A high-throughput ratiometric method for imaging hypertrophic growth in cultured primary cardiac myocytes

Aminah A Loonat,M Kate Curtis,Mark A Richards,Graciela Nunez-Alonso,Johanna Michl,Pawel Swietach

doi:10.1016/j.yjmcc.2019.04.001

Aminah A Loonat, M Kate Curtis + Show 4 more

Open Access

https://doi.org/10.1016/j.yjmcc.2019.04.001

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Abstract

Maladaptive hypertrophy of cardiac myocytes increases the risk of heart failure. The underlying signaling can be triggered and interrogated in cultured neonatal ventricular myocytes (NRVMs) using sophisticated pharmacological and genetic techniques. However, the methods for quantifying cell growth are, by comparison, inadequate. The lack of quantitative, calibratable and computationally-inexpensive high-throughput technology has limited the scope for using cultured myocytes in large-scale analyses. We present a ratiometric method for quantifying the hypertrophic growth of cultured myocytes, compatible with high-throughput imaging platforms. Protein biomass was assayed from sulforhodamine B (SRB) fluorescence, and image analysis calculated the quotient of signal from extra-nuclear and nuclear regions. The former readout relates to hypertrophic growth, whereas the latter is a reference for correcting protein-independent (e.g. equipment-related) variables. This ratiometric measure, when normalized to the number of cells, provides a robust quantification of cellular hypertrophy. The method was tested by comparing the efficacy of various chemical agonists to evoke hypertrophy, and verified using independent assays (myocyte area, transcripts of markers). The method's high resolving power and wide dynamic range were confirmed by the ability to generate concentration-response curves, track the time-course of hypertrophic responses with fine temporal resolution, describe drug/agonist interactions, and screen for novel anti-hypertrophic agents. The method can be implemented as an end-point in protocols investigating hypertrophy, and is compatible with automated plate-reader platforms for generating high-throughput data, thereby reducing investigator-bias. Finally, the computationally-minimal workflow required for obtaining measurements makes the method simple to implement in most laboratories.

Highlights

Cardiac myocytes can undergo hypertrophic growth in response to increased work-load
In comparison with isolated adult ventricular myocytes, neonatal rat ventricular myocytes (NRVMs) can survive in culture for extended periods of time, which is necessary for tracking hypertrophy
To visualize nuclear areas, fixed (4% paraformaldehyde, 10 min) and permeabilized (0.5% Triton X-100, 10 min) myocytes were stained with the nuclear dye Hoechst-33342 dissolved in PBS (10 μg/mL, 10 min) before staining with sulforhodamine B (SRB) dissolved in 1% acetic acid

Summary

Introduction

Cardiac myocytes can undergo hypertrophic growth in response to increased work-load. This can be physiological in response to regular exercise or pregnancy, or pathophysiological in conditions of cardiovascular disease such as ischemia, hypertension or cardiomyopathy [1]. Since hypertrophy is associated with an altered program of gene expression [4], controlled by transcription factors that respond to neuro-hormonal stimuli [4,5,6], a routine method for investigating pro-hypertrophic cascades is to apply such chemical triggers to myocytes in vivo using delivery devices [7,8,9], or in vitro by tissue culture methods [10,11]. In comparison with isolated adult ventricular myocytes, NRVMs can survive in culture for extended periods of time, which is necessary for tracking hypertrophy

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