Fig 2: 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 3: Altered gene and protein expression profiles indicate altered calcium signaling and regulation in VDAC2-KO.a, b Differential gene expression data derived from 16-week old cardiac ventricular tissue (n = 4); c–f Western blot image of PLN (p = 0.0042), pPLN (p = 0.0045), and NCX (p = 0.0121) along with lane-loading control GAPDH and respective quantification using Image Studio Lite (version 5.2.5) (n = 6); g, h Western blot image of PKA and pPKA and quantification using Image Studio Lite (version 5.2.5) (p = ns) (n = 6); i, j Western blot image of CASQ2 and lane-loading control GAPDH and respective quantification using Image Studio Lite (version 5.2.5) (p = 0.0111) (n = 6); k, l Western blot image of SERCA2 and lane-loading control VINCULIN and quantification using Image Studio Lite (version 5.2.5) (Mann–Whitney: p = 0.0260) (n = 6); m, n Western blot image of RYR2 and lane-loading control VINCULIN and quantification using Image Studio Lite (version 5.2.5) (p = 0.0018) (n = 6). p-value: unpaired two-tailed t-test and two-tailed Mann–Whitney test performed. Data are represented as mean ± SEM.

Fig 4: 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 5: Results of protein analyses. Representative examples of channel proteins (upper subpanels) and summarized results of channel proteins (lower subpanels) (n = 6, Student’s t-test for all comparisons). Non-phosphorylated connexin43 (NP-Cx43), calmodulin-dependent protein kinase II (CaMKII), and pThr287-CaMKII (CaMKII-p) expression was significantly upregulated, and Nav1.5, pThr17-phospholamban (PLB-t), pSer16-phospholamban (PLB-s), and ryanodine receptor (RYR2) expression was significantly downregulated in the ischemia–reperfusion (IR) zone in the non-therapeutic hypothermia (TH) group but not in the TH group. Because of technical issues and various protein sizes, Western blots protein analyses were conducted at different gels. The illustrative examples of Cx43 and NP-Cx43 were acquired from no-TH heart 1 and TH heart 1; the examples of CaMKII, CaMKII-P, PLB, PLB-t, PLB-s were from no-TH heart 2 and TH heart 2; the examples of Nav1.5 were from no-TH heart 3 and TH heart 3; the examples of sarcoplasmic reticulum Ca-ATPase (SERCA2a) were from no-TH heart 4 and TH heart 4; the examples of ryanodine receptor (RYR2) were from no-TH heart 5 and TH heart 5.