The overall goal of this procedure is to isolate healthy, intact papillary muscles for the study of in vitro cardiac contractile function in mice. This is accomplished by first isolating the heart, followed by interruption of the contractions, by immersion in a cardioplegic solution to avoid ischemic damage. In the second step, the intact left ventricular anterior papillary muscle is isolated and equipped with fixation sutures.
The muscle is then mounted onto the forced transducer and transferred into the organ bath chamber. In the final step, the papillary muscle is stimulated with field electric pulses to induce papillary muscle contractions in vitro. Ultimately, the effects of various physiological and pathological conditions on the contractility of the cardiac muscle can be transmitted to the forced transducer and recorded.
This method can be used to analyze the cardiac contractility of left ventricular popularity muscles in vitro in the absence of neuro humeral and vascular influences to allow the direct evaluation of the cardiac contractile. Properties of interest. Papillary muscle preparation serve as excellent models for studying cardiac physiology and pathophysiology, allowing the investigation of the effects of pharmacological agents on cardiac contractility.
We first had the idea for this method when we found a cardiovascular phenotype in mice deficient for a specific calcium activated, non-selective ion channel leading to the discovery of specific inotropic alterations in the beta AIC signaling pathway. Begin by placing an eight to 12 week old mouse in the dorsal position on a dissection board. Next, using sharp bone scissors, open the thorax laterally on both sides of the chest and cut through the diaphragm.
Then using blunt forceps, grab the heart on the vascular chunks and use scissors to separate the heart quickly from the lungs and the surrounding tissue. Transfer the beating heart to a Petri dish filled with cooled preparation solution, gassed with oxygen and gently touch the cardiac apex with forceps to stimulate heart contractions. As soon as it is completely ex sanguineous, transfer the heart to the dissection dish and place a small pin through the right ventricle to fix the heart in the dorsal position.
Now, transfer the dish under a stereo microscope and use microsurgical scissors to separate both of the atria on the atrial ventricular level. Turn the heart about 180 degrees and make a cut through the left ventricular wall from the atrial ventricular valve level to the apex of the heart. Taking care to avoid any mechanical pressure on the heart.
Then rotate the heart back. Then open the left ventricle and fix the left-sided free wall with forceps exposing both of the left ventricular papillary muscles. Carefully cut the ventricular tissue laterally on both sides of the left anterior papillary muscle.
Taking care to preserve a part of the valvular SA on the papillary muscle preparation. Then dissect the remaining ventricular wall tissue from the papillary muscle and attach one silk thread at the muscular part with two knots and one at the valvular sail of the papillary muscle. Preparation with one knot.
Fix the preparation in a previously prepared organ bath chamber. Then immediately after the fixation, stimulate the tissue with two millisecond rectangular pulses at a current of 100 milliamps and a stimulation frequency of one hertz. And start the recording.
Changing the solution as appropriate for the type of organ bath used. Gradually increase the mechanical pretension over a period of 45 to 60 minutes until the increase of the pretension is not followed by a further augmentation in the twitch force, and then initiate the desired downstream experimental analysis. Cardiac contractility can be characterized by a variety of parameters, including the twitch force, time to peak and half maximal relaxation.
The correlation between the beating rate and the twitch force is calculated by the force frequency relationship. For example, in this experiment, the stimulation frequency was increased in a stepwise fashion from 0.1 to five hertz leading to changes in the cardiac contractility. The post rest potentiation protocol illustrates the alterations of the twitch force Amplitude observed after a previous stimulation followed by a rest period of a defined duration.
By varying the calcium concentration of the physiological solution in the organ bath, the calcium sensitivity of the contractile apparatus can be evaluated to assess the inotropic effects mediated by the activation of beta recepts. The isolated papillary muscle can also be treated with isoprene as demonstrated in this representative experiment to simulate ischemia. In vitro, the muscles are exposed to a glucose and pyruvate free extracellular solution bubbled with 95%nitrogen and 5%carbon dioxide leading to a decline of the twitch force and the development of ischemic contracture as illustrated in the graph.
Once mastered the dissection and equilibration of one popularity, muscle preparation can be completed in about 19 minutes, even with taking the time to carefully isolate and mount the preparation without introducing mechanical or metabolic damage Following this procedure. Other methods like sharp electrode measurements, enzyme linked immunoassays and western blood analysis can be performed in order to answer additional questions After its development. This technique is a crucial approach for research in cardiac physiology and pharmacology, and for investigations of the cardiac phenotype in various transgenic rodent models.
