Interaction between vitamin D receptor with caveolin-3 and regulation by 1,25-dihydroxyvitamin D3 in adult rat cardiomyocytes

Abstract

We show that 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) and a synthetic non-genotropic vitamin D analog agonist, 1a,25(OH)2-lumisterol (JN), exhibit similar rapid effects on sarcomere shortening (contraction) of isolated adult cardiomyocyte. We also report that the vitamin D receptor (VDR) specifically interacts with caveolin-3 in the t-tubules and sarcolemma of isolated adult rat cardiac myocytes. Confocal immunofluorescence microscopy analysis showed co-localization of VDR and caveolin-3 in the t-tubules and sarcolemma of cardiomyocytes. Co-immunoprecipitation studies using VDR antibodies revealed that caveolin-3 specifically co-precipitates with the VDR and similarly the VDR is co-precipitated with caveolin-3 antibody. VDR is also in association with Serca-2, the sarcoplasmic reticulum Ca2+-ATPase, as demonstrated by co-immunoprecipitation, suggesting a role of VDR in regulating cardiac contractility by direct interaction with Serca-2. Treatment of isolated adult rat cardiomyocytes with 10 nM 1,25(OH)2D3 for 1 h caused decreased association between VDR and caveolin-3. These discoveries of the association between VDR and caveolin-3 and the regulation of this interaction by 1,25(OH)2D3 are fundamentally important in understanding 1,25(OH)2D3 signal transduction in heart cells and suggest a novel mechanism for VDR in the regulation of heart structure and function.

Figure 1

A, Percent change in cardiomyocyte contraction [peak of cell shortening] in response to treatment with 10^{− 9}M 1,25(OH)₂D₃ [Insert: Chemical structure of 1,25(OH)₂D₃; RB From Mizwicki et al. Sci Signal. 2, re4 (2009) RCI= relative competitive index ; MV=molecular volume]. Isolated individual rat cardiomyocytes were electrically stimulated and contraction recorded before and after 1,25(OH)₂D₃ treatment. Data shown are percent change in peak height after treatment compared to peak height at time zero (before treatment). Data shown were average of 4 cells. B, Percent change in cardiomyocyte contraction [peak shortening] in response to treatment 10⁻⁸M JN [Insert: chemical structure. From Mizwicki et al. Sci Signal. 2, re4 (2009) RCI= relative competitive index ; MV=molecular volume]. Data shown were average of 6 cells. JN (1,25(OH)2-lumisterol), is a non-genotropic 1,25(OH)₂D₃ analog with poor transcriptional activity. Thus, the effects of JN are reportedly through the membrane-associated VDR. The similarity of the effects between 1,25(OH)₂D₃ and JN in this figure suggests that vitamin D analogs can elicit non-genomic effects through a membrane-associated VDR.

Figure 2

Immunofluorescent confocal microscopy of adult rat cardiac myocytes. Double labeling using an antibody against VDR (sc-1008, rabbit polyclonal, a) and an antibody against caveolin-3 (sc-5310, mouse monoclonal, b) show similar transverse staining pattern, whereas a merge (c) shows association.

Figure 3

A, Co-immunoprecipitation of Caveolin-3 and serca 2 with VDR antibodies. 600 ug of cardiomyocyte and intestinal protein lysates were incubated with VDR monoclonal and polyclonal antibody-coupled gel resins, and control gel resin respectively. Eluted fractions of IPed protein samples (20 ul each) were subjected to Western blot analysis. The blot was first probed with Caveolin-3 antibody (sc-5310) and then striped, and reprobed with Serca 2 antibody (sc-8094) and VDR antibody (sc-1008), respectively. B, Co-immunoprecipitation of VDR with caveolin-3 antibody. 600 ug of cardiomyocyte lysates were incubated with caveolin-3 antibody-coupled gel resin and control resin. Eluted fractions of immuno-precipitated samples (20 ul each) were subjected to Western blot analysis. The blot was first probed with caveolin-3 antibody (sc-5310) and then striped, and reprobed with VDR antibody (sc-1008).

Figure 4

Effect of 1,25(OH)₂D₃ on interaction between VDR and Caveolin-3. Isolated cardiomyocytes from adult rat heart were treated in culture with 1 nM 1,25(OH)₂D₃ or ethanol (control) for 1 hour before protein extraction. 600 ug of protein lysates each were incubated with VDR monoclonal (MA1-710) and polyclonal (sc-1008) antibody-coupled gel resins, caveolin-3 polyclonal (PA1-066) antibody-coupled gel resin, and control gel resin respectively. Eluted fractions of IPed protein samples (20 ul each) were subjected to Western blot analysis. A, Western blot of VDR antibody IPed samples. The blot was first probed with Caveolin-3 antibody (sc-5310), then striped and reprobed with VDR antibody (sc-1008). B, Western blot of caveolin-3 antibody IPed samples. The blot was first probed with VDR antibody (sc-1008), then striped and reprobed with caveolin-3 antibody (sc-5310).