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Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

Balasubramanian S, Mani SK, Kasiganesan H, Baicu CC, Kuppuswamy D - PLoS ONE (2010)

Bottom Line: To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes.The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini.In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes.

View Article: PubMed Central - PubMed

Affiliation: Cardiology Division, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, United States of America. balasubr@musc.edu

ABSTRACT
The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation.

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Adult cardiomyocyte β-actin dynamics measurement by using fluorescence recovery after photobleaching.Adult cardiomyocytes were infected for 24–36 h with β-actin-GFP adenovirus and subjected to FRAP analysis as described under Materials and Methods. This figure shows the prebleach (control), bleached area (black rectangle at time 0) and the subsequent recovery of fluorescence within the bleached rectangular area (1, 5, 10 and 15 min). The recovery of fluorescence within this rectangular area is measured to calculate the t½ value as presented in Table 1. Scale bar  = 5 µm.
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pone-0011470-g004: Adult cardiomyocyte β-actin dynamics measurement by using fluorescence recovery after photobleaching.Adult cardiomyocytes were infected for 24–36 h with β-actin-GFP adenovirus and subjected to FRAP analysis as described under Materials and Methods. This figure shows the prebleach (control), bleached area (black rectangle at time 0) and the subsequent recovery of fluorescence within the bleached rectangular area (1, 5, 10 and 15 min). The recovery of fluorescence within this rectangular area is measured to calculate the t½ value as presented in Table 1. Scale bar  = 5 µm.

Mentions: Since we observe β-actin to exhibit specific localization in Z-discs of adult cardiomyocytes and since β-actin polymerization/depolymerization dynamics can be followed real-time using video microscopy as shown in other cell types [7], [8], we applied this approach to study the dynamics of β-actin in Z-discs. In these experiments, we expressed β-actin-GFP in cultured adult cardiomyocyte for 24–36 h and subjected them to FRAP analysis as described [7]. A rectangular area covering a portion of 4–6 sarcomeres was chosen and bleached. The recovery of β-actin-GFP fluorescence in the bleached area was monitored by imaging the cell at various time intervals (Figure 4). From the recovery of fluorescence data over time, the t½ (the time taken for half-maximal recovery) was deduced as described in Materials and Methods. Table 1 shows the t½ calculated for cells treated with insulin and endothelin for 30 min. The reduction in t½ under these conditions suggests that both endothelin and insulin enhance the β-actin dynamics upon hypertrophic stimulation.


Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

Balasubramanian S, Mani SK, Kasiganesan H, Baicu CC, Kuppuswamy D - PLoS ONE (2010)

Adult cardiomyocyte β-actin dynamics measurement by using fluorescence recovery after photobleaching.Adult cardiomyocytes were infected for 24–36 h with β-actin-GFP adenovirus and subjected to FRAP analysis as described under Materials and Methods. This figure shows the prebleach (control), bleached area (black rectangle at time 0) and the subsequent recovery of fluorescence within the bleached rectangular area (1, 5, 10 and 15 min). The recovery of fluorescence within this rectangular area is measured to calculate the t½ value as presented in Table 1. Scale bar  = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902504&req=5

pone-0011470-g004: Adult cardiomyocyte β-actin dynamics measurement by using fluorescence recovery after photobleaching.Adult cardiomyocytes were infected for 24–36 h with β-actin-GFP adenovirus and subjected to FRAP analysis as described under Materials and Methods. This figure shows the prebleach (control), bleached area (black rectangle at time 0) and the subsequent recovery of fluorescence within the bleached rectangular area (1, 5, 10 and 15 min). The recovery of fluorescence within this rectangular area is measured to calculate the t½ value as presented in Table 1. Scale bar  = 5 µm.
Mentions: Since we observe β-actin to exhibit specific localization in Z-discs of adult cardiomyocytes and since β-actin polymerization/depolymerization dynamics can be followed real-time using video microscopy as shown in other cell types [7], [8], we applied this approach to study the dynamics of β-actin in Z-discs. In these experiments, we expressed β-actin-GFP in cultured adult cardiomyocyte for 24–36 h and subjected them to FRAP analysis as described [7]. A rectangular area covering a portion of 4–6 sarcomeres was chosen and bleached. The recovery of β-actin-GFP fluorescence in the bleached area was monitored by imaging the cell at various time intervals (Figure 4). From the recovery of fluorescence data over time, the t½ (the time taken for half-maximal recovery) was deduced as described in Materials and Methods. Table 1 shows the t½ calculated for cells treated with insulin and endothelin for 30 min. The reduction in t½ under these conditions suggests that both endothelin and insulin enhance the β-actin dynamics upon hypertrophic stimulation.

Bottom Line: To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes.The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini.In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes.

View Article: PubMed Central - PubMed

Affiliation: Cardiology Division, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, United States of America. balasubr@musc.edu

ABSTRACT
The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation.

Show MeSH
Related in: MedlinePlus