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: 7732164
• 负责人: Edward Lakatta
• 金额: $ 12.33万
• 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/7732164
• 关键词: A kinase anchoring protein; AKAP13 gene; Action Potentials; Acute; Adenoviruses; Adrenergic Receptor; Adult; Affect; Artificial cardiac pacemaker; Binding; Binding Proteins; Ca(2+)-Transporting ATPase; Calmodulin; Cardiac; Cells; Characteristics; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; Data; Endoplasmic Reticulum; Fire - disasters; Fluorescence; Gene Transfer; Gene Transfer Techniques; Genes; Goals; Green Fluorescent Proteins; Heart; Human; Isoproterenol; Mediating; Methods; Modeling; Mus; Nodal; Oryctolagus cuniculus; Pacemakers; Peptides; Personal Satisfaction; Phosphorylation; Phosphotransferases; Process; Property; Protein Kinase A Inhibitor; Protein Overexpression; Proteins; Rate; Regulation; RyR2; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Sinoatrial Node; Sodium-Calcium Exchanger; Staining method; Stains; cyclic-nucleotide gated ion channels; day; density; genetic manipulation; genetic regulatory protein; indexing; mutant; phospholamban; protein expression

Genetic manipulation of key proteins involved in the 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 AP, global Ca2+ release transients and local Ca2+ releases just prior to the Ca2+ transients triggered by spontaneous AP, and the major characteristics of them are similar to f-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. 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 -adrenergic receptors with 1M isoproterenol 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 additionally, 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 (1.45 0.10, n=43) than that in f-SANC (3.57 0.13, n=56, p<0.001). Preliminary data of the immuno-staining of phosphorylated PLB at Ser16 suggests that this PKA-specific phosphorylation level is also lower in cultured vs. freshly isolated SANC. Inducing 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. We also successfully overexpressed some Ca2+ regulatory proteins in c-SANC, including wild type and mutant Ca2+ / calmodulin-dependent kinase IIC (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 conclude that adult c-SANC provide a reliable model to study the autonomic regulation by acute genetic manipulation of key proteins.

在兔子F-sant中不可能对参与该过程的关键蛋白的遗传操纵。因此，我们开发了一种稳定培养成人兔子（C-Sanc）的方法，已经表征了它们的特性，并通过腺病毒定向急性基因转移技术在C-SANC中成功表达了蛋白质。 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，这是一个合理的时期，这将使我们能够将外源性蛋白质引入C-Sanc。培养的SANC可以在34 0.5 OC下自发打击，并且动作电位（AP）发射速率在1.35 0.02 Hz的水平上稳定下来（n = 803，2到8天，培养为2至8天），其明显慢于F-SANC（2.79 0.04 Hz，n = 203，p <0.001）。 C-SANC在自发AP触发的Ca2+瞬变之前生成常规AP，全局Ca2+释放瞬变和局部CA2+释放，并且它们的主要特征与F-Sanc相似。 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和钠钙交换器。 有充分的文献证明，肽PKA抑制剂PKI可以大大降低或停止F-SANC的跳动率。我们假设，与F-SANC相比，C-SANC的跳动速率相对较低，可能是由于培养细胞中下调的蛋白激酶A（PKA）信号传导所致。用1M异丙肾上腺素刺激 - 肾上腺素能受体在C-SANC（3.34 0.05 Hz，n = 150）和F-SANC（3.55 0.06 Hz，n = 126）中将AP发射速率加速至类似的最大值。此外，我们观察到，RyR2的磷酸化水平是由Ser2809在Ser2809处的磷酸化RyR2的荧光密度归一化的，而C-Sanc（1.45 0.10，n = 43）比F-sanc（1.45 0.10，n = 43）均低于3.57 0.13 0.13 0.13 0.13 0.13，n = 56，n = 0.0056，n = 43）。 Ser16磷酸化PLB免疫染色的初步数据表明，在培养的与新鲜分离的Sanc中，这种PKA特异性磷酸化水平也较低。 通过腺病毒定向急性基因转移技术将绿色荧光蛋白（GFP）诱导到C-SANC中，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。 We also successfully overexpressed some Ca2+ regulatory proteins in c-SANC, including wild type and mutant Ca2+ / calmodulin-dependent kinase IIC (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中断AKAP介导的PKA锚定）及其不活动形式HT31P。 我们得出的结论是，成人C-Sanc提供了一个可靠的模型，以通过关键蛋白的急性遗传操纵来研究自主神经调节。