Pathogenesis of Cardiac Depression in Acute Destructive Pancreatitis

Материал и методы. Исследования проведены на 130 крысах самцах линии Вистар массой 292±4,0 г, разбитых на 4 группы. Животных наркотизировали этиловым эфиром. В трех экспериментальных группах моделировали острый деструктивный панкреатит путем введения в ткань поджелудочной железы желчи, взятой из желчного протока, из расчета 0,15 мл/кг массы тела. Через 24 часа, 7 дней и 1 месяц воспроизво дили модель изолированного изоволюмически сокращающегося сердца по E. L. Fallen et al. Давление в ле вом желудочке измеряли электроманометром ВМТ и регистрировали его вместе с первой производной на приборе Н338 4П, рассчитывая систолическое и диастолическое давление, скорости сокращения и расслаб ления. Одновременно брали пробы перфузата, прошедшего через коронарное русло, в котором унифициро ванными методами определяли активность аспартатаминотрансферазы (АсАТ) и глюкозы. Для выявления механизмов кардиодепрессии осуществляли навязывание высокого ритма сокращений, гиперкальциевую и гипоксическую перфузию.


Ключевые слова: острый панкреатит; сердечная недостаточность; кардиодепрессия
Objective is to identify the pathogenic factors for progression of pancreatogenic cardiac failure in the nearest and remote periods.

Materials and methods.
The study was carried out on 130 male Wistar rats (292±4.0g) divided into 4 groups.The animals were anesthetized with ethyl ether.Acute destructive pancreatitis was simulated in three experimental groups by infusion of bile (0.15 ml/kg body weight) taken from the bile duct into the pancreatic tissue.The isolated isovolumically contracting rat heart (according to E. L. Fallen et al) was simulated 24 hours, 7 days and 1 month after the bile infusion.The pressure in the left ventricle was measured by electric manometer BMT and registered along with the first derivative at the device N338 4P calculating the systolic and diastolic blood pressure, and the speed of

Introduction
The incidence rate of acute pancreatitis is steadily increasing [1,2], and the frequency of its destructive forms reaches 20-45% [3,4].Meanwhile, the mortality rate remains invariably high and depending on the area affected and the presence/absence of infected necrotic zones within ranges from 15% to 100% [5,6].The top position in the lethal outcomes in pancreatic necrosis is given to cardiovascular and multiple organ failure secondary to septic or cardiogenic shock [7].In previous stud ies we have detected the alterations in myocardial contractility early after the simulation of acute destructive pancreatitis [5].The aim of the study is to identify the pathogenic factors for progression of pancreatogenic cardiac failure in the nearest and remote periods.

Materials and Methods
The study was carried out on 130 male Wistar rats (292 ± 4.0g) divided into 4 groups.The animals were anes thetized with ethyl ether.The studies were conducted in compliance with the principles of humane treatment of animals.Acute destructive pancreatitis was simulated in three experimental groups.The animals were starved a day before the surgery and fed 30 minutes before the study launched that reinforced the digestive processes and facil itated the functional congestion of the pancreas.That allowed distinguishing it more accurately from peripancre atic mass and boosting the development of massive pancre atonecrosis, and the increase of bile secretion due to the activation of pancreatocytes and their enzymes.Midline laparotomy was chosen as a surgical approach.Acute destructive pancreatitis was induced in anesthetisized ani mals by infusion of bile (0.15 ml/kg body weight) taken from the bile duct into the pancreatic tissue followed by ligation of the common bile duct with absorbable suture below the confluence of the pancreatic duct [8].The ani mals were taken out of the experiment in 24 hours, 7 days and 1 month after it.The blood samples for biochemical analysis were collected at the same period.contraction and relaxation.At the same time the perfusate samples passed through the coronary arteries were har vested, and aspartate aminotransferase (AST) and glucose were determined by standard methods.To identify the car diac depression, the high contraction rhythm and hypercalcemic and hypoxic perfusion were applied.
Results.It was found that in acute destructive pancreatitis the power and speed parameters of the heart con tractile function were altered that led to lowering the systolic blood pressure and velocity of contraction and relax ation of the left ventricular myocardium and increased diastolic blood pressure as an indicator of cardyomyocyte contracture rate.These abnormalities were evidently manifested in increased heartbits, including hypercalcemic and hypoxic perfusion of the isolated hearts.Glucose consumption was raised per each mmHg generated by the ventricular pressure.w w w .r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2016 1 16 25 ревязкой общего желчного протока рассасывающимся шовным материалом ниже впадения в него панкреати ческого протока [8].Животных выводили из экспери мента через 24 часа, 7 дней и 1 месяц.В эти же сроки за бирали кровь для биохимических исследований.
The pressure in the left ventricle was measured by electric manometer BMT (Germany) and registered along with the first derivative at the device N338 4P.In 30 min after perfusion (the time required for cardiac function sta bilization) both the cardiac contractile function was recorded and the samples of perfusate passing through the coronary bed were harvested and AST and glucose levels were determined.AST leakage from cardiomyocytes into the coronary flow was calculated per 1g lean myocardium weight, and the glucose consumption was measured per each 1 mmHg developed by the ventricular pressure [10].
Group I included 32 rats, in which the total acute destructive pancreatitis was simulated.Lethal outcomes caused by acute cardio pulmonary failure and septic com plications were 37.5% (12 animals).Group II included ani mals with the follow up lasted for 7 days after the simula tion of acute destructive pancreatitis.Of 37 animals 14 rats died (37.8%).In group III the follow up lasted 30 days, of 41 animals 18 rats died (43.9%).
Upon follow up completed on simulated acute destructive pancreatitis in rats survived under the ether anesthesia the hearts were removed to study their contrac tile function [11].Perfusate sampling was carried out along with the registration of the parameters of the left ventricular contractile function.Glucose value and AST activity were determined with the unified methods calcu lating the glucose intake (1 g wet myocardial mass 1 min 1 mmHg).AST loss in cardiomyocytes was calculated as following (1g dry myocardial weight x 1 min of myocardial contraction).Isolated isovolumic rat hearts were perfused with oxygenated Krebs Henseleit buffer for 30 to elimi nate hypoxic damages and restore the systolic blood pres sure, and the contraction and relaxation rate to the values presented in the literature [10,11].Upon stabilization of the isolated heart work the series of techniques to assess the functional myocardial reserves and identify the most important pathogenetic factors for pancreatogenic cardiac depression: 1) Testing for high frequency rhythmic contractions.While the test performed the heart rate was increased up to 400 per minute.The development of the diastole failure dur ing the test allowed assessing the maintenance and capacity of the mechanisms responsible for calcium transportation from the sarcoplasm into the sarcoplasmic reticulum; 2) Testing for loading the isolated hearts with calcium ions by increasing Са 2+ level in the perfusate from 2.5 to 7.5 mmol/l.This test allowed evaluating the efficiency of Са 2+ pump function of the sarcolemma and the sarcoplas mic reticulum; 3) hypoxic cardiac perfusion with Krebs Henseleit buffer for 15
Снижение силовых и скоростных показате лей сократимости, а также повышение конечного to 150 mmHg) in the perfusate; cardiac reoxygenation with oxygen supply after the hypoxic test to estimate the resistance of the cell membranes to the reactive oxygen species.
Due to the functional limitations the hypoxic test was carried out in the group I (n=16), the group II (n=18), the group III (n=20), and the group IV (n=10).Tests for high frequently rhythmic contractions and hyper calcium test were conducted in the group I (n=16); in the group II (n=19), in the group III (n=21), and in group IV (n=10).
Twenty intact animals were included in the group IV (control).The animals from all groups underwent the same manipulation in one day.
Statistic data processing was conducted using the software Statistica 6.0.The normality of distribution of variables was assessed by (a) Kolmogorov Smirnov criteri um, or (b) the rule of two and three sigma (σ).The data, which disobeyed the Gaussian statistics, were presented as Me (LQ; HQ) and interquartile range (25 and 75 per centiles).Student's t test was exploited to compare the two sets of unrelated samples obeyed the normal distribu tion.Mann Whitney test was used when the compared variables from unrelated samples did not obey the rules of normal distribution.A Wilcoxon criterion was used to compare two dependent samples.The critical value for sta tistical hypothesis test was P=0.05, where the probability of differences was over 95%.

Results and Discussion
As shown in Table 1 pancreatic necrosis result ed in reducing the power and speed parameters of left ventricular contractility in the isolated heart both in the nearest and remote periods.Systolic blood pres sure at the end of the first day was reduced by 20% compared to the control group, and by 28% and 30% 7 and 30 days later, respectively.Diastolic ventricu lar pressure at the end of the first day exceeded 1.6 times the control value, and was 1.2 fold higher than the control values when determined on days 7 and 30 post induction.Contractility rate indices including rate of contractility and rate of relaxation have been similarly altered being decreased in different periods to 10-28% compared to control.
Decreases in contractility power and speed val ues and increases of left ventricular end diastolic pressure indicate a primary cardiac failure evidently expressed in the early period of the study.However, myocardium relaxation was considerably impaired particularly in the remote periods of the study (30 days) due to energy metabolism violation or damage of the membrane ionic pumps, especially Ca pump of sarcolemma and sarcoplasmic reticulum, or a conse quence of the combined effect of both factors [10].It was evidently manifested in inducing the high rate of contractions.Thus, in transition from 120 min 1to 400 min 1 in the control group, a positive inotropic effect (statistically significant increase of the systolic blood pressure by 16%) was observed, and a negative inotropic effect was registered in pancreatic necrosis with the lowering of systolic blood pressure in one w w w .r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2016 1 16 25 диастолического давления в левом желудочке ука зывают на первичную сердечную недостаточность, отчетливо выраженную уже в раннем периоде на блюдения.Однако в большей степени нарушается расслабление миокарда, особенно в отдаленные пе риоды наблюдения (30 суток), что может быть следствием нарушения его энергетического обме на или повреждения мембранных ионных насосов, в первую очередь Са насоса сарколеммы и сарко плазматического ретикулума, либо следствием со четанного действия обоих факторов [10].
In fact, the negative inotropic effect of high fre quency rate and a triple elevation of the diastolic pressure in the left ventricle in the sudden transition from 120 to 400 contractions in 1 minute evidence the damage of the membrane mechanisms for ion transportation.First of all, Ca pump of the sarcolem ma and sarcoplasmic reticulum and Na Ca ion exchange mechanisms responsible for the rapid removal of excessive Ca 2+ from the sarcoplasm and diastolic myocardium relaxation are damaged.
This was confirmed by experiments where the hearts were perfused with a buffer with the triple concentration of Ca 2+ .It is known, Ca 2+ plays a key role in ion transportation, energy metabolism and in interaction with contractile proteins, i.e. it deter mined the processes of contraction and relaxation of the cardiac muscle and, based on the detected viola tions of myocardial contractile function in necrotiz ing pancreatitis, it was reasonable to assess the sensi tivity of isolated isovolumic contracting hearts taken out at the different period after the simulation of pancreatic necrosis to the changes of Ca 2+ rate in the perfusate.This allows determining the damage of sarcolemma and sarcoplasmic reticulum Ca pump responsible for the transmembrane transportation of Ca 2+ , and the role of these alterations in the devel opment of pancreatogenic cardiac failure.
Increasing of Ca 2+ in the perfusate from 2.5 to 7.5 mmol/l accompanied by moderate positive inotropic effect in the hearts of the control group.In 15 minutes of hyper calciemic perfusion the systolic left ventricular pressure elevated from 96.2 (93.1; 98.3) mmHg to 114.4 (107.2;121.4) mmHg, and the diastolic pressure increased 1.6 fold from 3.8 (3.6; 3.9) to 6.3 (5.6; 6.8) mmHg.

Groups of animals
Thus, after the simulation of pancreatonecrosis, the dependence of the cardiac contractile function from Ca 2+ concentration in perfusate was increased.It is known that in the normal conditions the increase level of Ca 2+ in the ambient and in the sar coplasm activates Ca ATPase of sarcolemma and sar coplasmic reticulum and magnifies the absorption rate of Ca 2+ by the membrane structures.Alteration of this response in the hearts of rats with induced pancreatic necrosis demonstrate the damage of the Ca pump responsible for the contraction and relax ation of the heart muscle.
Hypoxia is another important pathogenetic fac tor of development of pancreatogenic cardiac depres sion.The validity of this point was verified by stud ies of isolated hearts perfusion with Krebs Henseleit buffer without glucose, in which рО 2 dropped down from 600 to 150 mmHg.In the control group during the first minute of the hypoxic perfusion the lower ing of the systolic blood pressure was not accompa nied by the raising the diastolic blood pressure, i.e., without the phenomena of contracture.2-3 minutes later the diastolic pressure started gradual elevation from 3.8 (3.6; 3.9) mmHg and in 15 minutes it reached 11.4 (10.5; 12.1) mmHg, indicating the grad ual development of hypoxic contracture.Systolic blood pressure dropped by 2.1 fold.
In the hearts of experimental animals with pan creatic necrosis the damaging effect of hypoxia was most significant; contractures developing from the first minutes of hypoxic perfusion stipulated the elevation of the diastolic blood pressure by 2.6 times in a day, by 2,9 times in 7 days, and by 2.3 times in a month.A 15 minute hypoxic perfusion significantly reduced the systolic pressure in the left ventricle by 1.8 fold in a day, by 1.9 fold in 7 days, and by 2.8 fold from 67.5 (63.1; 69.5) mmHg to 24.1 (22.5; 26.6) mm Hg in a month.Thus, pancreatic necrosis reduces the cardiac resistance to hypoxia, particularly at the level of Ca pump of sarcolemma and sarcoplasmic reticulum.
Recovery of myocardial contractile function of the left ventricle after hypoxia elimination went dif ferently.Reoxygenation after hypoxic perfusion enhanced the reduction of systolic and diastolic pres sure in the left ventricle of the isolated rat hearts.Analyzing the recovery process after hypoxic cardiac perfusion the dependence from the simulation of acute destructive pancreatitis was clearly observed.Elevation of the systolic blood pressure by 5%-10% in average was evidently registered.On the other hand, the diastolic pressure decreased in dynamics and tended to the parameters in the control group, however, it was less than the restoration of the car w w w .[12].
В этих же исследованиях одновременно с ре гистрацией сократительной функции миокарда diac systolic function, indicating on keeping of resid ual effects of hypoxic myocardial contraction.It has been supposed, that such multidirectional temporary changes of sensitivity to hypoxic isolated rat hearts, where acute destructive pancreatitis was simulated, were induced by additional pancreatogenic factors exposure (Figure), detected in the blood and tissues of animals and having a cardiotropic impact [12].
In these studies, myocardial contractile func tion and AST release from cardiomyocytes into the coronary flow and glucose consumption by 1 mmHg of the developed pressure were observed (Table .2).Leakage of enzymes into the coronary flow a day after the simulation of pancreatic necrosis was increased by 87%, and by 59% was over a control values a week later, reflecting alterations of sarcolemma of car diomyocytes and AST release in the perfusate, which did not differ from the control group a month later.
Under cardiac hypersensitivity to oxygen insufficiency, the deficit of energy supply for car diomyocytes, inadequate mechanisms for maintain ing of Ca optimum concentration in cytoplasma became evident.It could be assumed that the mito chondrial dysfunction and destabilization of mito chondrial membranes [13, 14 exacerbate the deficit of energy rich synthesis [15,16].Mitochondrial dys function did existed because of evidence of wasteful consumption of glucose by the rat hearts in necrotiz ing pancreatitis: a day intake of glucose by the left ventricle per each mmHg increased by 21% in 7 days and by 33% in a month later.

Conclusion
Most important pathogenetic factors of meta bolic alterations in the heart that led to development of prolong cardiac depression included hypoxia, car diomyocyte membrane destruction, mitochondrial dysfunction, early endotoxemia and reactive oxida tive species [12,14].These factors and their negative impact on the heart were discovered by assessing the impact of blood components of rats with acute destructive pancreatitis on the intact hearts.Presumably, the influence of various pathogenic fac tors on the metabolism and the structural integrity
Our study revealed the regularities in power and rate parameters in isolated isovolumic heart in rats under permanent monitoring of the main parameters of heart function during prolonged time frame following modeling of acute destruc tive pancreatitis.
The shifting of key pathogenetic factors effecting the cardiac tissue was registered.In acute pancreatitis.the signs of cardiomyocyte alterations by aggressive pancreatic enzymes, a myocardial depression factor, and toxins initially prevailed.Hypoxic damages and inflammation lesions of vascular wall manifested in excessive proliferation of the connective tissue cells in the myocardium seem to play a dominant role in acute pancreatitis [12,17].Then, the changing the domi nant dysfunction of myocardium in early acute destructive pancreatitis to systolic dysfunction occured, that could be considered as a consequence of a later inflammation within the pancreas.However, if the diastolic dysfunction was mainly associated with damage of calcium pumps and imbalance in the system of pro and anti oxidants [17,18], the cause of systolic dysfunction appeared to be alterations in oxidation reduction processes stipulating the inefficient metab olism of glucose as an energy substrate.
. r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2016 1 16 25 r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2016 1 16 25 Original Investigations Блок схема формирования порочного круга на клеточном уровне вследствие развития кислородной недостаточности кардиомиоцитов (на основе литературных данных и результатов собст венных исследований).Flow chart for a vicious circle formation at the cellular level due to the development of cardio myocytes hypoxia (based on the data published and the results of studies carried out).
. r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2016 1 16 25 min with рО 2 lowering in 4 times (from 600 w w w .r e a n i m a t o l o g y .c o m

Влияние острого деструктивного панкреатита на сократительную функцию сердца (Me, LQ; HQ). Table 1. Effect of acute destructive pancreatitis on the cardiac contractility (Me, LQ; HQ).
w w w .r e a n i m a t o l o g y .c o m

Table 2 . Effect of acute destructive pancreatitis on glucose intake and AST release into the coronary flow of isolat ed hearts (М±σ
σ).
w w w .r e a n i m a t o l o g y .c o m