Mechanisms Of An Increased Susceptibility To Apoptotic Stimulation In Cardiomyocytes Of Tail-suspended Rats

Objectives: With the rapid development of China’s manned space activities, medium-andlong-term spaceflights are increasing. Long-term spaceflight induces a wide range ofadaptive changes in astronauts. Therefore, astronauts are subjected to orthostaticintolerance and reduced exercise capacity when return to the ground1G gravityenvironment after logn-term spaceflight. The compensatory increase in circulationcatecholamine does not reverse orthostatic intolerance after returning from long-termspaceflight, but it is unclear whether or not high dose of catecholamine induces cardiacdamage. This becomes one of medical problems required to be resolved during the initialrecovery period after medium-and long-term spaceflight. In the present study, we wanted to investigate whether the apoptotic rates of cardiomyocytes were changed in the leftventricular myocardium in4-week of tail-suspended （SUS） and1-day recovery （SUS+R）rats from4-week tail-suspension, and molecular mechanisms of propensity towardapoptosis in cardiomyocytes.Methods: In the present study, the tail-suspended rat model was used to simulate theeffects of weightlessness on the heart. The noradrenaline （NA） content in plasma and leftventricular tissue was detected by ELISA. To evaluate the cardiomyocyte apoptosis, theTUNEL staining was performed. Adult rat left ventricular myocytes were isolated andcultured. Immunofluorescent cytochemical technique and western blot were carried out tovalidate the location and expression of various proteins. Calpain activity was measured bythe casein zymography. Calpain-1and calpain-2protein expression in cardiomyocytes wassilenced by small interfering RNA （siRNA） technology. The transient of intracellular Ca²⁺concentration was observed by a confocal microscope in the cardiomyocyte labeled byFluo3/AM. Reactive oxygen species （ROS） level in cardiomyocyte was measured by theDHE fluorescent probe. Co-immunoprecipitation was applied to validate the relationshipbetween calpain-2and CaMKⅡ B. Fluorescent dye JC-1was used to determinate thechanges in mitochondrial membrane potential （Δψm）.Results:（1） The apoptotic susceptibility increased in cardiomyocytes of tail-s uspended rats.The noradrenaline （NA） content in plasma and left ventricular tissue of SUS+R groupincreased significantly, but not in the synchronous control （CON） and SUS groups.Apoptotic rate of cardiomyocytes increased significantly in tail-suspended rats after ISOstimulation compared with the control group. One-day recovery increased apoptotic rate ofcardiomyocytes which was blocked by propranolol, a blocker of-adrenergic receptor orPD150606, an inhibitor of calpain-1and calpain-2.（2） Calpain-2was a key enzyme that regulated apoptotic susceptibility ofcardiomyocytes in tail-suspended rats.Calpain-2activity in the myocardium showed a marked increase in tail-suspendedrats compared with the control group, whereas there was no significant difference in activity and expression of calpain-1between the control and tail-suspended groups.Further more, calpain-2showed a greater amount of nuclear accumulation in thetail-suspended group than in the control. ISO treatment promoted the nuclear translocationof calpain-2in tail-suspended rat cardiomyocytes. The isolated nuclei of cardiomyocytesin tail-suspended rats showed a significant increase in activity and expression of calpain-2,but not in calpain-1compared with the control group. Moreover, the activity andexpression of calpain-2were increased under ISO treatment in the isolated nuclei ofcardiomyocytes from tail-suspended rats. PD150606reduced ISO-induced cardiomyocyteapoptosis to the level observed without ISO treatment in the tail-suspended group andreduced ISO-induced nuclear translocation of calpain-2. After using the siRNA silencecalpain-1or calpain-2gene expression, we found that ISO treatment significantlyincreased apoptotic rates of cardiomyocytes transefected with scramble or calpain-1siRNA in the tail-suspended group. In contrast, apoptotic rate of calpain-2siRNA-transfected cardiomyocytes was not increased during ISO treatment in thetail-suspended group.（3） Nucleoplas mic reticulum and reactive oxygen species jointly activated calpain-2of cardiomyocytes.L-type Ca²⁺channel1C subunit, ryanodine receptor2and sarcoplasmic reticulumCa²⁺-ATPase2were located along the T-tube in cardiomyocytes. The fluorescenceintensity of three proteins showed no significant difference between the SUS and CONgroups. After ISO stimulation, phospholamban （PLB） located at the nuclear envelope ofcardiomyocytes was significantly increased in control and tail-suspended groups, whilePLB accumulation at the nuclear envelope in tail-suspended rats was more than that in thecontrol rats. The ISO-induced increase in the phosphorylated PLB at the nuclear envelopein the tail-suspended group was also more than that in the control. ISO stimulation had noapparent effect on resting Ca²⁺fluorescence intensity in the tail-suspended and controlgroups, but ISO stimulation did increase cytoplasmic peak Ca²⁺fluorescence intensity inboth tail-suspended and control groups. ISO treatment also significantly increased peakintranuclear Ca²⁺fluorescence intensity in the tail-suspended group compared with the control. The ROS level in cardiomyocytes showed a significant increase in SUS+R groupcompared with the control group. ISO stimulation increased superoxide anion productionin cardiomyocytes in both SUS and SUS+R groups.（4） Calpain-2mediated cardiomyocytes apoptosis through the mitochondrialapoptosis pathway in tail-suspended rats.The isolated nuclei of cardiomyocytes in SUS+R and SUS+ISO groups showed asignificant increase in expression and activity of calpain-2, while the nuclear expression ofCaMKⅡ B decreased and the interaction between activated calpain-2and CaMKⅡ Bincreased. PD150606inhibited the degradation of CaMK Ⅱ B in nuclei ofcardiomyocytes. The cardiomyocytes in SUS+R and SUS+ISO groups showed asignificant increase in expression of activated caspase-3and caspase-9and cytochrome Crelease, a stronger mitochondrial depolarization and a marked decrease in the ratio ofBcl-2to Bax compared with the control group. These phenomena were blocked byPD150606.Conclusion: This study demonstrates that the apoptotic rate of cardiomyocytes increasesin1-day recovery rat. The mechanisms may be involved in the following components. Therecovery from4-week tail-suspension induces a high dose of NA in plasma and leftventricular tissue. ISO stimulation increases the phosphorylated PLB of the nuclearenvelope and elevated the intranuclear Ca2+peak of cardiomyocytes. High intranuclearCa2+concentration further activates nuclear calpain-2. The activated calpain-2in nucleoluscleaves its substrate CaMKⅡ B and induces downregulation of Bcl-2. Therefore, theapoptotic susceptibility in cardiomyocytes of tail-suspended rats is promoted via reducedinhibition of mitochondria-dependent apoptotic pathway.