Effects Of PLB-SERCA2a Trafficking On Spontanous Ca²⁺ Waves And Calcium Regulation In Rats Cardiomyocytes With And Without Myocardial Infarction

Part one Isolation and Culture of Adult Rat Cardiomyocytes for Cell Signaling and Generation of Recombinant Adenovirus Expresing Phospholamban and cardiac Ca-pump(SERCA2a)ObjectiveTo provide a well-established method for isolation of adult rat cardiomyocytes that can be implemented with little training.And to construct the recombinant adenovirus overexpressing phospholamban gene and cardiac Ca-pump(SERCA2a)for studying the biological functions of PLB and SERCA2 a.MethodsThe heart of rat is excised and cannulated to an isolated heart system,then perfused with a calcium-free and high potassium buffer followed by Liberase Blendzyme digestion in Langendorff retrograde perfusion mode.This protocol yields a consistent result for the collection of functional adult rat/mouse cardiomyocytes from a variety of genetically modified mice.Recombinants were selected by kanamyein resistance and confirmed by restriction endonuclease analysis.Then.the Pac I—linearized the recombinant adenovirus vector was transfected into human embryonic kidney cells 293 A for packaging adenoviruses.The resultant positive adenoviruses were amplified,purified by ultracentrifugation,and titered.Western blot was performed further to verify the protein expresion of recombinant PLB and SERCA2 a.ResultsWe have sucesfuly get enough cardiomyocytes and cultured for at least three to four days.We generated recombinant adenoviruses expressing canine PLB and SERCA2 a.The adenoviruses can effectively mediate PLB and SERCA2 a expression in rat myocardial cells.ConclusionThe obtained recombinant adenoviruses overexpresing PLB and SERCA2 a gene can be used as a useful tool for investigating the biological function of PLB and SERCA2 a.Part Two Accumulation of phospholamban at the nuclear envelope delays its trafficking along a transverse sarcoplasmic reticulum(SR)pathway common to SERCA2a and other SR proteinsObjectivePhospholamban(PLB)and cardiac Ca-pump(SERCA2a)are key to SR Ca handling.We recently showed that PLB is concentrated in the nuclear envelope(NE)of cardiomyocytes(CMs),with a roughly 2:1 NE to SR protein expression ratio.Whether this steady state distribution reflects the underlying dynamics of SR protein trafficking remains unclear,since the secretory pathways of SERCA2 a and PLB in CMs remain unknown.HypothesisPLB is retained in the NE of CMs after its biosynthesis.Subsequent anterograde trafficking of PLB follows the same recently identified pathway for junctional SR proteins,junctin and triadin,in CMs that delivers proteins to junctional SR directly from translocons in rough ER(Nuclear Envelope-to-SR along T-tubules or NEST).MethodsAdult rat CMs were infected with dog isoform PLB(d PLB)or d SERCA for 24 h and 48 h,then fixed.Confocal fluorescence microscopy was used to specifically visualize the accumulation of the newly-made d PLB or d SERCA2 a proteins,detected using species–specific monoclonal antibodies 1F1 or 3F1,respectively.ResultsWe examined localization of newly-synthesized d PLB(32CMs)or d SERCA2a(30 CMs)24 h post infection(n=8 rats).d PLB,detected by 1F1,was distributed continuously throughout the NE,but remained confined in the NE,without detectable transverse/radial trajectory.In contrast,d SERCA2a(3F1 fluorescence signals)showed discrete puncta in the NE,likely reflecting rough ER translocons.Further,d SERCA2 a protein exhibited intracellular distribution along transverse tracks emanating from translocons,with radially decreased amount.At the 3rd puncta away from the NE,3F1 fluorescence intensity reduced to 52 ± 12% of that in the NE.exhibited intracellular distribution along transverse tracks emanating from translocons,with radially decreased amountConclusionsAfter biosynthesis,unlike SERCA2 a,PLB initially populates NE uniformly before trafficking outward to SR.Trafficking of PLB/SERCA2 a to SR follows the NEST pathway,suggesting a common secretory pathway for cardiac SR protein trafficking.High NE retention rates for PLB may underlie its unique steady-state distribution compared to SERCA2 a,suggesting a possible new role in NE Ca regulation.Part Three Effects of the accumulation of phospholamban at the nuclear envelope delays its trafficking along a transverse sarcoplasmic reticulum(SR)pathway common to SERCA2 a and other SR proteins in myocardial infarction ratsObjectiveAfter 24 h,48h and 72 h of primary myocardial cells were cultured in rats with myocardial infarction,the transport pathway of neonatal PLB and SERCA from nucleus to cytoplasm was observed.The effects of this new transport pathway on spontaneous calcium activity and related electrophysiological properties of cardiomyocytes were investigated.Methods:Eight healthy adult SD rats were used to construct an infarct model before descending the ligation.After the model was successfully established and raised for four weeks,the primary cardiomyocytes of adult SD rats were isolated using Lan'gendorff perfusion method and Liberase enzymatic method.The SERCA adenovirus vector was transfected into cultured primary cardiomyocytes,and the viable cells were observed at 24 h,48h,and 72 h after transfection.The cells were observed by immunohistochemistry,western blot,calcium-imaging and patch clamp.The newly generated PLB and SERCA distribution in cardiomyocytes,the transport pathway from the nucleus to the cytoplasm,the effect on the spontaneous calcium activity of cardiomyocytes,and the related electrophysiological functions.The results were expressed as x±s.One-way analysis of variance was used to compare multiple groups.Variance analysis was used to compare the variances between the two groups.Variance analysis was performed using data from different distance points.p<0.05 was considered statistically significant.Significance,all statistical analyses were done using SPSS 24.0 software.Results:1.The primary myocardial cells were successfully isolated from 8 infarcted rats and cultured for 24 h,48h and 72 h after transfection.2.In the primary cardiomyocytes transfected with adenovirus of infarcted mice,PLB was mainly localized on the nuclear membrane after 24 h fluorescence staining,while SERCA2 a had begun to spread out from the nuclear membrane.The results of 48 h fluorescence staining showed that PLB was found.The nuclear membrane is still dominant,SERCA2 a has been completely distributed to the sarcoplasmic reticulum,close to the distribution of its own steady state;72h observation of both have reached a steady-state distribution,the results of western blot and fluorescence staining have been observed,intracellular neonatal PLB / SERCA2 a content increased with time;3.Comparing the results of calcium imaging experiments in myocardial cells of infarcted myocardium before and after transfection,the spontaneous calcium activity of cardiomyocytes was significantly increased after transfection,and the fluorescence intensity was higher than that of untransfected myocytes(p<0.05,the difference was statistically significant);Conclusions:Neonatal PLB and SERCA2 a in cardiomyocytes of infarcted rats also follow the NEST pathway.The retention of PLB on the nuclear membrane during transit is longer than that of normal rats.The spontaneous calcium activity of cardiomyocytes is significantly increased and may be related to the insufficiency of cardiacmyocytes NE Ca regulation.