Myocardial Function Section, Fourth Floor, Imperial Centre for Translational and Experimental Medicine, National Heart and Lung Institute, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.
Department of Electrophysiology, Imperial College Healthcare NHS Trust, Hammersmith Hospital, London W12 0HS, UK.
Cardiovasc Res. 2017 Dec 1;113(14):1743-1752. doi: 10.1093/cvr/cvx151.
It is generally accepted that post-MI heart failure (HF) changes a variety of aspects of sarcoplasmic reticular Ca2+ fluxes but for some aspects there is disagreement over whether there is an increase or decrease. The commonest statistical approach is to treat data collected from each cell as independent, even though they are really clustered with multiple likely similar cells from each heart. In this study, we test whether this statistical assumption of independence can lead the investigator to draw conclusions that would be considered erroneous if the analysis handled clustering with specific statistical techniques (hierarchical tests).
Ca2+ transients were recorded in cells loaded with Fura-2AM and sparks were recorded in cells loaded with Fluo-4AM. Data were analysed twice, once with the common statistical approach (assumption of independence) and once with hierarchical statistical methodologies designed to allow for any clustering. The statistical tests found that there was significant hierarchical clustering. This caused the common statistical approach to underestimate the standard error and report artificially small P values. For example, this would have led to the erroneous conclusion that time to 50% peak transient amplitude was significantly prolonged in HF. Spark analysis showed clustering, both within each cell and also within each rat, for morphological variables. This means that a three-level hierarchical model is sometimes required for such measures. Standard statistical methodologies, if used instead, erroneously suggest that spark amplitude is significantly greater in HF and spark duration is reduced in HF.
Ca2+ fluxes in isolated cardiomyocytes show so much clustering that the common statistical approach that assumes independence of each data point will frequently give the false appearance of statistically significant changes. Hierarchical statistical methodologies need a little more effort, but are necessary for reliable conclusions. We present cost-free simple tools for performing these analyses.
普遍认为，心肌梗死后心力衰竭（HF）改变了肌浆网 Ca2+流的多个方面，但对于某些方面，关于是否存在增加或减少仍存在分歧。最常见的统计方法是将从每个细胞收集的数据视为独立的，即使它们实际上是从每个心脏中多个可能相似的细胞聚类而来的。在这项研究中，我们检验了这种独立性的统计假设是否会导致研究人员得出结论，如果分析采用特定的统计技术（分层检验）处理聚类，则这些结论将被认为是错误的。
用 Fura-2AM 负载细胞记录 Ca2+瞬变，用 Fluo-4AM 负载细胞记录火花。数据进行了两次分析，一次使用常见的统计方法（独立性假设），一次使用分层统计方法，以允许任何聚类。统计检验发现存在显著的层次聚类。这导致常见的统计方法低估了标准误差并报告了人为的小 P 值。例如，这将导致错误的结论，即 HF 中 50%峰值瞬变幅度的时间显著延长。火花分析显示，形态学变量在每个细胞内和每个大鼠内都存在聚类。这意味着对于这些测量，有时需要使用三水平分层模型。标准统计方法，如果使用不当，会错误地表明 HF 中的火花幅度显著增加，HF 中的火花持续时间缩短。
分离的心肌细胞中的 Ca2+流显示出如此多的聚类，以至于假设每个数据点独立的常见统计方法通常会错误地显示出统计学上显著的变化。分层统计方法需要多做一些努力，但对于得出可靠的结论是必要的。我们提供了免费的简单工具，用于执行这些分析。
