Human CAMKK2 (DN) adenoviral particles

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates a variety of biological processes and is implicated in a variety of pathological processes. However, its role in myocardial ischemia/reperfusion (MI/R) injury remains unknown. Here, researchers show that MI/R in vivo or hypoxia/reoxygenation (H/R) in vitro induces a decrease in CaMKK2 levels. Upregulation of CaMKK2 in rats ameliorates MI/R-induced cardiac damage, while suppressing cardiac cell apoptosis, oxidative stress, and proinflammatory responses. CaMKK2-overexpressing rat cardiomyocytes are also protected from H/R injury by suppressing apoptosis, oxidative stress, and proinflammatory responses. CaMKK2 overexpression leads to increased AMPK, AKT, and GSK-3β phosphorylation and enhances Nrf2 activation under MI/R or H/R conditions. AMPK inhibition abolishes CaMKK2-mediated Nrf2 activation and the associated cardioprotective effects. Inhibition of Nrf2 also attenuated the associated cardioprotective effects mediated by CaMKK2. Thus, upregulation of CaMKK2 enhances the Nrf2 pathway through regulating AMPK/AKT/GSK-3β, thereby providing therapeutic benefits in a rat model of MI/R injury, suggesting that CaMKK2 is a novel molecular target for the treatment of MI/R injury.

The researchers investigated the effect of CaMKK2 overexpression on H/R injury in vitro. CaMKK2 was overexpressed in cardiomyocytes using a recombinant adenovirus carrying CAMKK2 (Ad-CaMKK2). Ad-CaMKK2 infection resulted in increased CaMKK2 levels in cultured cardiomyocytes (Figure 1A, B). CCK-8 assays showed that upregulation of CaMKK2 attenuated the deleterious effects of H/R on cell viability (Figure 1C). H/R led to a significant increase in apoptosis, which was reduced in CaMKK2-overexpressing cardiomyocytes (Figure 1D, E). The increased Bax levels and decreased Bcl2 levels caused by H/R were reversed by CaMKK2 overexpression (Figure 1F). In addition, overexpression of CaMKK2 also attenuated the H/R-induced overproduction of ROS (Figure 1G) and proinflammatory mediators in cultured cardiomyocytes.

Figure 1. The effect of CaMKK2 up-regulation on H/R injury. Cardiomyocytes were infected with Ad-CaMKK2 for 72 h before H/R injury. (Li C, et al., 2023)