Regulation of Cyclic Nucleotide Phosphodiesterase 1 by Endoplasmic Reticulum Stress Contributes to Homocysteine‐induced Cardiac Hypertrophy

Abstract

Objectives Mechanistic understanding of the role of homocysteine in the development of cardiac hypertrophy is inadequate at present. This study aimed to elucidate the association of cyclic nucleotide phosphodiesterase 1 (PDE1) and endoplasmic reticulum (ER) stress with cardiomyocyte hypertrophy induced by homocysteine. Methods H9c2 cardiomyocytes were allocated to control group and homocysteine-treated group in the presence or absence of ER stress inhibitor tauroursodeoxycholic acid (TUDCA) or PDE1 inhibitor Lu AF58027 for 72h. In another set of experiment, H9c2 cells were transfected with specific siRNA targeting PDE1 subtypes prior to homocysteine exposure. The cell surface area was measured by phase contrast microscope and image analysis software and ultrastructural changes of the cells were observed under transmission electron microscopy. Quantitative polymerase chain reaction and western blot were employed to detect the mRNA and protein expression of the hypertrophic markers beta-smooth muscle myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP), PDE1 subtypes (PDE1A, 1B, and 1C), and ER stress molecules (GRP78, phosphor-PERK, PERK, phosphor-IRE1, IRE1, and ATF6). The intracellular levels of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) were measured by enzyme linked immunosorbent assay. Results Homocysteine exposure resulted in an enlargement in cell size and an increase in β-MHC and ANP expression. Homocysteine induced ER stress in H9c2 cells, shown by increased expression of GRP78 and enhanced phosphorylation of PERK and IRE1, as well as dilatation and degranulation of rough ER. The expression of PDE1A and PDE1C was significantly upregulated by homocysteine. Inhibition of ER stress or PDE1 suppressed homocysteine-induced upregulation of β-MHC and ANP and alleviated cell enlargement. In addition, the protein expression of PDE1A and PDE1C in homocysteine-exposed H9c2 cells was downregulated by the ER stress inhibitor TUDCA. Silencing of PDE1C significantly inhibited homocysteine-induced cell hypertrophy whereas knockdown of PDE1A showed minor effect. Both cAMP and cGMP levels were lowered in H9c2 cells subjected to homocysteine exposure, while only cAMP content could be restored by Lu AF58027 and TUDCA. Conclusions ER stress mediates homocysteine-induced cardiomyocyte hypertrophy via upregulating PDE1 expression. Cyclic nucleotide, in particular cAMP, is the downstream mediator of the ER stress-PDE1 signaling axis in homocysteine-induced hypertrophy.