Hypertrophy of human embryonic stem cell–derived cardiomyocytes supported by positive feedback between Ca2+ and diacylglycerol signals

Abstract

Human embryonic stem cell–derived cardiomyocytes develop pronounced hypertrophy in response to angiotensin-2, endothelin-1, and a selected mix of three fatty acids. All three of these responses are accompanied by increases in both basal cytoplasmic Ca 2+ and diacylglycerol, quantified with the Ca 2+ sensor Fluo-4 and a FRET-based diacylglycerol sensor expressed in these cardiomyocytes. The heart glycoside, ouabain (30 nM), and a recently developed inhibitor of diacylglycerol lipases, DO34 (1 μM), cause similar hypertrophy responses, and both responses are accompanied by equivalent increases of basal Ca 2+ and diacylglycerol. These results together suggest that basal Ca 2+ and diacylglycerol form a positive feedback signaling loop that promotes execution of cardiac growth programs in these human myocytes. Given that basal Ca 2+ in myocytes depends strongly on the Na + gradient, we also tested whether nanomolar ouabain concentrations might stimulate Na + /K + pumps, as described by others, and thereby prevent hypertrophy. However, stimulatory effects of nanomolar ouabain (1.5 nM) were not verified on Na + /K + pump currents in stem cell–derived myocytes, nor did nanomolar ouabain block hypertrophy induced by endothelin-1. Thus, low-dose ouabain is not a “protective” intervention under the conditions of these experiments in this human myocyte model. To summarize, the major aim of this study has been to characterize the progression of hypertrophy in human embryonic stem cell–derived cardiac myocytes in dependence on diacylglycerol and Na + gradient changes, developing a case that positive feedback coupling between these mechanisms plays an important role in the initiation of hypertrophy programs.

Cardiac hypertrophy in a dish: A human stem cell based model

“…The NPPA gene codes for the ANP protein, which is a natriuretic peptide hormone that regulates cardiac homeostasis through regulation of natriuresis, diuresis and vasodilation. Notably, the NPPA gene is upregulated in ET-1 based in vitro models of cardiac hypertrophy (Carlson et al, 2013;Tanaka et al, 2014;Aggarwal et al, 2014;Deisl et al, 2019). The other four genes are not typical cardiac hypertrophy genes.…”

“…All for trying to maintain the cardiovascular homeostasis. Commonly used substances for cardiac hypertrophy induction are ET-1 and phenylephrine ( Foldes et al, 2011 ; Tanaka et al, 2014 ; Aggarwal et al, 2014 ; Deisl et al, 2019 ; Carlson et al, 2013 ). Both of these are vasoconstrictors; ET-1 being the most potent one ( Davenport et al, 2016 ).…”

“…Currently, the reports on neurohormonal in vitro models for cardiac hypertrophy show promising results with increased cell size and a hypertrophic gene expression pattern when using ET-1, phenylephrine or angiotensin II ( Aggarwal et al, 2014 ; Foldes et al, 2011 ; Ovchinnikova et al, 2018 ; Deisl et al, 2019 ; Carlson et al, 2013 ). However, the response varies depending on substance used.…”

“…For example, angiotensin II does not seem to result in the same cell size increase as caused by phenylephrine and ET-1 ( Foldes et al, 2011 ). Neurohormonal hypertrophy induction has been accomplished in hPSC-CMs derived from many different cell lines and the effect appears to be cell line independent ( Carlson et al, 2013 ; Aggarwal et al, 2014 ; Foldes et al, 2011 ; Deisl et al, 2019 ). Comparing in vitro changes of gene expression induced by ET-1 to the expression changes in left ventricular hypertrophy in vivo provides strong evidence that this type of model is reflecting the in vivo condition in humans ( Aggarwal et al, 2014 ).…”

A new stone for a new path, from “physiology to the bedside”

“…In this issue, Deisl et al from Donald W. Hilgemann's laboratory iterated the important hypertrophic signaling using human embryonic stem cell-derived and spontaneously beating cardiomyocytes (hESC) [1]. Cultured for 2-4 months after the differentiation, they have presented clear evidence that G protein-coupled, potent hypertrophic stimulators (endothelin-1 and angiotensin II) or fatty acid mixture (palmitic, myristic and palmitoleic acids) induced hypertrophy (assessed by greater α-actinin and β-myosin heavy chain protein expressions) mediated by intracellular DAG and Ca-dependent signaling.…”