Anneke Koeman

Disruption of Hexokinase II–Mitochondrial Binding Blocks Ischemic Preconditioning and Causes Rapid Cardiac Necrosis

Rationale: Isoforms I and II of the glycolytic enzyme hexokinase (HKI and HKII) are known to associate with mitochondria. It is unknown whether mitochondria-bound hexokinase is mandatory for ischemic preconditioning and normal functioning of the intact, beating heart. Objective: We hypothesized that reducing mitochondrial hexokinase would abrogate ischemic preconditioning and disrupt myocardial function. Methods and Results: Ex vivo perfused HKII+/− hearts exhibited increased cell death after ischemia and reperfusion injury compared with wild-type hearts; however, ischemic preconditioning was unaffected. To investigate acute reductions in mitochondrial HKII levels, wild-type hearts were treated with a TAT control peptide or a TAT-HK peptide that contained the binding motif of HKII to mitochondria, thereby disrupting the mitochondrial HKII association. Mitochondrial hexokinase was determined by HKI and HKII immunogold labeling and electron microscopy analysis. Low-dose (200 nmol/L) TAT-HK treatment significantly decreased mitochondrial HKII levels without affecting baseline cardiac function but dramatically increased ischemia-reperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 &mgr;mol/L) TAT-HK resulted in acute mitochondrial depolarization, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were mimicked by mitochondrial membrane depolarization after mild mitochondrial uncoupling that did not cause direct mitochondrial permeability transition opening. Conclusions: Acute low-dose dissociation of HKII from mitochondria in heart prevented ischemic preconditioning, whereas high-dose HKII dissociation caused cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarization mechanism. The results suggest that the association of HKII with mitochondria is essential for the protective effects of ischemic preconditioning and normal cardiac function through maintenance of mitochondrial potential.

Deletion of the innate immune NLRP3 receptor abolishes cardiac ischemic preconditioning and is associated with decreased Il-6/STAT3 signaling

Objective Recent studies indicate that the innate immune system is not only triggered by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As triggers for the innate immune NLRP3 inflammasome protein complex appear to overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NLRP3 inflammasome is involved in IPC and acute I/R injury of the heart. Principal Findings Baseline cardiac performance and acute I/R injury were investigated in isolated, Langendorff-perfused hearts from wild-type (WT), ASC−/− and NLRP3−/− mice. Deletion of NLRP3 inflammasome components ASC−/− or NLRP3−/− did not affect baseline performance. The deletions exacerbated I/R-induced mechanical dysfunction, but were without effect on I/R-induced cell death. When subjected to IPC, WT and ASC−/− hearts were protected against I/R injury (improved function and less cell death). However, IPC did not protect NLRP3−/− hearts against I/R injury. NLRP3−/− hearts had significantly decreased cardiac IL-6 levels with a trend towards lower IL-1β levels at end reperfusion, suggesting abrogation of IPC through diminished IL-6 and/or IL-1β signaling. Subsequent experiments showed that neutralising IL-6 using an antibody against IL-6 abrogated IPC in WT hearts. However, inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) did not abrogate IPC in WT hearts. Analysis of survival kinases after IPC demonstrated decreased STAT3 expression in NLRP3−/− hearts when compared to WT hearts. Conclusions The data suggest that the innate immune NLRP3 protein, in an NLRP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6/STAT3 dependent mechanism.

Optimizing anesthetic regimen for surgery in mice through minimization of hemodynamic, metabolic, and inflammatory perturbations:

The role of anesthetics in animal research models is crucial, yet often ignored, and is almost never the primary focus of examination. Here, we investigated the impact of anesthetic regimens on different parameters of hemodynamics (blood pressure (BP) and heart rate (HR)), metabolism (glucose, insulin, and free fatty acids (FFA)), and inflammation (IL-6 and TNF-α) in two frequently used mouse strains (C57BL/6 and FVB). All animals were at a similar surgical plane of anesthesia, mechanically ventilated, and monitored for 60u2009min. The following anesthetic regimens were studied: (1) fentanyl–ketamine–midazolam (FKM), (2) fentanyl–midazolam–haldol (FMH), (3) pentobarbital (P), (4) fentanyl–fluanisone–midazolam (FFM), (5) fentanyl–midazolam–acepromazine (FMA), (6) ketamine–medetomidine–atropine (KMA), (7) isoflurane (ISO), and (8) propofol–fentanyl–midazolam (PFM). Metabolic and inflammatory parameters were compared with those obtained from non-anesthetized animals. Hemodynamics: BP >80u2009mmu2009Hg were only obtained with KMA, whereas hypotension (BP <60u2009mmu2009Hg) was observed with FKM and P. HR >500 beats/min was observed with ISO and PFM, whereas HR <400 beats/min was induced with KMA, FMH (BL/6), P (BL/6), and FKM (FVB). Metabolism: Glucose and insulin were most disturbed by KMA and ISO and mildly disturbed by FMA, whereas FFM, PFM, and P did not have any effect. FFA increased largely by FMA, with ISO and FKM having no effects. Inflammation: Cytokines were increased least with ISO/FFM/FMA, whereas FKM and KMA induced the largest increases in cytokines. When aiming at achieving surgical anesthesia without large disturbances in hemodynamic, metabolic, and inflammatory profiles, FFM, ISO, or PFM may be the most neutral anesthetic regimens in mice.

Class effects of SGLT2 inhibitors in mouse cardiomyocytes and hearts: inhibition of Na+/H+ exchanger, lowering of cytosolic Na+ and vasodilation

Aims/hypothesisSodium–glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i) constitute a novel class of glucose-lowering (type 2) kidney-targeted agents. We recently reported that the SGLT2i empagliflozin (EMPA) reduced cardiac cytosolic Na+ ([Na+]c) and cytosolic Ca2+ ([Ca2+]c) concentrations through inhibition of Na+/H+ exchanger (NHE). Here, we examine (1) whether the SGLT2i dapagliflozin (DAPA) and canagliflozin (CANA) also inhibit NHE and reduce [Na+]c; (2) a structural model for the interaction of SGLT2i to NHE; (3) to what extent SGLT2i affect the haemodynamic and metabolic performance of isolated hearts of healthy mice.MethodsCardiac NHE activity and [Na+]c in mouse cardiomyocytes were measured in the presence of clinically relevant concentrations of EMPA (1xa0μmol/l), DAPA (1xa0μmol/l), CANA (3xa0μmol/l) or vehicle. NHE docking simulation studies were applied to explore potential binding sites for SGTL2i. Constant-flow Langendorff-perfused mouse hearts were subjected to SGLT2i for 30xa0min, and cardiovascular function, O2 consumption and energetics (phosphocreatine (PCr)/ATP) were determined.ResultsEMPA, DAPA and CANA inhibited NHE activity (measured through low pH recovery after NH4+ pulse: EMPA 6.69u2009±u20090.09, DAPA 6.77u2009±u20090.12 and CANA 6.80u2009±u20090.18 vs vehicle 7.09u2009±u20090.09; pu2009<u20090.001 for all three comparisons) and reduced [Na+]c (in mmol/l: EMPA 10.0u2009±u20090.5, DAPA 10.7u2009±u20090.7 and CANA 11.0u2009±u20090.9 vs vehicle 12.7u2009±u20090.7; pu2009<u20090.001). Docking studies provided high binding affinity of all three SGLT2i with the extracellular Na+-binding site of NHE. EMPA and CANA, but not DAPA, induced coronary vasodilation of the intact heart. PCr/ATP remained unaffected.Conclusions/interpretationEMPA, DAPA and CANA directly inhibit cardiac NHE flux and reduce [Na+]c, possibly by binding with the Na+-binding site of NHE-1. Furthermore, EMPA and CANA affect the healthy heart by inducing vasodilation. The [Na+]c-lowering class effect of SGLT2i is a potential approach to combat elevated [Na+]c that is known to occur in heart failure and diabetes.

Pathophysiological Consequences of TAT-HKII Peptide Administration Are Independent of Impaired Vascular Function and Ensuing Ischemia

Rationale: We have shown that partial dissociation of hexokinase II (HKII) from mitochondria in the intact heart using low-dose transactivating transcriptional factor (TAT)-HKII (200 nmol/L) prevents the cardioprotective effects of ischemic preconditioning, whereas high-dose TAT-HKII (10 &mgr;mol/L) administration results in rapid myocardial dysfunction, mitochondrial depolarization, and disintegration. In this issue of Circulation Research, Pasdois et al argue that the deleterious effects of TAT-HKII administration on cardiac function are likely because of vasoconstriction and ensuing ischemia. Objective: To investigate whether altered vascular function and ensuing ischemia recapitulate the deleterious effects of TAT-HKII in intact myocardium. Methods and Results: Using a variety of complementary techniques, including mitochondrial membrane potential (&Dgr;&psgr;m) imaging, high-resolution optical action potential mapping, analysis of lactate production, nicotinamide adenine dinucleotide epifluorescence, lactate dehydrogenase release, and electron microscopy, we provide direct evidence that refutes the notion that acute myocardial dysfunction by high-dose TAT-HKII peptide administration is a consequence of impaired vascular function. Moreover, we demonstrate that low-dose TAT-HKII treatment, which abrogates the protective effects of ischemic preconditioning, is not associated with ischemia or ischemic injury. Conclusions: Our findings challenge the notion that the effects of TAT-HKII are attributable to impaired vascular function and ensuing ischemia, thereby lending further credence to the role of mitochondria-bound HKII as a critical regulator of cardiac function, ischemia-reperfusion injury, and cardioprotection by ischemic preconditioning.

The effect of standard chow and reduced hexokinase II on growth, cardiac and skeletal muscle hexokinase and low-flow cardiac ischaemia–reperfusion injury

In the present study, we examined whether standard chow (SDS versus Purina 5001; both low fat, high carbohydrate) and reductions in hexokinase (HK) II (wild-type versus HKII+/− mice) affect (1) growth parameters, (2) HK levels in cardiac and skeletal muscle and (3) low-flow cardiac ischaemia–reperfusion (IR) injury. Total HK activity and HKI and HKII expressions were determined, and low-flow IR injury was examined in isolated hearts subjected to 40 min 5% low-flow ischaemia and 120 min reperfusion. Standard chow, but not HKII reductions, significantly affected body weight, heart weight and cardiac hypertrophy. Both standard chow and reduced HKII diminished total cardiac and skeletal muscle HK activity. For the heart, the Purina chow-induced decrease in total HK activity was through decreases in HKI expression, whereas for skeletal muscle post-translational mechanisms are suggested. Both standard chow and reduced HKII demonstrated a non-significant trend for affecting cardiac IR damage. However, the low-flow ischaemia model was associated with mild sublethal injury only (∼1% cell death). In conclusion, standard chow affects body weight, heart weight and HK activity and HKI expression in the heart, without altering HKII expression. This implicates standard chow as an important factor in genomic, physiological research models and demonstrates that large differences in fat or carbohydrates in the diet are not necessary to affect growth. In a cardiac low-flow IR model, resulting in only mild injury, standard chow or reduced HKII does not significantly affect IR damage.

In vivo mouse myocardial (31)P MRS using three-dimensional image-selected in vivo spectroscopy (3D ISIS): technical considerations and biochemical validations.

31P MRS provides a unique non‐invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of 31P MRS in the in vivo mouse heart has been limited. The small‐sized, fast‐beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three‐dimensional image‐selected in vivo spectroscopy (3D ISIS) for localized 31P MRS of the in vivo mouse heart at 9.4u2009T. Cardiac 31P MR spectra were acquired in vivo in healthy mice (n = 9) and in transverse aortic constricted (TAC) mice (n = 8) using respiratory‐gated, cardiac‐triggered 3D ISIS. Localization and potential signal contamination were assessed with 31P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5′‐triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one‐dimensional (1D) ISIS resulted in two‐fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3u2009mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo 31P MRS were within the normal physiological range. Our results show that respiratory‐gated, cardiac‐triggered 3D ISIS allows for non‐invasive assessments of in vivo mouse myocardial energy homeostasis with 31P MRS under physiological conditions. Copyright

Letter to the editor: Ketamine-only versus isoflurane effects on murine cardiac function: comparison at similar depths of anesthesia?

to the editor: In a recent issue of the American Journal of Physiology-Heart and Circulatory Physiology , we have read with much interest the article by Pachon and coworkers ([4][1]), where the authors report that a high dose of ketamine alone demonstrated the least deviations from the conscious

In vivomouse myocardial31P MRS using three-dimensional image-selectedin vivospectroscopy (3D ISIS): technical considerations and biochemical validations: IN VIVOMOUSE MYOCARDIAL31P MRS

31P MRS provides a unique non‐invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of 31P MRS in the in vivo mouse heart has been limited. The small‐sized, fast‐beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three‐dimensional image‐selected in vivo spectroscopy (3D ISIS) for localized 31P MRS of the in vivo mouse heart at 9.4u2009T. Cardiac 31P MR spectra were acquired in vivo in healthy mice (n = 9) and in transverse aortic constricted (TAC) mice (n = 8) using respiratory‐gated, cardiac‐triggered 3D ISIS. Localization and potential signal contamination were assessed with 31P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5′‐triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one‐dimensional (1D) ISIS resulted in two‐fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3u2009mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo 31P MRS were within the normal physiological range. Our results show that respiratory‐gated, cardiac‐triggered 3D ISIS allows for non‐invasive assessments of in vivo mouse myocardial energy homeostasis with 31P MRS under physiological conditions. Copyright

07 Mitochondrial hexokinase II is essential for cardiac function and ischaemic preconditioning

Rationale Isoforms I and II of the glycolytic enzyme hexokinase (HK) are known to associate with mitochondria. It is unknown whether mitochondrially bound hexokinase (mitoHK) is mandatory for ischaemic preconditioning and normal functioning of the intact, beating heart. Objective We hypothesise that reducing mitoHK abrogates ischaemic preconditioning and disrupts myocardial function. Methods and Results Ex vivo perfused HKII± hearts exhibited increased cell death following ischaemia (I) and reperfusion (R) injury as compared to WT hearts. However, IPC was unaffected. To investigate acute reductions in mitoHKII levels, WT hearts were treated with a TAT-control peptide or a TAT-HK peptide containing the binding motif of HKII to mitochondria, thereby disrupting mitoHKII association. MitoHK was determined by HKI and HKII immunogold labelling and EM analysis. Low-dose (200u2005nmol/l) TAT-HK treatment significantly decreased mitoHKII levels without affecting baseline cardiac function, but dramatically increased IR injury and prevented IPC protective effects. Treatment for 15u2005min with high-dose (10u2005μmol/l) TAT-HK resulted in acute mitochondrial depolarisation, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were re-capitulated by mitochondrial membrane depolarisation following mild mitochondrial uncoupling that does not directly cause mitochondrial permeability transition opening. Conclusion Acute low-dose dissociation of HKII from mitochondria in heart prevents IPC whereas high-dose HKII dissociation causes cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarisation mechanism. The results suggest that the association of HKII with mitochondria is essential for IPC protective effects and normal cardiac function through maintenance of mitochondrial potential.