Regulation of Ankyrin-G on Nav1.5 Channel in Hypoxic HL-1 Cardiac Muscle Cells

Hypoxia has a major regulatory impact on the electrical activity transmission in the myocardium, and it is involved in the development of tachyarrhythmia disease. Anchor protein G (ankyrin-G, ANK-G) is associated with voltage-gated Na⁺ channels (Nav1.5), but its specific role and mechanism have not been fully defined. In this experiment, we investigated the role and mechanism of hypoxia on cardiomyocyte electrophysiology of voltage-gated Na⁺ channel, as well as the intervention effect of ankyrin-G by simulating the environment of cardiomyocytes during hypoxia through hypoxia-treated murine atrial myocytes (HL-1).

The HL-1 cells were divided into 6 groups: normoxia group (NO), hypoxia group (HO), ANK-G-overexpressing hypoxia-negative group (ANK-G NC), ANK-G-overexpressing hypoxia group (ANK-G), ANK-G-silenced hypoxia-negative group (shANK-G NC), and ANK-G-silenced hypoxia group (shANK-G). ANK-G overexpression was induced using lentiviral vectors through the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. The characteristics of sodium ion channel current (I_Na) were observed through the whole-cell patch clamp technique. Western blotting was used to detect the expression of ANK-G and Nav1.5 channel proteins, and the distribution of Nav1.5 channel on HL-1 cells was observed by confocal microscope.

Under hypoxic conditions, the I_Na peak current amplitude (p < 0.01) and density (p < 0.01) of HL-1 cells increased. Compared with the normoxia group, the steady-state inactivation curve of the hypoxia group shifted to the right. The protein levels of ANK-G and Nav1.5 channels were increased under hypoxia (p < 0.001). In the ANK-G group, the upregulation of ANK-G protein increased the distribution of Nav1.5 channel in the cell membrane under the hypoxic condition (p < 0.01).

Hypoxia increases the I_Na amplitude and density of HL-1 cells, and the gating mechanism of I_Na is related to steady-state inactivation. Hypoxic condition triggers the upregulation of the ANK-G protein expression, which promotes the redistribution of Nav1.5 channel proteins in the cell membrane, thereby augmenting I_Na peak current amplitude and density.