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: 8335785
• 负责人: Edward Lakatta
• 金额: $ 15.38万
• 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/8335785
• 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

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 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). 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 varalibility 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, 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. Whats 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. 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.

为了对参与自主调节过程的关键蛋白质进行基因操作，我们必须开发一种培养兔窦房结细胞的技术，因为在新鲜分离的 SANC 中不可能做到这一点。我们已经能够获得稳定的成年兔培养 SANC (c-SANC)，以表征其特性，并通过腺病毒定向急性基因转移技术成功地在 c-SANC 中过表达蛋白质。 我们的结果表明，在原代 SANC 培养的第一天，大多数细胞倾向于扩散，并且可以存活长达 8 天，这使得我们能够将外源蛋白引入 c-SANC。通过免疫染色，我们检测到c-SANC中参与自主调节的重要蛋白，包括2型肌浆网Ca2+释放通道，即2型兰尼碱受体（RyR2）、L型Ca2+通道、超极化激活的环核苷酸门控通道4、受磷蛋白 (PLB)、Sarco/内质网 Ca2+-ATPase 2a 和钠钙交换器。在 34 ± 0.5 摄氏度时，c-SANC 产生自发的节律性动作电位 (AP)，但在一定水平（1.35 ± 0.02 Hz，n=804，培养 2 至 8 天后）约为 50% f-SANC 的值（2.79 加/减 0.04 Hz，n=203，p<0.001）。尽管c-和f-SANC都产生节律性AP并且AP触发全局Ca2+释放瞬变，但c-SANC AP的节律性不如f-SANC稳健，如c-中自相关函数的节律性指数较低所示。 SANC 与 f-SANC (p<0.001)。 c-SANC 中的自发局部 Ca2+ 释放 (LCR) 周期增加，并且与两种细胞类型中 AP 触发的全局 Ca2+ 释放瞬态的衰减时间相关，但与 f-SANC 相比，c-SANC 的变异性增加。 据充分证明，蛋白激酶 A (PKA) 的肽抑制剂 PKI 可以显着降低或停止 f-SANC 的跳动率。我们推测 c-SANC 相对较低的跳动率可能是由于培养细胞中 PKA 信号传导下调所致。事实上，用 1 微摩尔异丙肾上腺素 (ISO) 急性刺激 β-肾上腺素能受体 10 分钟可加速 AP 和 Ca2+ 瞬态动力学，缩短 LCR 周期，并将 AP 放电率加速至 c-SANC 中类似的最大值（3.34 加/负 0.05 Hz，n=150) 和 f-SANC（3.55 正/负 0.06 Hz， n=126）。此外，我们观察到 RyR2 的磷酸化水平（以 Ser2809 处磷酸化 RyR2 的荧光密度为索引，并以其自身的总 RyR2 荧光密度归一化）在 c-SANC 中显着较低（1.32 加/减 0.06，n=47） ）高于 f-SANC（1.66 加/减 0.15，n=24，p<0.01）。虽然急性 ISO 刺激将两种细胞类型中 Ser2809 处的 RyR2 磷酸化水平提高到相似水平，但 PKI 处理会降低磷酸化水平。更具体地说，c-SANC 中 PLB 在 Ser16（PKA 特异性位点）处的磷酸化水平也显着低于 f-SANC。同样，在培养的 SANC 和新鲜分离的 SANC 中，ISO 急性刺激增加，PKI 抑制 PKA 降低 Ser16 位点的 PLB 磷酸化，支持这样的解释：与新鲜分离的 SANC 相比，培养的 SANC 中 PKA 信号传导下调。 培养的起搏细胞中 PKA 下调的机制是什么？基于上述数据以及百日咳毒素（PTX）敏感Gi信号传导的激活参与f-SANC搏动率降低的事实，我们测量了G蛋白信号传导2型调节因子（RGS2）的蛋白表达水平，其作为 PTX 敏感 Gi 信号传导的强大负调节器。正如我们预期的那样，培养 2 天的 SANC 中的蛋白质水平（以沿细胞膜的免疫标记密度为索引）（149.9 加/减 4.0，n=100）显着低于 f-SANC（201.9 加/减 6.0） ，n=88，p<0.001）。 1 微摩尔 ISO 孵育 2 小时可增强 RGS2 的染色密度，而 PKI 完全抑制 ISO 效应。从功能上来说，通过腺病毒定向急性基因转移技术过度表达 RGS2 可将培养的 SANC 的自发搏动率从 1.35 ± 0.05 Hz (n=91) 增加到 1.86 ± 0.05 Hz (n=50，p<0.001) ），这是 f-SANC AP 发射的 66%。这种效应不是因为腺病毒感染，因为通过相同的技术将绿色荧光蛋白（GFP）引入c-SANC，不会影响细胞跳动率，并且AP发射率和GFP表达水平之间不存在相关性。此外，当培养的SANC用0.4微克/ml PTX处理过夜时，自发搏动率提高至2.38±0.11 Hz (n=45)，是f-SANCcs AP放电率的85%。 PTX 处理或 RGS2 过表达对 c-SANCs AP 放电率的部分挽救表明，Gi 依赖性 PKA 依赖性 Ca2+ 循环蛋白磷酸化的减少参与了 LCR 周期的延长和 c-SANC 自发 AP 放电率的降低，并且这种缺陷可以通过药理学或基因操作来逆转。 总之，我们定义了培养兔 SANC 模型的重要特征，其中改变了培养中形成的 cAMP/PKA 调制，降低了 AP 放电率和节律性。具体来说，PKA依赖性Ca2+循环蛋白磷酸化的Gi依赖性PTX敏感性减少，可能至少部分是由于RGS信号传导减少，增加了LCR周期的变异性并延长了平均LCR周期，并增加了变异性AP 周期长度并降低平均自发 AP 发射率。 c-SANC 的表型改变以及所采用的操作对其的拯救为起搏细胞的时钟耦合假说提供了额外的支持。具体来说，培养条件通过激活 Gi 信号传导，通过干扰 SR Ca2+ 循环以及干扰其与肌膜离子通道的耦合来干扰耦合时钟功能，从而导致自动性和节律性的改变。这种培养模型将使未来的研究成为可能，在这些研究中，可以利用成体起搏细胞的基因操作来收集对成体起搏细胞功能的新机制见解。