Fig. 3: Overexpression of SERCA2a inhibits TMAO-induced Ca²⁺ overload and cardiac hypertrophy.

a–c SERCA2a overexpression efficacy was examined by the RT-PCR (n = 4) and Western blot (n = 3). d, e Intracellular calcium concentration in Fura4-AM loaded H9c2 infected with Ad-control or Ad-SERCA2a (SERCA2a^OE) in the presence or absence of TMAO(1 mM for 48 h, n = 9), scale bar is 50 μm. Representative images of intracellular Ca²⁺ were detected with a Nikon microscope and the fluorescence ratio of intracellular Ca²⁺ was measured with a fluorescence microplate reader (BioTek). f–h Western blot analysis of protein levels of ANP, MYH7, and SERCA2a in H9c2, which was infected with Ad-control (NC) or Ad-SERCA2a (SERCA2a^OE) after TMAO treatment(1 mM for 48 h, n = 3). i, j H9C2 infected with Ad-control or Ad-SERCA2a in the presence or absence of TMAO (1 mM for 48 h, n = 4). Representative microscopic images of cells with rhodamine-phalloidin staining of the cytoskeleton and DAPI staining of the nucleus and cell surface area analysis. Statistical analyse was performed using the Student’s t-test vs. respective controls (a, c). One-way ANOVA with Tukey’s multiple comparisons test was used to compare groups (e, g–i). Error bars represent S.E.M.