Activation of sarcolipin expression and altered calcium cycling in LMNA cardiomyopathy

Figures

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  Fig. 1. Activation of SLN in LMNA cardiomyopathy Representative immunoblot showing SLN expression in hearts from 6 months old male LmnaH222P/H222P and wild type (WT) mice. GAPDH was shown as loading control.

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  Fig. 2. Expression of Serca2a and NCX in LMNA cardiomyopathy Representative immunoblots showing SLN, Serca2a and NCX expression in ventricles from 3 and 6 months old male LmnaH222P/H222P and wild type (WT) mice. GAPDH was shown as loading control.

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  Fig. 3. Expression of protein involved in calcium handling in LMNA cardiomyopathy. (A) Representative immunoblots showing total PLB, phosphorylated PLB(Ser16) and phosphorylated PLB(Thr17) expressions in hearts from 6 months-old male LmnaH222P/H222P and wild type (WT) mice. GAPDH was shown as loading control. (B) Schematic representation of the construct. (C) Representative immunoblots showing total PLB and phosphorylated PLB(Ser16) expressions in HL-1 cells stably expressing FLAG epitope-tagged SLN (FLAG-SLN). (D) Micrographs showing PLB labeling in HL-1 cells (empty) and HL-1 cells stably expressing FLAG epitope-tagged SLN (FLAG-SLN). Nuclei are counter-stained with 4′,6-diamidino-2-phenylindole (dapi).

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  Fig. 4. Alteration of calcium cycling in LMNA cardiomyopathy. (A) Line-scan images during field stimulation at 2 Hz of representative isolated cardiomyocytes from 3 months-old wild type (WT) and LmnaH222P/H222P mice. Below each image, there are represented the fluorescence traces in black for the cytosolic [Ca2+]i transient, and in gray for the ca transient inside the nucleus in the WT cell. The same for the LmnaH222P/H222P in red (cytosolic) and in dark red (nuclear). (B) Graph showing values of peak calcium transients (F/F0), peak nuclear calcium transients (F/F0), time to peak and decay time constant of steady-state Ca2+ transients from isolated cardiomyocytes from 3 months-old wild type (WT) and LmnaH222P/H222P mice. The number of cardiomyocytes is indicated. (t-test). (C) Line-scan images of caffeine-evoked intracellular Ca2+ transients after field stimulation at 2 Hz of representative isolated cardiomyocytes from 3 months-old wild type (WT) and LmnaH222P/H222P mice obtained by the application of 10 mmol/L caffeine. (D) Graph showing values of maximum amplitude of caffeine-induced SR Ca2+ release, indicative of SR Ca2+ load (F/F0) and decay time constant of caffeine-induced SR Ca2+…

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  Fig. 5. Electrophysiological characteristics in cardiomyocytes isolated from LmnaH222P/H222Pmice (A) Examples of action potentials (AP) recorded in wild type (WT) (black trace) and LmnaH222P/H222P (red trace) cells. Membrane resting potential measured as the Zero current potential (left) and AP amplitudes (right) (B), maximum AP upstroke velocity (measured as dV/dt) (C), and AP duration (APD) at 20, 50, and 90% of repolarization. (D). The number of cardiomyocytes is indicated. Data are represented as the mean ± standard deviation of individuals. (t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 6. Potassium currents density in cardiomyocytes isolated from LmnaH222P/H222Pmice. (A) Transient outward potassium current (Ito) recorded in 27 cells from wild type (WT) (black circles) and 22 LmnaH222P/H222P cells (red circles) from 3 mice for each genotype. Current amplitude (pA) was normalized by the membrane capacitance (pF), which is an indirect measurement of cell surface. (B) Transient ultra-rapid potassium current (IKur) recorded in 18 wild type (WT) and 15 LmnaH222P/H222P cells from 3 mice for each genotype. Current amplitude (pA) was normalized by the membrane capacitance (pF), which is an indirect measurement of cell surface. (C) Transient sustained potassium current (Iss) recorded in 20 wild type (WT) and 15 LmnaH222P/H222P cells from 3 mice for each genotype. Current amplitude (pA) was normalized by the membrane capacitance (pF), which is an indirect measurement of cell surface. (D) Membrane capacitance. The number of cardiomyocytes is indicated. (t-test).

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  Fig. 7. Inhibition of SLN expression delays cardiac dysfunction in a mouse model of LMNA cardiomyopathy. (A) Representative Sirius Red staining and 3D visualization of fibrosis (Fiji software) of cross sections of hearts from PBS-treated LmnaH222P/H222P and shSLN-treated LmnaH222P/H222P mice. Cross sections of hearts from wild type (WT) mice were shown as controls. (B) Graph showing fibrosis quantification from PBS-treated LmnaH222P/H222P and shSLN-treated LmnaH222P/H222P mice. (C) Fractional shortening evolution from 3 months to 5 months from PBS-treated LmnaH222P/H222P (n = 5) and shSLN-treated LmnaH222P/H222P mice (n = 5). Data are represented as the mean ± standard deviation of individuals. **P ≤ 0.01 between PBS-injected LmnaH222P/H222P mice and wild type mice at each time point (ANOVA). (D) Representative immunoblot SLN expression in heart from PBS-treated LmnaH222P/H222P and shSLN-treated LmnaH222P/H222P mice. GAPDH was shown as loading control. (E) Representative immunoblot showing GFP, total PLB, phosphorylated PLB(Ser16) and phosphorylated PLB(Thr17)expressions in heart from PBS-treated LmnaH222P/H222P and shSLN-treated LmnaH222P/H222P mice. GAPDH was shown as loading control.