Citation
- Authors: Röning T. et al.
- Year: 2021
- Journal: J Mol Cell Cardiol 164 148-155
- Applications: in vitro / siRNA / jetOPTIMUS
- Cell type: Neonatal rat ventricular cardiomyocytes
Method
Then, cells were transfected with Hey2 siRNA or control siRNA (100 nM) with jetOPTIMUS transfection reagent (2.2 μl/ml, Polyplus Transfection) and incubated for 24 h. Cells were rinsed with PBS and frozen at −70 °C for RNA extraction. Tritium [3H]-labelled leucine was used to measure protein synthesis. Neonatal rat ventricular cardiomyocytes cultured in 24-well plates were supplemented 24 h after the siRNA transfections with [3H]-leucine (1 μCi/ml, PerkinElmer) in DMEM/F12, 0.25% bovine serum albumin, 1 nM T3, 2.8 mM sodium pyruvate, insulin-transferrin‑selenium, penicillin-streptomycin and incubated for 24 h.
Abstract
Aims: We have previously demonstrated protection against obesity, metabolic dysfunction, atherosclerosis and cardiac ischemia in a hypoxia-inducible factor (HIF) prolyl 4-hydroxylase-2 (Hif-p4h-2) deficient mouse line, attributing these protective effects to activation of the hypoxia response pathway in a normoxic environment. We intended here to find out whether the Hif-p4h-2 deficiency affects the cardiac health of these mice upon aging.
Methods and results: When the Hif-p4h-2 deficient mice and their wild-type littermates were monitored during normal aging, the Hif-p4h-2 deficient mice had better preserved diastolic function than the wild type at one year of age and less cardiomyocyte hypertrophy at two years. On the mRNA level, downregulation of hypertrophy-associated genes was detected and shown to be associated with upregulation of Notch signaling, and especially of the Notch target gene and transcriptional repressor Hairy and enhancer-of-split-related basic helix-loop-helix (Hey2). Blocking of Notch signaling in cardiomyocytes isolated from Hif-p4h-2 deficient mice with a gamma-secretase inhibitor led to upregulation of the hypertrophy-associated genes. Also, targeting Hey2 in isolated wild-type rat neonatal cardiomyocytes with siRNA led to upregulation of hypertrophic genes and increased leucine incorporation indicative of increased protein synthesis and hypertrophy. Finally, oral treatment of wild-type mice with a small molecule inhibitor of HIF-P4Hs phenocopied the effects of Hif-p4h-2 deficiency with less cardiomyocyte hypertrophy, upregulation of Hey2 and downregulation of the hypertrophy-associated genes.
Conclusions: These results indicate that activation of the hypoxia response pathway upregulates Notch signaling and its target Hey2 resulting in transcriptional repression of hypertrophy-associated genes and less cardiomyocyte hypertrophy. This is eventually associated with better preserved cardiac function upon aging. Activation of the hypoxia response pathway thus has therapeutic potential for combating age-induced cardiac hypertrophy.