Cardiomyocyte Adaptation to Exercise: K+ Channels, Contractility and Ischemic Injury.

Cardiovascular disease is a leading cause of morbidity and mortality, and exercise-training (TRN) is known to reduce risk factors and protect the heart from ischemia and reperfusion injury. Though the cardioprotective effects of exercise are well-documented, underlying mechanisms are not well understood. This review highlights recent findings and focuses on cardiac factors with emphasis on K+ channel control of the action potential duration (APD), ß-adrenergic and adenosine regulation of cardiomyocyte function, and mitochondrial Ca2+ regulation. TRN-induced prolongation and shortening of the APD at low and high activation rates, respectively, is discussed in the context of a reduced response of the sarcolemma delayed rectifier potassium channel (IK) and increased content and activation of the sarcolemma KATP channel. A proposed mechanism underlying the latter is presented, including the phosphatidylinositol-3kinase/protein kinase B pathway. TRN induced increases in cardiomyocyte contractility and the response to adrenergic agonists are discussed. The TRN-induced protection from reperfusion injury is highlighted by the increased content and activation of the sarcolemma KATP channel and the increased phosphorylated glycogen synthase kinase-3ß, which aid in preventing mitochondrial Ca2+ overload and mitochondria-triggered apoptosis. Finally, a brief section is presented on the increased incidences of atrial fibrillation associated with age and in life-long exercisers.

Effects of Subnormothermic Regulated Hepatic Reperfusion on Mitochondrial and Transcriptomic Profiles in a Porcine Model.

OBJECTIVE: We sought to investigate the biological effects of pre-reperfusion treatments of the liver after warm and cold ischemic injuries in a porcine donation after circulatory death model. SUMMARY OF BACKGROUND DATA: Donation after circulatory death represents a severe form of liver ischemia and reperfusion injury that has a profound impact on graft function after liver transplantation. METHODS: Twenty donor pig livers underwent 60 minutes of in situ warm ischemia after circulatory arrest and 120 minutes of cold static preservation prior to simulated transplantation using an ex vivo perfusion machine. Four reperfusion treatments were compared: Control-Normothermic (N), Control- Subnormothermic (S), regulated hepatic reperfusion (RHR)-N, and RHR-S (n = 5 each). The biochemical, metabolic, and transcriptomic profiles, as well as mitochondrial function were analyzed. RESULTS: Compared to the other groups, RHR-S treated group showed significantly lower post-reperfusion aspartate aminotransferase levels in the reperfusion effluent and histologic findings of hepatocyte viability and lesser degree of congestion and necrosis. RHR-S resulted in a significantly higher mitochondrial respiratory control index and calcium retention capacity. Transcriptomic profile analysis showed that treatment with RHR-S activated cell survival and viability, cellular homeostasis as well as other biological functions involved in tissue repair such as cytoskeleton or cytoplasm organization, cell migration, transcription, and microtubule dynamics. Furthermore, RHR-S inhibited organismal death, morbidity and mortality, necrosis, and apoptosis. CONCLUSION: Subnormothermic RHR mitigates IRI and preserves hepatic mitochondrial function after warm and cold hepatic ischemia. This organ resuscitative therapy may also trigger the activation of protective genes against IRI. Sub- normothermic RHR has potential applicability to clinical liver transplantation.

Vascular regression in the kidney: changes in 3D vessel structure with time post-irradiation.

Though angiogenesis has been investigated in depth, vascular regression and rarefaction remain poorly understood. Regression of renal vasculature accompanies many pathological states such as diabetes, hypertension, atherosclerosis, and radiotherapy. Radiation decreases microvessel density in multiple organs, though the mechanism is not known. By using a whole animal (rat) model with a single dose of partial body irradiation to the kidney, changes in the volume of renal vasculature were recorded at two time points, 60 and 90 days after exposure. Next, a novel vascular and metabolic imaging (VMI) technique was used to computationally assess 3D vessel diameter, volume, branch depth, and density over multiple levels of branching down to 70 µm. Four groups of rats were studied, of which two groups received a single dose of 12.5 Gy X-rays. The kidneys were harvested after 60 or 90 days from one irradiated and one non-irradiated group at each time point. Measurements of the 3D vasculature showed that by day-90 post-radiation, when renal function is known to deteriorate, total vessel volume, vessel density, maximum branch depth, and the number of terminal points in the kidneys decreased by 55%, 57%, 28%, and 53%, respectively. Decreases in the same parameters were not statistically significant at 60 days post-irradiation. Smaller vessels with internal diameters of 70-450 µm as well as large vessels of diameter 451-850 µm, both decreased by 90 days post-radiation. Vascular regression in the lungs of the same strain of irradiated rats has been reported to occur before 60 days supporting the hypothesis that this process is regulated in an organ-specific manner and occurs by a concurrent decrease in luminal diameters of small as well as large blood vessels.

Time and charge/pH-dependent activation of K+ channel-mediated K+ influx and K+/H+ exchange in guinea pig heart isolated mitochondria; role in bioenergetic stability.

Mitochondria play an important role not only in producing energy for the cell but also for regulating mitochondrial and cell function depending on the cell's needs and environment. Uptake of cations, anions, and substrates requires a stable, polarized transmembrane charge potential (ΔΨm). Chemiosmosis requires ion exchangers to remove Na+, K+, Ca2+, PO43-, and other charged species that enter mitochondria. Knowledge of the kinetics of mitochondrial (m) cation channels and exchangers is important in understanding their roles in regulating mitochondrial chemiosmosis and bioenergetics. The influx/efflux of K+, the most abundant mitochondrial cation, alters mitochondrial volume and shape by bringing in anions and H2O by osmosis. The effects of K+ uptake through ligand-specific mK+ channels stimulated/inhibited by agonists/antagonists on mitochondrial volume (swelling/contraction) are well known. However, a more important role for K+ influx is likely its effects on H+ cycling and bioenergetics facilitated by mitochondrial (m) K+/H+ exchange (mKHE), though the kinetics and consequences of K+ efflux by KHE are not well described. We hypothesized that a major role of K+ influx/efflux is stimulation of respiration via the influx of H+ by KHE. We proposed to modulate KHE activity by energizing guinea pig heart isolated mitochondria and by altering the mK+ cycle to capture changes in mitochondrial volume, pHm, ΔΨm, and respiration that would reflect a role for H+ influx via KHE to regulate bioenergetics. To test this, mitochondria were suspended in a 150 mM K+ buffer at pH 6.9, or in a 140 mM Cs+ buffer at pH 7.6 or 6.9 with added 10 mM K+, minimal Ca2+ and free of Na+. O2 content was measured by a Clark electrode, and pHm, ΔΨm, and volume, were measured by fluorescence spectrophotometry and light-scattering. Adding pyruvic acid (PA) alone caused increases in volume and respiration and a rapid decrease in the transmembrane pH gradient (ΔpHm = pHin-pHext) at pHext 6.9> > 7.6, so that ΔΨm was charged and maintained. BKCa agonist NS1619 and antagonist paxilline modified these effects, and KHE inhibitor quinine and K+ ionophore valinomycin depolarized ΔΨm. We postulate that K+ efflux-induced H+ influx via KHE causes an inward H+ leak that stimulates respiration, but at buffer pH 6.9 also utilizes the energy of ΔpHm, the smaller component of the overall proton motive force, ΔµH+. Thus ΔpHm establishes and maintains the ΔΨm required for utilization of substrates, entry of all cations, and for oxidative phosphorylation. Thus, K+ influx/efflux appears to play a pivotal role in regulating energetics while maintaining mitochondrial ionic balance and volume homeostasis.

Hypothermia Prevents Cardiac Dysfunction during Acute Ischemia Reperfusion by Maintaining Mitochondrial Bioenergetics and by Promoting Hexokinase II Binding to Mitochondria.

Background: Hypothermia (H), cardioplegia (CP), and both combined (HCP) are known to be protective against myocardial ischemia reperfusion (IR) injury. Mitochondria have molecular signaling mechanisms that are associated with both cell survival and cell death. In this study, we investigated the dynamic changes in proapoptotic and prosurvival signaling pathways mediating H, CP, or HCP-induced protection of mitochondrial function after acute myocardial IR injury. Methods: Rats were divided into five groups. Each group consists of 3 subgroups based on a specific reperfusion time (5, 20, or 60 min) after a 25-min global ischemia. The time control (TC) groups were not subjected to IR but were perfused with 37 °C Krebs-Ringer's (KR) buffer, containing 4.5 mM K+, in a specific perfusion protocol that corresponded with the duration of each IR protocol. The IR group (control) was perfused for 20 min with KR, followed by 25-min global ischemia, and then KR reperfusion for 5, 20, or 60 min. The treatment groups were exposed to 17 °C H, 37 °C CP (16 mM K+), or HCP (17 °C + CP) for 5 min before ischemia and for 2 min on reperfusion before switching to 37 °C KR perfusion for the remainder of each of the reperfusion times. Cardiac function and mitochondrial redox state (NADH/FAD) were monitored online in the ex vivo hearts before, during, and after ischemia. Mitochondria were isolated at the end of each specified reperfusion time, and changes in O2 consumption, membrane potential (ΔΨ m), and Ca2+ retention capacity (CRC) were assessed using complex I and complex II substrates. In another set of hearts, mitochondrial and cytosolic fractions were isolated after a specified reperfusion time to conduct western blot assays to determine hexokinase II (HKII) and Bax binding/translocation to mitochondria, cytosolic pAkt levels, and cytochrome c (Cyto-c) release into the cytosol. Results: H and HCP were more protective of mitochondrial integrity and, concomitantly, cardiac function than CP alone; H and HCP improved post-ischemic cardiac function by (1) maintaining mitochondrial bioenergetics, (2) maintaining HKII binding to mitochondria with an increase in pAkt levels, (3) increasing CRC, and (4) decreasing Cyto-c release during reperfusion. Bax translocation/binding to mitochondria was unaffected by any treatment, regardless of cardiac functional recovery. Conclusions: Hypothermia preserved mitochondrial function and cardiac function, in part, by maintaining mitochondrial bioenergetics, by retaining HKII binding to mitochondria via upstream pAkt, and by reducing Cyto-c release independently of Bax binding to mitochondria.

Mitochondrial Calcium Handling in Isolated Mitochondria from a Guinea Pig Heart.

Mitochondrial calcium (Ca2+) plays a key role in regulating normal cardiac function. A physiological increase in mitochondrial matrix calcium [Ca2+]m drives mitochondrial ATP production to meet the high-energy demands during excitation-contraction coupling. However, a pathological increase in [Ca2+]m leads to increased oxidative stress, impaired bioenergetics, and the opening of mitochondrial permeability transition pore (mPTP), a hallmark of the failing heart. Therefore, a better understanding of the [Ca2+]m handling and its role in heart function and dysfunction is of great importance. Here, we describe a detailed protocol for measuring mitochondrial Ca2+ handling in the isolated functionally intact mitochondria from cardiac tissue of the guinea pig.

Repetitive Mild Traumatic Brain Injury in Rats Impairs Cognition, Enhances Prefrontal Cortex Neuronal Activity, and Reduces Pre-synaptic Mitochondrial Function.

A major hurdle preventing effective interventions for patients with mild traumatic brain injury (mTBI) is the lack of known mechanisms for the long-term cognitive impairment that follows mTBI. The closed head impact model of repeated engineered rotational acceleration (rCHIMERA), a non-surgical animal model of repeated mTBI (rmTBI), mimics key features of rmTBI in humans. Using the rCHIMERA in rats, this study was designed to characterize rmTBI-induced behavioral disruption, underlying electrophysiological changes in the medial prefrontal cortex (mPFC), and associated mitochondrial dysfunction. Rats received 6 closed-head impacts over 2 days at 2 Joules of energy. Behavioral testing included automated analysis of behavior in open field and home-cage environments, rotarod test for motor skills, novel object recognition, and fear conditioning. Following rmTBI, rats spent less time grooming and less time in the center of the open field arena. Rats in their home cage had reduced inactivity time 1 week after mTBI and increased exploration time 1 month after injury. Impaired associative fear learning and memory in fear conditioning test, and reduced short-term memory in novel object recognition test were found 4 weeks after rmTBI. Single-unit in vivo recordings showed increased neuronal activity in the mPFC after rmTBI, partially attributable to neuronal disinhibition from reduced inhibitory synaptic transmission, possibly secondary to impaired mitochondrial function. These findings help validate this rat rmTBI model as replicating clinical features, and point to impaired mitochondrial functions after injury as causing imbalanced synaptic transmission and consequent impaired long-term cognitive dysfunction.

Structural basis of complex formation between mitochondrial anion channel VDAC1 and Hexokinase-II.

Complex formation between hexokinase-II (HKII) and the mitochondrial VDAC1 is crucial to cell growth and survival. We hypothesize that HKII first inserts into the outer membrane of mitochondria (OMM) and then interacts with VDAC1 on the cytosolic leaflet of OMM to form a binary complex. To systematically investigate this process, we devised a hybrid approach. First, we describe membrane binding of HKII with molecular dynamics (MD) simulations employing a membrane mimetic model with enhanced lipid diffusion capturing membrane insertion of its H-anchor. The insertion depth of the H-anchor was then used to derive positional restraints in subsequent millisecond-scale Brownian dynamics (BD) simulations to preserve the membrane-bound pose of HKII during the formation of the HKII/VDAC1 binary complex. Multiple BD-derived structural models for the complex were further refined and their structural stability probed with additional MD simulations, resulting in one stable complex. A major feature in the complex is the partial (not complete) blockade of VDAC1's permeation pathway, a result supported by our comparative electrophysiological measurements of the channel in the presence and absence of HKII. We also show how VDAC1 phosphorylation disrupts HKII binding, a feature that is verified by our electrophysiology recordings and has implications in mitochondria-mediated cell death.

3D Optical Cryo-Imaging Method: A Novel Approach to Quantify Renal Mitochondrial Bioenergetics Dysfunction.

Mitochondrial dysfunction contributes to various injuries and diseases. A mechanistic understanding of how dysfunctional mitochondria modulates metabolism is of paramount importance. Three-dimensional (3D) optical cryo-imager is a custom-designed device that can quantify the volumetric bioenergetics of organs in small animal models. The instrument captures the autofluorescence of bioenergetics indices (NADH and FAD) from tissues at cryogenic temperature. The quantified redox ratio (NADH/FAD) is used as an optical indicator of mitochondrial redox state.

LETM1: A Single Entity With Diverse Impact on Mitochondrial Metabolism and Cellular Signaling.

Nearly 2 decades since its discovery as one of the genes responsible for the Wolf-Hirschhorn Syndrome (WHS), the primary function of the leucine-zipper EF-hand containing transmembrane 1 (LETM1) protein in the inner mitochondrial membrane (IMM) or the mechanism by which it regulates mitochondrial Ca2+ handling is unresolved. Meanwhile, LETM1 has been associated with the regulation of fundamental cellular processes, such as development, cellular respiration and metabolism, and apoptosis. This mini-review summarizes the diversity of cellular functions impacted by LETM1 and highlights the multiple roles of LETM1 in health and disease.

Total Matrix Ca2+ Modulates Ca2+ Efflux via the Ca2+/H+ Exchanger in Cardiac Mitochondria.

Mitochondrial Ca2+ handling is accomplished by balancing Ca2+ uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca2+ buffering in the matrix and Ca2+ efflux mainly via Ca2+ ion exchangers, such as the Na+/Ca2+ exchanger (NCLX) and the Ca2+/H+ exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca2+ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca2+ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca2+ load via repetitive application of a finite CaCl2 bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca2+ efflux at different matrix Ca2+ loads was revealed by inhibiting Ca2+ uptake or reuptake with Ru360 after increasing number of CaCl2 boluses. In Na+-free experimental buffer and with Ca2+ uptake inhibited, the rate of Ca2+ efflux and steady-state free matrix Ca2+ [mCa2+]ss increased as the number of administered CaCl2 boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca2+ buffering while maintaining a constant [mCa2+]ss, decreased the rate of Ca2+ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca2+ buffering rate and decreased [mCa2+]ss. A change in the pH of the external medium to a more acidic value from 7.15 to 6.8∼6.9 caused a twofold increase in the Ca2+ efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca2+ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca2+ efflux. The results indicate that Ca2+ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca2+. A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca2+ buffering system and the matrix free Ca2+ concentration.

Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress.

BACKGROUND/AIMS: The role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mouse embryonic fibroblasts (MEFs), the roles of VDAC1 knockout (VDAC1-/-) in apoptotic cell death are contradictory. The contribution and underlying mechanism of VDAC1-/- in oxidative stress-induced cell death in cardiac cells has not been established. We hypothesized that VDAC1 is an essential regulator of oxidative stress-induced cell death in H9c2 cells. METHODS: We knocked out VDAC1 in this rat cardiomyoblast cell line with CRISPR-Cas9 genome editing technique to produce VDAC1-/- H9c2 cells, and determined if VDAC1 is critical in promoting cell death via oxidative stress induced by tert-butylhydroperoxide (tBHP), an organic peroxide, or rotenone (ROT), an inhibitor of mitochondrial complex I by measuring cell viability with MTT assay, cell death with TUNEL stain and LDH release. The mitochondrial and glycolytic stress were examined by measuring O2 consumption rate (OCR) and extracellular acidification rate (ECAR) with a Seahorse XFp analyzer. RESULTS: We found that under control conditions, VDAC1-/- did not affect H9c2 cell proliferation or mitochondrial respiration. However, compared to the wildtype (WT) cells, exposure to either tBHP or ROT enhanced the production of ROS, ECAR, and the proton (H+) production rate (PPR) from glycolysis, as well as promoted apoptotic cell death in VDAC1-/- H9c2 cells. VDAC1-/- H9c2 cells also exhibited markedly reduced mitochondria-bound hexokinase II (HKII) and Bax. Restoration of VDAC1 in VDAC1-/- H9c2 cells reinstated mitochondria-bound HKII and concomitantly decreased tBHP and ROT-induced ROS production and cell death. Interestingly, mitochondrial respiration remained the same after tBHP treatment in VDAC1-/- and WT H9c2 cells. CONCLUSION: Our results suggest that VDAC1-/- in H9c2 cells enhances oxidative stress-mediated cell apoptosis that is directly linked to the reduction of mitochondria-bound HKII and concomitantly associated with enhanced ROS production, ECAR, and PPR.

Modulation of peroxynitrite produced via mitochondrial nitric oxide synthesis during Ca2+ and succinate-induced oxidative stress in cardiac isolated mitochondria.

We hypothesized that NO• is generated in isolated cardiac mitochondria as the source for ONOO- production during oxidative stress. We monitored generation of ONOO- from guinea pig isolated cardiac mitochondria subjected to excess Ca2+ uptake before adding succinate and determined if ONOO- production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl2 and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O2•-. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO- to form dityrosine (diTyr) in proportion to the ONOO- present. We found that exposing mitochondria to excess CaCl2 before succinate resulted in an increase in diTyr and amplex red fluorescence (H2O2) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa2+ and succinate, increased mitochondrial ONOO- production via NO• and O2•-. Changes in mitochondrial ONOO- production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO• scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO- production, PTIO decreased production of ONOO-, and TEMPOL decreased ONOO- levels by converting more O2•- to H2O2. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in ß-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO- into the buffer is dependent both on O2•- released from mitochondria and NO• derived from a mtCa2+-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.

Mitochondria in Health and Diseases.

Mitochondria are subcellular organelles evolved by endosymbiosis of bacteria with eukaryotic cells characteristics. They are the main source of ATP in the cell and play a pivotal role in cell life and cell death. Mitochondria are engaged in the pathogenesis of human diseases and aging directly or indirectly through a broad range of signaling pathways. However, despite an increased interest in mitochondria over the past decades, the mechanisms of mitochondria-mediated cell/organ dysfunction in response to pathological stimuli remain unknown. The Special Issue, "Mitochondria in Health and Diseases," organized by Cells includes 24 review and original articles that highlight the latest achievements in elucidating the role of mitochondria under physiological (healthy) conditions and, in various cell/animal models of human diseases and, in patients. Altogether, the Special Issue summarizes and discusses different aspects of mitochondrial metabolism and function that open new avenues in understanding mitochondrial biology.

Differences in Expression of Mitochondrial Complexes Due to Genetic Variants May Alter Sensitivity to Radiation-Induced Cardiac Dysfunction.

Radiation therapy is received by over half of all cancer patients. However, radiation doses may be constricted due to normal tissue side effects. In thoracic cancers, including breast and lung cancers, cardiac radiation is a major concern in treatment planning. There are currently no biomarkers of radiation-induced cardiotoxicity. Complex genetic modifiers can contribute to the risk of radiation-induced cardiotoxicities, yet these modifiers are largely unknown and poorly understood. We have previously reported the SS (Dahl salt-sensitive/Mcwi) rat strain is a highly sensitized model of radiation-induced cardiotoxicity compared to the more resistant Brown Norway (BN) rat strain. When rat chromosome 3 from the resistant BN rat strain is substituted into the SS background (SS.BN3 consomic), it significantly attenuates radiation-induced cardiotoxicity, demonstrating inherited genetic variants on rat chromosome 3 modify radiation sensitivity. Genes involved with mitochondrial function were differentially expressed in the hearts of SS and SS.BN3 rats 1 week after radiation. Here we further assessed differences in mitochondria-related genes between the sensitive SS and resistant SS.BN3 rats. We found mitochondrial-related gene expression differed in untreated hearts, while no differences in mitochondrial morphology were seen 1 week after localized heart radiation. At 12 weeks after localized cardiac radiation, differences in mitochondrial complex protein expression in the left ventricles were seen between the SS and SS.BN3 rats. These studies suggest that differences in mitochondrial gene expression caused by inherited genetic variants may contribute to differences in sensitivity to cardiac radiation.

PPARγ-Independent Side Effects of Thiazolidinediones on Mitochondrial Redox State in Rat Isolated Hearts.

The effect of anti-diabetic thiazolidinediones (TZDs) on contributing to heart failure and cardiac ischemia/reperfusion (IR) injury is controversial. In this study we investigated the effect of select TZDs on myocardial and mitochondrial function in Brown Norway rat isolated hearts. In a first set of experiments, the TZD rosiglitazone was given acutely before global myocardial IR, and pre- and post-IR function and infarct size were assessed. In a second set of experiments, different concentrations of rosiglitazone and pioglitazone were administered in the presence or absence of the specific PPARγ antagonist GW9662, and their effects on the mitochondrial redox state were measured by online NADH and FAD autofluorescence. The administration of rosiglitazone did not significantly affect myocardial function except for transiently increasing coronary flow, but it increased IR injury compared to the control hearts. Both TZDs resulted in dose-dependent, reversible increases in mitochondrial oxidation which was not attenuated by GW9662. Taken together, these data suggest that TZDs cause excessive mitochondrial uncoupling by a PPARγ-independent mechanism. Acute rosiglitazone administration before IR was associated with enhanced cardiac injury. If translated clinically, susceptible patients on PPARγ agonists may experience enhanced myocardial IR injury by mitochondrial dysfunction.

NOS cofactor tetrahydrobiopterin contributes to anesthetic preconditioning induced myocardial protection in the isolated ex vivo rat heart.

Anesthetic preconditioning (APC) may decrease the myocardium injury nearly 50% following ischemia/reperfusion (I/R) by enhancing recovery of cardiac function, reducing myocardial enzyme release and lowering infarct size when utilized as pretreatment or posttreatment agents. I/R increases nitric oxide (NO) production through endothelial NO synthase (NOS3) and heat shock protein 90 (HSP90). The present study aimed to observe the role of BH4 availability and the association of HSP90 with NOS3 in APC­mediated cardioprotection against I/R injury. Isolated rat hearts were subjected to no­flow ischemia for 30 min and reperfusion for 120 min. Sevoflurane (3.5%) was administered for 15 min followed by a 15 min washout prior to ischemia. 2,4-Diamino-6-hydroxypyrimidine (DAHP) or sepiapterin (SP) was administered for 40 min until the onset of ischemia. The results revealed that compared with pre­ischemic basal levels, BH4 levels decreased and BH2 levels increased following I/R. BH4 levels were significantly increased and BH2 levels were significantly decreased in the APC + I/R hearts compared with the I/R group hearts. The BH4:BH2 ratio in the APC­treated hearts was also increased compared with that in the I/R group hearts. SP increased the recovery of contractile function and the production of NO, and decreased the production of superoxide anion (O2·­) in I/R heart, but did not elicit these effects in APC­treated hearts. DAHP treatment inhibited the APC­mediated recovery of contractile function, increased O2·­ levels and decreased NO production, but had no effect in I/R hearts. The cardioprotection of APC was demonstrated to be modulated by the BH4 precursor SP, which increased BH4 levels, or DAHP, which inhibited GTP cyclohydrolase I. Both APC and SP treatments increased the combination of HSP90 and NOS3, which improved the NOS3 activity and function. The results suggested that BH4, which servesas a cofactor for NOS, mediated the resistance of APC to I/R injury by promoting the binding of HSP90 and NOS3.

miRNA551b-3p Activates an Oncostatin Signaling Module for the Progression of Triple-Negative Breast Cancer.

Genomic amplification of 3q26.2 locus leads to the increased expression of microRNA 551b-3p (miR551b-3p) in triple-negative breast cancer (TNBC). Our results demonstrate that miR551b-3p translocates to the nucleus with the aid of importin-8 (IPO8) and activates STAT3 transcription. As a consequence, miR551b upregulates the expression of oncostatin M receptor (OSMR) and interleukin-31 receptor-α (IL-31RA) as well as their ligands OSM and IL-31 through STAT3 transcription. We defined this set of genes induced by miR551b-3p as the "oncostatin signaling module," which provides oncogenic addictions in cancer cells. Notably, OSM is highly expressed in TNBC, and the elevated expression of OSM associates with poor outcome in estrogen-receptor-negative breast cancer patients. Conversely, targeting miR551b with anti-miR551b-3p reduced the expression of the OSM signaling module and reduced tumor growth, as well as migration and invasion of breast cancer cells.