TRPM2 channels protect against cardiac ischemia-reperfusion injury: role of mitochondria - PubMed (original) (raw)

. 2014 Mar 14;289(11):7615-29.

doi: 10.1074/jbc.M113.533851. Epub 2014 Feb 3.

Nicholas E Hoffman, Salim Merali, Xue-Qian Zhang, JuFang Wang, Sudarsan Rajan, Santhanam Shanmughapriya, Erhe Gao, Carlos A Barrero, Karthik Mallilankaraman, Jianliang Song, Tongda Gu, Iwona Hirschler-Laszkiewicz, Walter J Koch, Arthur M Feldman, Muniswamy Madesh, Joseph Y Cheung

Affiliations

TRPM2 channels protect against cardiac ischemia-reperfusion injury: role of mitochondria

Barbara A Miller et al. J Biol Chem. 2014.

Abstract

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca(2+) uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca(2+) uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca(2+) uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.

Keywords: Calcium Channels; Cardiac Ischemia; Cardiovascular Disease; Electrophysiology; Global Proteomics Analysis; Mitochondria; Mitochondrial Bioenergetics; TRP Channels.

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Figures

FIGURE 1.

FIGURE 1.

ADPR activates cationic currents in WT but not TRPM2 KO myocytes. LV myocytes were isolated from WT and KO hearts. The standard patch clamp whole cell configuration was used. Composition of pipette and external solutions and voltage-ramp protocols are given under “Experimental Procedures”. I-V relationships of cationic current (means ± S.E.) from WT + ADPR (300 μ

m

) (●; n = 5), WT (○; n = 4), KO + ADPR (■; n = 7) and KO (□; n = 3) myocytes are shown. Error bars are not shown if they fell within the boundaries of the symbol. Two-way analysis of variance indicate p < 0.0001 for WT + ADPR versus WT or KO + ADPR or KO myocytes. pF, picofarads.

FIGURE 2.

FIGURE 2.

WT TRPM2 currents do not inactivate and estimation of GCa and GNa. WT myocytes were held at −80 mV. ADPR was included in the pipette solution to activate TRPM2 channels. With 140 m

m

[Na+]o, after break-in, a large inward current that did not inactivate was observed. The current showed a linear increase with increasing hyperpolarization. After media change to a medium containing 110 m

m

[Ca2+]o, current became smaller and demonstrated a linear decrease with depolarization from −100 to −80 mV. GCa and GNa were estimated from the slope of I-V relationship (“Experimental Procedures”).

FIGURE 3.

FIGURE 3.

Top network functions generated using Ingenuity Pathways Analysis for KO-I/R versus WT-I/R hearts. Differentially expressed proteins between KO-I/R and WT-I/R hearts (n = 4 each) were determined using GeLC-MS/MS (“Experimental Procedures”). The graph represents cell functions with the highest score (y axis) based on the number of differentially regulated proteins. The orange squares and line in the graph show the ratio of the number of proteins altered identified from our dataset that are in the pathway relative to the total number of proteins in the pathway.

FIGURE 4.

FIGURE 4.

Ingenuity Pathways Analysis of mitochondrial proteins that are differentially expressed between KO-I/R and WT-I/R hearts. Mitochondrial network was generated from the differentially expressed proteins according to the Ingenuity Pathway Knowledge Criteria. Pink, significantly up-regulated proteins; green, significantly down-regulated proteins; white, proteins known to be in the network but were not identified in our analysis.

FIGURE 5.

FIGURE 5.

Expression of proteins associated with mitochondria. Homogenates of LV apices from WT-I/R and KO-I/R hearts were prepared and proteins subjected to Western blots (“Experimental Procedures”). Protein band intensities are quantified by densitometry, normalized to calsequestrin signals, and summarized in bar graphs at the right. *, p < 0.05.

FIGURE 6.

FIGURE 6.

Mitochondrial membrane potential and mitochondrial Ca2+ uptake are lower in KO myocytes subjected to hypoxia/reoxygenation. LV myocytes isolated from WT and KO mice were subjected to normoxia or hypoxia for 2 h followed by 30 min of reoxygenation (“Experimental Procedures”). Myocytes were permeabilized with digitonin and supplemented with succinate. A, the ratiometric indicator JC-1 was added at 20s and used to monitor Δψm. Arrows indicate the addition of JC-1, Ca2+ (10 μ

m

), and the mitochondrial uncoupler CCCP (2 μ

m

). B, summary of Δψm after Ca2+ addition but before CCCP addition (n = 3 each). C, the ratiometric dye Fura-FF was added at 0 s and used to monitor extra-mitochondrial Ca2+. Repeated pulses of Ca2+ (10 μ

m

) were added as indicated (arrows). The cytosolic Ca2+ clearance rate after the first Ca2+ pulse was measured. f.a.u., fluorescence arbitrary units. D, summary of cytosolic Ca2+ clearance (mitochondrial Ca2+ uptake) rates (n = 3 each). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

FIGURE 7.

Mitochondrial Ca2+ uniporter activity (_I_MCU) is lower in KO myocytes, but basal mitochondrial Ca2+ levels are similar between WT and KO myocytes. A, currents from cardiac mitoplasts (_I_MCU) were recorded before and after application of 5 m

m

Ca2+ to the bath medium. _I_MCU were recorded during a voltage-ramp as indicated. Traces are representative single recordings of _I_MCU from WT (black) and KO (blue) myocytes. B, current-time integral indicating the amount of Ca2+ influx during voltage-ramp (fmol/picofarads) in WT (black) and KO (blue) mitoplasts; n = 3 each. pF, picofarads. C, freshly isolated myocytes from WT and KO mice were permeabilized with digitonin and supplemented with succinate. The ratiometric dye Fura-FF was used to monitor extra-mitochondrial Ca2+. After steady-state Fura-FF signals were obtained, CGP37157 (10 μ

m

) was added to inhibit mitochondrial Ca2+ release via the mitochondrial Na+/Ca2+ exchanger. CCCP (2 μ

m

) was then added to release endogenous mitochondrial Ca2+. D, quantitation of mitochondrial Ca2+ contents (n = 4 each). **, p < 0.01.

FIGURE 8.

FIGURE 8.

O2 consumption is markedly reduced in KO-H/R myocytes. LV myocytes isolated from WT and KO mice were subjected to normoxia or hypoxia for 2 h followed by 30 min of reoxygenation. O2 consumption rate was measured in intact myocytes (“Experimental Procedures”). A, after basal OCR was obtained, oligomycin (1 μ

m

) was added to inhibit F0F1ATPase (Complex V). The uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 1 μ

m

) was then added, and maximal OCR was measured. Finally, antimycin A + rotenone (1 μ

m

each) were added to inhibit cytochrome bc1 complex (Complex III) and NADH dehydrogenase (Complex I), respectively. Each point in the traces represents the average of eight different wells. B and C, summary of basal (B) and maximal (C) OCR of 4 groups of myocytes (n = 3 each). ***, p < 0.001.

FIGURE 9.

FIGURE 9.

ATP levels and O2 consumption are lower in KO hearts. A, hearts from WT (black) and KO (blue) mice were homogenized, and ATP levels were measured by CellTiter-Glo assay. n = 3 each. B, similarly, freshly isolated myocytes from WT (black) and KO (blue) mice were used to measure the ATP levels. n = 6 each. C, heart slices generated from WT (black) and KO (blue) mice were subjected to OCR measurement, and the graph represents the basal OCR. n = 3 each. *, p < 0.05; ***, p < 0.001.

FIGURE 10.

FIGURE 10.

In situ ROS levels are prominent in KO-I/R heart slices. Hearts from WT and KO mice were subjected to sham operation or 30 min ischemia followed by 30 min reperfusion, after which ROS levels were measured in heart slices (“Experimental Procedures”). A, multi-photon confocal images of DHE-stained LV slices. B, 2.5-dimensional heatmap plots of mean DHE intensity are shown for WT-sham, KO-sham, WT-I/R, and KO-I/R heart slices. C, quantitation of DHE fluorescence in fluorescence arbitrary units (f.a.u.). DHE fluorescence from at least 3 slices from each heart are quantitated for WT-sham (2 mice), KO-sham (2 mice), WT-I/R (2 mice), and KO-I/R (5 mice) animals. *, p < 0.05; **, p < 0.01.

FIGURE 11.

FIGURE 11.

Mitochondrial membrane potential is lower and mitochondrial ROS level is higher in KO myocytes. A, myocytes isolated from WT and KO mice were loaded with the mitochondrial membrane potential indicator rhodamine 123 and mitochondrial superoxide indicator MitoSOX Red. Confocal images showed co-localization of MitoSOX Red and rhodamine 123 signals. B and C, quantitation of rhodamine 123 (ψm) and MitoSOX Red (mROS) signal intensities in fluorescence arbitrary units (f.a.u.) from WT (n = 21) and KO (n = 19) myocytes. **, p < 0.01.

FIGURE 12.

FIGURE 12.

Hypothetical mechanism by which cardiac TRPM2 channels modulate mitochondrial function and ROS production. Ca2+ influx via activated TRPM2 channels increases cytosolic Ca2+ concentration, thereby activating calcineurin. Calcineurin dephosphorylates RACK1 and blocks RACK1 dimerization. The net result is increased HIF-1α levels by impeding its ubiquitination and degradation. HIF-1α enhances many target gene transcription including FoxO3a, which leads to increased SOD2 expression, and mitochondrial NDUFA4L2 (Complex I) and other mitochondrial gene (BNIP3) expression. Both SOD2 and physiological Complex I activity result in reduced mitochondrial ROS levels. Differences in protein levels between WT-I/R and KO-I/R hearts that have been authenticated by experimental results from the present (Fig. 5) or previous (5) studies are in bold fonts, whereas hypothetical signaling molecules are in regular fonts.

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