Fig 2: The level of cardiac calcium channel proteins in the untrained and trained mice. The band intensities are shown relative to the overall protein amount for CACNA1C (a), RyR-2 (b), SERCA2 (c), and NCX1 (110kDa) (d). Whiskers indicate maximal and minimal values, without outliers, while the box upper and lower line indicate upper and lower inclusive quartiles respectively with the middle line indicating the group median and × the group mean. The images represent the bands of respective proteins and the overall protein band intensities of samples analyzed

Fig 3: Relationship Between MiR‐214‐3p and SLC8A1. (A) Interaction between miR‐214‐3p and the 3′‐untranslated region (UTR) of Slc8a1. (B) Luciferase activity of a reporter containing the wild‐type/mutant Slc8a1 3′‐UTR. ‘MUT‐SLC8A1’ indicates the Slc8a1 3′‐UTR with a mutation in the miR‐214‐3p binding site. Data are expressed as mean ± standard deviation of three independent experiments. **p < .01 vs. control (NC). (C) SLC8A1 protein levels in the brain tissue of hypoxic‐ischaemic encephalopathy (HIE) rats, as detected by western blotting. (D) SLC8A1 protein levels determined by western blotting in oxygen–glucose deprivation (OGD)‐treated neurones. (E, F) SLC8A1 protein levels determined by western blotting in OGD‐treated neurones in different groups. (G,H) Slc8a1 messenger RNA levels measured by quantitative reverse‐transcription polymerase chain reaction in OGD‐treated neurones in different groups. Data are expressed as mean ± standard deviation. **p < .01 vs. control or OGD (n = 6 rats/group). NCI, inhibitor control group; NCM, mimic control group.

Fig 4: Rad (Ras-related RGK GTP-binding protein)–dependent regulation of L-type Ca2+ current (ICaL) and phosphorylation of sarcoplasmic reticulum (SR) proteins. A, Sample of confocal line-scan images of baseline RyR2 (type 2 ryanodine receptor)-dependent Ca2+ release (top) and corresponding comparison between averaged frequencies of spontaneous Ca2+ transients (bottom) in n=17, N=5 wild-type (WT) and n=10, N=4 4SA-Rad sinoatrial node (SAN) myocytes. Statistics: Mann-Whitney U test. B, Comparison between averaged Ca2+ transient amplitude in n=14, N=5 WT and n=13, N=5 4SA-Rad in 3 Hz field-paced SAN myocytes. Statistics: Wilcoxon signed-rank test. Inset, Comparison between relative changes in Ca2+ transient amplitudes induced by isoprenaline (ISO) in WT and 4SA-Rad SAN myocytes shown in B. Statistics: Mann-Whitney test. C and D, Comparison between averaged SR Ca2+ load (C) and recovery time of cell-wide Ca2+ transient elicited by a 5-second puff of 10 mmol/L caffeine (D) in WT and 4SA-Rad SAN myocytes after field pacing at 3 Hz. Statistics: (C), n=17, N=3 WT in Tyrode solution (Tyr) and n=13, N=5 WT in ISO; n=14, N=5 4SA-Rad myocytes in Tyr and n=13, N=5 in ISO 4SA-Rad myocytes, Mann-Whitney U test; (D) n=16, N=3 WT in Tyr and n=8, N=3 WT in ISO; n=13, N=5 4SA-Rad myocytes in Tyr and n=11, N=5 4SA-Rad myocytes in ISO, Mann-Whitney U test. E, Sample western blots of expression and phosphorylation of RyR2 at PKA (protein kinase A)–dependent Ser2808 and CaMKII (Ca2+/calmodulin-dependent protein kinase II)–dependent Ser2815 phosphosites in WT and 4SA-Rad SANs, in control conditions and after 5-minute perfusion of ISO (0.1 µmol/L). F, Expression and phosphorylation of PLN (phospholamban) at PKA-dependent Ser16 (PS16) and CaMKII-dependent Thr17 (PT-17) phosphorylation sites in WT and 4SA-Rad SANs, in control conditions and after perfusion of ISO. G, Sample western blots of expression of NCX (Na+/Ca2+ exchanger) in WT and 4SA-Rad SANs. Expression of tubulin have been used as control. In E through G, 2 SANs have been pooled in each blot line. H, Averaged RyR2, PLN ratios expressed in arbirary units (AU), between total protein and protein phosphorylated at PKA-dependent and CaMKII-dependent phosphorylation sites are indicated and corresponding to E and F. NCX1 protein expression in SAN samples treated or not with ISO corresponding to G is also shown. I. Averaged fold changes in phosphorylation of RyR2 and PLN at PKA- and CaMKII-dependent phosphorylation sites induced by ISO in WT (black dots) and 4SA-Rad (blue dots) SANs, corresponding to E and F. Statistics: Multiple Mann-Whitney U test. J, Schematic figure summarizing the transition between basal pacemaker activity (left) and stimulated state (right) after activation of β-adrenoceptors by catecholamines. Ion channels, membrane receptors and the SR are depicted. Red lines indicate targets of phosphorylation by activated PKA, CaMKII, or increased ionic currents and exchange during the heart chronotropic response. P indicates phosphorylation site; and SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase. Created with BioRender.com.