Ultrasound assessment of heart remodelling affected by therapeutic hypothermia and MSC on myocardial infarction model

© Чиж М. О., Манченко А. О., Трофімова А. В., Бєлочкіна І. В., 2020 РЕЗЮМЕ Актуальність. Несвоєчасне звернення по медичну допомогу, обмежена кількість кардіохірургічних стаціонарів та не завжди результативна консервативна терапія стимулють пошук нових, більш ефективних методів лікування гострого інфаркту міокарда (ІМ). Останнім часом велику увагу дослідників привертають мезенхімальні стромальні клітини (МСК) завдяки їх перспективності для клінічного застосування. З іншого боку, за результатами багатоцентрових досліджень доведено, що терапевтична гіпотермія (ТГ) має нейроі кардіопротекторну дію і використовується як один із невідкладних методів при наданні первинної медичної допомоги. Мета роботи – проведення та аналіз ультразвукового дослідження серця щурів з експериментальним ІМ для визначення характеру ремоделювання серця під впливом поєднаного використання ТГ і введення алогенних МСК. Матеріали та методи. Дослідження було проведено на 90 безпорідних білих щурах масою 240–270 г. Інфаркт міокарда відтворювали шляхом перев’язки низхідної гілки лівої коронарної артерії на межі верхньої та середньої третини судини. Терапевтичну гіпотермію проводили в холодовій камері протягом 60 хв. Локальна температура шкіри комірцевої зони підтримувалася на рівні + 4 оС, при цьому ректальна і тимпанічна температура знижувалася до + 25 оС. Суспензію алогенних кріоконсервованих МСК плаценти з концентрацією 1,2 × 106 клітин/мл одноразово вводили внутрішньовенно через v. saphena magna. Сонографічне дослідження серця проводили на ультразвуковому ехотомоскопі «Сономед 500» («СПЕКТРОМЕД», Росія) у Ві М-режимі з використанням лінійного датчика 7,5L38 з частотою 7,5 МГц. Результати та їх обговорення. У контрольній групі з експериментальним ІМ встановлено значне пригнічення функції лівого шлуночка (ЛШ). Це відображалось у зниженні УО та ХО і в підсумку свідчило про зменшення ФВ до 46,04 %, що на 35 % менше відповідного показника норми. Ультразвукова характеристика ремоделювання серця під впливом терапевтичної гіпотермії та МСК на моделі інфаркту міокарда Чиж М. О.,1 ORCID ID: https://orcid.org/ 0000-0003-0085-296X, e-mail: n.chizh@ukr.net Манченко А. О.,1, 2 ORCID ID: https://orcid.org/ 0000-0001-5982-4504, e-mail: anna.gorlenko@gmail.com Трофімова A. В.,1 ORCID ID: https://orcid.org/ 0000-0002-4662-1318, e-mail: fortomi@rambler.ru Бєлочкіна І. В.,1 ORCID ID: https://orcid.org/ 0000-0003-0090-2971, e-mail: ibelochkina@ukr.net 1 Інститут проблем кріобіології і кріомедицини Національної академії наук України, Харків, Україна 2 Харківський національний університет ім. В. Н. Каразіна Міністерства освіти і науки України, Харків, Україна


Connection with scientific programs, plans and topics
The study has been carried out in the scope of the research project of Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine "Destructive and Regenerative Processes in Tissues in vivo after effect of Low Temperature and Biological Active Substances", state registration No 0117U000849.

ІNTRODUCTION
One of the most common disorders of modern cardiology is coronary heart disease (CHD). The prevalence of this pathology is rapidly increasing and high mortality rate results from one of its severe complications, i.e. myocardial infarction (MI). Annually, about 45 thous new cases of MI are recorded in Ukraine [1]. Stress, busy lifestyle, long-standing diseases of the cardiovascular Results and discussion. The control group with experimental MI showed significantly suppressed function of the left ventricle (LV). It resulted in decreasing stroke volume (SV) and cardiac output (CO) and on the whole indicated reduced ejection fraction (EF) to 46.04 %, that was 35 % less than the corresponding normal range. According to the values of relative wall thickness (RWT) and left ventricular mass (LVM) on day 7 and day 30 after ligating the left coronary artery, LV remodelling was proceeding via eccentric mode of LV alteration. Therapeutic hypothermia was not able to completely stop the pathophysiological processes associated with coronary ligation. EF was not significantly different from the control group, and was 51.08 ± ± 2.68 %. On day 7 of the experiment, heart remodelling in this group was proceeding according to the normal geometry model, and on day 30according to the eccentric model. In spite of the volume overload causing post-infarction extension of the left ventricular cavity, in the group with applying MSCs, on day 7 there was a compensatory increase of the stroke volume, 1.8 times over compared to the group with normal range values and 2.3 over compared to the control group. The ejection fraction was 17 % less than the normal range, but statistically significantly higher than the corresponding indicator of the control group of this observation period. Heart remodelling after applying allogeneic MSCs associated with MI at all stages of observation was proceeding by eccentric LV hypertrophy. Аfter therapeutic hypothermia and applying MSCs associated with experimental myocardial infarction on day 7 and day 30, the group recorded the best values of echo params of LV anatomical structures, indicating no dilatation along with occuring moderate myocardial hypertrophy. The ejection fraction showed the best outcome, i. e. 58.78 %, while LV remodelling was minimal, occurring according to normal heart geometry. Conclusions. Applying echocardiography in rats is a very informative diagnosis method which makes it possible to describe the type of structural and functional remodelling of the myocardium associated infarction at early and late observation stages. The ultrasound study showed that the closest to the normal range was the group of animals exposed to therapeutic hypothermia and MSC transplantation. According to LVM and RWT values, in the rats of that group on day 7 and day 30 of the experiment, LV remodelling was characterized by normal geometry. Ukrainian journal of radiology and oncology. 2020;28(3):191-328 ISSN 2708-7166 (Print) ISSN 2708-7174 (Online) Оригінальні дослідження 225 Original research захворювання серцево-судинної системи і несвоєчасне лікування -усе це стає причиною почастішання і «омолодження» гострого коронарного синдрому. Використання інтервенційних технологій в перший «золотий час» після інфаркту приводить до усунення механічної перешкоди (тромба) в коронарній судині, а встановлення стента сприяє відновленню кровопостачання серця [2]. На жаль, в Україні кількість кардіологічних центрів обмежена, а кількість втручань зі стентування, наприклад, за 2018 рік, складає лише 286 випадків на 1 млн населення [2,3]. Несвоєчасне звернення по медичну допомогу, обмежена кількість кардіохірургічних стаціонарів та не завжди результативна консервативна терапія стимулють пошук нових, більш ефективних методів лікування гострого ІМ.
Applying interventional technologies immediately during 1 "golden" hour after infarction results in the removal of a mechanical obstruction (thrombus) in the coronary vessel, and installing a stent helps to restore blood supply to the heart [2]. Unfortunately, the number of cardiac centers in Ukraine is limited, and the number of stenting interventions, for example, in 2018 is only 286 cases per 1 million population [2,3]. Late seeking medical advice, limited number of cardiac surgery hospitals and conservative treatment, which does not seem to be always efficacious, trigger the search for new, more effective mode therapy of acute myocardial infarction (MI).
Recently, mesenchymal stromal cells (MSCs) have come into sharp focus of scientists due to the prospects for clinical use. MSCs are the cells which have a potential to differentiate and form tissues of different types. Due to unique immune regulatory potential and ability to secret a wide range of trophic and growth factors, MSC are considered to be a perfect tool of gene and regenerative therapy of ischemic injuries of the myocardium [4].
On the other hand, multicenter studies have proved that therapeutic hypothermia (TH) has neuro-and cardioprotective effects, and it is administered as one of the urgent methods in providing primary health care [5]. Combining TH along with transplanting MSC seems to have good prospects in this regard.
In clinical experience, ultrasonography (US) of the heart, as a non-invasive and painless study, is an essential in diagnosis and control of the effectiveness of treating patients with acute coronary syndrome. In MI, LV dysfunction is accompanied by involvement of compensatory mechanisms aimed at maintaining cardiac output at an appropriate level. Cardiac remodelling, initiated during the cessation of blood supply to the coronary vessel, lasts throughout the treatment period and can be both adaptive and maladaptive. Adaptive changes in the heart aid to maintain normal cardiac output. The maladaptive nature of remodelling is characterized by progressive dilatation of the left ventricle, a significant change of geometry and reduced pumping function, which ultimately leads to chronic heart failure [6] and therefore, diagnosing postinfarction left ventricular remodelling and case monitoring along with assessing echocardiographic parameters make it possible to detect cardiac failure signs.
Purpose -рroviding and analyzing ultrasonography (US) of rat hearts with experimental MI in order to determine the nature of heart remodelling under combined use of TH and introducing allogeneic MSCs.
Myocardial infarction was reproduced by ligating the descending branch of the left coronary artery on the border of the upper and middle third of the vessel [7].
Therapeutic hypothermia was performed in a cold chamber, 60 minutes long. The local skin temperature of the neck area was maintained at +4 о C, while the rectal and tympanic temperature decreased to +25 о C [8].
A suspension of allogeneic cryopreserved MSCs of the placenta with a concentration of 1.2 × 10 6 cells / ml was administered once intravenously through v. saphena magna. Mesenchymal stromal cells of the placenta of rats were obtained and phenotyped according to the approach of G.M Svitina et al. [9].
After MI modelling, all animals were divided into 5 groups, represented by 15 animals each. The control group (MI) consisted of rats with experimental MI without treatment. Rats with induced TH associated with experimental MI were included in Group 2 (MI + TH). Group 3 there was represented by rats after administering MSCs associated with MI (MI + MSC). Group 4 consisted of animals after combined use of TH and applying MSCs associated with MI (MI + TH + MSC). The group with normal range values enrolled 15 intact rats.
Heart sonography was carried out by means of «Сономед 500» («СПЕКТРОМЕД», Russia) ultrasound scanner in B-and M-mode using a linear sensor 7.5L38 with frequency of 7.5 MHz. Ultrasound scanning was performed on the area perpendicular to the chest surface with parasternal access along the long axis of the heart. When researching in M-modal mode, the structures of heart cavities, i.e. diameters and distances, were measured: end-diastolic diameter of LV (EDD), end-systolic diameter of LV (ESD), interventricular septum thickness at end-diastole (IVSd), interventricular septum thickness at end-systole (IVSs), left ventricular posterior wall thickness at end-diastole (LVPWd), left ventricular posterior wall thickness at end-systole (LVPWs) and heart rate (HR).
After measuring the anatomical structures, automated data processing software, embedded in the ultrasound system, allows, on the basis of the obtained values, to calculate the main values of volume-velocity properties of the heart and LV myocardial contractile function according to the formulas presented in Table 1.
Based on the screening electrocardiographic study on a left coronary artery ligation model, we selected two observation periods that would reflect the processes of heart remodelling at different stages of the disease. At the acute stage of MI, ultrasound examination was carried out on day 7, when the echo params were reflecting the process of adaptation of the heart muscle to functional load, and on day 30 of the experiment, in the period процес ремоделювання серцевого м'яза знаходився в завершальній фазі (стадія рубцювання). Цифрові дані наведені у вигляді «M ± m» (M ± ± SE), де M -середнє арифметичне значення, m (SE)стандартна похибка середнього арифметичного. Статистичну обробку результатів проводили, використовуючи критерій Краскела -Уолліса за допомогою пакета програм STATISTICA 6.0 («StatSoft», USA).
The numeric data are presented in the form of "M ± m", where M is the arithmetic mean, m (SE) is the standard error of the arithmetic mean. Statistical processing of the outcomes was performed according to the Kruskel-Wallis test by means of STATISTICA 6.0 software package (StatSoft, USA).

RESULTS AND DISCUSSION
Echocardiographic params of rat hearts on day 7 of experimental myocardial infarction. Ultrasound examination of rats showed that normally the left ventricular cavity has an oval (conical) shape, the movement of the interventricular septum and posterior wall in at end systole is mutually directed, the anterior and posterior mitral valve leaflets are moving in antiphase (Fig.  1). The main ultrasonic measurements are presented in Table 2. Table 1 and Table 2 show that the difference between systolic and diastolic dimensions of the heart structures is of a great clinical importance; further, this fact is an essential for calculating volume and more integrative values of LV functional status: ejection fraction, fractional shortening as well as percentage of systole thickness of LV posterior wall and interventricular septum as well as LV relative wall thickness.
As far as is known, the removal of a part of viable cardiomyocytes from the general contractile process leads to a pronounced kinetic heterogeneity between the damaged, borderline and distant parts of the left ventricular wall. Ischemic and preserved areas differ significantly in terms of the level of systolic thickening and chronological sequence of contraction -relaxation of myocardial fibers. The decreased force developing by myocardial segments under their kinetic inhomogeneity is less than in the case of synchronous contraction, which potentiates the drop in contractility and pumping function of the left ventricle as a whole [11]. Significant suppression of LV function in the control group led to decreased SV and CO, and as a result, suggested a reduced ejection fraction to 46.04 %, Таблиця 3. Об'ємно-швидкісні характеристики лівого шлуночка щурів на 7-му добу після перев'язки лівої коронарної артерії

Notes:
Differences are statistically significant (p < 0.05): 1 -in comparison with intact animals; 2 -in comparison with the control group.
In this case, as a compensatory mechanism for the delivery of oxygen, nutrients and restoring the balance of volume and blood flow, the heart rate increased up to 430 contractions per minute. However, the cardiac output associated with heart rate is falling behind the normal range by 10 % (Table 3).
The thickness of the interventricular septum and posterior wall also tended to increase at end diastole as well as at end systole ( Table 2). This had an impact on the values of myocardial contractile function and indicated decreased systolic heart function ( Table 4).
When calculating the percentage of contractility of the interventricular septum (IVSsT), a decrease of this value by 20 % compared to animals of the intact group was observed, while the percentage of contraction of the posterior wall of the left ventricle was within normal range (Table 4).
Based on calculation data on left ventricular mass index and LV relative wall thickness, geometric models of the LV (remodelling types) were being assessed. According to the classification of Ganau A, et al. (1992), there are 4 types of structural and functional remodelling of the myocardium: concentric hypertrophy, eccentric hypertrophy, concentric remodelling and normal LV model [12]. In the analysis of experimental data, it was found that the relative thickness of the posterior wall of the left ventricle was statistically significantly different from the normal range value and was equal to 0.29 ± 0.01. Due to a minor increase of thickness of the interventricular septum and posterior wall of the left ventricle at end diastole, the mass of the left ventricle also increased and reached the level of 0.85 ± 0.03 g (Table 4).
According to RWT and LVM values, we could conclude that on day 7 after ligating the left coronary artery, the remodelling of the LV of rats as per the classification of LV hypertrophy models was proceeding due to eccentric remodelling mode of the LV.
Therapeutic hypothermia was not able to completely stop the pathophysiological processes associated with coronary ligation. Increasing EDD and ESD by 11 % and 35 %, respectively, on day 7 resulted in increased EDV by 36.5 % compared to normal range values, while the ESV value was 2.6 times increased (Tables 2, 3).
Despite the changes in the heart architecture, accompanied by dilatation of the left ventricular cavity, stroke volume and cardiac output were within the normal range (Table 3). However, the ejection fraction did not significantly differ from the control group, and was 51.08 ± 2.68 % (Table 3). At the same time, fractional shortening was 1.6 times less than the normal range values, and also it did not differ significantly from the control group (Table 4).
The relative wall thickness of the left ventricle and left ventricular mass did not differ significantly from the corresponding parameters of the normal group suggesting remodelling of the heart muscle in rats of this group according to the model of normal geometry, and as a consequence, the values of stroke volume and cardiac output did not differ from the values of intact animals. However, it is worth noting that on day 7 of experimental myocardial infarction, therapeutic hypothermia as a method improving energy metabolism in the myocardium, does not fully prevent the development of LV dilatation.
Post-infarction change in the geometry of the left ventricle is a compensatory process resulting in the maintenance of LV contractile function, which is achieved by balancing between myocardial hypertrophy and dilatation of the heart chambers. This is especially true for the group of rats injected with allogeneic cryopreserved MSCs.
Changes in the architecture of the heart on day 7 were primarily associated with increasing echo parameters of EDD and ESD, which were 1.35 and 1.6 times higher than the normal range suggesting dilatation of the left ventricle (Table 2). Despite the volume overload causing postinfarction enlargement of the left ventricular cavity, there was a compensatory increase in stroke volume 1.8 times over vs the normal group and 2.3 times over vs the control group (Table 3). It should be noted that the ejection fraction in animals of the group where MSCs are administered is 17 % less than the normal range, but statistically significantly higher than the corresponding value of the control group of this observation period.
Measuring the absolute values of LV wall thickness showed that the thickness of the interventricular septum did not differ significantly from the normal range, and the thickness of the posterior LV wall was increased at end diastole as well as at end systole: 1.39 ± 0.06 mm and 1.83 ± 0.08 mm respectively ( Table 2). The increase in the thickness of the posterior wall of the left ventricle, caused by the influence of MSCs on reparative processes in the myocardium, indicated hypertrophy of the heart muscle. This is confirmed by the fact that on day 7 the LV mass increased by 32 % vs the group with normal range values. Experimental and clinical outcomes show that when MSCs reach the lesion, they help to restore myocardial function and have a positive effect on LV remodelling [4]. It is known that after administering MSCs, the functional reserve of the myocardium increases due to stimulation of angiogenesis, development of collateral  [4]. Відомо, що після введення МСК функціональний резерв міокарда підвищується за рахунок стимуляції ангіогенезу, розвитку колатерального кровотоку, а також за рахунок підвищення якості роботи збережених скорочувальних елементів кардіоміоцитів [15]. На підставі проведеного дослідження на 7-му добу експерименту встановлено, що ремоделювання серця після перев'язки лівої коронарної артерії і введення алогенних МСК проходило шляхом ексцентричної гіпертрофії ЛШ, при якій збільшення маси ЛШ зумовлене його дилатацією з розвитком міокардіальної недостатності при нормальній відносній товщині стінки ЛШ.
Ultrasonography showed that the group of animals exposed to combination of therapeutic hypothermia and MSC transplantation was the closest to the normal range values. EDD and ESD values exceeded the corresponding values of the group with normal range by 10 % and 30 %. In spite of this fact, EDV and ESV, stroke volume value was max close to that one of intact animals,, 0.39 ± 0.04 ml ( Table 3). The ejection fraction and fractional shortening were also max close to the normal range. They did not differ significantly from the values of the group exposed to administering MSC (Tables 3, 4).
The relative wall thickness of the left ventricle also did not exceed the normal range, 0.35 ± 0.02, while the mass of the left ventricle was 0.87 ± 0.03g. Having analyzed the outcomes, we can conclude that the combined use of therapeutic hypothermia and allogeneic MSCs in terms of LV mass and RWT on day 7 of the experiment contributed to the remodelling of the LV under normal geometry. Regarding volume-velocity properties, the echo values of this group were max close to the normal range and, therefore, no signs of severe LV dilatation were observed.

Echocardiographic values of the heart of rats on day 30 of experimental myocardial infarction
Ultrasonography findings showed that on day 30 after ligating the left coronary artery in the control group of rats, increased EDD in comparison with the previous observation period in the setting of the preserved ESD was observed ( Table 5).
The increase in EDD, in its turn, led to increasing EDV by 17 % compared to this value on day 7, suggesting the enlargement of the left ventricular cavity, i.e. its dilatation (Table 6), and confirming the fact that longterm, continuously volume overload was causing the stress of compensatory mechanisms and dynamic progression of dilatation [16].
According to those changes, the stroke volume was increasing, 0.38 ± 0.03 ml. It affected the ejection fraction which within the period of 23 days increased only by 5 % and reached 50.95 ± 1.92 % being at the time quite low in comparison to the normal range and other groups involved in the experiment. The increase in stroke volume also influenced cardiac output velocity which increased 1.45 times compared to the previous observation period and exceeded the normal range by 10 % (Table  6). Due to the increased heart rate, cardiac output was increasing and so exceeding the normal range values by 26 % and 41 % compared to the previous observation period. Despite the increase in stroke volume, the lowest fractional shortening was observed among the groups (Table 7).
Adaptive tonogenic dilatation, under normal conditions, was combined with increasing muscle mass Ukrainian journal of radiology and oncology. 2020;28(

Notes:
Differences are statistically significant (p < 0.05): 1 -in comparison with intact animals; 2 -in comparison with the control group.
Thus, at the stage of scarring, after ligating the coronary artery, there was continuing LV dilatation, decreased contractility and signs of LV remodelling by eccentric type. In terms of prognosis, all these echocardiographic parameters suggest the likelihood of development of chronic heart failure, including the stage of decompensation, in rats of this group.
No pronounced differences in the echo parameters of anatomical structures in the group after therapeutic hypothermia associated with ligating coronary artery in comparison with the control group on day 30 were observed. First of all, it concerns ESD, so ESV was at the level of the control group as well (Tables 5, 6). The ejection fraction was 55 %, that was 4 % higher than the normal range (Table 6).
Regarding the relative wall thickness of the left ventricle, it was the smallest among the experimental groups, and the mass of the left ventricle did not differ statistically significantly from the normal range. Therefore, on day 30, LV remodelling was progressing in this group, not according to the normal geometry, but according to the eccentric model. Those changes resulted from the short-term effect of therapeutic hypothermia. Providing therapeutic hypothermia procedure for 60 minutes was fully aiding in short-term reduction of oxygen consumption in the area of ischemia and suppression of free radical reactions, that affected the echo parameters on day 7 of the experiment.
Quite high measurements of the anatomical structures of the heart after administering allogeneic mesenchymal MSCs associated with experimental myocardial infarction on day 30 showed a minor decrease compared to the previous observation period (Table 5). Despite this, they were quite high compared to the normal range. Thus, EDD and ESD were 1.27 and 1.6 times, respectively, higher than the normal range (Table 5). In this regard, increasing EDV and ESV 2 and 3 times over than the normal range lead to increasing stroke volume up to 0.57 ± 0.1 ml. It is worth noting that in this group the heart rate did not differ statistically significantly from the normal range, 390 ± 7.1 contractions per minute, that indicated rather saving activity of the heart due to the increase in cardiac output. The ejection fraction reached 57.45 % as well as the fractional shortening reached 26.53 % (Tables 6, 7). LV remodelling was progressing according to the eccentric type of hypertrophy, as evidenced by the ratio of the relative wall thickness and LV mass. It should be emphasized that MSCs have a paracrine effect, i.e. due to the release of a number of biologically active substances, there is the regulation not only of angiogenesis but also restorative morphogenesis of the myocardium [15].
Thus, the most pronounced dilatation of the left ventricle, caused by ligating the left coronary artery, was partially compensated by administering allogeneic MSCs, which aided in compensatory hypertrophy of the left ventricular wall affecting the restorating volume-velocity properties of the left ventricle. The increased LV mass in the setting of normal relative LV wall thickness confirmed that in animals of thas group, heart remodelling was progressing according to the model of eccentric LV hypertrophy.
Combination of therapeutic hypothermia and administering allogeneic MSCs aided in the most pronounced recovery of echo params on day 30 of the experiment. In comparison with the previous observation period and the control groups, EDD and ESD decreased ( Table 5). As a consequence of these changes, EDV and ESV values were 14 % and 29 % lower than the control group and were max close to the normal range values (Table 6).
Stroke volume and cardiac output did not differ statistically significantly from the normal range. Normalization of these params affected the ejection fraction exceeding the control group by 8 % and showing the best outcome -58.78 %, while the fractional shortening was 27.2 % ( Table 7).
The relative thickening of the left ventricular wall and the mass of the left ventricle did not differ significantly from the normal range, and therefore the remodelling of the heart was proceeding according to the normal geometry. Volume-velocity properties confirmed the preserved LV systolic function. MSCs act as inducers of regeneration processes in remodelling the damaged myocardium, providing reparative morphogenesis and increasing the adaptive reserves of the preserved myocardium. Increasing efficiency of myocardial reparative morphogenesis is enhanced by the production of MSCs of nonspecific and tissue-specific regulatory peptides, the action of which is aimed at optimizing the functioning of the preserved viable myocardium and reducing the volume of secondary myocardial damage.