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  Myocardin Induces Cardiomyocyte Hypertrophy

  Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson and Da-Zhi Wang

  Circulation Research. 2006;98:1089-1097, originally published April 27, 2006

  https://doi.org/10.1161/01.RES.0000218781.23144.3e

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  Myocardin Induces Cardiomyocyte Hypertrophy

  Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson, Da-Zhi Wang

  Circulation Research April 2006, 98 (8) 1089-1097; DOI: https://doi.org/10.1161/01.RES.0000218781.23144.3e

  

  By Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson and Da-Zhi Wang

  Figure 1. Upregulation of myocardin expression by hypertrophic signals. A, Neonatal rat cardiomyocytes grown in serum-free media were stimulated with...Show More

  Figure 1. Upregulation of myocardin expression by hypertrophic signals. A, Neonatal rat cardiomyocytes grown in serum-free media were stimulated with 10% serum or 100 μmol/L of PE for 48 hours or untreated to serve as control. Total RNAs were isolated and the expression of myocardin, ANF, and skeletal α-actin were examined by semiquantitative RT-PCR. GAPDH was used to serve as a loading control. B, The intensity of PCR products on the agarose gel image was quantified using the Photoshop Histogram Analysis and was plotted using Microsoft Excel and is shown as relative expression level after being normalized by GAPDH. C, Neonatal rat cardiomyocytes grown in serum-free media were stimulated with 10% FBS, 100 μmol/L of PE, or 104 units/mL of LIF for 24 hours and 48 hours, respectively. Untreated cardiomyocytes served as control. Total proteins were extracted, and the protein level for myocardin was detected by Western blot analysis. β-Tubulin was used as a loading control. D, The intensity of protein products from Western blot was quantified using the Photoshop Histogram Analysis and was plotted using Microsoft Excel and is shown as relative expression level after normalized by β-tubulin. E, Semiquantitative RT-PCR analysis of transcripts for myocardin, ANF, β-MHC, and skeletal α-actin (sk-α-actin) in TAB and control hearts. GAPDH was used to serve as a loading control. F, The intensity of PCR products on the agarose gel image was quantified using the Photoshop Histogram Analysis and was plotted using Microsoft Excel and is shown as relative expression level. G, Total proteins were extracted from wild-type (WT) and calcineurin (CnA) transgenic hearts, and the protein level for myocardin was detected by Western blot analysis. Protein extracts from wild-type mouse liver were used as a control. β-Tubulin was used as a loading control. H, Protein extracts from indicated human patient hearts with or without IDC were subjected to Western blot using antimyocardin antibody (top). Protein extracts from wild-type mouse liver were used as a control. β-Tubulin was used as a loading control. Error bars represent SD of two experiments. Statistical differences were determined using the Student t test; *P<0.05.Show Less
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  Myocardin Induces Cardiomyocyte Hypertrophy

  Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson, Da-Zhi Wang

  Circulation Research April 2006, 98 (8) 1089-1097; DOI: https://doi.org/10.1161/01.RES.0000218781.23144.3e

  

  By Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson and Da-Zhi Wang

  Figure 2. Association of myocardin with the ANF promoter detected by ChIP assay. ChIP assays were performed using chromatin prepared from primary neon...Show More

  Figure 2. Association of myocardin with the ANF promoter detected by ChIP assay. ChIP assays were performed using chromatin prepared from primary neonatal cardiomyocytes in serum-free medium or after stimulation with PE (100 μmol/L) for 24 hours. Immunoprecipitations were performed without primary antibody (no Ab) or using a mouse IgG (IgG) as a control and with antimyocardin antibody as indicated. PCR primers for sequences associated with the promoters of the ANF and GAPDH genes were used.Show Less
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  Myocardin Induces Cardiomyocyte Hypertrophy

  Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson, Da-Zhi Wang

  Circulation Research April 2006, 98 (8) 1089-1097; DOI: https://doi.org/10.1161/01.RES.0000218781.23144.3e

  

  By Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson and Da-Zhi Wang

  Figure 3. Myocardin activity is enhanced by hypertrophic stimuli in cardiomyocytes. Neonatal rat cardiomyocytes were transfected with the indicated lu...Show More

  Figure 3. Myocardin activity is enhanced by hypertrophic stimuli in cardiomyocytes. Neonatal rat cardiomyocytes were transfected with the indicated luciferase reporters and expression plasmids for myocardin. Twelve hours later, cells were treated with the indicated hypertrophic stimuli. Cells were harvested and luciferase activities were measured 36 hours after treatment. Values are expressed as the fold increase in luciferase activity in the presence of expression plasmid and hypertrophic stimuli above the level of activity with reporter plasmid alone. All experiments were repeated three times in duplicate. A, A pCDNA myocardin expression plasmid and ANF-luciferase reporter were transfected in the absence or presence of indicated hypertrophic agonists and luciferase activity was determined. B, A pCDNA myocardin expression plasmid and a luciferase reporter linked to the E1b basal promoter and four tandem copies of CArG box were transfected in the absence or presence of indicated hypertrophic agonists and luciferase activity was determined. C, An expression plasmid encoding full-length myocardin (1-935) fused to GAL4 (1-147) and the pL8G5-luciferase reporter were transfected in the absence or presence of indicated hypertrophic agonists, and luciferase activity was determined. D, An expression plasmid encoding myocardin TAD (713-935) fused to GAL4 (1-147) and the pL8G5-luciferase reporter were transfected in the absence or presence of indicated hypertrophic agonists, and luciferase activity was determined. Error bars represent SD of three independent experiments. Statistical differences were determined using the Student t test; *P<0.05.Show Less
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  Myocardin Induces Cardiomyocyte Hypertrophy

  Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson, Da-Zhi Wang

  Circulation Research April 2006, 98 (8) 1089-1097; DOI: https://doi.org/10.1161/01.RES.0000218781.23144.3e

  

  By Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson and Da-Zhi Wang

  Figure 4. Overexpression of myocardin in cardiomyocytes. Primary neonatal cardiomyocytes were infected with Ad-lacZ, Ad-myocardin (Ad-MYCD), which was...Show More

  Figure 4. Overexpression of myocardin in cardiomyocytes. Primary neonatal cardiomyocytes were infected with Ad-lacZ, Ad-myocardin (Ad-MYCD), which was FLAG tagged, or a dominant-negative mutant of myocardin (Ad-MYCD-dn), also FLAG tagged, or treated with the indicated hypertrophic agonists. A, Myocardin protein was detected in cardiomyocytes using immunohistology (top) or Western blot analysis using anti-FLAG antibody (bottom). B, Cell surface area measurement in cardiomyocytes. The results are presented as mean±SE compared with the control, which is assigned a value of 1. n=40. Statistical differences were determined using the Student t test; *P<0.05.Show Less
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  Myocardin Induces Cardiomyocyte Hypertrophy

  Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson, Da-Zhi Wang

  Circulation Research April 2006, 98 (8) 1089-1097; DOI: https://doi.org/10.1161/01.RES.0000218781.23144.3e

  

  By Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson and Da-Zhi Wang

  Figure 5. Induction of cardiomyocyte hypertrophy by myocardin. Primary neonatal cardiomyocytes were infected with Ad-lacZ, Ad-MYCD, Ad-MYCD-dn, or tre...Show More

  Figure 5. Induction of cardiomyocyte hypertrophy by myocardin. Primary neonatal cardiomyocytes were infected with Ad-lacZ, Ad-MYCD, Ad-MYCD-dn, or treated with indicated hypertrophic agonists. Two days later, cultures were fixed and stained with antibodies for α-actinin and ANF. Nontreated cardiomyocytes were used in control. Bar=50 μm.Show Less
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  Myocardin Induces Cardiomyocyte Hypertrophy

  Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson, Da-Zhi Wang

  Circulation Research April 2006, 98 (8) 1089-1097; DOI: https://doi.org/10.1161/01.RES.0000218781.23144.3e

  

  By Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson and Da-Zhi Wang

  Figure 6. Inhibition of cardiomyocyte hypertrophy by a dominant-negative myocardin mutant. A, Primary neonatal cardiomyocytes were infected with Ad-la...Show More

  Figure 6. Inhibition of cardiomyocyte hypertrophy by a dominant-negative myocardin mutant. A, Primary neonatal cardiomyocytes were infected with Ad-lacZ or Ad-MYCD as indicated. Two days later, total RNA was isolated and semiquantitative RT-PCR analysis was performed. GAPDH was used to serve as a loading control. B, The intensity of PCR products on the agarose gel image was quantified using the Photoshop Histogram Analysis and was plotted using Microsoft Excel and is shown as relative expression level. C, Primary neonatal cardiomyocytes were infected with Ad-lacZ or Ad-MYCD-dn at an moi of 100 in the presence or absence of PE, as indicated (see Materials and Methods). Two days later, total RNA was isolated, and transcripts were detected by dot-blot hybridization. D, Results from dot-blot hybridization were quantified by PhosphoImager analysis, which is shown as relative expression level after being normalized against that of GAPDH. Error bars represent SD of two experiments. Statistical differences were determined using the Student t test; *P<0.05. SM-α-A indicates smooth muscle alpha-actin; SM-α-MHC, smooth muscle alpha-myosin heavy chain; α-CA, alpha cardiac activity; BNP, B-type natriuretic peptide.Show Less
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  Myocardin Induces Cardiomyocyte Hypertrophy

  Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson, Da-Zhi Wang

  Circulation Research April 2006, 98 (8) 1089-1097; DOI: https://doi.org/10.1161/01.RES.0000218781.23144.3e

  

  By Weibing Xing, Tong-Cun Zhang, Dongsun Cao, Zhigao Wang, Christopher L. Antos, Shijie Li, Yibin Wang, Eric N. Olson and Da-Zhi Wang

  Figure 7. Inhibition of myocardin-induced cardiomyocyte hypertrophy by HDAC5. A, Primary neonatal cardiomyocytes were infected with Ad-MYCD or Ad-HDAC...Show More

  Figure 7. Inhibition of myocardin-induced cardiomyocyte hypertrophy by HDAC5. A, Primary neonatal cardiomyocytes were infected with Ad-MYCD or Ad-HDAC5 or both at an moi of 100, as indicated. Two days later, total RNA was isolated and transcripts were detected by dot-blot hybridization. B, Results from dot-blot hybridization were quantified by PhosphoImager analysis, which is shown as relative expression level after being normalized against that of GAPDH. Error bars represent SD of two experiments. C, Neonatal rat cardiomyocytes were transfected with ANF promoter luciferase reporters and expression plasmids for myocardin and HDAC5, as described in Materials and Methods. Forty-eight hours later, cells were harvested and luciferase activities were measured. Values are expressed as the fold increase in luciferase activity in the presence of expression plasmid and hypertrophic stimuli above the level of activity with reporter plasmid alone. All experiments were repeated four times in duplicate. Error bars represent SD of at least two experiments. Statistical differences were determined using the Student t test; *P<0.05.Show Less
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  Independent Regulation of Cardiac Kv4.3 Potassium Channel Expression by Angiotensin II and Phenylephrine

  Ting-Ting Zhang, Koichi Takimoto, Alexandre F. R. Stewart, Chongxue Zhu, Edwin S. Levitan

  Circulation Research March 2001, 88 (5) 476-482; DOI: https://doi.org/10.1161/01.RES.88.5.476

  

  Figure 1.

  By Ting-Ting Zhang, Koichi Takimoto, Alexandre F. R. Stewart, Chongxue Zhu and Edwin S. Levitan

  Ang II and PE induce hypertrophic responses. Rat neonatal cardiac myocytes were treated with Ang II (100 nmol/L), PE (100 μmol/L), or vehicle for 8 or...Show More

  Ang II and PE induce hypertrophic responses. Rat neonatal cardiac myocytes were treated with Ang II (100 nmol/L), PE (100 μmol/L), or vehicle for 8 or 24 hours. Medium containing the agent or vehicle was changed every 12 hours. Amount of β-MHC mRNA was measured with RNase protection assay and normalized with an internal control β-actin mRNA (n≥3).Show Less
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  Independent Regulation of Cardiac Kv4.3 Potassium Channel Expression by Angiotensin II and Phenylephrine

  Ting-Ting Zhang, Koichi Takimoto, Alexandre F. R. Stewart, Chongxue Zhu, Edwin S. Levitan

  Circulation Research March 2001, 88 (5) 476-482; DOI: https://doi.org/10.1161/01.RES.88.5.476

  

  Figure 2.

  By Ting-Ting Zhang, Koichi Takimoto, Alexandre F. R. Stewart, Chongxue Zhu and Edwin S. Levitan

  Ang II and PE decrease Kv4.3 mRNA and protein expression. A, Four days after plating, cells were treated with Ang II (100 nmol/L), PE (100 μmol/L), or...Show More

  Ang II and PE decrease Kv4.3 mRNA and protein expression. A, Four days after plating, cells were treated with Ang II (100 nmol/L), PE (100 μmol/L), or vehicle for 8 or 24 hours. Medium containing the agent or vehicle was changed every 12 hours. Myocytes were harvested, and Kv4.3 mRNAs were measured using RNase protection assay. In the upper 2 panels, β-actin mRNA was used as an internal control. Bottom panel shows results from an experiment with 8-hour treatments in which 28S ribosomal RNA was used as an internal control. P indicates probe; Y, yeast RNA; and C, control. B, Time courses of Ang II–induced and PE-induced Kv4.3 mRNA downregulation (n≥3 at each time point, normalization with β-actin). C, Three days after plating, cells were treated with Ang II (100 nmol/L), PE (100 μmol/L), or vehicle for 60 hours. Medium containing the agent or vehicle was changed every 24 hours. Total protein was isolated, and Kv4.3 proteins were detected using an anti-Kv4.3 antibody.Show Less

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