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. 2013 Jul 25;8(7):e70587.
doi: 10.1371/journal.pone.0070587. Print 2013.

9-Phenanthrol, a TRPM4 inhibitor, protects isolated rat hearts from ischemia-reperfusion injury

Affiliations

9-Phenanthrol, a TRPM4 inhibitor, protects isolated rat hearts from ischemia-reperfusion injury

Jing Wang et al. PLoS One. .

Abstract

Despite efforts to elucidate its pathophysiology, ischemia-reperfusion injury lacks an effective preventative intervention. Because transient receptor potential cation channel subfamily M member 4 (TRPM4) is functionally expressed by many cell types in the cardiovascular system and is involved in the pathogenesis of various cardiovascular diseases, we decided to assess its suitability as a target of therapy. Thus, the aim of this study was to examine the possible cardioprotective effect of 9-phenanthrol, a specific inhibitor of TRPM4. Isolated Langendorff-perfused rat hearts were pretreated with Krebs-Henseleit (K-H) solution (control), 9-phenanthrol, or 5-hydroxydecanoate (5-HD, a blocker of the ATP-sensitive potassium channel) and then subjected to global ischemia followed by reperfusion with the K-H solution. To evaluate the extent of heart damage, lactate dehydrogenase (LDH) activity in the effluent solution was measured, and the size of infarcted area of the heart was measured by 2,3,5-triphenyltetrazolium chloride staining. In controls, cardiac contractility decreased, and LDH activity and the infarcted area size increased. In contrast, in hearts pretreated with 9-phenanthrol, contractile function recovered dramatically, and the infarcted area size significantly decreased. The cardioprotective effects of 9-phenanthrol was not completely blocked by 5-HD. These findings show that 9-phenanthrol exerts a cardioprotective effect against ischemia in the isolated rat heart and suggest that its mechanism of action is largely independent of ATP-sensitive potassium channels.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Experimental protocol.
All experimental groups were first perfused for 20 min to allow the isolated hearts to stabilize. The hearts were then divided into groups as follows: ischemia–reperfusion (I/R), DMSO-treated, 9-Phe-treated, and 5-HD+9-Phe-treated. LDH measurement was carried out before drug application and after ischemia. Finally, the heart was used for TTC analysis.
Figure 2
Figure 2. Measurement of cardiac contractile function.
A. Representative recordings of left ventricular pressure (LVP). In the I/R and DMSO groups, the cardiac contraction decreased severely after the reperfusion. In contrast, LVDP recovered dramatically at the end of the reperfusion period in the 9-Phe-treated group. B. Contractile function of the heart indicated by the left ventricular developed pressure (LVDP). Impaired contractile function caused by I/R injury was significantly recovered by treatment with 9-Phe. Simultaneous application of the KATP channel-blocker, 5-HD, did not significantly change the cardioprotective effect of 9-Phe. Asterisks indicate a significant difference compared with the 9-Phe group as determined using Bonferroni post hoc tests.***p<0.001,**p<0.01.
Figure 3
Figure 3. Analysis of heart contractility.
Maximum value of the time derivative of LVP (dP/dtmax) was used as an indicator for cardiac contractility. In the I/R and DMSO groups, decrease in dP/dtmax is evident. In contrast, impaired contractility caused by I/R injury was significantly recovered by treatment with 9-Phe. Asterisks indicate a significant difference compared with the 9-Phe group as determined using Bonferroni post hoc tests.***p<0.001,**p<0.01,*p<0.05.
Figure 4
Figure 4. Representative epicardial ECGs from Langendorff excised hearts.
Three representative recordings are shown for the control and 9-Phe-treated groups. While ventricular fibrillation or tachycardia was evident at 15 min after ischemia–reperfusion in the control group, regular rhythm was maintained in the 9-Phe-treated group.
Figure 5
Figure 5. Measurement of heart rate.
While heart rate increased after ischemia–reperfusion in the control group, that in 9-Phe-treated group was relatively stable. Simultaneous application of KATP channel inhibitor 5-HD did not affect the action of 9-Phe. Asterisks indicate a significant difference compared with the 9-Phe group as determined using Bonferroni post hoc tests.**p<0.01,*p<0.05.
Figure 6
Figure 6. Determination of LDH activity to assess tissue damage.
The LDH activity of the 9-Phe group was significantly lower than that of the I/R and DMSO groups after ischemia–reperfusion. Simultaneous application of KATP channel inhibitor 5-HD did not affect the action of 9-Phe.*p<0.05, N.S. p>0.05.
Figure 7
Figure 7. Analysis of infarction size.
Images above the graph show the typical TTC-stained heart slices corresponding to the groups indicated below on the X-axis of the graph. The colored bar below the images indicates the brightness of the images. A fixed detection threshold for infarcted area is arbitrarily set and used throughout the analysis. The graph shows the percentage infarction in each group. The size of the infarcted area in the 9-Phe group was significantly smaller than that of the I/R and DMSO groups.*p<0.05, N.S. p>0.05.
Figure 8
Figure 8. Analysis of cardiac apoptosis.
A. Detection of apoptosis using the TUNEL assay. Representative microscopic pictures of fluorescein labeled apoptotic cells (green) and counter staining with DAPI (blue) from the I/R and the 9-Phe-treated groups, along with serial sections stained by HE and PAS. Circles show the cells of the cardiac conduction system, which are lightly stained in HE staining and purple in PAS staining. Apoptotic cells were not found in these areas either in the I/R group or in 9-Phe-treated group. Arrowhead: apoptotic cells in the endocardium. B. Comparison of apoptosis area between the I/R and 9-Phe-treated hearts. Percentage of apoptotic cells was determined for the whole area of the paraffin section and were expressed as a percentage of the DAPI-positive area. N.S.: p>0.05.

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