Langendorff-perfused Rat Hearts Research Articles

SGLT1 contributes to glucose-mediated exacerbation of ischemia-reperfusion injury in ex vivo rat heart.

Hyperglycaemia is common during acute coronary syndromes (ACS) irrespective of diabetic status and portends excess infarct size and mortality, but the mechanisms underlying this effect are poorly understood. We hypothesized that sodium/glucose linked transporter-1 (SGLT1) might contribute to the effect of high-glucose during ACS and examined this using an ex-vivo rodent heart model of ischaemia-reperfusion injury. Langendorff-perfused rat hearts were subjected to 35min ischemia and 2h reperfusion, with variable glucose and reciprocal mannitol given during reperfusion in the presence of pharmacological inhibitors of SGLT1. Myocardial SGLT1 expression was determined in rat by rtPCR, RNAscope and immunohistochemistry, as well as in human by single-cell transcriptomic analysis. High glucose in non-diabetic rat heart exacerbated reperfusion injury, significantly increasing infarct size from 45 ± 3 to 65 ± 4% at 11-22mmol/L glucose, respectively (p < 0.01), an association absent in diabetic heart (32 ± 1-37 ± 5%, p = NS). Rat heart expressed SGLT1 RNA and protein in vascular endothelium and cardiomyocytes, with similar expression found in human myocardium by single-nucleus RNA-sequencing. Rat SGLT1 expression was significantly reduced in diabetic versus non-diabetic heart (0.608 ± 0.08 compared with 1.116 ± 0.13 probe/nuclei, p < 0.01). Pharmacological inhibitors phlorizin, canagliflozin or mizagliflozoin in non-diabetic heart revealed that blockade of SGLT1 but not SGLT2, abrogated glucose-mediated excess reperfusion injury. Elevated glucose is injurious to the rat heart during reperfusion, exacerbating myocardial infarction in non-diabetic heart, whereas the diabetic heart is resistant to raised glucose, a finding which may be explained by lower myocardial SGLT1 expression. SGLT1 is expressed in vascular endothelium and cardiomyocytes and inhibiting SGLT1 abrogates excess glucose-mediated infarction. These data highlight SGLT1 as a potential clinical translational target to improve morbidity/mortality outcomes in hyperglycemic ACS patients.

Elevated Na is a dynamic and reversible modulator of mitochondrial metabolism in the heart

Elevated intracellular sodium Nai adversely affects mitochondrial metabolism and is a common feature of heart failure. The reversibility of acute Na induced metabolic changes is evaluated in Langendorff perfused rat hearts using the Na/K ATPase inhibitor ouabain and the myosin-uncoupler para-aminoblebbistatin to maintain constant energetic demand. Elevated Nai decreases Gibb’s free energy of ATP hydrolysis, increases the TCA cycle intermediates succinate and fumarate, decreases ETC activity at Complexes I, II and III, and causes a redox shift of CoQ to CoQH2, which are all reversed on lowering Nai to baseline levels. Pseudo hypoxia and stabilization of HIF-1α is observed despite normal tissue oxygenation. Inhibition of mitochondrial Na/Ca-exchange with CGP-37517 or treatment with the mitochondrial ROS scavenger MitoQ prevents the metabolic alterations during Nai elevation. Elevated Nai plays a reversible role in the metabolic and functional changes and is a novel therapeutic target to correct metabolic dysfunction in heart failure.

Bioresorbable molybdenum temporary epicardial pacing wires

Cardiac pacing with temporary epicardial pacing wires (TEPW) is used to treat rhythm disturbances after cardiac surgery. Occasionally, TEPW cannot be mechanically extracted and remain in the thorax, where they may rarely cause serious complications like migration and infection. We aim to develop bioresorbable TEPW that will dissolve over time even if postoperative removal is unsuccessful. In the present study, we demonstrate a completely bioresorbable design using molybdenum (Mo) as electric conductor and the resorbable polymers poly(D, L-lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) for electrically insulating double-coating. We compared the pacing properties of these Mo TEPW demonstrators to conventional steel TEPW in Langendorff-perfused rat hearts and observed similar functionality. In vitro, static immersion tests in simulated body fluid for up to 28 days elucidated the degradation behaviour of uncoated Mo strands and the influence of polymer coating thereon. Degradation was considerably reduced in double-coated Mo TEPW compared to the uncoated and the PLGA-coated condition. Furthermore, we confirmed good biocompatibility of Mo degradation products in the form of low cytotoxicity in cell cultures of human cardiomyocytes and cardiac fibroblasts. Statement of significanceTemporary pacing wires are routinely implanted on the heart surface to treat rhythm disturbances in the days following cardiac surgery. Subsequently, these wires are to be removed. When removal attempts are unsuccessful, wires are cut at skin level and the remainders are left inside the chest. Retained fragments may migrate within the body or become a centre of infection. These complications may be prevented using resorbable pacing wires. We manufactured completely resorbable temporary pacing wires using molybdenum as electrical conductor and assessed their function, degradation and biological compatibility. Our study represents an important step in the development of a safer approach to the treatment of rhythm disturbances after cardiac surgery.

Stimulation of soluble guanylyl cyclase in erythrocytes induces export of cGMP and cardioprotection in type 2 diabetes

Abstract Background Red blood cells (RBCs) are known to export cardioprotective nitric oxide (NO) bioactivity. Although, RBCs contain a functional soluble guanylyl cyclase (sGC), the signalling behind the export of NO bioactivity remains unclear. Impairment of NO signalling in RBCs has been suggested to contribute to augmented cardiac ischemia-reperfusion injury in type 2 diabetes (T2D). We hypothesized that increased NO signaling by stimulation of sGC in RBCs protects from myocardial ischemia-reperfusion injury by release of a cardioprotective signal. Purpose To investigate whether stimulation of sGC, the intracellular NO receptor, in RBCs from T2D patients induces export of a cardioprotective factor and to determine its nature. Methods RBCs were collected from patients with T2D and from gender- and age-matched healthy subjects and incubated with different pharmacological compounds affecting the NO-signalling pathway. The RBCs or their supernatant were then administered to isolated Langendorff-perfused rat hearts subjected to 25 min global ischemia and 60 min reperfusion. Left ventricular developed pressure and infarct size were measured. ELISA and immunofluorescence were used to determine levels of cyclic guanosine monophosphate (cGMP) and substrate of protein kinase G activation. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85-23, revised 1996). The present study was performed following The Code of Ethics of the World Medical Association outlined in the Declaration of Helsinki of 1975 and revised in 1983 for experiments that involve human subjects. Results RBCs from T2D patients impaired post-ischemic cardiac function and increased infarct size compared to RBCs from healthy subjects, and these effects were prevented by pre-incubation of the RBCs with the sGC stimulator CYR715. Administration of the supernatant from RBCs incubated with the sGC stimulator resulted in similar cardioprotection, indicating release of a cardioprotective factor from the RBCs (Figure 1A and B). The cardioprotection was abolished by inhibition of transport of cGMP with MK571, an inhibitor of cyclic nucleotide transport. Extracellular cGMP levels in the supernatant from RBCs were increased following sGC stimulation, and administration of exogenous cGMP induced cardioprotection (Figure 1C and D). Expression of protein kinase G-dependent phosphorylated vasodilator-specific phosphoprotein in cardiomyocytes was increased following administration of supernatant from RBCs incubated with the sGC stimulator (Figure 1E). Conclusions This study shows that stimulation of sGC in RBCs leads to release of a cardioprotective mediator, identified as cGMP, that activates cardiac protein kinase G and protects from ischemia-reperfusion injury. It further identifies RBC sGC as a novel therapeutic target to prevent cardiovascular injury in T2D.Figure 1

Abstract 15121: Bioresorbable Molybdenum Electrodes: Towards a Novel Generation of Temporary Epicardial Pacing Wires

Introduction: Cardiac pacing with temporary epicardial pacing wires (TEPW) is used to treat rhythm disturbances after cardiac surgery. Removal is recommended on day 4-7. Occasionally, TEPW cannot be mechanically extracted, have to stay in the thorax and may rarely cause serious complications like migration and infection. We aim to develop novel, bioresorbable TEPW that will dissolve over time even if postoperative removal is unsuccessful. Methods and Results: We manufactured prototypical braided molybdenum (Mo) leads (16 Mo wires of 40 μM diameter each, length 18 cm for ex-vivo, 7.5 cm for in-vivo trials) coated with the biodegradable polymers poly(lactide-co-glycolic acid) (PLGA, inner coating) and polycaprolactone (PCL, outer coating) for shaping and electrical insulation. Mo electrodes showed similar pacing and sensing properties to conventional steel electrodes (Osypka) in Langendorff-perfused rat hearts, even with somewhat lower stimulation thresholds. In artificial body fluid at 37°C, polymer-coated Mo electrodes dissolved at a rate of 1.6 ± 0.3 μg/cm 2 ·d (n=5) compared to uncoated electrodes with 30.3 ± 0.8 μg/cm 2 ·d (n = 4, p &lt; 0.001). Assessing apoptosis and necrosis in human cardiomyocytes and cardiac fibroblasts, we detected no toxicity at Mo concentrations up to 0.52 mM. To test Mo TEPW in vivo, we sutured them epicardially onto the anterior wall of hearts of female Wistar rats, led them out of the thorax and placed them in a subcutaneous pocket. We tested electrophysiological properties directly after implantation, after two weeks and one month. Mo electrodes showed similar pacing and sensing properties directly upon implantation and after two weeks. After one month, all but one pair of Mo electrodes were mechanically broken at their exit from the thorax, the site of assumedly highest mechanical stress. Conventional steel TEPW had similar electrical properties following implantation, after two weeks and one month with no signs of breakage. Conclusion: We demonstrate that Mo TEPW can feasibly be used for epicardial pacing in vivo for up to two weeks. These findings represent an important step in the development of bioresorbable TEPW as a novel and even safer approach to temporary epicardial pacing.

Evidence for the cardiodepressive effects of the plasticizer di-2-ethylhexyl phthalate.

Di-2-ethylhexyl phthalate (DEHP) is commonly used in the manufacturing of plastic materials, including intravenous bags, blood storage bags, and medical-grade tubing. DEHP can leach from plastic medical products, which can result in inadvertent patient exposure. DEHP concentrations were measured in red blood cell units stored between 7 and 42 days (17-119 μg/ml). Using these concentrations as a guide, Langendorff-perfused rat heart preparations were acutely exposed to DEHP. Sinus activity remained stable with lower doses of DEHP (25-50 μg/ml), but sinus rate declined by 43% and sinus node recovery time (SNRT) prolonged by 56.5% following 30-min exposure to 100 μg/ml DEHP. DEHP exposure also exerted a negative dromotropic response, as indicated by a 69.4% longer PR interval, 108.5% longer Wenckebach cycle length (WBCL), and increased incidence of atrioventricular (AV) uncoupling (60-min exposure). Pretreatment with doxycycline partially rescued the effects of DEHP on sinus activity, but did not ameliorate the effects on AV conduction. DEHP exposure also prolonged the ventricular action potential and effective refractory period, but had no measurable effect on intracellular calcium transient duration. Follow-up studies using human-induced pluripotent stem cell-derived cardiomyocytes confirmed that DEHP slows electrical conduction in a time (15 min-3 h) and dose-dependent manner (10-100 μg/ml). Previous studies have suggested that phthalate toxicity is specifically attributed to metabolites of DEHP, including mono-2-ethylhexylphthalate. This study demonstrates that DEHP exposure also contributes to cardiac dysfunction in a dose- and time-dependent manner. Future work is warranted to investigate the impact of DEHP (and its metabolites) on human health, with special consideration for clinical procedures that employ plastic materials.

Complexity analysis of electrical activity during endocardial and epicardial biventricular mapping of ventricular fibrillation.

Ventricular fibrillation (VF) is a lethal cardiac arrhythmia that is a significant cause of sudden cardiac death. Comprehensive studies of spatiotemporal characteristics of VF in situ are difficult to perform with current mapping systems and catheter technology. The goal of this study was to develop a computational approach to characterize VF using a commercially available technology in a large animal model. Prior data suggests that characterization of spatiotemporal organization of electrical activity during VF can be used to provide better mechanistic understanding and potential ablation targets to modify VF and its substrate. We therefore evaluated intracardiac electrograms during biventricular mapping of the endocardium (ENDO) and epicardium (EPI) in acute canine studies. To develop thresholds for organized and disorganized activity, a linear discriminant analysis (LDA)-based approach was performed to the known organized and disorganized activities recorded in ex vivo Langendorff-perfused rat and rabbit hearts using optical mapping experiments. Several frequency- and time-domain approaches were used as individual and paired features to identify the optimal thresholds for the LDA approach. Subsequently, VF was sequentially mapped in 4 canine hearts, using the CARTO mapping system with a multipolar mapping catheter in the ENDO left and right ventricles and EPI to capture the progression of VF at 3 discrete post-induction time intervals: VF period 1 (just after induction of VF to 15 min), VF period 2 (15 to 30 min), and VF period 3 (30 to 45 min). The developed LDA model, cycle lengths (CL), and regularity indices (RI) were applied to all recorded intracardiac electrograms to quantify the spatiotemporal organization of VF in canine hearts. We demonstrated the presence of organized activity in the EPI as VF progresses, in contrary to the ENDO, where the activity stays disorganized. The shortest CL always occurred in the ENDO, especially the RV, indicating a faster VF activity. The highest RI was found in the EPI in all hearts for all VF stages, indicating spatiotemporal consistency of RR intervals. We identified electrical organization and spatiotemporal differences throughout VF in canine hearts from induction to asystole. Notably, the RV ENDO is characterized by a high level of disorganization and faster VF frequency. In contrast, EPI has a high spatiotemporal organization of VF and consistently long RR intervals.

Amiodarone delivery using lipidic nanocapsules for the treatment of atrial fibrillation

Atrial fibrillation (AF) is the most common arrhythmia and is associated with significant morbidity and mortality. As AF progresses, controlling AF gets more and more challenging. Amiodarone (AMD) is considered as the most effective antiarrhythmic drug for maintaining sinus rhythm after cardioversion and it is also recommended for controlling AF in patient with congestive heart failure. However, its pharmacokinetic and metabolic profile require the use of high dosage resulting in many extracardiac side effects which limit prescription of AMD. Therefore, nanoencapsulation of AMD in lipid nanocapsules (LNC) with the ability to target the heart represents a promising strategy to enhance the efficiency of AMD and to limit its toxicity. This work focus on development of antiarrhythmic LNC-AMD nano-formulations and their in vitro toxicity. LNC-AMD are prepared by microfluidic using a low-energy patented process and purified by a tangential flow filtration process. LNC are characterized in terms of size and polydispersity (PDI) by dynamic light scattering, surface charge by electrophoretic mobility, drug loading (DL) and encapsulation efficiency (EE) by UPLC coupled to UV detector. Cell toxicity of LNC-AMD is evaluated on cardiac cells (H9C2 cardiomyoblastes, and isolated cardiomyocytes) and measured by MTT cell viability assay and lactate dehydrogenase (LDH) release. LNC-AMD have a size of 44 nm ± 5 nm with a PDI of 0.15 ± 0.3 and a zeta potential of +18 mV. After purification, DL is about 0.4 mg/mL of suspension of LNC and EE is of 60%. Formulation and purification of LNC-AMD have been optimized for their biological evaluation. Short-term perspectives are the co-encapsulation of AMD with an anti-fibrotic molecule, and the surface functionalization of LNC with a heart-targeting peptide (ZP1609) to ultimately establish proof-of-concept on Langendorff-perfused rat hearts.

Hidden Pool of Cardiac Adenine Nucleotides That Controls Adenosine Production.

Myocardial ischemic adenosine production decreases in subsequent events that may blunt its protective functions. To test the relation between total or mitochondrial cardiac adenine nucleotide pool (TAN) on the energy status with adenosine production, Langendorff perfused rat hearts were subjected to three protocols: 1 min ischemia at 40 min, 10 min ischemia at 50 min, and 1 min ischemia at 85 min in Group I; additional infusion of adenosine (30 µM) for 15 min after 10 min ischemia in Group I-Ado, and 1 min ischemia at 40 and 85 min in the controls (Group No I). A 31P NMR and an HPLC were used for the analysis of nucleotide and catabolite concentrations in the heart and coronary effluent. Cardiac adenosine production in Group I measured after 1 min ischemia at 85 min decreased to less than 15% of that at 40 min in Group I, accompanied by a decrease in cardiac ATP and TAN to 65% of the initial results. Adenosine production at 85 min was restored to 45% of that at 40 min in Group I-Ado, accompanied by a rebound of ATP and TAN by 10% vs. Group I. Mitochondrial TAN and free AMP concentrations paralleled that of total cardiac TAN. Changes in energy equilibrium or mitochondrial function were minor. This study highlights that only a fraction of the cardiac adenine nucleotide pool is available for adenosine production, but further studies are necessary to clarify its nature.

Generation of myocyte agonal Ca2+ waves and contraction bands in perfused rat hearts following irreversible membrane permeabilisation

Although irreversible cardiomyocyte injury provokes intracellular Ca2+ ([Ca2+]i) overload, the underlying dynamics of this response and its effects on cellular morphology remain unknown. We therefore visualised rapid-scanning confocal fluo4-[Ca2+]i dynamics and morphology of cardiomyocytes in Langendorff-perfused rat hearts following saponin-membrane permeabilisation. Our data demonstrate that 0.4% saponin-treated myocytes immediately exhibited high-frequency Ca2+ waves (131.3 waves/min/cell) with asynchronous, oscillatory contractions having a mean propagation velocity of 117.8 μm/s. These waves slowly decreased in frequency, developed a prolonged decay phase, and disappeared in 10 min resulting in high-static, fluo4-fluorescence intensity. The myocytes showing these waves displayed contraction bands, i.e., band-like actin-fibre aggregates with disruption of sarcomeric α-actinin. The contraction bands were not attenuated by the abolition of Ca2+ waves under pretreatment with ryanodine plus thapsigargin, but were partially attenuated by the calpain inhibitor MDL28170, while mechanical arrest of the myocytes by 2,3-butanedione monoxime completely attenuated contraction-band formation. The depletion of adenosine 5′-triphosphate by the mitochondrial electron uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone also attenuated Ca2+ waves and contraction bands. Overall, saponin-induced myocyte [Ca2+]i overload provokes agonal Ca2+ waves and contraction bands. Contraction bands are not the direct consequence of the waves but are caused by cross-bridge interactions of the myocytes under calpain-mediated proteolysis.

Anti-Ischemic Effect of Leptin in the Isolated Rat Heart Subjected to Global Ischemia-Reperfusion: Role of Cardiac-Specific miRNAs

Background: Leptin is an obesity-associated adipokine that has been implicated in cardiac protection against ischemia-reperfusion injury (IRI). In this study, concentration-dependent effects of leptin on myocardial IRI were investigated in the isolated rat heart. In addition, we analyzed myocardial miRNAs expression in order to investigate their potential involvement in leptin-mediated cardioprotection. Methods: The effect of leptin on IRI was examined in Langendorff-perfused rat hearts preconditioned with two leptin concentrations (1.0 nM and 3.1 nM) for 60 min. The hearts were subjected to 30 min global ischemia and 120 min reperfusion with buffer containing leptin in the respective concentration. Heart function and arrhythmia incidence were analyzed. Infarct size was assessed histochemically. Expression of miRNA-144, -208a, -378, and -499 was analyzed in the ventricular myocardium using RT-PCR. Results: The addition of 1.0 nM leptin to the buffer exerted an infarct-limiting effect, preserved post-ischemic ventricular function, and prevented reperfusion arrhythmia compared to 3.1 nM leptin. Myocardial expression of miRNA-208a was decreased after heart exposure to 1.0 nM leptin and significantly elevated in the hearts perfused with leptin at 3.1 nM. Conclusion: Acute administration of leptin at low dose (1.0 nM) results in cardiac protection against IRI. This effect is associated with reduced myocardial expression of miRNA-208a.

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection via the purinergic P2Y13 receptor and nitric oxide signalling

Abstract Background Red blood cells (RBC) are suggested to act as important mediators in the regulation of cardiovascular function by exporting nitric oxide (NO) bioactivity and ATP under hypoxic/ischemic conditions. In addition, RBCs are known to protect from ischemia-reperfusion injury via the export of NO bioactivity in experimental settings. However, it remains unknown if such beneficial effects of RBCs are protective in patients with acute myocardial infarction. Purpose To investigate whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia-reperfusion injury and whether such effect involves activation of purinergic and NO signalling in the RBCs. Methods RBCs were collected from patients with STEMI undergoing primary percutaneous coronary intervention and age- and gender-matched healthy controls. The RBCs were administered into the coronary circulation of isolated Langendorff-perfused rat hearts at the onset of global ischemia for 25 min followed by reperfusion of 60 min. Recovery of left ventricular developed pressure (LVDP) during reperfusion and infarct size were determined. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996). The present study was performed following The Code of Ethics of the World Medical Association outlined in the Declaration of Helsinki of 1975 and revised in 1983 for experiments that involve human subjects. Results Administration of RBCs from STEMI patients improved recovery of LVDP and reduced infarct size in hearts subjected to ischemia-reperfusion in comparison with RBCs from healthy controls (Figure 1A, B). Pre-incubation of the RBCs with the NO synthase (NOS) inhibitor L-NAME (Figure 1C, D) and the inhibitor of the NO receptor soluble guanylyl cyclase (sGC) ODQ abolished the cardioprotective effect of RBCs from STEMI patients. The cardioprotective effect was also attenuated by inhibition of cardiac cGMP-dependent protein kinase (PKG). Further, the purinergic P2Y13 receptor antagonist MRS2211 (Figure 1E, F), but not the P1 receptor antagonist 8PT applied to RBCs, attenuated the cardioprotection induced by RBCs from STEMI patients. Moreover, administration of RBCs from healthy subjects pre-incubated with a cell permeable ATP analogue improved post-ischemic recovery of LVDP and reduced infarct size. This cardioprotective effect was abolished by co-incubation of the RBCs with ODQ (Figure 2) and MRS2211. Conclusion Our findings demonstrate a novel function of RBCs in patients with STEMI that provides protection against myocardial ischemia-reperfusion injury via the activation of P2Y13 receptor and the NO-sGC pathway in RBCs and cardiac PKG. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation; Swedish Research Council

PI3K/Akt pathway mediates the positive inotropic effects of insulin in Langendorff-perfused rat hearts

Insulin exerts positive inotropic effects on cardiac muscle; however, the relationship between cardiac contractility and phosphoinositol-3-kinase/Akt (PI3K/Akt) activation remains unclear. We hypothesized that the positive inotropic effects of insulin are dose-dependent and mediated via the PI3K/Akt pathway in isolated normal rat hearts. The Institutional Animal Investigation Committee approved the use of hearts excised from rats under pentobarbital anesthesia. The hearts were perfused at a constant pressure using the Langendorff technique. After stabilization (baseline), the hearts were randomly divided into the following four insulin (Ins) groups: 1) Ins0 (0 IU/L), 2) Ins0.5 (0.5 IU/L), 3) Ins5 (5 IU/L), and 4) Ins50 (50 IU/L) (n = 8 in each group). To clarify the role of the PI3K/Akt pathway in insulin-dependent inotropic effects, we also treated the insulin groups with the PI3K inhibitor wortmannin (InsW): 5) InsW0 (0 IU/L), 6) InsW0.5 (0.5 IU/L), 7) InsW5 (5 IU/L), and 8) InsW50 (50 IU/L). Hearts were perfused with Krebs–Henseleit buffer solution with or without wortmannin for 10 min, followed by 20 min perfusion with the solution containing each concentration of insulin. The data were recorded as the maximum left ventricular derivative of pressure development (LV dP/dt max). Myocardial p-Akt levels were measured at 3 min, 5 min, and at the end of the perfusion. In the Ins groups, LV dP/dt max in Ins5 and Ins50 increased by 14% and 48%, respectively, 3 min after insulin perfusion compared with the baseline. Tachyphylaxis was observed after 10 min in the Ins5 and Ins50 treatment groups. Wortmannin partially inhibited the positive inotropic effect of insulin; although insulin enhanced p-Akt levels at all time points compared with the control group, this increase was suppressed in the presence of wortmannin. The positive inotropic effect of insulin is dose-dependent and consistent with Akt activation. This effect mediated by high doses of insulin on cardiac tissue was temporary and caused tachyphylaxis, potentially triggered by Akt overactivation, which leads beta 1 deactivation.

Metformin Protects Against Sunitinib-induced Cardiotoxicity: Investigating the Role of AMPK.

Sunitinib is associated with cardiotoxicity through inhibition of AMP-protein kinase (AMPK) signaling. By contrast, the common antidiabetic agent metformin has demonstrated cardioprotection through indirect AMPK activation. In this study, we investigate the effects of metformin during sunitinib-induced cytotoxicity. Left ventricular developed pressure, coronary flow, heart rate, and infarct size were measured in Langendorff-perfused rat hearts treated with 1 µM sunitinib ±50 µM metformin ±1 µM human equilibrative nucleoside transporter inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBTI). Western blot analysis was performed for p-AMPKα levels. Primary isolated cardiac myocytes from the left ventricular tissue were used to measure live cell population levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess adjunctive treatment of and metformin in human hepatoma G2 and promyelocytic leukemia (HL-60) cells treated with 0.1-100 µM sunitinib ±50 µM metformin. In the perfused hearts, coadministration of metformin attenuated the sunitinib-induced changes to left ventricular developed pressure, infarct size, and cardiac myocyte population. Western blot analysis revealed a significant decrease in p-AMPKα during sunitinib treatment, which was attenuated after coadministration with metformin. All metformin-induced effects were attenuated, and NBTI was coadministered. The MTT assay demonstrated an increase in the EC50 value during coadministration of metformin with sunitinib compared with sunitinib monotherapy in hepatoma G2 and HL-60 cell lines, demonstrating the impact and complexity of metformin coadministration and the possible role of AMPK signaling. This study highlights the novel cardioprotective properties of metformin and AMPK activation during sunitinib-induced cardiotoxicity when administered together in the Langendorff heart model.

Protective effects of trehalose preconditioning on cardiac and coronary endothelial function through eNOS signaling pathway in a rat model of ischemia-reperfusion injury.

Coronary endothelial dysfunction is a major cause of ischemia-reperfusion (I/R) injury. Trehalose, a natural disaccharide, has been reported to ameliorate endothelial dysfunction during aging by activating endothelial nitric oxide synthase (eNOS); however, its role in I/R injury is unknown. This study evaluated the effects of trehalose preconditioning on cardiac and coronary endothelial function after I/R. Langendorff-perfused rat hearts underwent 30min of global ischemia followed by 80min of reperfusion with or without trehalose preconditioning. Rate pressure product (RPP) and coronary flow (CF) were measured during reperfusion. Perivascular edema was assessed by hematoxylin and eosin staining. Myocardial oxidative stress and apoptosis were evaluated by immunohistochemistry and TUNEL staining, respectively. eNOS dimerization was determined by western blotting. An eNOS inhibitor was used to examine the role of eNOS. Trehalose preconditioning showed a higher recovery rate after I/R as indicated by high RPP (control vs. trehalose, 28 ± 6% vs. 46 ± 9%; P = 0.017, Cohen's d = 2.3) and CF values (35 ± 10% vs. 55 ± 9%; P = 0.025, d = 1.7). Furthermore, trehalose preconditioning reduced perivascular edema, myocardial oxidative stress, and apoptosis. The eNOS dimerization ratio was increased by trehalose (1.2 ± 0.2 vs. 1.6 ± 0.2; P = 0.023, d = 2.1), which was associated with the recovery of RPP and CF. These effects of trehalose were abolished by the eNOS inhibitor. Trehalose preconditioning showed protective effects on cardiac and coronary endothelial function after I/R through the eNOS signaling pathway.

Attenuated cardiac post-ischemic injury and endothelial dysfunction by stimulation of soluble guanylyl cyclase in red blood cells from patients with type 2 diabetes

Abstract Background Reduced bioavailability of nitric oxide (NO) contributes to ischemic heart disease in type 2 diabetes (T2D). Red blood cells (RBCs) are known to produce NO bioactivity and to contain a functional soluble guanylyl cyclase (sGC) that is activated by NO. Recent studies revealed that RBCs from patients with T2D exacerbates ischemia-reperfusion (I/R) injury and induces endothelial dysfunction via a mechanism that is dependent on reduced export of NO bioactivity from RBCs. It remains unknown whether stimulation of sGC in RBCs from patients with T2D protects against myocardial I/R injury and endothelial dysfunction. Purpose To test the hypothesis that stimulation of sGC in RBCs from T2D patients protects against myocardial I/R injury and improves endothelial function. Methods RBCs collected from T2D patients and healthy subjects were incubated with vehicle or the sGC stimulator CYR715 before being administered to isolated Langendorff-perfused rat hearts subjected to 25 min global ischemia and 60 min reperfusion. Left ventricular developed pressure (LVDP) and infarct size were determined. In addition, isolated rat aortic rings were incubated with RBCs subsequent determination of endothelium-dependent relaxation (EDR). Results Administration of RBCs from T2D patients impaired post-ischemic recovery of LVDP and induced endothelial dysfunction in comparison with RBCs from healthy subjects (P&amp;lt;0.001). Pre-incubation of RBCs from patients with T2D with CYR715 prior to administration to the isolated heart enhanced the recovery of LVDP (Fig.1A), reduced infarct size (Fig.1B), and attenuated endothelial dysfunction (Fig.1C). CYR715 did not induce cardioprotection in the absence of RBCs. The sGC inhibitor ODQ did not significantly affect cardiac recovery per se but totally abolished the protective effect of CYR715 (Fig.1A). Conclusions Stimulation of sGC in RBCs from patients with T2D protects against cardiac I/R injury and prevents development of endothelial dysfunction. Stimulation of the NO-sGC pathway in RBCs appears to be an attractive therapeutic strategy to prevent cardiovascular injury in T2D. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation, the Swedish Research Council

Inhibition of Cardiac RIP3 Mitigates Early Reperfusion Injury and Calcium-Induced Mitochondrial Swelling without Altering Necroptotic Signalling.

Receptor-interacting protein kinase 3 (RIP3) is a convergence point of multiple signalling pathways, including necroptosis, inflammation and oxidative stress; however, it is completely unknown whether it underlies acute myocardial ischemia/reperfusion (I/R) injury. Langendorff-perfused rat hearts subjected to 30 min ischemia followed by 10 min reperfusion exhibited compromised cardiac function which was not abrogated by pharmacological intervention of RIP3 inhibition. An immunoblotting analysis revealed that the detrimental effects of I/R were unlikely mediated by necroptotic cell death, since neither the canonical RIP3–MLKL pathway (mixed lineage kinase-like pseudokinase) nor the proposed non-canonical molecular axes involving CaMKIIδ–mPTP (calcium/calmodulin-dependent protein kinase IIδ–mitochondrial permeability transition pore), PGAM5–Drp1 (phosphoglycerate mutase 5–dynamin-related protein 1) and JNK–BNIP3 (c-Jun N-terminal kinase–BCL2-interacting protein 3) were activated. Similarly, we found no evidence of the involvement of NLRP3 inflammasome signalling (NOD-, LRR- and pyrin domain-containing protein 3) in such injury. RIP3 inhibition prevented the plasma membrane rupture and delayed mPTP opening which was associated with the modulation of xanthin oxidase (XO) and manganese superoxide dismutase (MnSOD). Taken together, this is the first study indicating that RIP3 regulates early reperfusion injury via oxidative stress- and mitochondrial activity-related effects, rather than cell loss due to necroptosis.

Selective Fatty Acid Amide Hydrolase Inhibitors as Potential Novel Antiepileptic Agents.

Temporal lobe epilepsy is the most common form of epilepsy, and current antiepileptic drugs are ineffective in many patients. The endocannabinoid system has been associated with an on-demand protective response to seizures. Blocking endocannabinoid catabolism would elicit antiepileptic effects, devoid of psychotropic effects. We herein report the discovery of selective anandamide catabolic enzyme fatty acid amide hydrolase (FAAH) inhibitors with promising antiepileptic efficacy, starting from a further investigation of our prototypical inhibitor 2a. When tested in two rodent models of epilepsy, 2a reduced the severity of the pilocarpine-induced status epilepticus and the elongation of the hippocampal maximal dentate activation. Notably, 2a did not affect hippocampal dentate gyrus long-term synaptic plasticity. These data prompted our further endeavor aiming at discovering new antiepileptic agents, developing a new set of FAAH inhibitors (3a-m). Biological studies highlighted 3h and 3m as the best performing analogues to be further investigated. In cell-based studies, using a neuroblastoma cell line, 3h and 3m could reduce the oxinflammation state by decreasing DNA-binding activity of NF-kB p65, devoid of cytotoxic effect. Unwanted cardiac effects were excluded for 3h (Langendorff perfused rat heart). Finally, the new analogue 3h reduced the severity of the pilocarpine-induced status epilepticus as observed for 2a.

Raman Spectroscopic Assessment of Myocardial Viability in Langendorff-Perfused Ischemic Rat Hearts.

Spontaneous Raman spectroscopy, which senses changes in cellular contents of reduced cytochrome c, could be a powerful tool for label-free evaluation of ischemic hearts. However, undetermined is whether it is applicable to evaluation of myocardial viability in ischemic hearts. To address this issue, we investigated sequential changes in Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart before and during ligation of the left coronary artery and its subsequent release and re-ligation. Under 532-nm wavelength excitation, the Raman peak intensity of reduced cytochrome c at 747 cm−1 increased quickly after the coronary ligation, and reached a quasi-steady state within 30 min. Subsequent reperfusion of the heart after a short-term (30-min) ligation that simulates reversible conditions resulted in quick recovery of the peak intensity to the baseline. Further re-ligation resulted in resurgence of the peak intensity to nearly the identical value to the first ischemia value. In contrast, reperfusion after prolonged (120-min) ligation that assumes irreversible states resulted in incomplete recovery of the peak intensity, and re-ligation resulted in inadequate resurgence. Electron microscopic observations confirmed the spectral findings. Together, the Raman spectroscopic measurement for cytochrome c could be applicable to evaluation of viability of the ischemic myocardium without labeling.

Pleiotropic, non-cell death-associated effects of inhibitors of receptor-interacting protein kinase 1 in the heart.

Inhibition of receptor-interacting protein kinase 1 (RIP1) has been recognized as a compelling tool for limiting necroptosis. Recent findings have indicated that RIP1 inhibitor, necrostatin-1 (Nec-1), is also able to modify heart function under non-cell death conditions. In this study, we investigated its underlying molecular mechanisms and compared with those of novel pharmacologically improved agents (Nec-1s and GSK'772) and its inactive analog (Nec-1i). Heart function was examined in Langendorff-perfused rat hearts. Certain proteins regulating myocardial contraction-relaxation cycle and oxidative stress (OS) were evaluated by immunoblotting and as the extent of lipid peroxidation, protein carbonylation and nitration, respectively. In spite of the increase of left ventricular developed pressure (LVDP) due to treatment by both Nec-1 and Nec-1i, only the former agent increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) at threonine 287 and cardiac myosin-binding protein-C (cMyBPc) at serine 282. In contrast, Nec-1s did not elicit such changes, while it also increased LVDP. GSK'772 activated CaMKIIδ-phospholamban (PLN) axis. Neither protein kinase A (PKA) nor its selected molecular targets, such as serine 16 phosphorylated PLN and sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), were affected by either RIP1 inhibitor. Nec-1, like other necrostatins (Nec-1i, Nec-1s), but not GSK'772, elevated protein tyrosine nitration without affecting other markers of OS. In conclusion, this study indicated for the first time that Nec-1 may affect basal heart function by the modulation of OS and activation of some proteins of contraction-relaxation cycle.