Modulation of human embryonic stem cell-derived cardiomyocyte growth: A testbed for studying human cardiac hypertrophy?

Abstract

Human embryonic stem cell-derived cardiomyocytes (hESC-CM) are being developed for tissue repair and as a model system for cardiac physiology and pathophysiology. However, the signaling requirements of their growth have not yet been fully characterized. We showed that hESC-CM retain their capacity for increase in size in long-term culture. Exposing hESC-CM to hypertrophic stimuli such as equiaxial cyclic stretch, angiotensin II, and phenylephrine (PE) increased cell size and volume, percentage of hESC-CM with organized sarcomeres, levels of ANF, and cytoskeletal assembly. PE effects on cell size were separable from those on cell cycle. Changes in cell size by PE were completely inhibited by p38–MAPK, calcineurin/FKBP, and mTOR blockers. p38–MAPK and calcineurin were also implicated in basal cell growth. Inhibitors of ERK, JNK, and CaMK II partially reduced PE effects; PKG or GSK3β inhibitors had no effect. The role of p38–MAPK was confirmed by an additional pharmacological inhibitor and adenoviral infection of hESC-CM with a dominant-inhibitory form of p38–MAPK. Infection of hESC-CM with constitutively active upstream MAP2K3b resulted in an increased cell size, sarcomere and cytoskeletal assembly, elongation of the cells, and induction of ANF mRNA levels. siRNA knockdown of p38–MAPK inhibited PE-induced effects on cell size. These results reveal an important role for active protein kinase signaling in hESC-CM growth and hypertrophy, with potential implications for hESC-CM as a novel in vitro test system. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

Nitric Oxide-cGMP-PKG Pathway Acts on Orai1 to Inhibit the Hypertrophy of Human Embryonic Stem Cell-Derived Cardiomyocytes

“…An a-adrenoceptor agonist PE (100 lM) was used to induce cardiomyocyte hypertrophy [3]. The hypertrophy was assessed by four cardiomyocyte hypertrophic indexes, including cell size enlargement [31], sarcomere organization [27], and expression of cardiac hypertrophic markers ANF and ACTA1 [27,32].…”

“…Recently, human embryonic stem cells (hESCs) and humaninduced pluripotent stem cells (hiPSCs) are used to generate cardiomyocytes. These hESC-or hiPSC-derived cardiomyocytes are suggested to have many of the properties of authentic cardiomyocytes [2,3]. hESC-and hiPSC-derived cardiomyocytes are relatively immature.…”

The case for induced pluripotent stem cell‐derived cardiomyocytes in pharmacological screening

“…Use of hiPSC-CMs for assessing apoptotic, proliferative and hypertrophic activity is much less explored, but assays are being developed using both live and fixed cells (Földes et al, 2011). Proliferative activity, as detected by increase in cell number, Ki67 + , phosphorylation of histone H3 and relative distribution of cells in G2 M -1 and G1/G0 phases of cell cycle, can be set up for automated analysis.…”

“…Proliferative activity, as detected by increase in cell number, Ki67 + , phosphorylation of histone H3 and relative distribution of cells in G2 M -1 and G1/G0 phases of cell cycle, can be set up for automated analysis. Assays for hypertrophic changes such as increases in cell size, protein content, sarcomere assembly and ANP/BNP have also been automated and may be useful markers of long-term cardiac toxicity (Földes et al, 2011).…”

Physiological aspects of cardiac tissue engineering

“…This immaturity is reflected by low maximal diastolic potentials because of insufficient inwardly rectifying K ϩ current and slow upstroke velocities (79,(103)(104). Evidence suggests that prolonged culturing (27,54), addition of nonmyocytes (45), and the 3-D format in EHT promote some aspects of maturation. Zhu et al (104) demonstrated that the differentiation in the presence of neuregulin increased the percentage of mature cardiomyocytes with slower firing rate, increased upstroke velocities, and more negative maximum diastolic potential.…”