Dynamic calcium regulation of the slowly delayed rectifier potassium current

 描述(由申请人提供)：心力衰竭(HF)是美国和发达国家成年人住院的主要原因，并增加了心律失常和心脏性猝死的风险。在心力衰竭中，心律失常通常继发于细胞内钙([Ca~(2+)]i)调节中断或心脏组织中涉及离子通道和离子通道调节蛋白表达或功能改变的电重构。特别是，慢激活延迟整流钾电流(Iks)在HF时被下调，有证据表明[Ca~(2+)]i可以调节Iks。IKs是心脏动作电位(AP)复极储备的关键成分，在β-肾上腺素能刺激时最为显著。在心性心动过速时，去极化通过L型钙通道(ICa)进入钙离子，进而导致肌浆网(SR)钙离子释放，导致[Ca~(2+)]i升高。在每个心动周期中，离子通道感受到的膜(膜下[Ca~(2+)]_i)[Ca~(2+)]_i的升高大于胞浆[Ca~(2+)]_i。因此，单个Ca~(2+)瞬变过程中[Ca~(2+)]_i的升高可能会动态地增加Iks，从而限制AP持续时间，从而限制Ca~(2+)进入心肌细胞的量。此外，在[Ca~(2+)]_i动力学受损的情况下(如HF)，IKs对Ca~(2+)的敏感性可降低复极储备。尽管IKs的钙依赖性似乎很重要，但动态变化的[Ca2+]i瞬变对IKs的调制还没有像ICa、Na-Ca交换器或SR钙离子释放那样深入研究。因此，这一建议的总体假设是，IKs的钙调节是动态的，并且是IKs对心脏复极的生理贡献的关键决定因素。 为了验证这一假设，目标1将确定稳态和动态变化的[Ca~(2+)]i对Iks的幅度和门控特性的影响。通过[Ca~(2+)]_i，特别是膜下[Ca~(2+)]_i对每个心动周期IKs的调节，将揭示IKs在正常心脏复极中的重要作用。这将在兔心室肌细胞中使用宽场荧光膜片钳技术进行评估，环境由一系列受控的[钙]i(稳态)组成，并在允许触发SR钙释放的AP波形(动态)中进行。目标2将在目标1的基础上扩展，通过测量β-肾上腺素能刺激期间iKs的稳态和动态[Ca~(2+)]i调节来确定这一调节是否增加iKs对[Ca~(2+)]i的敏感性。最后，目标3将使用与目标1和2类似的方法检验假设，即在兔心衰模型中iKs的减少既反映了iKs对[Ca~(2+)]i的敏感性降低，也反映了iKs对[Ca~(2+)]i刺激的反应性降低。

 DESCRIPTION (provided by applicant): Heart failure (HF) is the leading cause of hospitalization in the adult population of the United States and developed countries, and increases the risk for arrhythmias and sudden cardiac death. In HF, arrhythmias are typically secondary to disrupted regulation of intracellular calcium ([Ca2+]i) or electrical remodeling in cardiac tissue that involves altered expression or function of ion channels and ion channel regulatory proteins. In particular, the slowly activating delayed rectifier K+ current (IKs) is downregulated in HF, and evidence suggests [Ca2+]i can regulate IKs. IKs is a key component of the repolarization reserve of the cardiac action potential (AP), most prominently during β-adrenergic stimulation. During the cardiac AP, depolarization causes an increase in [Ca2+]i due to Ca2+ entry via L-type Ca channels (ICa) and subsequent sarcoplasmic reticulum (SR) Ca2+ release. The rise in [Ca2+]i at the membrane (submembrane [Ca2+]i) that is sensed by ion channels during each cardiac cycle is larger than cytosolic [Ca2+]i. Thus, the increase of [Ca2+]i during a single Ca2+ transient may dynamically increase IKs, and therefore limit the AP duration and consequently the amount of Ca2+ entry into a myocyte. Furthermore, Ca2+ sensitivity of IKs could reduce the repolarization reserve during conditions where [Ca2+]i dynamics are compromised, such as HF. Although the Ca2+ dependence of IKs is seemingly important, the modulation of IKs by the dynamically changing [Ca2+]i transient has not been studied as thoroughly as ICa, Na-Ca exchanger, or SR Ca2+ release. Therefore, the overall hypothesis of this proposal is that Ca2+ regulation of IKs is dynamic and a key determinant for the physiological contribution of IKs to cardiac repolarization. To examine this hypothesis, Aim 1 will determine the effects of steady-state and dynamic changes of [Ca2+]I on the amplitude and gating properties of IKs. The regulation of IKs during each cardiac cycle by [Ca2+]i, specifically submembrane [Ca2+]i, will reveal the prominent role of IKs within normal cardiac repolarization. This will be evaluated in rabbit ventricular myocytes using the patch-clamp technique with wide-field epifluorescence in an environment consisting of a range of controlled [Ca2+]i (steady-state) and during an AP waveform that allows for triggered SR Ca2+-release (dynamic). Aim 2 will expand upon Aim 1 by measuring the steady-state and dynamic [Ca2+]i regulation of IKs during β-adrenergic stimulation to determine if this regulation increases the sensitivity of IKs to [Ca2+]i. Lastly, Aim 3 will examine the hypothesis that decreases in IKs in a rabbit model of HF reflect both a decreased sensitivity of IKs to [Ca2+]i and a decreased responsiveness to β-adrenergic stimulation by using a similar approach as Aims 1 and 2.