Study Of The Influence Of CaMKIIδ Expression On Ca²⁺ In Rat Atrial Myocytes

BackgroundIt is supposed that Calcium (Ca²⁺) is an importent charge carrier and universal mediator of diverse cellular processes. In striated muscle cells of heart these processes include gene transcription, excitation–contraction coupling, and also apoptosis. As we know intracellular Ca²⁺ ions translate electrical signals into mechanical activity of the heart and result in the single myocytes'shortened and the whole heart's contracted.In recent studies,however, growing evidence has demonstrated that the overloading of intracellular Ca²⁺ can introduce many cardiac diseases,such as cardiomyopathy, cardiac dysfunction,and arrhythmias.Ca²⁺/calmodulin-dependent protein kinase II (CaMKâ…¡) is a serine/threonine protein kinase which phosphorylates many intracellular proteins in response to elevated intracellular Ca²⁺ concentrations. CaMKâ…¡plays in regulating Ca²⁺ cycling in normal cardiac myocytes and in pathophysiological states. Moreover, CaMKâ…¡was recently identified as a potential drug target in cardiac disease. CaMKâ…¡activity and expression are reported to be increased in cardiac hypertrophy, in human heart failure, as well as in arrhythmias. In heart disease, CaMKâ…¡protein and activity levels are increased along with changes in ion channel expression. There are four different CaMKâ…¡genes (α,β,γ,δ) with theδisoform being predominant in the heart.The cardiac action potential is unique in that it has a prolonged depolarized state termed the plateau phase where Ca²⁺ ion influx is balanced by potassium ion efflux. Electrical changes are seen in heart disease due to remodeling of Ca signaling proteins and other ion transport proteins. CaMKâ…¡is an enzyme known to decode the frequency and amplitude of intracellular Ca²⁺ transients. Activated by higher average intracellular Ca²⁺ concentrations, for example, at faster heart rates, CaMKâ…¡phosphorylates a variety of Ca²⁺-handling proteins including LTCC, RyR2, and the SERCA2a-inhibitory protein phospholamban (PLN). CaMKâ…¡alter cardiac action potential duration through modulating concentration of intracellular Ca²⁺ ions. So CaMKâ…¡is believed to contribute to the increased propensity for arrhythmias. Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with an increased risk of stroke and admission rate. The basic mechanisms proposed to underlie AF are rapid ectopic firing and reentry,which occurs principally by prolongation of action potential duration and determined by slowed conduction and/or shortened the atrial refractory period. In the atrial myocytes of AF, abnormally enhanced intracellular Ca²⁺ was observed and there were evidences suggesting that Ca²⁺ overload played an important role in its pathogenesis.Also increased CaMKâ…¡expression has been demonstrated in atrial myocytes of AF patients. Recently, more research concerned the relationship of CaMKâ…¡and cardiac arrhythmia.But more studies are in progressing to demonstrate that AF be relevant to CaMKâ…¡.Rapid electrical field stimulation applied on cultured primary atrial myocytes has been used to simulate the microenviroment of AF in vitro. It caused electrical remodeling in the intact atrium, with shortening of action potential duration and increased vulnerability to AF. But the function of CaMKâ…¡in this process remains not clear. We hypothesized that increased CaMKⅡδexpression in atrial myocytes influence the SR Ca²⁺ transient amplitude and L-type Ca²⁺ current (ICa), and rapid electrical field stimulation can active CaMKⅡδaltering intracellular Ca²⁺ homeostasis,then induced AF. In this study, we investigated the function of CaMKⅡδin electric field stimulation, through changing its expression. This study can provide some theory bases for AF treatment.MethodsConstruction of lentiviral vectors and virus preparation: The cDNA of rattus norvegicus CaMKⅡδ(NM₀12519) was purchased from Open Biosystems (7939424). The ORF of the CaMKⅡδwas cloned into lentiviral vector LV-IRES-RFP by PCR to generate the plasmid LV-CaMKⅡδ-IRES-RFP. In this vector, expression of CaMKⅡδwas driven by CMV promoter and RFP was coexpressed as a transfection marker. To downregulate the expression of CaMKⅡδ, three pairs of siRNA were designed and ligated into plasmid pFIV-H1/U6-RFP, in which the siRNA transcription was driven by double opposing promoters. All plasmids were verified by DNA sequencing.Lentiviral particles were produced by transfecting 293FT cells with calcium phosphate transfection methods. Crude viral solution was collected and concentrated by ultracentrifugation to generate viral stock with a titer of 108 TU/ml. Culture and transduction of primary atrial myocytes: To isolate atrial myocytes, hearts were rapidly excised from neonatal Sprague–Dawley rats (borned 1-2 days). Ventricles of the hearts were removed and digested with 0.125% trypsin (Amresco) and 0.1% CollagenaseI (Gibco). Single cells were collected and plated into 6-well plate or coverslips at a density of 0.5¹Ã—104/cm2. 48 hours later, atrial myocytes were transduced with lentivirus at a MOI of 10. Additional 24 hours later, virus was removed and cells were cultured in DMEM plus 10% FBS for further analysis.Electric field stimulation: The cultured primary atrial myocytes were placed in electrical field for 24-hour continual stimulation in a humidified incubator (5% CO2). Computer-controlled stimulus waveform is square wave with stimulation frequencies 10Hz and output voltage 1.5 V/cm.Measurement of cellular Ca²⁺: Caffeine-elicited Ca²⁺ transient was observed by laser scanning confocal microscope (LSM510 META, ZEISS), as described previously.ICa recording was carried out as reported previously. Atrial myocytes with red fluorescence were selected to be measured. To correct for cell size variability, current amplitudes were defined as current density by dividing the absolute current by cell capacitance (pA/pF).ResultsThe lentiviral vector for overexpressing CaMKⅡδwas constructed. High titer lentivirus was prepared by calcium phosphate transfection methods. The atrial myocytes were efficiently transduced by this virus. Three siRNA were designed and the RNAi efficiency was evaluated by Western blot. No.2 siRNA had highest efficiency for CaMKⅡδknockdown. The sequence was inserted into lentiviral vector to generated viral plasmid for downregulating CaMKⅡδ.The primary atrial myocytes were successfully isolated from hearts of neonatal Sprague-Dawley rats. Analyzed by Immunohistochemistry and immunofluorescence, the purity of cultured atrial myocytes was more than 90%. Lentivirus was added into cells at a MOI=10. About 70% cells were transduced, confirmed by Microscope and FACS. In the cells tranduced by overexpressing virus, the expression of CaMKⅡδwas about 2 times compared to normal cells. Thus in cells with RNAi virus, CaMKⅡδexpression was decreased about 50%. The CaMKⅡδexpression was successfully modulated in the primary atrial myocytes. After stimulated by electric field, activity of CaMKⅡδwas elevated, but its expression level was not affected. In steady state, changes of CaMKⅡδexpression altered the SR Ca²⁺ release and ICa. ICa was increased ²5% in high CaMKⅡδcells and decreased ²8% in low CaMKⅡδcells. Moreover, in the atrial myocytes stimulated by electrical field, SR Ca²⁺ release and ICa were also modulated by CaMKⅡδexpression. Elevation of SR Ca²⁺ release caused by stimulation was enhanced by overexpressing CaMKⅡδand suppressed by RNAi. Similar with unstimulated situation, ICa was increased ²3% in high CaMKⅡδmyocytes and decreased ²6% in low CaMKⅡδcells.ConclusionThe conclusion of this study:electric field stimulation applied on primary atrial myocytes can influence the cellular Ca²⁺. In this process, activity of CaMKⅡδwas changed, but not expression level. Overexpressing or downregulating CaMKⅡδcan modulate the Ca²⁺ changes, and improve the cellular Ca²⁺ in the abnormal electrical environment.