Fig 2: Loc14 restrains Ca2+ oscillation by affecting SOCE, Ca2+/Calm/calcineruin and MAPK pathways. (A) qPCR analysis of Calr, Stim1, Orai1, Trpc1, Calm, and Calcineurin transcript levels in control and Loc14 (5 μM)-treated groups. Data were normalized to GAPDH and presented as mean ± SD (n = 3). (B–G) WB showed expression levels of Stim1, Orai1, Calm and Calcineurin in control and Loc14 (5 μM)-treated groups. Full-length gels are presented in Supplementary material 5: Figs. S4 and S5. (H–J) WB showed expression levels of pERK, ERK, pJNK and JNK in control and Loc14(5 μM)-treated groups. Data were normalized to β-actin and presented as mean ± SD (n = 3). Full-length gels are presented in Supplementary material 5: Fig. S6. (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: The restoration of Lycium barbarum polysaccharides (LBPs) on reduced expression of key cardiac contractile regulatory proteins in miR‐1 Tg mice. A‐H, Representative examples of Western blot bands of each protein and statistical bar graph indicating the result of densitometric analysis of the bands as normalized to the quantity of GADPH protein. *P < .05, **P < .01 vs WT, # P < .05, ## P < .01 vs Tg; mean ± SEM, n = 5, 6, 6, 4, 5, 5, 7, 6 independent protein samples for each group in sequence from A to H. CaM, calmodulin; CaMKII, Ca2+/calmodulin‐dependent protein kinase II; cMyBP‐C, cardiac myosin binding protein C; cMLCK, cardiac myosin light chain kinase; MLC2v, myosin light chain 2v. I, Comparison of mRNA levels of CALM1,CALM2 and MYLK3 by qRT‐PCR in all groups. **P < .01 vs WT, # P < .05 vs Tg; mean ± SEM, n = 5 independent RNA samples for each group

Fig 4: Progressive CDI decrement of cardiac ICa.L with increase in Calm1N98S allele number.(A) Exemplar traces of whole-cell L-type Ca2+ current (ICa.L) recorded in left ventricular cardiomyocytes in response to 400 ms step depolarizations to +10 mV. Traces were normalized to their respective peaks to facilitate comparison of decay kinetics. Pipette solution contained 5 mM ryanodine and 10 mM BAPTA to restrict Ca2+ elevations to those in the nanodomains of individual L-type channels (6). CDI is evident as the accelerated decay in Ca2+ current as compared with Ba2+ in all 3 genotypes. Ca2+ currents exhibit progressively slower decays from Calm1+/+ to Calm1N98S/+ to Calm1N98S/N98S cardiomyocytes, while decay kinetics of Ba2+ currents are almost indistinguishable among genotypes. (B and C) Scatter dot plots of the fraction of peak ICa.L remaining at 50 ms after the peak (r50) using Ca2+ versus Ba2+ as charge carriers (B) and dot plots of f50 = (r50,Ca – r50,Ba)/r50,Ba, a measure of CDI (C). Horizontal lines superimposed on dots represent the median and interquartile ranges; n = 21 cells from N = 4 mice (Calm1+/+); n = 16, N = 5 (Calm1N98S/+); n = 14, N = 4 (Calm1N98S/N98S); *P ≤ 0.001 vs. Calm1+/+; †P = 0.004 vs. Calm1N98S/+. P values from a repeated measures linear model with independent terms of genotype. A compound symmetric covariance matrix was used, which assumes equal correlation from repeated measurements of the same experimental unit.

Fig 5: Quantification of CALM protein degradation.(A) Western blots of lysates from nontransfected HEK293T cells and HEK293T cells transfected with a vector encoding HA-tagged wild-type (HA CALM-WT) or mutant CALM (HA CALM-N98S). Cells were harvested before and after 6-hour, 12-hour, 24-hour, or 36-hour cycloheximide (CHX; 200 mg/mL) treatment. Control cells (labeled -) were harvested 36 hours after addition of the solvent (ethanol) only. Lysates were probed with an antibody recognizing the C-terminus of CALM or the HA-tag fused to the N-terminus of CALM. Samples were normalized to GAPDH protein expression. (B) Changes in normalized intensities of the endogenous CALM protein and HA-CALM protein bands expressed as percentages of their corresponding time 0 values and plotted as a function of CHX exposure time. Shown are dot plots with lines representing medians and interquartile ranges (N = 7–14 biological replicates per genotype). Each biological replicate is the average of 2 technical replicates. Numbers next to brackets denote P values by a repeated measures model with independent variables of plasmid, hour, and interaction of plasmid and hour. (C) Western blots of lysates from HEK293T cells transfected with HA CALM-WT, HA CALM-N98S, or control (labeled Empty) vectors. Lysates from cells following 24-hour exposure to MG132 (10 mmol/L) or lactacystin (10 mmol/L) were probed with the same anti-CALM or anti-HA antibodies as in A. Signal intensities were normalized to GAPDH protein expression. (D) Normalized intensities of HA-CALM protein bands expressed as fold-changes of normalized intensities in DMSO-treated cells. Lysates were probed with the anti-HA or anti-CALM antibody. Shown are dot plots with lines representing the median and interquartile ranges (N = 5 biological replicates per genotype per treatment). A linear model was used to compare mean CALM protein levels among treatment groups within the same genotype and treatment effects between genotypes.