Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Summary

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Abstract

Maintenance of steady-state calcium (Ca²⁺) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca²⁺ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca²⁺ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca²⁺-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca²⁺ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca²⁺ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca²⁺. This tool is useful for investigation into the nuanced changes in SR Ca²⁺ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca²⁺ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca²⁺ indicator, D1SR, is used to detect Ca²⁺ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca²⁺ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca²⁺ movement.

Introduction

Ca²⁺ is a very important ion found in abundance within every single cell in the body. It has significant roles in many different cellular functions, including growth, proliferation, migration, and apoptosis^1-4. It is well established that Ca²⁺ can affect these processes through both direct and indirect actions, and therefore, changes to the normal intracellular concentrations of this ion can easily result in negative outcomes for affected cells. The SR is considered as the largest intracellular Ca²⁺ store within the cell⁵. Steady state levels of Ca²⁺ in both the SR and cytoplasm are normally maintained ....

Protocol

Ethics statement: All experiments and procedures were conducted in agreement with the Laboratory Biosafety guidelines of the University of British Columbia, Vancouver, Canada.

1. Preparation of 35 mm Glass-bottom Culture Dishes for FRET-based Confocal Microscopy

1. Prepare plates using the following procedure 36-48 hr prior to experiments.
2. Retrieve chosen gelatinous protein mixture and 0.25% trypsin-EDTA from -20 °C storage and keep them in mobile ice bath until use.
   NOTE: The coating protein mixture should only be exposed to RT for a brief period of time to prevent the mixture from solidifying before use. Trypsin solution should ....

Results

This section provides examples of the results that can be obtained using the described method to capture SR luminal Ca²⁺ concentration data, compared to the commonly employed manner of indirectly measuring changes to organelle Ca²⁺ store concentrations by observing fluctuations in cytoplasmic Ca²⁺.

Figure 1A shows a snapshot of a region of interest (ROI) of the SMC culture transf.......

Discussion

This protocol describes the transfection of VSMCs with the D1SR adenovirus to study Ca²⁺ concentrations within the lumen of the SR. This method allows for the direct measurement of Ca²⁺ within this organelle as well as its movement into/out of the SR as a result of the application of different calcium-moving agents. This method has many advantages for investigators working towards a comprehensive understanding of how changes to SR and cytoplasmic Ca²⁺ levels affect overall cellular health.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle's Medium (high glucose, pyruvate)	Life technologies	11995-065	Warm in 37 °C water bath before use
Matrigel Basement Membrane Matrix Growth Factor Reduced, 5 ml	Corning	356230	Keep at -20 °C until right before use
Newborn Calf Serum	Sigma-Aldrich		Keep at -20 °C until right before use
35 mm glass-bottomed plates	MatTek Corporation	P35G-1.5-14-C
250 ml 0.2 μM vented blue plug seal cap flask	BD Falcon	353136
Trypsin/EDTA Solution	Life technologies	25200056	Keep at -20 °C until right before use
50 ml Polypropylene Conical Tube	BD Falcon	352070
D1SR	N/A	N/A	Gift
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8779
Pluronic F-127	Sigma-Aldrich	P2443
Fluo 4-AM	Life technologies	F-23917	Keep at -20 °C until right before use
1.5 ml Black Microcentrifuge Tubes	Argos Technologies	T7100BK
Leica TCS SP5 confocal microscope	Leica Microsystems
Leica Application Suite 2.6.3 (with FRET SE Wizard application)	Leica Microsystems
GraphPad Prism 5.0	GraphPad Software, Inc.