VLDL incubation induced a 17.2-fold increase in intracellular lipid accumulation (P=0.004) and increased VLDLR-II expression by 1.6-fold (P=0.030) in HL-1 cardiomyocytes.
VLDL induces lipid accumulation and modulates VLDLR alternative splicing in cardiomyocytes, with VLDLR-II showing the highest affinity for PCSK9, suggesting a mechanism for modulating VLDL/TG metabolism.
Effect estimate: 17.2 folds increase
p-value: p=0.004
Abstract Background/Introduction Triglyceride (TG)-enriched very–low–density lipoprotein (VLDL) binds to the ApoEApoE-recognition receptor, the VLDL receptor (VLDLR), for cellular internalisation of lipoproteins or interaction of lipoprotein lipase on the cell surface. Two of the four splicing variants of VLDLR are major splicing isoforms in cardiomyocytes. Proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates receptor degradation and recognises members of the LDLR family including VLDLR. Whether VLDL affects VLDLR splicing variant forms and whether VLDLR variants exhibit different affinities to PCSK9 remains unknown. Purpose To test the hypothesis that VLDLR splice variants have different PCSK9 affinities. VLDL-induced lipid accumulation can modulate alternative splicing of VLDLR. The aim of this study was to determine whether VLDLR splicing variants can be altered in cardiomyocytes and to compare the PCSK9 binding affinity to VLDLR variants. Methods Isolated and purified 50 μg/ ml VLDL was used to incubate murine atrial myocytes (HL-1), and RNA was extracted for cDNA and subsequent reverse transcriptase PCR to determine whether VLDL altered the alternative splicing of the VLDLR gene. VLDLR splicing variants I–IV were specifically cloned into GFP-tag plasmids and transfected into human embryonic kidney cells 293 (HEK 293T). BODIPY staining and immunofluorescence imaging were used to quantify the intracellular lipid content. In HEK 293T, co–immunoprecipitation was used to compare the binding of PCSK9 to different VLDLR variants. Results VLDL incubation induced intracellular lipid accumulation, as shown by BODIPY staining of HL-1 cells, which was increased by 17.2 ± 3.7 folds (P = 0.004). VLDL incubation also increased VLDLR-II in HL-1 by 1.6 ± 0.1 folds compared to the control (P = 0.030). Western blotting and immunofluorescence imaging of GFP confirmed that VLDLR-I–IV were specifically and evenly expressed in HEK293T cells. Using co-immunoprecipitation, the binding affinity of PCSK9 was found to differ among the four variants. VLDLR-II had the greatest affinity for PCSK9 (compared to VLDLR-I, 1.3 ± 0.3 folds, P 0.001). Conclusions VLDL incubation induced lipid accumulation in HL-1 cardiomyocytes and modulated alternative splicing of VLDLR to increase the VLDLR-II/VLDLR-I ratio. VLDLR-II had a significantly greater affinity for PCSK9 than other VLDLR variants. These findings suggest that alternative splicing of VLDLR is a cellular response to excessive lipid accumulation, and is an important mechanism for modulating VLDL/TG metabolism in cardiomyocytes.
Huang et al. (2025) studied Lipid accumulation and VLDLR alternative splicing. VLDL incubation vs. Control was evaluated on Intracellular lipid accumulation (17.2 folds increase, p=0.004). VLDL incubation induced a 17.2-fold increase in intracellular lipid accumulation (P=0.004) and increased VLDLR-II expression by 1.6-fold (P=0.030) in HL-1 cardiomyocytes.