**2. Materials and methods**

108 Front Lines of Thoracic Surgery

2002; Menicanti & Di Donato, 2004; Moreira et al., 2001; Popovic et al., 1998; Popovic et al.,

The efforts to find clinical and instrumental prognostic criteria of unfavorable late outcomes of surgical treatment in patients with ICMP have not resulted in anything. According to publications, the following preoperative values have been associated with higher postoperative mortality and morbidity of the patients with ICMP: size of left ventricle (LV) (Yamaguchi, 2005), LV end-systolic volume index (LVESVI) (especially > 80 ml/m2 (Athanasuleas, 2004) and > 100 ml/m2 (Yamaguchi, 2000)), LV ejection fraction (EF) (< 20% (Di Donato, 2001; Yamaguchi, 2005) or < 30%(Yamaguchi, 2000; Athanasuleas, 2004)), mitral regurgitation (Sartipy et al., 2006; Schroder, 2005), number of segments affected by dyssynergia (Di Donato, 1997), pulmonary hypertension >33mmHg (Di Donato, 1997), QRS>130ms (Yamaguchi, 2005), preoperative renal failure (Yamaguchi, 2005), time after previous myocardial infarction (Yamaguchi, 2005), age older than 75 years old (Athanasuleas, 2004). Nevertheless, there have not been any definite preoperative clinical

Myocardium is a unique tissue consisting of highly differentiated cells – cardiomyocites which possess a number of morphological features in norm responding by a set of nonspecific structural changes to pathomorphism of cardiovascular diseases. In our opinion, the degree of revensability/irreversibility of advanced pathological processes in myocardium plays a key role in the success of reconstructive cardiac surgical

It has been 10-12 years since researchers started their first search for morphological predictors of postoperative heart remodeling in patients with cardiomyopathies, carrying out the analysis of the postoperative period course and evaluating morphofunctional condition of LV myocardium by the data of intraoperative biopsies (Gradinac, 1998; Moreira et al., 2001; Popovic et al., 2001; Stolf, 1998). However, the results of these solitary studies have been quite controversial (Moreira et al., 2001; Popovic et al., 2001). We have not found data about any attempts to search for morphological predictors of progressive postoperative LV remodeling in patients with ICMP. In the available Russian publications there are separate articles devoted to studies of morphofunctional condition of LV myocardium and myocardium of RA auricle in patients with coronary artery disease of different functional classes (Kuznetsov, 2003; Salikova et al., 2002). These works can hardly boast wide analysis of morphological parameters (Kuznetsov, 2003) and some authors only provide descriptive morphology without deep investigation of the mechanisms of possible pathogenesis of heart

At the same time, identification of morphological predictors of postoperative LV remodeling will not solve all the problems which cardiac surgeons face when choose the tactics for surgical intervention and think of the prognosis for each individual patient since pre- and postoperative morphological diagnostics of the pathological processes reversibility degree in ischemic myocardium in reality is limited very much by a definite degree of a risk associated with harvesting biopsies from heart walls which very often becomes a reason to refuse from this diagnostics. In the light of this, one of the perspective directions of scientific research is the finding of molecular predictors of postoperative heart remodeling in peripheral blood of patients together with tissue and cellular aspects of this phenomenon for

the blood is always available for laboratory testing and monitoring of its content.

2001; Ratcliffe, 1998; Shah, 2003; Soo, 2005; Stolf, 1998).

predictors of postoperative LV remodeling offered.

interventions.

remodeling (Salikova et al., 2002).

### **2.1 Design of the study. Object**

One hundred and ninety five patients with ICMP and with previous myocardial infarctions have become the object of the study. All the patients were admitted to the cardiovascular surgery department at Tomsk Institute of Cardiology during the period from 2002 to 2009. Preoperative diagnostics included transthoracic EchoCG, Halter ECG monitoring, coronaroventriculography with manometry, SPECT imaging with 99mTc-technetril, MRI imaging of the heart with dye.

The clinical inclusion criteria for the patients enrolled into the study were the following parameters: LV end-diastolic volume index (LVEDVI) > 90 ml/m2, LVESVI > 70 ml/m2 , LV end-diastolic pressure (LVEDP) > 30 mmHg, EF LV < 40%, akinetic and dyskinetic areas of LV, angina II-IV CCS FC, heart failure (HF) II-IV NYHA, coronary artery disease from 1 - 10 years, lesions of coronary arteries – stenosis of more than 75% of LAD or of the trunk, or not less than 75% stenosis in at least two coronary arteries. The age of the patients included into the study was between 37 and 68 years (53.6±8.3), mean number of affected coronary arteries was 2.7±0.4. Lack of organic lesions of heart valves apart from ischemic mitral valve regurgitation was also a clinical criterion for patients' selection.

The reason for the development of ICMP in all the patients was an extensive transmural myocardial infarction. In 135 patients (69.2%) heart insufficiency appeared after their first myocardial infarction. All the patients in conditions of bypass and cardioplegia underwent surgical reconstruction of LV by V.Dor and L.Menicanti methods in different combinations with myocardial revascularization, reduction and reconstruction of LV volume and shape due to exclusion of its scarred septal, anterior and basal parts by endoventriculoplasty and by restoration of the mitral valve (MV) function.

Morphological Predictors and Molecular Markers of Progressing

revascularization.

enrolled into the study

examination and control EchoCG.

remodeling – group II) dynamics.

pathology.

myocardial structures.

**2.2 Histological study methods** 

Postoperative Remodeling of Left Ventricle in Patients with Ischemic Cardiomyopathy 111

Spectrum of surgical interventions Absolute value (n = 195) Relative value (%)

*Note:* CABG – coronary artery bypass grafting; SVR – surgical ventricular reconstruction; MV repair –

Biopsy samples of RA auricle and LV were taken from the border area of endocardial scar and from the area of the myocardium without visual changes from all the patients (n=195, 100%) with ICMP. RA auricle biopsy was performed during the period of RA cannulation. LV biopsy samples were taken during surgical ventricular reconstruction from the transient

All the patients signed an informed consent form for the participation in the study; the

In order to find noninvasive molecular markers of postoperative LV remodeling blood samples were taken from 53 patients with ICMP (27.2%) to identify the content of natriuretic peptides and matrix metalloproteinases in blood plasma and serum correspondingly. In 37

In the early postoperative period (1 month) control transthoracic EcoCG was made. In 12 months after the surgical treatment the patients were hospitalized again for clinical

In the late follow-up period repeated LV remodeling and HF progressing took place in a part of the patients which resulted in assignment of the patients into two groups: with positive (regressive remodeling – group I) and negative (progressing postoperative

To achieve the objectives of identification tissue, cellular and molecular markers of postoperative LV remodeling we have used histological, electron-microscopic,

To compare morphometrical parameters, as a control group we took autopsy samples of the identical sites of LV myocardium and myocardium of RA auricle from 25 cadavers of both sexes and of a comparable age died from an acute trauma with no signs of cardio-vascular

Seventeen healthy male and female volunteers of comparable age comprised a control group for the evaluation of the content of natriuretic peptide and matrix metalloproteinases in blood plasma and serum, respectively, as well as for identification of the antibodies titre to

Histological methods include preliminary treatment of the studied material necessary for its further microscopic evaluation. Preparation of histological samples was performed as follows (Krivolapov, 2006): the samples of myocardium were being fixed in 10% solution of

study was approved by the local ethical committee of Tomsk Institute of Cardiology.

patients antibodies titre to myocardial structures was identified in blood serum.

CABG+SVR 132 67.6 CABG+SVR+MV repair 39 20.0 CABG+SVR+MV prosthesis 12 6.2 SVR+TMLR 6 3.1 SVR+CABG+TMLR 6 3.1

mitral valve repair; MV prosthesis – mitral valve prosthesis; TMLR – transmyocardial laser

zone on the border between scarred tissue and unchanged myocardium.

morphometrical, biochemical and statistical methods of study.

Table 2. The spectrum of surgical interventions carried out in the patients with ICMP


Table 1. Initial clinical characteristics of the patients enrolled into the study

Intraoperative control of the remaining LV cavity was performed with the help of special devices (sizers) and satisfied a physiological norm for each patient (55-60 ml/m2). Reduction of LV cavity had to be combined with giving it elliptical shape, which was performed with the use of endovascular patch and retraction of papillary muscles. Interventions on mitral valve were performed in 51 patients (26.2 %). MV repair was made in 36 cases (18.5 %); MV prosthesis was placed in 12 cases (6.2 %). The spectrum of surgical interventions is shown in table 2.

Number of the patients 195 (100 %) Men 177 (90.8 %) Women 18 (9.2 %) Mean age (years) 53.6±8.3

NYHA functional class 2.86±0.40

LVEDVI (ml/m2) 114.9±28.4 LVESVI (ml/m2) 76.7±23.0 LV EF (%) 32.1±5.5

Hypertensive disease 126 (64.6 %) Diabetes mellitus 30 (15.4 %) Obesity 39 (20.0 %) Peripheral atherosclerosis 33 (16.9 %)

Intraoperative control of the remaining LV cavity was performed with the help of special devices (sizers) and satisfied a physiological norm for each patient (55-60 ml/m2). Reduction of LV cavity had to be combined with giving it elliptical shape, which was performed with the use of endovascular patch and retraction of papillary muscles. Interventions on mitral valve were performed in 51 patients (26.2 %). MV repair was made in 36 cases (18.5 %); MV prosthesis was placed in 12 cases (6.2 %). The spectrum of surgical

Table 1. Initial clinical characteristics of the patients enrolled into the study

Number of previous MI

Angina functional class (CSS)

atherosclerotic lesions

Degree of mitral regurgitation

interventions is shown in table 2.

Number of the coronary arteries with

Clinical characteristics Value

Without

1 135 (69.2 %) 2 39 (20.0 %) 3 15 (7.7 %) 4 6 (3.1 %)

angina 9 (4.6 %) I 15 (7.7 %) II 36 (18.5 %) III 132 (67.7 %) IV 3 (1.5 %)

1 72 (36.9 %) 2 54 (27.7 %) 3 69 (35.4 %)

0 36 (18.5 %) I 51 (26.2 %) II 63 (32.3 %) III 36 (18.4 %) IV 9 (4.6 %)


*Note:* CABG – coronary artery bypass grafting; SVR – surgical ventricular reconstruction; MV repair – mitral valve repair; MV prosthesis – mitral valve prosthesis; TMLR – transmyocardial laser revascularization.

Table 2. The spectrum of surgical interventions carried out in the patients with ICMP enrolled into the study

Biopsy samples of RA auricle and LV were taken from the border area of endocardial scar and from the area of the myocardium without visual changes from all the patients (n=195, 100%) with ICMP. RA auricle biopsy was performed during the period of RA cannulation. LV biopsy samples were taken during surgical ventricular reconstruction from the transient zone on the border between scarred tissue and unchanged myocardium.

All the patients signed an informed consent form for the participation in the study; the study was approved by the local ethical committee of Tomsk Institute of Cardiology.

In order to find noninvasive molecular markers of postoperative LV remodeling blood samples were taken from 53 patients with ICMP (27.2%) to identify the content of natriuretic peptides and matrix metalloproteinases in blood plasma and serum correspondingly. In 37 patients antibodies titre to myocardial structures was identified in blood serum.

In the early postoperative period (1 month) control transthoracic EcoCG was made. In 12 months after the surgical treatment the patients were hospitalized again for clinical examination and control EchoCG.

In the late follow-up period repeated LV remodeling and HF progressing took place in a part of the patients which resulted in assignment of the patients into two groups: with positive (regressive remodeling – group I) and negative (progressing postoperative remodeling – group II) dynamics.

To achieve the objectives of identification tissue, cellular and molecular markers of postoperative LV remodeling we have used histological, electron-microscopic, morphometrical, biochemical and statistical methods of study.

To compare morphometrical parameters, as a control group we took autopsy samples of the identical sites of LV myocardium and myocardium of RA auricle from 25 cadavers of both sexes and of a comparable age died from an acute trauma with no signs of cardio-vascular pathology.

Seventeen healthy male and female volunteers of comparable age comprised a control group for the evaluation of the content of natriuretic peptide and matrix metalloproteinases in blood plasma and serum, respectively, as well as for identification of the antibodies titre to myocardial structures.

## **2.2 Histological study methods**

Histological methods include preliminary treatment of the studied material necessary for its further microscopic evaluation. Preparation of histological samples was performed as follows (Krivolapov, 2006): the samples of myocardium were being fixed in 10% solution of

Morphological Predictors and Molecular Markers of Progressing

as the ratio of mitochondrial SV to the SV of miofibrills.

endothelial cells cytoplasm.

Postoperative Remodeling of Left Ventricle in Patients with Ischemic Cardiomyopathy 113

of atrial cardiomyocytes. One mcm3 of tissue was taken as a unit volume for the study on a light-microscopic level (Avtandilov, 1990). Mitochondrial-myofibrillar ratio was evaluated

Electron-microscopic study of myocardial microcirculatory bed allowed for evaluation of the ratio between open (functioning) and closed (not functioning) capillaries. For the open capillaries we performed quantitative evaluation of their lumen and active transport through endothelium with the help of micropinocytic vesicles. For that, we identified the number of pinocytic vesicles associated with a length unit of a luminal contour on an area unit of capillary lumen, as well as density of free pinocytic vesicles per a volume unit of

**2.5 Biochemical methods of study of blood plasma and serum of the ICMP patients**  The content of natriuretic peptides (pro-ANP и NT-proBNP), matrix metalloproteinases (pro-ММР-1, MMP-3, MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in blood plasma and serum of the ICMP patients was identified by immunoenzyme method with standard kits by: pro-ANP and NT-proBNP – Biomedica (Austria); ММP-1 and MMP-9 – Quantikine® (R&D Systems, USA); MMP-3 and TIMP-1 – Biosource (Belgium). Evaluation of circulating antibodies to myocardium in blood serum had been performed in 37 patients with ICMP by the method based on the indirect immunofluorescence reaction («IMMCO Diagnostics» set, USA): we registered the presence of antifibrillar, antisarcolemmic and antinuclear antibodies by localization of fluorochrome on histological preparations by the method of fluorescent microscopy using the Axio Scope A1 microscope (Carl Zeiss,

Every patient underwent twelve-lead ECG study (Schiller АТ-6 machine, Switzerland) before and after the surgery. The degree of cardiomegalia and the condition of pulmonary circulation was evaluated by radiographic methods of study. Each patient underwent EchoCG examination with color dopplerography on Асuson 128 XP/10 (Japan). To study global systolic function of LV the following parameters were evaluated: end-diastolic size of LV, end-systolic size of LV, LV stroke volume and their indexes, LV EF, thickness of ventricular septum and that of posterior LV wall. Contractility of 16 segments was evaluated by a 4-score system and the index of local contractility disturbance was calculated to study disturbances of local LV myocardial contractility. To study LV diastolic function we evaluated transmitral blood flow by dopplerography. We also assessed the sizes of left atrium, degree of mitral regurgitation, estimate pressure in pulmonary artery. All the patients underwent EchoCG study before the surgery and in the postoperative period. Using the obtained data of echocardiography we calculated an estimate indicator – the index of specific thickness of LV wall by the following formula: thickness of ventricular septum + thickness of LV posterior wall/end-diastolic LV size. To assess this indicator we used the following grading: with remodeling index <0.30 – maladaptive remodeling; with the index

Germany). The concentration of the antibodies was expressed in titre.

from 0.30 to 0.45 – adaptive remodeling; >0.45 – asymptomatic remodeling.

performed by Judkins method (1967) with freezing monitoring images. To calculate changes of ESVI expressed as a percentage we used the formula:

Coronary angiography and left ventriculargraphy were performed on Philips maximus C1250, Philips polydiagnost C20 angiographic units. Selective coronary angiography was

**2.6 Clinical and instrumental methods of study** 

neutral formalin during 24 hours, and then they were washed in running water and dehydrated in the solution for histological treatment (dehydration and clearing) based on absolute isopropyl alcohol IsoPrep (BioVitrum, Saint Petersburg, Russia). After dehydration the myocardial samples were placed into homogenized paraffin media HISTOMIX® (BioVitrum, Saint Petersburg, Russia). Paraffin section of about 5-7mcm thick obtained with the use of a sliding microtome MC-2 were stained by hematoxylin and eosine and by Mallori method (stains and staining kits by BioOptica, Italy). The stained samples were place into synthetic monitoring media BioMount (BioOptica, Italy).

Histological samples were studied with a routine light and polarization microscopy on Axioskop 40 microscope (Carl Zeiss, Germany). Microimages of histological samples were taken with the Canon G10 camera (Japan).

#### **2.3 Methods of electron-microscopic study**

We took LV myocardium and RA auricle myocardium samples from 58 ICMP patients (50 men, 8 women) for electron-microscopy study. Myocardial samples of not more than 2mm3 were fixed in 2.5% glutaric aldehyde solution on 0.2M cacodylate buffer with pH=7.2 with the temperature of +4 °C and postfixed in 1% OsO4 solution in cold during 4 hours. The bioptates were then dehydrated in ethanol of rising concentration and placed into the mixture of epone and araldite. Semifine and ultrafine sections were prepared on ultratome LKB III (Sweden). The semifine sections were stained by 1% azure II solution and evaluated visually through a light microscope. The ultrafine sections were contrasted by lead citrate and uranyl acetate and studied in an electron microscope JEM-100 CX (Japan).

#### **2.4 Morphological methods of study**

For quantitative characteristic of the changes morphometrical methods were applied such as measurement of specific volume (SV) of edema, vessels, parenchyma and myocardial stroma by the point counting methods (Avtandilov, 1990; Glagolev & Chepulin, 1968). Measurement of parenchyma, stroma, vessels and edema SV was performed in 5-7 random microscopic fields of each section with the use of the software for graphic images procession (AxioVision by Carl Zeiss, ImageJ, Germany). One mm3 of the tissue was considered as a unit volume for the study on the light-optical level (Avtandilov, 1990). Ocular micrometer МОВ-1-16х («ЛОМО», Saint Petersburg, Russia) was used to measure diameter of cardiomyocytes on longitudinal sections on the level of myocardial cells nuclei. For the quantitative characteristics of the interrelation among myocardial parenchyma, stroma of the organ and exchange link of microcirculatory bed the following morphometrical parameters were evaluated to reveal risk factors of postoperative heart remodeling: parenchyma-stromal ratio (PSR), trophic index (TI) and pericapillar diffusion zone (PcDZ); and for the quantitative characteristics of microvasculature and their capacity Kernogan index (KI) was calculated. PSR is a ratio between myocardial parenchyma SV and stromal SV; TI (the best index reflecting the condition of myocardial trophy) – is the ratio between capillary SV and parenchyma SV; PcDZ (the area of tissue supplied with blood by one capillar) – the ratio between the capillary diameter and their SV; KI (the index of carrying capacity of microcirculatory bed) – is the ratio between arterioles vascular wall and the radius of their lumens (Avtandilov, 1990).

Morphometry of the ultrastructures was performed on digitized negative photoplates with initial magnitude 4800–10000. We calculated SV of myofibrils, mitochondrias and granules

neutral formalin during 24 hours, and then they were washed in running water and dehydrated in the solution for histological treatment (dehydration and clearing) based on absolute isopropyl alcohol IsoPrep (BioVitrum, Saint Petersburg, Russia). After dehydration the myocardial samples were placed into homogenized paraffin media HISTOMIX® (BioVitrum, Saint Petersburg, Russia). Paraffin section of about 5-7mcm thick obtained with the use of a sliding microtome MC-2 were stained by hematoxylin and eosine and by Mallori method (stains and staining kits by BioOptica, Italy). The stained samples were

Histological samples were studied with a routine light and polarization microscopy on Axioskop 40 microscope (Carl Zeiss, Germany). Microimages of histological samples were

We took LV myocardium and RA auricle myocardium samples from 58 ICMP patients (50 men, 8 women) for electron-microscopy study. Myocardial samples of not more than 2mm3 were fixed in 2.5% glutaric aldehyde solution on 0.2M cacodylate buffer with pH=7.2 with the temperature of +4 °C and postfixed in 1% OsO4 solution in cold during 4 hours. The bioptates were then dehydrated in ethanol of rising concentration and placed into the mixture of epone and araldite. Semifine and ultrafine sections were prepared on ultratome LKB III (Sweden). The semifine sections were stained by 1% azure II solution and evaluated visually through a light microscope. The ultrafine sections were contrasted by lead citrate

For quantitative characteristic of the changes morphometrical methods were applied such as measurement of specific volume (SV) of edema, vessels, parenchyma and myocardial stroma by the point counting methods (Avtandilov, 1990; Glagolev & Chepulin, 1968). Measurement of parenchyma, stroma, vessels and edema SV was performed in 5-7 random microscopic fields of each section with the use of the software for graphic images procession (AxioVision by Carl Zeiss, ImageJ, Germany). One mm3 of the tissue was considered as a unit volume for the study on the light-optical level (Avtandilov, 1990). Ocular micrometer МОВ-1-16х («ЛОМО», Saint Petersburg, Russia) was used to measure diameter of cardiomyocytes on longitudinal sections on the level of myocardial cells nuclei. For the quantitative characteristics of the interrelation among myocardial parenchyma, stroma of the organ and exchange link of microcirculatory bed the following morphometrical parameters were evaluated to reveal risk factors of postoperative heart remodeling: parenchyma-stromal ratio (PSR), trophic index (TI) and pericapillar diffusion zone (PcDZ); and for the quantitative characteristics of microvasculature and their capacity Kernogan index (KI) was calculated. PSR is a ratio between myocardial parenchyma SV and stromal SV; TI (the best index reflecting the condition of myocardial trophy) – is the ratio between capillary SV and parenchyma SV; PcDZ (the area of tissue supplied with blood by one capillar) – the ratio between the capillary diameter and their SV; KI (the index of carrying capacity of microcirculatory bed) – is the ratio between arterioles vascular wall and the

Morphometry of the ultrastructures was performed on digitized negative photoplates with initial magnitude 4800–10000. We calculated SV of myofibrils, mitochondrias and granules

and uranyl acetate and studied in an electron microscope JEM-100 CX (Japan).

place into synthetic monitoring media BioMount (BioOptica, Italy).

taken with the Canon G10 camera (Japan).

**2.4 Morphological methods of study** 

radius of their lumens (Avtandilov, 1990).

**2.3 Methods of electron-microscopic study** 

of atrial cardiomyocytes. One mcm3 of tissue was taken as a unit volume for the study on a light-microscopic level (Avtandilov, 1990). Mitochondrial-myofibrillar ratio was evaluated as the ratio of mitochondrial SV to the SV of miofibrills.

Electron-microscopic study of myocardial microcirculatory bed allowed for evaluation of the ratio between open (functioning) and closed (not functioning) capillaries. For the open capillaries we performed quantitative evaluation of their lumen and active transport through endothelium with the help of micropinocytic vesicles. For that, we identified the number of pinocytic vesicles associated with a length unit of a luminal contour on an area unit of capillary lumen, as well as density of free pinocytic vesicles per a volume unit of endothelial cells cytoplasm.

### **2.5 Biochemical methods of study of blood plasma and serum of the ICMP patients**

The content of natriuretic peptides (pro-ANP и NT-proBNP), matrix metalloproteinases (pro-ММР-1, MMP-3, MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in blood plasma and serum of the ICMP patients was identified by immunoenzyme method with standard kits by: pro-ANP and NT-proBNP – Biomedica (Austria); ММP-1 and MMP-9 – Quantikine® (R&D Systems, USA); MMP-3 and TIMP-1 – Biosource (Belgium). Evaluation of circulating antibodies to myocardium in blood serum had been performed in 37 patients with ICMP by the method based on the indirect immunofluorescence reaction («IMMCO Diagnostics» set, USA): we registered the presence of antifibrillar, antisarcolemmic and antinuclear antibodies by localization of fluorochrome on histological preparations by the method of fluorescent microscopy using the Axio Scope A1 microscope (Carl Zeiss, Germany). The concentration of the antibodies was expressed in titre.

#### **2.6 Clinical and instrumental methods of study**

Every patient underwent twelve-lead ECG study (Schiller АТ-6 machine, Switzerland) before and after the surgery. The degree of cardiomegalia and the condition of pulmonary circulation was evaluated by radiographic methods of study. Each patient underwent EchoCG examination with color dopplerography on Асuson 128 XP/10 (Japan). To study global systolic function of LV the following parameters were evaluated: end-diastolic size of LV, end-systolic size of LV, LV stroke volume and their indexes, LV EF, thickness of ventricular septum and that of posterior LV wall. Contractility of 16 segments was evaluated by a 4-score system and the index of local contractility disturbance was calculated to study disturbances of local LV myocardial contractility. To study LV diastolic function we evaluated transmitral blood flow by dopplerography. We also assessed the sizes of left atrium, degree of mitral regurgitation, estimate pressure in pulmonary artery. All the patients underwent EchoCG study before the surgery and in the postoperative period. Using the obtained data of echocardiography we calculated an estimate indicator – the index of specific thickness of LV wall by the following formula: thickness of ventricular septum + thickness of LV posterior wall/end-diastolic LV size. To assess this indicator we used the following grading: with remodeling index <0.30 – maladaptive remodeling; with the index from 0.30 to 0.45 – adaptive remodeling; >0.45 – asymptomatic remodeling.

Coronary angiography and left ventriculargraphy were performed on Philips maximus C1250, Philips polydiagnost C20 angiographic units. Selective coronary angiography was performed by Judkins method (1967) with freezing monitoring images.

To calculate changes of ESVI expressed as a percentage we used the formula:

Morphological Predictors and Molecular Markers of Progressing

Postoperative Remodeling of Left Ventricle in Patients with Ischemic Cardiomyopathy 115

polymorphism of myocardial cells nuclei: their enlargement, changing shape and tinctoral properties. In the most cases the shape of cardiomyocytes was oval with fuzzy contours. Quite often the nuclei had the shapes of "eights", "bow", "spring", etc. Chromatin in such the nuclei was condensed and located mostly along the nuclei periphery. Oxyphilous inclusions looking like apoptotic bodies were noticed either close to some nuclei or inside them. Such cardiomyocytes had eosinophilic cytoplasm and irregular contours of the cells.

Fig. 1. LV myocardium of ICMP patients: a – perivascular edema; b –venous plethora.

Fig. 2. LV myocardium of ICMP patients: а – mixed (lymphocytic-macrophage) focal infiltration; b – diffuse lymphocytic infiltration of myocardial stroma. Stained with

Stained with hematoxylin and eosin. X 450 (a) and 400 (b)

hematoxylin and eosin. X 500 (a) and 300 (b)

ΔESVI = preoperative ESVI /ESVI in a year after the sur ( ) gery x 100 – 100.

To study the values of central hemodynamics the patients were subjected to catheterization of heart chambers with measurements of LVEDP and pressure in the pulmonary artery.

#### **2.7 Statistical analysis of the results**

The results were statistically analyzed with the software package SSPS 11.5 for Windows. Normality of a distribution law of quantitative values was assessed by Shapiro-Wilk test. Parameters which obey the normal distribution law were described with the help of the mean value (M) and standard deviation (m); those which do not obey the normal distribution law - with the use of median (Me) and interquintile interval (Q25–Q75). Qualitative data were described by the frequency of occurrence or its percentage. If the distribution law was normal, Student t-test was used for the assessment of reliability of quantitative values differences in the compared groups; Mann-Whitney test – in the case of not normal distribution law. To evaluate reliability of quantitative data *<sup>2</sup> χ* criterion was used (or Fisher exact test in cases when *<sup>2</sup> χ* test was not possible). To find statistical dependences of linear character, to identify their strength and direction Pearson correlation coefficient (r) (among quantitative values obeying the law of normal distribution) and Spearsman correlation coefficient (for quantitative values not obeying the law of normal distribution and for qualitative values in the ordinal scale) were calculated. All statistical values were considered significant with p<0.05.

#### **3. Results and discussion**

#### **3.1 Morphological predictors of postoperative LV remodeling in ICMP patients**

During the study of morphofunctional condition of LV myocardium and myocardium of RA auricle in the patients with ICMP there was found that the density of vessels distribution was significantly decreased in comparison with that in the control group. Irrespectively of the blood vessels diameter the signs of hemodynamic disturbances were noticed everywhere: perivascular edema, venous plethora, desolation and spasm of arterioles and small arteries (fig. 1). Nuclei of endothelial cells in spasm arterioles were visually "extruded" into the vessels lumen. Microcirculatory link of vascular bed was plethoric; phenomena of erythrocyte stasis in capillaries, pericapillaries and arterioles were observed quite often. In separate capillaries we noticed rounding of endothelial cells manifesting as extrusion of endothelial cells nuclei into the capillaries lumen which, without any doubt, lowered their carrying capacity and the level of myocardial trophy.

In LV myocardium and in that of RA auricle of the ICMP patients stroma had enlarged volume and was edematous; its collagen fibers were curved and sometimes swollen. Mixed (lymphocytic-macrophage) infiltrate (> 14 per mm2 of tissue by Marburg classification (World Heart Federation Consensus Conferences Definition of Inflammatory Cardiomyopathy (Myocarditis), 1997) was found in some patients' LV myocardial stroma and RA auricle which was considered as myocarditis (fig. 2).

In the ICMP patients LV myocardial cardiomyocytes were, as a rule, hypertrophic and located either singly or in small foci surrounded by the areas of scarred tissue which had been formed at the sites of previous infarctions. It is worth mentioning a pronounced

ΔESVI = preoperative ESVI /ESVI in a year after the sur ( ) gery x 100 – 100.

To study the values of central hemodynamics the patients were subjected to catheterization of heart chambers with measurements of LVEDP and pressure in the pulmonary artery.

The results were statistically analyzed with the software package SSPS 11.5 for Windows. Normality of a distribution law of quantitative values was assessed by Shapiro-Wilk test. Parameters which obey the normal distribution law were described with the help of the mean value (M) and standard deviation (m); those which do not obey the normal distribution law - with the use of median (Me) and interquintile interval (Q25–Q75). Qualitative data were described by the frequency of occurrence or its percentage. If the distribution law was normal, Student t-test was used for the assessment of reliability of quantitative values differences in the compared groups; Mann-Whitney test – in the case of not normal distribution law. To evaluate reliability of quantitative data *<sup>2</sup> χ* criterion was used (or Fisher exact test in cases when *<sup>2</sup> χ* test was not possible). To find statistical dependences of linear character, to identify their strength and direction Pearson correlation coefficient (r) (among quantitative values obeying the law of normal distribution) and Spearsman correlation coefficient (for quantitative values not obeying the law of normal distribution and for qualitative values in the ordinal scale) were calculated. All statistical

**3.1 Morphological predictors of postoperative LV remodeling in ICMP patients** 

lowered their carrying capacity and the level of myocardial trophy.

and RA auricle which was considered as myocarditis (fig. 2).

During the study of morphofunctional condition of LV myocardium and myocardium of RA auricle in the patients with ICMP there was found that the density of vessels distribution was significantly decreased in comparison with that in the control group. Irrespectively of the blood vessels diameter the signs of hemodynamic disturbances were noticed everywhere: perivascular edema, venous plethora, desolation and spasm of arterioles and small arteries (fig. 1). Nuclei of endothelial cells in spasm arterioles were visually "extruded" into the vessels lumen. Microcirculatory link of vascular bed was plethoric; phenomena of erythrocyte stasis in capillaries, pericapillaries and arterioles were observed quite often. In separate capillaries we noticed rounding of endothelial cells manifesting as extrusion of endothelial cells nuclei into the capillaries lumen which, without any doubt,

In LV myocardium and in that of RA auricle of the ICMP patients stroma had enlarged volume and was edematous; its collagen fibers were curved and sometimes swollen. Mixed (lymphocytic-macrophage) infiltrate (> 14 per mm2 of tissue by Marburg classification (World Heart Federation Consensus Conferences Definition of Inflammatory Cardiomyopathy (Myocarditis), 1997) was found in some patients' LV myocardial stroma

In the ICMP patients LV myocardial cardiomyocytes were, as a rule, hypertrophic and located either singly or in small foci surrounded by the areas of scarred tissue which had been formed at the sites of previous infarctions. It is worth mentioning a pronounced

**2.7 Statistical analysis of the results** 

values were considered significant with p<0.05.

**3. Results and discussion** 

polymorphism of myocardial cells nuclei: their enlargement, changing shape and tinctoral properties. In the most cases the shape of cardiomyocytes was oval with fuzzy contours. Quite often the nuclei had the shapes of "eights", "bow", "spring", etc. Chromatin in such the nuclei was condensed and located mostly along the nuclei periphery. Oxyphilous inclusions looking like apoptotic bodies were noticed either close to some nuclei or inside them. Such cardiomyocytes had eosinophilic cytoplasm and irregular contours of the cells.

Fig. 1. LV myocardium of ICMP patients: a – perivascular edema; b –venous plethora. Stained with hematoxylin and eosin. X 450 (a) and 400 (b)

Fig. 2. LV myocardium of ICMP patients: а – mixed (lymphocytic-macrophage) focal infiltration; b – diffuse lymphocytic infiltration of myocardial stroma. Stained with hematoxylin and eosin. X 500 (a) and 300 (b)

Morphological Predictors and Molecular Markers of Progressing

β


cardiomyocyte; MF – myofibrils

Postoperative Remodeling of Left Ventricle in Patients with Ischemic Cardiomyopathy 117

Perinuclear space was dilated; it was not filled with mitochondrias, granular reticulum and

and having round or elongated shape (monogranular glycogen) (fig. 4). Rosettes of

Fig. 4. A fragment of a left ventricular cardiomyocyte of an ICMP patient: vacuolization (V) of a nucleus (N) with its further desolation; dilatation of perinuclear space filled with

Dispersed nucleoli, segregation of fibrillar and granular components of nucleolonema, ringshaped nucleoli were noticed which evidenced suppression of rRNA biosynthesis (fig. 5).

Fig. 5. A fragment of a left ventricular cardiomyocyte of an ICMP patient. Dispersion of nucleoles (Nu), segregation of granular and fibrillar components. N- a nucleus of a

β-shape

elements of Golgi organ but consisted mostly of rare matrix containing glycogen of


Intracellular edema in cardiomyocytes of LV was noticed everywhere and its degree varied very much from cell to cell. In a polarized light on histological preparations stained with hematoxyline and eosine alongside with unchanged areas of cardiomyocytes' cytoplasm we observed damaged areas of sarcoplasm with predominantly subsegmental contractures, contracture lesions of the Ist, IInd and less often IIId degree which differed by the enhancement of luminescence of disdiaclasts with different degrees of isotropic discs shortening; we also noticed isolated areas of intracellular myocytolysis and primary clump disintegration of myofibrillas and cytolysis of cardiomyocytes. The described changes of mosaic nature were also noticed in RA auricle myocardium.

Study of morphofunctional condition of LV myocardium in patients with ICMP revealed very interesting peculiarities: cardiomyocytes organization disturbance in muscular fibers and disturbance of fibers orientation relative to each other, disintegration of myocardial cells on intercalated disks, elimination of cardiomyocytes along muscular fibers (fig. 3). Myocardium in these cases had not fibrous but cellular structure which resulted in its poor contractility (Anderson, 2005). Sometimes cardiac myocytes were star-shaped. Besides, on the longitudinal sections of LV myocardium of ICMP patients one could observe waveshape deformation of cardiomyocytes along myocardial fibers.

Fig. 3. LV myocardium of ICMP patients: а- disturbance of muscular fiber orientation, starshaped cardiomyocytes; b- wave-shaped deformation of muscular fibers. Stained with hematoxylin and eosin. X 370 (a) and 330 (b)

During electron-microscopic study of myocardial cardiomyocytes of LV and RA auricle our attention was drawn to the polymorphism of ultrastructures of myocardial cells. Nuclei often having irregular scalloped shape with multiple intussusceptums and outgrowths of nuclear membrane were located in the center of the cells, but in some cardiomyocytes they were displaced into subsarcolemmic zone. Genetic material was observed mostly as euchromatin which took a central position. In some cardiomyocytes' nuclei, vice versa, heterochromatin prevailed and was situated mostly in juxtamembrane zone. Chromatin aggregation (compaction) was noticed quite often. Nuclear envelope was continuous throughout and had pores.

Intracellular edema in cardiomyocytes of LV was noticed everywhere and its degree varied very much from cell to cell. In a polarized light on histological preparations stained with hematoxyline and eosine alongside with unchanged areas of cardiomyocytes' cytoplasm we observed damaged areas of sarcoplasm with predominantly subsegmental contractures, contracture lesions of the Ist, IInd and less often IIId degree which differed by the enhancement of luminescence of disdiaclasts with different degrees of isotropic discs shortening; we also noticed isolated areas of intracellular myocytolysis and primary clump disintegration of myofibrillas and cytolysis of cardiomyocytes. The described changes of

Study of morphofunctional condition of LV myocardium in patients with ICMP revealed very interesting peculiarities: cardiomyocytes organization disturbance in muscular fibers and disturbance of fibers orientation relative to each other, disintegration of myocardial cells on intercalated disks, elimination of cardiomyocytes along muscular fibers (fig. 3). Myocardium in these cases had not fibrous but cellular structure which resulted in its poor contractility (Anderson, 2005). Sometimes cardiac myocytes were star-shaped. Besides, on the longitudinal sections of LV myocardium of ICMP patients one could observe wave-

Fig. 3. LV myocardium of ICMP patients: а- disturbance of muscular fiber orientation, starshaped cardiomyocytes; b- wave-shaped deformation of muscular fibers. Stained with

During electron-microscopic study of myocardial cardiomyocytes of LV and RA auricle our attention was drawn to the polymorphism of ultrastructures of myocardial cells. Nuclei often having irregular scalloped shape with multiple intussusceptums and outgrowths of nuclear membrane were located in the center of the cells, but in some cardiomyocytes they were displaced into subsarcolemmic zone. Genetic material was observed mostly as euchromatin which took a central position. In some cardiomyocytes' nuclei, vice versa, heterochromatin prevailed and was situated mostly in juxtamembrane zone. Chromatin aggregation (compaction) was noticed quite often. Nuclear envelope was continuous

mosaic nature were also noticed in RA auricle myocardium.

shape deformation of cardiomyocytes along myocardial fibers.

hematoxylin and eosin. X 370 (a) and 330 (b)

throughout and had pores.

Perinuclear space was dilated; it was not filled with mitochondrias, granular reticulum and elements of Golgi organ but consisted mostly of rare matrix containing glycogen of β-shape and having round or elongated shape (monogranular glycogen) (fig. 4). Rosettes of -glycogen were present in a very insignificant number in intermiofibrillar spaces.

Fig. 4. A fragment of a left ventricular cardiomyocyte of an ICMP patient: vacuolization (V) of a nucleus (N) with its further desolation; dilatation of perinuclear space filled with β- glycogen (G)

Dispersed nucleoli, segregation of fibrillar and granular components of nucleolonema, ringshaped nucleoli were noticed which evidenced suppression of rRNA biosynthesis (fig. 5).

Fig. 5. A fragment of a left ventricular cardiomyocyte of an ICMP patient. Dispersion of nucleoles (Nu), segregation of granular and fibrillar components. N- a nucleus of a cardiomyocyte; MF – myofibrils

Morphological Predictors and Molecular Markers of Progressing

space between fibrils (in the area of Z-bands) and mitochondrias.

insufficiency of cardiomyocytes in both LV and RA auricle.

cells circulation were considered closed (non-functioning).

condition of vacuolization.

capillaries was very insignificant.

surface of the cells.

mitochondrias.

various sizes.

Postoperative Remodeling of Left Ventricle in Patients with Ischemic Cardiomyopathy 119

Polymorphism of mitochondrias attracted our attention. We observed large mitochondrias reaching a length of 2-3 sarcomeres in the space among myofibrils. On the contrary, more often we were observing small round mitochondrias located randomly or in clusters. Sometimes we saw mitochondrias with destructive and degenerative changes, with cleared matrix, destroyed and reduced cristas, few mitochondrias with electron-dense matrix in

Cisterns and vacuoles of cytoplasmic reticulum and Golgi apparatus in LV and RA auricle myocardium cardiomyocytes were deduced and sometimes dilated in the samples of ICMP patients. Dilation of the cisterns was found mostly in perinuclear zone and less often in the

Atrial cardiomyocytes contained electron-dense granules of atrial natriuretic peptide of

Thus, electron-microscopic study revealed mixed, alterative and regenerative-plastic

Electron-microscopic study of peculiarities of microvasculature functional morphology was performed for intraoperative samples of LV and RA auricle from 47 ICMP patients (44 men, 3 women). Hemocapillaries having a sufficient lumen for passage of blood cells or containing these cells in their lumen were considered open (functioning). Hemocapillaries with a minimal lumen between plasmalemmas of endothelial cells insufficient for blood

In endothelial cells of myocardial capillaries a large number of micropinocytic vesicles of different diameters (from 30 to 120nm) was found; these vesicles were present in both closed and open capillaries. The content of small vesicles was electron-optically low dense. On the contrary, large vesicles were electron-optically transparent. The number of micropinocytic vesicles in endotheliocytes in different capillaries was variable and their number in closed

In cytoplasm of endothelial cells unexpanded canaliculi of granular and granular endoplasmic reticulum were observed as well as multiple crests and dints on a luminal

Electron-microscopic study of microvasculatory link of LV and RA auricle myocardial vascular bed one could notice a bulging endotheliocytes into the capillaries lumen, reduction of their lumen and their decreased capacity. Rounding of endotheliocytes, probably, has adaptive meaning and is directed toward slowing blood flow in capillaries and more efficient use of blood mass in transcapillar exchange (Kawamura et al., 1974). Matrix of endothelial cells cytoplasm in one and the same capillary is vividly cleared in one cells evidencing swelling of cytoplasm and in other cells it becomes electron-optically dense. If an endothelial cell matrix was cleared, we could visualize mostly singular small size micropinocytic vesicles situated predominantly on free and basal edges of cytoplasm. In cytoplasm of dark endotheliocytes larger pinocytic vesicles were present quite often (fig. 7). Often one could see mitochondrias in capillary endotheliocytes of LV and RA auricle myocardium. Usually no changes in functional morphology of cellular energy apparatus were noticed, but sometimes we observed mitochondrias with cleared matrix, dilated intracristal spaces and destroyed crysts. Myelin-like structures were found in some

In clear endotheliocytes were swallow and chromatin was loose. In osmium dark cells nuclei remained unchanged. Chromatin aggregation and pyknotic nuclei were noticed eventually. In nuclei of singular endotheliocytes we noticed the lost connection between heterochromatin and nuclear membrain. Heterochromatin was displaced deeper into the

nuclei of endothelial cells and was present there as ring-shaped structures.

We also found wide variety of myofibril lesion forms in LV and RA auricle myocardial cardiomyocytes in ICMP patients such as: contracture lesions of myofibrils of the Ist, less often of the IInd and IIId degrees, isolated areas of primary clump of fibrillar disaggregation (fig. 6). The presence of contracture lesions of the IIId degree in a cell was associated with formation of sarcolemma festoons, contraction and deformation of a nucleus and mitochondrias due to overcontraction of myofibrils and with displacement of mitochondrias into the space between myofibrils and formation of compact assemblies.

Fig. 6. Primary clump disintegration of LV myocardial cardiomyocite's myofibrils of an ICMP patient: alteration of the areas of mosaic lysis (L) and contractures (C) in isolated groups of myofibrillar (MF) sarcomeres; MCh- mitochondrias

In the most cardiomyocytes small-focal and diffuse lysis of myofibrillar bundles, myofibril "melting" were registered. I-disks with thin (actin) filaments in them were lysed to a greater extent. Myofibrils were becoming less dense, cavities appeared in some sarcomeres, total lysis of myofilaments within a sarcomere was noticed. Sarcomeres in the area of intercalated disks and in perinuclear zone were significantly destructed.

It was very seldom when foci of intracellular regeneration of ultrastructures were found in LV and RA auricle myocardial cardiomyocytes. The foci were evaluated by the accumulation of free ribosomes on the stumps of survived myofibrils contributing to the synthesis and neoformation of contractile proteins. As the newly formed myofilaments synthesized on polyribosomes and got matured they gathered into the bundles of myofibrils. But in the process of their neoformation their normal orientation is disturbed, they elongate excessively and it results in growing distance between Z-bands.

Apart from disturbance of normal orientation of the newly formed myofibrils we observed chaotic orientation of "mature" contractile proteins. Myofibrilar bundles and even individual myofilaments were oriented at different angles in relation to each other. Besides, wave-shaped deformation of a contractile apparatus of LV and RA auricle myocardial cardiomyocytes took place in the patients with ICMP. All these circumstances, with no doubt, made their contribution into desynchronization of contractile processes, thus preconditioning systolic dysfunction of myocardium (Anderson, 2005).

We also found wide variety of myofibril lesion forms in LV and RA auricle myocardial cardiomyocytes in ICMP patients such as: contracture lesions of myofibrils of the Ist, less often of the IInd and IIId degrees, isolated areas of primary clump of fibrillar disaggregation (fig. 6). The presence of contracture lesions of the IIId degree in a cell was associated with formation of sarcolemma festoons, contraction and deformation of a nucleus and mitochondrias due to overcontraction of myofibrils and with displacement of mitochondrias

Fig. 6. Primary clump disintegration of LV myocardial cardiomyocite's myofibrils of an ICMP patient: alteration of the areas of mosaic lysis (L) and contractures (C) in isolated

In the most cardiomyocytes small-focal and diffuse lysis of myofibrillar bundles, myofibril "melting" were registered. I-disks with thin (actin) filaments in them were lysed to a greater extent. Myofibrils were becoming less dense, cavities appeared in some sarcomeres, total lysis of myofilaments within a sarcomere was noticed. Sarcomeres in the area of intercalated

It was very seldom when foci of intracellular regeneration of ultrastructures were found in LV and RA auricle myocardial cardiomyocytes. The foci were evaluated by the accumulation of free ribosomes on the stumps of survived myofibrils contributing to the synthesis and neoformation of contractile proteins. As the newly formed myofilaments synthesized on polyribosomes and got matured they gathered into the bundles of myofibrils. But in the process of their neoformation their normal orientation is disturbed,

Apart from disturbance of normal orientation of the newly formed myofibrils we observed chaotic orientation of "mature" contractile proteins. Myofibrilar bundles and even individual myofilaments were oriented at different angles in relation to each other. Besides, wave-shaped deformation of a contractile apparatus of LV and RA auricle myocardial cardiomyocytes took place in the patients with ICMP. All these circumstances, with no doubt, made their contribution into desynchronization of contractile processes, thus

they elongate excessively and it results in growing distance between Z-bands.

preconditioning systolic dysfunction of myocardium (Anderson, 2005).

groups of myofibrillar (MF) sarcomeres; MCh- mitochondrias

disks and in perinuclear zone were significantly destructed.

into the space between myofibrils and formation of compact assemblies.

Polymorphism of mitochondrias attracted our attention. We observed large mitochondrias reaching a length of 2-3 sarcomeres in the space among myofibrils. On the contrary, more often we were observing small round mitochondrias located randomly or in clusters. Sometimes we saw mitochondrias with destructive and degenerative changes, with cleared matrix, destroyed and reduced cristas, few mitochondrias with electron-dense matrix in condition of vacuolization.

Cisterns and vacuoles of cytoplasmic reticulum and Golgi apparatus in LV and RA auricle myocardium cardiomyocytes were deduced and sometimes dilated in the samples of ICMP patients. Dilation of the cisterns was found mostly in perinuclear zone and less often in the space between fibrils (in the area of Z-bands) and mitochondrias.

Atrial cardiomyocytes contained electron-dense granules of atrial natriuretic peptide of various sizes.

Thus, electron-microscopic study revealed mixed, alterative and regenerative-plastic insufficiency of cardiomyocytes in both LV and RA auricle.

Electron-microscopic study of peculiarities of microvasculature functional morphology was performed for intraoperative samples of LV and RA auricle from 47 ICMP patients (44 men, 3 women). Hemocapillaries having a sufficient lumen for passage of blood cells or containing these cells in their lumen were considered open (functioning). Hemocapillaries with a minimal lumen between plasmalemmas of endothelial cells insufficient for blood cells circulation were considered closed (non-functioning).

In endothelial cells of myocardial capillaries a large number of micropinocytic vesicles of different diameters (from 30 to 120nm) was found; these vesicles were present in both closed and open capillaries. The content of small vesicles was electron-optically low dense. On the contrary, large vesicles were electron-optically transparent. The number of micropinocytic vesicles in endotheliocytes in different capillaries was variable and their number in closed capillaries was very insignificant.

In cytoplasm of endothelial cells unexpanded canaliculi of granular and granular endoplasmic reticulum were observed as well as multiple crests and dints on a luminal surface of the cells.

Electron-microscopic study of microvasculatory link of LV and RA auricle myocardial vascular bed one could notice a bulging endotheliocytes into the capillaries lumen, reduction of their lumen and their decreased capacity. Rounding of endotheliocytes, probably, has adaptive meaning and is directed toward slowing blood flow in capillaries and more efficient use of blood mass in transcapillar exchange (Kawamura et al., 1974).

Matrix of endothelial cells cytoplasm in one and the same capillary is vividly cleared in one cells evidencing swelling of cytoplasm and in other cells it becomes electron-optically dense. If an endothelial cell matrix was cleared, we could visualize mostly singular small size micropinocytic vesicles situated predominantly on free and basal edges of cytoplasm. In cytoplasm of dark endotheliocytes larger pinocytic vesicles were present quite often (fig. 7).

Often one could see mitochondrias in capillary endotheliocytes of LV and RA auricle myocardium. Usually no changes in functional morphology of cellular energy apparatus were noticed, but sometimes we observed mitochondrias with cleared matrix, dilated intracristal spaces and destroyed crysts. Myelin-like structures were found in some mitochondrias.

In clear endotheliocytes were swallow and chromatin was loose. In osmium dark cells nuclei remained unchanged. Chromatin aggregation and pyknotic nuclei were noticed eventually. In nuclei of singular endotheliocytes we noticed the lost connection between heterochromatin and nuclear membrain. Heterochromatin was displaced deeper into the nuclei of endothelial cells and was present there as ring-shaped structures.

Morphological Predictors and Molecular Markers of Progressing

29.4% (n=45), 27.5% (n=42) died from cardiac diseases.

surgical treatment outcomes was performed.

(Geidel, 2005; Gelsomino, 2008).

value of ∆ESVI was < minus 20%.

value of ∆ESVI was > minus 20%.

postoperative period).

LVEDVI – significantly lower.

Postoperative Remodeling of Left Ventricle in Patients with Ischemic Cardiomyopathy 121

examination (in a year after the surgery) clinical and echocardiography evaluation of the

In January 2009 all the patients who had underwent reexamination (n=153) were interviewed over the telephone. Mean follow-up period was 4.24 years, maximal follow-up period – 6 years. We managed to acquire information about all the patients who had had control examination. By the time of the interview there had been 108 alive patients of all included into the study. Mortality in the group during all the follow-up period comprised

Echocardiography was used as an instrumental method of the evaluation of heart cavities sizes and for the calculation of their indexed values. Despite obvious benefits of this method of study, it has low reproducibility of results, which varies within 20% from obtained values

With an artificially introduced efficacy endpoint (∆ESVI – delta ESVI- postoperatively less than 20% from preoperative values) we managed to divide the initially homogeneous group of the patients into two subgroups, different by the character of clinical course of HF after

ΔESVI = postoperative ESVI /preoperative ESVI x 100 – 100 ( )

To evaluate changes of LV ESVI 20% interval in comparison with preoperative values was used: changes > 20% into positive or negative side were considered sufficient to take these changes as true. The patients in which LV ESVI was <80% from preoperative values were assigned to group I. In these patients the process of LV remodeling was stopped and reversed by surgical intervention i.e. reverse (regressive) remodeling type. The calculated

The patients in which ESVI was > 80% from preoperative values were assigned to group II of the study. In these patients the course of HF remained unchanged due to the complex surgical treatment of ICMP: remodeling process was either resistant to the exposure or went on progressing after surgical treatment, i.e. had progressing remodeling type. The calculated

The distribution of the patients between the groups basing on the ∆ESVI value was the following: the 1st group consisted of 97 patients (63.4%) with the reverse remodeling type (i.e. with positive dynamics of the late postoperative period); the 2nd group consisted of 56 patients (36.6%) with progressing remodeling type (i.e. with negative dynamics of the late

Analysis of the control EchoCG data showed that in the early postoperative period in all the patients end-diastolic and end-systolic heart volumes decreased: LV EDVI from (114.9±28.4) ml/m2 to (98.0±25.3) ml/m2, LV ESVI from (83.9 ±21.6) ml/m2 to (64.3±21.0) ml/m2; and LV EF significantly increased from (32.1±5.5) % to (36.7±8.9) %.The values of echocardiography

Performed comparison of EchoCG data in the group of the patients with reverse LV remodeling showed that LV EF was significantly higher and the values of LVESVI and

It should be noted that the great majority of the patients continued medical treatment recommended at the time of their dismissal from the hospital after the surgery. The groups with reverse and progressing types of remodeling were comparable in respect to the

study performed during control follow-up period are shown in Table 3.

frequency of taking of different drug groups and their mean dosages.

surgical treatment. To calculate ∆ESVI parameter we used the following formula:

Fig. 7. A capillary from LV myocardium of an ICMP patient: Capillary lumen (CL) is limited by an endothelial cell with osmium dark matrix (a dark cell - DC) and by an endothelial cell with cleared matrix (a clear cell - CC). PCS – pericapillar space

Sometimes large lipid inclusions were noticed in cytoplasm of endothelial cells of capillaries in both LV and RV auricle myocardium; large vacuoles – only in singular cardiomyocytes. Glycogen granules were eventually found in insignificant amount.

Subendocardial capillary zone as a rule was not changed. Sometimes on longitudinal sections one could see isolated local dilations. Basal membrane was uninterrupted throughout and in most cases had the same thickness with only rare local thickened parts. Noncellular element of a basal layer was thickened locally and loose, occasionally thinned and interrupted.

The space between basal capillary membrane and cardiomyocytes (adventitious layer) was, as a rule, dilated and filled mostly with fibrous component of a loose connective tissue (with the prevalence of collagen fibers) and less often – with electron-transparent amorphous substance of low electron density (with separate fibrous structures like collagen fibers). In the latter case pericapillar space was sharply cleared; areas of a substance with low electronoptical density identical to that of plasma in a capillary lumen were found. The contours of these areas were not clear.

Cytoplasmic crest of pericytes were close to cytoplasm adjusting immediately to it. There were no any pronounced changes of functional morphology in pericytes found.

Pericapillar edema was registered in almost all the patients with ICMP, its degree varied significantly.

Thus, a thorough analysis of functional morphology of microvasculature in ICMP patients revealed specific changes of endothelial capillaries situated in foci of chronic ischemia.

Early postoperative mortality during 30 days after the surgery in the group of ICMP patients was 7.7% (n=15). First year postoperative mortality comprised 9.2% (n=18). Mean period of the control follow-up comprised 369±147 days. Only 153 (78.5 %) patients took part in the further study since 33 patients (16.9%) died in the early and mid-term postoperative period and communication with 9 patients (4.6%) was lost. During control

Fig. 7. A capillary from LV myocardium of an ICMP patient: Capillary lumen (CL) is limited by an endothelial cell with osmium dark matrix (a dark cell - DC) and by an endothelial cell

Sometimes large lipid inclusions were noticed in cytoplasm of endothelial cells of capillaries in both LV and RV auricle myocardium; large vacuoles – only in singular cardiomyocytes.

Subendocardial capillary zone as a rule was not changed. Sometimes on longitudinal sections one could see isolated local dilations. Basal membrane was uninterrupted throughout and in most cases had the same thickness with only rare local thickened parts. Noncellular element of a basal layer was thickened locally and loose, occasionally thinned

The space between basal capillary membrane and cardiomyocytes (adventitious layer) was, as a rule, dilated and filled mostly with fibrous component of a loose connective tissue (with the prevalence of collagen fibers) and less often – with electron-transparent amorphous substance of low electron density (with separate fibrous structures like collagen fibers). In the latter case pericapillar space was sharply cleared; areas of a substance with low electronoptical density identical to that of plasma in a capillary lumen were found. The contours of

Cytoplasmic crest of pericytes were close to cytoplasm adjusting immediately to it. There

Pericapillar edema was registered in almost all the patients with ICMP, its degree varied

Thus, a thorough analysis of functional morphology of microvasculature in ICMP patients revealed specific changes of endothelial capillaries situated in foci of chronic ischemia. Early postoperative mortality during 30 days after the surgery in the group of ICMP patients was 7.7% (n=15). First year postoperative mortality comprised 9.2% (n=18). Mean period of the control follow-up comprised 369±147 days. Only 153 (78.5 %) patients took part in the further study since 33 patients (16.9%) died in the early and mid-term postoperative period and communication with 9 patients (4.6%) was lost. During control

were no any pronounced changes of functional morphology in pericytes found.

with cleared matrix (a clear cell - CC). PCS – pericapillar space

and interrupted.

significantly.

these areas were not clear.

Glycogen granules were eventually found in insignificant amount.

examination (in a year after the surgery) clinical and echocardiography evaluation of the surgical treatment outcomes was performed.

In January 2009 all the patients who had underwent reexamination (n=153) were interviewed over the telephone. Mean follow-up period was 4.24 years, maximal follow-up period – 6 years. We managed to acquire information about all the patients who had had control examination. By the time of the interview there had been 108 alive patients of all included into the study. Mortality in the group during all the follow-up period comprised 29.4% (n=45), 27.5% (n=42) died from cardiac diseases.

Echocardiography was used as an instrumental method of the evaluation of heart cavities sizes and for the calculation of their indexed values. Despite obvious benefits of this method of study, it has low reproducibility of results, which varies within 20% from obtained values (Geidel, 2005; Gelsomino, 2008).

With an artificially introduced efficacy endpoint (∆ESVI – delta ESVI- postoperatively less than 20% from preoperative values) we managed to divide the initially homogeneous group of the patients into two subgroups, different by the character of clinical course of HF after surgical treatment. To calculate ∆ESVI parameter we used the following formula:
