Langendorff Heart Preparation Research Articles

Defining cardiac stability during Langendorff perfusion

The Langendorff heart preparation is a valuable ex vivo technique for studying cardiac electrophysiology. For the past century it has been used in toxicology studies, drug testing, and disease modeling and has contributed significantly to understanding cardiac physiology, development, and pathological states. This study aimed to investigate the stability and reproducibility of the modern Langendorff heart preparation under various experimental conditions and to establish cardiac metric standards with corresponding exclusion criteria. Isolated hearts were extracted from adult Sprague Dawley rats and retrograde perfused with a modified Krebs-Henseleit buffer. A cohort of isolated hearts were used to establish a control baseline of multiple physiological parameters measured over time (e.g., electrocardiogram, flow rate, electrophysiology, electrical and calcium dynamics). These control baseline values were then compared to hearts exposed to the most common media additives in Langendorff studies (e.g., blebbistatin(−/−), bovine serum albumin, and voltage and calcium indicator dye). Over a three-hour period, cardiac metrics were monitored in stable Langendorff-perfused hearts. The addition of blebbistatin, DMSO or dyes did not change EP or ECG measurements as compared to control baseline. The most significant drift in all the EP values was ventricular effective refractory period (VERP). With or without blebbistatin, VERP increased from 40 to 60ms over the course of three hours. In that time heart rate and flow also decreased slightly. Blebbistatin was associated with an increased action potential duration but did not alter calcium transient duration. It was also observed that 1% circulating bovine serum albumin quenched the rh237 voltage dye signal 80% as measured by signal to noise. Constant pressure Langendorff-perfusion with a crystalloid buffer provides a reliable and reproducible model for cardiac electrophysiology research with a few caveats. The total length of study should be carefully considered to account for adaptations in cardiac physiology during longer perfusion times. As an excitation-contraction uncoupler, blebbistatin reduces motion artifact but delays action potential duration. Although BSA is used to minimize cardiac edema, it interferes with membrane bound voltage dyes. This study sets physiological parameters as a useful guide for future cardiac studies that use the isolated heart as a research model. American Physiological Society, R01HD108839 to N.G.P. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Utilization of the genetically encoded calcium indicator Salsa6F in cardiac applications

Calcium signaling is a critical process required for cellular mechanisms such as cardiomyocyte contraction. The inability of the cell to properly activate or regulate calcium signaling can lead to contractile dysfunction. In isolated cardiomyocytes, calcium signaling has been primarily studied using calcium fluorescent dyes, however these dyes have limited applicability to whole organs. Here, we crossed the Salsa6f mouse which expresses a genetically encoded ratiometric cytosolic calcium indicator with a cardiomyocyte specific inducible cre to temporally-induce expression and studied cytosolic calcium transients in isolated cardiomyocytes and modified Langendorff heart preparations. Isolated cardiomyocytes expressing Salsa6f or Fluo-4AM loaded were compared. We also crossed the Salsa6f mouse with a floxed Polycystin 2 (PC2) mouse to test the feasibility of using the Salsa6f mouse to measure calcium transients in PC2 heterozygous or homozygous knock out mice. Although there are caveats in the applicability of the Salsa6f mouse, there are clear advantages to using the Salsa6f mouse to measure whole heart calcium signals.

Endothelium-Derived Dopamine and 6-Nitrodopamine in the Cardiovascular System.

The review deals with the release of endothelium-derived dopamine and 6-nitrodopamine (6-ND) and its effects on isolated vascular tissues and isolated hearts. Basal release of both dopamine and 6-ND is present in human isolated umbilical cord vessels, human popliteal vessels, nonhuman primate vessels, and reptilia aortas. The 6-ND basal release was significantly reduced when the tissues were treated with Nω-nitro-l-arginine methyl ester and virtually abolished when the endothelium was mechanically removed. 6-Nitrodopamine is a potent vasodilator, and the mechanism of action responsible for this effect is the antagonism of dopamine D2-like receptors. As a vasodilator, 6-ND constitutes a novel mechanism by which nitric oxide modulates vascular tone. The basal release of 6-ND was substantially decreased in endothelial nitric oxide synthase knockout (eNOS-/-) mice and not altered in neuronal nitric oxide synthase knockout (nNOS-/-) mice, indicating a nonneurogenic source for 6-ND in the heart. Indeed, in rat isolated right atrium, the release of 6-ND was not affected when the atria were treated with tetrodotoxin. In the rat isolated right atrium, 6-ND is the most potent endogenous positive chronotropic agent, and in Langendorff's heart preparation, it is the most potent endogenous positive inotropic agent. The positive chronotropic and inotropic effects of 6-ND are antagonized by β1-adrenoceptor antagonists at concentrations that do not affect the effects induced by noradrenaline, adrenaline, and dopamine, indicating that blockade of the 6-ND receptor is the major modulator of heart chronotropism and inotropism. The review proposes that endothelium-derived catecholamines may constitute a major mechanism for control of vascular tone and heart functions, in contrast to the overrated role attributed to the autonomic nervous system.

Abstract 12951: Electrical and Calcium-Dependent Mechanisms That Contribute to Increase the Risk for Atrial Fibrillation in Obesity

Obesity (OB) is an independent predictor for atrial fibrillation (AF), but mechanisms by which OB increases AF risk remain poorly characterized. We hypothesized that OB modulates AF risk through electrical remodeling and impaired Ca 2+ dynamics that can be reversed after weight loss (WL). Using transesophageal rapid pacing we characterized AF inducibility and burden in lean, OB, and OB mice that lost 20% of body weight after modified vertical sleeve gastrectomy. Local field fluorescence microscopy was used to simultaneously record action potentials (APs) (di-8-ANEPPS or glass microelectrodes) and Ca 2+ transients (CaT) (Rhod-2) in Langendorff heart preparations. We evaluated AP duration (APD) dependency of frequency from 4 to 14 Hz (1 Hz incremental steps). AP parameters analyzed included APD30, APD50, APD90 and spontaneous arrhythmogenic events. We assessed Ca 2+ dynamics by studying CaT and fractional sarcoplasmic reticulum Ca 2+ release in the absence and presence of ryanodine (Ry) and thapsigargin (Tg). Since CaT Ry+Tg represents sarcolemmal Ca 2+ influx, we calculated the gain of Ca 2+ induced Ca 2+ release (CICR gain ) as the ratio between CaT in the absence and presence of Ry+Tg. Experiments were done in the presence of blebbistatin at 37° C. Mean±SE, t-test. After burst pacing, both AF prevalence and burden were higher in OB mice vs lean (94 % vs 47 %, 227±19 vs 20±1.2 s, n=16 mice/group, p<0.0001). About 76 % of OB hearts showed afterdepolarizations and fibrillation vs no events in lean. APD50 and APD90 significantly decreased as stimulation frequency increased from 4 to 14 Hz in OB hearts vs lean (n=6 mice/group, p<0.05). Peak CaT from OB hearts were significantly decreased compared to peak CaT from lean (0.3±0.05 vs. 0.8±0.06, n=9, p<0.001). CICR gain in OB hearts was also significantly compromised compared to lean (5.2±1 vs 23.3±1.4, n=7 mice/group, p<0.0001). After WL, OB mice showed a decreased AF burden (21.7±8 s), increased APD, increased peak CaT (0.8±0.15) and increased CICR gain (20±2.3) back to lean levels (n=8, p<0.001). In conclusion, shortened APs, spontaneous afterdepolarizations and impaired CICR significantly contribute to the development of AF in OB. WL reversed the electrical phenotype and the impaired Ca 2+ dynamics and prevented AF.

Cardiovascular and hemodynamic consequences of recombinant placental growth factor administration in Guinea pigs

ABSTRACT Placental growth factor (PlGF), a member of the vascular endothelial growth factor family of proteins regulating angiogenesis, has been shown to have acute vasodilatory effects on human resistance arteries. However, the acute hemodynamic effects of PlGF in vivo are not known. The aim of this study was to report acute cardiovascular changes induced by recombinant human PlGF administered intravenously in male Guinea Pigs with implanted telemeters. PlGF decreased mean arterial blood pressure by 10–20% within minutes. The magnitude of reduction was similar at three dose levels; however, the duration of relative hypotension was dose-dependent. Blood pressure reduction resulted in a compensatory increase in heart rate, or reflex tachycardia. To rule out any direct effect on the heart, PlGF was tested in the ex vivo Langendorff heart preparation, and no cardiac changes were found. Together these results suggest that the PlGF-related changes in blood pressure are largely mediated by its actions in the vasculature.

Ryanodine receptor inhibition prevents ventricular arrhythmia in a murine model of hypokalaemia

Abstract Background Hypokalaemia-induced arrhythmia is a major cause of morbidity and mortality, affecting approximately 14% of outpatients and 20% of inpatients. Hypokalaemic inpatients have a mortality 10 times higher than their counterparts. This is due to ventricular arrhythmias initiated by early afterdepolarisations (EADs) and triggered activity. There is no antiarrhythmic drug approved for the prevention of hypokalaemia-induced ventricular arrhythmia. Recent evidence suggests that EADs can arise because of the synchronised release of Ca2+ from the sarcoplasmic reticulum, rather than the reactivation of L-type Ca2+ channels during action potential prolongation. Therefore, ryanodine receptor inhibition should prevent hypokalaemia-induced ventricular arrhythmia. Purpose To test the hypothesis that the ryanodine receptor inhibitors dantrolene and VKII-86 (a carvedilol analogue) are effective in preventing hypokalaemia-induced ventricular arrhythmia. Methods Whole heart Langendorff preparations isolated from C57BL/J6 mice were perfused with Krebs-Henseleit solution containing normal (4mM) or low (2mM) concentrations of [K+]. Surface ECG and subepicardial ventricular action potentials were simultaneously recorded. After establishing that the model reproduces EAD-mediated triggered arrhythmias in low extracellular [K+], hearts in the treatment groups were pre-treated with dantrolene or VKII-86 for 30 minutes prior to being exposed to low extracellular [K+]. The frequency of non-sustained and sustained ventricular arrhythmias occurring during a 30-minute period of exposure to low extracellular [K+] were compared between the treatment groups and controls treated with the solvent DMSO (0.1% v/v) (n=6 in each group). Results The mean number of non-sustained arrhythmias was 29.3±9.6 (Mean±SEM) in the control group, 3.2±1.2 in the dantrolene group and 0 in the VKII-86 group; p<0.05 for both treatments vs. control. The mean number of sustained arrhythmias was 1.7±0.4 in the control group, 0.17±0.17 in the dantrolene group and 0 in the VKII-86 group; p<0.05 for both treatments vs. control. Thus, dantrolene significantly reduced arrhythmia frequency by approximately 90%, whereas VKII-86 prevented all hypokalaemia-induced ventricular arrhythmias. Conclusions These results provide the first demonstration of ryanodine receptor inhibition as an effective treatment for the prevention of ventricular arrhythmia in a murine model of hypokalaemia. This study is consistent with recent studies indicating that intracellular Ca2+ load is an important mechanism underlying the development of EAD-mediated triggered arrythmia. Further studies are needed to determine whether these drugs could be re-purposed as antiarrhythmics in the setting of hypokalemia. Further development of the carvedilol analogue VKII-86 should also be considered. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): The British Heart Foundation, The Lankenau Institute for Medical Research.

Elevated perfusate [Na+] increases contractile dysfunction during ischemia and reperfusion

Recent studies revealed that relatively small changes in perfusate sodium ([Na+]o) composition significantly affect cardiac electrical conduction and stability in contraction arrested ex vivo Langendorff heart preparations before and during simulated ischemia. Additionally, [Na+]o modulates cardiomyocyte contractility via a sodium-calcium exchanger (NCX) mediated pathway. It remains unknown, however, whether modest changes to [Na+]o that promote electrophysiologic stability similarly improve mechanical function during baseline and ischemia–reperfusion conditions. The purpose of this study was to quantify cardiac mechanical function during ischemia–reperfusion with perfusates containing 145 or 155 mM Na+ in Langendorff perfused isolated rat heart preparations. Relative to 145 mM Na+, perfusion with 155 mM [Na+]o decreased the amplitude of left-ventricular developed pressure (LVDP) at baseline and accelerated the onset of ischemic contracture. Inhibiting NCX with SEA0400 abolished LVDP depression caused by increasing [Na+]o at baseline and reduced the time to peak ischemic contracture. Ischemia–reperfusion decreased LVDP in all hearts with return of intrinsic activity, and reperfusion with 155 mM [Na+]o further depressed mechanical function. In summary, elevating [Na+]o by as little as 10 mM can significantly modulate mechanical function under baseline conditions, as well as during ischemia and reperfusion. Importantly, clinical use of Normal Saline, which contains 155 mM [Na+]o, with cardiac ischemia may require further investigation.

Involvement of miR-665 in protection effect of dexmedetomidine against Oxidative Stress Injury in myocardial cells via CB2 and CK1

BackgroundDexmedetomidine (Dex) can confer cardioprotective effects against ischemia/reperfusion (I/R) injury. While there are no studies addressing cardioprotection of Dex via regulation of microRNAs. The purpose of this study was to examine the roles and mechanisms of microRNA in cardioprotection of dexmedetomidine. MethodsRat heart Langendorff preparation was established. We assayed expression profiling of miRNAs in perfused rat hearts and predicted Target genes using MiRanda, MiRDB, and TargetScan. Oxide stress (H2O2) was employed to simulate I/R injury. miR-665 mimic, inhibitor, and siRNA of AK1 and Cnr2 were transfected to H9C2. The real-time quantitative polymerase chain reaction was used to quantify miR-665 and Ak1 and Cnr2 mRNA. The apoptosis of the cells was examined. The expression levels of cleaved caspase-3, Bcl-2, Bax, AK1, and Cnr2 were detected by Western blot. The combination between miR-665 and the 3′-untranslated region of AK1 and Cnr2 was validated by a luciferase reporter assay. ResultsDex precondition down-regulated miR-665 expression in hearts compared to I/R group. Dex reduced miR-665 expression and apoptosis increased by oxide stress. However, up-regulation of miR-665 exacerbated the changes caused by oxide stress and inhibited the effects of Dex. Down-regulation of miR-665 also reduced apoptosis, but inhibition of AK1 and Cnr2 aggravated apoptosis. The luciferase reporter assay indicated that miR-665 could down-regulate expression levels of AK1 and Cnr2. ConclusionsDex precondition confers hearts protective effect against I/R injury by down-regulating expression of miR-665 and up-regulating expression of AK1 and Cnr2.

Synthesis and biological evaluation of pyrazolo[3,4-b]pyridine-3-yl pyrimidine derivatives as sGC stimulators for the treatment of pulmonary hypertension

A series of new pyrazolo[3,4-b]pyridin-3-yl pyrimidine derivatives were synthesized and evaluated for the activation of sGC. Compared with riociguat, compound 13a exhibited equivalent in vitro activity on preconstricted rat thoracic aorta rings and in Rat heart Langendorff preparation. Compound 13a also showed acceptable PK profiles, which might become a promising candidate for the treatment of pulmonary hypertension.

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.

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.

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.

Noise enhanced the electrical stimulation-contractile response coupling in isolated mouse heart

BackgroundStochastic resonance is a phenomenon that allows a system to improve its capability to detect stimulus when a limited amount of noise is added to the stimuli. It has experimentally been shown that noise enhances the homeostatic function of the blood pressure regulatory system. This study aimed to investigate whether the noise can enhance the contractile response in the whole heart. MethodsExperiments were conducted in isolated mouse hearts (0.040kg, n=8), a Langendorff heart preparation is used to obtain two variables of the contractile response contraction force and heart rate. The contractile response due to an electrical stimulation perturbed with Gaussian noise was recorded. ResultsThe results show that the intensity of noise induced in the electrical stimuli has an effect on the electrical stimulation-contractile response coupling. With 10% noise induced, the bandwidth where the synchronization effect is presented was increased from (7–11Hz) to (6–12Hz), and the irregular dynamic threshold was changed to 13Hz. ConclusionsWe find that the noise increases the synchronization bandwidth in the electrical stimulation-contractile response coupling. We have experimentally demonstrated the stochastic resonance in isolated mouse heart.

Contractile function assessment by intraventricular balloon alters the ability of regional ischaemia to evoke ventricular fibrillation.

In drug research using the rat Langendorff heart preparation, it is possible to study left ventricular (LV) contractility using an intraventricular balloon (IVB), and arrhythmogenesis during coronary ligation-induced regional ischaemia. Assessing both concurrently would halve animal requirements. We aimed to test the validity of this approach. The electrocardiogram (ECG) and LV function (IVB) were recorded during regional ischaemia of different extents in a randomized and blinded study. IVB-induced proarrhythmia was anticipated, but in hearts with an ischaemic zone (IZ) made deliberately small, an inflated IVB reduced ischaemia-induced ventricular fibrillation (VF) incidence as a trend. Repeating studies in hearts with large IZs revealed the effect to be significant. There were no changes in QT interval or other variables that might explain the effect. Insertion of an IVB that was minimally inflated had no effect on any variable compared with 'no IVB' controls. The antiarrhythmic effect of verapamil (a positive control drug) was unaffected by IVB inflation. Removal of an inflated (but not a non-inflated) IVB caused a release of lactate commensurate with reperfusion of an endocardial/subendocardial layer of IVB-induced ischaemia. This was confirmed by intracellular (31) phosphorus ((31) P) nuclear magnetic resonance (NMR) spectroscopy. IVB inflation does not inhibit VF suppression by a standard drug, but it has profound antiarrhythmic effects of its own, likely to be due to inflation-induced localized ischaemia. This means rhythm and contractility cannot be assessed concurrently by this approach, with implications for drug discovery and safety assessment.

From Metabolomics to Fluxomics: A Computational Procedure to Translate Metabolite Profiles into Metabolic Fluxes

We describe a believed-novel procedure for translating metabolite profiles (metabolome) into the set of metabolic fluxes (fluxome) from which they originated. Methodologically, computational modeling is integrated with an analytical platform comprising linear optimization, continuation and dynamic analyses, and metabolic control. The procedure was tested with metabolite profiles obtained from ex vivo mice Langendorff-heart preparations perfused with glucose. The metabolic profiles were analyzed using a detailed kinetic model of the glucose catabolic pathways including glycolysis, pentose phosphate (PP), glycogenolysis, and polyols to translate the glucose metabolome of the heart into the fluxome. After optimization, the ability of the model to simulate the initial metabolite profile was confirmed, and metabolic fluxes as well as the structure of control and regulation of the glucose catabolic network could be calculated. We show that the step catalyzed by phosphofructokinase together with ATP demand and glycogenolysis exert the highest control on the glycolytic flux. The negative flux control exerted by phosphofructokinase on the PP and polyol pathways revealed that the extent of glycolytic flux directly affects flux redirection through these pathways, i.e., the higher the glycolytic flux the lower the PP and polyols. This believed-novel methodological approach represents a step forward that may help in designing therapeutic strategies targeted to diagnose, prevent, and treat metabolic diseases.

The heart's ‘little brain’ controlling cardiac function in the rabbit

New Findings•What is the topic of this review?The topic of the review is the intrinsic cardiac nervous system in the rabbit.•What advances does it highlight?The anatomy of rabbit intrinsic ganglia is similar to that of other species, including humans. Immunohistochemistry confirms the presence of cholinergic and adrenergic neurones, with a striking arrangement of neuronal nitric oxide synthase-positive cell bodies. Activation of atrial ganglia produces effects on local and remote regions.Heart disease is a primary cause of mortality in the developed world, and it is well recognized that neural mechanisms play an important role in many cardiac pathologies. The role of extrinsic autonomic nerves has traditionally attracted the most attention. However, there is a rich intrinsic innervation of the heart, including numerous cardiac ganglia (ganglionic plexuses), that has the potential to affect cardiac function independently as well as to influence the actions of the extrinsic nerves. To investigate this, an isolated, perfused, innervated rabbit Langendorff heart preparation was considered the best option. Although ganglionic plexuses have been well described for several species, there was no full description of the anatomy and histochemistry of rabbit hearts. To this end, rabbit intrinsic ganglia were located using acetylcholinesterase histology (n = 33 hearts). This revealed six generalized ganglionic regions, defined as a left neuronal complex above the left pulmonary vein, a right neuronal complex around the base of right cranial vein, three scattered in the dorsal right atrium and a region containing numerous ventricular ganglia located on the conus arteriosus. Using immunohistochemistry, neurons were found to contain choline acetyltransferase or tyrosine hydroxylase and/or neuronal nitric oxide synthase in differing amounts (choline acetyltransferase > tyrosine hydroxylase > neuronal nitric oxide synthase). The function of rabbit intrinsic ganglia was investigated using a bolus application of nicotine or electrical stimulation at each of the above sites whilst measuring heart rate and atrioventricular conduction. Nicotine applied to different ganglionic plexuses caused a bradycardia, a tachycardia or a mixture of the two, with the right atrial plexus producing the largest chronotropic responses. Electrical stimulation at these sites induced only a bradycardia. Atrioventricular conduction was modestly changed by nicotine, the main response being a prolongation. Electrical stimulation produced significant prolongation of atrioventricular conduction, particularly when the right neuronal complex was stimulated. These studies show that the intrinsic plexuses of the heart are important and could be crucial for understanding impairments of cardiac function. Additionally, they provide a strong basis from which to progress using the isolated, innervated rabbit heart preparation.

Ultrasound current source density imaging of the cardiac activation wave using a clinical cardiac catheter.

Ultrasound current source density imaging (UCSDI), based on the acoustoelectric (AE) effect, is a noninvasive method for mapping electrical current in 4-D (space + time). This technique potentially overcomes limitations with conventional electrical mapping procedures typically used during treatment of sustained arrhythmias. However, the weak AE signal associated with the electrocardiogram is a major challenge for advancing this technology. In this study, we examined the effects of the electrode configuration and ultrasound frequency on the magnitude of the AE signal and quality of UCSDI using a rabbit Langendorff heart preparation. The AE signal was much stronger at 0.5 MHz (2.99 μV/MPa) than 1.0 MHz (0.42 μV/MPa). Also, a clinical lasso catheter placed on the epicardium exhibited excellent sensitivity without penetrating the tissue. We also present, for the first time, 3-D cardiac activation maps of the live rabbit heart using only one pair of recording electrodes. Activation maps were used to calculate the cardiac conduction velocity for atrial (1.31 m/s) and apical (0.67 m/s) pacing. This study demonstrated that UCSDI is potentially capable of real-time 3-D cardiac activation wave mapping, which would greatly facilitate ablation procedures for treatment of arrhythmias.

Abstract 215: The Redox Activation of DUSP4 by N-Acetyl Cysteine Protects Endothelial Cells and the Myocardium against Oxidative Damage

Background: Redox imbalance is the primary cause for endothelial dysfunction (ED), obstructed blood flow, and subsequent heart attack and failure. Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of glutathione (GSH), the primary reducing source, can significantly regulate cell function via reversible protein thiol modification. N-Acetyl cysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. Methods: We employed EPR spin-trapping, HPLC, fluorescent microscopy, immunoblotting, and qPCR of both in vitro and ex vivo experiments using either cultured cells or the Langendorff heart preparation. Results: NAC treatment increases NO generation from endothelial cells, as well as the enzyme and cofactor responsible for its production, ie eNOS and BH4. Interestingly, NAC treatment also increased the expression of DUSP4, an inducible nuclear dual-specificity phosphatase implicated in cardiovascular function. We hereby establish that DUSP4 redox modulates two important kinases (p38 and ERK1/2) of MAPK signaling pathways and provides protection against Cd2+-induced ROS damage as well as hypoxia-reoxygenation insult to endothelial cells. Furthermore, a four week oral NAC pre-treatment promotes DUSP4 both protein and mRNA expression in the rat myocardium and renders the heart less susceptible to ex vivo ischemia-reperfusion injury. Protein expression profiles in the myocardium closely mimic those observed with cultured endothelial cells. The infarct size of NAC-treated hearts is significantly reduced. The myocardial rate pressure product is much improved above vehicle treated rats. Conclusion: NAC serves as a direct antioxidant and as a regulator of transcription and translation of DUSP4, modulating the activity of its downstream effectors: ERK1/2 and p38, and thus protects against ROS-induced damage both in vitro and ex vivo. As such, NAC-derived drugs can provide a novel therapy to oxidant-induced diseases via the specific up-regulation of protective proteins such as DUSP4 and eNOS.

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes – a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user’s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

An isolated working heart system for large animal models.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes – a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user’s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.