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Cyclosporine A Treatment Inhibits Abcc6-Dependent Cardiac Necrosis and Calcification following Coxsackievirus B3 Infection in Mice.

Marton J, Albert D, Wiltshire SA, Park R, Bergen A, Qureshi S, Malo D, Burelle Y, Vidal SM - PLoS ONE (2015)

Bottom Line: The damaged tissue that results is replaced with fibrotic or calcified tissue, which can lead to permanently altered cardiac function.Microarray expression profiling identified genotype-dependent regulation of genes associated with mitochondria.Cyclosporine A (CsA) inhibits mitochondrial permeability transition pore opening by inhibiting cyclophilin D (CypD).

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics and Complex Traits Group, McGill University, Montreal, Canada.

ABSTRACT
Coxsackievirus type B3 (CVB3) is a cardiotropic enterovirus. Infection causes cardiomyocyte necrosis and myocardial inflammation. The damaged tissue that results is replaced with fibrotic or calcified tissue, which can lead to permanently altered cardiac function. The extent of pathogenesis among individuals exposed to CVB3 is dictated by a combination of host genetics, viral virulence, and the environment. Here, we aimed to identify genes that modulate cardiopathology following CVB3 infection. 129S1 mice infected with CVB3 developed increased cardiac pathology compared to 129X1 substrain mice despite no difference in viral burden. Linkage analysis identified a major locus on chromosome 7 (LOD: 8.307, P<0.0001) that controlled the severity of cardiac calcification and necrosis following infection. Sub-phenotyping and genetic complementation assays identified Abcc6 as the underlying gene. Microarray expression profiling identified genotype-dependent regulation of genes associated with mitochondria. Electron microscopy examination showed elevated deposition of hydroxyapatite-like material in the mitochondrial matrices of infected Abcc6 knockout (Abcc6-/-) mice but not in wildtype littermates. Cyclosporine A (CsA) inhibits mitochondrial permeability transition pore opening by inhibiting cyclophilin D (CypD). Treatment of Abcc6 -/- mice with CsA reduced cardiac necrosis and calcification by more than half. Furthermore, CsA had no effect on the CVB3-induced phenotype of doubly deficient CypD-/-Abcc6-/- mice. Altogether, our work demonstrates that mutations in Abcc6 render mice more susceptible to cardiac calcification following CVB3 infection. Moreover, we implicate CypD in the control of cardiac necrosis and calcification in Abcc6-deficient mice, whereby CypD inhibition is required for cardioprotection.

No MeSH data available.


Related in: MedlinePlus

Causative gene identification by allelic complementation tests.Cardiac calcification was quantified in Abcc6 KO, Het, WT, (Abcc6 KO x 129S1)F1, (Abcc6 WTxS1)F1, 129S1, and 129X1 mice before infection and at day 8 post-infection. Abcc6 KO, Het, and WT mice were infected with 50pfu/g CVB3; the rest were infected with 500pfu/g CVB3. The concentration of calcium in uninfected mice was equal between all strains (A, B). At day 8 post-infection, increased calcification was consistently observed only in Abcc6 KO, (Abcc6 KO x 129S1)F1, and 129S1 mice (A,B). The severity of inflammation was evaluated in the same mice. Although differences between 129S1 and 129X1 were apparent, inflammation severity did not differ significantly between the other groups (C, D). The statistical test for panels A and B was a two-way ANOVA followed by Bonferonni post tests. The statistical test for panels C and D was a one-way ANOVA followed by a Dunn’s post-test. *: P<0.05, ***: P<0.001, ns: not significant.
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pone.0138222.g004: Causative gene identification by allelic complementation tests.Cardiac calcification was quantified in Abcc6 KO, Het, WT, (Abcc6 KO x 129S1)F1, (Abcc6 WTxS1)F1, 129S1, and 129X1 mice before infection and at day 8 post-infection. Abcc6 KO, Het, and WT mice were infected with 50pfu/g CVB3; the rest were infected with 500pfu/g CVB3. The concentration of calcium in uninfected mice was equal between all strains (A, B). At day 8 post-infection, increased calcification was consistently observed only in Abcc6 KO, (Abcc6 KO x 129S1)F1, and 129S1 mice (A,B). The severity of inflammation was evaluated in the same mice. Although differences between 129S1 and 129X1 were apparent, inflammation severity did not differ significantly between the other groups (C, D). The statistical test for panels A and B was a two-way ANOVA followed by Bonferonni post tests. The statistical test for panels C and D was a one-way ANOVA followed by a Dunn’s post-test. *: P<0.05, ***: P<0.001, ns: not significant.

Mentions: Abcc6 was a promising candidate gene because of its genomic location (chr 7: 45.9Mbp) and its known role in dystrophic cardiac calcification. Meng et al showed that a splice site mutation in Abcc6 (c.1866C>T, p.R621C, rs32756904) causes dystrophic cardiac calcification in C3H/HeJ mice[31]. The mutation creates a new splice donor site, which causes a 5bp deletion, leads to a premature stop codon and results in a 60% reduction in protein expression [32]. Interestingly, genetic contamination of 129S1 DNA with C3H/HeJ DNA has been described [33]. Moreover, C3H/HeJ and 129S1 mice share a common haplotype, which is distinct from the 129X1 haplotype, in the chr. 7 QTL region (Fig 3A). We therefore sequenced Abcc6 in 129S1 and 129X1 animals and found that 129S1 mice possess the mutant allele whereas 129X1 mice possess the wildtype allele (Fig 3B). To determine whether this mutation affects Abcc6 expression, we measured mRNA levels in 129S1 and 129X1 livers. Abcc6 mRNA expression was reduced by twofold (P = 5.15e-08) in 129S1 mice compared to 129X1 mice (Fig 3C). This was comparable to the reduction in protein expression observed in the C3H/HeJ mice. Given this, we assessed whether Abcc6 was the gene underlying our locus using Abcc6 KO mice (Fig 4A and 4C) and genetic complementation (Fig 4B and 4D). We first evaluated calcification in Abcc6 KO, Het and WT mice at days 0 and 8 post infection. No genotype-dependent differences in calcification were observed in uninfected mice (Fig 4A). Upon infection, levels of calcium in Abcc6 WT hearts remained constant with infection whereas levels in Abcc6 KO hearts increased drastically with infection (Fig 4A). We next evaluated calcium levels in two genetic complementation groups, (Abcc6 KO x 129S1)F1 and (Abcc6 WT x 129S1)F1 mice, at days 0 and 8 post infection (Fig 4B). Increased calcification was observed in (Abcc6 KO x 129S1)F1 but not in (Abcc6 WT x 129S1)F1 mice (p<0.001), thereby confirming that Abcc6 is responsible for the CVB3-induced calcification in 129S1 mice. Interestingly, inflammation severity remained unaffected by genotype in both experiments suggesting a role for genetic background in modulating this phenotype (Fig 4C and 4D).


Cyclosporine A Treatment Inhibits Abcc6-Dependent Cardiac Necrosis and Calcification following Coxsackievirus B3 Infection in Mice.

Marton J, Albert D, Wiltshire SA, Park R, Bergen A, Qureshi S, Malo D, Burelle Y, Vidal SM - PLoS ONE (2015)

Causative gene identification by allelic complementation tests.Cardiac calcification was quantified in Abcc6 KO, Het, WT, (Abcc6 KO x 129S1)F1, (Abcc6 WTxS1)F1, 129S1, and 129X1 mice before infection and at day 8 post-infection. Abcc6 KO, Het, and WT mice were infected with 50pfu/g CVB3; the rest were infected with 500pfu/g CVB3. The concentration of calcium in uninfected mice was equal between all strains (A, B). At day 8 post-infection, increased calcification was consistently observed only in Abcc6 KO, (Abcc6 KO x 129S1)F1, and 129S1 mice (A,B). The severity of inflammation was evaluated in the same mice. Although differences between 129S1 and 129X1 were apparent, inflammation severity did not differ significantly between the other groups (C, D). The statistical test for panels A and B was a two-way ANOVA followed by Bonferonni post tests. The statistical test for panels C and D was a one-way ANOVA followed by a Dunn’s post-test. *: P<0.05, ***: P<0.001, ns: not significant.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4574283&req=5

pone.0138222.g004: Causative gene identification by allelic complementation tests.Cardiac calcification was quantified in Abcc6 KO, Het, WT, (Abcc6 KO x 129S1)F1, (Abcc6 WTxS1)F1, 129S1, and 129X1 mice before infection and at day 8 post-infection. Abcc6 KO, Het, and WT mice were infected with 50pfu/g CVB3; the rest were infected with 500pfu/g CVB3. The concentration of calcium in uninfected mice was equal between all strains (A, B). At day 8 post-infection, increased calcification was consistently observed only in Abcc6 KO, (Abcc6 KO x 129S1)F1, and 129S1 mice (A,B). The severity of inflammation was evaluated in the same mice. Although differences between 129S1 and 129X1 were apparent, inflammation severity did not differ significantly between the other groups (C, D). The statistical test for panels A and B was a two-way ANOVA followed by Bonferonni post tests. The statistical test for panels C and D was a one-way ANOVA followed by a Dunn’s post-test. *: P<0.05, ***: P<0.001, ns: not significant.
Mentions: Abcc6 was a promising candidate gene because of its genomic location (chr 7: 45.9Mbp) and its known role in dystrophic cardiac calcification. Meng et al showed that a splice site mutation in Abcc6 (c.1866C>T, p.R621C, rs32756904) causes dystrophic cardiac calcification in C3H/HeJ mice[31]. The mutation creates a new splice donor site, which causes a 5bp deletion, leads to a premature stop codon and results in a 60% reduction in protein expression [32]. Interestingly, genetic contamination of 129S1 DNA with C3H/HeJ DNA has been described [33]. Moreover, C3H/HeJ and 129S1 mice share a common haplotype, which is distinct from the 129X1 haplotype, in the chr. 7 QTL region (Fig 3A). We therefore sequenced Abcc6 in 129S1 and 129X1 animals and found that 129S1 mice possess the mutant allele whereas 129X1 mice possess the wildtype allele (Fig 3B). To determine whether this mutation affects Abcc6 expression, we measured mRNA levels in 129S1 and 129X1 livers. Abcc6 mRNA expression was reduced by twofold (P = 5.15e-08) in 129S1 mice compared to 129X1 mice (Fig 3C). This was comparable to the reduction in protein expression observed in the C3H/HeJ mice. Given this, we assessed whether Abcc6 was the gene underlying our locus using Abcc6 KO mice (Fig 4A and 4C) and genetic complementation (Fig 4B and 4D). We first evaluated calcification in Abcc6 KO, Het and WT mice at days 0 and 8 post infection. No genotype-dependent differences in calcification were observed in uninfected mice (Fig 4A). Upon infection, levels of calcium in Abcc6 WT hearts remained constant with infection whereas levels in Abcc6 KO hearts increased drastically with infection (Fig 4A). We next evaluated calcium levels in two genetic complementation groups, (Abcc6 KO x 129S1)F1 and (Abcc6 WT x 129S1)F1 mice, at days 0 and 8 post infection (Fig 4B). Increased calcification was observed in (Abcc6 KO x 129S1)F1 but not in (Abcc6 WT x 129S1)F1 mice (p<0.001), thereby confirming that Abcc6 is responsible for the CVB3-induced calcification in 129S1 mice. Interestingly, inflammation severity remained unaffected by genotype in both experiments suggesting a role for genetic background in modulating this phenotype (Fig 4C and 4D).

Bottom Line: The damaged tissue that results is replaced with fibrotic or calcified tissue, which can lead to permanently altered cardiac function.Microarray expression profiling identified genotype-dependent regulation of genes associated with mitochondria.Cyclosporine A (CsA) inhibits mitochondrial permeability transition pore opening by inhibiting cyclophilin D (CypD).

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics and Complex Traits Group, McGill University, Montreal, Canada.

ABSTRACT
Coxsackievirus type B3 (CVB3) is a cardiotropic enterovirus. Infection causes cardiomyocyte necrosis and myocardial inflammation. The damaged tissue that results is replaced with fibrotic or calcified tissue, which can lead to permanently altered cardiac function. The extent of pathogenesis among individuals exposed to CVB3 is dictated by a combination of host genetics, viral virulence, and the environment. Here, we aimed to identify genes that modulate cardiopathology following CVB3 infection. 129S1 mice infected with CVB3 developed increased cardiac pathology compared to 129X1 substrain mice despite no difference in viral burden. Linkage analysis identified a major locus on chromosome 7 (LOD: 8.307, P<0.0001) that controlled the severity of cardiac calcification and necrosis following infection. Sub-phenotyping and genetic complementation assays identified Abcc6 as the underlying gene. Microarray expression profiling identified genotype-dependent regulation of genes associated with mitochondria. Electron microscopy examination showed elevated deposition of hydroxyapatite-like material in the mitochondrial matrices of infected Abcc6 knockout (Abcc6-/-) mice but not in wildtype littermates. Cyclosporine A (CsA) inhibits mitochondrial permeability transition pore opening by inhibiting cyclophilin D (CypD). Treatment of Abcc6 -/- mice with CsA reduced cardiac necrosis and calcification by more than half. Furthermore, CsA had no effect on the CVB3-induced phenotype of doubly deficient CypD-/-Abcc6-/- mice. Altogether, our work demonstrates that mutations in Abcc6 render mice more susceptible to cardiac calcification following CVB3 infection. Moreover, we implicate CypD in the control of cardiac necrosis and calcification in Abcc6-deficient mice, whereby CypD inhibition is required for cardioprotection.

No MeSH data available.


Related in: MedlinePlus