Identification and functional analysis of a new putative caveolin-3 variant found in a patient with sudden unexplained death
- Vincenzo Lariccia†1,
- Annamaria Assunta Nasti†1,
- Federica Alessandrini†1,
- Mauro Pesaresi1,
- Santo Gratteri2,
- Adriano Tagliabracci1 and
- Salvatore Amoroso1Email author
© Lariccia et al.; licensee BioMed Central Ltd. 2014
Received: 10 January 2014
Accepted: 3 June 2014
Published: 10 June 2014
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.
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.
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.
The most challenging type of SCD occurs when it cannot be traced to any evident cause other than a lethal arrhythmia, which may represent the sole sentinel event of an otherwise silent disease[1, 2]. Congenital cardiac channelopathies and cardiomyopathies are known to play significant roles in SCD[2–4]. In the first cases genotype-phenotype correlation studies have identified most of the known disease-causing defects in genes encoding cardiac ion channels or other membrane components, while in the second cases cardiac structural abnormalities have been detected in subjects carrying mutations in sarcomeric proteins. However, the identified variants commonly have reduced penetrance and are associated with heterogeneous clinical manifestations. Such variability in disease expression suggests that other modifying and triggering causes must be considered. For instance, genetic predisposition and environmental factors may negatively influence the arrhythmic risk by increasing heart susceptibility to stressing conditions[5–8]. Aberrant cell death can potentially provoke at least three changes in the heart that are relevant to lethal arrhythmic processes, namely cardiac contractility compromise, conduction disturbance and cardiac remodeling[9–11].
Recent studies have discovered mutations in CAV3 gene encoding caveolin-3 (Cav-3) in subject affected by hereditary arrhythmias - such as long QT syndrome (LQTS) and sudden infant death syndrome (SIDS)[12, 13] - or congenital cardiomyopathies[14, 15]. Cardiac manifestations are not commonly observed among individuals affected by caveolinopathies, which often display abnormal skeletal muscle phenotypes[15–19].
Cav-3 is predominantly expressed in cells experiencing cyclic mechanical stress (such as striated muscle myocytes) and is one of the three major isoforms of caveolins. These proteins localize within caveolae, plasma membrane microdomains considered as key platforms for numerous cellular processes such as endocytosis, lipid metabolism, mechanosensing and survival response to stressful stimuli[20–27]. Caveolar membranes contain and regulate different signalling enzymes, including ERKs (Extracellular-signal-Regualted Kinases, also known as p44/42 MAP kinases). Caveolins bind to and negatively regulate ERK activity[28, 29]. ERKs can be considered as master regulatory kinases, critically involved in cell fate determination processes in response to various stressful stimuli. In particular, alterations in ERK signalling negatively impact on cell viability under stressed conditions such as hyperosmotic shock[30, 31].
In the present study, we investigated whether a new putative Cav-3 variant, Cav-3 V82I, found in a patient with SCD in adulthood, renders cells more vulnerable to osmotic stress. In particular, we collected evidence that this variant accumulates within the cell, impairs ERK activation and increases cell death susceptibility to sub-lethal osmotic stress.
Analysis of sequence
Primers used for CAV3 PCR and sequencing reaction
Sequenza 5′ > 3′
All experiments were carried out in strict accordance with the Clinical Practice Guidelines of A.O.U. “Ospedali Riuniti di Ancona”, and with the Oviedo Convention on Human Rights and Biomedicine, ratified by Italian law 145 of 28 March 2001.
Plasmid containing Cav-3 wild-type cDNA was purchased from Origene. In vitro site-direct mutagenesis was performed by QuickChange Site-Direct Mutagenesis kit (Agilent Technologies, Milan, Italy) following manufacturer’s instructions. The presence of the G244A mutation (V82I) was confirmed by sequence analysis.
BHK cells were cultured in a humified 5% CO2 atmosphere in DMEM supplemented with 10% fetal bovine serum, 6 mM glutamine, 1 mM pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin. For immunofluorescence studies, cells were plated on glass coverslips 16 h before transfection.
Transient transfection and western blot
The plasmids containing respectively the Cav-3 WT and Cav-3 V82I were transfected to BHK cells with Attractene (Qiagen, Milan, Italy) following manufacturer’s instructions. Transfection efficiency, quantified by co-transfecting Cav-3 and EGFP expressing plasmids, was ~70-80% both for the WT and mutant. Twenty-four hours post transfection, whole cell lysates for western blot analysis were obtained using a cell lysis solution containing (in mM): NaCl, 150; Tris–HCl (pH 7.4), 10; EDTA (pH 8.0), 1; SDS 1%, and a protease inhibitor cocktail mixture. All samples were prepared in buffer containing SDS and 2-mercaptoethanol and boiled at 100°C for 10 min prior to loading. Immunoblotting on 8% polyacrylamide was performed as described previously and the following primary antibodies were used: mouse anti-CAV3 (1:500), purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA) and mouse anti-tubulin (1:10000; Sigma). The band images were digitally captured and band intensities were quantified using a ChemiDoc station and the Quantity one analysis software. The relative amount of caveolin-3 protein was normalized on tubulin. In some experiments, 10 μg/ml cycloheximide (stock solution 100 mg/ml in DMSO; Millipore, Billerica, MA, USA) was added directly to BHK cells 24 h after transfection with Cav-3 WT or Cav-3 V82I and the relative amount of caveolin-3 protein was determined by collecting cell lysates at the indicated time points[33, 34].
Caveolin-3 Triton solubility
Caveolin-3 partitions into Triton-soluble fraction were determined 24 h after transfection with the indicated plasmid as described before[14, 35, 36]. Briefly, transfected BHK cells were washed twice with ice-cold PBS and lysed for 30 min on ice in 1% Triton X-100 buffer (pH 6.5) containing 150 mM NaCl, 25 mM MES, protease inhibitors. Samples were then centrifuged at 14,000 × g for 10 min at 4°C and pellet (the insoluble fraction; I) and supernatant (soluble fraction; S) were resolved and analyzed by caveolin-3 immunoblotting as described before.
24 hours after transfection, BHK cells plated on glass coverslips were washed with PBS and fixed for 15 min with ice-cold PBS 4% paraformaldehyde, 2% sucrose. Cells were then incubated with PBS 3% BSA and 0.1% Triton for 30 min at RT and following incubated over night with the mouse antibody anti-Cav-3 (Santa Cruz Biotechnology) 1:500 in PBS 3% BSA. Finally, cells were incubated for 1 h in the dark with the Alexa Fluor 488 anti-mouse IgG (Invitrogen, Monza, Italy) 1:500 in PBS 3% BSA and then analyzed on a Zeiss LSM510 META confocal microscope (Zeiss, Arese, MI, Italy). For Nile red staining, the dye was added directly to fixed cells (1:100 dilution in PBS from a 1 mg/ml stock solution in acetone) after Cav-3 immunolabelling, and samples were incubated in the dark for 10 min at room temperature.
Determination of mitochondrial activity and cell death
Mitochondrial activity was quantified by measuring dehydrogenase activity retained in the cultured cells, using the MTT assay. The assay is based on the ability of living cells to convert dissolved MTT into insoluble formazan. Briefly, 24 h after transfection, BHK cells were washed with PBS and treated for 30 min in the cell culture incubator with 1 ml culture medium or mannitol solution (1.1 M in culture medium). After an additional wash with PBS, cells were then incubated with 1 ml of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (0.5 mg/ml in PBS). After 1 h incubation at 37°C, cells were finally washed with PBS and the formazan produced was dissolved in 1 ml of dimethylsulfoxide (DMSO). The absorbance was read at 540 nm. Data were expressed as the percentage of cell injury to sham-treated cultures.
To evaluate cell death under basal and mannitol stress conditions, cells were treated as described above and then double stained with 36 μM Fluorescein diacetate and 7 μM Propidium iodide for 20 min at 37°C in PBS. Stained cells were examined immediately with a standard inverse fluorescence microscope and analyzed as described before.
Determination of ERK activity
ERK activity was evaluated by in vitro phosphorylation of Elk-1 protein (an ERK substrate) in cell lysates using a commercially available nonradioactive assay kit (Cell Signaling, Boston, MA, USA). Briefly, 1 × 106 BHK cells were plated into 100 mm dishes 16 h before transfection with EGFP, Cav-3 WT or Cav-3 V82I expressing vectors. Transfected cells were exposed to standard or mannitol-containing culture media as previously described. pERK affinity precipitation was performed incubating cell extracts (overnight at 4°C) with sepharose bead conjugate phospho-p44/42 MAP Kinase monoclonal antibody. The immunoprecipitates were then used in an in vitro kinase assay using Elk-1 as substrate. pERK activity was finally evaluated quantifying by immunoblotting the level of Elk-1 phosphorylation with anti-phospho-Elk-1 (pElk) mouse antibody.
Since the doubly phosphorylated forms of ERK represent the active enzymes, we also used anti-phospho-ERK immunoblotting as direct ERK activation marker. To detect the active form of ERKs, cell lysates were subjected to western blot analysis using pERK and ERK antibodies (Santa Cruz, California, USA). The amount of ERK phosphorylation was quantified by densitometry and normalized by expressing the data as a ratio of pERK over the total ERK.
Data were analysed by t-test for paired or unpaired data as appropriate. For multiple group comparisons, two way ANOVA followed Dunnett post-hoc test was used as appropriate.
All the other chemicals were of analytical grade and were purchased from Sigma, Italy.
Identification of a novel CAV3 mutation
Expression and distribution of wild type and V82I mutant of Caveolin-3
Effect of Cav-3 V82I on ERK activity and cell viability
In this report, we identified and characterized a novel heterozygous mutation in caveolin-3 gene. This mutation (V82I) was found in an individual adult with suspected LQTS who suddenly died prior to completion of comprehensive cardiological examination. No mutations were found in the entire coding regions of the major LQTS associated genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, and KCNJ2) and in ANK2 and RyR2 genes[2, 3].
Valine-to-isoleucine substitution is considered as a conservative change in that the two amino acids differ by only a methylene group, and a priori it would not be expected to substantially alter the properties of the protein. However, despite their similarity, their side-chains differ in size, shape and hydrophobicity, and these differences account for the fact that valine and isoleucine are not always interchangeable in proteins. Moreover, V82I mutation alters a conserved amino acid of the Cav-3 membrane-spanning segment (Figure 1 and).
In humans, the majority of caveolinopathies often refers to a wide spectrum of skeletal muscle disorders[19, 46] and only recently mutations in caveolin-3 were associated to cardiac pathologies including LQTS and SIDS[12, 13] and cardiomyopayhies[14, 15]. Several mechanisms have been proposed for the skeletal muscle degenerative processes, whereas the molecular bases for the cardiac phenotypes are largely unknown[14, 19, 46].
Biochemical analysis of transiently transfected cell line showed that Cav-3 V82I is expressed at higher level than Cav-3 WT (Figure 2 and Additional file3) probably as consequence of higher protein stability as revealed by CHX assay (Figure 3). Moreover, V82I mutant did not alter the detergent solubility of caveolin-3 protein, and a significant increase of caveolin-3 expression was found both in the Triton-insoluble (I) and in the Triton-soluble (S) fractions (Figure 7). These results are in strike contrast to the majority of the known caveolin-3 mutants which cause 1- severe loss of protein, 2- decrease of protein half-life, 3- increased expression in the Triton-soluble fraction[14, 19, 35, 36, 41].
To study the functional alterations of Cav-3 due to V82I mutation, we examined the intracellular distribution of the mutant protein by confocal microscopy. Immunofluorescence studies revealed that Cav-3 V82I tends to accumulate within the cells, suggesting that the V82I mutation affects a critical residue required for correct cell surface expression as observed with caveolin-3 WT (Figures 4,5,6 and Additional files2,3). It is interesting to note that the threonine 78, a residue very close to the valine 82, is also critical for proper targeting of caveolin-3 protein to the plasma membrane, since Cav-3 T78K (but not Cav-3 T78M) mutant fails to distribute to cellular surface. The retention in intracellular compartments is a common phenotype observed with different pathogenetic caveolin-3 mutants that often form aggregates within Golgi complex[15, 19, 35, 36, 41, 47]. In contrast to the WT form, Cav-3 V82I showed a particular cellular distribution, characterized by reduced targeting to the plasma membrane and, in most transfected cells, by increased staining of ring-like LB structures (Figures 4,5 and Additional files2,3). A very similar labelling pattern was reported for Cav-3DGV, an amino-terminal truncation mutant of caveolin-3, which fails to reach plasma membrane and highly localizes into LB[40, 47]. Since Cav-3 V82I mutant is not properly distributed to cell surface, we suggest that Cav-3 V82I found in the Triton insoluble fraction does not primarily come from plasma membrane caveolae/lipid rafts but instead from intracellular compartments such as LB where Cav-3 V82I accumulates (Figure 5). To this regard, it is interesting to note that Cav-3DGV LB pool is Triton-insoluble.
When proteasome activity is inhibited, intracellular retained and unstable Cav-3 mutants are expressed at significantly higher level and display a characteristic redistribution to ER (but not to plasma membrane). Considering that V82I mutation increased Cav-3 cell expression and that LBs have an ER origin, we hypothesized that Cav-3 V82I would also display a similar ER retention. However, no significant accumulation of Cav-3 V82I protein was observed within the ER (Figure 6).
Several studies have proposed a critical role for caveolin-3 in cell life and death decision mechanisms. In fact, when caveolin-3 dependent signalling pathways are disrupted, critical cellular functions are significantly affected, including proliferation, survival response to stressful stimuli, preservation of plasma membrane integrity during mechanical stresses or toxin-induced membrane injury. Based on these findings, we compared the survival response to brief hyperosmotic stress elicited in cells transfected with Cav-3 WT or with Cav-3 V82I. Under resting conditions, neither WT nor V82I form had any effect on cell viability (Figure 9). However, V82I mutant significantly increased cell death susceptibility under a mannitol stress that was sub-lethal in Cav-3 WT transfected cells (Figure 9). Such increased cell vulnerability may be related to the altered caveolin-3 distribution observed in Cav-3 V82I transfected cells (Figures 4,5). In fact, the reduced plasma membrane targeting of V82I mutant would impair caveolae formation (a process that require caveolin protein) and consequently affect caveolae-mediated protective response to plasma membrane stress[24, 27].
Since ERK signalling pathways are regulated by Cav-3 proteins and influence the survival rate of the cells[30, 49], we also explored the effect of Cav-3 V82I on ERK activation. We found that V82I significantly reduced ERK activation both under resting condition and after hyperosmotic stress (Figure 8). The negative effects on ERK activation exerted by V82I mutant may be simply related to the higher caveolin-3 expression level observed in Cav-3 V82I transfected cells. Collectively, our data suggest that the expression of Cav-3 V82I is detrimental for the survival response to mechanical stress.
Signs of apoptosis among myocytes have been documented as characteristic histological signature in various arrhythmic patients[11, 50, 51] and abnormal cardiomyocyte death may be clinically relevant for the development of life-threatening arrhythmia in terms of associated (if not causative) mechanism[9–11, 50, 51]. It is interesting to note that for Cav-3 the so-called “TFT mutation” causes apoptosis in skeletal muscle cells and decreases myotube resistance to oxidative stress. In connection to these data, we propose that the Cav-3 V82I variant increases cell vulnerability to stress conditions and may participate in determining the susceptibility to cardiac death.
The sodium/calcium exchanger 1 (NCX1) is an electrogenic cardiac Ca2+ transporter protein that regulates cardiac excitation-contraction coupling. NCX1 normally contributes to the ion currents responsible for the action potential but can trigger arrhythmias under pathological conditions. Based on previous data showing Cav-3/NCX1 interaction, we hypothesize that Cav-3 V82I could also affect NCX1 activity. However, in biochemical and functional experiments using BHK cells stable expressing NCX1, we found no interaction between NCX1 and WT or V82I Cav-3 (Additional file4).
The biochemical and functional findings presented here provided evidence that the new putative caveolin-3 V82I variant discovered in our patient has a cell expression profile significantly different not only from the WT form (Figures 2,3,4,5,6,7,8,9 and Additional files2,3) but also from most of the mutants analyzed so far[14, 15, 19, 35, 36, 41, 47]. Cells expressing Cav-3 V82I displayed altered caveolin-3 distribution and were more vulnerable to sub-lethal osmotic stress. These defects may potentially be of clinical relevance considering that abnormal loss of myocyte can deadly impair people’s health[9–11, 50, 51].
Some Cav-3 mutants have been reported to influence the activity of ion channels localized within caveolae[12, 13, 55], but it is still not fully understood whether and how these mutants impact on the action potential as a whole[56, 57]. Therefore, we cannot exclude the possibility that Cav-3 V82I may alter the currents carried by these channels and further work is required to address this issue. However, we provided evidence that the V82I mutant has no effect on NCX1, a key plasma membrane transporter that under normal and pathological conditions influences multiple properties of cardiomyocytes, including cell excitability.
Adriano Tagliabracci and Salvatore Amoroso are senior authors.
Annamaria A. Nasti’s present address: Department of Molecular Medicine, University of Padua, Italy.
The authors thank Carlo Alfredo Violet, Loredana Macri’, Alessandra Matteucci and Gerardo Galeazzi for their excellent technical support. This study was supported by “Ricerca Scientifica di Ateneo” grant from the University “Politecnica delle Marche” to VL, AT, MP and to SA. The funding source had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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