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The role of mAKAPβ in the process of cardiomyocyte hypertrophy induced by angiotensin II.

Guo H, Liu B, Hou L, The E, Li G, Wang D, Jie Q, Che W, Wei Y - Int. J. Mol. Med. (2015)

Bottom Line: The cell size of the AngII-treated cardiaomyocytes was significantly larger than that of the untreated cardiomyocytes.The expression of hypertrophic markers and p-ERK2, the cell surface area and the [3H]Leucine incorporation rate were all significantly increased in the AngII‑treated cells.However, the expression of mAKAPβ remained unaltered in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China.

ABSTRACT
Angiotensin II (AngII) is the central product of the renin-angiotensin system (RAS) and this octapeptide contributes to the pathophysiology of cardiac hypertrophy and remodeling. mAKAPβ is an A-kinase anchoring protein (AKAP) that has the function of binding to the regulatory subunit of protein kinase A (PKA) and confining the holoenzyme to discrete locations within the cell. In this study, we aimed to investigate the role of mAKAPβ in AngII‑induced cardiomyocyte hypertrophy and the possible mechanisms involved. Cultured cardiomyocytes from neonatal rats were treated with AngII. Subsequently, the morphology of the cardiomyocytes was observed and the expression of mAKAPβ and cardiomyocyte hypertrophic markers was measured. mAKAPβ‑shRNA was constructed for RNA interference; the expression of mAKAPβ and hypertrophic markers, the cell surface area and the [3H]Leucine incorporation rate in the AngII‑treated rat cardiomyocytes were detected following RNA interference. Simultaneously, changes in the expression levels of phosphorylated extracellular signal-regulated kinase (p-ERK)2 in the cardiomyocytes were assessed. The cell size of the AngII-treated cardiaomyocytes was significantly larger than that of the untreated cardiomyocytes. The expression of hypertrophic markers and p-ERK2, the cell surface area and the [3H]Leucine incorporation rate were all significantly increased in the AngII‑treated cells. However, the expression of mAKAPβ remained unaltered in this process. RNA interference simultaneously inhibited the protein expression of mAKAPβ and p‑ERK2, and the hypertrophy of the cardiomyocytes induced by AngII was attenuated. These results demonstrate that AngII induces hypertrophy in cardiomyocytes, and mAKAPβ is possibly involved in this process. The effects of mAKAPβ on AngII‑induced cardiomyocyte hypertrophy may be associated with p-ERK2 expression.

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Expression of mAKAPβ in cardiomyocytes following the different treatments. Six sets of cardiomyocytes were divided into 3 groups: i) blank group, no virus; ii) mock group, control virus, and iii) pLvx-mAKAPβ-shRNA lentivirus group. (A) Representative results of assays of mAKAPβ and GAPDH abundance in the 3 groups by western blot analysis. (B) The protein expression levels of mAKAPβ and GAPDH were analyzed by western blot analysis using polyclonal antibodies to mAKAPβ and GAPDH to quantify the expression in these groups. Following treatment with angiotensin II (AngII), the expression of mAKAPβ in the pLvx-mAKAPβ-shRNA lentivirus group was significantly lower when compared with that of the AngII-treated cells in the blank and mock groups (both P<0.05). No statistically significant differences was observed in the expression of mAKAPβ in the blank and mock groups following treatment with AngII compared with the controls (P=0.08 and P=0.06, respectively). GAPDH was used as an equal loading control. The band value was quantified by densitometric analysis. Experiments were repeated at least 3 times. Data are expressed as the means ± SD in the corresponding bar graph and statistical significance was determined by the Student’s t-test. Ctrl, control. Columns, mean; error bars, ± SD; *P<0.05.
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f5-ijmm-35-05-1159: Expression of mAKAPβ in cardiomyocytes following the different treatments. Six sets of cardiomyocytes were divided into 3 groups: i) blank group, no virus; ii) mock group, control virus, and iii) pLvx-mAKAPβ-shRNA lentivirus group. (A) Representative results of assays of mAKAPβ and GAPDH abundance in the 3 groups by western blot analysis. (B) The protein expression levels of mAKAPβ and GAPDH were analyzed by western blot analysis using polyclonal antibodies to mAKAPβ and GAPDH to quantify the expression in these groups. Following treatment with angiotensin II (AngII), the expression of mAKAPβ in the pLvx-mAKAPβ-shRNA lentivirus group was significantly lower when compared with that of the AngII-treated cells in the blank and mock groups (both P<0.05). No statistically significant differences was observed in the expression of mAKAPβ in the blank and mock groups following treatment with AngII compared with the controls (P=0.08 and P=0.06, respectively). GAPDH was used as an equal loading control. The band value was quantified by densitometric analysis. Experiments were repeated at least 3 times. Data are expressed as the means ± SD in the corresponding bar graph and statistical significance was determined by the Student’s t-test. Ctrl, control. Columns, mean; error bars, ± SD; *P<0.05.

Mentions: The requirement for mAKAPβ in AngII-induced cardiomyocyte hypertrophy was examined by RNA interference using mAKAPβ-shRNA which was constructed as aforementioned (see Materials and methods). Six sets of cardiomyocytes were divided into 3 groups: i) the blank group, no virus; ii) the mock group, control virus; and iii) the pLvx-mAKAPβ-shRNA lentivirus group. After 24 h, the culture medium was changed to maintenance medium which does not contain serum. One set from each group was treated with 10−8 mol/l AngII, and the other set was used as a control. All myocardial cells were then cultured for 72 h. The results from western blot analysis revealed that the expression of mAKAPβ in the blank and mock groups showed no statistically significant difference following treatment with AngII compared with the control (P=0.08 and P=0.06, respectively; Fig. 5). However, the expression of mAKAPβ was significantly reduced in the pLvx-mAKAPβ-shRNA lentivirus group following RNA interference. Moreover, even following treatment with AngII, the expression of mAKAPβ in the pLvx-mAKAPβ-shRNA lentivirus group was significantly lower when compared with that of the AngII-treated cells in the blank and mock groups (both P<0.05; Fig. 5).


The role of mAKAPβ in the process of cardiomyocyte hypertrophy induced by angiotensin II.

Guo H, Liu B, Hou L, The E, Li G, Wang D, Jie Q, Che W, Wei Y - Int. J. Mol. Med. (2015)

Expression of mAKAPβ in cardiomyocytes following the different treatments. Six sets of cardiomyocytes were divided into 3 groups: i) blank group, no virus; ii) mock group, control virus, and iii) pLvx-mAKAPβ-shRNA lentivirus group. (A) Representative results of assays of mAKAPβ and GAPDH abundance in the 3 groups by western blot analysis. (B) The protein expression levels of mAKAPβ and GAPDH were analyzed by western blot analysis using polyclonal antibodies to mAKAPβ and GAPDH to quantify the expression in these groups. Following treatment with angiotensin II (AngII), the expression of mAKAPβ in the pLvx-mAKAPβ-shRNA lentivirus group was significantly lower when compared with that of the AngII-treated cells in the blank and mock groups (both P<0.05). No statistically significant differences was observed in the expression of mAKAPβ in the blank and mock groups following treatment with AngII compared with the controls (P=0.08 and P=0.06, respectively). GAPDH was used as an equal loading control. The band value was quantified by densitometric analysis. Experiments were repeated at least 3 times. Data are expressed as the means ± SD in the corresponding bar graph and statistical significance was determined by the Student’s t-test. Ctrl, control. Columns, mean; error bars, ± SD; *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380120&req=5

f5-ijmm-35-05-1159: Expression of mAKAPβ in cardiomyocytes following the different treatments. Six sets of cardiomyocytes were divided into 3 groups: i) blank group, no virus; ii) mock group, control virus, and iii) pLvx-mAKAPβ-shRNA lentivirus group. (A) Representative results of assays of mAKAPβ and GAPDH abundance in the 3 groups by western blot analysis. (B) The protein expression levels of mAKAPβ and GAPDH were analyzed by western blot analysis using polyclonal antibodies to mAKAPβ and GAPDH to quantify the expression in these groups. Following treatment with angiotensin II (AngII), the expression of mAKAPβ in the pLvx-mAKAPβ-shRNA lentivirus group was significantly lower when compared with that of the AngII-treated cells in the blank and mock groups (both P<0.05). No statistically significant differences was observed in the expression of mAKAPβ in the blank and mock groups following treatment with AngII compared with the controls (P=0.08 and P=0.06, respectively). GAPDH was used as an equal loading control. The band value was quantified by densitometric analysis. Experiments were repeated at least 3 times. Data are expressed as the means ± SD in the corresponding bar graph and statistical significance was determined by the Student’s t-test. Ctrl, control. Columns, mean; error bars, ± SD; *P<0.05.
Mentions: The requirement for mAKAPβ in AngII-induced cardiomyocyte hypertrophy was examined by RNA interference using mAKAPβ-shRNA which was constructed as aforementioned (see Materials and methods). Six sets of cardiomyocytes were divided into 3 groups: i) the blank group, no virus; ii) the mock group, control virus; and iii) the pLvx-mAKAPβ-shRNA lentivirus group. After 24 h, the culture medium was changed to maintenance medium which does not contain serum. One set from each group was treated with 10−8 mol/l AngII, and the other set was used as a control. All myocardial cells were then cultured for 72 h. The results from western blot analysis revealed that the expression of mAKAPβ in the blank and mock groups showed no statistically significant difference following treatment with AngII compared with the control (P=0.08 and P=0.06, respectively; Fig. 5). However, the expression of mAKAPβ was significantly reduced in the pLvx-mAKAPβ-shRNA lentivirus group following RNA interference. Moreover, even following treatment with AngII, the expression of mAKAPβ in the pLvx-mAKAPβ-shRNA lentivirus group was significantly lower when compared with that of the AngII-treated cells in the blank and mock groups (both P<0.05; Fig. 5).

Bottom Line: The cell size of the AngII-treated cardiaomyocytes was significantly larger than that of the untreated cardiomyocytes.The expression of hypertrophic markers and p-ERK2, the cell surface area and the [3H]Leucine incorporation rate were all significantly increased in the AngII‑treated cells.However, the expression of mAKAPβ remained unaltered in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China.

ABSTRACT
Angiotensin II (AngII) is the central product of the renin-angiotensin system (RAS) and this octapeptide contributes to the pathophysiology of cardiac hypertrophy and remodeling. mAKAPβ is an A-kinase anchoring protein (AKAP) that has the function of binding to the regulatory subunit of protein kinase A (PKA) and confining the holoenzyme to discrete locations within the cell. In this study, we aimed to investigate the role of mAKAPβ in AngII‑induced cardiomyocyte hypertrophy and the possible mechanisms involved. Cultured cardiomyocytes from neonatal rats were treated with AngII. Subsequently, the morphology of the cardiomyocytes was observed and the expression of mAKAPβ and cardiomyocyte hypertrophic markers was measured. mAKAPβ‑shRNA was constructed for RNA interference; the expression of mAKAPβ and hypertrophic markers, the cell surface area and the [3H]Leucine incorporation rate in the AngII‑treated rat cardiomyocytes were detected following RNA interference. Simultaneously, changes in the expression levels of phosphorylated extracellular signal-regulated kinase (p-ERK)2 in the cardiomyocytes were assessed. The cell size of the AngII-treated cardiaomyocytes was significantly larger than that of the untreated cardiomyocytes. The expression of hypertrophic markers and p-ERK2, the cell surface area and the [3H]Leucine incorporation rate were all significantly increased in the AngII‑treated cells. However, the expression of mAKAPβ remained unaltered in this process. RNA interference simultaneously inhibited the protein expression of mAKAPβ and p‑ERK2, and the hypertrophy of the cardiomyocytes induced by AngII was attenuated. These results demonstrate that AngII induces hypertrophy in cardiomyocytes, and mAKAPβ is possibly involved in this process. The effects of mAKAPβ on AngII‑induced cardiomyocyte hypertrophy may be associated with p-ERK2 expression.

Show MeSH
Related in: MedlinePlus