Fig 2: 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.

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).

Fig 4: Testosterone activates CaMKII in cardiac myocytes. Cardiac myocytes were stimulated with testosterone and then subjected to Western blotting to determine the levels of phosphorylated CaMKII (pCaMKII) and phospholamban (pPLN). CaMKII phosphorylation kinetics were assessed over short and long periods of testosterone stimulation: CaMKII phosphorylation was evaluated from (A) 5 to 60 min (n = 5) and (B) 3 to 24 h (n = 6). (C) Cardiac myocytes were stimulated for 15 min with the indicated concentrations of testosterone (n = 6). (D) PLN Thr17 phosphorylation and total protein levels in cells treated for 5–60 min with 100 nM testosterone (n = 5). The results of densitometric analyses are shown as ratios of phosphorylated to total protein. Data are presented as means ± SEM. P-values were determined using ANOVA followed by Bonferroni post hoc test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control.

Fig 5: Adrenergic signaling pathways are fully functional in 4SA-Rad mice.a, Graph of amplitude of Ca2+ transient after caffeine in control, isoproterenoland forskolin-treated cardiomyocytes. Data are the mean ± s.e.m. P < 0.0001 by one-way ANOVA; ****P < 0.0001, **P < 0.01 by Sidak’s multiple-comparison test (n = 121, 120, 66, 129, 165 and 129 cardiomyocytes from 3, 4, 5, 3, 4 and 5 mice, respectively). b, Anti-SR Ca2+-ATPase (SERCA) antibody western blot of protein lysates of cardiomyocytes isolated from WT and 4SA-Rad mice. Lower: graph of density normalized to β-actin. Data are the mean ± s.e.m. Unpaired, two-tailed Student’s t-test. P > 0.99 (n = 3 mice in each group). c, Same as b except with anti-NCX antibody western blot. Lower: graph of density normalized to β-actin. Data are the mean ± s.e.m. Unpaired, two-tailed Student’s t-test P = 0.38 (n = 3 mice in each group). d, Graph of time constant of Ca2+ transient relaxation under basal conditions and after either isoproterenol or forskolin. Data are the mean ± s.e.m. P < 0.0001 by one-way ANOVA, ****P < 0.0001 by Tukey’s multiple-comparison test (n = 462, 225, 109, 478, 183, 149 cardiomyocytes from 12, 4, 3, 13, 5 and 5 mice, respectively, from left to right). e, Anti-phospho-PLN (P-PLN) antibody (upper) and anti-PLN antibody (middle) western blots of protein lysates of cardiomyocytes. Lower: graph of P-PLN density normalized to PLN density. Data are the mean ± s.e.m. P = 0.43 and P = 0.89, from left to right, by unpaired, two-tailed Student’s t-test (n = 7 mice in each group). f, Same as in e except with anti-phospho-S2808 RyR2 antibody (upper) and anti-RyR2 antibody (middle) western blots. Lower: graph of P-RyR2 density normalized to RyR2 density. Data are the mean ± s.e.m. P = 0.66 and P = 0.97, from left to right, by unpaired, two-tailed Student’s t-test (n = 3 mice in each group). g, Same as in e except with anti-S23/S24 TnI antibody (upper) and anti-TnI antibody (middle) western blots. Lower: graph of P-TnI density normalized to TnI density. Data are mean ± s.e.m. P = 0.46 and P = 0.33, from left to right, by unpaired, two-tailed Student’s t-test (n = 4 mice in each group).