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CAP2 in cardiac conduction, sudden cardiac death and eye development.

Field J, Ye DZ, Shinde M, Liu F, Schillinger KJ, Lu M, Wang T, Skettini M, Xiong Y, Brice AK, Chung DC, Patel VV - Sci Rep (2015)

Bottom Line: One gene at 6p22 is CAP2, which encodes a cytoskeletal protein that regulates actin dynamics.To address the mechanisms underlying these phenotypes, we used Cre-mediated recombination to knock out CAP2 in cardiomyocytes.We found that the mice developed CCD, leading to sudden cardiac death from complete heart block, but no longer developed DCM or the other phenotypes, including sex bias.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine Philadelphia, Pennsylvania 19041 USA.

ABSTRACT
Sudden cardiac death kills 180,000 to 450,000 Americans annually, predominantly males. A locus that confers a risk for sudden cardiac death, cardiac conduction disease, and a newly described developmental disorder (6p22 syndrome) is located at 6p22. One gene at 6p22 is CAP2, which encodes a cytoskeletal protein that regulates actin dynamics. To determine the role of CAP2 in vivo, we generated knockout (KO) mice. cap2(-)/cap2(-) males were underrepresented at weaning and ~70% died by 12 weeks of age, but cap2(-)/cap2(-) females survived at close to the expected levels and lived normal life spans. CAP2 knockouts resembled patients with 6p22 syndrome in that mice were smaller and they developed microphthalmia and cardiac disease. The cardiac disease included cardiac conduction disease (CCD) and, after six months of age, dilated cardiomyopathy (DCM), most noticeably in the males. To address the mechanisms underlying these phenotypes, we used Cre-mediated recombination to knock out CAP2 in cardiomyocytes. We found that the mice developed CCD, leading to sudden cardiac death from complete heart block, but no longer developed DCM or the other phenotypes, including sex bias. These studies establish a direct role for CAP2 and actin dynamics in sudden cardiac death and cardiac conduction disease.

No MeSH data available.


Related in: MedlinePlus

CAP2 knockout strains and survival curves.(a) the strategy for creating the CAP2 knockout mice used in this study and an example of a genotyping gel, (b) upper panel, expression of CAP2 in heart, muscle and in brain analyzed by qPCR; lower panel, expression in hearts analyzed by qPCR. The ages of mice in the upper panel are 3 wks. Three mice of varying ages were used for each genotype in the lower panel. Although the ages ranged from 3 weeks to 69 weeks, expression levels did not change significantly between mice of the different ages (c) Western blots of CAP2 in heart and muscle. (d) Kaplan-Meier survival curve for males (e) Kaplan-Meier survival curve for females. The genotypes are: wild type (+/+), whole body knockout (cap2−/cap2− or −/−), rescued mice (cap2loxp/cap2loxp or L/L) and cardiac specific knockouts (Myh6Cre-cap2loxp/cap2loxp or L/L, Cre+). Error bars ± S.E.M.
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f1: CAP2 knockout strains and survival curves.(a) the strategy for creating the CAP2 knockout mice used in this study and an example of a genotyping gel, (b) upper panel, expression of CAP2 in heart, muscle and in brain analyzed by qPCR; lower panel, expression in hearts analyzed by qPCR. The ages of mice in the upper panel are 3 wks. Three mice of varying ages were used for each genotype in the lower panel. Although the ages ranged from 3 weeks to 69 weeks, expression levels did not change significantly between mice of the different ages (c) Western blots of CAP2 in heart and muscle. (d) Kaplan-Meier survival curve for males (e) Kaplan-Meier survival curve for females. The genotypes are: wild type (+/+), whole body knockout (cap2−/cap2− or −/−), rescued mice (cap2loxp/cap2loxp or L/L) and cardiac specific knockouts (Myh6Cre-cap2loxp/cap2loxp or L/L, Cre+). Error bars ± S.E.M.

Mentions: To determine the role of CAP2 in mice, we knocked out CAP2 in C57/B6 mice by insertional mutagenesis. The European Conditional Mouse Consortium (EUCOMM) generated the targeting construct and ES clones. Figure 1a shows the strategy for creating three transgenic genotypes, cap2−, cap2loxp and Myh6Cre-cap2loxp/cap2loxp as well as an example of a genotyping experiment. The cap2− construct introduced an insert within intron 2 that results in a truncated fusion protein containing 40 amino acids of CAP2. The construct also introduced FRT sites flanking the insert and loxP sites flanking exon 3. Crossing cap2− mice to actin-FLP mice to create cap2loxp deleted most of the insert and restored CAP2 expression, but left the two loxP sites flanking exon 3. Crossing the cap2loxp mice to appropriate Cre-recombinase expressing mice creates tissue-specific deletions of CAP2. Deletion of the cap2 gene was tested by extensive PCR mapping of the ES cell lines. To analyze expression, we extracted RNA from heart, muscle and brain of wild-type controls (+/+), heterozygotes (+/−) and knockout mice (−/−), and performed q-PCR reactions (Fig. 1b upper panel). cap2 expression was significantly reduced in all three tissues in the −/− mice, while there was a partial loss of expression in heterozygotes. Additionally, crossing cap2loxp/cap2loxp with the Myh6Cre reduced the message in the heart to knockout levels (Fig. 1b, lower panel). We also tested the expression of CAP2 in heart, muscle and brain on western blots probed with a polyclonal antibody (Fig. 1c; data not shown for brain). CAP2 protein levels were diminished in cap2−/cap2− mice, restored in cap2loxp/cap2loxp mice and not detected in the hearts Myh6Cre-cap2loxp/cap2loxp mice (Fig. 1c).


CAP2 in cardiac conduction, sudden cardiac death and eye development.

Field J, Ye DZ, Shinde M, Liu F, Schillinger KJ, Lu M, Wang T, Skettini M, Xiong Y, Brice AK, Chung DC, Patel VV - Sci Rep (2015)

CAP2 knockout strains and survival curves.(a) the strategy for creating the CAP2 knockout mice used in this study and an example of a genotyping gel, (b) upper panel, expression of CAP2 in heart, muscle and in brain analyzed by qPCR; lower panel, expression in hearts analyzed by qPCR. The ages of mice in the upper panel are 3 wks. Three mice of varying ages were used for each genotype in the lower panel. Although the ages ranged from 3 weeks to 69 weeks, expression levels did not change significantly between mice of the different ages (c) Western blots of CAP2 in heart and muscle. (d) Kaplan-Meier survival curve for males (e) Kaplan-Meier survival curve for females. The genotypes are: wild type (+/+), whole body knockout (cap2−/cap2− or −/−), rescued mice (cap2loxp/cap2loxp or L/L) and cardiac specific knockouts (Myh6Cre-cap2loxp/cap2loxp or L/L, Cre+). Error bars ± S.E.M.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663486&req=5

f1: CAP2 knockout strains and survival curves.(a) the strategy for creating the CAP2 knockout mice used in this study and an example of a genotyping gel, (b) upper panel, expression of CAP2 in heart, muscle and in brain analyzed by qPCR; lower panel, expression in hearts analyzed by qPCR. The ages of mice in the upper panel are 3 wks. Three mice of varying ages were used for each genotype in the lower panel. Although the ages ranged from 3 weeks to 69 weeks, expression levels did not change significantly between mice of the different ages (c) Western blots of CAP2 in heart and muscle. (d) Kaplan-Meier survival curve for males (e) Kaplan-Meier survival curve for females. The genotypes are: wild type (+/+), whole body knockout (cap2−/cap2− or −/−), rescued mice (cap2loxp/cap2loxp or L/L) and cardiac specific knockouts (Myh6Cre-cap2loxp/cap2loxp or L/L, Cre+). Error bars ± S.E.M.
Mentions: To determine the role of CAP2 in mice, we knocked out CAP2 in C57/B6 mice by insertional mutagenesis. The European Conditional Mouse Consortium (EUCOMM) generated the targeting construct and ES clones. Figure 1a shows the strategy for creating three transgenic genotypes, cap2−, cap2loxp and Myh6Cre-cap2loxp/cap2loxp as well as an example of a genotyping experiment. The cap2− construct introduced an insert within intron 2 that results in a truncated fusion protein containing 40 amino acids of CAP2. The construct also introduced FRT sites flanking the insert and loxP sites flanking exon 3. Crossing cap2− mice to actin-FLP mice to create cap2loxp deleted most of the insert and restored CAP2 expression, but left the two loxP sites flanking exon 3. Crossing the cap2loxp mice to appropriate Cre-recombinase expressing mice creates tissue-specific deletions of CAP2. Deletion of the cap2 gene was tested by extensive PCR mapping of the ES cell lines. To analyze expression, we extracted RNA from heart, muscle and brain of wild-type controls (+/+), heterozygotes (+/−) and knockout mice (−/−), and performed q-PCR reactions (Fig. 1b upper panel). cap2 expression was significantly reduced in all three tissues in the −/− mice, while there was a partial loss of expression in heterozygotes. Additionally, crossing cap2loxp/cap2loxp with the Myh6Cre reduced the message in the heart to knockout levels (Fig. 1b, lower panel). We also tested the expression of CAP2 in heart, muscle and brain on western blots probed with a polyclonal antibody (Fig. 1c; data not shown for brain). CAP2 protein levels were diminished in cap2−/cap2− mice, restored in cap2loxp/cap2loxp mice and not detected in the hearts Myh6Cre-cap2loxp/cap2loxp mice (Fig. 1c).

Bottom Line: One gene at 6p22 is CAP2, which encodes a cytoskeletal protein that regulates actin dynamics.To address the mechanisms underlying these phenotypes, we used Cre-mediated recombination to knock out CAP2 in cardiomyocytes.We found that the mice developed CCD, leading to sudden cardiac death from complete heart block, but no longer developed DCM or the other phenotypes, including sex bias.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine Philadelphia, Pennsylvania 19041 USA.

ABSTRACT
Sudden cardiac death kills 180,000 to 450,000 Americans annually, predominantly males. A locus that confers a risk for sudden cardiac death, cardiac conduction disease, and a newly described developmental disorder (6p22 syndrome) is located at 6p22. One gene at 6p22 is CAP2, which encodes a cytoskeletal protein that regulates actin dynamics. To determine the role of CAP2 in vivo, we generated knockout (KO) mice. cap2(-)/cap2(-) males were underrepresented at weaning and ~70% died by 12 weeks of age, but cap2(-)/cap2(-) females survived at close to the expected levels and lived normal life spans. CAP2 knockouts resembled patients with 6p22 syndrome in that mice were smaller and they developed microphthalmia and cardiac disease. The cardiac disease included cardiac conduction disease (CCD) and, after six months of age, dilated cardiomyopathy (DCM), most noticeably in the males. To address the mechanisms underlying these phenotypes, we used Cre-mediated recombination to knock out CAP2 in cardiomyocytes. We found that the mice developed CCD, leading to sudden cardiac death from complete heart block, but no longer developed DCM or the other phenotypes, including sex bias. These studies establish a direct role for CAP2 and actin dynamics in sudden cardiac death and cardiac conduction disease.

No MeSH data available.


Related in: MedlinePlus