized ASMC mitochondria and even damaged mitochondria,which might be related to inducing MPT with concentration effect and without chronergy.2.Effects of ATR on membrane potential and ATP level in normal and shock ASMCs.Laser confocal scanning microscopy and biochemical assay were used to detect effects of 7.5μM ATR on membrane potential and ATP level in normal and shock ASMCs for 10min.Relative content of ATP in normal ASMCs significantly reduced by 25%that in ATR-treated cells(t=3.389,P=0.01).However,ATR had no effects on ASMC membrane potential.Relative content of ATP in shock ASMCs significantly reduced to 59%that in ATR-treated cells(t=3.904,P=0.005) and the intensity of DiBAC₄(3) fluorescent decreased in ATR-treated cells(t=14.550,P= 0.000).It suggested in this condition decrease in ATP level in shock ASMCs might contribute to hyperpolization of ASMCs.3.Effects of ATR on ASMC contratile reaction.The above results showed ATR as MPTP opener could induce mitochondria dysfunction in ASMCs in vitro,which leaded to decrease in cellular ATP level and even hyperpolization of shock cells.In order to investigate changes of ASMC contratile reaction,effects of 7.5μM ATR on ASMC reactivity to NE(normal group 1μg/ml NE,shock group 60μg/ml NE) were studied in normal and shock cells respectively.It was observed that at the time of 10min of administration of ATR the area of normal ASMCs decreased by 37.83±5.74 %less than that of normal cells without ATR treatment(44.10±3.30%)(t=2.318,P =0.043).The area of shock ASMCs decreased by 10.94±2.78%less than that of shock cells without ATR treatment(25.59±2.37%)(t=9.816,P=0.000),which demonstrated that depression of mitochondrial function could directly weaken ASMC contratile reaction,which might be related to decrease in cellular ATP content.Part three:The possible protection of CsA from low vascular reactivity and hypotension in severe shock in vivo.Two variables were used to determine the change of vasoreactivity in shock:one was the NE threshold concentration for contraction of the arterioles in muscle microcirculatory preparation,and the other was MAP alterations in vivo after administration of dopamine and reinfusion of half volume of shed blood in shock rat.It was shown that the NE concentration increased to 40.9 times that of prebleeding and MAP only increased by 11.7 mmHg at 60 min after administration of dopamine and reinfusion of shed blood in shock group. Meanwhile,in shock+CsA group,the NE concentration increased to 20.6 times that of prebleeding and MAP increased by 34.2 mmHg at time-matched point.These indicated that the low vasoreactivity and MAP was partially recovered by CsA. However,pretreatment of ATR could abolish protection effect of CsA.These results showed that inhibition of MPT and protection of mitochondria make for vasoreactivity recovery and the prevention of hypotension during severe shock,which is a new approach to treatment of persistent hypotension in severe shock.CsA could significantly prolong survival time of animals suffering severe shock,however,at the same time could not significantly improve 24-hour survival rate of animals,which was only 3/8.It suggests that there might be other mechanisms underlying low vasoreactivity as well as side effects of CsA,such as immunosuppression,so it is not an ideal anti-shock drug.Conclusions:1.In vivo and vitro models of severe hemorrhagic shock were constructed to investigate the relationship between mitochondria and vasoconstriction.2.Based on the morphological and physiological changes,includingΔψm, intracellular ATP level,ASMCs membrane potential and K_ATP channels,and vasoresponsiveness,ASMCs mitochondria dysfunction existed in vivo model and played a role in the genesis of low vasoreactivity after resuscitation following severe shock.3.Protection against mitochondria injury might be a potential pharmaceutical target in refractory hypotension therapy...