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 Poincaré 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.

Keywords: Atomic force microscopy; Beating; Biological sciences; Biomechanics; Cardiology; Cardiomyocytes; Cardiomyopathy; Cx43; Cytoskeleton; Lamin A/C; Mechanical property; Membrane.

Figure 1

Mechanobiological cardiomyocyte properties: frequency, contraction force and peak duration (A) An example of the AFM trace during NT cardiomyocyte beating, (B) Box and whisker plots showing beating frequency of NT, WT and LMNA^E161K, LMNA^N195K, and LMNA^D192G mutations. ***Represents p < 0.0001, (t-test) (C) Beating force. ***Represents p < 0.0001, (t-test) (D) Peak duration of LMNA mutant cardiomyocytes described as full width at the half-maximum (FWHM, namely the width of the beating pulse measured between those points on the y-axis which are half the maximum amplitude). ***Represents p < 0.0001, **p < 0.001, (t-test).

Figure 2

Beating rate variability by Poincaré plot of LMNA mutant cardiomyocytes. (A) A typical Poincaré Plot of RR intervals for NT cardiomyocyte. The peak interval (RR n) is displayed on the x-axis and the extent of the following interval (RR n+1) on the y-axis. All points described by successive beats of equal duration (RR n = RR n+1) are situated on the identity line. The points below this line represent all shortenings of the interval between 2 consecutive beats (RR n > RR n+1) while the points above the identity line correspond to all prolongations (RR n < RR n+1). (B) Analysis of the beating rate variability of LMNA mutant cardiomyocytes. SD1 (the standard deviation of the instantaneous - short term - beat-to-beat variability and (C) SD2 (the standard deviation of the long-term interval variability) for NT, WT and LMNA^E161K, LMNA^N195K, and LMNA^D192G mutations. ***Represents p < 0.0001, (t-test).

Figure 3

Cx43 protein expression in LMNA mutant cardiomyocytes. (A) Western blot showing Cx43 expression (left) and the results from western blots (n = 4) indicating no significant difference between the groups (right), p = 0.5038. (B) Cx43 localization in control (NT) cardiomyocytes. Fluorescence images of Connexin 43 (red dots), sarcomeric α-actinin (gray), LMNA GFP (green) and Hoechst (blue) staining of NRVMs (C) Cx43 localization in (from left to right): wild-type LMNA, LMNA^E161K, LMNA^D192G, LMNA^N195K. Upper panels: Sarcomeric alpha-actinin and connexin 43. Lower panels LMNA GFP expression and connexin 43. (D) Quantification of Cx43 gap junction area: significant differences on Cx43 gap-junction were observed between LMNA^E161K, LMNA^D192G and LMNA^N195K and the non-transfected NT cells and WT. Data are presented as mean ± S.D. (n = 5). 161 vs non-treatment: ***p value: <0.0005; 192 vs non treatment: **p value: <0.0045; 195 vs non treatment: ***p value: <0.0001; 161 vs WT: ***p value: <0.0009; 192 vs WT: **p value: <0.0086; 195 vs WT: ***p value: <0.0002. Non-significant differences were observed between the mutant cells and between WT and NT. A full, non-adjusted image is provided in supplementary material (Figure S1).

Figure 4

Spontaneous calcium transients of NRVMs. (A) representative spontaneous calcium transient phenotype. From left to right: Control (NT), WT, LMNA^E161K, LMNA^N195K and LMNA^D192G. (B) quantification of peak amplitude. (C) Quantification of Ca⁺⁺ transient frequency. ***Represents p < 0.0001, **p < 0.001, (t-test).

Figure 5

Microtubule network organization. (A) Example of microtubule organization used to calculate the Haralick features. (B) Haralick texture features: Contrast, Angular second moment (energy), Inverse difference moment (homogeneity), Entropy and Correlation are shown only for an angle of O° (complete set of data in Table 1); Data are presented as mean ± S.D. (n = 4 independent experiments). *p < 0.05; **p < 0.005; ***p < 0.0001.