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The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression.

Kelloniemi A, Szabo Z, Serpi R, Näpänkangas J, Ohukainen P, Tenhunen O, Kaikkonen L, Koivisto E, Bagyura Z, Kerkelä R, Leosdottir M, Hedner T, Melander O, Ruskoaho H, Rysä J - PLoS ONE (2015)

Bottom Line: We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects.When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle.In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550).

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine, Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland.

ABSTRACT
The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks' follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio.

No MeSH data available.


Related in: MedlinePlus

Phactr1 mRNA and protein levels increase dose-dependently after adenovirus-mediated Phactr1 gene delivery into left ventricle of rat.A) Phactr1 mRNA levels measured by RT-PCR and B) Phactr1 total protein levels analyzed by Western blot from the LV apex samples 3 days after gene delivery (n = 3). *P< 0.05 versus Phactr1 5×108 (one-way ANOVA followed by least significance difference post hoc test). C) Phactr1 mRNA levels 3 days, 1 week and 2 weeks after adenoviral gene transfer. Open bars represent LacZ 5×108 IFU/rat and solid bars Phactr1 5×108 IFU/rat (n = 6–9). *P< 0.05, **P<0.01, ***P<0.001 versus LacZ (Student’s t-test). D) Phactr1 total protein levels 3 days, 1 week and 2 weeks after adenoviral gene transfer (n = 6–10). *P< 0.05, ***P<0.001 versus group at 2 weeks (one-way ANOVA followed by least significance difference post hoc test). ND indicates not detected. E) Efficiency of gene transfer was confirmed by immunohistochemical staining against Phactr1 at day 3 after gene transfer. The results are expressed as mean±SEM. Representative Western blots are shown.
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pone.0130502.g002: Phactr1 mRNA and protein levels increase dose-dependently after adenovirus-mediated Phactr1 gene delivery into left ventricle of rat.A) Phactr1 mRNA levels measured by RT-PCR and B) Phactr1 total protein levels analyzed by Western blot from the LV apex samples 3 days after gene delivery (n = 3). *P< 0.05 versus Phactr1 5×108 (one-way ANOVA followed by least significance difference post hoc test). C) Phactr1 mRNA levels 3 days, 1 week and 2 weeks after adenoviral gene transfer. Open bars represent LacZ 5×108 IFU/rat and solid bars Phactr1 5×108 IFU/rat (n = 6–9). *P< 0.05, **P<0.01, ***P<0.001 versus LacZ (Student’s t-test). D) Phactr1 total protein levels 3 days, 1 week and 2 weeks after adenoviral gene transfer (n = 6–10). *P< 0.05, ***P<0.001 versus group at 2 weeks (one-way ANOVA followed by least significance difference post hoc test). ND indicates not detected. E) Efficiency of gene transfer was confirmed by immunohistochemical staining against Phactr1 at day 3 after gene transfer. The results are expressed as mean±SEM. Representative Western blots are shown.

Mentions: To examine the direct myocardial effects of Phactr1, we established an in vivo adenoviral gene transfer protocol to restore the decreased Phactr1 levels after MI in the LV of the adult rat heart. Three different doses (5×108, 1×109 and 2×109 infectious units) of adenoviral constructs were first tested to increase LV Phactr1 mRNA levels at day 3. Both Phactr1 mRNA and protein levels were elevated in a dose-dependent manner, with mRNA levels being nine times higher after the lowest dose (Fig 2A and 2B). Next, we studied the time-course of Phactr1 expression in response to adenovirus mediated delivery of Phactr1 into adult rat LV at a dose of 5×108 infectious units, the control animals receiving virus expressing LacZ also at 5×108 infectious units. Both the LV Phactr1 mRNA and protein levels were significantly elevated for up to 2 weeks (Fig 2C and 2D). When the localization of the Phactr1 expression was evaluated by immunohistochemistry, strong cytoplasmic and nuclear Phactr1 immunoreactivity localized to cardiomyocytes was observed, whereas endocardial endothelium, perivascular connective tissue and smooth muscle stained negative. No Phactr1 immunoreactivity was detected in LacZ-injected hearts (Fig 2E).


The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression.

Kelloniemi A, Szabo Z, Serpi R, Näpänkangas J, Ohukainen P, Tenhunen O, Kaikkonen L, Koivisto E, Bagyura Z, Kerkelä R, Leosdottir M, Hedner T, Melander O, Ruskoaho H, Rysä J - PLoS ONE (2015)

Phactr1 mRNA and protein levels increase dose-dependently after adenovirus-mediated Phactr1 gene delivery into left ventricle of rat.A) Phactr1 mRNA levels measured by RT-PCR and B) Phactr1 total protein levels analyzed by Western blot from the LV apex samples 3 days after gene delivery (n = 3). *P< 0.05 versus Phactr1 5×108 (one-way ANOVA followed by least significance difference post hoc test). C) Phactr1 mRNA levels 3 days, 1 week and 2 weeks after adenoviral gene transfer. Open bars represent LacZ 5×108 IFU/rat and solid bars Phactr1 5×108 IFU/rat (n = 6–9). *P< 0.05, **P<0.01, ***P<0.001 versus LacZ (Student’s t-test). D) Phactr1 total protein levels 3 days, 1 week and 2 weeks after adenoviral gene transfer (n = 6–10). *P< 0.05, ***P<0.001 versus group at 2 weeks (one-way ANOVA followed by least significance difference post hoc test). ND indicates not detected. E) Efficiency of gene transfer was confirmed by immunohistochemical staining against Phactr1 at day 3 after gene transfer. The results are expressed as mean±SEM. Representative Western blots are shown.
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pone.0130502.g002: Phactr1 mRNA and protein levels increase dose-dependently after adenovirus-mediated Phactr1 gene delivery into left ventricle of rat.A) Phactr1 mRNA levels measured by RT-PCR and B) Phactr1 total protein levels analyzed by Western blot from the LV apex samples 3 days after gene delivery (n = 3). *P< 0.05 versus Phactr1 5×108 (one-way ANOVA followed by least significance difference post hoc test). C) Phactr1 mRNA levels 3 days, 1 week and 2 weeks after adenoviral gene transfer. Open bars represent LacZ 5×108 IFU/rat and solid bars Phactr1 5×108 IFU/rat (n = 6–9). *P< 0.05, **P<0.01, ***P<0.001 versus LacZ (Student’s t-test). D) Phactr1 total protein levels 3 days, 1 week and 2 weeks after adenoviral gene transfer (n = 6–10). *P< 0.05, ***P<0.001 versus group at 2 weeks (one-way ANOVA followed by least significance difference post hoc test). ND indicates not detected. E) Efficiency of gene transfer was confirmed by immunohistochemical staining against Phactr1 at day 3 after gene transfer. The results are expressed as mean±SEM. Representative Western blots are shown.
Mentions: To examine the direct myocardial effects of Phactr1, we established an in vivo adenoviral gene transfer protocol to restore the decreased Phactr1 levels after MI in the LV of the adult rat heart. Three different doses (5×108, 1×109 and 2×109 infectious units) of adenoviral constructs were first tested to increase LV Phactr1 mRNA levels at day 3. Both Phactr1 mRNA and protein levels were elevated in a dose-dependent manner, with mRNA levels being nine times higher after the lowest dose (Fig 2A and 2B). Next, we studied the time-course of Phactr1 expression in response to adenovirus mediated delivery of Phactr1 into adult rat LV at a dose of 5×108 infectious units, the control animals receiving virus expressing LacZ also at 5×108 infectious units. Both the LV Phactr1 mRNA and protein levels were significantly elevated for up to 2 weeks (Fig 2C and 2D). When the localization of the Phactr1 expression was evaluated by immunohistochemistry, strong cytoplasmic and nuclear Phactr1 immunoreactivity localized to cardiomyocytes was observed, whereas endocardial endothelium, perivascular connective tissue and smooth muscle stained negative. No Phactr1 immunoreactivity was detected in LacZ-injected hearts (Fig 2E).

Bottom Line: We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects.When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle.In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550).

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine, Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland.

ABSTRACT
The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks' follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio.

No MeSH data available.


Related in: MedlinePlus