The LMNA mutation R321X associated with dilated cardiomyopathy leads to 2+ reorganization of the endoplasmic reticulum and aberrant Ca signaling in HEK 293 cells Silvia
1 Torretta ,
Giorgia
2 Schena ,
Giuseppe
CHOP
Unfolded protein response 600
Expression of the transcription factor C/EBP-homologous protein (CHOP)
A
400 300 200 100 0
1
2
3
4
ER
Ca2+ 120
C
+ FRET-based D1ER probe
D
100
° vs lmna WT * vs MOCK
80
*** °°°
60
[Ca2+]i 40 20 0
MOCK
100
§§§
80
*** °°°
60 40 20 0
cytosol
-
120
is reduced in LMNA-R321X expressing cells B
netFRET (% of control)
A-type lamins are encoded by LMNA gene whose alternative splicing events produce two principal isoforms, Lamin A and C. Both proteins polymerize to form the nuclear lamina, a filamentous scaffold underlying the inner nuclear membrane which maintain cellular and nuclear integrity. This role is particularly important in contracting cells such as cardiomyocytes which are continuously subjected to tension. In addition, lamins A/C interaction with a complex network of intranuclear membrane proteins allow:
ER
***
Confocal laser microscopy experiments showed that the localization of the newly characterized mutant LMNA-DUP (green, anti-GFP), albeit profoundly impaired if compared with LMNA WT, was nuclear and did not diffuse outside the nuclear envelope. In the insets, a merged image of LMNA and Phalloidin-TRITC is shown. Nor ER Ca2+-levels nor the refill/leak rate were affected by LMNA-DUP expression as measured with ERD1.
LMNA WT LMNA R321X
(A) Schematic drawing of the dynamic equilibrium responsible for mantaining ER calcium levels. (B) Schematic model of the FRET-based probe (D1ER, kindly provided by Prof. R. Tsien) used to monitor ER calcium levels. Binding of Ca2+ to the calmodulin sequence results in an intramolecular rearrangement of the probe with a consequent increase in the FRET signal. (C) The histogram of changes in netFRET reveals that ER Ca2+ levels are significantly reduced in LMNA-R321X expressing cells (° vs lmna WT).(D) The FRET signal (emission ratio 530/470 nm) is depicted in pseudocolor.
LMNA-R321X cells have slower ER Ca2+ leak/refill rate
Slope (ratio/min)
0,06
MOCK LMNA-WT LMNA-R321X
LMNA
LMNA-R321X
**
0,06 0,04 0,02
0,04
0 Ca2+EGTA 100μM
Ca2+ 2 mM CPA 30μM
350 300
i
150 100
***
50 300
200
250
150 100
200
***
150 100
50
50
0
0
MOCK LMNA-WT LMNA-R321X LMNA-DUP
LEAK RATE
homeostasis?
200
250
ER-Ca2+ release and capacitative Ca2+ entry from the plasma membrane were studied in Fura-2 loaded HEK293 transfected with either LMNA WT or LMNA-R321X. As expected, Ca2+ release from the ER of LMNA-R321X was significantly reduced when compared with LMNA WT cells. Surprisignly, capacitative Ca2+ entry was also affected in the presence of the mutant, suggesting a putative involvmente of STIM-1. * vs LMNA WT
LMNA-R321X
?
-
?
Bak/Bax
Contraction?
? Ca2+
Apoptotic cell death
0,02
Cyt C Caspase 9 Apaf1
0
ER LEAK RATE
Dynamic FRET. SERCA pump specific blockade unmasked the passive Ca2+ leak. The leak rate was measured with D1ER as the initial slope of the ratio decrease after CPA addition (*vs LMNA-WT) .
Caspase 3
LMNA-dilated cardiomyopathy
Schematic drawing of the mechanism elicited by LMNA-R321X expression in HEK293 cells. ER stress induced by dislocalization of the truncated mutant might affect Ca2+ homeostasis eventually leading to DCM via apoptotic cell death and/or impaired contaction in contractile cells.
LMNA-R321X does not affect spontaneous Ca2+i oscillations in Fluo-4 loaded HL-1 cardiomyocytes
ER Ca2+
0,16
97
MOCK LMNA-WT LMNA-R321X
Slope (ratio/min)
0,12
10s
0,10 0,08 0,06
**
0,04 0,02 0,00
ER REFILL RATE
Dynamic FRET. Ca2+ was depleted from the ER. The speed of Ca2+ re-uptake into the ER after Ca2+ readdition was measured with D1ER as the initial slope of the ratio increase (**vs LMNA-WT) .
[Ca2+]i (nM)
(A) GFP-LMNA-R321X had abnormal nucleoplasmic distribution and diffuse cytoplasmic localization in HeLa cells. Lamin B2 and Emerin were not affected by the expression of the mutant protein. (B) Western blot analysis using an anti-GFP monoclonal antibody confirmed the correct expression of either GFP-LMNA WT or -R321X in HEK293 cells. (C) Confocal laser microscopy experiments showed that the expression of LMNAR321X (green, anti-GFP) colocalized with the endoplasmic reticulum (red, anti-SERCA) in HEK 293 cells (right panel). Note the typical perinuclear rim staining of LMNA –WT (left panel).
0,01
250
0,14
J Mol Med (2008) 86:281-289
0,02
0
0,03
GFP-LMNA-WT and -R321X transfected HEK293 cells
64
0,03
0
2+ Ca
*
0,04
REFILL RATE
Fura-2
ER
Ca2+
cytosol
C
0,08
[Ca2+]i (nM)
KDa
0,05
0,01
Expression of GFP-tagged LMNA-R321X caused retention of lamin A in the ER of HEK293 cells B
0,10
0,05
300
ER stress
Disease models and Mechanisms 4,562-568(2011)
A
0,12
Ca2+
*
Accelerated aging disorders (progerias)
0,06
Working hypothesis
0,07
Peripheral neuropathy
0,14
Does LMNA-R321X affect
ER
Clinical entities caused by LMNA mutations
LMNA-dilated cardiomyopathy (DCM) Early conduction defects and arrhythmias Left ventricular dilatation Systolic dysfunction Myocyte death Myocardial fibrosis
0,07
0,00
ER-Ca2+ LEVEL
cytosol
1) proper physical and functional connections between the nucleus, cytoskeleton and contractile elements which are important for effective mechanotransduction in contractile cells, 2) anchorage of chromatin to the nuclear envelope and organization of its higher order structure 3) regulation of gene expression, DNA replication and transcription and 4) nuclear pore complex spatial organization.
0,16
CCE (nM)
2+ [Ca ]
***
500
Proapoptotic BCl2 family proteins (BAX/BAK)
Antiapoptoti c BCl2 family proteins
***
(A) Schematic diagram of the ER stressinduced activation of C/EBPhomologous protein (CHOP). (B) Western blotting analysys. LMNAWT and LMNA-R321X cell lysates either in control condition or after CPA treatment were immunoblotted and probed with anti-CHOP. Densitometry of immunoreactive bands revealed that CHOP expression, normalized against GAPDH, was significantly increased in LMNA-R321X HEK293 cells.
Slope (ratio/min)
Chronic ER stress
Background
Lipodystrophy syndromes
Maria
1 Svelto
Is ER-stress caused by LMNA-R321X ER dislocalization?
Ca2+ release (nM)
BACKGROUND: The lamin A/C (LMNA) are nuclear intermediate filament proteins composing the nuclear lamina. Mutations in LMNA are associated with a broad range of tissue-specific laminopathies. In particular, dilated cardiomyopathy (DCM) with conduction system disease and sudden death is frequently caused by a premature termination codon mutation in LMNA resulting in an aberrant expression of a truncated Lamin A on the nuclear envelope. AIM: In this study, we used a known LMNA nonsense mutation, R321X, to gain more insight into the pathophysiological mechanisms induced by LMNA-R321X in human embryonic kidney (HEK293) cells. RESULTS: Western blot analysis of homogenate from HEK293 cells transfected with LMNA-R321X showed a 65kDa band corresponding to the truncated LMNA. Also, immunofluorescence confocal analysis demonstrated that expression of GFP-tagged LMNA-R321X, but not GFP-tagged LMNA-WT, caused (I) loss of nuclear envelope shape and integrity (II) retention of lamin A in the endoplasmic reticulum (ER) and (III) dramatic reorganization of the ER. Interestingly, LMNA-R321X-induced ER reorganization dramatically perturbed Ca2+ homeostasis since both cyclopiazonic acid (CPA)-induced Ca2+ release and capacitative Ca2+ entry were perturbed in HEK293 cells loaded with the Ca2+ dye Rhod-2. CONCLUSIONS: Here, we proposed for the first time that the aberrant geometry of the ER induced by the expression of truncated LMNA may potentially lead to the development of DCM, rhythm disturbances and cardiac sudden death also via perturbance of Ca2+ homeostasis. CONTACTS:
[email protected]
Dislocalization of LMNA-R321X leads to ER stress
Slope (ratio/min)
Program Nº 1038.7 - ABSTRACT
Diseases of striated muscle •Autosomal Emery-Dreifuss muscular dystrophy •Cardiomyopathy dilated 1A •Limb-girdle muscular dystrophy type 1B •Congenital muscular dystrophy
1 Procino ,
of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “Aldo Moro”, Bari, Italy 2Department of Sciences, University of Basilicata, Potenza, Italy
netFRET (% of control)
1Department
Francesca Romana Del
1 Vecchio ,
[Ca2+]i (nM)
Monica
2 Carmosino ,
O.D.
Andrea
1 Gerbino ,
The frequency of spontaneous Ca2+i oscillation was studied in Fluo-4 loaded HL-1 cardiomyocytes transfected with either LMNA WT or LMNAR321X. The expression of the truncated LMNA-R321X did not affect intracellular Ca2+ oscillation (0,56±0.08 spikes/s, n=3) when compared with both LMNA WT (0,52±0.10 spikes/s, n=3) and mock (0,58±0.6 spikes/s, n=6) HL-1 cardiomyocytes.
10s
1038.7
