23 research outputs found
Cyclosporine-insensitive mode of cell death after prolonged myocardial ischemia: Evidence for sarcolemmal permeabilization as the pivotal step
<div><p>A prominent theory of cell death in myocardial ischemia/reperfusion (I/R) posits that the primary and pivotal step of irreversible cell injury is the opening of the mitochondrial permeability transition (MPT) pore. However, the predominantly positive evidence of protection against infarct afforded by the MPT inhibitor, Cyclosporine A (CsA), in experimental studies is in stark contrast with the overall lack of benefit found in clinical trials of CsA. One reason for the discrepancy might be the fact that relatively short experimental ischemic episodes (<1 hour) do not represent clinically-realistic durations, usually exceeding one hour. Here we tested the hypothesis that MPT is not the primary event of cell death after prolonged (60–80 min) episodes of global ischemia. We used confocal microcopy in Langendorff-perfused rabbit hearts treated with the electromechanical uncoupler, 2,3-Butanedione monoxime (BDM, 20 mM) to allow tracking of MPT and sarcolemmal permeabilization (SP) in individual ventricular myocytes. The time of the steepest drop in fluorescence of mitochondrial membrane potential (ΔΨ<sub>m</sub>)-sensitive dye, TMRM, was used as the time of MPT (T<sub>MPT</sub>). The time of 20% uptake of the normally cell-impermeable dye, YO-PRO1, was used as the time of SP (T<sub>SP</sub>). We found that during reperfusion MPT and SP were tightly coupled, with MPT trending slightly ahead of SP (T<sub>SP</sub>-T<sub>MPT</sub> = 0.76±1.31 min; p = 0.07). These coupled MPT/SP events occurred in discrete myocytes without crossing cell boundaries. CsA (0.2 μM) did not reduce the infarct size, but separated SP and MPT events, such that detectable SP was significantly ahead of MPT (T<sub>SP</sub> -T<sub>MPT</sub> = -1.75±1.28 min, p = 0.006). Mild permeabilization of cells with digitonin (2.5–20 μM) caused coupled MPT/SP events which occurred in discrete myocytes similar to those observed in Control and CsA groups. In contrast, deliberate induction of MPT by titration with H<sub>2</sub>O<sub>2</sub> (200–800 μM), caused propagating waves of MPT which crossed cell boundaries and were uncoupled from SP. Taken together, these findings suggest that after prolonged episodes of ischemia, SP is the primary step in myocyte death, of which MPT is an immediate and unavoidable consequence.</p></div
Detailed spatiotemporal analysis of F<sub>TMRM</sub> loss and F<sub>YO-PRO1</sub> gain during reperfusion in a representative myocyte from a control heart.
<p><b>A</b>, each row shows F<sub>TMRM</sub> (green), F<sub>YO-PRO1</sub> (orange) and the merged image of the cell undergoing the critical transition (outlined with white) for different time points (<i>a</i> to <i>e</i>) indicated in <b>B</b>. <b>B</b>, the cell-averaged F<sub>TMRM</sub> (green) and F<sub>YO-PRO1</sub> (orange) as the function of time shown as 0–100% of the dynamic range of each signal. The vertical green and orange dashed lines indicate T<sub>MPT</sub> and T<sub>SP</sub>, respectively. Note the spatiotemporal overlap between the processes of F<sub>TMRM</sub> loss (indicator of MPT) and F<sub>YO-PRO1</sub> gain (indicator of SP), such that at time point <i>b</i> there is a clear uptake of YO-PRO1 while the majority of the cell interior still shows polarized mitochondria.</p
Detailed spatiotemporal analysis of F<sub>TMRM</sub> loss and F<sub>YO-PRO1</sub> gain during reperfusion in a myocyte from a heart treated with CsA (0.2 μM).
<p>Notations are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.g001" target="_blank">Fig 1</a>. Note that compared to a myocyte from a control heart shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.g001" target="_blank">Fig 1</a>, the earliest detectable F<sub>YO-PRO1</sub> gain (indicator of SP), which is observed at the time point <i>b</i>, clearly occurs before the processes of F<sub>TMRM</sub> loss (indicator of MPT).</p
The summary of the time difference between the MPT and SP events (T<sub>SP</sub>-T<sub>MPT</sub>) in individual myocytes from the three experimental groups as indicated.
<p>Thick horizontal bars show average values. Note that CsA increases separation between MPT and SP, such that T<sub>SP</sub>-T<sub>MPT</sub> is significantly negative. *, p < 0.01.</p
Assessment of infarct size using TTC staining.
<p><b>A</b>, <i>left to right</i>, examples of original scanned images of TTC staining, the respective green channel signal, and the thresholded regions, respectively (see detailed description of TTC staining procedure in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s002" target="_blank">S1 Fig</a>). Rows from top to bottom in <b>A</b> represent <i>No_BDM</i>, <i>Control</i>, and <i>CsA</i> groups, respectively. <b>B</b>, average percent of severe infarct (white), intermediate infarct (pink) and viable tissue (red) in the three experimental groups. The error bars are omitted for clarity, but none of the regions were significantly different between the groups by 2-way ANOVA. At least in part this is due to the large variation in TTC staining data between hearts in each group (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s007" target="_blank">S6 Fig</a>).</p
Wave-like propagation of MPT caused by H<sub>2</sub>O<sub>2</sub>.
<p>The time is shown according to the onset of observation starting 6 min after the end of H<sub>2</sub>O<sub>2</sub> application (200 μM). <b>A</b> to <b>D</b>, consecutive snapshots taken every 2 minutes. Green, fluorescence of TMRM (F<sub>TMRM</sub>); orange, fluorescence of YO-PRO1 (F<sub>YO-PRO1</sub>). The nature of objects brightly stained with YO-PRO1 is unknown, but they may represent dead fibroblasts or other cell types which are usually abundant in the most superficial layers and are visible immediately after YO-PRO1 delivery to the heart. White arrows point to two such bright objects, which were used as fiducial points to track myocytes amid slight changes in the field of view and the focal plane. Note that unlike the naturally occurring MPT/SP events, H<sub>2</sub>O<sub>2</sub>-induced MPT propagated as a wide front (white line), easily crossing cell boundaries, and was not associated with immediate cellular YO-PRO1 uptake. <b>E</b> and <b>F</b>, Different structure of the propagating front of <sub>FTMRM</sub> loss during naturally occurring MPT (<b>E</b>; same cell as #13 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s005" target="_blank">S4 Fig</a> as well as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s014" target="_blank">S5 Movie</a>) and H<sub>2</sub>O<sub>2</sub>-induced MPT (<b>F</b>). In each Panel, F<sub>TMRM</sub> was computed as the function of the distance along the long axis of the rectangular region of interest (ROI) approximating the selected cell (white rectangle). The magnified image of the ROI is shown at the top of each Panel. For each x-position along the cell, F<sub>TMRM</sub> was averaged over all pixels in the respective column (across the width) of the rectangle. This yielded an instantaneous profile of the propagating wave (green curve). The minimal value on the y-axis of mean F<sub>TMRM</sub> represents the minimal value in the field of view (extracellular level, or background). Note that during naturally occurring MPT (<b>E</b>) the level of F<sub>TMRM</sub> in the wake of the MPT front is very close to the background level indicating complete loss of the TMRM in that part of the cell. In contrast, during H<sub>2</sub>O<sub>2</sub>-induced MPT (<b>F</b>) the level of F<sub>TMRM</sub> in the wake of the MPT front remains at about 50% of the dynamic range.</p
Perm1 regulates cardiac energetics as a downstream target of the histone methyltransferase Smyd1.
The transcriptional regulatory machinery in mitochondrial bioenergetics is complex and is still not completely understood. We previously demonstrated that the histone methyltransferase Smyd1 regulates mitochondrial energetics. Here, we identified Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) as a downstream target of Smyd1 through RNA-seq. Chromatin immunoprecipitation assay showed that Smyd1 directly interacts with the promoter of Perm1 in the mouse heart, and this interaction was significantly reduced in mouse hearts failing due to pressure overload for 4 weeks, where Perm1 was downregulated (24.4 ± 5.9% of sham, p<0.05). Similarly, the Perm1 protein level was significantly decreased in patients with advanced heart failure (55.2 ± 13.1% of donors, p<0.05). Phenylephrine (PE)-induced hypertrophic stress in cardiomyocytes also led to downregulation of Perm1 (55.7 ± 5.7% of control, p<0.05), and adenovirus-mediated overexpression of Perm1 rescued PE-induced downregulation of estrogen-related receptor alpha (ERRα), a key transcriptional regulator of mitochondrial energetics, and its target gene, Ndufv1 (Complex I). Pathway enrichment analysis of cardiomyocytes in which Perm1 was knocked-down by siRNA (siPerm1), revealed that the most downregulated pathway was metabolism. Cell stress tests using the Seahorse XF analyzer showed that basal respiration and ATP production were significantly reduced in siPerm1 cardiomyocytes (40.7% and 23.6% of scrambled-siRNA, respectively, both p<0.05). Luciferase reporter gene assay further revealed that Perm1 dose-dependently increased the promoter activity of the ERRα gene and known target of ERRα, Ndufv1 (Complex I). Overall, our study demonstrates that Perm1 is an essential regulator of cardiac energetics through ERRα, as part of the Smyd1 regulatory network
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Histone methyltransferase Smyd1 regulates mitochondrial energetics in the heart
Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1's involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α