The video demonstrates how to use CytoSolver software for analyzing contractility and calcium measurements in human iPSC cardiomyocytes. It explains importing data, exporting results to Excel, and the effects of the beta-adrenergic agonist ISO on beating frequency and contractility kinetics. It concludes with instructions for shutting down equipment.

contractility and calcium transient measurements data analysis

hiPSC-CMにおけるカルシウムおよび収縮性パラメータのリアルタイム測定

Heart failure is the leading cause of death worldwide. In 2019, cardiovascular disease caused 18.6 million deaths globally, which reflects a 17.1% increase over the past decade1. Despite the efforts of researchers to identify drug targets to prevent and cure heart failure, patient outcomes are still poor2. The difference in the pathophysiology of animal models with respect to humans could be one of the underlying factors in the limited success for the optimization of heart failure therapy3. Additional human-like models are warranted to model disease and test the toxicity and effectiveness of novel drug compounds.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool to define cellular pathomechanisms induced by stressors, ischemia, altered metabolism, or pathogenic gene variants, and the effectiveness and toxicity of drug interventions4. To define effects on the functional properties of hiPSC-CMs, a high-speed system that measures the systolic and diastolic properties of cellular contractions in an unbiased and reproducible manner is warranted. This overall goal of the current study is to demonstrate that an optics-based system is a powerful tool to perform real-time analyses of the functional parameters of hiPSC-CMs.
Currently, there are multiple platforms to evaluate the contractility of hiPSC-CMs. However, current systems either offer a slow readout, or the required number of cells might be a challenge. Label-free video measurement systems5,6 rely on post-hoc analysis of videos, which requires a lot of storage and lacks direct feedback during video acquisition. In addition, sufficient temporal and spatial resolution is hard to achieve, and may result in undersampling. Other methods to determine cardiomyocyte properties, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-edited hiPSC-CMs7 with fluorescent reporters might interfere with the gene stability of the cells and require specialized laboratory expertise.
To overcome the limitations mentioned above, a unique optics-based measurement system was developed and introduced in this study. This platform enables contractility measurements on unmodified hiPSC-CMs by simply replating them on any required plate format, without any limitation in plate size. Moreover, real-time measurements allow direct observation and analyses of the functional parameters of hiPSC-CMs, and thereby provide an experimental setting to instantly adjust and optimize protocols. Moreover, the location of each measurement is stored, which enables paired measurements on the same sample, thereby increasing the power of the experiments.
To demonstrate the optics-based system, contractility measurements were performed on a control hiPSC-CM line. This control hiPSC line was generated from the dermal fibroblast of a healthy male donor with a normal karyotype8. Contraction kinetics were measured at 37 &#176;C, based on pixel correlation changes relative to a reference frame taken during relaxation at a 250 Hz sampling frequency. As the exact start of contraction cannot always be unambiguously determined, the time point at which 20% of the peak height was reached (time to peak 20%) was taken as the starting point for measurements of time to peak. By doing so, less variability was found for this parameter within one sample. Similarly, as the exact moment at which the signal returns to baseline is difficult to assess, the time it took to reach 80% of the return to baseline from peak (time to baseline 80% ) was used to describe the relaxation time.
Overall contractility measurements included resting beating frequencies, the time from 20% peak to peak contraction (TP.Con), and the time from peak contraction to 80% of baseline (TB.Con80) (Figure 1B). To test the effect of a stressor, cells were incubated with isoprenaline (ISO). In addition, coincidentally with contractility measurements, Ca2+ transients were measured by loading the cells with Fura-2-acetoxymethyl ester (Fura-2 AM) at a sampling frequency of 250 Hz. Ratiometric Ca2+ measurements9 using a hyperswitch were done on a 50 &#181;m diameter illumination area, corresponding to the area of the contractility measurements. Ca2+ measurement data are depicted as time from 20% peak to peak in the Ca2+ transient (TP.Ca) and the time from peak to 80% of the baseline (TB.Ca80) (Figure 1C). A fast, automatic data analysis tool was used to yield average contractile and calcium kinetic parameters for each area.

著者スポットライト:ヒト誘導多能性幹細胞由来心筋細胞におけるカルシウムおよび収縮性パラメータのリアルタイム測定  

ヒト人工多能性幹細胞由来心筋細胞におけるカルシウムおよび収縮性パラメータのリアルタイム測定

Watch this Scientific Journal Video about Contractility and Calcium Transient Measurements Data Analysis at JoVE.com

Contractility and Calcium Transient Measurements Data Analysis  

収縮性およびカルシウム過渡測定データ解析

データを解析するには、デスクトップの「サイトソルバー」をクリックします。ファイルメニューからインポートをクリックし、分析するファイルを選択します。解析後、許容されるトランジェントと拒否されるトランジェントを画面上で観察します。画面の左上にある [エクスポート] をクリックし、平均過渡データを有効にします。Excel ファイルにエクスポートし、[エクスポート] をクリックします。スプレッドシートに表示される分析済みデータを検査します。コンピュータと他のすべての機器の電源を切ります。収縮性の測定をCytoSolverプログラムで分析し、対照ヒトIPSC心筋細胞の静止、拍動頻度、ピーク収縮性までの時間、およびベースライン収縮性までの時間のパラメーターを報告しました。非選択的ベータアドレナリン受容体アゴニストである500ナノモルISOの適用は、すべてのウェルにおいて拍動頻度を有意に増加させた。ISOはまた、収縮性のピークまでの時間およびベースライン収縮性までの時間の減少によって証明される動態を増加させた。収縮性と同様に、カルシウム測定値も分析され、パラメータが報告されました。

contractility-and-calcium-transient-measurements-data-analysis

Research

JoVE Journal

Biology

Contractility and Calcium Transient Measurements Data Analysis

351 ビュー. データを解析するには、デスクトップの「サイトソルバー」をクリックします。ファイルメニューからインポートをクリックし、分析するファイルを選択します。解析後、許容されるトランジェントと拒否されるトランジェントを画面上で観察します。画面の左上にある [エクスポート] をクリックし、平均過渡データを有効にします。Excel ファイルにエクスポート...

ビデオ: Contractility and Calcium Transient Measurements Data Analysis  

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. In this study, it is demonstrated that this optics-based system is a powerful tool to assess the functional parameters of hiPSC-CMs in 2D. By using this platform, it is possible to perform paired measurements in a well-preserved temperature environment on different plate layouts. Moreover, this system provides researchers with instant data analysis.
This paper describes a method for measuring the contractility of unmodified hiPSC-CMs. Contraction kinetics are measured at 37 &#176;C based on pixel correlation changes relative to a reference frame taken at relaxation at a 250 Hz sampling frequency. Additionally, simultaneous measurements of intracellular calcium transients can be acquired by loading the cell with a calcium-sensitive fluorophore, such as Fura-2. Using a hyperswitch, ratiometric calcium measurements can be performed on a 50 &#181;m diameter illumination spot, corresponding to the area of the contractility measurements.

Here, an established method is described to perform contractility and calcium measurements in human induced pluripotent stem cell-derived cardiomyocytes using an optics-based platform. This platform enables researchers to study the effect of mutations and the response to various stimuli in a rapid and reproducible manner.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte ...