Exploring calcium signaling in the heart

背景：为了有效地将血液泵送到全身，心脏必须有节奏地跳动。每一次心跳都是由一个穿过心肌的电脉冲触发的，使心房和心室同步收缩。心肌细胞是构成心肌的细胞，其功能是将电激活与肌肉收缩耦合。参与偶联过程的关键离子之一是Ca 2+。细胞内Ca 2+水平直接激活肌丝，导致心肌收缩。Ca 2+必须迅速从细胞质中清除，以使心脏放松并充满血液。Ca 2+进出之间的任何不平衡都可能导致心率、心律和心输出量的变化，从而对细胞稳态和心脏功能产生深远的影响。解读Ca 2+动力学如何调节以及Ca 2+微结构域如何在心肌细胞内发挥作用是我的NSERC研究计划的长期目标。 原理/假设：虽然肌浆网是心肌细胞中主要的Ca 2+储存，但最近的数据表明，酸性细胞器，包括内体和溶酶体，可能充当由阳离子可渗透TRPML通道调节的额外Ca 2+储存。内溶体在支持细胞生物学，包括细胞信号传导和能量代谢方面发挥着非常不同的作用。然而，这些细胞器是否有助于心肌细胞中的Ca 2+动力学尚不清楚。这个建议建立在我的初步工作和测试的假设，TRPML通道调节Ca 2+的动态和功能，以维持心肌细胞中的Ca 2+稳态。特定的短期目标/方法：在目标1中，我们将通过定义发育中脊椎动物胚胎中TRPML基因表达和通过跳动的斑马鱼心脏的高速成像分析Ca 2+动力学来表征酸性Ca 2+储存。每个TRPML通道的功能作用将在目标2中使用遗传功能丧失模型进行测试。目的3将采取双管齐下的方法来剖析的因素，调节内溶酶体钙离子通过分析上游转录调节因子（TFEB），并进行化学筛选，使用一种新的生物传感器，报告钙离子动力学的内溶酶体。 影响：定义酸性Ca 2+商店的功能，确定调节其功能的通道，并探索如何从这些商店动员Ca 2+将描述一种研究心肌中Ca 2+稳态的新范式。解决这一知识差距将使我们能够全面了解心脏的功能。 HQP：在这个提议中，我将在五年内培训15名HQP。HQP将使用遗传工具，分子技术，先进的实时成像，细胞和发育研究，以产生Ca 2+稳态机制的基础知识。我的培训计划将使HQP在这些领域发展技术专长，以及磨练他们在沟通，批判性思维和教学方面的专业知识。

Background: To efficiently pump blood throughout the body, the heart must beat rhythmically. Each heartbeat is triggered by an electrical impulse that travels through the heart muscle, causing the atrial and ventricular chambers to contract in synchrony. Cardiomyocytes, the cells that make up the cardiac muscle, function by coupling electrical activation to muscle contraction. One of the key ions involved in the coupling process is Ca2+. Intracellular Ca2+ levels directly activate the myofilaments, causing the heart muscle to contract. Ca2+ must then be rapidly removed from the cytoplasm to allow the heart to relax and fill with blood. Any imbalance between Ca2+ entry and exit can lead to changes in heart rate, heart rhythm, and cardiac output, thus having profound consequences on cell homeostasis and heart function. Deciphering how Ca2+ dynamics are regulated and how Ca2+ microdomains function within the cardiomyocyte is the long-term goal of my NSERC research program. Rationale/Hypothesis: While the sarcoplasmic reticulum is the major Ca2+ store in cardiomyocytes, recent data indicate that acidic organelles, including the endosomes and lysosomes, may act as additional Ca2+ stores that are regulated by cation permeable TRPML channels. Endolysosomes play remarkably diverse roles supporting cell biology, including cell signaling and energy metabolism. However, whether these organelles contribute to Ca2+ dynamics in cardiomyocytes is unknown. This proposal builds upon my preliminary work and tests the hypothesis that TRPML channels regulate Ca2+ dynamics and function to maintain Ca2+ homeostasis in cardiomyocytes. Specific Short-Term Aims/Methods: In Aim 1, we will characterize acidic Ca2+ stores by defining TRPML gene expression in developing vertebrate embryos and analyzing Ca2+ dynamics by high-speed imaging of beating zebrafish hearts. Functional roles for each TRPML channel will be tested in Aim 2 using genetic loss-of-function models. Aim 3 will take a two-pronged approach to dissect the factors that regulate endolysosomal Ca2+ by analyzing an upstream transcriptional regulator (TFEB) and by performing a chemical screen using a novel biosensor that reports Ca2+ dynamics from the endolysosomes.   Impact:Defining the function of acidic Ca2+ stores, identifying the channels that regulate their function, and exploring how Ca2+ can be mobilized from these stores will describe a novel paradigm for studying Ca2+ homeostasis in cardiac muscle. Addressing this gap in knowledge will allow us to comprehensively understand how the heart functions.  HQP: Within this proposal, I will train 15 HQP over five years. HQP will use genetic tools, molecular techniques, advanced live imaging, and cell and developmental studies to generate fundamental knowledge on mechanisms of Ca2+ homeostasis. My training program will allow HQP to develop technical expertise in these areas, as well as hone their expertise in communication, critical thinking, and teaching.