Name: Calcium Transient Measurements in hiPSC-CMs
Uploaded: 2023-05-26T14:50:00
Duration: 1 min 29 s
Description: Watch this Scientific Journal Video about Calcium Transient Measurements in hiPSC-CMs at JoVE.com

The video outlines a protocol for preparing and measuring cardiac myocyte contractility using Fura-2 AM. It details the dissolution of Fura-2 AM in Tyrode's Solution, cell incubation, washing, and final measurements using an optics-based system, highlighting real-time calcium transients alongside contractility traces.

calcium transient measurements in hipsc-cms

מדידות בזמן אמת של פרמטרים של סידן והתכווצות ב-hiPSC-CMs

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.

מחבר זרקור: מדידות בזמן אמת של פרמטרים של סידן והתכווצות בקרדיומיוציטים שמקורם בתאי גזע פלוריפוטנטיים המושרים על ידי בני אדם  

מדידות בזמן אמת של פרמטרים של סידן והתכווצות בקרדיומיוציטים פלוריפוטנטיים המושרים על ידי תאי גזע המושרים על ידי בני אדם

Human induced pluripotent stem cell-derived cardiomyocytes represent a powerful tool for studying mutation-mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. There's a need for a platform in which contractility and calcium handling of human induced pluripotent stem cell-derived cardiomyocytes can be measured in a user-friendly and reproducible manner. This method enables researchers to study contractility of human-induced pluripotent stem cell-derived cardiomyocytes by pixel correlation and measuring intracellular calcium transients simultaneously by loading the cells with calcium-sensitive fluorophore.By using this platform, it's possible to perform paired measurements in a well-preserved temperature environment and different plate formats, and to access the data instantly. This method enables the study of cellular pathomechanisms and evaluates the effect of compounds and the functionality of human iPSC-derived cardiomyocytes, that can be helpful in the preclinical drug screening process.

Watch this Scientific Journal Video about Calcium Transient Measurements in hiPSC-CMs at JoVE.com

Calcium Transient Measurements in hiPSC-CMs  

מדידות סידן חולפות ב-hiPSC-CMs

להמיס את aliquots Fura-2 AM בתמיסת Tyrode כדי לקבל ריכוז סופי של 1 מיקרומולרי Fura-2 AM להוסיף לתאים. שאפו את המדיום והחליפו אותו בתמיסה של טירודה המכילה Fura-2 AM. דגירה במשך 15 דקות בטמפרטורה של 37 מעלות צלזיוס באינקובטור או במערכת מדידה מבוססת אופטיקה. הסר את הפתרון של Tyrode המכיל Fura-2 AM ושטוף את הבארות פעמיים עם פתרון Tyrode טרי.לבסוף, לדגור על התאים בתמיסת Tyrode למשך חמש דקות נוספות בטמפרטורה של 37 מעלות צלזיוס כדי לאפשר דה-אסטריפיקציה של Fura-2 AM. מקם את הצלחת בתוך מערכת מדידה מבוססת אופטיקה כדי להמשיך במדידות ההתכווצות. בחר באר, בחר אזור בתוך הבאר כדי לצפות בהתכווצות והרפייה מסונכרנות, ולאחר מכן לחץ על התחל, והוסף מדידה. בנוסף לעקבות ההתכווצות, סידן ארעי בזמן אמת מופיע על המסך.

Research

JoVE Journal

Biology

Calcium Transient Measurements in hiPSC-CMs

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 ...