Cardiac Slo2.1 Is Required for Volatile Anesthetic Stimulation of K+ Transport and Anesthetic Preconditioning.

BACKGROUND: Anesthetic preconditioning (APC) is a clinically important phenomenon in which volatile anesthetics (VAs) protect tissues such as heart against ischemic injury. The mechanism of APC is thought to involve K channels encoded by the Slo gene family, and the authors showed previously that slo-2 is required for APC in Caenorhabditis elegans. Thus, the authors hypothesized that a slo-2 ortholog may mediate APC-induced cardioprotection in mammals. METHODS: A perfused heart model of ischemia-reperfusion injury, a fluorescent assay for K flux, and mice lacking Slo2.1 (Slick), Slo2.2 (Slack), or both (double knockouts, Slo2.x dKO) were used to test whether these channels are required for APC-induced cardioprotection and for cardiomyocyte or mitochondrial K transport. RESULTS: In wild-type (WT) hearts, APC improved post-ischemia-reperfusion functional recovery (APC = 39.5 ± 3.7% of preischemic rate × pressure product vs. 20.3 ± 2.3% in controls, means ± SEM, P = 0.00051, unpaired two-tailed t test, n = 8) and lowered infarct size (APC = 29.0 ± 4.8% of LV area vs. 51.4 ± 4.5% in controls, P = 0.0043, n = 8). Protection by APC was absent in hearts from Slo2.1 mice (% recovery APC = 14.6 ± 2.6% vs. 16.5 ± 2.1% in controls, P = 0.569, n = 8 to 9, infarct APC = 52.2 ± 5.4% vs. 53.5 ± 4.7% in controls, P = 0.865, n = 8 to 9). APC protection was also absent in Slo2.x dKO hearts (% recovery APC = 11.0 ± 1.7% vs. 11.9 ± 2.2% in controls, P = 0.725, n = 8, infarct APC = 51.6 ± 4.4% vs. 50.5 ± 3.9% in controls, P = 0.855, n = 8). Meanwhile, Slo2.2 hearts responded similar to WT (% recovery APC = 41.9 ± 4.0% vs. 18.0 ± 2.5% in controls, P = 0.00016, n = 8, infarct APC = 25.2 ± 1.3% vs. 50.8 ± 3.3% in controls, P < 0.000005, n = 8). Furthermore, VA-stimulated K transport seen in cardiomyocytes or mitochondria from WT or Slo2.2 mice was absent in Slo2.1 or Slo2.x dKO. CONCLUSION: Slick (Slo2.1) is required for both VA-stimulated K flux and for the APC-induced cardioprotection.

The cardioprotective compound cloxyquin uncouples mitochondria and induces autophagy.

Mitochondrial quality control mechanisms have been implicated in protection against cardiac ischemia-reperfusion (IR) injury. Previously, cloxyquin (5-chloroquinolin-8-ol) was identified via phenotypic screening as a cardioprotective compound. Herein, cloxyquin was identified as a mitochondrial uncoupler in both isolated heart mitochondria and adult cardiomyocytes. Additionally, cardiomyocytes isolated from transgenic mice expressing green fluorescent protein-tagged microtubule-associated protein light chain 3 showed increased autophagosome formation with cloxyquin treatment. The autophagy inhibitor chloroquine abolished cloxyquin-induced cardioprotection in both cellular and perfused heart (Langendorff) models of IR injury. Finally, in an in vivo murine left anterior descending coronary artery occlusion model of IR injury, cloxyquin significantly reduced infarct size from 31.4 ± 3.4% to 16.1 ± 2.2%. In conclusion, the cardioprotective compound cloxyquin simultaneously uncoupled mitochondria and induced autophagy. Importantly, autophagy appears to be required for cloxyquin-induced cardioprotection.

Metabolomic profiling of the heart during acute ischemic preconditioning reveals a role for SIRT1 in rapid cardioprotective metabolic adaptation.

Ischemic preconditioning (IPC) protects tissues such as the heart from prolonged ischemia-reperfusion (IR) injury. We previously showed that the lysine deacetylase SIRT1 is required for acute IPC, and has numerous metabolic targets. While it is known that metabolism is altered during IPC, the underlying metabolic regulatory mechanisms are unknown, including the relative importance of SIRT1. Thus, we sought to test the hypothesis that some of the metabolic adaptations that occur in IPC may require SIRT1 as a regulatory mediator. Using both ex-vivo-perfused and in-vivo mouse hearts, LC-MS/MS based metabolomics and (13)C-labeled substrate tracing, we found that acute IPC altered several metabolic pathways including: (i) stimulation of glycolysis, (ii) increased synthesis of glycogen and several amino acids, (iii) increased reduced glutathione levels, (iv) elevation in the oncometabolite 2-hydroxyglutarate, and (v) inhibition of fatty-acid dependent respiration. The majority (83%) of metabolic alterations induced by IPC were ablated when SIRT1 was acutely inhibited with splitomicin, and a principal component analysis revealed that metabolic changes in response to IPC were fundamentally different in nature when SIRT1 was inhibited. Furthermore, the protective benefit of IPC was abrogated by eliminating glucose from perfusion media while sustaining normal cardiac function by burning fat, thus indicating that glucose dependency is required for acute IPC. Together, these data suggest that SIRT1 signaling is required for rapid cardioprotective metabolic adaptation in acute IPC.

SIRT3 deficiency exacerbates ischemia-reperfusion injury: implication for aged hearts.

Ischemia-reperfusion (IR) injury is significantly worse in aged hearts, but the underlying mechanisms are poorly understood. Age-related damage to mitochondria may be a critical feature, which manifests in an exacerbation of IR injury. Silent information regulator of transcription 3 (SIRT3), the major mitochondrial NAD(+)-dependent lysine deacetylase, regulates a variety of functions, and its inhibition may disrupt mitochondrial function to impact recovery from IR injury. In this study, the role of SIRT3 in mediating the response to cardiac IR injury was examined using an in vitro model of SIRT3 knockdown (SIRT3(kd)) in H9c2 cardiac-derived cells and in Langendorff preparations from adult (7 mo old) wild-type (WT) and SIRT3(+/-) hearts and aged (18 mo old) WT hearts. SIRT3(kd) cells were more vulnerable to simulated IR injury and exhibited a 46% decrease in mitochondrial complex I (Cx I) activity with low O2 consumption rates compared with controls. In the Langendorff model, SIRT3(+/-) adult hearts showed less functional recovery and greater infarct vs. WT, which recapitulates the in vitro results. In WT aged hearts, recovery from IR injury was similar to SIRT3(+/-) adult hearts. Mitochondrial protein acetylation was increased in both SIRT3(+/-) adult and WT aged hearts (relative to WT adult), suggesting similar activities of SIRT3. Also, enzymatic activities of two SIRT3 targets, Cx I and MnSOD, were similarly and significantly inhibited in SIRT3(+/-) adult and WT aged cardiac mitochondria. In conclusion, decreased SIRT3 may increase the susceptibility of cardiac-derived cells and adult hearts to IR injury and may contribute to a greater level of IR injury in the aged heart.

A non-cardiomyocyte autonomous mechanism of cardioprotection involving the SLO1 BK channel.

Opening of BK-type Ca(2+) activated K(+) channels protects the heart against ischemia-reperfusion (IR) injury. However, the location of BK channels responsible for cardioprotection is debated. Herein we confirmed that openers of the SLO1 BK channel, NS1619 and NS11021, were protective in a mouse perfused heart model of IR injury. As anticipated, deletion of the Slo1 gene blocked this protection. However, in an isolated cardiomyocyte model of IR injury, protection by NS1619 and NS11021 was insensitive to Slo1 deletion. These data suggest that protection in intact hearts occurs by a non-cardiomyocyte autonomous, SLO1-dependent, mechanism. In this regard, an in-situ assay of intrinsic cardiac neuronal function (tachycardic response to nicotine) revealed that NS1619 preserved cardiac neurons following IR injury. Furthermore, blockade of synaptic transmission by hexamethonium suppressed cardioprotection by NS1619 in intact hearts. These results suggest that opening SLO1 protects the heart during IR injury, via a mechanism that involves intrinsic cardiac neurons. Cardiac neuronal ion channels may be useful therapeutic targets for eliciting cardioprotection.

Kir6.2 is not the mitochondrial KATP channel but is required for cardioprotection by ischemic preconditioning.

ATP-sensitive K(+) (KATP) channels that contain K(+) inward rectifier subunits of the 6.2 isotype (Kir6.2) are important regulators of the cardiac response to ischemia-reperfusion (I/R) injury. Opening of these channels is implicated in the cardioprotective mechanism of ischemic preconditioning (IPC), but debate surrounds the contribution of surface KATP (sKATP) versus mitochondrial KATP (mKATP) channels. While responses to I/R injury and IPC have been examined in Kir6.2(-/-) mice before, breeding methods and other technical obstacles may have confounded interpretations. The aim of this study was to elucidate the role of Kir6.2 in cardioprotection and mKATP activity, using conventionally bred Kir6.2(-/-) mice with wild-type littermates as controls. We found that perfused hearts from Kir6.2(-/-) mice exhibited a normal baseline response to I/R injury, were not protected by IPC, and showed a blunted response to the IPC mimetic drug diazoxide. These data suggest that the loss of IPC in Kir6.2(-/-) hearts is not due to an underlying difference in I/R sensitivity. Furthermore, mKATP channel activity was identical in cardiac mitochondria isolated from wild-type versus Kir6.2(-/-) mice, suggesting no role for Kir6.2 in the mKATP. Collectively, these data indicate that Kir6.2 is required for the full response to IPC or diazoxide but is not involved in mKATP formation.

A cell-based phenotypic assay to identify cardioprotective agents.

RATIONALE: Tissue ischemia/reperfusion (IR) injury underlies several leading causes of death such as heart-attack and stroke. The lack of clinical therapies for IR injury may be partly due to the difficulty of adapting IR injury models to high-throughput screening (HTS). OBJECTIVE: To develop a model of IR injury that is both physiologically relevant and amenable to HTS. METHODS AND RESULTS: A microplate-based respirometry apparatus was used. Controlling gas flow in the plate head space, coupled with the instrument's mechanical systems, yielded a 24-well model of IR injury in which H9c2 cardiomyocytes were transiently trapped in a small volume, rendering them ischemic. After initial validation with known protective molecules, the model was used to screen a 2000-molecule library, with post-IR cell death as an end point. Po2 and pH monitoring in each well also afforded metabolic data. Ten protective, detrimental, and inert molecules from the screen were subsequently tested in a Langendorff-perfused heart model of IR injury, revealing strong correlations between the screening end point and both recovery of cardiac function (negative, r2=0.66) and infarct size (positive, r2=0.62). Relationships between the effects of added molecules on cellular bioenergetics and protection against IR injury were also studied. CONCLUSIONS: This novel cell-based assay can predict either protective or detrimental effects on IR injury in the intact heart. Its application may help identify therapeutic or harmful molecules.

Nitroalkenes confer acute cardioprotection via adenine nucleotide translocase 1.

Electrophilic nitrated lipids (nitroalkenes) are emerging as an important class of protective cardiovascular signaling molecules. Although species such as nitro-linoleate (LNO(2)) and nitro-oleate can confer acute protection against cardiac ischemic injury, their mechanism of action is unclear. Mild uncoupling of mitochondria is known to be cardioprotective, and adenine nucleotide translocase 1 (ANT1) is a key mediator of mitochondrial uncoupling. ANT1 also contains redox-sensitive cysteines that may be targets for modification by nitroalkenes. Therefore, in this study we tested the hypothesis that nitroalkenes directly modify ANT1 and that nitroalkene-mediated cardioprotection requires ANT1. Using biotin-tagged LNO(2) infused into intact perfused hearts, we obtained mass spectrometric (MALDI-TOF-TOF) evidence for direct modification (nitroalkylation) of ANT1 on cysteine 57. Furthermore, in a cell model of ischemia-reperfusion injury, siRNA knockdown of ANT1 inhibited the cardioprotective effect of LNO(2). Although the molecular mechanism linking ANT1-Cys(57) nitroalkylation and uncoupling is not yet known, these data suggest that ANT1-mediated uncoupling may be a mechanism for nitroalkene-induced cardioprotection.

SLO-2 is cytoprotective and contributes to mitochondrial potassium transport.

Mitochondrial potassium channels are important mediators of cell protection against stress. The mitochondrial large-conductance "big" K(+) channel (mBK) mediates the evolutionarily-conserved process of anesthetic preconditioning (APC), wherein exposure to volatile anesthetics initiates protection against ischemic injury. Despite the role of the mBK in cardioprotection, the molecular identity of the channel remains unknown. We investigated the attributes of the mBK using C. elegans and mouse genetic models coupled with measurements of mitochondrial K(+) transport and APC. The canonical Ca(2+)-activated BK (or "maxi-K") channel SLO1 was dispensable for both mitochondrial K(+) transport and APC in both organisms. Instead, we found that the related but physiologically-distinct K(+) channel SLO2 was required, and that SLO2-dependent mitochondrial K(+) transport was triggered directly by volatile anesthetics. In addition, a SLO2 channel activator mimicked the protective effects of volatile anesthetics. These findings suggest that SLO2 contributes to protection from hypoxic injury by increasing the permeability of the mitochondrial inner membrane to K(+).