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Published on: September 27, 2018
Recording and Interpretation of Active Calcium Transients in Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Maedeh Mozneb1,2,3,4, Jemima Moses1,2,3,4,5, Madelyn Arzt1,2,3,4
1Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California.
Insights
This study introduces a standardized method using genetically encoded calcium indicators (GCaMP) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to accurately measure calcium transients. The protocols ensure reliable data processing and analysis for cardiomyocyte electrophysiology research.
Area of Science:
- Cardiovascular Sciences
- Cellular Electrophysiology
- Biomedical Imaging
Background:
- Calcium signaling is crucial for cardiomyocyte excitation-contraction coupling and rhythmic contraction.
- Existing methods for tracing intracellular calcium lack standardization, leading to signal processing biases and interpretation challenges.
- Accurate interpretation of calcium transient signals is vital for understanding cardiomyocyte electrophysiology.
Purpose of the Study:
- To establish a standardized methodology for recording and analyzing calcium transients in cardiomyocytes.
- To utilize genetically encoded calcium indicators (GCaMP) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for precise calcium signal detection.
- To provide protocols for data extraction, processing, and visualization, minimizing signal processing biases.
Main Methods:
- Employing genetically encoded calcium indicator (GCaMP) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for fluorescence-based calcium recording.
- Utilizing ImageJ and MATLAB software for the extraction and processing of calcium transient waveforms.
- Developing protocols for hiPSC maintenance, expansion, differentiation, video recording, and signal analysis.
- Implementing a support protocol for replating hiPSC-CMs onto diverse culture platforms for data multiplexing.
Main Results:
- Demonstrated a comprehensive approach for maintaining, expanding, and differentiating GCaMP hiPSCs.
- Outlined video recording techniques for capturing calcium transients in GCaMP hiPSC-CMs.
- Provided detailed methods for signal extraction, preprocessing, analysis, and visualization of calcium transient data.
- Facilitated the categorization of waveform features based on physiological relevance to cardiomyocyte function.
Conclusions:
- The presented protocols offer a standardized and reliable method for studying calcium dynamics in cardiomyocytes.
- This approach enables accurate interpretation of calcium transient signals, crucial for advancing cardiovascular sciences.
- The methodology supports data multiplexing by allowing cardiomyocyte culture on various platforms, enhancing research capabilities.
Abstract:
Calcium plays a pivotal role in the excitation-contraction coupling process in cardiomyocytes, a critical multi-parametric event leading to rhythmic contraction. Over the past few decades, calcium signaling in cardiomyocytes has been extensively studied in cardiovascular sciences. However, a standard methodology is needed not only to trace the calcium within cells but also to remove signal processing biases and to accurately interpret the features of calcium transient signals in relation to cardiomyocyte electrophysiology. This article outlines the use of genetically encoded calcium indicator (GCaMP) human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to record calcium transients. These cells express a green fluorescent signal when calcium binds to intracellular calmodulin, a key regulator of calcium signaling. The extraction and processing of calcium transient waveforms are performed using ImageJ and MATLAB software. Key features of these waveforms are then identified and categorized based on their physiological relevance to cardiomyocyte function. Additionally, this work includes a Support Protocol for the successful replating of cardiomyocytes onto non-traditional culture platforms, such as metallic sensors and polymer-based substrates, to facilitate data multiplexing. The three Basic Protocols outlined here provide a comprehensive approach for maintaining, expanding, and differentiating the GCaMP hiPSCs, video recording of calcium transients, and the subsequent signal extraction, preprocessing, analysis, and data visualization. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance, expansion, and differentiation of genetically encoded calcium indicator hiPSCs Support Protocol: Replating GCaMP hiPSC-CMs for stimulation and multielectrode array studies Basic Protocol 2: Video recording from calcium transients of GCaMP hiPSC-CMs Basic Protocol 3: Signal extraction, preprocessing, analysis, and data visualization.
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