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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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Related in: MedlinePlus

Colocalization analysis of Cav-3 WT and Cav-3 V82I with the ER marker calnexin. BHK cells were transiently transfected with Cav-3 WT (top) or Cav-3 V82I (bottom) and subjected to double labelling with antibodies raised against caveolin-3 (green) and calnexin (red). As shown in the merged images, no significant colocalization with calnexin was observed for both wilt-type and mutated caveolin-3 proteins. Data are representative of three independent cultures.
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Figure 6: Colocalization analysis of Cav-3 WT and Cav-3 V82I with the ER marker calnexin. BHK cells were transiently transfected with Cav-3 WT (top) or Cav-3 V82I (bottom) and subjected to double labelling with antibodies raised against caveolin-3 (green) and calnexin (red). As shown in the merged images, no significant colocalization with calnexin was observed for both wilt-type and mutated caveolin-3 proteins. Data are representative of three independent cultures.

Mentions: It has been reported that unstable Cav-3 mutants, usually characterized by a marked reduction in their expression and retained in the Golgi complex[15,19,35,36], are expressed at higher levels and accumulate within the endoplasmic reticulum (ER) after treatment with proteasome inhibitors[41]. Considering that the V82I mutation increased Cav-3 expression and that LB have an ER origin[39], we sought to investigate whether the distribution of Cav-3 V82I protein was also consistent with the retention of the protein in the ER. To address this hypothesis, transiently transfected BHK cells were subjected to double labeling with antibodies directed against caveolin-3 and calnexin, an ER specific marker protein[42]. Despite the increased expression and intracellular caveolin-3 retention observed in Cav-3 V82I transfected cells, V82I mutant was not distributed to ER since no significant colocalization was observed between V82I and calnexin (Figure 6). In cells transfected with the WT form, caveolin-3 was mostly expressed at cell surface and did not colocalize with calnexin (Figure 6), in line with previous results[41].


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)

Colocalization analysis of Cav-3 WT and Cav-3 V82I with the ER marker calnexin. BHK cells were transiently transfected with Cav-3 WT (top) or Cav-3 V82I (bottom) and subjected to double labelling with antibodies raised against caveolin-3 (green) and calnexin (red). As shown in the merged images, no significant colocalization with calnexin was observed for both wilt-type and mutated caveolin-3 proteins. Data are representative of three independent cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Colocalization analysis of Cav-3 WT and Cav-3 V82I with the ER marker calnexin. BHK cells were transiently transfected with Cav-3 WT (top) or Cav-3 V82I (bottom) and subjected to double labelling with antibodies raised against caveolin-3 (green) and calnexin (red). As shown in the merged images, no significant colocalization with calnexin was observed for both wilt-type and mutated caveolin-3 proteins. Data are representative of three independent cultures.
Mentions: It has been reported that unstable Cav-3 mutants, usually characterized by a marked reduction in their expression and retained in the Golgi complex[15,19,35,36], are expressed at higher levels and accumulate within the endoplasmic reticulum (ER) after treatment with proteasome inhibitors[41]. Considering that the V82I mutation increased Cav-3 expression and that LB have an ER origin[39], we sought to investigate whether the distribution of Cav-3 V82I protein was also consistent with the retention of the protein in the ER. To address this hypothesis, transiently transfected BHK cells were subjected to double labeling with antibodies directed against caveolin-3 and calnexin, an ER specific marker protein[42]. Despite the increased expression and intracellular caveolin-3 retention observed in Cav-3 V82I transfected cells, V82I mutant was not distributed to ER since no significant colocalization was observed between V82I and calnexin (Figure 6). In cells transfected with the WT form, caveolin-3 was mostly expressed at cell surface and did not colocalize with calnexin (Figure 6), in line with previous results[41].

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