Limits...
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.

Show MeSH

Related in: MedlinePlus

Stability of Cav-3 WT and Cav-3 V82I. Transfected BHK cells were treated with cycloheximide (10 μg/ml; CHX) for the indicated length of time. Cells were then collected in same volumes of lysis buffer and equal volumes of extracts were analyzed by immunoblotting. Representative blot is shown in (a). Levels of residual caveolin-3 at the indicated time points (% of time 0) for Cav-3 WT and Cav-3 V82I are shown in (b). Data are representative of four independent experiments. *, P < 0.05 vs WT at the respective time point; **, P < 0.01 vs WT at the respective time points.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Stability of Cav-3 WT and Cav-3 V82I. Transfected BHK cells were treated with cycloheximide (10 μg/ml; CHX) for the indicated length of time. Cells were then collected in same volumes of lysis buffer and equal volumes of extracts were analyzed by immunoblotting. Representative blot is shown in (a). Levels of residual caveolin-3 at the indicated time points (% of time 0) for Cav-3 WT and Cav-3 V82I are shown in (b). Data are representative of four independent experiments. *, P < 0.05 vs WT at the respective time point; **, P < 0.01 vs WT at the respective time points.

Mentions: To investigate the impact of V82I mutation on Cav-3 protein level and cellular localization, we introduced the V82I substitution detected in the patient into the human Cav-3 WT sequence. We next transfected BHK cells with Cav-3 WT or Cav-3 V82I and compared levels of wild-type and mutant in SDS lysates, which represent the total caveolin-3 pool. As shown in Figure 2, Cav-3 V82I mutant was expressed at significantly higher level (about 1.4 fold) than Cav-3 WT. The increased expression in caveolin-3 protein caused by V82I mutation is somehow a new finding since most of caveolin-3 mutations often cause a severe loss of Cav-3 protein[19]. To examine whether differences in protein stability between WT or V82I mutant accounted for the higher expression level of Cav-3 V82I, we performed cycloheximide (CHX) block experiments[33,34]. WT or V82I transfected BHK cells were treated with CHX for 1–6 h to inhibit protein synthesis, harvested at different time points, and resulting lysates were probed with anti-Cav-3 antibody to monitor caveolin-3 levels, or anti-tubulin antibody as a loading control. As shown in Figure 3, during CHX chase, Cav-3 WT protein declined over time, decreasing to 70% levels by 6 h, while Cav-3 V82I levels were significantly more stable over the 6 h time course. These results suggest that V82I mutation confers increased stability to caveolin-3 and are consistent with the higher protein expression level observed with Cav-3 V82I as compared to Cav-3 WT. It is interesting to note that 1 h of CHX treatment significantly increased the expression of the V82I mutant (but not of the WT form): the reason of such increase is currently unknown.


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)

Stability of Cav-3 WT and Cav-3 V82I. Transfected BHK cells were treated with cycloheximide (10 μg/ml; CHX) for the indicated length of time. Cells were then collected in same volumes of lysis buffer and equal volumes of extracts were analyzed by immunoblotting. Representative blot is shown in (a). Levels of residual caveolin-3 at the indicated time points (% of time 0) for Cav-3 WT and Cav-3 V82I are shown in (b). Data are representative of four independent experiments. *, P < 0.05 vs WT at the respective time point; **, P < 0.01 vs WT at the respective time points.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Stability of Cav-3 WT and Cav-3 V82I. Transfected BHK cells were treated with cycloheximide (10 μg/ml; CHX) for the indicated length of time. Cells were then collected in same volumes of lysis buffer and equal volumes of extracts were analyzed by immunoblotting. Representative blot is shown in (a). Levels of residual caveolin-3 at the indicated time points (% of time 0) for Cav-3 WT and Cav-3 V82I are shown in (b). Data are representative of four independent experiments. *, P < 0.05 vs WT at the respective time point; **, P < 0.01 vs WT at the respective time points.
Mentions: To investigate the impact of V82I mutation on Cav-3 protein level and cellular localization, we introduced the V82I substitution detected in the patient into the human Cav-3 WT sequence. We next transfected BHK cells with Cav-3 WT or Cav-3 V82I and compared levels of wild-type and mutant in SDS lysates, which represent the total caveolin-3 pool. As shown in Figure 2, Cav-3 V82I mutant was expressed at significantly higher level (about 1.4 fold) than Cav-3 WT. The increased expression in caveolin-3 protein caused by V82I mutation is somehow a new finding since most of caveolin-3 mutations often cause a severe loss of Cav-3 protein[19]. To examine whether differences in protein stability between WT or V82I mutant accounted for the higher expression level of Cav-3 V82I, we performed cycloheximide (CHX) block experiments[33,34]. WT or V82I transfected BHK cells were treated with CHX for 1–6 h to inhibit protein synthesis, harvested at different time points, and resulting lysates were probed with anti-Cav-3 antibody to monitor caveolin-3 levels, or anti-tubulin antibody as a loading control. As shown in Figure 3, during CHX chase, Cav-3 WT protein declined over time, decreasing to 70% levels by 6 h, while Cav-3 V82I levels were significantly more stable over the 6 h time course. These results suggest that V82I mutation confers increased stability to caveolin-3 and are consistent with the higher protein expression level observed with Cav-3 V82I as compared to Cav-3 WT. It is interesting to note that 1 h of CHX treatment significantly increased the expression of the V82I mutant (but not of the WT form): the reason of such increase is currently unknown.

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