Fig 2: TCA abrogated PE-induced cardiac hypertrophy in vivo.A Representative myocardium pictures. (a) Pictures of heart material object; (b) H&E staining heart sections from paraffin embedding samples; (c) WGA staining from heart frozen sections. B Quantification of heart weight/body weight ratio, heart weight/tibial length ratio and cross-section with WGA staining. C mRNA expression of Nppa, Nppb and Mhy7 in myocardium tissues. D Diagram of echocardiography. E Quantification of EF, FS, LVPWd and LVPWs. F Immunoblotting in detection of p-CaMKII, CaMKII, p-MEK, MEK, p-ERK and ERK in myocardial tissue. G Quantification of p-CaMKII/CaMKII, p-MEK/MEK, p-ERK/ERK. *P < 0.05 versus control; #P < 0.05 versus PE. EF: ejection fraction; FS: fractional shorting

Fig 3: CaMKII interacts with and regulates MYMK and Rac1 during fusion(A) Representative IF images of myoblasts infected with control retrovirus or virus expressing Myomaker, and treated with DMSO (Ctrl), 1 µM ERKi, 5 µM CaMKIIi, or co-treated with ERKi and CaMKIIi for 18 h.(B) Stratified fusion index of (A).(D) Representative images showing proximity ligation assay (PLA) between CaMKII and MYMK for DMSO (Ctrl) or ERKi-treated myoblasts at 24 h post treatment. Top panel shows the PLA signal (red), and bottom panel shows the overlay of PLA signal (red), membrane marker (green), and nuclei (blue).(E) Quantification of the PLA assay in (D) shown as the mean fluorescent intensity normalized to nuclei number per field. (E) Representative WB analysis of Rac1 S71 phosphorylation following treatment with ERKi and co-treatment with CaMKIIi.(F) Representative images showing results of the PLA between CaMKII and Rac1 for DMSO (Ctrl) or ERKi-treated myoblasts at 24 h post treatment. Top panel for each shows the PLA signal (red) and the bottom panel shows the overlay of PLA signal (red), phalloidin (green), and nuclei (blue).(G) Quantification of the results of the PLA assay, shown as the mean fluorescent intensity normalized to nuclei number per field. All data are representative of at least 3 biological repeats. Error bars indicate SEM. Scale bars: 100 µm.

Fig 4: Protective effects and possible mechanism of TCA on PE-induced cardiac hypertrophy. TCA protects PE-induced cardiac hypertrophy via blocking CaMKII-ERK activation and nuclear localization. TCA prevents PE-induced CaMKII activation which also may contribute to restoring cardiac excitation-contraction coupling. CaMKII: calcium/calmodulin-dependent protein kinase II; ERK: extracellular regulated protein kinases; MAPK: mitogen activated protein kinase; PE: phenylephrine; PLN: phospholamban; RyR2: ryanodine receptor type 2; SERCA: sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; TCA: trans-cinnamaldehyde

Fig 5: CaMKII function during muscle regeneration and ERK-CaMKII pathway conservation(A) WB of analysis of indicated proteins from CTX-induced injured muscle. Line indicates where a lane was purposely removed.(B) Schematic illustration of the SC-specific double CaMKII KO mouse model.(C) Schematic illustration depicting the timeline of the repeat-injury experimental design.(D) WB validation of CaMKII depletion in WT or scDKO primary myoblasts isolated for 2 weeks following initial injury.(E) IF staining of WT or scDKO primary myoblasts following ERKi-induced fusion at 24 h post treatment. Insets are enlarged to the right.(F) Fusion index comparison between WT (n = 4) and scDKO (n = 4) primary myoblasts stratified by number of nuclei per fiber. Total number of nuclei assayed, n = 12,743.(G) Representative field of WT and scDKO muscle 14 days after CTX-induced reinjury.(H) Quantification of myofiber cross-sectional areas of WT (n = 4) and scDKO (n = 4) mice 14 days following reinjury.(I) Average percentage of central nuclei in WT (n = 4) and scDKO (n = 4) mice 14 days following reinjury. At least 9,000 fibers per mouse were measured for (H) and (I).(J) Representative IF staining of primary chicken myoblasts over 72 h of treatment either with ERKi in proliferation medium, or in conventional DM.(K) Fusion index for the 48-h time point of (J).(L) Representative WB analysis of CaMKII activation in chicken myoblasts, following treatment with ERKi or co-treatment with CaMKIIi. Error bars indicate SEM. All scale bars, 100 µm.