The Role Of The “abcc6 Pathway” And Dietary Pyrophosphate In Dystrophic Calcification

Abstract

The Abcc6-/- mice display calcification symptoms similar to human PXE patients in addition to an acute and inducible dystrophic muscle calcification phenotype. We hypothesize 1) the lack of Abcc6 lowers ATP efflux and influences the expression of Enpp1 and Nt5e, thereby impacting plasma PPi levels and calcification susceptibility and 2) ABCC6 deficiency causes dystrophic calcification in cardiac tissues and skeletal muscle. The aims of this study were to 1) characterize the muscle calcification phenotype, 2) explore gene expression of the “Abcc6 pathway” and its molecular players, and 3) supplement PPi to counteract skeletal muscle calcification. Our inability to reproduce previous gastrocnemius calcification data for Aim 1 despite multiple trials and attempting protocol optimization led to the discovery of elevated levels of PPi in the rodent chow. Surprisingly, reversion to a low PPi diet did not restore the calcification phenotype and instead reduced calcium deposits in muscle tissues. The testing of an acceleration diet designed to enhance calcification was infructuous as it produced mineralization even in wild type mice. Our results demonstrated the impact that animal chow can have on phenotypic outcome. Moreover, our data may provide an explanation for the phenotype variability in PXE patients and further suggested that dietary intervention to slow the progression of the calcification phenotype of PXE in patients may be possible. Gene expression studies showed positive correlation between the Abcc6 status and Enpp1 and Nt5e mRNA levels in heart tissues. However, since we showed that PPi supplementation influences the expression of Enpp1, the presence of high levels of PPi in mouse chow probably negatively influenced our data. The Aim 3 experiments could not be completed as planned since we discovered elevated levels of PPi in the animal diet. However, results of specific Aim 1 provided some indications that indeed supplementation with PPi can effectively suppress dystrophic calcification in muscle tissues. Overall, the discovery of the elevated levels of PPi in the diet prevented us from achieving some of the goals of our specific aims and produced unanticipated but valuable data. Collectively, these studies have improved our understanding of the role of Abcc6 and PPi in the inhibition of soft-tissue calcification.