PERK Overexpression-Mediated Nrf2/HO-1 Pathway Alleviates Hypoxia/Reoxygenation-Induced Injury in Neonatal Murine Cardiomyocytes via Improving Endoplasmic Reticulum Stress

Abstract

Reperfusion processes following acute myocardial infarction (AMI) have been reported to induce additional cardiomyocyte death, known as ischemia-reperfusion (I/R) injury. Endoplasmic reticulum (ER) stress is reported to be involved in the development of I/R injury. There is evidence that PERK exerts beneficial roles in alleviating ER stress. Here, we investigated whether upregulation of PERK improved cardiomyocytes injury induced by I/R. Specific siRNAs or adenovirus vectors were incubated with isolated neonatal cardiomyocytes (NCMs) to regulate expression levels of target genes including PERK, Nrf2, and HO-1. Afterwards, hypoxia and subsequent reoxygenation (H/R) administration was performed as the in vitro model of I/R injury. MTT assay showed that H/R intervention decreased the viability of cells, yet PERK overexpression increased the cellular proliferative rate. Moreover, the upregulation of Nrf2 or HO-1 elevated the growth rate of cells, while gene silencing of Nrf2 or HO-1 reduced the viability of NCMs treated with PERK-rAAV9. In addition, we observed that the apoptotic index of cells with H/R stimulation was reduced when NCMs were pretreated with PERK-rAAV9, Nrf2-rAAV9, or HO-1-rAAV9. After cells were incubated with Nrf2-siRNA or HO-1-siRNA, the upregulation of PERK had no roles in affecting the apoptosis rate of NCMs damaged by H/R. Then, our findings indicated that there was a level decrease of GRP78, CRT, CHOP, and Caspase-12 in NCMs of the PERK-rAAV9 group compared to that of the H/R group. Both Nrf2 overexpression and HO-1 upregulation reduced the expression of ER stress-related proapoptotic factors, yet the expression suppression of Nrf2 and HO-1 increased levels of GRP78, CRT, CHOP, and Caspase-12 in NCMs treated with PERK-rAAV9. Taken together, our results suggested that the effects of PERK against H/R injury might be attributed to the upregulation of Nrf2/HO-1 cascade, followed by the inhibition of ER stress-related apoptotic pathway.

A review on herbal Nrf2 activators with preclinical evidence in cardiovascular diseases

“…In various preclinical ischemia and reperfusion experimental models, it has been demonstrated the downregulation of Nrf2 and associated antioxidant genes expression in cardiac tissue (Silva‐Palacios et al, 2019; Yang, Ding, et al, 2020; Yang, Liu, et al, 2020; Yang, Mao, et al, 2020; Zhao et al, 2020; Zhao, Chen, Wang, & Cai, 2020; Zheng et al, 2020). Recently, Wang et al (2020) demonstrated that the silensing of the gene, Nrf2 or HO‐1 reduces the viability of neonatal cardiomyocytes isolated from neonatal mice following hypoxia/reoxygenation. They found that upregulation of Nrf2/HO‐1 (through protein kinase‐like endoplasmic reticulum kinase overexpression) reduces the expression of endoplasmic reticulum stress related pro‐apoptotic markers, GRP78, CHOP, calreticulin (CRT) and caspase‐12 in neonatal cardiomyocytes following hypoxia/reoxygenation, and prevents the cardiomyocytes death (Wang et al, 2020).…”

MicroRNA‑24 protects against myocardial ischemia‑reperfusion injury via the NF‑κB/TNF‑α pathway

“…Acute myocardial infarction (AMI) is one of the leading causes of mortality in patients with cardiovascular disease worldwide ( 1 ), accounting for 80% of cardiogenic shock ( 2 ). AMI is caused by coronary artery blockage and myocardial ischemic necrosis, which eventually leads to heart failure ( 3 ). Therefore, early reperfusion therapy can save the dying myocardium, which is essential for blood supply and nutritional support of ischemic myocardium, as well as improving the prognosis of patients with AMI ( 1 , 3 ).…”

“…AMI is caused by coronary artery blockage and myocardial ischemic necrosis, which eventually leads to heart failure ( 3 ). Therefore, early reperfusion therapy can save the dying myocardium, which is essential for blood supply and nutritional support of ischemic myocardium, as well as improving the prognosis of patients with AMI ( 1 , 3 ). However, there is evidence that traditional reperfusion therapy can cause additional damage to myocardial structure and function, namely myocardial ischemia-reperfusion injury (IRI) ( 3 , 4 ).…”

“…Therefore, early reperfusion therapy can save the dying myocardium, which is essential for blood supply and nutritional support of ischemic myocardium, as well as improving the prognosis of patients with AMI ( 1 , 3 ). However, there is evidence that traditional reperfusion therapy can cause additional damage to myocardial structure and function, namely myocardial ischemia-reperfusion injury (IRI) ( 3 , 4 ). IRI is a pathological phenomenon in which the reintroduction of oxygen and other nutrients into the myocardium further aggravates pathological damage to the myocardium, resulting in decreased cardiac function, arrhythmias and irreversible death of cardiomyocytes ( 1 , 3 , 4 ).…”

“…However, there is evidence that traditional reperfusion therapy can cause additional damage to myocardial structure and function, namely myocardial ischemia-reperfusion injury (IRI) ( 3 , 4 ). IRI is a pathological phenomenon in which the reintroduction of oxygen and other nutrients into the myocardium further aggravates pathological damage to the myocardium, resulting in decreased cardiac function, arrhythmias and irreversible death of cardiomyocytes ( 1 , 3 , 4 ). Therefore, elucidating the mechanism of IRI and improving myocardial damage caused by IRI is important for the clinical treatment of patients with AMI to restore coronary blood flow.…”