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

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.

Alkaline nucleoplasm facilitates contractile gene expression in the mammalian heart

“…Myocyte isolation and culture was performed as described previously [ 42 ]. Animals were killed by cervical dislocation according to Schedule 1 of the Animals (Scientific Procedures) Acts 1986.…”

New Insights and Current Approaches in Cardiac Hypertrophy Cell Culture, Tissue Engineering Models, and Novel Pathways Involving Non-Coding RNA

“…Induction of hypertrophy by all of these chemical agents show a similar modeling time of 24 h, which may be extended to 48 h depending on the used cell type ( Zhang et al, 2016 ; Zlabinger et al, 2019 ; Zhao et al, 2020 ). The concentration needs to be optimized by testing different dilutions before the experiment, however the general used concentrations are 20 µM phenylephrine ( Gelinas et al, 2018 ), 100 nM angiotensin II ( Zlabinger et al, 2019 ), 10 μM isoproterenol ( Zhang et al, 2016 ), and 10 nM endothelin-1 ( Loonat et al, 2019 ).…”

“…This development combined with the relative paucity of direct treatment options for cardiac hypertrophy makes continued research and the identification of novel therapeutic target molecules absolutely vital. Cardiac hypertrophy is an adaptive process which develops in response to physiological but also pathological processes, leading to heart muscle and cell hypertrophic growth with increased rigidity of the heart structures, and impaired diastolic function leading to heart failure with preserved ejection fraction (HFpEF) ( Loonat et al, 2019 ; Zhao et al, 2020 ). Hypertrophic growth involves cardiomyocyte enlargement rather than division, as adult cardiomyocytes have lost the ability to divide ( Porrello et al, 2013 ).…”

The specific inhibition of the cardiac electrogenic sodium/bicarbonate cotransporter leads to cardiac hypertrophy

“…Our and other laboratories have demonstrated a differential activity and/or expression of cardiac NBC isoform in different in vivo hypertrophy models [16][17][18][19] . Moreover, we showed that the activity of NBCe1 is decreased while NBCn1 is over-expressed and over-activated in the hypertrophied myocytes of spontaneous hypertensive rats (SHR) 17 .…”