Isolated Perfused Hearts Research Articles

Secretoneurin Is an Endogenous Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor That Attenuates Ca2+-Dependent Arrhythmia.

Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes. SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.

Non-invasive Measurement of Dynamic Myocardial Stiffness Using Acoustic Radiation Force Impulse Imaging

Myocardial stiffness exhibits cyclic variations over the course of the cardiac cycle. These trends are closely tied to the electromechanical and hemodynamic changes in the heart. Characterization of dynamic myocardialstiffness can provide insights into the functional state of the myocardium, as well as allow for differentiation between the underlying physiologic mechanisms that lead to congestive heart failure. Previous work has revealed the potential of acoustic radiation force impulse (ARFI) imaging to capture temporal trends in myocardial stiffness in experimental preparations such as the Langendorff heart, as well as on animals in open-chest and intracardiac settings. This study was aimed at investigating the potential of ARFI to measure dynamic myocardial stiffness in human subjects, in a non-invasive manner through transthoracic imaging windows. ARFI imaging was performed on 12 healthy volunteers to track stiffness changes within the interventricular septum in parasternal long-axis and short-axis views. Myocardial stiffness dynamics over the cardiac cycle was quantified using five indices: stiffness ratio, rates of relaxation and contraction and time constants of relaxation and contraction. The yield of ARFI acquisitions was evaluated based on metrics of signal strength and tracking fidelity such as displacement signal-to-noise ratio, signal-to-clutter level, temporal coherence of speckle and spatial similarity within the region of excitation. These were quantified using the mean ARF-induced displacements over the cardiac cycle, the contrast between the myocardium and the cardiac chambers, the minimum correlation coefficients of radiofrequency signals and the correlation between displacement traces across simultaneously acquired azimuthal beams, respectively. Forty-one percent of ARFI acquisitions were determined to be “successful” using a mean ARF-induced displacement threshold of 1.5 μm. “Successful” acquisitions were found to have higher (i) signal-to-clutter levels, (ii) temporal coherence and (iii) spatial similarity compared with “unsuccessful” acquisitions. Median values of these three metrics, between the two groups, were measured to be 13.42dB versus 5.42dB, 0.988 versus 0.976 and 0.984 versus 0.849, respectively. Signal-to-clutter level, temporal coherence and spatial similarity were also found to correlate with each other. Across the cohort of healthy volunteers, the stiffness ratio measured was 2.74 ± 0.86; the rate of relaxation, 7.82 ± 4.69/s; and the rate of contraction, –7.31±3.79 /s. The time constant of relaxation was 35.90 ± 20.04ms, and that of contraction was 37.24 ± 19.85ms. ARFI-derived indices of myocardial stiffness were found to be similar in both views. These results indicate the feasibility of using ARFI to measure dynamic myocardial stiffness trends in a non-invasive manner and also highlightthe technical challenges of implementing this method in the transthoracic imaging environment.

Abnormal synchronization patterns in the electrical stimulation-contractile response coupling decrease with noise

Synchronization theory predicts that if an oscillator interacts with a rhythmical external force, then it should react to a rhythmical force by adjusting its frequency. Furthermore, noise is present in nature, and it affects the nervous and cardiovascular systems. In this paper, we analyze the heart as an oscillator, where noisy periodic electrical stimulation can be regarded as an external forcing. This study aimed to investigate, from an experimental point of view, whether noise can induce synchronization of higher order in the mechanical heart response. A Langendorff heart preparation was used to obtain two variables of the mechanical response, intensity of contractile force and heart rate. The experiments show frequency locking in the electrical stimulation-contractile response coupling with and without noise induced. The role of noise in the response of effector organs invites further investigation.

Neuropeptide-Y causes coronary microvascular constriction and is associated with reduced ejection fraction following ST-elevation myocardial infarction.

AimsThe co-transmitter neuropeptide-Y (NPY) is released during high sympathetic drive, including ST-elevation myocardial infarction (STEMI), and can be a potent vasoconstrictor. We hypothesized that myocardial NPY levels correlate with reperfusion and subsequent recovery following primary percutaneous coronary intervention (PPCI), and sought to determine if and how NPY constricts the coronary microvasculature.Methods and resultsPeripheral venous NPY levels were significantly higher in patients with STEMI (n = 45) compared to acute coronary syndromes/stable angina ( n = 48) or with normal coronary arteries (NC, n = 16). Overall coronary sinus (CS) and peripheral venous NPY levels were significantly positively correlated (r = 0.79). STEMI patients with the highest CS NPY levels had significantly lower coronary flow reserve, and higher index of microvascular resistance measured with a coronary flow wire. After 2 days they also had significantly higher levels of myocardial oedema and microvascular obstruction on cardiac magnetic resonance imaging, and significantly lower ejection fractions and ventricular dilatation 6 months later. NPY (100–250 nM) caused significant vasoconstriction of rat microvascular coronary arteries via increasing vascular smooth muscle calcium waves, and also significantly increased coronary vascular resistance and infarct size in Langendorff hearts. These effects were blocked by the Y1 receptor antagonist BIBO3304 (1 μM). Immunohistochemistry of the human coronary microvasculature demonstrated the presence of vascular smooth muscle Y1 receptors.ConclusionHigh CS NPY levels immediately after reperfusion correlate with microvascular dysfunction, greater myocardial injury, and reduced ejection fraction 6 months after STEMI. NPY constricts the coronary microcirculation via the Y1 receptor, and antagonists may be a useful PPCI adjunct therapy.

SRPIN340 protects heart muscle from oxidative damage via SRPK1/2 inhibition-mediated AKT activation

SRPIN340, a selective serine-arginine protein kinase 1/2 (SRPK1/2) inhibitor, has been shown to have antiviral and anti-angiogenesis effects. However, its role in the heart is unknown. The present study explored the role of SRPIN340 in myocardial protection and the related mechanisms. During challenge with H2O2, cardiomyocytes (CMs) pretreated with SRPIN340 showed strikingly more injury tolerance, which was manifested as reduced lactate dehydrogenase (LDH) release and lower apoptotic index. Further research showed that SRPIN340 activated AKT under basal conditions, and AKT inhibition abolished the protective effects of SRPIN340 treatment during H2O2 stress. The protective effect of SRPIN340 was also demonstrated in perfused rat hearts subjected to ischemia/reperfusion (I/R). Collectively, our results reveal the beneficial effects of SRPIN340 against H2O2-induced oxidative damage in CMs and I/R-induced injury in a Langendorff heart model, supporting a potential application of SRPIN340 in the clinically relevant context of reperfusion. The effectiveness of SRPIN340 may be attributed to AKT signal activation.

Cardiac safety assessment with motion field imaging analysis of human iPS cell-derived cardiomyocytes is improved by an integrated evaluation with cardiac ion channel profiling.

We validated a motion field imaging (MFI) assay with human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) as a model to assess multiple cardiac liabilities by comparing the guinea-pig Langendorff heart with hiPS-CMs using 4 reference compounds and 9 internal compounds. We investigated repolarization duration, beating rate (BR), conduction speed, contractility, and inhibitory profile of three cardiac ion channels: hERG, Cav1.2, and Nav1.5. For repolarization, the contraction-relaxation duration (CRDc) of hiPS-CMs was generally consistent with the QTc interval of Langendorff heart. However, 2 internal compounds shortened CRDc despite QTc prolongation in Langendorff heart. Cardiac ion channel profiling revealed that hiPS-CMs could not be used to detect QTc prolongation when the value of Cav1.2 IC50 / hERG IC50 for a compound was between 1 and 10, whereas hiPS-CMs showed responses largely consistent with Langendorff heart when Cav1.2 IC50 / hERG IC50 was below 1 or above 10. The accuracy of hiPS-CMs for the BR was not high, mainly because the BR of hiPS-CMs was increased by an inhibition of Cav1.2. The hiPS-CMs were highly sensitive to conduction speed and contractility, able to detect QRS widening caused by Nav1.5-inhibition, as well as decreased LVdP/dtmax caused by the inhibition of Cav1.2 and/or Nav1.5. In conclusion, the MFI assay with hiPS-CMs would be useful for evaluating multiple cardiac liabilities. The ion channel profile helps to interpret the results of MFI assay and correctly evaluate cardiac risks. Therefore, an integrated cardiac safety assessment with MFI and ion channel profiling is recommended.

S-Nitrosylation as a Post-Translational Modification of CaMKIIδ and its Effects on Cardiac Function

Nitric Oxide (NO) is a gaseous signalling molecule that plays a role in many biological processes especially in the heart. S-nitrosylation involves the covalent attachment of NO-related species to specific cysteine residues of a protein to form S-nitrosothiol, and this process is known to modify protein kinases, such as Calcium/Calmodulin- (Ca2+/CaM) dependent protein kinase (CaMKII). The cardiac-specific isoform, CaMKIIδ, is a key Ca2+-handling kinase involved in excitation-contraction coupling in the heart. In pathophysiological conditions, CaMKIIδ can undergo autonomous activity and cause progression of arrhythmias, diabetes and heart failure. S-nitrosylation has been found to modify CaMKIIδ, however, it is unknown how this affects cardiac function in the whole heart and how inhibition of CaMKIIδ alters NO signaling in the heart. We hypothesised that S-Nitrosylation of CaMKIIδ will lead to impaired cardiac function in mice. Using Langendorff isolated heart perfusion technique, C57BL/6 wild-type (WT) and CaMKIIδ knock-out (KO) mice hearts were treated acutely with a nitric oxide donor, S-nitrosoglutathione (GSNO) at different concentrations. Prior to excision of the heart for Langendorff perfusion, echocardiography measurements were taken and results showed no changes in cardiac structure and function between WT and KO mice. Our data from Langendorff perfusion showed a trend towards increased left ventricular developed pressure in GSNO-treated KO hearts compared to GSNO-treated WT hearts. Notably, our data also showed that GSNO reduced arrhythmia frequency by 26% in KO hearts compared to WT hearts. These results suggest that NO may be contributing to whole heart impairment via a CaMKIIδ-mediated signalling pathway.

Clock Changes in the Type 2 Diabetic Sinoatrial Node

Diabetic patients have impaired heart rate control. In diabetic rats ex vivo heart rate was significantly decreased compared to non-diabetic animals. Why this is the case remains unknown. Within sinoatrial nodal cardiomyocytes heart rate is determined by rhythmic oscillations in Ca2+ and other ions, the so called Ca2+ and membrane clocks. The Ca2+ clock primarily involves the intracellular Ca2+ store, the sarcoplasmic reticulum, and ‘Ca2+-handling’ proteins such as the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) and its regulator phospholamban (PLB). The membrane clock involves membrane ion transporters such as the hyperpolarisation-activated cyclic nucleotide-gated channel (HCN4) and Na+-Ca2+ exchanger (NCX). The aim of this research was to investigate whether the reduced intrinsic heart rate in diabetes is due to changes in the Ca2+ and/ or membrane clocks. The sinoatrial node of non-diabetic and type 2 diabetic rats was isolated and protein expression was determined. A significant 1.6-fold increase in PLB expression was found in DM (p < 0.05), with no change to SERCA2a expression (p > 0.05). A significant 2.2-fold increase in NCX expression (p < 0.05) and a trend towards an increase in HCN4 (p = 0.08) in DM was found. The functional effects on heart rate were investigated by increasing external Ca2+ and ivabradine (HCN4 inhibitor) concentrations in isolated Langendorff hearts. Changes in external Ca2+ did not change intrinsic heart rate in both groups, however ivabradine reduced intrinsic heart rate in nDM, but not in DM. We conclude the lower intrinsic heart rate in diabetic rats is, in part, a result of change in membrane clock proteins (non-functional HCN4).

Abstract 16722: L-2-Hydroxyglutarate Protects Against Myocardial Ischemia-Reperfusion Injury

Background: L-2-hydroxyglutarate (L2HG) couples mitochondrial (Mito) and cytoplasmic energy metabolism in a model of cellular redox regulation. Under conditions of limited oxygen availability, mammalian cells generate L2HG to counteract the adverse effects of Mito reductive stress induced by hypoxia. L2HG is oxidized to α-ketoglutarate by L2HG dehydrogenase (L2HGDH) in a variety of mammalian tissues, including the heart. Myocardial ischemia/reperfusion injury (MIRI) is a consequence of coronary vascular disease and vascular procedures. Mito oxidative stress plays a critical role in MIRI. Here we hypothesized that L2HG protects against MIRI. Methods and Results: We induced accumulation of L2HG by heterozygous (HET) and homozygous (KO) deletion of L2HGDH gene in mice. The hearts isolated from HET, KO, and wild-type (WT) littermate mice were subjected to 30 min global thermal ischemia followed by 60 min reperfusion. Cardiac function and energy metabolism were simultaneously measured using a Langendorff heart perfusion model and 31 P-NMR spectroscopy. We found that female HET and KO exhibited better recovery of cardiac function post-MIRI in consistent with better preservation of intracellular ATP and phosphocreatine (Table). Using HPLC, we measured compounds representative of cellular redox state (GSG, GSSG, NAD, NADH, NADP, NADPH, coenzyme A and ascorbate), cellular energy state (ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP, CTP, CDP, CMP, IMP, malonyl-CoA and acetyl-CoA), and nucleotide catabolism (adenosine, hypoxanthine, xanthine and inosine) in freeze-clamped hearts. We found that ratios of GSH/GSSG and ATP/ADP increased in HET and KO hearts (Table). Interestingly, the ratios of NADH/NAD and NADPH/NADP increased only in KO but not in HET hearts. Consistent with the increased ratio of ATP/AMP, hypoxanthine and xanthine decreased only in KO hearts but not in HET hearts (Table). Conclusion: The accumulation of L2HG in a genetic mouse model protects against MIRI though increasing cellular antioxidative capacity and preserving high energy phosphates. The underlying mechanism of these beneficial adaptive phenomena warrants further study. Targeting L2HG levels and L2HGDH expression may serve as new therapeutic strategies for MIRI protection.

Leadless multisite pacing: A feasibility study using wireless power transfer based on Langendorff rodent heart models.

Fifteen to thirty percent of patients with impaired cardiac function have ventricular dyssynchrony and warrant cardiac resynchronization therapy (CRT). While leadless pacemakers eliminate lead-related complications, their current form factor is limited to single-chamber pacing. In this study, we demonstrate the feasibility of multisite, simultaneous pacing using miniaturized pacing nodes powered through wireless power transfer (WPT). A wireless energy transfer system was developed based on resonant coupling at approximately 200 MHz to power multiple pacing nodes. The pacing node comprises circuitry to efficiently convert the harvested energy to output stimuli. To validate the use of these pacing nodes, ex vivo studies were carried out on Langendorff rodent heart models (n = 4). To mimic biventricular pacing, two beating Langendorff rodent heart models, kept 10 cm apart, were paced using two distinct pacing nodes, each attached on the ventricular epicardial surface of a given heart. All ex vivo Langendorff heart models were successfully paced with a simple coil antenna at 2 to 3 cm from the pacing node. The coil was operated at 198 MHz and 0.3 W. Subsequently, simultaneous pacing of two Langendorff heart models 30 cm apart using an output power of 5 W was reliably demonstrated. WPT provides a feasible option for multisite, wireless cardiac pacing. While the current system remains limited in design, it offers support and a conceptual framework for future iterations and eventual clinical utility.

Isolated perfused working hearts provide valuable additional information during phenotypic assessment of the diabetic mouse heart.

Although murine models for studying the development of cardiac dysfunction in diabetes mellitus are well established, their reported cardiac phenotypes vary. These reported divergences may, in addition to the severity of different models, also be linked to the methods used for cardiac functional assessment. In the present study, we examined the functional changes using conventional transthoracic echocardiography (in vivo) and isolated heart perfusion techniques (ex vivo), in hearts from two mouse models; one with an overt type 2 diabetes (the db/db mouse) and one with a prediabetic state, where obesity was induced by a high-fat diet (HFD). Analysis of left ventricular function in the isolated working hearts from HFD-fed mice, suggested that these hearts develop diastolic dysfunction with preserved systolic function. Accordingly, in vivo examination demonstrated maintained systolic function, but we did not find parameters of diastolic function to be altered. In db/db mice, ex vivo working hearts showed both diastolic and systolic dysfunction. Although in vivo functional assessment revealed signs of diastolic dysfunction, the hearts did not display reduced systolic function. The contrasting results between ex vivo and in vivo function could be due to systemic changes that may sustain in vivo function, or a lack of sensitivity using conventional transthoracic echocardiography. Thus, this study demonstrates that the isolated perfused working heart preparation provides unique additional information related to the development of cardiomyopathy, which might otherwise go unnoticed when only using conventional echocardiographic assessment.

Knockout of TRPV1 Exacerbates Left Ventricular Diastolic Dysfunction Induced by A High-fat Diet in Mice.

Transient Receptor Potential Vanilloid 1 (TRPV1) channels in sensory nerves have anti-oxidative properties and counteract obesity and diabetes that are associated with diastolic dysfunction with preserved ejection fraction. We tested the hypothesis that TRPV1 knockout exacerbates high-fat diet (HFD)-induced glucose intolerance and diastolic dysfunction. Trpv1-/- and wild-type (WT) mice were fed chow diet or HFD for 20 weeks. Then, we performed the intraperitoneal glucose tolerance test, measured the heart function through transthoracic echocardiography and Langendorff heart perfusion system, analyzed cardiac histology, and measured the myocardial superoxide production and the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. HFD increased body weight, heart weight, and levels of fasting glucose, insulin, and leptin in both strains, with no differences between two strains. HFD impaired glucose tolerance in both strains with a more profound effect on Trpv1-/- than WT mice. HFD increased left ventricular (LV) internal diameter in diastole in both strains, while increased LV posterior wall thickness in diastole in Trpv1-/- but not in WT mice. HFD increased LV end-diastolic pressure in both strains with a further increase in Trpv1-/- mice, while decreased -dP/dt in Trpv1-/- but not in WT mice. HFDinduced cardiac collagen deposition and superoxide production were enhanced in Trpv1-/- mice. HFD upregulated cardiac p22phox in both strains, while increased p47phox in Trpv1-/- but not in WT mice. In summary, TRPV1 knockout exacerbates HFD-induced glucose intolerance, cardiac oxidative stress and collagen deposition, leading to aggravated LV diastolic dysfunction.

Paclitaxel enhances the protective effect of myocardial ischemia preconditioning on ischemia/reperfusion injury in aged rat

Objective: To investigate if paclitaxel can enhance the protective effect of myocardial ischemia preconditioning on ischemia/reperfusion injury in aged ratand explore related mechanism. Methods: Primary cardiomyocytes of Sprague-Dawley rats were isolated by trypsin and divided into 5 groups(n=6 each): control group, hypoxia injury group, hypoxia preconditioning group, paclitaxel group,and paclitaxel+hypoxia preconditioning group. The structure of microtubules and the expression of hypoxia-inducible factor-1α(HIF-1α) were analyzed by immunofluorescence staining. The Langendorff isolated heart perfusion model was applied in 4 groups: hypoxia reperfusion injury group, hypoxia preconditioning group, paclitaxel group, and paclitaxel+hypoxia preconditioning group. Each group was further divided into elderly subgroup and adult subgroup (n=6 each). Left ventricular developed pressure and maximum rate of rise in left ventricular pressure were analyzed. Results: (1) Primary cardiomyocyte experiments showed that the myocardial tubular microtubule structure in control group was intact and evenly stained; most of the microtubules in the hypoxia-injured group were absent and the tubular tissue was broken; the hypoxia-induced damage on microtubule structure was smaller in the hypoxic preconditioning group compared with the hypoxic injury group (microtubule staining was not uniform, and the lattice structure was broken, but not that obvious as in the hypoxia group); the tubular structure of the microtubules of the paclitaxel group was basically complete, and the staining was basically uniform.The integrity of tubular structure was maintained to some extent, similar to a normal microtubule structure in paclitaxel+hypoxia preconditioning group. The expression of HIF-1α in the cytoplasm and nucleus was very low in the control group, which was evidenced in both cytoplasm and nucleus in the hypoxic injury group.The expression was further increased in hypoxic preconditioning group, significant nuclear HIF-1 expression was found in the paclitaxel group, the expression was aggregated in the nucleus in the Paclitaxel+ hypoxia preconditioning group. (2)In Langendorff isolated heart perfusion model, left ventricular developed pressure was similar between the elderly subgroup and the adult subgroup at the end of the infusion,after precondition, 5 minutes of reperfusion, 30 minutes of reperfusion, and 60 minutes of reperfusion in the hypoxic injury group (all P> 0.05).In the hypoxic injury group, both the elderly subgroup and the adult subgroup had lower left ventricular developed pressure at 30 minutes of reperfusion when compared with the end of the infusion((15.63±4.88) mmHg (1 mmHg=0.133 kPa) vs. (95.63±22.14)mmHg and (17.31±2.75)mmHg vs. (91.00±9.58)mmHg, respectively,all P<0.05). In the hypoxic preconditioning group, the adult subgroup had higher left ventricular developed pressure at 5 and 30 minutes of reperfusion when compared with the elderly subgroup((7.13±1.02) mmHg vs. (3.75±1.06)mmHg and (43.94±3.21)mmHg vs.(16.31±1.54)mmHg, respectively,all P<0.01). In the paclitaxel group, the adult subgroup had higher left ventricular developed pressure at 30 and 60 minutes of reperfusion when compared with the elderly subgroup((44.31±7.59)mmHg vs. (5.44±1.21)mmHg, (51.56±6.03)mmHg vs. (22.19±5.14)mmHg, respectively, all P<0.01). In the paclitaxel+hypoxia preconditioning group, both the elderly subgroup and the adult subgroup had lower left ventricular developed pressure at 30 minutes of reperfusion when compared with the end of the infusion((18.63±4.30)mmHg vs. (99.94±8.23) mmHg, P<0.01; (49.69±5.34)mmHg vs. (95.31±5.26)mmHg, P<0.05). Meanwhile, the adult subgroup had higher left ventricular developed pressure at 30 minutes of reperfusion when compared with the elderly subgroup((49.69±5.34)mmHg vs. (18.63±4.33)mmHg, P<0.01).The adult subgroup had higher change rate of maximum rate of rise in left ventricular pressure at 60 minutes of reperfusion when compared with the elderly subgroup in hypoxia preconditioning group, paclitaxel group, and paclitaxel combined hypoxia preconditioning group((62.83±3.92)% vs. (33.33±3.20)%, (44.17±2.32)% vs. (36.67±2.88)%, (72.50±2.66)% vs. (53.17±2.56)%, respectively,all P<0.01). Conclusion: Paclitaxel can enhance the myocardial protective effect of myocardial ischemia preconditioning through stabilizing microtubules of cardiomyocytes and promoting HIF-1α localization in the nucleus.

Integration of cardiac energetics, function and histology from isolated rat hearts perfused with doxorubicin and doxorubicin-ol; a model for use in drug safety evaluations

The isolated rat heart (Langendorff) assay combined with NMR spectroscopy and histology were used to elucidate functional, metabolic, and histological signs of cardiotoxicity resulting from acute exposure to clinically relevant concentrations of doxorubicin and its metabolite dox-ol. Doxorubicin blood concentrations and pharmacokinetic parameters were assessed following a clinically relevant dose of 2 mg/kg in order to select concentrations for isolated heart perfusions. Isolated rat hearts were exposed to 1 or 10 μM of doxorubicin or 0.3 μM dox-ol for at least 60 min using the Langendorff perfusion method. Effects on heart function were monitored using ECGs, left ventricular contraction parameters, and microscopic histology. Cardiac energetics (PCr, ATP, and Pi) were evaluated before, during, and after exposure to doxorubicin/dox-ol in perfused hearts using NMR spectroscopy. Cardiac effects were evident following clinically relevant concentrations of doxorubicin and dox-ol in isolated rat hearts demonstrated by altered heart function, energetic reserve, and microscopic lesions. A cardiac stress test utilizing isoproterenol resulted in enhanced functional response and reductions in PCr in doxorubicin versus vehicle treated hearts indicating possible alterations in the isoproterenol mediated pathway. Dox-ol treated hearts were similar to control with regard to function, but exhibited histologic findings. The use of combined Langendorff/NMR/histology methodologies allowed for comparison of multiple indices of cardiac function at one time in which cardiac effects were evident in multiple parameters. Short abstractThe isolated rat heart assay combined with NMR spectroscopy and histology was used to elucidate functional, metabolic, and histological signs of cardiotoxicity resulting from acute exposure to clinically relevant concentrations of doxorubicin and its metabolite dox-ol. Heart function was altered and microscopic signs of toxicity were evident with dox and dox-ol exposures. The use of combined Langendorff/NMR/histology assays allowed for comparison of multiple indices of cardiac function at one time in which cardiac effects were evident in multiple parameters.

A Langendorff-like system to quantify cardiac pump function in adult zebrafish.

ABSTRACTZebrafish are increasingly used as a vertebrate model to study human cardiovascular disorders. Although heart structure and function are readily visualized in zebrafish embryos because of their optical transparency, the lack of effective tools for evaluating the hearts of older, nontransparent fish has been a major limiting factor. The recent development of high-frequency echocardiography has been an important advance for in vivo cardiac assessment, but it necessitates anesthesia and has limited ability to study acute interventions. We report the development of an alternative experimental ex vivo technique for quantifying heart size and function that resembles the Langendorff heart preparations that have been widely used in mammalian models. Dissected adult zebrafish hearts were perfused with a calcium-containing buffer, and a beat frequency was maintained with electrical stimulation. The impact of pacing frequency, flow rate and perfusate calcium concentration on ventricular performance (including end-diastolic and end-systolic volumes, ejection fraction, radial strain, and maximal velocities of shortening and relaxation) were evaluated and optimal conditions defined. We determined the effects of age on heart function in wild-type male and female zebrafish, and successfully detected hypercontractile and hypocontractile responses after adrenergic stimulation or doxorubicin treatment, respectively. Good correlations were found between indices of cardiac contractility obtained with high-frequency echocardiography and with the ex vivo technique in a subset of fish studied with both methods. The ex vivo beating heart preparation is a valuable addition to the cardiac function tool kit that will expand the use of adult zebrafish for cardiovascular research.

Pharmacological postconditioning with Neuregulin-1 mimics the cardioprotective effects of ischaemic postconditioning via ErbB4-dependent activation of reperfusion injury salvage kinase pathway

BackgroundThe protective effect of Neuregulin-1 (NRG-1) on heart failure is well established. In this study, we assessed whether NRG-1 could protect the heart by mimicking the cardioprotective effects of ischaemic postconditioning (IP).MethodsWe used a myocardial reperfusion injury rat model in vivo to compare the cardioprotective effects of NRG-1(3 μg/kg, iv. at the onset of reperfusion) and IP. In Langendorff isolated heart perfusion experiments, we used the erythroblastic leukaemia viral oncogene homolog 4 (ErbB4) inhibitor AG1478, a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and a mitogen-activated protein/extracellular signal regulated kinase (MEK) inhibitor PD98059 to clarify whether the protective effects of NRG-1and IP depend on the NRG-1/ErbB4 signals and the reperfusion injury salvage kinase (RISK) pathway. Infarct size was detected by Evans blue and TTC. Apoptosis was detected by TUNEL assays. The expression of NRG-1/ErbB4 and downstream ERK1/2, AKT, AMPK and p70s6K were detected by western blotting. Hematoxylin/eosin (H&E) staining was used for histological analysis.ResultsWe found that NRG-1 and IP had similar effects on reducing myocardial infarct size and apoptosis in vivo. NRG-1 heart protein levels were upregulated in the IP group. Phosphorylation of AKT, ERK1/2 and ErbB4 were also increased in both the IP and NRG-1 groups. Furthermore, in Langendorff analyses, the ErbB4 inhibitor AG1478 suppressed the phosphorylation of ErbB4 and the RISK pathway and aggravated myocardial edema and fiber fracture, thereby inhibited the cardioprotective effects in both the IP and NRG-1 groups. For assessment of downstream signals, the PI3K inhibitor LY294002 and the MEK inhibitor PD98059 suppressed the phosphorylation of AKT and ERK1/2 respectively and abolished the cardioprotective effects induced by IP and NRG-1.ConclusionIn conclusion, both IP and NRG-1 could reduce infarct size and apoptosis through ErbB4-dependent activation of the RISK pathway in the same model; these results indicated the therapeutic potential of NRG-1 as a pharmacological postconditioning agent against myocardial reperfusion injury.

A Multi-site Heart Pacing Study Using Wirelessly Powered Leadless Pacemakers.

In this work, we report an energy-efficient switched capacitor based millimeter-scale pacemaker (5 mm ×7.5 mm) and a multi-receiver wireless energy transfer system operating at around 200 MHz, and use them in a proof-of-concept multi-site heart pacing study. Two pacemakers were placed on two beating Langendorff rodent heart models separately. By utilizing a single transmitter positioned 20-30 cm away, both Langendorff hearts captured the stimuli simultaneously and were electromechanically coupled. This study provides an insight for future energy-efficient and distributed cardiac pacemakers that can offer cardiac resynchronization therapies.

Melatonin improves cardiac and mitochondrial function during doxorubicin-induced cardiotoxicity: A possible role for peroxisome proliferator-activated receptor gamma coactivator 1-alpha and sirtuin activity?

Mitochondrial dysfunction is a central element in the development of doxorubicin (DXR)-induced cardiotoxicity. In this context, melatonin is known to influence mitochondrial homeostasis and function. This study aimed to investigate the effects of melatonin on cardiac function, tumor growth, mitochondrial fission and fusion, PGC1-α and sirtuin activity in an acute model of DXR-induced cardiotoxicity. During the in vitro study, H9c2 rat cardiomyoblasts were pre-treated with melatonin (10 μM, 24 h) followed by DXR exposure (3 μM, 24 h). Following treatment, cellular ATP levels and mitochondrial morphology were assessed. In the in vivo study, female Sprague Dawley rats (16 weeks old), were inoculated with a LA7 rat mammary tumor cell line and tumors were measure daily. Animals were injected with DXR (3 × 4 mg/kg) and/or received melatonin (6 mg/kg) for 14 days in their drinking water. Rat hearts were used to conduct isolated heart perfusions to assess cardiac function and thereafter, heart tissue was used for immunoblot analysis. DXR treatment increased cell death and mitochondrial fission which were reduced with melatonin treatment. Cardiac output increased in rats treated with DXR + melatonin compared to DXR-treated rats. Tumor volumes was significantly reduced in DXR + melatonin-treated rats on Day 8 in comparison to DXR-treated rats. Furthermore, DXR + melatonin treatment increased cellular ATP levels, PGC1-α and SIRT1 expression which was attenuated by DXR treatment. These results indicate that melatonin treatment confers a dual cardio-protective and oncostatic effect by improving mitochondrial function and cardiac function whilst simultaneously retarding tumor growth during DXR-induced cardiotoxicity.

Matrixmetalloproteinases and tissue inhibitors of metalloproteinases: Immunhistochemical markers in the diagnosis of lethal myocardial infarctions?

Matrixmetalloproteinases (MMP) 2 and 9 as well as tissue inhibitor of metalloproteinases (TIMP) 1 were tested as markers of myocardial early ischemia/infarctions. Experiments with an animal model, the isolated Langendorff heart, and analysis of human tissue samples drawn during autopsies were performed. Results of the experiments with the Langendorff model implied that the detectable amount of the markers might increase early after the onset of ischemia, in less than one hour, under ideal conditions. The results of the examined human cases showed that MMP-2 is constantly detectable in human myocardial tissue with an increased amount in case of an infarction with longer survival times. MMP-9 and TIMP-1 were negative in control cases, distinct positive staining results were obtained mainly in cases of infarctions with longer survival times and only rarely in those with a short survival time. According to these results MMPs and TIMPs do not qualify as first choice markers of myocardial infarctions. As an interesting side finding in the Langendorff experiments, positive staining results for all three markers were seen in myocardial areas that were mechanically traumatized by ECG-electrodes or ligation of blood vessels. These findings make the markers interesting for forensic wound age estimation.

Upregulation of vascular endothelial growth factor receptor-1 contributes to sevoflurane preconditioning-mediated cardioprotection.

PurposeSevoflurane preconditioning (SPC) can provide myocardial protective effects similar to ischemic preconditioning. However, the exact mechanism of SPC remains unclear. Previous studies indicate that vascular endothelial growth factor receptor 1 (VEGFR-1) is involved in ischemic preconditioning-mediated cardioprotection. This study was designed to determine the significance of VEGFR-1 signaling in SPC-mediated cardioprotection.Materials and methodsMyocardial ischemia–reperfusion (I/R) rat model was established using the Langendorff isolated heart perfusion apparatus. Additionally, after 15 min of baseline equilibration, the isolated hearts were pretreated with 2.5% sevoflurane, 2.5% sevoflurane+MF1 10 μmol/L, or 2.5% sevoflurane+placental growth factor 10 μmol/L, and then subjected to 30 min of global ischemia and 120 min of reperfusion. The changes in hemodynamic parameters, myocardial infarct size, and the levels of creatine kinase-MB, lactate dehydrogenase, cardiac troponin-I, tumor necrosis factor-α, and interleukin 6 in the myocardium were evaluated.ResultsCompared to the I/R group, pretreatment with 2.5% sevoflurane significantly improved the cardiac function, limited myocardial infarct size, reduced cardiac enzyme release, upregulated VEGFR-1 expression, and decreased inflammation. In addition, the selective VEGFR-1 agonist, placental growth factor, did not enhance the cardioprotection and anti-inflammation effects of sevoflurane, while the specific VEGFR-1 inhibitor, MF1, completely reversed these effects.ConclusionOur data have demonstrated that 2.5% sevoflurane preconditioning alleviates heart I/R injury, which is probably mediated by the anti-inflammatory property and upregulation of VEGFR-1.