Endoplasmic reticulum stress mediates homocysteine-induced hypertrophy of cardiac cells through activation of cyclic nucleotide phosphodiesterase 1C

Abstract

Although the association of elevated homocysteine level with cardiac hypertrophy has been reported, the molecular mechanisms by which homocysteine induces cardiac hypertrophy remain inadequately understood. In this study we aim to uncover the roles of cyclic nucleotide phosphodiesterase 1 (PDE1) and endoplasmic reticulum (ER) stress and their relationship to advance the mechanistic understanding of homocysteine-induced cardiac cell hypertrophy. H9c2 cells and primary neonatal rat cardiomyocytes are exposed to homocysteine with or without ER stress inhibitor TUDCA or PDE1-specific inhibitor Lu AF58027, or transfected with siRNAs targeting PDE1 isoforms prior to homocysteine-exposure. Cell surface area is measured and ultrastructure is examined by transmission electron microscopy. Hypertrophic markers, PDE1 isoforms, and ER stress molecules are detected by q-PCR and western blot analysis. Intracellular cGMP and cAMP are measured by ELISA. The results show that homocysteine causes the enlargement of H9c2 cells, increases the expressions of hypertrophic markers β-MHC and ANP, upregulates PDE1A and PDE1C, promotes the expressions of ER stress molecules, and causes ER dilatation and degranulation. TUDCA and Lu AF58027 downregulate β-MHC and ANP, and alleviate cell enlargement. TUDCA decreases PDE1A and PDE1C levels. Silencing of PDE1C inhibits homocysteine-induced hypertrophy, whereas PDE1A knockdown has minor effect. Both cAMP and cGMP are decreased after homocysteine-exposure, while only cAMP is restored by Lu AF58027 and TUDCA. TUDCA and Lu AF58027 also inhibit cell enlargement, downregulate ANP, β-MHC and PDE1C, and enhance cAMP level in homocysteine-exposed primary cardiomyocytes. ER stress mediates homocysteine-induced hypertrophy of cardiac cells via upregulating PDE1C expression Cyclic nucleotide, especially cAMP, is the downstream mediator of the ER stress-PDE1C signaling axis in homocysteine-induced cell hypertrophy.

Keywords: cardiac hypertrophy; endoplasmic reticulum stress; homocysteine; phosphodiesterase.

Figure1

Hypertrophic effect of homocysteine (Hcy) on H9c2 cardiac cells Exposure to 100 μM Hcy for 72 h significantly upregulated the expressions of cardiac hypertrophy markers β-MHC and ANP in H9c2 cells at both the mRNA level (A, n=6) and protein level (B, n=5), and enlarged the cell surface area (C, n=5). Such exposure showed no cytotoxicity and barely affected cell viability (D, n=6). Data are presented as the mean±SEM. *P<0.05, **P<0.01, ***P<0.01 vs control.

Figure2

The role of endoplasmic reticulum (ER) stress in homocysteine (Hcy)-induced hypertrophy of H9c2 cardiac cells Representative blots and expression levels of ER stress molecules from 5 independent experiments showed that inhibition of ER stress by TUDCA suppressed the expression/activation of ER stress molecules (A). Transmission electron microscopy examination of the H9c2 cardiac cells, showing dilatation and degranulation of rough ER and loss of mitochondrial cristae after Hcy exposure, which could be ameliorated by TUDCA. Red arrows denote the rough ER. M, N, and PM represent mitochondrion, nucleus, and plasma membrane, respectively. Scale bar=1 μm (B). Representative immunofluorescence microscopy images of control H9c2 cardiac cells and cells treated with Hcy in the presence or absence of the ER stress inhibitor TUDCA. Scale bar=20 μm. The increase of cell surface area caused by Hcy was reversed by TUDCA, as calculated from 5 independent experiments (C). Inhibition of ER stress with TUDCA suppressed homocysteine-induced upregulation of β-MHC and ANP expression at both the mRNA level and protein level (D, n=4). Data are presented as the mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs control; #P<0.05, ##P<0.01 vs Hcy. TUDCA: tauroursodeoxycholic acid.

Figure3

The role of PDE1 in homocysteine (Hcy)-induced hypertrophy of H9c2 cardiac cells Western blot analysis showed the upregulation of PDE1A and PDE1C protein expressions after Hcy exposure (A, n=4). Inhibition of PDE1 with Lu AF58027 suppressed Hcy-induced expressions of β-MHC and ANP, along with a downregulation of PDE1A and PDE1C proteins (B, n=5). Representative immunofluorescence microscopy images of control H9c2 cardiac cells and cells treated with Hcy in the presence or absence of the PDE1 inhibitor Lu AF58027. Scale bar=20 μm. The increase of cell surface area induced by Hcy was reversed by Lu AF58027, as calculated from 5 independent experiments (C). Data are presented as the mean±SEM. *P<0.05, **P<0.01 vs control; #P<0.05, ##P<0.01 vs Hcy.

Figure4

Effect of silencing of PDE1A and PDE1C isoforms on homocysteine (Hcy)-induced hypertrophy of H9c2 cardiac cells The knockdown efficiency and specificity of siRNAs (siPDE1A and siPDE1C) for target genes. The PDE1A targeting siRNA (siPDE1A) significantly suppressed the mRNA and protein expressions of PDE1A in both control and Hcy-exposed cells but showed no influence on the expression of the PDE1C isoform, and vice versa (A, n=5). Selective knockdown of the PDE1C but not PDE1A isoform in H9c2 cardiac cells significantly suppressed the increase of cell surface area induced by Hcy (B, n=5). PDE1C silencing inhibited Hcy-induced expressions of the hypertrophic markers β-MHC and ANP in H9c2 cells, while PDE1A knockdown only decreased the β-MHC level (C, n=5). Data are presented as the mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs control (vector); #P<0.05, ##P<0.01 vs Hcy (vector).

Figure5

Association between ER stress and PDE1 in homocysteine (Hcy)-induced hypertrophy of H9c2 cardiac cells Inhibition of ER stress with TUDCA significantly decreased the protein expressions of PDE1A and PDE1C in Hcy-exposed H9c2 cells (A, n=4). Hcy-induced upregulation/activation of ER stress molecules was not affected by the PDE1 inhibitor Lu AF58027 (B, upper panel, n=5). Inhibition of PDE1 showed no protection against Hcy-induced dilatation and degranulation of the rough ER (B, lower panel). Hcy-induced decrease of cAMP level was restored by the PDE1 inhibitor Lu AF58027 and the ER stress inhibitor TUDCA. Neither Lu AF58027 nor TUDCA restored the cGMP content (C, n=5–7). Data are presented as the mean±SEM. *P<0.05, ***P<0.001, ****P<0.0001 vs control; #P<0.05, ##P<0.01 vs Hcy. TUDCA: tauroursodeoxycholic acid.

Figure6

Association between ER stress and PDE1C in homocysteine (Hcy)-induced hypertrophy of primary neonatal rat cardiomyocytes ER stress inhibitor TUDCA and PDE1 inhibitor Lu AF58027 suppressed homocysteine-induced cell enlargement and expressions of β-MHC and ANP (A, n=5). Both TUDCA and Lu AF58027 inhibited homocysteine-induced expression of PDE1C (B, n=5) and elevated the intracellular cAMP content (C, n=5). Data are presented as the mean±SEM. **P<0.01, ***P<0.001 vs control; #P<0.05, ##P<0.01, ###P<0.001 vs Hcy. TUDCA: tauroursodeoxycholic acid.