Isolated Mouse Hearts Research Articles

Lipopolysaccharides Improve Mesenchymal Stem Cell-Mediated Cardioprotection by MyD88 and stat3 Signaling in a Mouse Model of Cardiac Ischemia/Reperfusion Injury.

Bone marrow-derived mesenchymal stem cells (MSCs) improve cardiac function after ischemia/reperfusion injury, in part, due to the release of cytoprotective paracrine factors. Toll-like receptor 4 (TLR4) is expressed in MSCs and regulates the expression of cytoprotective factors, cytokines, and chemokines. Lipopolysaccharide (LPS) stimulation of TLR4 activates two distinct signaling pathways that are either MyD88 dependent or MyD88 independent/TIR-domain-containing adapter-inducing interferon-β (TRIF) dependent. While it was reported previously that LPS treatment improved MSC-mediated cardioprotection, the mechanism underlying such improved effect remains unknown. To study the role of MyD88 signaling in MSC cardioprotective activity, wild type (WT) and MyD88-/- MSCs were treated with LPS (200 ng/mL) for 24 h. WT and MyD88-/- MSCs with or without LPS pretreatment were infused into the coronary circulation of isolated mouse hearts (Langendorff model) and then subjected to ischemia (25 min) and reperfusion (50 min). Saline served as a negative control. Both untreated and LPS-pretreated WT MSCs significantly improved postischemic recovery of myocardial function of isolated mouse hearts, as evidenced by improved left ventricular developed pressure and ventricular contractility assessment (ie, the rate of left ventricle pressure change over time, ± dp/dt). LPS-pretreated WT MSCs conferred better cardiac function recovery than untreated MSCs; however, such effect of LPS was abolished when using MyD88-/- MSCs. In addition, LPS stimulated stat3 activity in WT MSCs, but not MyD88-/- MSCs. stat3 small interfering RNA abolished the effect of LPS in improving the cardioprotection of WT MSCs. In conclusion, this study demonstrates that LPS improves MSC-mediated cardioprotection by MyD88-dependent activation of stat3.

Inhibition of CD38 with the Thiazoloquin(az)olin(on)e 78c Protects the Heart against Postischemic Injury.

Inhibition of and genetic deletion of the NAD(P)+ hydrolase [NAD(P)ase] CD38 have been shown to protect against ischemia/reperfusion (I/R) injury in rat and mouse hearts. CD38 has been shown to enhance salvage of NADP(H), which in turn prevents impairment of endothelial nitric oxide synthase function, a hallmark of endothelial dysfunction. Despite growing evidence for a role of CD38 in postischemic injury, until recently there had been a lack of potent CD38 inhibitors. Recently, a new class of thiazoloquin(az)olin(on)e compounds were identified as highly potent and specific CD38 inhibitors. Herein, we investigate the ability of one of these compounds, 78c, to inhibit CD38 and protect the heart in an ex vivo model of myocardial I/R injury. The potency and mechanism of CD38 inhibition by 78c was assessed in vitro using recombinant CD38. The dose-dependent tissue uptake of 78c in isolated mouse hearts was determined, and high tissue permeability of 78c was observed when delivered in perfusate. Treatment of hearts with 78c was protective against both postischemic endothelial and cardiac myocyte injury, with preserved nitric oxide synthase-dependent vasodilatory and contractile function, respectively. Myocardial infarction was also significantly decreased in 78c-treated hearts, with preserved levels of high-energy phosphates. Protective effects peaked at 10 μM treatment, and similar protection without toxicity was seen at 5-fold higher doses. Overall, 78c was shown to be a potent and biologically active CD38 inhibitor with favorable tissue uptake and marked protective effects against I/R injury with enhanced preservation of contractile function, coronary flow, and decreased infarction.

Direct Cardiac Actions of Sodium Glucose Cotransporter 2 Inhibitors Target Pathogenic Mechanisms Underlying Heart Failure in Diabetic Patients.

Sodium glucose cotransporter 2 inhibitors (SGLT2i) are the first antidiabetic compounds that effectively reduce heart failure hospitalization and cardiovascular death in type 2 diabetics. Being explicitly designed to inhibit SGLT2 in the kidney, SGLT2i have lately been investigated for their off-target cardiac actions. Here, we review the direct effects of SGLT2i Empagliflozin (Empa), Dapagliflozin (Dapa), and Canagliflozin (Cana) on various cardiac cell types and cardiac function, and how these may contribute to the cardiovascular benefits observed in large clinical trials. SGLT2i impaired the Na+/H+ exchanger 1 (NHE-1), reduced cytosolic [Ca2+] and [Na+] and increased mitochondrial [Ca2+] in healthy cardiomyocytes. Empa, one of the best studied SGLT2i, maintained cell viability and ATP content following hypoxia/reoxygenation in cardiomyocytes and endothelial cells. SGLT2i recovered vasoreactivity of hyperglycemic and TNF-α-stimulated aortic rings and of hyperglycemic endothelial cells. Anti-inflammatory actions of Cana in IL-1β-treated HUVEC and of Dapa in LPS-treated cardiofibroblast were mediated by AMPK activation. In isolated mouse hearts, Empa and Cana, but not Dapa, induced vasodilation. In ischemia-reperfusion studies of the isolated heart, Empa delayed contracture development during ischemia and increased mitochondrial respiration post-ischemia. Direct cardiac effects of SGLT2i target well-known drivers of diabetes and heart failure (elevated cardiac cytosolic [Ca2+] and [Na+], activated NHE-1, elevated inflammation, impaired vasorelaxation, and reduced AMPK activity). These cardiac effects may contribute to the large beneficial clinical effects of these antidiabetic drugs.

Cardioprotective effects of CYP-derived epoxy metabolites of docosahexaenoic acid involve limiting NLRP3 inflammasome activation 1.

Impaired mitochondrial function and activation of NLRP3 inflammasome cascade has a significant role in the pathogenesis of myocardial ischemia-reperfusion (IR) injury. The current study investigated whether eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or their corresponding CYP epoxygenase metabolites 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) protect against IR injury. Isolated mouse hearts were perfused in the Langendorff mode with vehicle, DHA, 19,20-EDP, EPA, or 17,18-EEQ and subjected to 30 min of ischemia and followed by 40 min of reperfusion. In contrast with EPA and 17,18-EEQ, DHA and 19,20-EDP exerted cardioprotection, as shown by a significant improvement in postischemic functional recovery associated with significant attenuation of NLRP3 inflammasome complex activation and preserved mitochondrial function. Hearts perfused with DHA or 19,20-EDP displayed a marked reduction in localization of mitochondrial Drp-1 and Mfn-2 as well as maintained Opa-1 levels. DHA and 19,20-EDP preserved the activities of both the cytosolic Trx-1 and mitochondrial Trx-2. DHA cardioprotective effect was attenuated by the CYP epoxygenase inhibitor N-(methysulfonyl)-2-(2-propynyloxy)-benzenehexanamide. In conclusion, our data indicate a differential cardioprotective response between DHA, EPA, and their active metabolites toward IR injury. Interestingly, 19,20-EDP provided the best protection against IR injury via maintaining mitochondrial function and thereby reducing the detrimental NLRP3 inflammasome responses.

Ultrafast Volumetric Optoacoustic Imaging of Whole Isolated Beating Mouse Heart

The Langendorff-perfused heart technique has become the model of choice for multiparametric optical mapping of cardiac function and electrophysiology. However, photon scattering in tissues represents a significant drawback of the optical imaging approach, fundamentally limiting its mapping capacity to the heart surface. This work presents the first implementation of the optoacoustic approach for 4D imaging of the entire beating isolated mouse heart. The method combines optical excitation and acoustic detection to simultaneously render rich optical contrast and high spatio-temporal resolution at centimeter-scale depths. We demonstrate volumetric imaging of deeply located cardiac features, including the interventricular septum, chordae tendineae, and papillary muscles while further tracking the heart beat cycle and the motion of the pulmonary, mitral, and tricuspid valves in real time. The technique possesses a powerful combination between high imaging depth, fast volumetric imaging speed, functional and molecular imaging capacities not available with other imaging modalities currently used in cardiac research.

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis-in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before-and provide some representative results.

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Abstract 546: GJA1-20k Protects the Heart From Ischemic Injury by Inducing Mitochondrial Biogenesis and Metabolic Quiescence

Connexin 43 (Cx43) is a cell-cell communication gap junction protein that is encoded by the GJA1 gene and is expressed in all organ systems. It was recently found that the GJA1 mRNA undergoes alternative translation to generate N-terminally truncated isoforms, of which GJA1-20k is the most abundant. We have recently identified that GJA1-20k has a strong tropism for mitochondria and that GJA1-20k overexpression is sufficient to rescue mitochondrial localization to the cell periphery, limiting organelle network collapse upon exposure to oxidative stress. Given the protective role of GJA1-20k on mitochondrial network and the importance of preserving mitochondrial integrity in preventing cardiac ischemic injury, we therefore asked the question whether exogenous GJA1-20k can increase myocardial survival when subjected to stress. Using high resolution confocal imaging, we find that H 2 O 2 -induced oxidative stress in cardiomyocytes causes an upregulation of GJA1-20k independent of full length Cx43 expression. Furthermore, we find that GJA1-20k is upregulated and preferentially enriched in mitochondria after acute ischemia/reperfusion injury in isolated mouse hearts. We also find that exogenous GJA1-20k, when delivered by adenovirus to isolated adult mouse cardiomyocytes, was sufficient to improve cell survival and viability during H 2 O 2 -induced oxidative stress. When delivered in vivo using an Adeno-associated virus serotype 9 (AAV9), GJA1-20k exhibited potent cardioprotective effects by reducing ischemic injury size in a mouse model of myocardial infarction. AAV9-mediated gene transfer of GJA1-20k also resulted in increased cardiac mitochondrial content as observed by electron microscopy. Yet 20k-increased mitochondrial content in cardiomyocytes is associated with reduced mitochondrial membrane potential as well as respiration as measured by Seahorse assay, indicating a quiescent metabolic state resistant to stress. In conclusion, we have identified GJA1-20k as a stress response protein that increases mitochondrial biogenesis and reduces metabolic reactivity, providing powerful cardioprotection against ischemic insults.

Empagliflozin effects on ischemic contracture and I/R injury in isolated mouse hearts perfused with or without insulin

Background: Activation of sodium hydrogen exchanger (NHE) contributes to cardiac ischemia reperfusion (I/R) injury. We recently reported NHE inhibition properties of the diabetic drug SGLT2 inhibitor empagliflozin (EMPA). Classic NHE inhibitors delay ischemic contracture and reduce I/R injury in the heart, dependent on the degree of ischemic glycogen depletion. We examined in normal-/low-glycogen hearts whether EMPA delays ischemic contracture and reduces I/R injury.

Chances and limitations of isolated mouse heart models for investigating the endothelial glycocalyx1.

The endothelial glycocalyx plays a decisive role in maintaining vascular homeostasis. Previous animal models have mainly focused on in-vitro experiments or the isolated beating guinea pig heart. To further evaluate underlying mechanisms of up- and down regulation, knock-out animals seem to be a promising option. Aim of the present study was to evaluate if an isolated mouse-heart model is suitable for glycocalyx research. Isolated beating mouse hearts (C57/Bl6J) underwent warm, no-flow ischemia and successive reperfusion. Coronary effluent was analyzed by ELISA and Western blot for the glycocalyx core protein: syndecan-1. Hearts were prepared for either immunofluorescence or electron microscopy and lysed for Western blot analysis. An endothelial glycocalyx covering the total capillary circumference and syndecan-1 were detected by electron and immunofluorescence microscopy. Ischemia/reperfusion seriously deteriorated both findings. Confoundingly, syndecan-1 was not detectable either in the coronary effluent or in the lysates of blood-free hearts by ELISA or Western blot technique. Blood vessels of mouse hearts contain an endothelial glycocalyx comparable to that of other animals also with respect to its core protein syndecan-1. But, for studies including quantification of intravascular soluble glycocalyx constituents, the amount of syndecan-1 in mouse hearts seems to be too low.

CaMKII-dependent late Na+ current increases electrical dispersion and arrhythmia in ischemia-reperfusion.

The mechanisms underlying Ca2+/calmodulin-dependent protein kinase II (CaMKII)-induced arrhythmias in ischemia-reperfusion (I/R) are not fully understood. We tested the hypothesis that CaMKII increases late Na+ current ( INa,L) via phosphorylation of Nav1.5 at Ser571 during I/R, thereby increasing arrhythmia susceptibility. To test our hypothesis, we studied isolated, Langendorff-perfused hearts from wild-type (WT) mice and mice expressing Nav channel variants Nav1.5-Ser571E (S571E) and Nav1.5-Ser571A (S571A). WT hearts showed a significant increase in the levels of phosphorylated CaMKII and Nav1.5 at Ser571 [p-Nav1.5(S571)] after 15 min of global ischemia (just before the onset of reperfusion). Optical mapping experiments revealed an increase in action potential duration (APD) and APD dispersion without changes in conduction velocity during I/R in WT and S571E compared with S571A hearts. At the same time, WT and S571E hearts showed an increase in spontaneous arrhythmia events (e.g., premature ventricular contractions) and an increase in the inducibility of reentrant arrhythmias during reperfusion. Pretreatment of WT hearts with the Na+ channel blocker mexiletine (10 μM) normalized APD dispersion and reduced arrhythmia susceptibility during I/R. We conclude that CaMKII-dependent phosphorylation of Nav1.5 is a crucial driver for increased INa,L, arrhythmia triggers, and substrate during I/R. Selective targeting of this CaMKII-dependent pathway may have therapeutic potential for reducing arrhythmias in the setting of I/R. NEW & NOTEWORTHY Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of Nav1.5 at Ser571 leads to a prolongation of action potential duration (APD), increased APD dispersion, and increased arrhythmia susceptibility after ischemia-reperfusion in isolated mouse hearts. Genetic ablation of the CaMKII-dependent phosphorylation site Ser571 on Nav1.5 or low-dose mexiletine (to inhibit late Na+ current) reduced APD dispersion, arrhythmia triggers, and ventricular tachycardia inducibility.

Empagliflozin effects on ischemic contracture and I/R injury in isolated mouse hearts perfused with or without insulin

IntroductionThe kidney‐targeted Empagliflozin (EMPA) has shown large unexpected cardiovascular benefits, which cannot be explained by general cardiovascular risk factors. We recently reported that EMPA has sodium hydrogen exchanger (NHE) inhibition properties in isolated cardiomyocytes. Classic NHE inhibitors such as Cariporide (CARI) delay ischemic contracture onset and reduce ischemia reperfusion (I/R) injury in the heart. Here we examined whether EMPA delays contracture onset and reduces I/R injury. Both normal‐ and low‐glycogen hearts were used, because the rate of glycogen depletion during ischemia affects contracture onset and IR injury.Methods/ResultsIsolated mouse hearts were perfused with and without 50 mU/L insulin, to create normal‐ and low‐glycogen hearts respectively, and subjected to 25 min I and 120 min R. 1μM EMPA, 10μM cariporide (CARI; positive control) or vehicle was administered prior to ischemia until 10 minutes post‐reperfusion. In normal‐glycogen hearts, EMPA and CARI did not change ischemic contracture (in seconds, vehicle 1056±56, EMPA 900±44, CARI 880±84) and IR injury (infarct size (%): vehicle 59±6, Empa 62±10, Cari 68±4), however in low‐glycogen hearts, both EMPA and CARI delayed ischemic contracture onset (vehicle 459±25, EMPA 559±22, CARI 588±31). Higher EMPA concentration further delayed contracture onset. Only CARI reduced I/R injury (from 51±6 to 34±5). The presence of insulin (same concentration used) did not alter NHE activity, nor the ability of CARI or EMPA to inhibit NHE, in isolated cardiomyocytes. The increased IR injury in normal‐glycogen hearts was associated with increased end‐ischemic lactate and glucose‐6‐phosphate accumulation, and consequently decreased mitochondrial hexokinase ll.ConclusionEMPA does not protect against I/R injury in isolated hearts, yet EMPA delayed ischemic contracture onset in low‐glycogen hearts, suggesting a direct cardiac, ATP preserving, effect of EMPA during ischemia. Cardiac glycogen content interferes with the effects of NHE inhibitors on I/R injury in isolated hearts. These data indicate that empagliflozin and cariporide may inhibit NHE in different manners.Support or Funding InformationNone.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of Mitochondrial Calpain 1 Leads to Degradation of PDH

INTODUCTIONCalpain 1 (CPN1) is a ubiquitous calpain including one large subunit and a small regulatory subunit (calpain 4, CPN4). Genetic removal of CPN4 eliminates the CPN1 activity. CPN1 exists in both cytosol and mitochondria. Activation of CPN1 contributes to mitochondrial damage and cardiac injury during ischemia (ISC)‐reperfusion (REP). Pyruvate dehydrogenase (PDH) is degraded in mitochondria following ISC‐REP. Since mitochondrial CPN1 (mit‐CPN1) is found in the matrix, we propose that activation of mit‐CPN1 contributes to PDH degradation. Purified heart mitochondria were used to exclude potential cytosolic CPN1 contamination. CPN4 knockout mice were used to eliminate mit‐CPN1.HYPOTHESISWe hypothesize that activation of the mit‐CPN1 impairs oxidative phosphorylation (OXPHOS) through degradation of PDH in isolated mouse heart mitochondria.METHODSPurified cardiac mitochondria were isolated from wild‐type and CPN4 knockout mice. Exogenous calcium (25 uM) was used to activate mit‐CPN1. OXPHOS was determined in mitochondria with or without calcium treatment. Protein content of PDH E1 subunit was assessed by Western blotting.RESULTSIn wild type mice, calcium treatment significantly decreased the rate of OXPHOS using pyruvate + malate as complex I substrates compared to non‐calcium‐treated control. Succinate oxidation was also slightly decreased in calcium treated mitochondria from wild type hearts (Table). In contrast, calcium treatment did not affect OXPHOS in mitochondria from CPN4 knockout mice in both substrates. PDH E1 subunit content was decreased in calcium‐treated mitochondria from wild type mice but not from CPN4 knockout mice. A representative image is shown in Figure 1.CONCLUSIONKnockout of CPN4 prevents exogenous calcium‐mediated PDH degradation. Inhibition of CPN4 during calcium over load conditions including heart failure and endoplasmic reticulum stress may decrease cardiac injury by protecting mitochondria.Support or Funding InformationA Grant‐in‐aid (15GRNT24480123) from the American Heart Association (QC), R21AG054975‐01 (QC), and Merit Review Award (2IO1BX001355‐01, EL)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synchronization modes of the electrical stimulation-contractile response coupling in isolated mouse heart

The heart is an organ with a continuous activity whose heart rate and strength contractions are modulated by several physiological subsystems. These subsystems can be seen as weakly coupled oscillators. The electrical and mechanical oscillators are considered in this manuscript. 1:1 synchronization mode is a typical response of the healthy heart. However, although the heart rhythm as a system of weakly coupled oscillators was studied recently, little attention has been paid to the synchronization modes of the electrical stimulation-contractile response coupling in isolated mouse heart. This study aimed to describe with a minimalistic mathematical model the synchronization modes on the electrical stimulation-contractile response coupling in the whole heart. In this paper, we propose a minimal linear coupled oscillator model to study the synchronization modes. It contains four variables, and it is validated with experimental results. The local stability is studied. We predict through in-silico experiments the presence of several synchronization modes, and these could be associated with arrhythmias.

Cyclophilin D ablation is associated with increased end-ischemic mitochondrial hexokinase activity

Both the absence of cyclophilin D (CypD) and the presence of mitochondrial bound hexokinase II (mtHKII) protect the heart against ischemia/reperfusion (I/R) injury. It is unknown whether CypD determines the amount of mtHKII in the heart. We examined whether CypD affects mtHK in normoxic, ischemic and preconditioned isolated mouse hearts. Wild type (WT) and CypD−/− mouse hearts were perfused with glucose only and subjected to 25 min ischemia and reperfusion. At baseline, cytosolic and mtHK was similar between hearts. CypD ablation protected against I/R injury and increased ischemic preconditioning (IPC) effects, without affecting end-ischemic mtHK. When hearts were perfused with glucose, glutamine, pyruvate and lactate, the preparation was more stable and CypD ablation−resulted in more protection that was associated with increased mtHK activity, leaving little room for additional protection by IPC. In conclusion, in glucose only-perfused hearts, deletion of CypD is not associated with end-ischemic mitochondrial-HK binding. In contrast, in the physiologically more relevant multiple-substrate perfusion model, deletion of CypD is associated with an increased mtHK activity, possibly explaining the increased protection against I/R injury.

Divergent coronary flow responses to uridine adenosine tetraphosphate in atherosclerotic ApoE knockout mice

Uridine adenosine tetraphosphate (Up4A) exerts potent relaxation in porcine coronary arteries that is reduced following myocardial infarction, suggesting a crucial role for Up4A in the regulation of coronary flow (CF) in cardiovascular disorders. We evaluated the vasoactive effects of Up4A on CF in atherosclerosis using ApoE knockout (KO) mice ex vivo and in vivo. Functional studies were conducted in isolated mouse hearts using the Langendorff technique. Immunofluorescence was performed to assess purinergic P2X1 receptor (P2X1R) expression in isolated mouse coronary arteries. In vivo effects of Up4A on coronary blood flow (CBF) were assessed using ultrasound. Infusion of Up4A (10−9–10−5 M) into isolated mouse hearts resulted in a concentration-dependent reduction in CF in WT and ApoE KO mice to a similar extent; this effect was exacerbated in ApoE KO mice fed a high-fat diet (HFD). The P2X1R antagonist MRS2159 restored Up4A-mediated decreases in CF more so in ApoE KO + HFD than ApoE KO mice. The smooth muscle to endothelial cell ratio of coronary P2X1R expression was greater in ApoE KO + HFD than ApoE KO or WT mice, suggesting a net vasoconstrictor potential of P2X1R in ApoE KO + HFD mice. In contrast, Up4A (1.6 mg/kg) increased CBF to a similar extent among the three groups. In conclusion, Up4A decreases CF more in ApoE KO + HFD mice, likely through a net upregulation of vasoconstrictor P2X1R. In contrast, Up4A increases CBF in vivo regardless of the atherosclerotic model.

Ischemia/reperfusion injury study in isolated mouse hearts using a pressure-volume curve

Objective: To establish and assess the feasibility and sensitivity of left ventricular elasticity, compliance and stiffness for study of ischemia/reperfusion injury in an isolated mouse heart model utilizing the pressure-volume curve framework. Methods: An isolated, balloon-in-ventricle, isovolumically contracting, crystalloid-perfused Langendorff heart preparation was set up from 15 male C57/6BL mice aged 12-14 weeks. End-systolic pressure-volume relationship (ESPVR) and end-diastolic pressure-volume relationship (EDPVR) were obtained by measuring left ventricular (LV) systolic pressure (LVSP) and diastolic pressure under different balloon volumes. End-systolic elasticity (E(es)), end-diastolic stiffness (S(ed)) and compliance (C(ed)) were calculated from the slope of ESPVR and EDPVR. Measurements of LVSP, developed pressure (LVDP), end of diastolic pressure (EDP), the rate of pressure development dp/dt(max) and dp/dt(min) were monitored during 30 min stabilization, 20 min global ischemia and 40 min reperfusion. The reliability and coefficient of variation (CV) of these parameters were compared with E(es) and C(ed). Results: During stabilization, the heart rate was (395±40) bpm, LVDP was (126±25) mmHg, + dp/dt(max) was (5 590±625) mmHg/s, -dp/dt(min) was (-4 128±625) mmHg/s, E(es) was 5.7±0.3, C(ed) was 0.7±0.2, S(ed) was 1.4±0.1. Ischemia/reperfusion injury resulted in significant decrease in contractile function parameters. The recovery level of LVSP, LVDP, dp/dt(max) and dp/dt(min) were (57±19)%, (23±6)%, (23±7)% and (21±5)% (all P<0.001), respectively. The EDP increased to (5.4±2.0) times than the baseline after ischemia/reperfusion injury. The E(es) and C(ed) was deceased to (42±2)% and (33±2)%, compared with baseline. The stiffness was increased up to (3.1±0.2) times higher than the baseline. The CV of E(es,)S(ed) and C(ed) were lower than LVSP, LVDP, EDP, dp/dt(max,)dp/dt(min,)while the reliability of E(es), S(ed) and C(ed) were higher than the classic contractile function parameters. Conclusion: Pressure-volume curves are feasible during ischemia/reperfusion injury in the isolated mouse heart model with acceptable reliability and sensitivity.

Vascular endothelial overexpression of human CYP2J2 (Tie2-CYP2J2 Tr) modulates cardiac oxylipin profiles and enhances coronary reactive hyperemia in mice.

Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) by cytochrome (CYP) P450 epoxygenases, and to ω-terminal hydroxyeicosatetraenoic acids (HETEs) by ω-hydroxylases. EETs and HETEs often have opposite biologic effects; EETs are vasodilatory and protect against ischemia/reperfusion injury, while ω-terminal HETEs are vasoconstrictive and cause vascular dysfunction. Other oxylipins, such as epoxyoctadecaenoic acids (EpOMEs), hydroxyoctadecadienoic acids (HODEs), and prostanoids also have varied vascular effects. Post-ischemic vasodilation in the heart, known as coronary reactive hyperemia (CRH), protects against potential damage to the heart muscle caused by ischemia. The relationship among CRH response to ischemia, in mice with altered levels of CYP2J epoxygenases has not yet been investigated. Therefore, we evaluated the effect of endothelial overexpression of the human cytochrome P450 epoxygenase CYP2J2 in mice (Tie2-CYP2J2 Tr) on oxylipin profiles and CRH. Additionally, we evaluated the effect of pharmacologic inhibition of CYP-epoxygenases and inhibition of ω-hydroxylases on CRH. We hypothesized that CRH would be enhanced in isolated mouse hearts with vascular endothelial overexpression of human CYP2J2 through modulation of oxylipin profiles. Similarly, we expected that inhibition of CYP-epoxygenases would reduce CRH, whereas inhibition of ω-hydroxylases would enhance CRH. Compared to WT mice, Tie2-CYP2J2 Tr mice had enhanced CRH, including repayment volume, repayment duration, and repayment/debt ratio (P < 0.05). Similarly, inhibition of ω-hydroxylases increased repayment volume and repayment duration, in Tie2-CYP2J2 Tr compared to WT mice (P < 0.05). Endothelial overexpression of CYP2J2 significantly changed oxylipin profiles, including increased EETs (P < 0.05), increased EpOMEs (P < 0.05), and decreased 8-iso-PGF2α (P < 0.05). Inhibition of CYP epoxygenases with MS-PPOH attenuated CRH (P < 0.05). Ischemia caused a decrease in mid-chain HETEs (5-, 11-, 12-, 15-HETEs P < 0.05) and HODEs (P < 0.05). These data demonstrate that vascular endothelial overexpression of CYP2J2, through changing the oxylipin profiles, enhances CRH. Inhibition of CYP epoxygenases decreases CRH, whereas inhibition of ω-hydroxylases enhances CRH.

Crizotinib inhibits hyperpolarization-activated cyclic nucleotide-gated channel 4 activity

BackgroundSinus bradycardia is frequently observed in patients treated with crizotinib, a receptor tyrosine kinase inhibitor used for the treatment of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). We investigated whether crizotinib could influence heart rate (HR) through direct cardiac effects.MethodsThe direct effect of crizotinib on HR was studied using ECG analysis of Langendorff-perfused mouse hearts. The whole-cell patch clamp technique was used to measure the effects of crizotinib on the hyperpolarization-activated funny current, If, in mouse sinoatrial node cells (SANCs) and hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) activity in HEK-293 cells stably expressing human HCN4.ResultsCrizotinib resulted in a dose-dependent reduction in HR in isolated intact mouse hearts with a half maximal inhibitory concentration (IC50) of 1.7 ± 0.4 μmol/L. Because ECG analysis revealed that crizotinib (0–5 μmol/L) resulted in significant reductions in HR in isolated mouse hearts without changes in PR, QRS, or QT intervals, we performed whole-cell patch clamp recordings of SANCs which showed that crizotinib inhibited If which regulates cardiac pacemaker activity. Crizotinib resulted in diminished current density of HCN4, the major molecular determinant of If, with an IC50 of 1.4 ± 0.3 μmol/L. Crizotinib also slowed HCN4 activation and shifted the activation curve to the left towards more hyperpolarized potentials.ConclusionsOur results suggest that crizotinib’s effects on HCN4 channels play a significant role in mediating its observed effects on HR.

Vascular Endothelial Over-Expression of Human Soluble Epoxide Hydrolase (Tie2-sEH Tr) Attenuates Coronary Reactive Hyperemia in Mice: Role of Oxylipins and ω-Hydroxylases.

Cytochromes P450 metabolize arachidonic acid (AA) into two vasoactive oxylipins with opposing biologic effects: epoxyeicosatrienoic acids (EETs) and omega-(ω)-terminal hydroxyeicosatetraenoic acids (HETEs). EETs have numerous beneficial physiological effects, including vasodilation and protection against ischemia/reperfusion injury, whereas ω-terminal HETEs induce vasoconstriction and vascular dysfunction. We evaluated the effect of these oxylipins on post-ischemic vasodilation known as coronary reactive hyperemia (CRH). CRH prevents the potential harm associated with transient ischemia. The beneficial effects of EETs are reduced after their hydrolysis to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). ω-terminal HETEs are formed by ω-hydroxylase family members. The relationship among endothelial over-expression of sEH (Tie2-sEH Tr), the changes in oxylipins it may produce, the pharmacologic inhibition of ω-hydroxylases, activation of PPARγ, and CRH response to a brief ischemia is not known. We hypothesized that CRH is attenuated in isolated mouse hearts with endothelial sEH over-expression through modulation of oxylipin profiles, whereas both inhibition of ω-hydroxylases and activation of PPARγ enhance CRH. Compared to WT mice, Tie2-sEH Tr mice had decreased CRH, including repayment volume, repayment duration, and repayment/debt ratio (P < 0.05), whereas inhibition of ω-hydroxylases increased these same CRH parameters in Tie2-sEH Tr mice. Inhibition of sEH with t-AUCB reversed the decreased CRH in Tie2-sEH Tr mice. Endothelial over-expression of sEH significantly changed oxylipin profiles, including decreases in DHETs, mid-chain HETEs, and prostaglandins (P < 0.05). Treatment with rosiglitazone, PPARγ-agonist, enhanced CRH (P < 0.05) in both Tie2-sEH Tr and wild type (WT) mice. These data demonstrate that endothelial over-expression of sEH (through changing the oxylipin profiles) attenuates CRH, whereas inhibition of ω-hydroxylases and activation of PPARγ enhance it.