ИСТИНА

Рестриктивная кардиомиопатия, клинически единое заболевание, представляет собой гетерогенную группу заболеваний. В связи с этим выяснение причины и механизма возникновения заболевания у пациента может играть ключевую роль для подбора необходимой терапии. Тем не менее, в большинстве случаев причины развития генетически-обусловленных кардиомиопатий остаются невыясненными. Целью нашего проекта является изучение роли кардиопротекторного малого белка теплового шока человека HspB7 (cvHsp, cardiovascular Hsp), принадлежащего системе белков-шаперонов, в поддержании нормального функционирования клеток сердца и возможной роли этого белка в развитии рестриктивной кардиомиопатии, ассоциированной с мутацией в гене филамина С. В результате реализации проекта мы планируем подробно изучить взаимодействие между HspB7 и филамином С и возможную роль этого взаимодействия в поддержании нормального функционирования кардиомиоцитов и в процессе развития рестриктивной кардиомиопатии. Это фундаментальное исследование может иметь прикладное значение, поскольку в ходе выполнения проекта будет исследован конкретный случай рестриктивной кардиомиопатии, связанной с мутацией филамина С.

Project actuality. The data of the WHO indicate that the cardio-vascular diseases are the main reason of death over the world (18.6 million deaths in 2019). The frequency of child cardiomyopathies is close to 1 per 100000. This disease is the leading reason of heart transplantation in childhood. About 40% of children with cardiomyopathy require transplantation otherwise die in the first two years of their life. The largest part of cardiomyopathies has a genetic origin and is induced by mutations in the main contractile and/or regulatory proteins or proteins maintaining cardiomyocytes in the active state. Very often these mutations induce accumulation of protein aggregates in cardiomyocytes and/or incorrect protein folding or inability to assemble functionally active myofibrils. Therefore, investigation of proteins involved in protein homeostasis, i.e. heat shock proteins (chaperones) are of great importance. Chaperones (or heat shock proteins, Hsp) is a large group of proteins expressed in all domains of life including archaea, bacteria, plants and animals. All Hsp can be divided in two large groups. The small heat shock proteins (sHsp, HspB) form the first line of protection against different unfavorable factors. They bind and prevent aggregation of partially denatured proteins later transferring them to the second group of Hsp. This second group is presented by “large” predominantly ATP-dependent heat shock proteins that can refold and reconstruct functional activity of partially denatured proteins. HspB7 (or cvHsp) small heat shock protein is specifically expressed in the heart in addition to the other ubiquitously expressed small heat shock proteins such as HspB1, HspB5, HspB6 and HspB8. The data of literature indicate that the single nucleotide polymorphism (SNP) of HspB7 correlates with development of cardiomyopathy. Therefore, this project deals with detailed investigation of fundamental basis of cardioprotective activity of HspB7 under normal conditions and in pathology, associated with restricted cardiomyopathy. HspB7 belongs to the family of small heat shock proteins that possess the so-called chaperone-like activity, i.e. ability to prevent aggregation of partially denatured proteins. However, up to now this activity of HspB7 was poorly analyzed. In experiments performed with commonly used model protein substrates HspB7 demonstrated either very low chaperone-like activity or unexpectedly induced aggregation instead of its inhibition. HspB7 effectively prevented aggregation of polyGln-containing proteins such as huntingtin fragments. However, huntingtin is a neuronal protein, that is not expressed in heart. Therefore, potential substrates of HspB7 in heart remain unknown. At the same time there are no doubts in cardioprotective activity of HspB7. For instance, decrease of HspB7 expression is accompanied by disturbance of myofibril assembly and accumulation of aggregates of filamin C, important protein involved in myofibrils assembly and their interaction with plasma membrane. In addition, filamin C is one of few potential protein partner for HspB7. Therefore, the first problem that we would like to address is analysis of HspB7 as a genuine small heat shock protein and comparison of its cardioprotective activity with the corresponding activity of other small heat shock proteins that are also expressed in cardiomyocytes. To solve this problem, we plan to obtain recombinant fragment containing 22-24th immunoglobulin domains of filamin C containing putative sites of HspB7 binding. We will analyze interaction of this filamin fragment with HspB7 and other small heat shock proteins (HspB1, HspB5, HspB6, HspB8) by using spectral methods, size-exclusion chromatography, chemical crosslinking and native gel-electrophoresis. Afterwards we plan to determine chaperone-like activity of isolated HspB7 and isolated HspB1, HspB5, HspB6, HspB8 and their heterooligomeric complexes using filamin C fragment as a model protein substrate. Thus we hope to understand the nature of HspB7-filamin C interaction and to determine chaperone-like activity of HspB7 as one of important properties providing for cardioprotective activity of HspB7. If interaction of filamin C fragment and HspB7 is estimated, we will turn to determination of HspB7 sites involved in this kind of interaction. For this purpose, we will obtain α-crystallin domains of HspB7 and other small heat shock proteins and analyze their interaction with filamin C fragment under normal conditions and under conditions leading to partial denaturation and aggregation of filamin C fragment. Afterwards by using bioinformatics approaches we will try to localize key sites of HspB7 involved in interaction with filamin C. After analyzing interaction of HspB7 and filamin C under in vitro conditions, we will turn to analyzing this interaction in cell model under normal conditions and in restrictive cardiomyopathy that is characterized by increased stiffness of cardiac walls. Filamin C mutations are associated with cardiomyopathy and probably lead to accumulation of filamin C aggregates in cardiomyocytes. Recently new filamin C mutation was described where deletion of three nucleotides results in amino acid replacement of Glu2472 and Asn2473 by two Asp residues (c.7416_7418delGAA/p.Glu2472_Asn2473delAsp). This mutation is located in the 22th immunoglobulin-like domain of filamin C, i.e. close to the putative HspB7-binding site of filamin C. Detailed mechanism of this pathology remains enigmatic. If the problems planed in the first part of our project are successively solved than we can turn to analysis of the wild type and mutated fragments of filamin C interaction with HspB7. These experiments will be performed both under normal conditions as well as under conditions inducing partial denaturation and aggregation of filamin C fragments. After finishing in vitro experiments we are planning to run experiments using cell culture. We will use inducible pluripotent stem cells (iPSC) obtained from patient suffering from restrictive cardiomyopathy caused by the above mentioned mutation. By using CRISPR-Cas technology we will repair filamin C mutation in iPSC and thus obtain two cell lines, one containing recovered wild type filamin C gene and another containing mutated form of filamin C. These cells will be differentiated and two lines of cardiomyocytes will be obtained. Thus we will be able to analyze the effect of above mentioned mutations on the level of filamin C and HspB7 expression and compare the level of these two protein in normal cardiomyocytes and cardiomyocytes expressing mutated form of filamin C. We also will analyze co-localization of filamin C and its mutated form with HspB7 in two types of cardiomyocytes. In addition, with the method of co-immunoprecipitation we will measure the effect of the above mentioned mutations on the interaction of full-size filamin C with HspB7. We hope that solving this problems will be useful for understanding molecular mechanisms underlying functioning of HspB7 and will explain mechanism of cardioprotective activity of HspB7. Scientific novelty of this project development consists of complex approach to understanding of cardioprotective activity of HspB7 and comparison of this activity with the corresponding function of other small heat shock proteins. In the course of this project we will analyze cardioprotective activity both in in vitro experiments and in cell cultures. We will analyze interaction of HspB7 and other small heat shock proteins with filamin C one of the regulatory protein of cardiomyocytes, playing crucial role in myofibril assembly and sarcomere functioning. Successful fulfillment of this project will provide important conclusions concerning mechanism of cardioprotective activity of HspB7. To our knowledge analogous investigations were not undertaken earlier.

В результате реализации проекта мы планируем решить ряд взаимосвязанных задач. Во-первых, нам предстоит ответить на ключевой вопрос - способен ли HspB7 предотвращать агрегацию физиологичных белков-субстратов. Поэтому первой задачей нашего проекта является исследование функциональной активности HspB7 как классического малого белка теплового шока и сопоставление его активности с аналогичной активностью других малых белков теплового шока, также экспрессирующихся в сердце. Решение этой задачи с использованием в качестве партнера и субстрата филамин С позволит приблизиться к пониманию того, каким образом HspB7 может участвовать в поддержании сократительной активности сердца. Если в ходе выполнения проекта удастся выявить образование прочного комплекса HspB7 (или других малых белков теплового шока) с филамином C, то следующей задачей станет локализация участков связывания филамина С в структуре HspB7 в нативных условиях и условиях частичной денатурации филамина С. Изучив взаимодействие и функционирование HspB7 в норме, мы планируем исследовать его роль при развитии кардиомиопатии. Для этого мы планируем перейти к третьей задаче и изучить влияния точечной мутации c.7416_7418delGAA в последовательности гена филамина С, ассоциированной с кардиомиопатией, на его взаимодействие с HspB7. Решение этого вопроса позволит перейти к четвертой задаче, в рамках которой мы в условиях, приближенных к in vivo, планируем оценить влияние описанной выше мутации на уровни экспрессии филамина С и HspB7, а также на возможность формирования агрегатов филамина С в кардиомиоцитах. Изучить влияние аминокислотных замен на распределение филамина С и HspB7 в кардиомиоцитах. Таким образом, в нашем проекте мы планируем всесторонне, используя комплексный подход в системах in vitro и клеточных моделях, изучить взаимодействие HspB7 и филамина С. Данное исследование позволит выяснить один из возможных механизмов кардиопротекторной активности HspB7 как в нормальных условиях, так и в условиях кардиомиопатии.