Peptides isolated from the heart of cattle with a molecular weight of less than 15 kD have a biologically active effect on the heart of laboratory animals. This action shows itself in high doses in the normal state, and in hypoxia and ischemia in very small doses. Previously, we have noted the protective effect of peptide in isadrinum myocardial necrosis, experimental myocardial infarction caused by ligation of the left coronary artery, and found that there is a dependence of the effects of the drug on the course of myocardial infarction on the concentration and method of its introduction .
In this paper we studied the correlation of doses and pharmacological effects of shot peptide on different models in connection with the need to develop a scheme of application in clinical settings.
Research methods
Myocardial infarction was modeled on 120 white outbred rats weighing 160-180 g by ligation of the left coronary artery. Parts of the rats after surgery were injected by peptide by intra—addominale way. The size of myocardial infarction (MI) was determined gravimetrically. Hearts of Guinea pigs weighing 230-250 g for retrograde Langendorf perfusion were taken under thiopental anesthesia (75 mg/kg). Pre-pigs administered 500 ED heparin. Perfusion was carried out with a solution Tyrode without electrostimulation at a constant flow rate of solution 10 ml/min, a temperature of +30’C. and saturated with Carbogen. The pH of the solution was brought to 7.4 by sodium bicarbon. The peptide added impulsively in the initial concentration of 2х10-8 -2х10-4 g/ml in the upper reservoir (air trap) with subsequent permanent breeding and cleaning of the medicine. The registration of contractions was carried out by latex balloon placed in the left ventricle, which was connected with the pulse recorder, and then the signal was received by the electrocardiograph «Malysh». Cardiomyocytes were received by Vahouny. Oxygen consumption by isolated cardiomyocytes was recorded polarographically with the application of Clark electrode at a temperature of +30 °C. The volume of the polarographic cell was 1 ml. The Rate of oxygen consumption by cardio — myocytes was expressed in ng oxygen atoms/min/mg of protein. The protein concentration was determined by the Lowry method. Oxygenation of the suspension Cardiomyocytes carried out by saturating it with Carbogen at normal temperature.
Research result
The isolated heart of the Guinea pig reacted in a dependent way to the impulse addition of different concentrations of shot peptide during their subsequent dilution and washing, the effect began to show itself with a concentration of 2-10-8 g/ml or more and was expressed by a decrease in the amplitude of reductions. Then decreased the heart rate, which after the cleaning from the medicine was restored earlier than the amplitude. After 15 minutes of cleaning, parameters of contractile function were restored (Figure 1). If the heart was subjected to total ischemia for 25 minutes and began reperfusion in the presence of peptide, then also noted the dependence of contractile function on the concentration of the medicine. Thus, at a concentration of 10-6 and, in particular, 5×10-6 g/ml, dose-dependent negative inotropic action was observed, and at a concentration of shot peptide in perfusate of 5-10-7 g/ml or less, the effect reversal was noted — improvement of the parameters of contractile function: increase in the amplitude of contractions, decrease in post-ischemic contracture. That is, the use of small doses of the drug led to an improvement in the parameters of contractile function, which was especially clearly shown in the conditions of ischemic exposure. The use of high doses of peptide leads to inhibition of myocardial contractile activity, that is, it can be assumed that with an increase in the dose of the medicine there is an overdose. It is also evident that ischemic myocardium is more sensitive to increased peptide concentration.
Most likely, the explanation of the observed effects of shot peptide on the isolated heart can be the data on their influence on the bio-energetic processes in cardiomyocytes. Thus, rat cardiomyocytes, which are in conditions of oxygen deficiency, become more sensitive to additives of exogenous succinate. In other words, the rate of oxygen consumption by the suspension of hypoxic cardiomyocytes increases in response to succinate additives, the greater their hypoxic damage. Such hyperactive oxidation of succinic acid is fraught not only with depletion of substrates in low-energy mitochondria, but also with further separation of oxidative phosphorylation. Therefore, inhibition of oxidation of succinic acid becomes necessary for energy deficiency, which develops with deep hypoxia. Stimulation of oxygen consumption, causing the addition of succinate to the suspension of cardiomyocytes, is considered an index of cell damage. Peptide begin to show a depressing effect on the breathing of cells under hypoxia, against the background of the introduction of succinate concentration of 10-13 g/ml and with a further increase in the concentration of their effects are progressively enhanced (Figure 2). It is important to note that the oxygenation of nom cell suspension relieves the effect of breath stimulation by succinate. So the action of peptide is comparable with the action of oxygen and can be considered as a positive fact. However, with an increase in the concentration of peptide in the medium to 10-6 g/ml, their depressing effect not only on succinate-dependent, but also on shot peptide -dependent oxidation begins to appear, which leads to almost complete respiratory inhibition in high concentrations of the medicine (Figure 2, a).
With these concentrations NADH-stimulated respiration is not regulated by the medicine on the contrary succinate-dependent does. (Fig.2,b). Thus, there is a parallelism of dose-dependent effects of shot peptide in two different ways. The concentration of peptide 5×10-7 g/ml and less has a positive effect on the contractile function of the heart and helps to optimize breathing processes in conditions of oxygen deficiency. Peptide in a concentration, and more clearly, dose-dependent decrease of contractile function, and inhibitors of respiration of cardiomyocytes until a full brake application.
The data obtained are consistent with the results of studies of the effect of peptide on the course of myocardial infarction in rats (see table). When receiving Peptide in doses 5×10-4 and 10-3 g/kg there is a tendency to reduce the focus of ischemic myocardial damage, and at a dose this effect is leveled. The effects of higher peptide concentrations on these models have not been studied. However for mice the doses causing their death were found: 255+12 mg/kg with intraperitoneal introduction, which is more than 50 times higher than the therapeutic dose. Nevertheless, the results obtained in experiments with IT indicate that there may be an overdose of the medicine, that is, with an increase in the dose of peptide, the positive effects first increase, then decrease, and in the long run — there may have the negative consequences. It was found that repeated introduction of the medicine at a dose of 5×10-4 g/kg gave the most pronounced positive effect and led to a significant reduction in the area of myocardial damage (see table).
Peptide bio-regulators contained in the peptide in certain concentrations favorably affect the processes of oxidative phosphorylation in the cells of the ischemic area, normalize contractile function and slow down the rate of death of the ischemic myocardium cells. However, the increase in doses leads to the disappearance of positive effects, followed by increased negative effects of such therapy.
The repeatability and unidirectionality of the effects observed on different models indicates the possibility of its use in clinical conditions. However, the use of shot peptide in the clinic for the correction of ischemic compounds requires the establishment of the most optimal doses and methods of administration.
As the peptide show the effect on the function of the myocardium in the result of each injection and repeated injection is not potentiated negative effects (negative effects are simultaneously at high doses or in a steady increase of medicine concentration), in our view, it is advisable to use small doses of the medicine at certain intervals. Treatment is desirable at a time when there are local myocardial ischemic damage as a result of acute (myocardial infarction) or chronic (ischemic heart disease) course of the disease.