Phospholamban Is Downregulated by pVHL-Mediated Degradation through Oxidative Stress in Failing Heart

Figures

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  Figure 1. pVHL expression was increased and associated with PLN in hearts from TgPLNR9C and NHE1-Tg mice. (a,b) Wild type (WT) and TgPLNR9C (a) or NHE1-Tg mice (b) were sacrificed at 16 weeks of age and the hearts were homogenized. The lysates were subjected to Western blot analysis with antibodies against pVHL, HIF-1α, BNP, PLN, ubiquitin (Ub), and α-tubulin. The data designated indicate representative blots (n = 5); (c) The lysates from WT and TgPLNR9C or NHE1-Tg mice hearts were subjected to co-immunoprecipitation with anti-Ub antibody, followed by Western blot analysis using anti-PLN antibody. * and ** indicate mono-ubiquitinated PLN (14.5 kDa) and di-ubiquitinated PLN (23 kDa), respectively; (d) The lysates from WT and TgPLNR9C or NHE1-Tg mice hearts were subjected to co-immunoprecipitation with anti-pVHL antibody, followed by Western blot analysis using anti-PLN antibody. “p” and “m” indicate PLN pentamers and PLN monomers, respectively.

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  Figure 2. Ubiquitinated PLN was decreased after VHL knockdown. (a,b) VHL silencing using siRNA was performed in PLN-transfected HEK293 cells. Cells were treated with 10 μM MG132 or 100 nM bafilomycin for 3 h. Western blot analysis was performed using antibodies against pVHL and α-tubulin (a). Cell lysates were subjected to co-immunoprecipitation with anti-Ub antibody. After boiling to dissociate the non-covalent bonds, Western blot analysis were performed using anti-PLN antibody. * and ** indicate mono-ubiquitinated PLN (14.5 kDa) and di-ubiquitinated PLN (23 kDa), respectively (b).

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  Figure 3. PLN was degraded by the disturbance in the mitochondrial membrane potential through an increase in the oxidative stress. (a,b) PLN-transfected HEK293 cells were harvested 4 h after treatment with 20 μM carbonylcyanide m-chlorophenylhydrazone (CCCP) with or without 5 mM N-acetyl-l-cysteine (NAC). Western blot analysis of whole cell lysates (a) and pVHL pull-down fractions (b) from PLN-transfected HEK293 cells was performed using an anti-PLN antibody. The bar graph indicates the intensity of PLN bands, as measured by ImageJ (Version 1.50i, National Institutes of Health, Bethesda, MD, USA) (n = 3). A value of p <0.05 (*) was considered significant. (c) PLN-transfected HEK293 cells were harvested 4 h after treatment with 125 μM CoCl2. Western blot analysis of whole cell lysates was performed using an anti-PLN antibody. The bar graph indicates the intensity of PLN bands, as measured by ImageJ (n = 3). NS: not significant.

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  Figure 4. The expression of PLN is regulated by VHL. (a,b) VHL silencing or overexpression using siRNA or HA-VHL expression vector was performed in PLN-transfected HEK293 cells. The cells were harvested with (b) or without (a) 100 μM H2O2 treatment for 1 h. Western blot analysis was performed using antibodies against pVHL and PLN using whole cell lysates. The bar graph indicates the intensity of PLN bands, as measured by ImageJ (n = 3). A value of p <0.05 (*) was considered significant. (c) VHL silencing was performed in PLN-transfected HEK293 cells. The cells were harvested 1 h after treatment with 1 μM isoproterenol (Iso). Western blot analysis was performed using antibodies against pVHL and PLN using whole cell lysates. The bar graph indicates the intensity of PLN bands, as measured by ImageJ (n = 3). A value of p <0.05 (*) was considered significant.