Pcsk6 Deficiency Promotes Cardiomyocyte Senescence by Modulating Ddit3-Mediated ER Stress

Abstract

Cardiac aging is a critical determinant of cardiac dysfunction, which contributes to cardiovascular disease in the elderly. Proprotein convertase subtilisin/kexin 6 (PCSK6) is a proteolytic enzyme important for the maintenance of cardiac function and vascular homeostasis. To date, the involvement of PCSK6 in cardiac aging remains unknown. Here we report that PCSK6 expression decreased in the hearts of aged mice, where high levels cyclin dependent kinase inhibitor 2A (P16) and cyclin dependent kinase inhibitor 1A (P21) (senescence markers) were observed. Moreover, PCSK6 protein expression was significantly reduced in senescent rat embryonic cardiomyocytes (H9c2) induced by D-galactose. Pcsk6 knockdown in H9c2 cells increased P16 and P21 expression levels and senescence-associated beta-galactosidase activity. Pcsk6 knockdown also impaired cardiomyocyte function, as indicated by increased advanced glycation end products, reactive oxygen species level, and apoptosis. Overexpression of PCSK6 blunted the senescence phenotype and cellular dysfunction. Furthermore, RNA sequencing analysis in Pcsk6-knockdown H9c2 cells identified the up-regulated DNA-damage inducible transcript 3 (Ddit3) gene involved in endoplasmic reticulum (ER) protein processing. Additionally, DDIT3 protein levels were remarkably increased in aged mouse hearts. In the presence of tunicamycin, an ER stress inducer, DDIT3 expression increased in Pcsk6-deficient H9c2 cells but reduced in PCSK6-overexpressing cells. In conclusion, our findings indicate that PCSK6 modulates cardiomyocyte senescence possibly via DDIT3-mediated ER stress.

Therapeutic potential of the Proprotein Convertase Subtilisin/Kexin family in vascular disease

“…Not only were PCSK6 plaque levels increased in patients with previous myocardial infarction and patients undergoing carotid endarterectomy with stroke symptoms, but the positive correlation observed with MATX components and LRNC volume suggests a possible role of PCSK6 in plaque vulnerability ( Testa et al, 2021 ; Perisic et al, 2013 ). Our data aligns with a recent report by Kuhn et al identifying PCSK6 as a novel player in cardiac remodeling after myocardial infarction ( Kuhn et al, 2020 ), but also with a more recent report showing that Pcsk6 deficiency results in cardiomyocyte senescence ( Zhan et al, 2022 ) that confirmed our findings in primary vascular SMCs isolated from Pcsk6 knockouts ( Rykaczewska et al, 2020 ). Interestingly, PCSK6 expression also associated with serum creatinine levels suggesting an unexplored role in renal function.…”

Alteration of E2F2 Expression in Governing Endothelial Cell Senescence

“…The concentration of protein was determined with a BCA Protein Assay Kit (Beyotime, Shanghai, China). Western blot analysis was performed as described previously [ 24 ]. The primary and secondary antibodies used in this study include E2F2 (1:3000, Abcam, Cambridge, UK), P21 (1:3000, Proteintech, Wuhan, Hubei, China), p-eNOS (1:2000, Abcam, Cambridge, UK), SRIT1 (1:2000, Thermo Fisher Scientific, Waltham, MA, USA), GAPDH (1:10,000, Proteintech, Wuhan, Hubei, China), HRP-conjugated goat anti-rabbit IgG (1:10,000, Proteintech, Wuhan, Hubei, China), and HRP-conjugated goat anti-mouse IgG (1:10,000, Proteintech, Wuhan, Hubei, China).…”

“…The inhibition of E2F2 activity reduces cell proliferation in cancer development [ 22 ]. E2F2 also plays an important role in regulating the expression of PCSK6, which modulates cardiomyocyte senescence [ 23 , 24 ]. In vivo, E2F2 knockout mice display impaired endothelial function, increased vessel contraction, and high blood pressure [ 25 ].…”

Proprotein Convertase Subtilisin/Kexin 6 in Cardiovascular Biology and Disease

“…PCSK6 is necessary for corin activation and ANP generation in the heart [ 53 ]. A recent study indicates that PCSK6 deficiency may contribute to senescence in cardiomyocytes [ 144 ]. In aged mouse hearts and senescent cardiomyocytes, Pcsk6 expression is reduced.…”

“…In aged mouse hearts and senescent cardiomyocytes, Pcsk6 expression is reduced. Moreover, Pcsk6 downregulation causes senescence in cultured cardiomyocytes, as indicated by increased advanced glycation end products, oxidative stress, and apoptosis [ 144 ]. Conversely, Pcsk6 overexpression prevents senescence and dysfunction in cultured cardiomyocytes under similar experimental conditions [ 144 ].…”