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Identification and functional analysis of a new putative caveolin-3 variant found in a patient with sudden unexplained death.

Lariccia V, Nasti AA, Alessandrini F, Pesaresi M, Gratteri S, Tagliabracci A, Amoroso S - J. Biomed. Sci. (2014)

Bottom Line: Here we characterized a new putative Cav-3 variant, Cav-3 V82I, found in a patient with SCD.In heterologous systems Cav-3 V82I was expressed at significantly higher level than Cav-3 WT and accumulated within the cells.Cells expressing Cav-3 V82I exhibited a decreased activation of extracellular-signal-regulated kinases (ERKs) and were more vulnerable to sub-lethal osmotic stress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences and Public Health, School of Medicine, University "Politecnica delle Marche", Ancona, Italy. s.amoroso@univpm.it.

ABSTRACT

Background: Sudden cardiac death (SCD) is the clinical outcome of a lethal arrhythmia that can develop on the background of unrecognized channelopathies or cardiomyopathies. Several susceptibility genes have been identified for the congenital forms of these cardiac diseases, including caveolin-3 (Cav-3) gene. In the heart Cav-3 is the main component of caveolae, plasma membrane domains that regulate multiple cellular processes highly relevant for cardiac excitability, such as trafficking, calcium homeostasis, signal transduction and cellular response to injury. Here we characterized a new putative Cav-3 variant, Cav-3 V82I, found in a patient with SCD.

Results: In heterologous systems Cav-3 V82I was expressed at significantly higher level than Cav-3 WT and accumulated within the cells. Cells expressing Cav-3 V82I exhibited a decreased activation of extracellular-signal-regulated kinases (ERKs) and were more vulnerable to sub-lethal osmotic stress.

Conclusion: Considering that abnormal loss of myocytes can play a mechanistic role in lethal cardiac diseases, we suggest that the detrimental effect of Cav-3 V82I variant on cell viability may participate in determining the susceptibility to cardiac death.

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Cav-3 V82I partitions into Triton-soluble (S) and Triton-insoluble (I) cell fractions. Twenty-four hours after transfection, the Triton-soluble and insoluble fractions were collected from Cav-3 WT and Cav-3 V82I transfected BHK cells as described in Materials and Methods. Equal volumes of the soluble and insoluble fractions were analyzed by immunoblotting. Representative blot is shown in (a). Cav-3 V82I was significantly more abundant than the WT both in the S and in the I fractions (b). Data are representative of five independent experiments. **, P < 0.01 V82I vs WT in the soluble fraction; ***, P < 0.001 V82I vs WT in the insoluble fraction.
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Figure 7: Cav-3 V82I partitions into Triton-soluble (S) and Triton-insoluble (I) cell fractions. Twenty-four hours after transfection, the Triton-soluble and insoluble fractions were collected from Cav-3 WT and Cav-3 V82I transfected BHK cells as described in Materials and Methods. Equal volumes of the soluble and insoluble fractions were analyzed by immunoblotting. Representative blot is shown in (a). Cav-3 V82I was significantly more abundant than the WT both in the S and in the I fractions (b). Data are representative of five independent experiments. **, P < 0.01 V82I vs WT in the soluble fraction; ***, P < 0.001 V82I vs WT in the insoluble fraction.

Mentions: We next characterized the biochemical properties of the Cav-3 V82I assessing its detergent solubility pattern. Caveolins mainly reside within caveolar lipid rafts which are insoluble in non-ionic detergents such as Triton X-100, while several caveolin-3 mutants with decreased surface expression usually display an increased Triton X-100 solubility[14,35,36]. By western blot analysis we found that in Cav-3 WT transfected cells caveolin-3 protein was mainly distributed in the Triton-insoluble fraction (I) (Figure 7), consistent with data reported before[36,43]. For Cav-3 V82I, we observed a significant increased of caveolin-3 expression both in the Triton-insoluble and in the Triton-soluble (S) fractions (respectively 2.6 and 3.5 fold as compared to WT; Figure 7). The increased distribution of Cav-3 V82I in the Triton-soluble fraction (expressed as caveolin-3 soluble/pellet ratio) was not statistically different (42.4 ± 10.1% vs 29.1 ± 5.9%, V82I vs WT, respectively; n = 5, P = 0.29).


Identification and functional analysis of a new putative caveolin-3 variant found in a patient with sudden unexplained death.

Lariccia V, Nasti AA, Alessandrini F, Pesaresi M, Gratteri S, Tagliabracci A, Amoroso S - J. Biomed. Sci. (2014)

Cav-3 V82I partitions into Triton-soluble (S) and Triton-insoluble (I) cell fractions. Twenty-four hours after transfection, the Triton-soluble and insoluble fractions were collected from Cav-3 WT and Cav-3 V82I transfected BHK cells as described in Materials and Methods. Equal volumes of the soluble and insoluble fractions were analyzed by immunoblotting. Representative blot is shown in (a). Cav-3 V82I was significantly more abundant than the WT both in the S and in the I fractions (b). Data are representative of five independent experiments. **, P < 0.01 V82I vs WT in the soluble fraction; ***, P < 0.001 V82I vs WT in the insoluble fraction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4109384&req=5

Figure 7: Cav-3 V82I partitions into Triton-soluble (S) and Triton-insoluble (I) cell fractions. Twenty-four hours after transfection, the Triton-soluble and insoluble fractions were collected from Cav-3 WT and Cav-3 V82I transfected BHK cells as described in Materials and Methods. Equal volumes of the soluble and insoluble fractions were analyzed by immunoblotting. Representative blot is shown in (a). Cav-3 V82I was significantly more abundant than the WT both in the S and in the I fractions (b). Data are representative of five independent experiments. **, P < 0.01 V82I vs WT in the soluble fraction; ***, P < 0.001 V82I vs WT in the insoluble fraction.
Mentions: We next characterized the biochemical properties of the Cav-3 V82I assessing its detergent solubility pattern. Caveolins mainly reside within caveolar lipid rafts which are insoluble in non-ionic detergents such as Triton X-100, while several caveolin-3 mutants with decreased surface expression usually display an increased Triton X-100 solubility[14,35,36]. By western blot analysis we found that in Cav-3 WT transfected cells caveolin-3 protein was mainly distributed in the Triton-insoluble fraction (I) (Figure 7), consistent with data reported before[36,43]. For Cav-3 V82I, we observed a significant increased of caveolin-3 expression both in the Triton-insoluble and in the Triton-soluble (S) fractions (respectively 2.6 and 3.5 fold as compared to WT; Figure 7). The increased distribution of Cav-3 V82I in the Triton-soluble fraction (expressed as caveolin-3 soluble/pellet ratio) was not statistically different (42.4 ± 10.1% vs 29.1 ± 5.9%, V82I vs WT, respectively; n = 5, P = 0.29).

Bottom Line: Here we characterized a new putative Cav-3 variant, Cav-3 V82I, found in a patient with SCD.In heterologous systems Cav-3 V82I was expressed at significantly higher level than Cav-3 WT and accumulated within the cells.Cells expressing Cav-3 V82I exhibited a decreased activation of extracellular-signal-regulated kinases (ERKs) and were more vulnerable to sub-lethal osmotic stress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences and Public Health, School of Medicine, University "Politecnica delle Marche", Ancona, Italy. s.amoroso@univpm.it.

ABSTRACT

Background: Sudden cardiac death (SCD) is the clinical outcome of a lethal arrhythmia that can develop on the background of unrecognized channelopathies or cardiomyopathies. Several susceptibility genes have been identified for the congenital forms of these cardiac diseases, including caveolin-3 (Cav-3) gene. In the heart Cav-3 is the main component of caveolae, plasma membrane domains that regulate multiple cellular processes highly relevant for cardiac excitability, such as trafficking, calcium homeostasis, signal transduction and cellular response to injury. Here we characterized a new putative Cav-3 variant, Cav-3 V82I, found in a patient with SCD.

Results: In heterologous systems Cav-3 V82I was expressed at significantly higher level than Cav-3 WT and accumulated within the cells. Cells expressing Cav-3 V82I exhibited a decreased activation of extracellular-signal-regulated kinases (ERKs) and were more vulnerable to sub-lethal osmotic stress.

Conclusion: Considering that abnormal loss of myocytes can play a mechanistic role in lethal cardiac diseases, we suggest that the detrimental effect of Cav-3 V82I variant on cell viability may participate in determining the susceptibility to cardiac death.

Show MeSH
Related in: MedlinePlus