Inhibition of HIPK2 protects stress-induced pathological cardiac remodeling

Abstract

Background: Homeodomain-Interacting Protein Kinase 2 (HIPK2) has been reported to maintain basal cardiac function, however, its role in pathological cardiac remodeling remains unclear.

Methods: HIPK2 inhibitors (tBID and PKI1H) treated mice and two lines of HIPK2^-/- mice were subjected to transverse aortic constriction (TAC). HIPK2 knockdown were performed in neonatal rat cardiomyocytes (NRCMs), neonatal rat cardiac fibroblasts (NRCFs), and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Microarray analysis was used to screen HIPK2 targets. Overexpression of early growth response 3 (EGR3) and C-type lectin receptor 4D (CLEC4D) were performed in NRCMs, while an activator of Smad3 was used in NRCFs, to rescue the effects of HIPK2 knockdown. Finally, the effects of EGR3 and CLEC4D knockdown by AAV9 in TAC were determined.

Findings: HIPK2 was elevated in TAC mice model, as well as cardiomyocyte hypertrophy and NRCFs fibrosis model. Pharmacological and genetic inhibition of HIPK2 improved cardiac function and suppressed cardiac hypertrophy and fibrosis induced by TAC. In vitro, HIPK2 inhibition prevented cardiomyocyte hypertrophic growth and NRCFs proliferation and differentiation. At the mechanistic level, we identified EGR3 and CLEC4D as new targets of HIPK2, which were regulated by ERK1/2-CREB and mediated the protective function of HIPK2 inhibition in cardiomyocytes. Meanwhile, inhibition of phosphorylation of Smad3 was responsible for the suppression of cardiac fibroblasts proliferation and differentiation by HIPK2 inhibition. Finally, we found that inhibition of EGR3 or CLEC4D protected against TAC.

Interpretation: HIPK2 inhibition protects against pathological cardiac remodeling by reducing EGR3 and CLEC4D with ERK1/2-CREB inhibition in cardiomyocytes, and by suppressing the phosphorylation of Smad3 in cardiac fibroblasts.

Funding: This work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to J.X.), National Natural Science Foundation of China (82020108002 and 81911540486 to J.X., 81400647 to MJ Xu), the grant from Science and Technology Commission of Shanghai Municipality (21XD1421300 and 20DZ2255400 to J.X.), the "Dawn" Program of Shanghai Education Commission (19SG34 to J.X.), and Shanghai Sailing Program (21YF1413200 to Q.Z.).

Keywords: CLEC4D; EGR3; HIPK2; Pathological cardiac remodeling; Smad3.