Isolation of Adult Mouse Cardiomyocytes Using Langendorff Perfusion Apparatus.

Heart disease is one of the leading causes of death in the United States. Isolation and culture adult cardiomyocytes are important for studying cardiomyocyte contractility, heart hypertrophy, and cardiac failure. In contrast to neonatal cardiomyocyte isolation, adult mice cardiomyocytes isolation is challenging due to firm connections among cardiomyocytes through intercalated discs. The availability of newly generated genetically modified mouse lines requires to establish protocols to isolation and culture adult mouse cardiomyocyte for in vitro studies. In this manuscript, we described a straightforward method of isolating adult mouse cardiomyocytes using Langendorff perfusion apparatus. Briefly, the hearts were harvested from adult mice and the heart was mounted to Lagendorff apparatus. After perfusion with calcium depletion and collagenase digestion, the left ventricles were minced and filtered. Lastly, the separated cardiomyocytes were treated with CaCl2. The isolated cardiac myocytes can be utilized in a broad range of experiments including screening for drugs.

Development of a pro-arrhythmic ex vivo intact human and porcine model: cardiac electrophysiological changes associated with cellular uncoupling.

We describe a human and large animal Langendorff experimental apparatus for live electrophysiological studies and measure the electrophysiological changes due to gap junction uncoupling in human and porcine hearts. The resultant ex vivo intact human and porcine model can bridge the translational gap between smaller simple laboratory models and clinical research. In particular, electrophysiological models would benefit from the greater myocardial mass of a large heart due to its effects on far-field signal, electrode contact issues and motion artefacts, consequently more closely mimicking the clinical setting. Porcine (n = 9) and human (n = 4) donor hearts were perfused on a custom-designed Langendorff apparatus. Epicardial electrograms were collected at 16 sites across the left atrium and left ventricle. A total of 1 mM of carbenoxolone was administered at 5 ml/min to induce cellular uncoupling, and then recordings were repeated at the same sites. Changes in electrogram characteristics were analysed. We demonstrate the viability of a controlled ex vivo model of intact porcine and human hearts for electrophysiology with pharmacological modulation. Carbenoxolone reduces cellular coupling and changes contact electrogram features. The time from stimulus artefact to (-dV/dt)max increased between baseline and carbenoxolone (47.9 ± 4.1-67.2 ± 2.7 ms) indicating conduction slowing. The features with the largest percentage change between baseline and carbenoxolone were fractionation + 185.3%, endpoint amplitude - 106.9%, S-endpoint gradient + 54.9%, S point - 39.4%, RS ratio + 38.6% and (-dV/dt)max - 20.9%. The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically induced pro-arrhythmic substrates.

Primed Left Ventricle Heart Perfusion Creates Physiological Aortic Pressure in Porcine Hearts.

This article presents a primed left ventricle heart perfusion method to generate physiologic aortic pressure (AoP) and perform functional assessment. Isolated hearts of male Yorkshire pigs were used to study the hemodynamic behaviors of AoPs generated in the primed left ventricle heart perfusion (n = 6) and conventional (zero-loaded left ventricle) Langendorff perfusion (n = 6). The measurement results show that left ventricular pressure generated in the primed left ventricle heart perfusion is a determinant of physiologic AoP (i.e. systolic and diastolic pressures within physiologic range). The aortic pulse pressure (systolic pressure = 124.5 ± 1.7 mm Hg, diastolic pressure = 87.8 ± 0.9 mm Hg, aortic pulse pressure = 36.7 ± 2.6 mm Hg) from the primed left ventricle heart perfusion represents close match with the in vivo physiologic data. The volume in the left ventricle remains constant throughout the primed left ventricle heart perfusion, which allows us to perform isovolumetric left ventricular pressure measurement in ex vivo heart perfusion (EVHP). Left ventricular contractility measurements (maximum and minimum rates of left ventricular pressure change) were derived for cardiac assessment. In summary, the proposed primed left ventricle heart perfusion method is able to create physiologic AoP and enables left ventricular functional assessment in EVHP in porcine hearts.

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

INTRODUCTION: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The practicalities of establishing a porcine isolated heart model.

BACKGROUND: The isolated heart apparatus is over 100 years old, but remains a useful research tool today. While designs of many large animal systems have been described in the literature, trouble-shooting and refining such a model to yield a stable, workable system has not been previously described. This paper outlines the issues, in tabular form, that our group encountered in developing our own porcine isolated heart rig with the aim of assisting other workers in the field planning similar work. The paper also highlights some of the modern applications of the isolated heart apparatus. Methods Landrace pigs (50-80 kg) were used in a pilot project to develop the model. The model was then used in a study examining the effects of various cardioplegic solutions on function after reanimation of porcine hearts. During the two projects, non-protocol issues were documented as well as their solutions. These were aggregated in this paper. RESULTS: Issues faced by the group without explicit literature solutions included pig size selection, animal acclimatisation, porcine transoesophageal echocardiography, cannulation and phlebotomy for cross-clamping, cardioplegia delivery, heart suspension and rig tuning. CONCLUSION: Prior recognition of issues and possible solutions faced by workers establishing a porcine isolated heart system will speed progress towards a useable system for research. The isolated heart apparatus remains applicable in transplant, ischaemia reperfusion, heart failure and organ preservation research.

Design and validation of a tissue bath 3-D printed with PLA for optically mapping suspended whole heart preparations.

With the sudden increase in affordable manufacturing technologies, the relationship between experimentalists and the designing process for laboratory equipment is rapidly changing. While experimentalists are still dependent on engineers and manufacturers for precision electrical, mechanical, and optical equipment, it has become a realistic option for in house manufacturing of other laboratory equipment with less precise design requirements. This is possible due to decreasing costs and increasing functionality of desktop three-dimensional (3-D) printers and 3-D design software. With traditional manufacturing methods, iterative design processes are expensive and time consuming, and making more than one copy of a custom piece of equipment is prohibitive. Here, we provide an overview to design a tissue bath and stabilizer for a customizable, suspended, whole heart optical mapping apparatus that can be produced significantly faster and less expensive than conventional manufacturing techniques. This was accomplished through a series of design steps to prevent fluid leakage in the areas where the optical imaging glass was attached to the 3-D printed bath. A combination of an acetone dip along with adhesive was found to create a water tight bath. Optical mapping was used to quantify cardiac conduction velocity and action potential duration to compare 3-D printed baths to a bath that was designed and manufactured in a machine shop. Importantly, the manufacturing method did not significantly affect conduction, action potential duration, or contraction, suggesting that 3-D printed baths are equally effective for optical mapping experiments.NEW & NOTEWORTHY This article details three-dimensional printable equipment for use in suspended whole heart optical mapping experiments. This equipment is less expensive than conventional manufactured equipment as well as easily customizable to the experimentalist. The baths can be waterproofed using only a three-dimensional printer, acetone, a glass microscope slide, c-clamps, and adhesive.

Mode of perfusion influences infarct size, coronary flow and stress kinases in the isolated mouse heart.

AIM: The isolated, retrogradely perfused heart (modified Langendorff model) is a widely used method in experimental heart research. The presence of an intraventricular balloon is necessary to get functional measurements. We have previously shown that the balloon induces phosphorylation of some suggested cardioprotective mitogen-activated protein kinases (MAPK): P38-MAPK, ERK 1/2 and JNK. We hypothesized that the balloon could influence cardioprotection, protect against ischaemia reperfusion injury and interfere with coronary flow. METHODS AND RESULTS: Isolated mouse hearts were perfused for 5, 10, 20, 40 and 60 min with a balloon in the left ventricle. We found a wavelike phosphorylation of all MAPK while AKT displayed a gradual dephosphorylation when compared to non-perfused hearts. Hearts were subjected to 20 min of stabilization with or without the balloon, followed by 35 min of ischaemia and 120 min of reperfusion. Although the MAPK were phosphorylated, the infarcts were larger in the balloon group. When the balloon was present during the entire protocol, compared to removal at the end of ischaemia, the infarct size was also larger, especially in the endocardial layer. The balloon reduced post-ischaemic endocardial coronary flow, despite a higher average flow, indicating a hyperperfused epicard. Blocking the balloon-induced ERK 1/2 phosphorylation during stabilization did not affect infarct size. The effect of post-conditioning was influenced by the balloon, showing reduced infarct size when the balloon was present. CONCLUSION: The balloon used for pressure measurements may contributes to cell death possibly by reducing endocardial coronary flow.

In Search of the Optimal Heart Perfusion Ultrasound Imaging Platform.

OBJECTIVES: Quantification of myocardial perfusion by contrast echocardiography remains a challenge. Existing imaging phantoms used to evaluate the performance of ultrasound scanners do not comply with perfusion basics in the myocardium, where perfusion and motion are inherently coupled. To contribute toward an improvement, we developed a contrast echocardiographic perfusion imaging platform based on an isolated rat heart coupled to an ultrasound scanner. METHODS: Perfusion was assessed by using 3 different types of contrast agents: dextran-based Promiten (Meda AB, Solna, Sweden), phospholipid-shelled SonoVue (Bracco Diagnostics, Inc, Princeton, NJ), and polymer-shelled MB-pH5-RT, developed in-house. The myocardial video intensity was monitored over time from contrast agent administration to peak, and 2 characteristic constants were calculated by using an exponential fit: A, representing capillary volume; and ß, representing inflow velocity. RESULTS: Acquired experimental evidence demonstrates that the application of all 3 contrast agents allows sonographic estimation of myocardial perfusion in the isolated rat heart. Video intensity maps show that an increase in contrast concentration increases the late-plateau values, A, mimicking increased capillary volume. Estimated values of the flow, proportional to A × ß, increase when the pressure of the perfusate column increases from 80 to 110 cm of water. This finding is in agreement with the true values of the coronary flow increase measured by a flowmeter attached to the aortic cannula. CONCLUSIONS: The contrast echocardiographic perfusion imaging platform described holds promise for standardized evaluation and optimization of contrast perfusion ultrasound imaging in which real-time inflow curves at low acoustic power semiquantitatively reflect coronary flow.