Cardiac Response to β-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Our open-chest approach, in which we include ventilation with positive end-expiratory pressure, is significant to assess and analyze cardiac function in vivo, and the increase in beta-adrenergic stimulation. The procedure can be used to address both baseline parameters of load-independent cardiac function, and the beta-adrenergic reserve in genetically modified mice or upon interventions. After confirming a lack of response to pedal reflex, disinfect the left hind limb of the anesthetized mouse with 70%ethanol and make an incision to expose the left femoral vein.

Blast the epigastric artery and vein with a cautery. To better see the femoral vein, the field of vision is spanned by a suture through the abdomen. Then, a suture is placed distal to the catheter access, to ligate the femoral vein.

Pass a suture under the femoral vein, and prepare a knot cranial to the puncture site. Puncture the femoral vein with a prepared micro tube attached to a 1-milliliter syringe and secure the tube inside the vessel with the knotted suture. To counteract any fluid loss, use an automatic syringe pump to infuse 0.9%sodium chloride, supplemented with 12.5%albumin, at a 15 microliter infusion rate, and hydrate the exposed tissue with pre-warmed 0.9%sodium chloride.

To access the thorax, rinse the thorax with 70%ethanol, and incise the skin just beneath the xiphoid process. Bluntly separate the pectoral muscles from the chest wall, and use forceps to lift the xiphoid process to allow the chest wall to be cut, until the diaphragm is fully visible. Incise the diaphragm from below to expose the cardiac apex, and use forceps to carefully remove the pericardium.

Perform a limited costotomy on the left side, and pass a suture beneath the inferior caval vein to allow downstream preload reduction. Using a 25 gauge cannula, gently puncture the cardiac apex, and replace the cannula with a pressure-volume catheter, until all the electrodes are within the ventricle. Then, gently adjust the position of the catheter until rectangular-shaped loops are obtained.

To acquire pressure-volume loop measurements, perform an online analysis of the cardiac function parameters of interest, and wait until a steady-state cardiac function is obtained. Stop the respirator at the end-expiratory position, and record the baseline parameters. After 3 to 5 seconds, use forceps to lift the suture beneath the inferior caval vein to obtain pre-load independent parameters, and turn on the ventilator.

Wait at least 30 seconds for the second occlusion until the hemodynamic parameters are stabilized. After obtaining the measurement under basal conditions, mount an isoproterenol-loaded syringe onto the syringe pump. Wait at least two minutes, until a new steady-state cardiac function is observed.

Before stopping the respirator at the end-expiratory position, and recording the baseline parameters. After 3 to 5 seconds, lift the suture beneath the inferior caval vein to obtain preload independent parameters, and wait at least 30 seconds for the second occlusion. Then mount the syringe containing the next isoproterenol concentration, and repeat the baseline, and pre-load independent parameters recordings.

To perform a parallel-conductance calibration, after the last isoproterenol dose-response measurement, connect a syringe loaded with the 15%sodium chloride solution to the femoral cannula, and carefully infuse 5 microliters of the hypertonic solution until the pressure-volume loop slightly shifts to the right. Wait until the loops return to the steady-state, before stopping the respirator at end-expiration, and immediately injecting a 10 microliter bolus of 15%sodium chloride. Then check whether the pressure-volume loop has largely broadened, and shifted to the right.

To perform a conductance-to-volume ratio, wait 5 minutes until the hypertonic saline bolus is completely diluted before removing the catheter, and using a 1-milliliter syringe, equipped with a 21 gauge cannula to remove at least 600 microliters of blood from the left ventricle of the beating heart. Then transfer the blood into a pre-warmed calibration cuvette in a 37-degree celsius water bath, with cylinders of a known volume, and place the pressure-volume catheter centrally into each cylinder to allow recording of the conductance. Using this open chest pressure-volume-loop measurement method, if the catheter is correctly placed within the ventricle, a full cardiac cycle will be represented by a rectangular-shaped pressure-volume-loop.

As illustrated, systole begins with a phase of isovolumetric contraction, during which both cardiac valves are closed. When the ventricular pressure, exceeds the aortic pressure, the aortic valve opens, and blood is pumped into the aorta during the ejection phase. Subsequently, when the aortic pressure exceeds the ventricular pressure, the aortic valve closes, and diastole begins.

During the isovolumetric relaxation, the ventricular pressure falls, until the atrial pressure exceeds the ventricular pressure, and the mitral valve opens. Passive diastolic filling, characterized by the end-diastolic pressure-volume relationship then takes place until the next cardiac cycle begins. Since it is capable of determining cardiac function independent of preload, pressure-volume analysis can provide detailed insight into cardiac function, in cardiac contractility.

For example, in this analysis isoproterenol induced a significant effect on each measured cardiac function, and cardiac contractility parameter. In contrast, a reduction in the diastolic parameters was observed in response to, increasing isoproterenol concentrations. Precise catheter placement within the left ventricle, is crucial to obtain reproducible loops without artifacts.

To optimize catheter placement, we prefer the open chest approach. Following PV loop measurements, allometric endysis can be determined, and tissue can be isolated, to identify molecular and histopathological alterations, caused by treatments, and or genetic modifications. This technique paves the way to analyze beta-adrenergic reserve in mouse models under normal and pathological conditions and may be used as a template for testing other stimuli.