Limits...
Interaction of annexin A6 with alpha actinin in cardiomyocytes.

Mishra S, Chander V, Banerjee P, Oh JG, Lifirsu E, Park WJ, Kim do H, Bandyopadhyay A - BMC Cell Biol. (2011)

Bottom Line: In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal.Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Kolkata, India.

ABSTRACT

Background: Annexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state.

Results: To identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST)- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α)-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP) tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.

Conclusions: In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

Show MeSH

Related in: MedlinePlus

Colocalization of AnxA6 with a-actinin in cardiomyocytes. The representative images shown are endogenous expression of AnxA6 (a) and α-actinin (b) in the same cell and their colocalization pattern (c). The line profile for quantification of colocalization (d) was done using the merged image (inset) of AnxA6 and sarcomeric α-actinin. The images represent results of three different experiments with separate batches of myocyte preparation. Scale bar represents 10 μm (100×).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3037912&req=5

Figure 4: Colocalization of AnxA6 with a-actinin in cardiomyocytes. The representative images shown are endogenous expression of AnxA6 (a) and α-actinin (b) in the same cell and their colocalization pattern (c). The line profile for quantification of colocalization (d) was done using the merged image (inset) of AnxA6 and sarcomeric α-actinin. The images represent results of three different experiments with separate batches of myocyte preparation. Scale bar represents 10 μm (100×).

Mentions: The interaction of AnxA6 with α-actinin in situ was examined in NRVM by immunofluorescence microscopy. The pattern of AnxA6 localization in cardiomyocytes was similar to α-actinin and both displayed striated distribution similar to z-discs of sarcomere (Figure 4). The extent of co-localization was enumerated by generating the line profiles of fluorescence intensities of tetramethylrhodamine isothiocyanate (TRITC) for α-actinin and fluorescein isothiocyanate (FITC) for AnxA6 (Figure 4 lower panel). These fluorescence profiles demonstrates (through randomly chosen line over the merged images) the parallel pattern of spatial distribution of AnxA6 and α-actinin signals, indicating a high degree of co-localization of these two proteins, which was further strengthened by Pearson's correlation coefficient as high as 0.898 ± 0.014, obtained from co-localization analysis.


Interaction of annexin A6 with alpha actinin in cardiomyocytes.

Mishra S, Chander V, Banerjee P, Oh JG, Lifirsu E, Park WJ, Kim do H, Bandyopadhyay A - BMC Cell Biol. (2011)

Colocalization of AnxA6 with a-actinin in cardiomyocytes. The representative images shown are endogenous expression of AnxA6 (a) and α-actinin (b) in the same cell and their colocalization pattern (c). The line profile for quantification of colocalization (d) was done using the merged image (inset) of AnxA6 and sarcomeric α-actinin. The images represent results of three different experiments with separate batches of myocyte preparation. Scale bar represents 10 μm (100×).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037912&req=5

Figure 4: Colocalization of AnxA6 with a-actinin in cardiomyocytes. The representative images shown are endogenous expression of AnxA6 (a) and α-actinin (b) in the same cell and their colocalization pattern (c). The line profile for quantification of colocalization (d) was done using the merged image (inset) of AnxA6 and sarcomeric α-actinin. The images represent results of three different experiments with separate batches of myocyte preparation. Scale bar represents 10 μm (100×).
Mentions: The interaction of AnxA6 with α-actinin in situ was examined in NRVM by immunofluorescence microscopy. The pattern of AnxA6 localization in cardiomyocytes was similar to α-actinin and both displayed striated distribution similar to z-discs of sarcomere (Figure 4). The extent of co-localization was enumerated by generating the line profiles of fluorescence intensities of tetramethylrhodamine isothiocyanate (TRITC) for α-actinin and fluorescein isothiocyanate (FITC) for AnxA6 (Figure 4 lower panel). These fluorescence profiles demonstrates (through randomly chosen line over the merged images) the parallel pattern of spatial distribution of AnxA6 and α-actinin signals, indicating a high degree of co-localization of these two proteins, which was further strengthened by Pearson's correlation coefficient as high as 0.898 ± 0.014, obtained from co-localization analysis.

Bottom Line: In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal.Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Kolkata, India.

ABSTRACT

Background: Annexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state.

Results: To identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST)- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α)-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP) tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.

Conclusions: In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

Show MeSH
Related in: MedlinePlus