Fig 1.
Rats were treated with the vehicle (DMSO; Groups 1 and 3) or 9-Phe (1 mg/kg in DMSO; Groups 2 and 4). The bolus injections into the jugular vein were given before (preconditioning; Groups 1 and 2) or after (postconditioning; Groups 3 and 4) ischemia. The 30-min ischemia period was followed by 120 min of reperfusion, and then the hearts were collected for TTC staining.
Fig 2.
Impact of 9-Phe on the size of myocardial infarction.
Rats received a bolus injection of DMSO (control) or 9-Phe before (preconditioning) or after (postconditioning) ischemia during an ischemia/reperfusion (I/R) protocol. (A) Impact on the percentage of area at risk (AAR) caused by I/R. (B) Impact on the percent infarct size over AAR. A fixed detection threshold for infarcted area was arbitrarily set, and used throughout the analysis. Only 9-Phe preconditioning significantly reduced the percent infarcted size, compared to DMSO (n = 5–6; p < 0.01). (C) Typical TTC-stained heart slices after preconditioning with DMSO or 9-Phe. The blue region indicates cardiac tissue that received normal blood flow, whereas the red region indicates ischemic tissue due to LAD occlusion. The light red region encircled by a dotted line indicates the infarcted tissue.
Fig 3.
Immunofluorescence imaging of TRPM4 in the healthy rat heart.
(A) Adult rat heart sections were labeled with anti-TRPM4 antibodies (green) and stained with Hoechst 33342 for the nuclei (blue). The green and blue fluorescence images were overlaid with the DIC image. Staining specificity was confirmed by the lack of signal when anti-TRPM4 antibodies were preincubated with the antigenic blocking peptide (ab65597). Scale bar: 100 μm. (B) Double immunofluorescence showing TRPM4 (green) and SERCA2 (red) expression in ventricular cardiomyocytes. Scale bar: 20 μm. The overlaid image (left bottom) reveals alternating positions for the TRPM4 and SERCA2 proteins on the longitudinal axis of cardiomyocytes. Arrowheads indicate the intercalated disc. Fluorescence intensity profile (right bottom) confirmed the alternating expression of TRPM4 and SERCA2.
Fig 4.
Protective effect of 9-Phe against H2O2-induced death in H9c2 cardiomyocytes.
(A) Dose-dependent impact of H2O2 on cell viability. Cultures of H9c2 cells were exposed 4 h to DMEM medium containing 0, 100, 200, 300, 400, or 500 μM H2O2, then viability was measured by MTT assay. Asterisks indicate significantly lower viability (absorbance) than for the control (0 μM H2O2). n = 4 for each concentration. (B) Impact of 9-Phe on the H2O2 challenge. Cultures were incubated 4 h with 200 μM H2O2 in the presence of DMSO or 20 μM 9-Phe. The 9-Phe completely prevented the damage caused by H2O2. n = 5 for each condition. * p < 0.05, ** p < 0.01, N.S.: p > 0.05.
Fig 5.
Protective effect of 9-Phe against hypoxia-reperfusion-induced death in H9c2 cardiomyocytes.
Cultures of H9c2 cells, incubated with DMSO or 9-Phe (10 or 20 μM), were subjected to hypoxia-reperfusion (H/R; 4 h anoxia followed by 1 h reoxygenation). Viability was measured by the MTT assay, and absorbance was normalized to that of the normoxic condition. The presence of 9-Phe (≥10 μM) completely prevented the damage caused by H/R (n = 3 for each condition). Dunnett’s post hoc test was performed. * p < 0.05, N.S.: p > 0.05.
Fig 6.
Knockdown of TRPM4 prevents cell death caused by H2O2 challenge in H9c2 cardiomyocytes.
(A) Quantitative RT-PCR confirming the gene silencing of TRPM4. siNEG, cells transfected with control siRNA; siTRPM4, cells transfected with TRPM4-targeting siRNA. n = 5 for each group. (B) Confirmation of suppressed TRPM4 protein expression by immunocytochemistry 48 h after siRNA transfection. Green, anti-TRPM4, Blue, Hoechst 33342 dye (nuclei). The fluorescent images were overlaid with DIC images of the cultures. (C) Confirmation of suppressed TRPM4 protein expression by Western blot 24 h after siRNA transfection. (D) Impact of gene silencing on the loss of viability induced by 200 μM H2O2. Cell viability was measured by the MTT assay. n = 5 for each group. (E) Impact of TRPM4 knockdown on the hypoxia/reoxygenation (H/R) challenge. Cell viability was measured by the MTT assay. n = 6 for each group. Statistical analysis was performed using Dunnett’s test as post hoc. *: p < 0.05, **: p < 0.01, N.S.: p > 0.05.