Culture and adenoviral infection of sinoatrial node myocytes from adult mice.
Culture and adenoviral infection of sinoatrial node myocytes from adult mice.
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DOI：
10.1152/ajpheart.00068.2015
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发表时间：
2015-08
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影响因子：
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通讯作者：
J. S. St. Clair;Emily J. Sharpe;C. Proenza
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文献类型：
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作者：
J. S. St. Clair;Emily J. Sharpe;C. Proenza
心脏窦房结的起搏器肌细胞通过激发自发动作电位来启动每一次心跳。然而，起搏背后的分子过程还不完全清楚，部分原因是我们在窦房结肌细胞(SAM)的天然细胞环境中操纵蛋白质表达的能力有限。在这里，我们描述了一种培养成年小鼠完全分化的SAM的新方法，我们证明了通过腺病毒基因转移，引入的蛋白可以在体外24-48小时内获得强劲的表达。比较培养48h和急性分离的SAMs的形态和电生理特性，在体外只有很小的变化。具体地说，我们发现细胞在培养中趋于扁平，但保持了总体正常的形态，细胞尺寸或膜电容没有显著变化。培养的细胞自发搏动，在膜片钳记录中，尽管动作电位波形参数的子集发生了轻微变化，但培养的细胞和急性分离的细胞的自发动作电位放电率没有差异。在培养和急性分离的细胞中，对起搏活动至关重要的两种膜电流--“滑稽电流”(IF)和电压门控钙电流(ICA)--的生物物理特性也无法区分。这种从成年小鼠心脏培养和腺病毒感染完全分化的SAM的新方法扩大了可用于研究心脏起搏分子生理学的实验技术范围，因为它将使研究蛋白质表达水平可以改变或SAM中表达的基因编码报告分子成为可能。
Pacemaker myocytes in the sinoatrial node of the heart initiate each heartbeat by firing spontaneous action potentials. However, the molecular processes that underlie pacemaking are incompletely understood, in part because of our limited ability to manipulate protein expression within the native cellular context of sinoatrial node myocytes (SAMs). Here we describe a new method for the culture of fully differentiated SAMs from adult mice, and we demonstrate that robust expression of introduced proteins can be achieved within 24-48 h in vitro via adenoviral gene transfer. Comparison of morphological and electrophysiological characteristics of 48 h-cultured versus acutely isolated SAMs revealed only minor changes in vitro. Specifically, we found that cells tended to flatten in culture but retained an overall normal morphology, with no significant changes in cellular dimensions or membrane capacitance. Cultured cells beat spontaneously and, in patch-clamp recordings, the spontaneous action potential firing rate did not differ between cultured and acutely isolated cells, despite modest changes in a subset of action potential waveform parameters. The biophysical properties of two membrane currents that are critical for pacemaker activity in SAMs, the "funny current" (If) and voltage-gated Ca(2+) currents (ICa), were also indistinguishable between cultured and acutely isolated cells. This new method for culture and adenoviral infection of fully-differentiated SAMs from the adult mouse heart expands the range of experimental techniques that can be applied to study the molecular physiology of cardiac pacemaking because it will enable studies in which protein expression levels can be modified or genetically encoded reporter molecules expressed within SAMs.