Altered microtubule structure, hemichannel localization and beating activity in cardiomyocytes expressing pathologic nuclear lamin A/C

Abstract

Given the clinical effect of laminopathies, understanding lamin mechanical properties will benefit the treatment of heart failure. Here we report a mechano-dynamic study of LMNA mutations in neonatal rat ventricular myocytes (NRVM) using single cell spectroscopy with Atomic Force Microscopy (AFM) and measured changes in beating force, frequency and contractile amplitude of selected mutant-expressing cells within cell clusters. Furthermore, since beat-to-beat variations can provide clues on the origin of arrhythmias, we analyzed the beating rate variability using a time-domain method which provides a Poincar e plot. Data were further correlated to cell phenotypes. Immunofluorescence and calcium imaging analysis showed that mutant lamin changed NRVMs beating force and frequency. Additionally, we noted an altered microtubule network organization with shorter filament length, and defective hemichannel membrane localization (Connexin 43). These data highlight the interconnection between nucleoskeleton, cytoskeleton and sarcolemmal structures, and the transcellular consequences of mutant lamin protein in the pathogenesis of the cardiac laminopathies.

Peptidic Connexin43 Therapeutics in Cardiac Reparative Medicine

“…Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from loss-of-function of dystrophin [ 64 ]. In skeletal and cardiac myocytes cells, dystrophin is a membrane-associated protein that links the cytoskeleton and the surrounding extracellular matrix, thereby providing mechanical integrity during muscle cell work [ 65 ].…”

Compromised Biomechanical Properties, Cell–Cell Adhesion and Nanotubes Communication in Cardiac Fibroblasts Carrying the Lamin A/C D192G Mutation

“…In 2015, Lanzicher et al investigated the biomechanical impact of LMNA missense mutations in neonatal rat ventricular myocytes (NRVM), and found damages on the actin cytoskeleton, suggesting loss of actin filament anchorage by dysfunction of the laminar proteins at the nuclear membrane as the initiating alteration [7,8]. Their results were further confirmed by different studies, in which it was demonstrated that lamina network mutations induce an alteration of actin filaments, microtubule, and intermediate filament anchorage in the nuclear membrane, a region critical for cytoskeleton organization [9,10]. Moreover, the link between the lamina network and the cytoskeleton filaments is ensured by a protein complex: LInker of Nucleoskeleton and Cytoskeleton (LINC).…”

“…Finally, other mechanisms may be implicated in the defective mechanotransduction of LMNA mutant cardiac cells. In cardiomyocytes with three different LMNA missense mutations, we previously reported defective adhesion, altered actin, and microtubule networks and dislocated connexin 43 [10] leading to higher frequency of beating but with a reduced beating force. However, future studies should investigate the complex molecular changes in LMNA mutant cardiac fibroblasts.…”

“…To verify the aforementioned hypothesis, a mimetic protein of Cx43, Gap 27, was used to block the link between two Cx43 and subsequently, the TNTs' formation. In previous work [10], we examined the levels and localization of Cx43 in cells with LMNA D192G mutation, since Cx43 protein is important for ensuring coordinated contractile action and its amount and localization might be one of the leading mechanisms for arrhythmias. We found that in the presence of this mutation, there was an altered Cx43 localization even though its overall cellular levels were unchanged.…”

Preclinical Advances of Therapies for Laminopathies

“…Impairment of the microtubule network triggered abnormal electrical communication between cardiomyocytes and induced cardiac conduction defects in Lmna H222P / H222P , Lmna N195K / N195K and Lmna Δ 8–11 /Δ 8–11 mice [ 95 , 96 , 97 ]. Increased phosphorylation and aberrant localization of Cx43 have been reported, in vivo and in vitro, due to microtubule instability [ 96 , 97 , 98 ]. Stabilization of microtubules using paclitaxel, a microtubule-stabilization agent commonly used in chemotherapy, improved intraventricular conduction defects in Lmna H222P / H222P mice, demonstrating a novel pathophysiological mechanism based on microtubule network and Cx43 displacement [ 97 ].…”