ANALYSES OF MOUSE HEARTS: CELLULAR ELECTROPHYSIOLOGY, PHARMACOLOGY AND ISOLATED PERFUSED HEART HEMODYNAMICS

General description and purpose of the system: The system allows semiautomatic studies of isolated perfused hearts, with specific reference to studies of contractility and pharmacological responses in wild type and transgenic animals. The system has several service components, that allow to maintain performance of isolated heart under constant conditions of temperature, ion and gas composition and to measure coronary flow flow, cardiac output, pressure in heart chambers under desired drug composition/or concentration. Data are recorded and processed using an A/D board, stored in computer memory and further processed by custom designed software. The system allows one to perform 2-3 studies in different hearts per day by one trained operator.

The setup performs investigation of contractility under different stimuli and automatically processes the following variables: Cardiac output, coronary flow, pressure in left ventricle, pressure in aorta and left atrium, systolic pressure, diastolic pressure, heart work, oxygen consumption, heart rate, contractility (dP/dt max), relaxation (-dP/dt min), time to peak pressure, half time of relaxation, response to pharmacological or electrical stimulation or change in ion composition, and responses to work load (Starling relationship).

The animal is anesthetized. The heart is removed from the chest and the aorta is cannulated with a 20-gauge stainless steel cannula. The general scheme is illustrated to the right:

A retrograde perfusion with 37.7ºC Krebs-Henseleit solution is started immediately (Langendorff mode), at a hydrostatic pressure of ~55 mmHg. The reservoir containing the perfusion media is oxygenated with 95% O₂-5% CO₂ via glass frits and is located 75 cm above the level of the heart. A PE-50 catheter is inserted into the left atrium through the pulmonary vein, advanced further through the mitral valve into the left ventricle and forced through the ventricular apex. The proximal end of the catheter with wide edges is fixed in the left ventricle. The distal end of the catheter is connected through a larger catheter to a Baxter pressure transducer (PX260) for the recording of the intraventricular pressure. Pressures are recorded using a Grass amplifier P122 (Grass, West Warwick, RI). Aortic pressure in the aortic outflow catheter is measured through a sidearm. A steel cannula (18 gauge) inserted into the pulmonary vein, is fixed with surgical suture to prevent retrograde outflow of the perfusate and is then connected to the working heart line of the apparatus. The working heart line is a separate perfusion line with the same source of oxygenated solution, but with a separate bubble trap and pressure valve to maintain the driving pressure at 8 cm H₂0. The pressure in the left atrium is measured through a side port at the very entrance to the steel cannula. Flow through the left atrial cannula is adjusted to a level, that maintains pressure inside the atrium at 6-8 cm H₂0 (flow approximately ~5 ml/min). Perfusion is then completely switched from retrograde to antegrade (work-performing heart preparation). In this manner we can assess physiology and pharmacology in the Langendorff as well as the working modes.

The heart is then submerged into a bath with isotonic solution, that contains heated coils and a means for constant stirring. The fluid passes into the aorta through a bubble chamber that serves also as"elastic recoil" of the "arterial" system. The temperature and gas composition are maintained by circulating perfusate through the heat exchanger and gas bubbler, and temperature is continuously monitored before it enters the heart. Coronary flow is allowed to drip through the open pulmonary artery onto a weighing pan. Alternatively a flow meter may be used. The fluid is collected and measured using an electronic balance. A micrometer clamp is attached to the outflow catheter so that the "Starling resistance" aortic pressure (afterload) can be modulated. Atrial and aortic pressure are monitored. We generally measure the coronary and the aortic flows by collecting and weighing fluid from the aorta and right atrium every 30 sec by a custom made automatic computer- based weight analyzer, that displays coronary flow and cardiac output in real time. The electrocardiogram is monitored via connections to the stainless steel cannulas attached to the aorta and left atrium. Electrodes are used for pacing with an electro stimulator E9 (Grass instruments) with pulse duration of 10 ms, applied voltage 2.5 V and frequency range of 300-600 imp/min. Microperfusion pumps either directly in the aorta, or into the pulmonary venous inflow canulla can deliver drugs of interest at desired concentrations. The actual drug concentration delivered is calculated from the drug concentration in the syringe multiplied by the delivery rate divided by coronary flow.

The overall appearance is shown in the following picture:

All pressure parameters were recorded simultaneously using an AT-MIO-16XE data acquisition board and acquired by the software package "Biobench" (National Instruments, Austin, TX), or by "Labview" data acquisition software. Data are stored in the computer memory and back-upped on CDs.

An example of a pressure curve is shown below:

Further processing allows us to determine the derivatives of the intraventricular pressure: contractility (dP/dt max) and relaxation (-dP/dt min), time to peak pressure (TPP) and half time of relaxation (RT ½), frequency of heartbeats, average pressures and peak pressures in the heart chambers. Our software uses a moving rectangle algorithm generally applied to process the periodic curve with multiple peaks. If a peak is to be found inside a rectangle, the difference in height between the local data maximum inside the rectangle and the data values at both rectangle ends has to be no less than the rectangle height. The height of the rectangle is the percentage of the total amplitude of the data in the range (the amplitude is defined as the difference between the maximum and the minimum of the data). The width of the rectangle is the total number of points included in the rectangle. The software package allows to process and average these variables from 5-15 heartbeats; time required to process data from one experiment is approximately 10-20 min. The analyses are performed using a double-blinded method.

An example of a study is illustrated below: Contractility (circles) and relaxation (squares) dose-response curve to Isoproterenol. Open symbols [Ca²⁺] = 2.0 mM, closed symbols [Ca²⁺] = 1.5 mM. Data are shown as percent change from baseline (Mean ± SE) in an a1 subunit of the L-type calcium channel over expressing mice. Due to the increase in the number of L-type Ca channels, the heart cells are subjected to a modest but sustained increase in calcium influx [Circulation: 2001; 103: 140-147]

Our laboratory is completely equipped to carry about a panel of electrophysiological (EP) quantitative studies on single cells, such as cardiomyocytes, smooth muscle cells, transfected cells such as HEK, Xenopus oocytes, etc. Whole-cell mode of the patch-clamp method and single channel analysis are among the methods we employ. As an example, the routine is as follows: After excision of the heart from your mouse, we use a system that isolates viable cardiac cells. This is done in our laboratory, CVRC: G- 940. The cells are then transferred to one of our EP set-ups, in the same laboratory. We routinely quantitate, Na, K, and Ca channel currents and subject the cells to a standard pharmacological protocol, e.g., isoproterenol, forskolin, a variety of inhibitors as needed. We provide, as an example, Ca channel density, activation and inactivation kinetics, etc. in a form ready for publication. Please read some of recent publications: to get an idea of the quality of our work: Muth et al: Circulation. 2001; 103: 140-147 and Muth et al: J. Biol. Chem 1999; 274: 21503-21506.

We have focused on the α₁ subunit of the L-type calcium channel. This subunit harbors the pore of the channel as well as the receptor sites for the three major classes of calcium antagonist drugs. We have generated a series of point, deletion and chimeric mutants of the rabbit and human cardiac a1 subunit. We investigated the contribution of motif IV and motif III to nifedipine-like dyhydropyridine drugs (DHP) action by testing chimeric α₁ subunits engineered between the DHP -sensitive rabbit heart LVDCC and the DHP-insensitive brain (α_1A) subunits. Please refer to: Motoike et al: J. Biol. Chem. 1999; 274: 9409-9420 for details of the planar structure of the channel and the numbering. For example, the Q1010M and T1006Y mutant channels had a decreased sensitivity to agonists and antagonists. To further extend this work, we studied the voltage dependent action of DHPs with mutants of the IIIS6 and IVS6 segments of cardiac calcium channel. These data showed that the agonist and antagonist interaction sites for DHPs with L-type calcium channels might overlap, some amino acids in this site might exhibit a preference for either DHP enantiomers. The cysteine accessibility method was used to explore calcium channel pore topology. Recent studies have demonstrated the role of inactivation in the mechanism of use-dependent calcium block by phenylalkylamines and benzothiazepines. In order to determine the structural features of the calcium channel responsible for use-dependent block, we introduced mutations in motif IVS5 of the human heart α_1C calcium channel utilizing homologous regions of the rat brain sodium channel 2a. In order to see whether the mutations reflect changes in the channel function and maintained normal biophysical properties and pharmacology to calcium channel antagonists and agonists, we expressed cRNAs for the full-length channels in Xenopus oocytes. Our electrophysiological data provided information about the involvement of a number of amino acid residue in the IVS5 transmembrane segments of the cardiac calcium channel in regard to use-dependency. Since the human and rabbit α₁-subunit clone expressed very well, it provided an experimental tool to investigate the above outlined research programs using site directed mutagenesis followed by oocyte expression. The electrophysiological measurements were done using the two-microelectrode voltage-clamp technique (analyses of voltage-dependent activation and inactivation, recovery from inactivation, use-dependent block by calcium antagonists) and single-channel recording in oocytes (channel conductance, open time, latency).

Mouse models of cardiac hypertrophy-failure are also available in our laboratory. For example, we overexpressed the α₁ subunit of the L-type human voltage-dependent Ca²⁺ channel (L-VDDC) in hearts of transgenic mice. To determine possible functional changes in the L-VDCC, whole cell voltage clamp recordings were carried out on isolated ventricular cardiomyocytes. It is thought that heart failure is associated with prolongation of action potential duration, the result of down-regulation of one or more of the K⁺-currents. We recorded action potential, transient outward and inward rectifier potassium current, calcium current and carried out kinetic and pharmacological analysis of these channels. Similar studies have been done with the calcineurin overexpressed failing heart, with the Gαq, and with other mouse models of hypertrophy and failure or with models that exhibit an early uncoupling or blunting of the β-adrenergic receptor signaling system, but do not develop hypertrophy or failure.

Mice can be bred against different TG disease backgrounds. The hypothesis is that the transition from compensated hypertrophy to heart failure, or the time course and severity of the final phenotype may be affected by these crossbreedings. The EP data obtained thus far provides us with the complexity of electrical re-modeling in the developing normal and genetically engineered mice.

We generally combine our data with those obtained from the animal hearts from the same transgenic line analyzed by our Physiology/Pharmacology Core facility (described above) component in the same laboratory.

Examples of typical traces obtained from several experiments are presented on the next page: