An alternative splice variant in Abcc6, the gene causing dystrophic calcification, leads to protein deficiency in C3H/He mice.

Dystrophic cardiac calcification (DCC) is an autosomal recessive trait characterized by calcium phosphate deposits in myocardial tissue. The Abcc6 gene locus was recently found to mediate DCC; however, at the molecular level the causative variants remain to be determined. Examining the sequences of Abcc6 cDNA in DCC-resistant C57BL/6 and DCC-susceptible C3H/He mice, we identified a missense mutation (Cys to Thr at codon 619, rs32756904) at the 3'-border of exon 14 that creates an additional donor splice site (GT). Accordingly, an alternative transcript variant was detected, lacking the last 5 bp of exon 14 (-AGG(C/T)GCTgtga-) in DCC-susceptible C3H/He mice that carry the Thr allele. The 5-bp deletion was found to result in premature termination at codon 684, in turn leading to protein deficiency in DCC-susceptible mouse tissue as well as in cells transfected with Abcc6 cDNA lacking the last 5 bp of exon 14. All mouse strains that were found to carry the Thr allele, including C3H/He, DBA/2J, and 129S1/SvJ, were also found to be positive for DCC. In summary, we identified a splice variant leading to a 5-bp deletion in the Abcc6 transcript that gives rise to protein deficiency both in vivo and in vitro. The fact that all mouse strains that carry the deletion also develop dystrophic calcifications further suggests that the underlying splice variant affects the biological function of MRP6 protein and is a cause of DCC in mice.

Association between arterial pressure and coronary artery calcification.

OBJECTIVES: Coronary artery calcification (CAC) determined by electron beam computed tomography is a predictor of future cardiovascular events. This study investigates conditions affecting CAC severity in patients with coronary artery disease (CAD) undergoing coronary angiography. METHODS: Presence and degree of CAC were assessed angiographically in 877 CAD patients grouped into no visible CAC (n = 333), mild to moderate CAC (n = 321), and severe CAC (n = 223). Regression analyses investigated relationships between CAC and demographic data, cardiovascular risk factors, and coronary anatomy. RESULTS: Prevalences of hypertension and systolic blood pressure (SBP) values were higher in individuals with CAC (moderate CAC: 49.5%, 137.5 +/- 18.6 mmHg; severe CAC: 58.3%, 142.1 +/- 20.4 mmHg) compared to individuals with CAD but no CAC (42.0%, 134.0 +/- 18.4 mmHg; both P < 0.001). Likewise, pulse pressure was significantly elevated with increasing degree of CAC (no CAC, 52.3 +/- 13.6 mmHg vs moderate CAC, 55.7 +/- 14.4 mmHg vs severe CAC, 59.1 +/- 15.4 mmHg; P < 0.001). Further determinants of CAC were age, positive family history for CAC and severity of CAD. No differences in CAC severity were found in relation to body mass index, low-density lipoprotein-cholesterol, diabetes, and smoking habits. In multivariate analysis, CAC was independently related to age, SBP or pulse pressure, respectively, positive family history for CAC, and the severity of CAD. CONCLUSIONS: Of the cardiovascular risk factors, SBP and pulse pressure display the strongest relationship with angiographic detection of CAC. Mechanistic studies need to clarify whether hypertension causes CAC, or whether coronary calcium deposition serves as a marker for a higher degree of vascular calcification and, thus, impaired vascular compliance and higher blood pressure levels.

Ultrafine mapping of Dyscalc1 to an 80-kb chromosomal segment on chromosome 7 in mice susceptible for dystrophic calcification.

In mice, dystrophic cardiovascular calcification (DCC) is controlled by a major locus on proximal mouse chromosome 7 named Dyscalc1. Here we present a strategy that combines in silico analysis, expression analysis, and extensive sequencing for ultrafine mapping of the Dyscalc1 locus. We subjected 15 laboratory mouse strains to freeze-thaw injury of the heart, and association with respective genotypes allowed condensation of the Dyscalc1 locus to 1 Mb. Within this region, 51 known and predicted genes were studied in DCC-susceptible C3H/He and DCC-resistant C57BL/6 mice with respect to mRNA expression in response to injury. Five genes displayed differential expression. Genotyping of seven novel single nucleotide polymorphisms (SNPs) within these genes revealed an 80-Kb region in NZB mice that were found positive for calcification though carrying otherwise alleles from DCC-resistant mice. This microheterogeneity in NZB mice was evolutionary conserved in all DCC-susceptible mouse strains and contains the genes EMP-3, BC013491, and Abcc6 (partially). The flanking SNPs are rs3703247 and NT_039420.5_2757991. mRNA levels of EMP-3 were found to be upregulated in response to injury in both C57BL/6 and C3H/He mice. Sequencing of EMP-3 revealed an SNP leading to an amino acid substitution (p.T153I) that was found in all mouse strains susceptible for DCC but not in resistant strains such as C57BL/6 mice. Thus, the p.T153I changes might affect the biological function of EMP-3 gene product after injury. Using this combined approach, we ultrafine-mapped the Dyscalc1 locus to an 80-Kb region and identified EMP-3 as a new candidate gene for DCC.

Arterial calcification in mice after freeze-thaw injury.

Vascular calcification is highly correlated with atherosclerosis and cardiovascular disease and is a significant predictor of cardiovascular morbidity and mortality. Studies in mice indicate a genetic contribution to this dystrophic extra osseous calcification. We sought to elaborate a method to induce dystrophic arterial calcification in mice and further examine the pathogenetical mechanisms involved in the phenotype. We established a method of freeze-thaw injury of the infrarenal aorta producing a limited tissue necrosis and histologically investigated the occurrence of dystrophic calcification within the aortic wall 1, 3 and 7 days after injury in C57BL/6 (a mouse strain shown to be resistant to dystrophic cardiac calcification after injury) and C3H/He (susceptible to dystrophic cardiac calcification). C57BL/6 mice exhibited no dystrophic calcification at all within the vessel wall upon injury of the infrarenal aorta (0/5 mice 1 day after injury and 0/10 animals 7 days after injury). By contrast C3H/He mice displayed a remarkable extent of calcification mainly present within the media of the infrarenal aorta which was evident as early as 24 h (three out of five animals 1 day after injury) and reached its maximum extent 7 days after injury (10 out of 10 animals at the seventh postoperative day, p<0.001 compared to C57BL/6 mice). Upon immuno-histological analysis calcification was accompanied by the occurrence of certain bone-matrix associated proteins. Osteopontin and Bone Morphogenetic Protein 2/4 expression was detected co-localized with the calcified lesions. Our results demonstrate that freeze-thaw injury of the infrarenal aorta is a sufficient method to induce dystrophic arterial calcification in mice. We present evidence that the occurrence of arterial calcification in C3H/He mice seems to be actively regulated by certain bone-matrix associated proteins.

A locus on chromosome 7 determines dramatic up-regulation of osteopontin in dystrophic cardiac calcification in mice.

Calcification of necrotic tissue is frequently observed in chronic inflammation and atherosclerosis. A similar response of myocardium to injury, referred to as dystrophic cardiac calcinosis (DCC), occurs in certain inbred strains of mice. We now examined a putative inhibitor of calcification, osteopontin, in DCC after transdiaphragmal myocardial freeze-thaw injury. Strong osteopontin expression was found co-localizing with calcification in DCC-susceptible strain C3H/HeNCrlBr, which exhibited low osteopontin plasma concentrations otherwise. Osteopontin mRNA induction was 20-fold higher than in resistant strain C57BL/6NCrlBr, which exhibited fibrous lesions without calcification and little osteopontin expression. Sequence analysis identified several polymorphisms in calcium-binding and phosphorylation sites in osteopontin cDNA. Their potential relevance for DCC was tested in congenic mice, which shared the osteopontin locus with C57BL/6NCrlBr, but retained a chromosomal segment from C3H/HeNCrlBr on proximal chromosome 7. These mice exhibited strong osteopontin expression and DCC comparable to C3H/HeNCrlBr suggesting that a trans-activator of osteopontin transcription residing on chromosome 7 and not the osteopontin gene on chromosome 5 was responsible for the genetic differences in osteopontin expression. A known osteopontin activator encoded by a gene on chromosome 7 is the transforming growth factor-beta1, which was more induced (3.5x) in C3H/HeNCrlBr than in C57BL/6NCrlBr mice.