1.
Implication of TRPM4 channel in ischemia-reperfusion injury
Ken Takahashi, Keiji Naruse
Cardiovascular Physiology, Okayama Univ. Grad. Sch. Med. Dent. Pharmaceut. Sci., Okayama, Japan
Abstract
Ischemic heart disease often causes heart failure.
During ischemia-reperfusion (I/R), excessive reactive
oxygen species (ROS), including hydrogen peroxide
(H2O2), are produced in cardiac tissue, where they
induce cell death. We previously reported that a
TRPM4 channel inhibitor 9-Phenanthrol protects
cardiac I/R injury in the excised rat heart. Based on
this finding, we hypothesized that TRPM4 channels
are involved in the pathophysiology of the cardiac I/R
injury. We confirmed that intravenous application of
9-Phenanthrol mitigated the development of
myocardial infarction caused by the occlusion of the
left anterior descending artery in rats. Positive
expression of TRPM4 channels in the ventricular
cardiomyocytes was confirmed by immuno-
histochemistry. To evaluate the toxic effect of ROS
on cardiac cells, we measured cellular viability of
H9c2 cardiomyocytes underwent H2O2 challenge.
Pretreatment of 9-Phenanthrol preserved cellular
viability. Furthermore, knockdown of TRPM4
channels preserved the viability of H9c2
cardiomyocytes exposed to H2O2. These results
suggest that TRPM4 channels are involved in
development of cardiac ischemia-reperfusion injury.
Figure 1. Animal experimental protocol
Group 1 was pretreated with the vehicle (DMSO), and
Group 2 was pretreated with 9-Phe (1 mg/kg i.v.). Both
groups were exposed to 30 min of ischemia by occluding
left anterior descending artery (LAD), followed by 120
min of reperfusion. Then the hearts were collected for
TTC staining.
Figure 2. Impact of 9-Phe on the size of myocardial infarction
(A), percentage of area at risk (AAR) caused by ischemia-reperfusion injury. (B), percent infarct size to
ischemic region (AAR). The percent infarcted area in the 9-Phe group (n = 6) was 3.5-fold smaller than that
of the DMSO group (n = 5; *p < 0.01). (C), typical TTC-stained heart slices, corresponding to the groups
indicated on the X axis. Blue region indicates cardiac tissue that received normal blood flow. In contrast, red
region indicates ischemic tissue due to LAD occlusion. The light red region encircled by a dotted line
indicates infarcted tissue.
Figure 3. Expression of TRPM4 in the rat heart
(A), Specificity of the TRPM4 staining (green) was confirmed by pre-
incubation of blocking peptide for TRPM4 antibody. Scale bar: 100 μm. (B),
Expression of TRPM4 (green) and SERCA2 (red). Overlaid image (left
bottom) indicates that TRPM4 and SERCA2 proteins are alternately located
in the longitudinal direction of cardiomyocytes. Arrowheads indicate the
intercalated disc. Scale bar: 20 μm.
Figure 4. Protective effect of 9-Phe against H2O2- and hypoxia/reoxygenation-induced death of
H9c2 cardiomyocytes
Cellular viability was quantified by dissolving the formazan crystals, and reading the absorbance
of the samples (MTT assay). (A), Cells were exposed to D-MEM medium containing 200 µM
H2O2 for 4 h. 9-Phe protected the cells from H2O2 challenge. n = 5 for each condition. (B), Cells
were subjected to 4 h anoxia, followed by 1 h reoxygenation. Absorbance was normalized to
that of normoxic condition. n = 3 for each condition. *:p < 0.05, **: p < 0.01, N.S.: p > 0.05.
Figure 5. Knockdown of TRPM4 prevents cell death caused by H2O2- and hypoxia/reoxygenation-challenge in H9c2 cardiomyocytes
(A), Quantitative RT-PCR confirming 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: TRPM4, Blue: Hoechst 33342. Fluorescent images were overlaid with
DIC images of the tissue. (C), Confirmation of suppressed TRPM4 protein expression by Western blot 24 h after siRNA transfection.
(D, E), Impact of gene silencing and pharmacological block of TRPM4 on the 200 µM-H2O2 (D)- and hypoxia/reoxygenation (E)-
challenge. Cell viability was measured by the MTT assay. n = 5 for each group. *: p < 0.05, **: p < 0.01, N.S.: p > 0.05.
Figure S1. Analysis of TRPM4 expression in organs.
TRPM4 protein detected by immunohistochemistry. Organ sections were
stained with anti-TRPM4 antibodies. Brown regions indicate positive TRPM4
staining. (A) TRPM4 expression in the rat ventricle, atrium, kidney, and liver.
Ab (−): without primary antibody, Ab (+): with primary antibody. Scale for x5
magnification: 200 μm, Scale for x20 magnification: 50 μm.
A B
A B
A
B
C
D E ventricle atrium kidney liver
x5
Ab(-)
x5
Ab(+)
x20
Ab(+)
LAD
occlusion
Adult male SD rats
in vivo surgery