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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

Hypothesis on the role of defective lamin in altering cell adhesion behavior.Speculative cartoon underlying the potential role of mutated LMNA in cell adhesion properties. As recently reported42, we hypothesized that lamin mutant cells cause altered actin dynamics and cytoskeletal actin polymerization. The defective nuclear-cytoskeletal connection may lead to adhesion proteins dysfunction, and ultimately to defective adhesion-detachment properties in mutant cells.
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f8: Hypothesis on the role of defective lamin in altering cell adhesion behavior.Speculative cartoon underlying the potential role of mutated LMNA in cell adhesion properties. As recently reported42, we hypothesized that lamin mutant cells cause altered actin dynamics and cytoskeletal actin polymerization. The defective nuclear-cytoskeletal connection may lead to adhesion proteins dysfunction, and ultimately to defective adhesion-detachment properties in mutant cells.

Mentions: In conclusion, to our knowledge, this is the first comprehensive report on abnormalities in AFM-derived mechanical properties of cardiomyocytes expressing a LMNA mutation associated with a severe form of familial dilated cardiomyopathy (LMNA D192G). These data were derived from analysis of a series of loading and unloading curves of living cardiomyocytes in which expression of both wild-type and mutant proteins were accomplished using an adenoviral expression system that resulted in a time-dependent expression of exogenous protein mirrored by alterations in both stiffness and fragility of the cells. Specifically, LMNA D192G transduced cardiomyocytes showed an increase in the nuclear Young modulus and greater nuclear brittleness that were clearly apparent as expression of mutant protein increases. We hypothesize that the concurrent change in both elasticity and brittleness might be explained by a shape change in the nuclear lamin structure going from a simple flat mesh to a more dimpled structure. Moreover, the loading-unloading curves showed: (i) differences in the maximum load required to deform the nucleus where mutant cells required higher loads, (ii) sphere/cell membrane detachment area where mutant cells show almost no detachment area. These results confirm that AFM is a very good tool for cell mechanical perturbation and force measurements. Furthermore, simple models may be utilized to rationalize the observed force-deformation measurements. Similar AFM measurement of the aforementioned parameters in control cells in which the actin cytoskeleton has been chemically disrupted suggest that the alterations in the mechanical properties seen in mutant cells reflects a fundamental defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion proteins expressed at the cell-surface (Fig. 8). Structural anomalies resulting from mutation of lamin A/C therefore, seems to extend far beyond the nucleus to affect the cytoskeleton as shown in our case for the actin network. Our descriptive experiments clearly demonstrate that there is an adhesion defect in LMNA mutant NRVMs. We speculate that this biomechanical behavior could be driven by defective membrane adhesion proteins, such as integrins, as suggested by previous investigations194142434445464748. While a comprehensive functional study of membrane proteins, such as integrins, dystrophin complex proteins, and other specialized complexes such as the desmosome, is beyond the scope of this work, the role of membrane adhesion in laminopathies is intriguing and prompt future investigations.


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)

Hypothesis on the role of defective lamin in altering cell adhesion behavior.Speculative cartoon underlying the potential role of mutated LMNA in cell adhesion properties. As recently reported42, we hypothesized that lamin mutant cells cause altered actin dynamics and cytoskeletal actin polymerization. The defective nuclear-cytoskeletal connection may lead to adhesion proteins dysfunction, and ultimately to defective adhesion-detachment properties in mutant cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555041&req=5

f8: Hypothesis on the role of defective lamin in altering cell adhesion behavior.Speculative cartoon underlying the potential role of mutated LMNA in cell adhesion properties. As recently reported42, we hypothesized that lamin mutant cells cause altered actin dynamics and cytoskeletal actin polymerization. The defective nuclear-cytoskeletal connection may lead to adhesion proteins dysfunction, and ultimately to defective adhesion-detachment properties in mutant cells.
Mentions: In conclusion, to our knowledge, this is the first comprehensive report on abnormalities in AFM-derived mechanical properties of cardiomyocytes expressing a LMNA mutation associated with a severe form of familial dilated cardiomyopathy (LMNA D192G). These data were derived from analysis of a series of loading and unloading curves of living cardiomyocytes in which expression of both wild-type and mutant proteins were accomplished using an adenoviral expression system that resulted in a time-dependent expression of exogenous protein mirrored by alterations in both stiffness and fragility of the cells. Specifically, LMNA D192G transduced cardiomyocytes showed an increase in the nuclear Young modulus and greater nuclear brittleness that were clearly apparent as expression of mutant protein increases. We hypothesize that the concurrent change in both elasticity and brittleness might be explained by a shape change in the nuclear lamin structure going from a simple flat mesh to a more dimpled structure. Moreover, the loading-unloading curves showed: (i) differences in the maximum load required to deform the nucleus where mutant cells required higher loads, (ii) sphere/cell membrane detachment area where mutant cells show almost no detachment area. These results confirm that AFM is a very good tool for cell mechanical perturbation and force measurements. Furthermore, simple models may be utilized to rationalize the observed force-deformation measurements. Similar AFM measurement of the aforementioned parameters in control cells in which the actin cytoskeleton has been chemically disrupted suggest that the alterations in the mechanical properties seen in mutant cells reflects a fundamental defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion proteins expressed at the cell-surface (Fig. 8). Structural anomalies resulting from mutation of lamin A/C therefore, seems to extend far beyond the nucleus to affect the cytoskeleton as shown in our case for the actin network. Our descriptive experiments clearly demonstrate that there is an adhesion defect in LMNA mutant NRVMs. We speculate that this biomechanical behavior could be driven by defective membrane adhesion proteins, such as integrins, as suggested by previous investigations194142434445464748. While a comprehensive functional study of membrane proteins, such as integrins, dystrophin complex proteins, and other specialized complexes such as the desmosome, is beyond the scope of this work, the role of membrane adhesion in laminopathies is intriguing and prompt future investigations.

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