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The Cardiomyopathy Lamin A/C D192G Mutation Disrupts Whole-Cell Biomechanics in Cardiomyocytes as Measured by Atomic Force Microscopy Loading-Unloading Curve Analysis.

Lanzicher T, Martinelli V, Puzzi L, Del Favero G, Codan B, Long CS, Mestroni L, Taylor MR, Sbaizero O - Sci Rep (2015)

Bottom Line: Our results suggested that the LMNA D192G mutation increased maximum nuclear deformation load, nuclear stiffness and fragility as compared to controls.Furthermore, chemical disruption of the actin cytoskeleton by cytochalasin D in control cardiomyocytes mirrored the alterations in the mechanical properties seen in mutant cells, suggesting a defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion molecules in cells expressing the mutant protein.These data add to our understanding of potential mechanisms responsible for this fatal cardiomyopathy, and show that the biomechanical effects of mutant lamin extend beyond nuclear mechanics to include interference of whole-cell biomechanical properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Engineering and Architecture, University of Trieste, Via Valerio 2, 34127, Trieste Italy.

ABSTRACT
Atomic force microscopy (AFM) cell loading/unloading curves were used to provide comprehensive insights into biomechanical behavior of cardiomyocytes carrying the lamin A/C (LMNA) D192G mutation known to cause defective nuclear wall, myopathy and severe cardiomyopathy. Our results suggested that the LMNA D192G mutation increased maximum nuclear deformation load, nuclear stiffness and fragility as compared to controls. Furthermore, there seems to be a connection between this lamin nuclear mutation and cell adhesion behavior since LMNA D192G cardiomyocytes displayed loss of AFM probe-to-cell membrane adhesion. We believe that this loss of adhesion involves the cytoskeletal architecture since our microscopic analyses highlighted that mutant LMNA may also lead to a morphological alteration in the cytoskeleton. Furthermore, chemical disruption of the actin cytoskeleton by cytochalasin D in control cardiomyocytes mirrored the alterations in the mechanical properties seen in mutant cells, suggesting a defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion molecules in cells expressing the mutant protein. These data add to our understanding of potential mechanisms responsible for this fatal cardiomyopathy, and show that the biomechanical effects of mutant lamin extend beyond nuclear mechanics to include interference of whole-cell biomechanical properties.

No MeSH data available.


Related in: MedlinePlus

(A) EGFP and human LMNA expression detected by fluorescence light microscopy 24 hours post-infection with adenoviral WT and MT NRVM’s constructs. (B) Expression of the transduced human LMNA protein detected by human specific anti-LMNA antibody by western blot: human LMNA and rat Calnexin at 24 and 48 hours post-infection. (C) Indirect immunofluorescence showing co-localization between GFP (green, upper-left panel) and human LMNA (red, right-upper panel) signals in D192G LMNA NRVMs, transduced by adenoviral bicistronic GFP-LMNA construct. The nuclei are stained in blue (TOTO3, lower-left column). The fluorescence is represented in three channels. Bars: 5 μ. As seen in the merged panel (lower-right), human LMNA is perfectly co-localized in the area of the nuclear wall. (D) AFM loading-unloading curves for CT, WT LMNA and MT LMNA D192G.
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f1: (A) EGFP and human LMNA expression detected by fluorescence light microscopy 24 hours post-infection with adenoviral WT and MT NRVM’s constructs. (B) Expression of the transduced human LMNA protein detected by human specific anti-LMNA antibody by western blot: human LMNA and rat Calnexin at 24 and 48 hours post-infection. (C) Indirect immunofluorescence showing co-localization between GFP (green, upper-left panel) and human LMNA (red, right-upper panel) signals in D192G LMNA NRVMs, transduced by adenoviral bicistronic GFP-LMNA construct. The nuclei are stained in blue (TOTO3, lower-left column). The fluorescence is represented in three channels. Bars: 5 μ. As seen in the merged panel (lower-right), human LMNA is perfectly co-localized in the area of the nuclear wall. (D) AFM loading-unloading curves for CT, WT LMNA and MT LMNA D192G.

Mentions: Neonatal rat ventricular myocytes (NRVMs) were isolated and enriched (> 90% purity) over non-myocytes as previously reported11121314 and subjected to infection with an adenoviral construct carrying either the wild type or the mutant D192G LMNA A cDNA as well as the Enhanced Green Fluorescent Protein (EGFP) which can be used to identify LMNA expressing cells (described in detail in the Methods section)1516171819. NRVMs were infected on culture day 1 and the expression of both EGFP and human LMNA (wild-type and mutant) examined after 24 and 48 hours. As indicated in Fig. 1A,B, although EGFP expression was always detected visually by 24 hour of infection, the expression of the LMNA proteins, as determined by Western blotting with a human-specific anti-lamin A antibody, appeared at 24 hours and was clearly expressed at 48 hours. Furthermore, as shown in Fig. 1C, immunofluorescence confirmed the localization of the exogenous human LMNA (in red, right panels) in the nuclear wall of D192G LMNA NRVMs, when transduced by adenoviral bicistronic GFP-LMNA constructs. Increases in cell-turnover or senescence during the time in culture or in response to adenoviral infection were not observed (data not shown).


The Cardiomyopathy Lamin A/C D192G Mutation Disrupts Whole-Cell Biomechanics in Cardiomyocytes as Measured by Atomic Force Microscopy Loading-Unloading Curve Analysis.

Lanzicher T, Martinelli V, Puzzi L, Del Favero G, Codan B, Long CS, Mestroni L, Taylor MR, Sbaizero O - Sci Rep (2015)

(A) EGFP and human LMNA expression detected by fluorescence light microscopy 24 hours post-infection with adenoviral WT and MT NRVM’s constructs. (B) Expression of the transduced human LMNA protein detected by human specific anti-LMNA antibody by western blot: human LMNA and rat Calnexin at 24 and 48 hours post-infection. (C) Indirect immunofluorescence showing co-localization between GFP (green, upper-left panel) and human LMNA (red, right-upper panel) signals in D192G LMNA NRVMs, transduced by adenoviral bicistronic GFP-LMNA construct. The nuclei are stained in blue (TOTO3, lower-left column). The fluorescence is represented in three channels. Bars: 5 μ. As seen in the merged panel (lower-right), human LMNA is perfectly co-localized in the area of the nuclear wall. (D) AFM loading-unloading curves for CT, WT LMNA and MT LMNA D192G.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: (A) EGFP and human LMNA expression detected by fluorescence light microscopy 24 hours post-infection with adenoviral WT and MT NRVM’s constructs. (B) Expression of the transduced human LMNA protein detected by human specific anti-LMNA antibody by western blot: human LMNA and rat Calnexin at 24 and 48 hours post-infection. (C) Indirect immunofluorescence showing co-localization between GFP (green, upper-left panel) and human LMNA (red, right-upper panel) signals in D192G LMNA NRVMs, transduced by adenoviral bicistronic GFP-LMNA construct. The nuclei are stained in blue (TOTO3, lower-left column). The fluorescence is represented in three channels. Bars: 5 μ. As seen in the merged panel (lower-right), human LMNA is perfectly co-localized in the area of the nuclear wall. (D) AFM loading-unloading curves for CT, WT LMNA and MT LMNA D192G.
Mentions: Neonatal rat ventricular myocytes (NRVMs) were isolated and enriched (> 90% purity) over non-myocytes as previously reported11121314 and subjected to infection with an adenoviral construct carrying either the wild type or the mutant D192G LMNA A cDNA as well as the Enhanced Green Fluorescent Protein (EGFP) which can be used to identify LMNA expressing cells (described in detail in the Methods section)1516171819. NRVMs were infected on culture day 1 and the expression of both EGFP and human LMNA (wild-type and mutant) examined after 24 and 48 hours. As indicated in Fig. 1A,B, although EGFP expression was always detected visually by 24 hour of infection, the expression of the LMNA proteins, as determined by Western blotting with a human-specific anti-lamin A antibody, appeared at 24 hours and was clearly expressed at 48 hours. Furthermore, as shown in Fig. 1C, immunofluorescence confirmed the localization of the exogenous human LMNA (in red, right panels) in the nuclear wall of D192G LMNA NRVMs, when transduced by adenoviral bicistronic GFP-LMNA constructs. Increases in cell-turnover or senescence during the time in culture or in response to adenoviral infection were not observed (data not shown).

Bottom Line: Our results suggested that the LMNA D192G mutation increased maximum nuclear deformation load, nuclear stiffness and fragility as compared to controls.Furthermore, chemical disruption of the actin cytoskeleton by cytochalasin D in control cardiomyocytes mirrored the alterations in the mechanical properties seen in mutant cells, suggesting a defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion molecules in cells expressing the mutant protein.These data add to our understanding of potential mechanisms responsible for this fatal cardiomyopathy, and show that the biomechanical effects of mutant lamin extend beyond nuclear mechanics to include interference of whole-cell biomechanical properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Engineering and Architecture, University of Trieste, Via Valerio 2, 34127, Trieste Italy.

ABSTRACT
Atomic force microscopy (AFM) cell loading/unloading curves were used to provide comprehensive insights into biomechanical behavior of cardiomyocytes carrying the lamin A/C (LMNA) D192G mutation known to cause defective nuclear wall, myopathy and severe cardiomyopathy. Our results suggested that the LMNA D192G mutation increased maximum nuclear deformation load, nuclear stiffness and fragility as compared to controls. Furthermore, there seems to be a connection between this lamin nuclear mutation and cell adhesion behavior since LMNA D192G cardiomyocytes displayed loss of AFM probe-to-cell membrane adhesion. We believe that this loss of adhesion involves the cytoskeletal architecture since our microscopic analyses highlighted that mutant LMNA may also lead to a morphological alteration in the cytoskeleton. Furthermore, chemical disruption of the actin cytoskeleton by cytochalasin D in control cardiomyocytes mirrored the alterations in the mechanical properties seen in mutant cells, suggesting a defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion molecules in cells expressing the mutant protein. These data add to our understanding of potential mechanisms responsible for this fatal cardiomyopathy, and show that the biomechanical effects of mutant lamin extend beyond nuclear mechanics to include interference of whole-cell biomechanical properties.

No MeSH data available.


Related in: MedlinePlus