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

Medições em tempo real de parâmetros de cálcio e contratilidade em 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.

Autor em destaque: Medições em tempo real de parâmetros de cálcio e contratilidade em cardiomiócitos derivados de células-tronco pluripotentes induzidas por humanos  

Medidas em Tempo Real de Parâmetros de Cálcio e Contratilidade em Cardiomiócitos Derivados de Células-Tronco Pluripotentes Induzidos Humanos

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  

Medidas Transitórias de Cálcio em MC-hiPSC

Dissolva as alíquotas de Fura-2 AM na Solução de Tyrode para obter uma concentração final de 1 Fura-2 AM micromolar para adicionar às células. Aspirar o meio e substituí-lo por Solução de Tyrode, contendo Fura-2 AM. Incubar por 15 minutos a 37 graus Celsius em uma incubadora ou no sistema de medição baseado em ótica. Retire a Solução de Tyrode contendo Fura-2 AM e lave os poços duas vezes com a Solução de Tyrode fresca.Finalmente, incube as células na Solução de Tyrode por mais cinco minutos a 37 graus Celsius para permitir a desesterificação do Fura-2 AM. Coloque a placa dentro do sistema de medição baseado em óptica para prosseguir com as medições de contratilidade. Selecione um poço, escolha uma área dentro do poço para observar a contração e o relaxamento sincronizados e, em seguida, clique em Iniciar e adicione medição. Além dos traços de contratilidade, o transiente de cálcio ratiométrico em tempo real aparece na tela.

calcium-transient-measurements-in-hipsc-cms

Research

JoVE Journal

Biology

Calcium Transient Measurements in hiPSC-CMs

308 visualizações.  Vrije University. Dissolva as alíquotas de Fura-2 AM na Solução de Tyrode para obter uma concentração final de 1 Fura-2 AM micromolar para adicionar às células. Aspirar o meio e substituí-lo por Solução de Tyrode, contendo Fura-2 AM. Incubar por 15 minutos a 37 graus Celsius em uma incubadora ou no sistema de medição baseado em ótica. Retire a Solução de Tyrode contendo Fura-2 AM e lave os poços duas vezes com a Solução de Tyrode fresca.Finalmente, incube as células na Solução de Ty...

Vídeo: 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 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 ...

Biologia

Contractility Measurements in hiPSC-CMs

To begin, switch on the optics based measurement system, microscope light emitting diode source, and the computer. Start the program on the computer and open a new file by clicking new under the file in the top left of the screen. Click on collect, choose experiments and then select IPSC and calcium measurements.Next, replace the human IPSC cardiomyocyte culture medium with 0.5 milliliters per well of Tyrode's solution. In a 24 well plate. Replace the plate lid with the climate control lid and place the plate inside the optics based measurement system in the device adjusted to 37 degrees Celsius temperature.Click on open cell finder under the toolbar and wait for a new screen to appear. Select the plate format under chamber type and well at the top of the screen. Select a well and choose an area within the well to observe synchronized contraction and relaxation.Press start on the bottom left of the screen. Then click add measurement to measure the area. Measure approximately 10 areas per well in a 24 well plate.For only baseline measurements, continue the measurement from the next well. For testing the compounds, press complete once the areas within a well are measured. Open the optics based measurement instrument and add the desired stressor or compound to the well.After incubating at the desired time period, click on automatic measurement to perform paired measurements in the same areas of the well where the baseline measurements were performed. Press complete after all the measurements are performed. Choose the next well from the top right of the screen and continue measurements for all the required wells.After measuring all the required wells, click stop and save the file to the desired location.

Medidas de Contratilidade em MC-hiPSC

Dissolve the Fura-2 AM aliquots in Tyrode's Solution to get a final concentration of 1 micromolar Fura-2 AM to add to the cells. Aspirate the medium and replace it with Tyrode's Solution containing Fura-2 AM.Incubate for 15 minutes at 37 degrees Celsius in an incubator or the optics based measurement system. Remove the Tyrode's Solution containing Fura-2 AM and wash the wells two times with fresh Tyrode's Solution.Finally, incubate the cells in Tyrode's Solution for another five minutes at 37 degrees Celsius to allow deesterification of Fura-2 AM.Place the plate inside the optics based measurement system to proceed with the contractility measurements. Select a well, choose an area within the well to observe synchronized contraction and relaxation, and then click Start, and add measurement. In addition to the contractility traces, real-time ratiometric calcium transient appears on the screen.

Contractility and Calcium Transient Measurements Data Analysis

To analyze the data, click CytoSolver on the desktop. Click on import from the file menu and select the files to be analyzed. After the analysis, observe the accepted and rejected transients on the screen.Click export on the top left of the screen and enable average transient data. Export to Excel file and then click export. Inspect the analyzed data presented in a spreadsheet.Turn off the computer and all the other instruments. Contractility measurements were analyzed with the CytoSolver program and the parameters of resting, beating frequency, time to peak contractility and time to baseline contractility of the control human IPSC cardiomyocytes were reported. The application of 500 nano molar ISO, a non-selective beta adrenergic receptor agonist, significantly increased the beating frequency in all wells.ISO also increased the kinetics evidenced by a decrease in time to peak contractility and time to baseline contractility. Like contractility, the calcium measurements were also analyzed, and the parameters were reported.