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.

为了操纵参与自主调节过程的关键蛋白质，我们为兔子辛里氏淋巴结细胞的培养提供了一种技术，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）来表征其性质，并通过腺病毒指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，在初级萨克培养的第一天，大多数细胞倾向于散布，并且可以存活长达8天。通过免疫染色，我们检测到C-SANC中参与自主神经调节的必需蛋白质，包括2型Ryanodine受体（RYR2），L型Ca2+通道，高极化激活的环核苷酸门控通道4，Phospholamban（PLB），phospholamban（PLB），sarco/sarco/popolallasm-crasmic-calc-cal CAS2+ -ADPase 2A。在34 plus/sinus 0.5度c时，c-sanc产生自发性的节奏动作电位（APS），但在A培养的2至8天的水平上（1.35 + 0.02 Hz，n = 804），大约50％的F-sanc（2.79 plus/sig 0.04 Hz，n = 203，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).在C-Sanc与F-Sanc中，经典的简历和AP特性的其他分析也增加了。 C-SANC中自发的局部Ca2+释放（LCR）周期增加了，并且与AP触发的全局Ca2+释放瞬变的跳动间隔相关，两种细胞类型中Ca2+瞬变的衰减时间，但C-Sanc cv中的CV与F-Sanc的CV增加。 C-SANC APBI的节奏性降低与舒张期去极化期间自发LCR周期的延长以及其变化系数的增加（0.199 plus/sinus 0.014（n = 41）C-Sanc vs. 0.122 vs. 0.122 vs. 0.122 plus/Minus 0.009（n = 32）（n = 32）（n = 32）（n = 32），f-Sanc，p <0.001）。 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的低节奏率是由于培养细胞中下调的PKA信号传导引起的。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。 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）。此外，我们观察到，RyR2的磷酸化水平由Ser2809磷酸化的RyR2的荧光密度通过C-SANC（1.32 plus/sinus 0.06，n = 47）归一化的Ser2809归一化的磷酸化水平，比F-Sanc（1.66 plus 0.06，n = 47）（1.66 plus 0.66 plus/minus plus/minus n = 47）。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。 培养的起搏器细胞中PKA下调的基础机制是什么？基于以下事实：百日咳毒素（PTX）敏感的GI信号的激活参与F-SANC的跳动速率降低，我们测量了G蛋白信号2型调节剂的蛋白质表达水平（RGS2）的蛋白质表达水平（RGS2），该蛋白质表达水平是PTX敏感GI信号的强大负调节剂。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）中比F-Sanc（201.9 plus/sinus 6.0，n = 88，p <0.001）低。 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，通过腺病毒导向的急性基因转移技术对RGS2的过表达使培养的SANC的自发殴打率从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。这种作用不是因为腺病毒感染，而是通过相同的技术将绿色荧光蛋白（GFP）引入C-SANC，不会影响细胞的殴打率。此外，当培养的SANC用0.4microgram/ml PTX处理过夜时，自发的跳动速率将提高到2.38 plus/sinus 0.11 Hz（n = 45），占F-SAP 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性地进行药物。 单一SANC的早期研究表明，内在的起搏器刺激不仅改变了平均作用APBI，而且还会决定APBI的可变性。我们测试了以下想法：两种自发性局部舒张性RYR激活的时空同步的程度是由Ca2+循环蛋白在完整sanc中磷酸化诱导的，这是APBI和APBIV的决定因素。我们观察到，Sr Ca2+自动蛋白磷酸化的磷酸化降低，平均APBI增加，这是培养中SANC（C-SANC）的特征（C-Sanc）的特征（C-SANC），时空DE同步的自发性局部RYR激活的时空同步，不仅会增加平均LCR尺寸和增加的定期性能，还增加了平均lcr的变化，lcr的变化增加了。由舒张期LCR调节的参数。 β-肾上腺素能受体刺激增加了蛋白质磷酸化，并降低了C-SANC和F-SANC的平均APBI，增加了LCR周期和大小的时空同步，从而降低了舒张期去极化参数的差异和降低的CV。详细的分析还应用于F-SAN的动作潜在参数，干预措施增加了APBI，例如PKI或CCH，并且变异性也增加了。 因此，孤立的SANC的自发性AP发射节奏和平均AP触发速率都与随机局部自发的RYR激活与APBI同步以及SR Ca2+ Cycling蛋白磷酸化的程度有关。