Distinct molecular features of FLNC mutations, associated with different clinical phenotypes

Abstract

Filamin С is a key an actin-binding protein of muscle cells playing a critical role in maintaining structural integrity and sarcomere organization. FLNC mutations contribute to various types of cardiomyopathies and myopathies through potentially different molecular mechanisms. Here, we described the impact of two clinically distinct FLNC variants (R1267Q associated with arrhythmogenic cardiomyopathy and V2264M associated with restrictive cardiomyopathy) on calcium homeostasis, electrophysiology, and gene expression profile of iPSC-derived patient-specific cardiomyocytes. We demonstrated that R1267Q FLNC variant leads to greater disturbances in calcium dynamics, Nav1.5 kinetics and action potentials compared to V2264M variant. These functional characteristics were accompanied by transcriptome changes in genes linked to action potential and sodium transport as well as structural cardiomyocyte genes. We suggest distinct molecular effects of two FLNC variants linked to different types of cardiomyopathies in terms of myofilament structure, electrophysiology, ion channel function and intracellular calcium homeostasis providing the molecular the bases for their different clinical phenotypes.

Keywords: cardiomyocyte; cardiomyopathy; electrophysiology; filamin; iPSC; transcriptome.

FIGURE 1

Summary of transcriptome analysis results. (a) Results of a principal component analysis (PCA) of transformed count data from RNA‐seq using DESeq2 (varianceStabilizingtransformation). Samples are colored according to FLNC mutation status. (b) Venn diagram of downregulated and upregulated differentially expressed genes of two sample comparisons: R1267Q hiPSC‐CMC versus Donor hiPSC‐CMC and V2264M hiPSC‐CMC versus Donor hiPSC‐CMC.

FIGURE 2

Calcium transients of Donor hiPSC‐CMC and R1267Q hiPSC‐CMC. Black plot—health donor hiPSC‐CMC (n = 129); blue plot—R1267Q hiPSC‐CMC (n = 78). (a) Dynamics of calcium transients. Dotted lines—time points (10%, 50%, 90%, and peak). A—calcium transient amplitude; V—velocity. Different properties are specified; p‐value marked under each parameter. (b) Parameters of calcium transient. AUC—area under curve. p‐value: * −<0.05; ** −<0.01; *** −<0.001; **** − <0.0001. The columns are presented as the mean and the bars correspond to SD.

FIGURE 3

Biophysical characteristics of sodium current and action potential in R1267Q hiPSC‐CMC. Black plot—health donor hiPSC‐CMC (n = 23); blue plot—R1267Q hiPSC‐CMC (n = 36). (a) Volt–Ampere characteristics of sodium current. (b) Peak of sodium current density. (c) Steady‐state activation of sodium current. (d) Steady‐state inactivation of sodium current. (e) Representative traces of action potentials. (f) Parameters of action potential. p‐value: * −<0.05; ** −<0.01; *** −<0.001; **** −<0.0001. The columns are presented as the mean and the bars correspond to SD.

FIGURE 4

Results for gene set enrichment analysis (GSEA). Significant activated and suppressed GO terms were considered if p‐adjust values were ≤0.05. The dot size represents the number of genes associated with the GO term, the dot color represents adjusted p‐values.

FIGURE 5

Calcium transients of V2264M hiPSC‐CMC and Donor hiPSC‐CMC. Black plot—health donor hiPSC‐CMC (n = 129); red plot—V2264M hiPSC‐CMC (n = 65). (a) Dynamics of calcium transients; dotted lines—time points of calcium transient (10%, 50%, 90%, and peak); A—calcium transient amplitude; V—velocity; different properties are specified; p‐value marked under each parameter. (b) Parameters of calcium transient. AUC—area under curve. p‐value: * −<0.05; ** −<0.01; *** −<0.001; **** −<0.0001. The columns are presented as the mean and the bars correspond to SD.

FIGURE 6

Biophysical characteristics of sodium current and action potential in V2264M hiPSC‐CMC. Black plot—health donor hiPSC‐CMC (n = 23); red plot—V2264M hiPSC‐CMC (n = 14). (a) Volt–Ampere characteristics of sodium current. (b) Peak of sodium current density. (c) Steady‐state activation of sodium current. (d) Steady‐state inactivation of sodium current. (e) Representative traces of action potentials. (f) Parameters of action potential. p‐value: *−0.05; **−<0.01; ***−<0.001; ****−<0.0001. The columns are presented as the mean and the bars correspond to SD.

FIGURE 7

Results for gene set enrichment analysis (GSEA) for differentially expressed genes. Pathway analysis was performed on downregulated and upregulated DEGs in V2264M/Donor comparison.

FIGURE 8

Heatmap comparison of DEGs related to ion channel function, action potential, and calcium homeostasis for V2264M hiPSC‐CMC, R1267Q hiPSC‐CMC, and Donor hiPSC‐CMC.

FIGURE 9

(a) Representative immunofluorescence staining of cardiac alpha‐Actin (ACTC1—Red) and phalloidin staining (Phalloidin—Green). The top row—Hipsc‐CMC cells of Health Donor; The middle row—Hipsc‐CMC cells from patient with R1267Q mutation. The bottom row—Hipsc‐CMC cells from patients with V2264M mutation. (b) Representative sample of the intensity profile (red graph—Cardiac alpha‐Actin; green graph—Phalloidin) in donor cells and in hiPSC‐CMC cells from patients with V2264M mutation. Caliper X between channels' extremums reflects actin shift in absolute value. (c) Measured actin shifts in donor and patient hiPSC‐CMC cells. (D) Width of phalloidin hyperfluorescent stained (n = 71; 103; 59 respectively). Images were acquired using consistent shooting parameters. The columns are presented as the mean and the bars correspond to SD.**p‐value <0.01, ****p‐value <0.0001.

FIGURE 10

Length of TNNI3 and TPM1 fluorescent stained zone. (a)–(c) Representative immunofluorescent staining of tropomyosin with fluorescent intensity profile in control cells, FLNC‐R1267Q and ‐V2264M hiPSC‐CMC respectively; (e)–(g) representative immunofluorescent staining of tropomyosin with fluorescent intensity profile in control cells, FLNC‐R1267Q and ‐V2264M hiPSC‐CMC respectively; (d), (h) length of tropomyosin (n = 122; 89; 109, respectively) and troponin I (n = 54; 74; 59, respectively) peaks. The columns are presented as the mean and the bars correspond to SD. ***p‐value <0.001; ****p‐value <0.0001.

FIGURE 11

(a) Representative immunofluorescence staining of Filamin C (FLNC—red) and sarcomeric α‐actinin (ACTN2—green). The top row—hiPSC‐CMC cells of Health Donor; the middle row—hiPSC‐CMC cells of patient with R1267Q mutation; the bottom row—hiPSC‐CMC cells of patient with V2264M mutation, white square regions highlight FLNC striates co‐localized with ACTN2. The areas of filamin C visualization are highlighted by white rectangles. (b) Intensity of filamin C fluorescence staining normalized to α‐actinin staining. (c) Width of α‐actinin fluorescent stained (n = 82; 75; 93 respectively). Images were acquired using consistent shooting parameters. The columns are presented as the mean and the bars correspond to SD. ****p‐value <0.0001.