The liver regulates ectopic calcification in Abcc6-deficient models of pseudoxanthoma elasticum
Yijie Wang, Baiming Sun, Feiyang Ma, Bo Tao, Yiqian Gu, Zhiqiang Zhou, Jason Kim, Linlin Zhang, Zhihao Liu, Johanna ten Hoeve, Linsey Stiles, Lucia Fernandez del Rio, Calvin Pan, Orian Shirihai, Shili Xu, Thomas G. Graeber, Tamer Sallam, Matteo Pellegrini, Aldons J. Lusis, Arjun Deb
Yijie Wang, Baiming Sun, Feiyang Ma, Bo Tao, Yiqian Gu, Zhiqiang Zhou, Jason Kim, Linlin Zhang, Zhihao Liu, Johanna ten Hoeve, Linsey Stiles, Lucia Fernandez del Rio, Calvin Pan, Orian Shirihai, Shili Xu, Thomas G. Graeber, Tamer Sallam, Matteo Pellegrini, Aldons J. Lusis, Arjun Deb
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Research Article Cardiology Metabolism

The liver regulates ectopic calcification in Abcc6-deficient models of pseudoxanthoma elasticum

Abstract

Pseudoxanthoma Elasticum (PXE) is a rare disease caused by loss of function of the ATP-binding cassette C (ABC) member 6 (Abcc6) gene and characterized by ectopic calcification of multiple tissues, but the physiological reasons underlying ectopic calcification in PXE remain unclear. In a murine model of Abcc6-deficient PXE in which animals developed robust cardiac calcification after heart injury, we show the critical importance of the liver in mediating ectopic cardiac calcification. Tissue-specific deletion of Abcc6 in the liver, but not in the heart, was sufficient to cause post-injury cardiac calcification. Metabolomics and gene expression analysis demonstrated deficiencies in nucleotide metabolism, cellular energetics, and defects in cellular respiration underlying ectopic calcification in PXE. Functional abnormalities in cellular respiration in the injured heart were similar in animals with global or liver-specific Abcc6 deficiency, showing that hepatic Abcc6 expression regulated cellular respiration in the injured heart. We show that ectopic calcification in PXE was primarily dystrophic and that treatment with clodronate or etidronate, which prevent the growth of calcium hydroxyapatite mineralization, was sufficient to rescue the phenotype of ectopic cardiac calcification in Abcc6-deficient states. Taken together, these observations highlight the role of the liver in regulating target tissue metabolic and mitochondrial function in causing ectopic calcification in Abcc6-deficient states.

Authors

Yijie Wang, Baiming Sun, Feiyang Ma, Bo Tao, Yiqian Gu, Zhiqiang Zhou, Jason Kim, Linlin Zhang, Zhihao Liu, Johanna ten Hoeve, Linsey Stiles, Lucia Fernandez del Rio, Calvin Pan, Orian Shirihai, Shili Xu, Thomas G. Graeber, Tamer Sallam, Matteo Pellegrini, Aldons J. Lusis, Arjun Deb

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Figure 1

Genetic deletion of Abcc6 leads to persistent myocardial calcification after heart injury.

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Genetic deletion of Abcc6 leads to persistent myocardial calcification a...
(A) Schematic of the experimental design with cryo-induced injury of WT or Abcc6-KO animals and harvesting 3 days after injury. A high-phosphate, low-magnesium diet was administered starting 3 days before surgery. (B) Cryo-injured WT and Abcc6-KO animals demonstrating cardiac calcification (black arrow) on gross inspection and calcification in the Abcc6-KO heart, visualized with (C) CT scan and (D) following 3D rendering (red circles and insets indicate the calcific lesions). (E) Quantitative analysis of calcium content in scar tissue as measured by CT imaging (n = 6 in each group, mean ± SD, **P < 0.01, by 2-tailed, unpaired t test, WT vs. Abcc6-KO group). (F and G) Histological staining of cryo-injured myocardium in WT and Abcc6-KO animals with (F) Von-Kossa staining (scale bars: 100 μm) and (G) corresponding quantitative analysis showing calcium deposits (black arrows) (n = 6 in each group, mean ± SD, ***P < 0.001, by 2-tailed, unpaired t test, WT vs. Abcc6-KO group). (H) Immunostaining for hydroxyapatite (white arrows, scale bars: 10 μm) and (I) corresponding quantitative analysis. (n = 6 in each group, mean ± SD, ***P < 0.001 WT versus Abcc6-KO group, P value was calculated by 2-tailed, unpaired t test). (J and K) Biochemical measurements of (J) myocardial calcium and (K) phosphate deposits. All staining and measurements were performed on day 3 following injury (n = 6 in each group, mean ± SD, ***P < 0.001, by 2-tailed, unpaired t test, WT vs. Abcc6-KO group). (L–P) Persistence of cardiac calcification with (L) a gross image of the heart 4 months after cryo-injury (black arrowhead indicates the calcific lesion), (M) CT scan (white arrowhead), and (N) 3D rendering showing the calcification region. Red circles (L) and insets (N) indicate the calcific lesions. (O and P) Histological analysis of cryo-injured myocardium 4 months after surgery. (O) Von-Kossa staining (scale bar: 100 μm) and (P) immunostaining for hydroxyapatite (black and white arrowheads; scale bar: 50 μm). (n = 6 per group; WT vs. Abcc6-KO).