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".

Fig. 1

(A) Representative immunofluorescence images showing differentiated hESC-CM-enriched culture and single hESC-CM stained positive for cardiac-specific atrial natriuretic factor (ANF), troponin I, sarcomeric myosin MF20, and myosin heavy chain α/β (MHCα/β) as well as brightfield (BF) image at 30 days after differentiation. Nuclei are stained with DAPI (blue). Scale bar represents 50 μm. Bar graphs showing cell size (B), cell shape (length/width ratio) (C), and beating rate (D), and hESC-CM with organized sarcomere structure as a percentage of all MHCα/β-positive cells (E) in early (15 to 40 days after differentiation, n = 6 preparations; light grey), intermediate (41 to 60 days, n = 6; grey), and late cultures (> 60 days, n = 6; dark grey). Results are shown as mean ± SEM (one-way ANOVA **P ≤ 0.01, ***P < 0.001 vs. early culture groups).

Fig. 2

Phenylephrine-induced hypertrophy of hESC-CM. The hESC-CM underwent phenylephrine treatment (10 μM, 48 h) 30 days after differentiation. (A) Representative scanning ion conductance microscopy images of control (left image) and PE-treated (right image) hESC-CM. (B) Representative immunofluorescence image showing hESC-CM stained with Rhodamine–phalloidin (red), MHC (green), and DAPI. Scale bar represents 50 μm. (C) Bar graphs showing fold change in cell area, estimated whole cell volume, hESC-CM with organized sarcomeres, ANF mRNA and intensity levels, F-actin distribution, and total protein/DNA content ratio in hESC-CM treated with PE: results are shown as fold changes vs. control group. The percent of MHC-positive cells with organized sarcomeres was 19.5 ± 4.3 in control vs. 65.2 ± 5.6 in phenylephrine-treated group, from n = 6 preparations. (n > 100 MHC-positive cells analyzed per well, mean ± SEM of triplicate wells, repeated in n = 3 preparations. *P ≤ 0.05, **P < 0.01, ***P < 0.001 vs. control group).

Fig. 3

Cyclic stretch and angiotensin II-induced increase in cell size, sarcomere alignment, and hypertrophic gene activation. The hESC-CM underwent cyclic stretch (0.5 Hz with pulsation of 10–25% elongation of cells, 24 h, (A) and angiotensin II treatment (1 μM, 48 h; (B) 30 days after differentiation. Bar graphs show fold change in cell size, hESC-CM with organized sarcomeres and ANF, αMHC, and βMHC mRNA levels measured by quantitative PCR. mRNA results are expressed as ratio of mRNA to GAPDH (n > 100 MHC-positive cells analyzed per well, mean ± SEM of triplicate wells, repeated in n = 3 preparations, *P < 0.05, ***P < 0.001 vs. control).

Fig. 4

Small molecule inhibitors on phenylephrine-induced hESC-CM hypertrophy. Bar graphs showing cell size relative to control (A) and ANF mRNA (B) of hESC-CM treated with PE in the presence of selective p38–MAPK inhibitor SB202190 (1 μM), PKG inhibitor KT5823 (1 μM), HDAC II inhibitor trichostatin A (0.25 μM), ERK inhibitor PD98059 (10 μM), JNK inhibitor SP600125 (1 μM), GSK3β inhibitor 1-azakenpaullone (10 μM), CAMK II inhibitor KN93 (10 μM), calcineurin inhibitor cyclosporine A (0.2 μM), mTOR inhibitor rapamycin (10 ng/ml), and calcineurin/FKBP inhibitor FK506 (0.1 μM) for 48 h. (For cell size, n > 100 MHC-positive cells analyzed per well, mean ± SEM of triplicate wells, repeated in n = 3 preparations. For mRNA levels, triplicate wells, repeated in n = 2 preparations. One-way ANOVA **P < 0.01, ***P < 0.001 vs. PE-group; ^#P < 0.001 vs. DMSO-control group.)

Fig. 5

(A) Bar graphs showing cell size of hESC-CM (~ 30 days) treated with phenylephrine (10 μM, 48 h) in the presence of p38–MAPK or scrambled control (NT) siRNA. Mean ± SEM of triplicate wells, repeated in n = 3 preparations. ***P < 0.001 vs. mock control group; ^#P < 0.001 vs. phenylephrine-treated group. (B) Infection of hESC-CM with constitutively active MAP2K3b recombinant adenovirus. Bar graphs showing fold changes in cell area, hESC-CM with organized sarcomeres, F-actin distribution, ANF mRNA levels, percentage of binuclear, and Ki67-positive hESC-CM infected with constitutively active MAP2K3b-adenovirus vs. control GFP adenovirus group. (n > 100 MHC-positive cells analyzed per well, mean ± SEM of triplicate wells, repeated in n = 3 preparations. One-way ANOVA.*P < 0.05, **P < 0.01, ***P < 0.001 vs. control group.)

Fig. 6

Phenylephrine modulates cell size independently of cell cycle. (A) Representative immunofluorescence image showing hESC-CM stained positive for the myosin heavy chain α/β (MHCα/β, green), DAPI (blue), Ki67 (nuclear, red), and atrial natriuretic factor (ANF, perinuclear, orange) in the presence of cell cycle inhibitor blebbistatin (10 μM) at 30 days after differentiation. Scale bar represents 50 μm. Bar graphs showing cell size of hESC-CM (B), percentage of binucleated hESC-CM (C), and percentage of Ki67-positive hESC-CM (D) treated with phenylephrine (PE) in the presence of blebbistatin (Bleb, solid bar) or vehicle (light grey bar). Results are shown as mean ± SEM (n > 100 MHC-positive cells per well, in triplicate, n = 2 preparations). The Cellomics Cell Cycle BioApplication classified hESC-CM treated with blebbistatin, nocodazole, or control medium into their cell cycle phase based on the total nuclear intensity of DNA binding DAPI (E). Cell cycle distribution presented as histogram where the Y-axis represents the number of instances and the X-axis represents the total nuclear intensity. The positions of the 2 N and 4 N DNA contents as well G0/G1, G2/M, and S phases are indicated (n = 600 from 3 experiments). *P < 0.05 vs. control, ***P < 0.001 vs. control, ^#P < 0.001 vs. respective vehicle-treated group.