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: 9147245
• 负责人: Edward Lakatta
• 金额: $ 17.72万
• 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/9147245
• 关键词: Action Potentials; Acute; Adenovirus Infections; Adenoviruses; Adult; Affect; Ca(2+)-Transporting ATPase; Cardiac; Cell Culture Techniques; Cell membrane; Cells; Characteristics; Culture Techniques; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Eating; Endoplasmic Reticulum; Fluorescence; Gene Transfer; Gene Transfer Techniques; Goals; Green Fluorescent Proteins; Heart; Hour; Intervention; Isoproterenol; Kinetics; Link; Measures; Modeling; Nodal; Oryctolagus cuniculus; Pacemakers; Peptides; Periodicity; Pertussis Toxin; Phosphorylation; Process; Property; Proteins; RGS2 gene; Regulation; RyR2; Ryanodine Receptors; Signal Transduction; Sinoatrial Node; Site; Sodium-Calcium Exchanger; Staining method; Stains; Techniques; Testing; Time; Variant; base; beta-adrenergic receptor; cell type; cyclic-nucleotide gated ion channels; density; genetic manipulation; human RGS2 protein; indexing; inhibitor/antagonist; nodal myocyte; overexpression; phospholamban; protein expression; spatiotemporal

To genetically manipulate key proteins involved in the autonomic regulation process, we have developed a technique for the culture of rabbit sinoatrial node cells, as it is 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. By immunostaining, we detected essential proteins involved in autonomic regulation in c-SANC, including 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 (APs), but at a level (1.35 plus/minus 0.02 Hz, n=804, over 2 to 8 days into culture) 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, the rhythmicity of c-SANC AP beating interval (APBI) is less robust than that of f-SANC, as indicated by a lower rhythmicity index of the autocorrelation function, a lower power spectrum amplitude and a bigger coefficient of variation (CV) in c-SANC versus f-SANC (p<0.001). The CVs of classic and additional analysis of AP characteristics are also increased in c-SANC vs. f-SANC. Spontaneous Local Ca2+ Releases (LCR) period are increased in c-SANC, and are correlated with the beating intervals of AP triggered global Ca2+ release transients the decay time of Ca2+ transients in both cell types, but with an increased CV in c-SANC vs. f-SANC. The reduced rhythmicity in c-SANC APBI is associated with prolongation of spontaneous LCR period during diastolic depolarization and an increase in its coefficient of variation (0.199 plus/minus 0.014 (n=41) for c-SANC vs. 0.122 plus/minus 0.009 (n=32) for f-SANC, p<0.001). 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 low beating rate of c-SANC is 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, indexed by the fluorescence density of phosphorylated RyR2 at Ser2809 normalized by 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. What is the mechanism underlying the PKA down-regulation in cultured pacemaker cells? Based upon 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 immunolabeling 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, overexpression 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. 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-SANCs AP firing rate. Partial rescue of c-SANCs AP firing rate by PTX treatment or RGS2 overexpression indicates 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. Early studies in single SANC indicate that intrinsic pacemaker stimulation not only change the average action APBI but also determine APBI variability. We tested the idea that the extent of spatiotemporal synchronization of both spontaneous local diastolic RyR activation induced by phosphorylation of Ca2+ cycling proteins in intact SANC are determinants of both APBI and APBIV. We observed that reduced phosphorylation of SR Ca2+ cycling protein phosphorylation and increased average APBI that are characteristic of SANC in culture (c-SANC) were accompanied by spatiotemporal de-synchronization of spontaneous local RyR activation, manifest not only as a reduced average LCR size and increased average LCR period, but also in increased variability of LCR periods and increased variability of diastolic depolarization parameters that is modulated by diastolic LCR occurrence. beta-adrenergic receptor stimulation increased protein phosphorylation and reduced the average APBI in both c-SANC and f-SANC, increased the spatiotemporal synchronization of LCR periods and sizes, reduced the variability of diastolic depolarization parameters and reduced CV. Detailed analysis was also applied to action potential parameters in f-SANC with interventions increasing the APBI, e.g. PKI or Cch, and the variabilities were also increased. Thus, both the spontaneous AP firing rhythm and average AP firing rate of isolated SANC are linked to the extent to which random local spontaneous RyR activation become synchronized by the APBI and by SR Ca2+ cycling protein phosphorylation.

为了对参与自主调节过程的关键蛋白质进行基因操作，我们开发了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) 来表征其特性，并通过腺病毒引导的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。通过免疫染色，我们检测到 c-SANC 中参与自主调节的必需蛋白，包括 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)、肌/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)，但其水平（1.35 ± 0.02 Hz，n=804，培养 2 至 8 天后）大约为 50% f-SANC 的值（2.79 加/减 0.04 Hz，n=203，p<0.001）。虽然 c- 和 f-SANC 都生成有节律的 AP，但 c-SANC AP 搏动间隔 (APBI) 的节律性不如 f-SANC 的稳健，如自相关函数的节律性指数较低、功率谱较低所示与 f-SANC 相比，c-SANC 的振幅和变异系数 (CV) 更大 (p<0.001)。与 f-SANC 相比，c-SANC 中 AP 特征的经典分析和附加分析的 CV 也有所增加。自发性局部 Ca2+ 释放 (LCR) 周期在 c-SANC 中增加，并且与 AP 触发的全局 Ca2+ 释放瞬变的搏动间隔相关，即两种细胞类型中 Ca2+ 瞬变的衰减时间，但与 c-SANC 相比，c-SANC 中的 CV 增加.f-SANC。 c-SANC APBI 节律性的降低与舒张期除极期间自发 LCR 期的延长及其变异系数的增加相关（c-SANC 为 0.199 ± 0.014 (n=41)，而 c-SANC 为 0.122 ± 0.009 (n=41)） n=32) 对于 f-SANC，p<0.001)。 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。我们假设 c-SANC 的低搏动率是由于培养细胞中 PKA 信号传导下调所致。事实上，非特异性 PDE 抑制剂 IBMX（100 微摩尔，10 分钟）将 c-SANC 的 AP 放电率提高到与 f-SANC 治疗相似的最大值。此外，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/减） 0.05 Hz，n=150）和 f-SANC（3.55 正/负 0.06 Hz， n=126）。此外，我们观察到，c-SANC 中的 RyR2 磷酸化水平（以 Ser2809 处磷酸化 RyR2 的荧光密度（以总 RyR2 荧光密度归一化）为索引）显着低于 f 中的磷酸化水平（1.32 加/减 0.06，n=47）。 -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。 培养的起搏细胞中 PKA 下调的机制是什么？基于百日咳毒素 (PTX) 敏感的 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 蛋白信号传导的 2 型调节因子 (RGS2) 的蛋白表达水平，其功能为PTX 敏感 Gi 信号传导的强大负调节剂。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。从功能上讲，通过腺病毒定向的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-SANCs 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。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-SANCs 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。 单一 SANC 的早期研究表明，内在起搏器刺激不仅改变平均动作 APBI，还决定 APBI 变异性。我们测试了以下观点：完整 SANC 中 Ca2+ 循环蛋白磷酸化诱导的自发局部舒张 RyR 激活的时空同步程度是 APBI 和 APBIV 的决定因素。我们观察到，SR Ca2+ 循环蛋白磷酸化的磷酸化减少和平均 APBI 增加是 SANC 培养物 (c-SANC) 的特征，伴随着自发局部 RyR 激活的时空去同步，不仅表现为平均 LCR 大小减少，而且平均 LCR 周期增加，而且 LCR 周期变异性增加以及由舒张期 LCR 发生调节的舒张期去极化参数变异性增加。 β-肾上腺素能受体刺激增加了 c-SANC 和 f-SANC 中的蛋白质磷酸化并降低了平均 APBI，增加了 LCR 周期和大小的时空同步性，减少了舒张期去极化参数的变异性并降低了 CV。还对 f-SANC 中的动作电位参数进行了详细分析，并通过增加 APBI 的干预措施，例如PKI或Cch，变异性也增加。 因此，孤立 SANC 的自发 AP 放电节律和平均 AP 放电率与随机局部自发 RyR 激活通过 APBI 和 SR Ca2+ 循环蛋白磷酸化同步的程度相关。