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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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Effects of Cav-3 WT or Cav-3 V82I expression on cells viability. BHK cells were transiently transfected with Cav-3 WT or Cav-3 V82I and mitochondrial activity and cell death were evaluated by MTT and Propidie/Fluorosceine assays, respectively. (a) Expression of Cav-3 WT or Cav-3 V82I did not modified mitochondrial function under basal conditions. On the contrary, a significant decrease of mitochondrial activity was observed in Cav-3 V82I expressing cells subjected to hyperosmotic stress. Data are representative of five independent experimental sessions. *, P < 0.05. (b) Images show the intravital staining that yields green yellow fluorescence for vital cells and red fluorescence for dead cells under basal and hyperosmolar conditions. Images are representative of three independent experiments.
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Figure 9: Effects of Cav-3 WT or Cav-3 V82I expression on cells viability. BHK cells were transiently transfected with Cav-3 WT or Cav-3 V82I and mitochondrial activity and cell death were evaluated by MTT and Propidie/Fluorosceine assays, respectively. (a) Expression of Cav-3 WT or Cav-3 V82I did not modified mitochondrial function under basal conditions. On the contrary, a significant decrease of mitochondrial activity was observed in Cav-3 V82I expressing cells subjected to hyperosmotic stress. Data are representative of five independent experimental sessions. *, P < 0.05. (b) Images show the intravital staining that yields green yellow fluorescence for vital cells and red fluorescence for dead cells under basal and hyperosmolar conditions. Images are representative of three independent experiments.

Mentions: As previously reported, caveolin proteins negatively regulate ERK activity[28,29,44]. Considering that cell viability is compromised when ERK signalling pathways are disrupted[30,31], we first tested the effects of Cav-3 V82I on ERK activation. Lysates from BHK cells transfected, either with Cav-3 WT or Cav-3 V82I, were analyzed by western blot using specific monoclonal antibodies directed against total ERKs or their phosphorylated (active) form. Both Cav-3 WT and Cav-3 V82I did not modify total ERK expression. On the contrary V82I mutant significantly impaired both basal and mannitol-stimulated ERK activation as compared to Cav-3 WT (Figure 8). Similar results were obtained when we measured p44/42 MAP kinase activity using a nonradioactive assay kit that detects the specific phosphorylation of the p44/42 MAP kinase substrate Elk-1 (Figure 8). Interestingly, cells transfected with Cav-3 V82I were more vulnerable to the mannitol challenge used to activate ERK, since both mitochondrial activity (measured by MTT assay) and cell viability (measured by the Propidium/Fluoroscein assay) were significantly affected (Figure 9).


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)

Effects of Cav-3 WT or Cav-3 V82I expression on cells viability. BHK cells were transiently transfected with Cav-3 WT or Cav-3 V82I and mitochondrial activity and cell death were evaluated by MTT and Propidie/Fluorosceine assays, respectively. (a) Expression of Cav-3 WT or Cav-3 V82I did not modified mitochondrial function under basal conditions. On the contrary, a significant decrease of mitochondrial activity was observed in Cav-3 V82I expressing cells subjected to hyperosmotic stress. Data are representative of five independent experimental sessions. *, P < 0.05. (b) Images show the intravital staining that yields green yellow fluorescence for vital cells and red fluorescence for dead cells under basal and hyperosmolar conditions. Images are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Effects of Cav-3 WT or Cav-3 V82I expression on cells viability. BHK cells were transiently transfected with Cav-3 WT or Cav-3 V82I and mitochondrial activity and cell death were evaluated by MTT and Propidie/Fluorosceine assays, respectively. (a) Expression of Cav-3 WT or Cav-3 V82I did not modified mitochondrial function under basal conditions. On the contrary, a significant decrease of mitochondrial activity was observed in Cav-3 V82I expressing cells subjected to hyperosmotic stress. Data are representative of five independent experimental sessions. *, P < 0.05. (b) Images show the intravital staining that yields green yellow fluorescence for vital cells and red fluorescence for dead cells under basal and hyperosmolar conditions. Images are representative of three independent experiments.
Mentions: As previously reported, caveolin proteins negatively regulate ERK activity[28,29,44]. Considering that cell viability is compromised when ERK signalling pathways are disrupted[30,31], we first tested the effects of Cav-3 V82I on ERK activation. Lysates from BHK cells transfected, either with Cav-3 WT or Cav-3 V82I, were analyzed by western blot using specific monoclonal antibodies directed against total ERKs or their phosphorylated (active) form. Both Cav-3 WT and Cav-3 V82I did not modify total ERK expression. On the contrary V82I mutant significantly impaired both basal and mannitol-stimulated ERK activation as compared to Cav-3 WT (Figure 8). Similar results were obtained when we measured p44/42 MAP kinase activity using a nonradioactive assay kit that detects the specific phosphorylation of the p44/42 MAP kinase substrate Elk-1 (Figure 8). Interestingly, cells transfected with Cav-3 V82I were more vulnerable to the mannitol challenge used to activate ERK, since both mitochondrial activity (measured by MTT assay) and cell viability (measured by the Propidium/Fluoroscein assay) were significantly affected (Figure 9).

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