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

Immonolocalization of Cav-3 WT and Cav-3 V82I. BHK cells were transiently transfected with Cav-3 WT (a) or Cav-3 V82I (b) and immunostained with the antibodies against caveolin-3 followed by Alexa Fluor 488 conjugated secondary antibodies. Cav-3 V82I mutant was retained intracellularly and not properly targeted to the plasma membrane as Cav-3 WT (arrowheads). Vesicular-like structures (LB) stained with the caveolin-3 antibodies were also observed, especially for Cav-3 V82I mutant (arrows); Scale bar: 10 μm. Four independent transfected cultures were analyzed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Immonolocalization of Cav-3 WT and Cav-3 V82I. BHK cells were transiently transfected with Cav-3 WT (a) or Cav-3 V82I (b) and immunostained with the antibodies against caveolin-3 followed by Alexa Fluor 488 conjugated secondary antibodies. Cav-3 V82I mutant was retained intracellularly and not properly targeted to the plasma membrane as Cav-3 WT (arrowheads). Vesicular-like structures (LB) stained with the caveolin-3 antibodies were also observed, especially for Cav-3 V82I mutant (arrows); Scale bar: 10 μm. Four independent transfected cultures were analyzed.

Mentions: We further investigated the sub-cellular distribution of wild type and mutant V82I caveolin-3 by means of confocal microscopic analysis. In BHK cells, the wild type caveolin-3 was localized both at the plasma membrane and within the cells, while Cav-3 V82I was mainly retained within the cell (Figure 4; see Additional file2 for complete Z-stack image series). In most cells, a ring-like labelling was also observed, especially for Cav-3 V82I mutant (Figure 4). These intracellular structures stained with the Cav-3 antibody varied in size and were often clustered together. To determine whether these spherical structures were the so-called lipid bodies (LB), organelles targeted by caveolins (both wild types and mutant forms)[34,39,40], we carried out double-labelling analysis with antibodies to caveolin-3 proteins and with Nile red, a marker for LB[34,39,40]. As reported in Figure 5, both in Cav-3 WT and in Cav-3 V82I expressing cells, the intracellular Cav-3 pools highly localized on the surface of Nile red stained vesicles. However, we found that not all the Cav-3 positive structures were also positive for Nile red, as well as that some LB were stained only with Nile red. Similar results were obtained in a rat cardiomyoblast cell line, H9c2 cells (Additional file3), suggesting that the observed differences in protein expression between Cav-3 V82I and WT forms were not related to the cell type used.


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)

Immonolocalization of Cav-3 WT and Cav-3 V82I. BHK cells were transiently transfected with Cav-3 WT (a) or Cav-3 V82I (b) and immunostained with the antibodies against caveolin-3 followed by Alexa Fluor 488 conjugated secondary antibodies. Cav-3 V82I mutant was retained intracellularly and not properly targeted to the plasma membrane as Cav-3 WT (arrowheads). Vesicular-like structures (LB) stained with the caveolin-3 antibodies were also observed, especially for Cav-3 V82I mutant (arrows); Scale bar: 10 μm. Four independent transfected cultures were analyzed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Immonolocalization of Cav-3 WT and Cav-3 V82I. BHK cells were transiently transfected with Cav-3 WT (a) or Cav-3 V82I (b) and immunostained with the antibodies against caveolin-3 followed by Alexa Fluor 488 conjugated secondary antibodies. Cav-3 V82I mutant was retained intracellularly and not properly targeted to the plasma membrane as Cav-3 WT (arrowheads). Vesicular-like structures (LB) stained with the caveolin-3 antibodies were also observed, especially for Cav-3 V82I mutant (arrows); Scale bar: 10 μm. Four independent transfected cultures were analyzed.
Mentions: We further investigated the sub-cellular distribution of wild type and mutant V82I caveolin-3 by means of confocal microscopic analysis. In BHK cells, the wild type caveolin-3 was localized both at the plasma membrane and within the cells, while Cav-3 V82I was mainly retained within the cell (Figure 4; see Additional file2 for complete Z-stack image series). In most cells, a ring-like labelling was also observed, especially for Cav-3 V82I mutant (Figure 4). These intracellular structures stained with the Cav-3 antibody varied in size and were often clustered together. To determine whether these spherical structures were the so-called lipid bodies (LB), organelles targeted by caveolins (both wild types and mutant forms)[34,39,40], we carried out double-labelling analysis with antibodies to caveolin-3 proteins and with Nile red, a marker for LB[34,39,40]. As reported in Figure 5, both in Cav-3 WT and in Cav-3 V82I expressing cells, the intracellular Cav-3 pools highly localized on the surface of Nile red stained vesicles. However, we found that not all the Cav-3 positive structures were also positive for Nile red, as well as that some LB were stained only with Nile red. Similar results were obtained in a rat cardiomyoblast cell line, H9c2 cells (Additional file3), suggesting that the observed differences in protein expression between Cav-3 V82I and WT forms were not related to the cell type used.

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