Knock Down of Plakophillin 2 Dysregulates Adhesion Pathway through Upregulation of miR200b and Alters the Mechanical Properties in Cardiac Cells

Background: Mutations in genes encoding intercalated disk/desmosome proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (ACM). Desmosomes are responsible for myocyte–myocyte attachment and maintaining mechanical integrity of the myocardium. Methods: We knocked down Pkp2 in HL-1 mouse atrial cardiomyocytes (HL-1Pkp2-shRNA) and characterized their biomechanical properties. Gene expression was analyzed by RNA-Sequencing, microarray, and qPCR. Immunofluorescence was used to detect changes in cytoskeleton and focal adhesion. Antagomirs were used to knock down expression of selected microRNA (miR) in the rescue experiments. Results: Knockdown of Pkp2 was associated with decreased cardiomyocyte stiffness and work of detachment, and increased plasticity index. Altered mechanical properties were associated with impaired actin cytoskeleton in HL-1Pkp2-shRNA cells. Analysis of differentially expressed genes identified focal adhesion and actin cytoskeleton amongst the most dysregulated pathways, and miR200 family (a, b, and 429) as the most upregulated miRs in HL-1Pkp2-shRNA cells. Knockdown of miR-200b but not miR-200a, miR-429, by sequence-specific shRNAs partially rescued integrin-α1 (Itga1) levels, actin organization, cell adhesion (on collagen), and stiffness. Conclusions: PKP2 deficiency alters cardiomyocytes adhesion through a mechanism that involves upregulation of miR-200b and suppression of Itga1 expression. These findings provide new insights into the molecular basis of altered mechanosensing in ACM.


Single cell spectroscopy experiments using atomic force microscopy (AFM)
Biomechanical experiments were carried out through a Solver Pro-M AFM from NT-MDT (Moscow Russia), as previously reported (1,2). For these measurements, sQube CP-PNPL-Au-C cantilevers were used, with a nominal spring constant of 0.08 N/m, which was checked prior each experiment by Sader method. A spherical gold probe of about 5 μm-diameter was glued at the cantilever's apex.
Measurements on single living cells were performed in physiological conditions of medium and temperature, within one hour. Since the nuclear elasticity is correlated with the stages of cell division (3), cells with nuclei optically showing mitosis were excluded. For each investigated area, a preliminary scan was made to assess the cell morphology and the nuclear position, which corresponds to the highest portion of the cell. In order to avoid possible artefacts due to substrate stiffness and/or due to hydrodynamic forces, indentations were performed above the nucleus, at the constant speed of 1 μm/s for approach and withdrawal of the cantilever.
Cell elasticity was calculated from the first portion of the indentation curve (10% of cell deformation).
Here, the experimental data were fitted with the model proposed by Sneddon for spherical probes (4): Where F is the loading force, E is the Young's modulus, the Poisson's ratio, R the radius of the probe, a is the contact radius (function of the tip penetration) and  is the probe penetration into the cell. Approximating the cell to an incompressible body (i.e. a flexible object filled with liquid), the Poisson's ratio was assumed as  = 0.5 (5). All the curves analysis was performed using AtomicJ software (6).
The model used for estimating the Young's modulus value is conventionally used to yield a general idea of cell elasticity (7). Other models could be used for this task. One fairly new and accurate is the "Brush Model" developed by Sokolov et al. (8,9). In the present study, it has been decided to use a model which does not consider the brush since (i) this model is the one most commonly used, (ii) the cell line was the same throughout the all research and (iii) the same protocol/methodology and the same model has been used for all cells and indentations. Therefore, it has been considered that the calculated Young's modulus values might be compared for all specimens within the presented experiments. Furthermore, resulting Young's modulus data are reasonably similar to others reported by another group for the same cell line (10).
To describe the cell viscoelastic behaviour towards an external applied force, we used a parameter introduced by Klymenko et al. (11) and indicated as "plasticity index"  (even though "plastic" stands for non-recoverable deformation). This was assessed from the hysteresis between the approach and withdrawal curves as: where A 1 and A 2 are the areas under the loading and unloading curves (green box in Figure S1), respectively. Intermediate values between a fully elastic (η=0) and a fully plastic behaviour (η=1), indicate mixed viscoelastic properties.
For both the Young's modulus and the plasticity index assessment, each cell was subjected to three consecutive indentations at the same position and the mean of the results was considered as a single cell value (n=1).
Cell-to-ECM protein interaction was assessed through a JPK NanoWizard II AFM equipped with a CellHesion module, using tipless V-shaped silicon nitride gold covered cantilevers having nominal spring constant value of 0.32 N/m (NanoWorld, Innovative Technologies). O2 plasma treated cantilevers were functionalized with fibronectin (Thermo Fisher Scientific) at the final concentration of 20 µg/ml for 15 hrs at 4° C, and stored in PBS (12). Before each experiment, the cantilever spring constant was calibrated using the thermal noise method. Measurements were performed according to published protocols(13-15) ( Figure S2). Briefly, HL-1 cell suspension was overlaid on a BSA coated glass coverslip inserted into a petri dish previously coated with type I collagen or fibronectin (both from Thermo Fisher Scientific) at the final concentration of 50 and 20 µg/ml, respectively. A single 5 cell from the suspension was captured by pressing it with the fibronectin-functionalized cantilever against the glass for 30 sec with a contact force of 0.5 nN. Next, the cell was lifted up from the surface and allowed establishing a firm adhesion to the cantilever for about 20 minutes. Afterwards, the cantilever with the cell was moved far away reaching a coated plastic surface and the adhesion measurements were performed at a constant force of 0.5 nN for 20 seconds. After each force measurement, the cell was retracted to recover for 60 seconds before repeating the measure on the same spot or adhering to a different spot on the surface. During contact, the force exerted was kept constant using the AFM closed loop feedback mode. The cantilever was withdrawn at constant speed of 5 μm/s over pulling ranges of 80 μm to ensure complete detachment of the cell from substrate.
During this step, the cantilever deflection, which is proportional to the vertical force that exists between the cell and substrate, is recorded in a force-distance curve. This curve provides information regarding the cell adhesion. The work that is required to detach the cell can be used to describe the adhesion strength of the cell. It is calculated from the area that is enclosed by the retraction-forcedistance curve. This curve also carries other information: after the cell starts to detach from the substrate, individual force steps can be observed. During this phase, the receptors either detaches from the substrate surface or are pulled away from the cell cortex as a membrane tether. While cell membrane is still in contact with the substrate, either of these processes can occur. During the final phase of detachment, the cell body is no longer in contact with the substrate and, thus, attachment is due only to the tethers. Since the receptors are anchored in the cell cortex they unbind as the force increases (in the AFM curve they are denoted as jumps or ruptures). The second type of unbinding event occurs when membrane tethers are pulled out of the cell. In this case, long plateaus of constant force characterize tethers. Our withdrawn (unloading) curves were analyzed classifying receptors detachment events as "rupture" or "tether" based on the slope of the curve preceding the force step.
Withdrawn curves were analysed using the JPK Data Processing software. Positive steps at the right of the minimum force were automatically identified (using default fit parameters values: "Smoothing" = 5; "Significance" = 0.001). Detachment events were classified as "rupture" or "tether" based on the 6 slope of the curve preceding the force step as follows: values ≤ -0.15mN/m, corresponding to the 15% derivation were associated to ruptures, while slopes between -0.15 and 0.15 mN/m were classified as tethers (14). Intervals between steps lower than 12.5 nm were discarded, as fit was made only on two experimental points. For these experiments, each cell was subjected to 4-6 consecutive indentations at the same collagen or fibronectin spot and the mean of the results was considered as a single cell-ECM interaction (n=1).

Actin aggregates
A quantification of the actin aggregates amount has been done using imageJ software. The calculations were performed for every cell line on 20 cells from 4 independent experiment. The results are shown in the figure below and presented as the amount of actin aggregates on the total cytoskeleton area.