LITAF (Lipopolysaccharide-Induced Tumor Necrosis Factor) Regulates Cardiac L-Type Calcium Channels by Modulating NEDD (Neural Precursor Cell Expressed Developmentally Downregulated Protein) 4-1 Ubiquitin Ligase

Abstract

Background: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. Methods: Optical mapping was performed in zebrafish hearts to determine Ca 2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca 2+ handling. L-type calcium channel (LTCC) current ( I Ca,L ) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. Results: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in I Ca,L and Cavα1c abundance, whereas LITAF knockdown increased I Ca,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD ( neural precursor cell expressed developmentally downregulated protein ) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on I Ca,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of I Ca,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. Conclusions: LITAF acts as an adaptor protein promoting NEDD4-1–mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.

The endosomal trafficking regulator LITAF controls the cardiac Nav1.5 channel via the ubiquitin ligase NEDD4-2

“…Notably, there is a significant increase in the variations of the APD of the KO zebrafish as compared to WT fish (p<0.05). Of note, we previously observed insignificant shortening of APD in 3-week-old rabbit cardiomyocytes overexpressing LITAF in agreement with computer modeling results (data not shown), whereas morpholino-mediated downregulation of LITAF in zebrafish embryos resulted in prolongation of APD that did not reach statistical significance (27). This suggests that decreased [Na + ] i and I NaK as a knock-on effect of decreased I Ca,L current density may be a common mechanism for the modest change of APD in both rabbit and zebrafish cardiomyocytes.…”

“…Increased calcium concentrations in the cytosol due to malfunctioning RYR2 increased NEDD4-2 expression lowering Nav1.5 levels, as data from volume-overload heart failure rat hearts suggested (41). In agreement with the experiments associated with LITAF overexpression in 3-week-old rabbit cardiomyocytes and with LITAF KO in zebrafish (27), the computational simulation shows that LITAF overexpression shortens APD (Fig. 7).…”

“…7A). The results further show that this modest change of APD can be attributed to the subtle knock-on effect of I Ca,L on other currents mediated by the changes in [Ca 2+ ] i transient and [Na + ] i , preliminarily explored in Moshal et al (27). More specifically, the decrease in the [Ca 2+ ] i transient amplitude ( Fig.…”

“…Thus, it is possible that LITAF and the small NEDD4 family-interacting proteins (NDFIPs), NDFIP1 and NDFIP2 (40) may share common mechanisms in regulating members of the NEDD4 family. Interestingly, we have recently published that LITAF is also a regulator of L-type calcium channels in zebrafish and rabbit cardiomyocytes (27). Here, LITAF acts as an adaptor protein and activator of the HECT ubiquitin ligase NEDD4-1.…”

“…To this end, we used a physiologically detailed model of rabbit ventricular myocyte with membrane voltage coupled to spatially distributed subcellular calcium dynamics. This model is improved from Moshal et al (27) through several modifications. First, we included the late sodium current (I NaL ) from Hwang et al (34).…”