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: 7963887
• 负责人: Edward Lakatta
• 金额: $ 14.46万
• 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/7963887
• 关键词: A kinase anchoring protein; AKAP13 gene; Action Potentials; Acute; Adenoviruses; Adult; Affect; Artificial cardiac pacemaker; Binding; Binding Proteins; Ca(2+)-Transporting ATPase; Calmodulin; Cardiac; Cell Culture Techniques; Cells; Characteristics; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Endoplasmic Reticulum; Fluorescence; Gene Transfer; Gene Transfer Techniques; Genes; Goals; Green Fluorescent Proteins; Heart; Hour; Human; Isoproterenol; Measures; Mediating; Methods; Modeling; Mus; Nodal; Oryctolagus cuniculus; Pacemakers; Peptides; Pertussis Toxin; Phosphorylation; Phosphotransferases; Process; Property; Protein Kinase A Inhibitor; Proteins; RGS2 gene; Regulation; RyR2; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcoplasmic Reticulum; Signal Pathway; Signal Transduction; Sinoatrial Node; Site; Sodium-Calcium Exchanger; Staining method; Stains; Supporting Cell; beta-adrenergic receptor; cell type; cyclic-nucleotide gated ion channels; density; genetic manipulation; genetic regulatory protein; human RGS2 protein; indexing; mutant; overexpression; phospholamban; protein expression

Genetic manipulation of key proteins involved in the autonomic regulation process is impossible in rabbit f-SANC. So, we have developed a method to stable culture adult rabbit SANC (c-SANC), have characterized their properties, and have successfully overexpressed proteins in c-SANC via adenovirus directed acute gene-transfer technique. 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 reasonable period which would allow us to introduce exogenous proteins into c-SANC. The cultured SANC can beat spontaneously at 34 0.5 oC, and the action potential (AP) firing rate stabilizes at a level of 1.35 +/- 0.02 Hz (n=803, 2 to 8 days into culture), which is significantly slower than f-SANC (2.79 +/- 0.04 Hz, n=203, p<0.001). c-SANC generate regular and rhythmic AP, global Ca2+ release transients and local Ca2+ releases (LCRs) just prior to the Ca2+ transients triggered by spontaneous AP. The major characteristics of AP, Ca2+ transients and LCRs in c-SANC are similar to those from f-SANC, with some difference correlated with the spontaneous AP firing rate. By immuno-staining, we detected essential proteins involved in autonomic regulation in c-SANC, including type 2 sarcoplasmic reticulum Ca2+ release channel, i.e. 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. It is well documented that the peptide PKA inhibitor, PKI can dramatically reduce or stop the beating rate of f-SANC. We hypothesized that the relatively low beating rate of c-SANC compared to f-SANC, is possibly due to the down-regulated protein kinase A (PKA) signaling in the cultured cells. Acute stimulation of beta-adrenergic receptors with 1microM isoproterenol (ISO) accelerates the AP firing rate to a similar maximum in c-SANC (3.34 +/- 0.05 Hz, n=150) and in f-SANC (3.55 +/- 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 the cells total RyR2 fluorescence density, is substantially lower in c-SANC (1.32 +/- 0.06, n=47) than that in f-SANC (1.66 +/- 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, we measured the phosphorylation level PLB at Ser16, a PKA specific site, indexed by the fluorescence density of phosphorylated PLB at Ser16 normalized by total PLB fluorescence density. The results obtained with ISO acute stimulation and PKI inhibition in both cultured and freshly isolated SANC, support 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? We chose to measure the protein expression level of type 2 regulator of G protein signaling (RGS2), which functions as a powerful negative regulator of pertussis toxin (PTX)-sensitive Gi signaling. As we expected, the protein level is dramatically lower in c-SANC (149.9 +/- 4.0, n=100) than in f-SANC (201.9 +/- 6.0, n=88, p<0.001). Again, ISO (1microM, 2 hours incubation) enhances the staining density of RGS2 and PKI completely blocks ISOs effect. Furthermore, over-expression of RGS2 via adenovirus directed acute gene-transfer technique partially rescues the spontaneous beating rate of cultured SANC from 1.35 +/- 0.05 Hz (n=91) to 1.86 +/- 0.05 Hz (n=50, p<0.001). Introducing the green fluorescent protein (GFP) into c-SANC via adenovirus directed acute gene-transfer technique, does not affect the cell beating rate, and there is no correlation between AP firing rate and GFP expression level. When we treated cultured SANC with 0.4g/ml PTX overnight, the spontaneous beating rate promoted to 2.38 +/- 0.11 Hz (n=45). Partial rescue of s-SANC beating rate by PTX or RGS2 overexpression suggests the involvement of up-regulated Gi- signaling pathway in cultured SANC. We also successfully overexpressed some Ca2+ regulatory proteins in c-SANC, including wild type and mutant Ca2+ / calmodulin-dependent kinase IIgammaC (a multifunctional Ca2+ dependent kinase), Ht31 (a peptide that binds the PKA regulatory subunit type II (RII) and competes with endogenous A-kinase anchoring protein (AKAP) for RII binding, thus interrupts AKAP mediated PKA anchoring) and its inactive form Ht31p. We concluded that adult c-SANC provide a reliable model to study the autonomic regulation by acute genetic manipulation of key proteins. Moreover, understanding the difference between freshly isolated and cultured SANC itself broaden our view of autonomic regulation.

在兔 f-SANC 中，对参与自主调节过程的关键蛋白进行基因操作是不可能的。因此，我们开发了一种稳定培养成年兔 SANC (c-SANC) 的方法，表征了其特性，并通过腺病毒定向急性基因转移技术成功地在 c-SANC 中过表达了蛋白质。 在原代 SANC 培养的第一天，大多数细胞倾向于扩散并可存活长达 8 天，这是一个合理的期限，允许我们将外源蛋白引入 c-SANC。培养的 SANC 可在 34±0.5 oC 下自发跳动，动作电位 (AP) 放电率稳定在 1.35 +/- 0.02 Hz 的水平（n=803，培养 2 至 8 天），明显慢于 f -SANC（2.79+/-0.04Hz，n=203，p<0.001）。 c-SANC 在自发 AP 触发的 Ca2+ 瞬变之前生成规则且有节奏的 AP、全局 Ca2+ 释放瞬变和局部 Ca2+ 释放 (LCR)。 c-SANC 中的 AP、Ca2+ 瞬变和 LCR 的主要特征与 f-SANC 相似，但与自发 AP 放电率相关的一些差异。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+-ATP 酶 2a 和钠钙交换器。 据充分证明，肽 PKA 抑制剂 PKI 可以显着降低或停止 f-SANC 的跳动率。我们假设与 f-SANC 相比，c-SANC 的搏动率相对较低，可能是由于培养细胞中蛋白激酶 A (PKA) 信号传导下调所致。用 1μM 异丙肾上腺素 (ISO) 急性刺激 β-肾上腺素能受体可将 c-SANC（3.34 +/- 0.05 Hz，n=150）和 f-SANC（3.55 +/- 0.06 Hz）中的 AP 放电率加速至类似的最大值，n=126）。此外，我们观察到 RyR2 的磷酸化水平（以细胞总 RyR2 荧光密度归一化的 Ser2809 处磷酸化 RyR2 的荧光密度为索引）在 c-SANC 中显着较低（1.32 +/- 0.06，n=47） ）比 f-SANC 中的值（1.66 +/- 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，我们测量了 PKA 特异性位点 Ser16 处的 PLB 磷酸化水平，通过总 PLB 荧光密度归一化的 Ser16 处磷酸化 PLB 的荧光密度来索引。在培养的和新鲜分离的 SANC 中通过 ISO 急性刺激和 PKI 抑制获得的结果支持这样的解释：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？我们选择测量 G 蛋白信号传导 2 型调节因子 (RGS2) 的蛋白表达水平，该调节因子是百日咳毒素 (PTX) 敏感 Gi 信号传导的强大负调节因子。正如我们预期的那样，c-SANC 中的蛋白质水平 (149.9 +/- 4.0，n=100) 显着低于 f-SANC 中的蛋白质水平 (201.9 +/- 6.0，n=88，p<0.001)。同样，ISO（1μM，孵育 2 小时）增强了 RGS2 的染色密度，而 PKI 完全阻断了 ISO 的效应。此外，通过腺病毒定向急性基因转移技术过度表达 RGS2 可部分挽救培养 SANC 的自发搏动率，从 1.35 +/- 0.05 Hz (n=91) 至 1.86 +/- 0.05 Hz (n=50，p< 0.001）。通过腺病毒定向急性基因转移技术将绿色荧光蛋白（GFP）引入c-SANC，不影响细胞跳动率，并且AP发射率与GFP表达水平之间不存在相关性。当我们用 0.4g/ml PTX 处理培养的 SANC 过夜时，自发搏动率提升至 2.38 +/- 0.11 Hz (n=45)。 PTX 或 RGS2 过表达对 s-SANC 搏动率的部分挽救表明培养的 SANC 中涉及上调的 Gi 信号通路。 我们还成功地在 c-SANC 中过表达了一些 Ca2+ 调节蛋白，包括野生型和突变型 Ca2+/钙调蛋白依赖性激酶 IIgammaC（一种多功能 Ca2+ 依赖性激酶）、Ht31（一种结合 PKA II 型调节亚基 (RII) 并竞争的肽）与内源性 A 激酶锚定蛋白 (AKAP) 结合 RII，从而中断 AKAP 介导的 PKA 锚定）及其失活形式 Ht31p。 我们得出的结论是，成人 c-SANC 提供了一个可靠的模型来研究通过关键蛋白质的急性遗传操作进行的自主调节。此外，了解新鲜分离的 SANC 和培养的 SANC 本身之间的差异拓宽了我们对自主调节的看法。