Cultured adult rabbit pacemaker cells for gene transfer studies

用于基因转移研究的培养成年兔起搏细胞

• 批准号: 8552335
• 负责人: Edward Lakatta
• 金额: $ 17.61万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552335
• 关键词: Action Potentials; Acute; Adenovirus Infections; Adenoviruses; Adult; Affect; Ca(2+)-Transporting ATPase; Cardiac; Cell Culture Techniques; Cell membrane; Cell model; Cell physiology; Cells; Characteristics; Coupled; Coupling; Culture Techniques; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Down-Regulation; Eating; Endoplasmic Reticulum; Fluorescence; Future; Gene Transfer; Gene Transfer Techniques; Glean; Goals; Green Fluorescent Proteins; Heart; Hour; Human; Ion Channel; Isoproterenol; Kinetics; Label; Length; Measures; Modeling; Mus; Nodal; Oryctolagus cuniculus; Pacemakers; Peptides; Periodicity; Pertussis Toxin; Phenotype; Phosphorylation; Process; Property; Proteins; RGS2 gene; Rattus; Regulation; Rodent; RyR2; Ryanodine Receptors; Sarcolemma; Sarcoplasmic Reticulum; Signal Transduction; Sinoatrial Node; Site; Sodium-Calcium Exchanger; Staining method; Stains; Supporting Cell; Techniques; Time; base; beta-adrenergic receptor; cell type; cyclic-nucleotide gated ion channels; density; genetic manipulation; human RGS2 protein; indexing; inhibitor/antagonist; insight; nodal myocyte; novel; overexpression; phospholamban; protein expression; Action Potentials; Acute; Adenovirus Infections; Adenoviruses; Adult; Affect; Ca(2+)-Transporting ATPase; Cardiac; Cell Culture Techniques; Cell membrane; Cell model; Cell physiology; Cells; Characteristics; Coupled; Coupling; Culture Techniques; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Down-Regulation; Eating; Endoplasmic Reticulum; Fluorescence; Future; Gene Transfer; Gene Transfer Techniques; Glean; Goals; Green Fluorescent Proteins; Heart; Hour; Human; Ion Channel; Isoproterenol; Kinetics; Label; Length; Measures; Modeling; Mus; Nodal; Oryctolagus cuniculus; Pacemakers; Peptides; Periodicity; Pertussis Toxin; Phenotype; Phosphorylation; Process; Property; Proteins; RGS2 gene; Rattus; Regulation; Rodent; RyR2; Ryanodine Receptors; Sarcolemma; Sarcoplasmic Reticulum; Signal Transduction; Sinoatrial Node; Site; Sodium-Calcium Exchanger; Staining method; Stains; Supporting Cell; Techniques; Time; base; beta-adrenergic receptor; cell type; cyclic-nucleotide gated ion channels; density; genetic manipulation; human RGS2 protein; indexing; inhibitor/antagonist; insight; nodal myocyte; novel; overexpression; phospholamban; protein expression

In order to genetically manipulate key proteins involved in the autonomic regulation process, we had to develop a technique for the culture of rabbit Sinoatrial node cells, as its impossible to do so in freshly isolated SANC. We have been able to obtain stable adult rabbit cultured SANC (c-SANC), to characterize their properties, and have successfully overexpressed proteins in c-SANC via adenovirus directed acute gene-transfer technique. Our results show that on the first day of primary SANC culture, most of the cells tend to spread out and could stay alive for up to 8 days, a period which would allow us to introduce exogenous proteins into c-SANC. By immuno-staining, we detected essential proteins involved in autonomic regulation in c-SANC, including type 2 sarcoplasmic reticulum Ca2+ release channel, i.e. type 2 ryanodine receptors (RyR2), L-type Ca2+ channel, hyperpolarization-activated cyclic nucleotide-gated channel 4, phospholamban (PLB), Sarco/Endoplasmic Reticulum Ca2+-ATPase 2a and Sodium-Calcium exchanger. At 34 plus/minus 0.5 degrees C, c-SANC generate spontaneous, rhythmic action potentials (AP), but at a level (1.35 plus/minus 0.02 Hz, n=804, over 2 to 8 days into culture) is roughly 50% of that of f-SANC (2.79 plus/minus 0.04 Hz, n=203, p<0.001). Although both c-and f-SANC generate rhythmic APs and AP triggered global Ca2+ release transients, the rhythmicity of c-SANC AP or Ca2+ transient is less robust than that of f-SANC, as indicated by a lower rhythmicity index of the autocorrelation function in c-SANC versus f-SANC (p<0.001). By comparing the rhythmicity index of c-SANC and f-SANC at control or different treatments, the rhythmicity of AP is positively correlated with the AP firing rate (r2=0.75). Spontaneous Local Ca2+ Releases (LCR) period are increased in c-SANC, and are correlated with the decay time of AP triggered global Ca2+ release transients in both cell types, but with an increased variability in c-SANC vs. f-SANC. It is well documented that the peptide inhibitor of protein kinase A (PKA), PKI, can dramatically reduce or stop the beating rate of f-SANC. We hypothesized that the relatively low beating rate of c-SANC, is possibly due to the down-regulated PKA signaling in the cultured cells. Indeed, non-specific PDE inhibitor IBMX (100 microMolar, 10min) increases the AP firing rate of c-SANC to a similar maximum to the treatment of f-SANC. Furthermore, acute stimulation of beta-adrenergic receptors with 1 microMolar isoproterenol (ISO) for 10 min accelerates AP and Ca2+-transient kinetics, reduces the LCR period, and accelerates the AP firing rate to a similar maximum in c-SANC (3.34 plus/minus 0.05 Hz, n=150) and f-SANC (3.55 plus/minus 0.06 Hz, n=126). In addition, we observed that the phosphorylation level of RyR2, which is indexed by the fluorescence density of phosphorylated RyR2 at Ser2809 normalized by its own total RyR2 fluorescence density, is substantially lower in c-SANC (1.32 plus/minus 0.06, n=47) than in f-SANC (1.66 plus/minus 0.15, n=24, p<0.01). While acute ISO stimulation raises the RyR2 phosphorylaiton at Ser2809 to a similar level in both cell types, PKI treatment reduces the phosphorylation level. More specifically, the phosphorylation level of PLB at Ser16, a PKA specific site, is also significantly lower in c-SANC than f-SANC. Similarly, ISO acute stimulation increases and PKA inhibition by PKI decreases PLB phosphorylation at Ser16 in both cultured and freshly isolated SANC, supporting the interpretation that PKA signaling is down-regulated in cultured SANC compared with freshly isolated SANC. What is the mechanism underlying the PKA down-regulation in cultured pacemaker cells? Based upon the above data and the fact that the activation of pertussis toxin (PTX)-sensitive Gi signaling is involved in the beating rate reduction of f-SANC, we measured the protein expression level of type 2 regulator of G protein signaling (RGS2), which functions as a powerful negative regulator of PTX-sensitive Gi signaling. As we expected, the protein level, indexed by the immuno-labeling density along the cell membrane, is substantially lower in 2 day cultured SANC (149.9 plus/minus 4.0, n=100) than in f-SANC (201.9 plus/minus 6.0, n=88, p<0.001). 2 hours incubation of 1 microMolar ISO enhances the staining density of RGS2 and PKI completely inhibits ISOs effect. Functionally, over-expression of RGS2 via adenovirus directed acute gene-transfer technique increases the spontaneous beating rate of cultured SANC from 1.35 plus/minus 0.05 Hz (n=91) to 1.86 plus/minus 0.05 Hz (n=50, p<0.001), which is 66% of f-SANCs AP firing ate. This effect is not because of adenovirus infection, as introducing the green fluorescent protein (GFP) into c-SANC via the same technique, does not affect the cell beating rate, and there is no correlation between AP firing rate and GFP expression level. To our surprise, over-expression of RGS2 in c-SANC increased the phosphorylation level of PLB at Ser16 but not the phosphorylation level of RyR2 at Ser2809. Furthermore, when cultured SANC were treated with 0.4micrograms/ml PTX overnight, the spontaneous beating rate is boosted to 2.38 plus/minus 0.11 Hz (n=45), 85% of f-SANCcs AP firing rate. Partial rescue of c-SANCs AP firing rate by PTX treatment or RGS2 overexpression indicate that a reduction in PKA-dependent Ca2+-cycling protein phosphorylation that is Gi-dependent is involved in prolongation of LCR period and reduced spontaneous AP firing rate of c-SANC, and that this deficit can be reversed by pharmacologic or genetic manipulation. In summary, we have defined important characteristics of a cultured rabbit SANC model, in which altered cAMP/PKA modulation that develops in culture, reduces the AP firing rate and rhythmicity. Specifically, a Gi-dependent PTX-sensitive reduction in PKA-dependent Ca2+-cycling protein phosphorylation, likely due, in part at least, to reduced RGS signaling, increases the variability of LCR period and prolongs the average LCR period, and increases the variability of AP cycle length and reduces the average spontaneous AP firing rate. The altered phenotypes of c-SANC, and its rescue by the maneuvers employed provide additional support for the coupled-clock hypothesis of pacemaker cells. Specifically, culture conditions, via activation of Gi signaling, interfere with coupled-clock function by interfering with SR Ca2+ cycling and also interfering with its coupling to sarcolemma ion channels, leading to alteration in automaticity and rhythmicity. This culture model will enable future studies in which genetic manipulation of adult pacemaker cells can be employed to glean novel mechanistic insights into adult pacemaker cell function.

为了通过遗传操纵参与自主调节过程的关键蛋白，我们必须开发一种针对兔子辛里型节点细胞培养的技术，因为在新鲜分离的sanc中不可能这样做。我们已经能够获得稳定的成年兔培养的sanc（C-sanc），以表征其性质，并通过腺病毒定向急性基因转移技术成功地表达了C-SANC中的过度表达蛋白质。 我们的结果表明，在原发性圣培养的第一天，大多数细胞倾向于散布，并且可以生存长达8天，这将使我们能够将外源性蛋白质引入C-Sanc。 By immuno-staining, we detected essential proteins involved in autonomic regulation in c-SANC, including type 2 sarcoplasmic reticulum Ca2+ release channel, i.e. type 2 ryanodine receptors (RyR2), L-type Ca2+ channel, hyperpolarization-activated cyclic nucleotide-gated channel 4, phospholamban (PLB), Sarco/Endoplasmic Reticulum Ca2+-ATPase 2a和钠钙交换器。在34 plus/sinus 0.5度c时，c-sanc产生自发性的节奏动作电位（AP），但在A级水平（1.35 plus/sinus 0.02 Hz，n = 804，在文化中2至8天内）是F-SANC的50％（2.79 plus/nim Minus 0.04 Hz，n = 203，p <0.001）。尽管C-and F-Sanc都会产生节奏AP和AP触发了全局Ca2+释放瞬变，但C-Sanc AP或Ca2+瞬态的节奏性比F-SANC的节奏性较低，如C-Sanc-Sanc versus v-Sanc（p <0.001）中自相关功能的较低节律性索引所指出。通过比较C-SANC和F-SANC在对照或不同处理时的节奏指数，AP的节奏性与AP发射速率呈正相关（R2 = 0.75）。 C-SANC中自发的局部Ca2+释放（LCR）周期增加了，并且与两种细胞类型中AP触发的全局Ca2+释放瞬变的衰减时间相关，但C-Sanc and cancanc vs.F-sanc的可变性增加。 有充分的文献证明，PKI蛋白激酶A（PKA）的肽抑制剂可以大大降低或停止F-SANC的跳动率。我们假设，C-Sanc的比较率相对较低，这可能是由于培养细胞中的PKA信号下调。实际上，非特异性PDE抑制剂IBMX（100微摩尔，10分钟）将C-SANC的AP发射速率提高到与F-SANC治疗相似的最大值。此外，用1分钟的1分钟急性刺激β-肾上腺肾上腺素受体（ISO）加速AP和Ca2+过量动力学，降低了LCR周期，并使AP发射速率加速至C-SANC相似的最大值（3.34 Plus/Minus 0.05 Hz，N = 15 Hz，n = 150），并加速。 n = 126）。此外，我们观察到，RyR2的磷酸化水平是由Ser2809磷酸化的RyR2的荧光密度通过其自身的RYR2荧光密度归一化的，C-Sanc中的磷酸化水平在C-SANC中大大低（1.32 plus/situs 0.06，n = 47，n = 47）（n = 47）（n = 47）（n = 47）（1.66 plus/n = 1.66 plus n 0.66 plus n = <0.66 plus n = 0.66 pl <0.66 pl <0.15，= 0.15 = 0.15 = 0.15 = 0.15。尽管在两种细胞类型中，急性ISO刺激将Ser2809的RyR2磷酸磷酸化剂提高到相似的水平，但PKI治疗降低了磷酸化水平。更具体地说，在pKA特异性位点Ser16处PLB的磷酸化水平也明显低于f-sanc。同样，在培养和新鲜分离的SANC中，PKI的ISO急性刺激增加和PKA抑制作用Ser16的PLB磷酸化降低，这支持了与新隔离的Sanc相比，PKA信号在培养的SANC中下调下调的解释。 培养的起搏器细胞中PKA下调的基础机制是什么？基于上述数据以及百日咳毒素（PTX）敏感的GI信号的激活与F-SANC的降低速率降低有关，我们测量了G蛋白信号传导的蛋白质表达水平（RGS2）的蛋白质表达水平（RGS2），该蛋白质表达水平（RGS2）是PTX敏感信号gi敏感的gi信号的强大调节剂。正如我们所预期的，由沿细胞膜的免疫标记密度索引的蛋白质水平在2天培养的SANC（149.9 plus/sinus 4.0，n = 100）中大大低于F-sanc（201.9 plus/sinus 6.0，n = 88，p <0.001）。 2小时的1个微摩尔ISO孵育可增强RGS2和PKI的染色密度完全抑制了ISOS效应。在功能上，通过腺病毒对RGS2的过表达将急性基因转移技术提高了培养的SANC的自发跳动率从1.35 plus/sinus 0.05 Hz（n = 91）增加到1.86加/减0.05 Hz（n = 50，p <0.001），f-SAPS ap firn firn firn firn firn firn。这种作用不是因为腺病毒感染，因为将绿色荧光蛋白（GFP）通过相同的技术引入了C-SANC，不会影响细胞的殴打率，并且AP发射速率与GFP表达水平之间没有相关性。令我们惊讶的是，C-SANC中RGS2的过表达增加了Ser16时PLB的磷酸化水平，而不是Ser2809的RYR2的磷酸化水平。此外，当培养的SANC用0.4microgram/ml PTX处理过夜时，自发的跳动率将提高至2.38 plus/sinus 0.11 Hz（n = 45），占F-Sanccs AP触发率的85％。通过PTX治疗或RGS2过表达对C-SANCS AP发射率的部分营救表明，依赖GI依赖性的PKA依赖性Ca2+Cyccling蛋白磷酸化的降低与LCR期间的延长有关，并降低了c-S-S-SCANC的自发性发射速率，而这种抗拒的人可以通过药物为代表性或概括。 总而言之，我们定义了培养的兔子SANC模型的重要特征，在该模型中，在文化中发展的CAMP/PKA调制改变，降低了AP的射击率和节奏性。具体而言，依赖于GI的PTX敏感性降低了PKA依赖性Ca2+循环蛋白磷酸化的磷酸化，可能至少部分归因于RGS信号的降低，可提高LCR周期的变异性并延长平均LCR周期，并增加AP周期的变异性，并增加平均自AP型号的自动化速率。 C-Sanc的表型改变及其被采用的操作的营救为起搏器细胞的耦合锁定假设提供了额外的支持。具体而言，通过激活GI信号传导，培养条件通过干扰SR Ca2+循环的耦合锁定功能，并干扰其与肌膜离子通道的耦合，从而导致自动性和节奏性的改变。这种培养模型将实现未来的研究，其中可以利用对成年起搏器细胞的遗传操纵来收集新的机械洞察力，以了解成人起搏器细胞功能。