Morphological assessment of actin and desmin expression at different cold myocardial ischemia times: observational study

INTRODUCTION

One of the main problems faced by global health care today is terminal heart failure, for which heart transplantation remains the leading treatment [1]. The critical shortage of donor organs with the increasing need for heart transplantation indicates the inevitability of expanding donor selection criteria; among these, one of the most important criteria is the ischemia time of the donor heart. Increasing the ischemia time to over 240 minutes, according to some authors, raises the risk of developing transplant dysfunction and may lead to a lethal outcome [2][3].

Noteworthy is that normothermic ischemic myocardial damage is associated with the destructive effect of hypoxia on the structures of cellular proteins comprising the cellular framework of cardiomyocytes. Together with myosin, actin, which constitutes an integral part of microfilaments, creates the contractile elements of muscles, i.e., actomyosin complexes of sarcomeres. The reduction in sarcomeric actin intensity in the cytoplasm of muscle cells in the damaged areas indicates a decrease in the contractile function of cardiomyocytes¹ [4]. Another representative of the main structural proteins of muscle cells is desmin [5][6], which binds cytoplasmic components creating a matrix near the sarcomere Z-disk and links it to the cytoplasmic zone of the cardiomyocyte membrane [7][8], thus linking both adjacent myofibrils and Z-disks [9][10]. In order to maintain longitudinal loading during contraction, desmin, through its own contact with the sarcomere binds the contractile part to the cell nucleus, organelles in the cell cytoplasm that provide cellular respiration, and the postsynaptic region [11][12]. This mechanism is most likely associated with the phosphorylation pathway disruption followed by the destruction of proteins in the cardiomyocyte cytoskeleton that are bound to them, as well as an increase in the number of calcium and hydrogen ions in the cells, which initiates calcium-dependent protease activity with the subsequent breakdown of actin and desmin.

Some studies report that prolonged cold ischemia of the transplant cannot be a factor in its dysfunction and has no effect on surgical outcomes [3][13]. The determination of myocardial damage using light optical microscopy is associated with certain difficulties if a short period of time has passed since its onset. In practice, it is reasonable to use an immunohistochemical method or polarized light microscopy in the diagnostics of myocardial damage [10][14–16], which provide a means to draw conclusions about the macromolecular structure status of cardiac muscle cells when studying areas of myocardial ischemia.

The article aims to comparatively assess the features of cardiomyocyte pathomorphology and expression of protein markers (actin and desmin) in the myocardium of donor hearts prior to the main stage of orthotopic heart transplantation.

METHODS

Study design

The work adopted the design of an observational clinical study, which was prospective in nature. Seventeen myocardial samples from the left atrial appendage (LAA) of donor hearts were included in the study.

Study conditions

The study was conducted at the Meshalkin National Medical Research Center, Ministry of Health of the Russian Federation. The heart transplantation procedure was carried out using a classical bicaval technique. Heart extraction was performed using the standard method, with preservation performed using a cold Bretschneider cardioplegic solution (Dr. Franz Köhler Chemie GmbH, Germany). In some cases, donor hearts from remote regions were used: Altai Krai and Kemerovo Oblast.

Eligibility criteria

Inclusion criteria

Hearts from donors aged up to 60 years. Absence of chest trauma, prolonged hypotension and hypoxemia; stable hemodynamics; mean arterial pressure (MAP) of >60 mmHg; central venous pressure (CVP) of 8–12 mmHg; inotropic support of below 10 mg/kg/min (dopamine); electrocardiography (ECG) and echocardiography (Echo-CG) revealing no pathological changes.

Exclusion criteria

Donors aged under 18. Complex cardiac rhythm disorders; need for excessive inotropic support (dopamine at a dose of 20 µg/kg/min or similar doses of other adrenergic preparations despite aggressive pre- and post-load optimization); contractile dysfunction according to echocardiography or left ventricular ejection fraction of < 40% despite the optimization of hemodynamics with inotropic drugs.

Removal criteria

Intravitam refusal of the patient to have internal organs removed.

Description of the eligibility criteria (diagnostic criteria)

The selection of donors for participation in the study involved establishing brain death on the basis of the clinical picture and results of laboratory and instrumental studies, which were conducted collegially in accordance with the national recommendations and accepted clinical protocols.²

Selection of group members

The participants were assigned to groups according to the time of preoperative cold ischemia of the transplant. The first group included ten cases with cold ischemia of the transplant lasting up to 240 minutes, while the second group consisted of seven cases with cold ischemia of the transplant lasting over 240 minutes.

Target parameters in the study

Main parameter in the study

The nature of pathomorphological myocardial rearrangements in the LAA of the donor heart was determined at different cold ischemia times.

Additional parameters in the study

Additional parameters are not provided in this study.

Methods for measuring the target parameters

In an operation theatre setting during the main stage of the operation, myocardial samples were collected from the LAA of the donor heart following cold ischemia with the use of Bretschneider solution (Dr. Franz Köhler Chemie GmbH, Germany). The fixation of myocardial slices, their histological processing, and subsequent hematoxylin-eosin staining were conducted according to available guidelines. The study of the obtained slide mounts was conducted by means of an Axio Scope.A1 universal microscope (Zeiss, Germany), equipped with an analyzer and polarizer, an AxioCam MRc5 camera (Zeiss, Germany), and ZEN blue software (Zeiss, Germany). The staining of myocardial slices for immunohistochemical analysis was performed according to the available recommendations of the antibody manufacturer and standards outlined in the guidelines for immunohistochemical studies [15]. The slices were dewaxed, and the tissue antigens were unmasked in a PT Link module (DAKO, Denmark) in citrate buffer (pH 6.0) at 95 °C for 60 minutes. Endogenous peroxidase was blocked with a 3% H₂O₂ solution, followed by protein blocking with a serum. After that, the obtained slices were incubated with antibodies to Actin, Muscle Specific Ab-4 (HHF35) and Desmin 43/GJA1 (clone D33, mouse monoclonal, DAKO, Denmark). Immunostaining was performed using a peroxidase-labeled polymer detection system (EnVision FLEX, DAKO, Denmark). At the final stage, cell nuclei were stained with hematoxylin.

Morphometry was performed using the ImageJ 1.48v software (USA). In the analysis of actin and desmin amount, the area of DAB-positive products of the immunohistochemical reaction was estimated as a percentage of the image area. The volume density (Vv,%) of immunohistochemically detectable actin and desmin was determined using 20 images at a magnification of 40×10. In order to study the intensity of the immune reaction, a semiquantitative method was used: (+/+++) counting the number of cells in 25 randomly selected fields of view; (-) no staining; (+) weak, only the cytoplasm of cells is stained; (++) moderate, with staining of cell cytoplasm and focal staining of extracellular components (<50%); (+++) marked expression, with staining of both cell cytoplasm and most of the extracellular components (>50%) [17]. The types of myocardial contracture damage were assessed via polarization microscopy [18].

Variables (predictors, confounders, and effect modifiers)

Previous congenital or acquired cardiac pathology could be a confounding factor independently affecting the study result. This factor was eliminated at the sampling stage by adding them to the exclusion criteria.

Statistical procedures

Principles behind sample size determination

The sample size was not determined in advance.

Statistical methods

A statistical analysis of the obtained results was performed using the STATISTICA 10.0 software (StatSoft, Inc., USA). Quantitative variables are presented as a median and interquartile range Me [Q1—Q3], while qualitative variables are given as the frequency of occurrence and/or percentage. An intergroup comparison of parameters was conducted using the Mann–Whitney test or Fisher’s exact test. The p-value of < 0.05 was considered statistically significant for all types of analysis.

RESULTS

Sampling

Two groups were formed for the study: Group 1 — ten cases with cold ischemia of the transplant lasting up to 240 minutes, i.e., 140 [ 140;150] minutes; Group 2 — seven cases with cold ischemia of the transplant lasting over 240 minutes, i.e., 375 [ 245;375] minutes (p ≤ 0.05) (Fig. 1).

Characteristics of the study sample (groups)

Patients included in the first and second groups were comparable in terms of mean age and anthropometric indices. The mean age of patients amounted to 50 [ 44;59] years in Group 1 and 50 [ 49;50] years in Group 2, р = 0.193. The body mass index (BMI) was 25 [ 22;27] in Group 1 and 25 [ 21;31] in Group 2, р = 0.288. Both groups showed male predominance: 8 (80%) in Group 1 and 6 (85.7%) in Group 2, р = 0.256.

Main study results

In the study of the material taken from the Group 1 patients, LAA myocardial preparations stained with hematoxylin and eosin revealed features characteristic of the fragmentation of individual muscle fibers in the stroma, as well as signs of non-uniform vascular plethora with moderate intermuscular edema and areas of erythrocytic sludges in microcirculatory vessels. A spasm of arterioles was noted, with veinous dilatation and plethora. Some vessels revealed a rod-shaped elongation of endotheliocytes. In general, most of the cardiomyocytes retained a regular structure with a slight sign of non-uniform oxyphilic staining characteristic of cells with myofibril contracture (Fig. 2).

The use of polarization microscopy showed the presence of contractural changes in LAA muscle cells in both groups. Generally, such changes did not exceed subsegmental (contractions of individual groups of sarcomeres in the myofibrils of cardiomyocytes) and segmental Class I–II contractures (which involve the entire muscle cell) and were characterized by the increased anisotropy in the A-disk of sarcomeres with a varying degree of shortening of isotropic I-disks (Fig. 3). Cardiomyocytes having merged A-disks in the form of homogeneous anisotropy (Class III contracture) were observed sporadically in both groups.

The study of actin and desmin expression revealed that most muscle cells were separated from each other by clearly outlined intercalated disks with preserved near-disk intensity in the form of dark-brown staining, which was revealed in the immunohistochemical reaction. In general, the cytoplasm had a uniform light brown color with increased reaction intensity in the Z-band region (Figs. 4 A, B). The preserved uniform staining of actin and desmin in cardiomyocytes both in the cytoplasm itself and in the region of intercalated disks, characterizing the sarcomeric cytoskeleton and the extra-sarcomeric cytoskeleton associated with the sarcolemma, indicates sufficient protection of the myocardium during prolonged ischemia. With unprotected myocardium, prolonged ischemia leads to the dissociation of the actin cytoskeleton from the cardiomyocyte membrane, followed by cell degeneration and apoptosis, which was not observed in the present study.

In the histological study of the material obtained from Group 2 patients with cold myocardial ischemia lasting over 240 minutes, dilation was observed in the perivascular spaces with wall edema of intramural arteries and preserved paretic changes in some of them, which involved venous plethora and dilation. Some arteries exhibited spastic changes with luminal narrowing; the endothelial lining retained its integrity. In microcirculatory vessels, erythrocytic sludges were observed in some places. Muscle fibers were mostly uniform in diameter with weak non-uniform oxyphilic staining of cardiomyocytes; only in some places, individual fibers were observed showing thinning and a wavy contour. Cytoplasmic edema was found in cardiomyocytes, which was accompanied by non-uniform staining. Along with the contractural changes of muscle cells, which did not exceed subsegmental and segmental Class I–II contractures, another type of change in cardiomyocytes—intracellular myocytolysis—was observed in polarized light. It was characterized by a decrease in the A-disk anisotropy or the absence of anisotropic structures within individual muscle cells due to the disappearance of one or another part of myofibrils. The preserved myofibrils were present as weakly anisotropic inclusions (Fig. 5).

In the immunohistochemical study, the expression of actin and desmin generally remained the same in the form of a light-brown staining reaction of cardiomyocyte cytoplasm while slightly increasing in some regions. In some fields of view, a decrease in intercalated disks was detected, with the emergence of deformations in some of them in the form of a “polyline band,” while a slight cross-striation blurring and moderate changes in the structure of the cytoplasm of muscle cells were observed in some places (Figs. 6 A, B).

Thus, the immunohistochemical analysis aimed at studying the expression of actin and desmin showed that in both studied groups, LAA cardiomyocytes exhibit a uniform light-brown staining reaction with its abrupt changes to dark-brown color in the region of the intercalated disks of muscle cells and throughout the muscle fiber in the region of Z-bands.

At different cold ischemia times (up to 240 minutes and over 240 minutes), fairly stable morphological dynamics of actin and desmin expression in the myocardium are observed according to the volume density parameter. The determination of this parameter in myocardial slices from the LAA in comparison of groups revealed its slight but statistically significant increase in Group 2: desmin, by 1.13 times; actin, by 1.07 times (Table), which may be associated with the continuing swelling of muscle cells due to the lysis changes in myofibrils, as well as with the absence of cellular-interstitial exchange during complete cold myocardial ischemia. In the comparison of two cold ischemia groups (up to 240 minutes and over 240 minutes) in terms of the intensity of actin and desmin expression, the morphological dynamics were reversed. With the parameter of immunohistochemically detectable actin and desmin increasing simultaneously, their expression intensities were reverse in nature. The intensity of desmin expression decreased by 1.6 times due to an increase in cardiomyocytes exhibiting a low level of intensity. Conversely, the intensity of actin expression increased by 1.5 times due to the increase in cardiomyocytes showing moderate and strong immunohistochemical reaction intensity (Table 1). For actin, this change in the expression intensity of the studied cellular proteins can be attributed to the contractile structure of the muscle cell—sarcomere, which is the main structural unit of the heart muscle and plays an important role in the effective regulation of cardiac rhythm and hemodynamic maintenance, while for desmin, to the involvement of compartments only beyond the sarcomeric cytoskeleton that provide physical support and structural integrity for heart cells.

Additional study results

No additional results were obtained during the study.

DISCUSSION

Main findings of the study

The comprehensive morphological state assessment of LAA myocardium showed stabile actin and desmin expression in patients with cold ischemia lasting up to 240 minutes and more. Structural changes developing in the form of contractures did not exceed Class I–II. Lysis changes were noted in the individual cardiomyocytes of Group 2 patients.

Research limitations

The results of the present study were obtained on small samples of patients. In order to further develop the completed work, a study using a larger sample size is required.

Interpretation of the study results

Several works [19–22] examine the nature of the cellular response in case of damage to the molecular structures of the cell that realize the interaction of its membrane with intracellular elements comprising the cytoskeleton structure. The structure of cardiomyocytes, including the arrangement of cellular proteins in it, is strictly determined. Cellular proteins are polyfunctional, performing a variety of functions and interacting with different structures in the extracellular and intracellular space. This allows them to ensure that the cell remains preserved and stable during cardiac cycles. By interacting with the extracellular matrix components, cellular proteins provide mechanical strength and support to cells and help in the formation and stabilization of tissue structures. Within the cell, proteins interact with various organelles, thus regulating cellular metabolism, cellular energy supply, protein transport and sorting, as well as signaling pathways within the cell [21–26]. In literary sources, heart diseases involving damage to the structure of muscle cells are mostly cardiomyopathies [23]. The use of the immunohistochemical method in diagnosing damage to the molecular structures of the cell enables assessment of both the dynamics of changes in their number and the degree of cardiomyocyte integrity.

The analysis of actin and desmin expression in the study of myocardium during cold ischemia showed that they are reversed in the groups. The intensity of desmin expression decreased with increasing ischemia time (on the point scale, the group with one point characteristic significantly increased) (Table 1). A significant decrease in desmin expression in various forms of ischemic myocardial damage was reported in a large number of studies. Ischemic myocardial damage leads to impaired blood supply to the heart and subsequent cardiomyocyte death. This can cause changes in the expression of different proteins, including desmin. Studies using immunohistochemistry, immunoblotting, and other methods also confirm that ischemic myocardial damage results in decreased desmin expression, which is associated with the activation of pathological signaling pathways, altered gene expression, and other mechanisms. Decreased desmin expression in ischemic myocardial damage has important implications for myocardial structure and function. Under these conditions, a decrease in desmin level leads to impaired cell integrity, as well as the disorganization of sarcomeres and other structural components. However, of note is that the nature and extent of changes in desmin expression may differ at different stages and in different forms of ischemic myocardial damage. For example, I.V. Zadnipryanyi et al. (2017) report a significant decrease in desmin expression in various forms of ischemic myocardial damage while noting the its heterogeneous presence in different regions—from no response of cardiomyocytes to desmin to its strengthening in the region of Z-bands and intercalated disks [27].

With prolonged complete ischemia of the LAA myocardium, the present study also showed different expression intensities during cold ischemia in both groups; however, no regions exhibiting no reaction to desmin were observed. The expression intensity of actin, unlike that of desmin, increased in Group 2 patients (Table 1) and was non-uniform. Taking into account that during complete cold myocardial ischemia, the repair of damaged myofibrils via activation of synthetic processes is impossible, another mechanism behind enhancing actin expression should be assumed, which is associated with the increased availability of reactive groups in actin filaments under conditions of protein denaturation, with the development of mixed damages in the form of contractures and myocytolysis [19]. This may also be attributed to the inclusion of additional receptors during the fragmentation of contractures, spirally twisted actin filaments in foci of myofibril overcontraction and overstretching.

CONCLUSION

The study revealed that the use of Bretschneider solution during cold myocardial ischemia effectively protects the donor heart during its transportation (with the myocardial cold ischemia lasting up to 240 min and more) due to its balanced elemental composition that determines the metabolic protection of cells and their ionic balance. The comprehensive morphological state assessment of the LAA myocardium in patients with cold ischemia lasting up to 240 minutes and more, which involved the use of light and polarization microscopy and immunohistochemical studies, showed stable actin and desmin expression in the myocardium, which indicates the reversibility of structural changes in the form of contractures not exceeding Class I–II. Lysis changes in individual cardiomyocytes (only in Group 2), as well as preserved expression of actin and desmin in both groups at their average intensity, indicate a sufficiently high degree of preservation of their macromolecular structure in both groups to restore adequate cardiac function following heart transplantation.