CapZ dynamics are altered by endothelin-1 and phenylephrine via PIP2- and PKC-dependent mechanisms

Abstract

One of the unanswered questions in muscle hypertrophy is how new contractile units are inserted into a stable existing cytoskeletal meshwork. Regulation of actin capping by CapZ may play a role in remodeling processes, therefore, CapZ dynamics are determined during rapid growth of cardiac cells in vitro. Neonatal rat ventricular myocytes were infected with adenovirus expressing green fluorescent protein-CapZ beta1 and responded normally to hypertrophic stimuli. CapZ dynamics were analyzed by fluorescence recovery after photobleaching in cultured myocytes treated with endothelin-1 (100 nM) or phenylephrine (10 muM). Recovery by 30 s was greater with endothelin treatment. Analysis 30 min postbleach showed CapZ-infected cells treated with endothelin recovered more completely than controls (77 +/- 9% vs. 50 +/- 6%, P < 0.001). Similar results were found with phenylephrine (77 +/- 5%, P < 0.05). A potential mechanism for phosphatidylinositol bisphosphate (PIP2) mediation of increased CapZ exchange in endothelin- and phenylephrine-treated cells was tested. PIP2 sequestration with neomycin (500 muM) blocked both endothelin- (43 +/- 6%, P < 0.001) and phenylephrine (36 +/- 4%, P < 0.001)-mediated recovery. The protein kinase C inhibitor chelerythrine chloride (10 muM) also blocked endothelin- (53 +/- 10%, P < 0.001) and phenylephrine (42 +/- 3%, P < 0.001)-mediated recovery. This study demonstrates for the first time that endothelin and phenylephrine alter CapZ dynamics through PIP2- and PKC-dependent pathways, which might destabilize the existing framework and permit sarcomeric remodelling to proceed.

Fig. 1.

Wild-type (WT) GFP-CapZ fusion proteins localize appropriately to Z-disk. Cells were cultured for 24 h, infected for 1 h, and cultured for another 24 h at which point they were fixed and subjected to either staining with phalloidin for actin (A) or immunostaining for antibody for α-actinin (B). Cells stained for actin and infected with CapZ adenovirus demonstrate proper localization of CapZ at the Z-disk and actin localization adjacent to Z-disks in the I-band. Inset, higher magnification images of area delineated by the white box in the lower magnification image. Scale bar = 10 μm.

Fig. 2.

Fluorescence recovery after photobleaching (FRAP) recovery curves for WT GFP-CapZ fusion protein treated with endothelin (ET). FRAP analysis was performed on cells expressing WT green fluorescence protein (GFP)-tagged CapZ protein. Recovery percentages were reported as a percentage of prebleach intensity. A: cells were cultured for 24 h, infected for 1 h, and cultured for an additional 44 h at which time they were either left untreated for 4 h, treated with ET for 4 h, treated with ET and neomycin (Neo), an inhibitor of PIP2 signaling, for 4 h, or treated with ET and chelerythrine chloride (CC), an inhibitor of PKC signaling, for 4 h. B: confocal images of cells analyzed in each of the experimental conditions in A at time points immediately before photobleaching, immediately after photobleaching, and 30 min postphotobleaching.

Fig. 3.

FRAP recovery curves for WT GFP-CapZ fusion protein treated with phenylephrine (PE). FRAP analysis was performed on cells expressing GFP-tagged CapZ protein. Recovery percentages were reported as a percentage of prebleach intensity. A: cells were cultured for 24 h, infected for 1 h, and cultured for an additional 44 h at which time they were left untreated for 4 h, treated with PE for 24 h, treated with PE for 24 h and Neo for 4 h, or treated with PE for 24 h and CC for 4 h. B: confocal images of cells analyzed in each of the experimental conditions in A at time points immediately before photobleaching, immediately after photobleaching, and 30 min postphotobleaching.