Autophagy in molecular pathogenesis of inherited and acquired cardiovascular diseases

Autophagy is a major catabolic process, maintaining cellular homeostasis and providing the cell with building blocks and energy under stress conditions and during cellular remodeling. Autophagy is responsible for the on-time elimination of misfolded, aggregated proteins and damaged organelles from the cell. Autophagy dysfunction has been associated with neurodegenerative diseases, neuromuscular disorders, and cardiovascular pathologies. It has been demonstrated that in the pathogenesis of muscular disorders caused by mutations in Z-disk proteins, autophagic activity was altered. Mutations in the DES gene encoding for desmin, a key component of Z-disk, have been linked with the development of desmin-related myopathy (DRM). Severe cases of DRM resulting from aggregate-prone DES mutations were characterized by protein aggregate accumulation in cardiomyocytes and myofibrils. The exact mechanism underlying autophagy dysfunction in the presence of DES mutations in muscle cells still needs to be clarified. Nowadays, steps of the autophagy pathway are actively studied as potential therapeutic targets in treating neurodegenerative disorders, cancer, and infectious diseases. Knowledge about the precise autophagy step affected by distinct DES mutations may contribute to future studies on a search for novel therapeutic agents. Thus, the autophagy process is an attractive target for therapy during cardiac remodeling. However, there is no consensus on whether autophagy is an adaptive response and improves cell survival during cardiac remodeling or is a part of programmed cell death and results in cardiomyocyte death. Furthermore, autophagy’s role in reverse cardiac remodeling in patients with heart failure (HF) receiving heart assist device therapy is constantly under discussion. Therefore, this study aimed to estimate the molecular changes in the autophagy pathway in the model of mouse myoblasts C2C12 expressing various Des mutations and in myocardial samples from patients with chronic HF who underwent implantation of assist device as a part of reverse cardiac remodeling therapy. The objectives of the study to achieve the desired aim are: To assess the autophagy dynamics by western blot and immunocytochemistry in C2C12 cells under the effect of Des mutations: DesS12F, DesL345P, DesA357P, DesL370P, and DesD399Y, associated with severe DRM cases. To decipher by western blot the distinct autophagy stages and to determine the pattern of autophagy alteration specific to each Des mutation using chloroquine (CQ) treatment to inhibit autophagy. Using RNA sequencing to determine genes and molecular pathways involved in the processes of muscle cell homeostasis and affected by the presence of Des mutations. To evaluate the Des mutations’ effect on mitochondria network distribution by immunofluorescence staining and estimate mitophagy activation by RNA sequencing. To prove a key role of Bag3-mediated CASA autophagy in the clearance of desmin induced aggregates using Bag3 silencing approach, immunocytochemistry and RNA sequencing Using RNA sequencing to evaluate the autophagy contribution in myocardium recovery in patients with chronic HF who underwent assist device implantation as a part of reverse remodeling therapy. We sought to assess autophagy dynamics by western blot and immunocytochemistry in C2C12 cells under the effect of Des mutations: DesS12F, DesL345P, DesA357P, DesL370P, and DesD399Y, associated with severe DRM cases. We applied an autophagy inhibitor, chloroquine, to estimate distinct autophagy stages and to determine the pattern of autophagy alterations specific to each Des mutation. Genes involved in muscle cell homeostasis and affected by the presence of Des mutations were analyzed by RNA sequencing. Des mutations’ effect on mitochondria network distribution and mitophagy activation was estimated by immunofluorescence staining and RNA sequencing. The hypothesis of increased autophagy flux in muscle cells and the capacity of muscle cells to accumulate protein aggregates was tested by downregulation of Bag3-mediated selective autophagy (CASA) using the shBag3 transduction. To detect obtained aggregates, immunocytochemistry was used, and RNA sequencing was applied to estimate transcriptional changes under shBag3 effect. Furthermore, RNA sequencing was applied to evaluate the autophagy contribution to myocardium recovery in patients with chronic HF who underwent assist device implantation as a part of reverse remodeling therapy. Throughout the study, we revealed that autophagy flux was increased in basal conditions in muscle cells. Further, we determined that Des mutations affected autophagy dynamics in a mutation-specific manner, and we were able to define the autophagy alteration pattern for each studied Des mutation. Then, we confirmed the disruption of the mitochondrial network in the presence of Des aggregate-prone mutations and mitophagy activation as an adaptive response to impaired mitochondrial functions. Downregulation of CASA allowed us to prove an increased rate of protein degradation by autophagy machinery in muscle cells. In addition, we determined the involvement of Hspb7, Hspb8, Wipi1, Pink1, and Atg16l1 genes in protein homeostasis in C2C12 cells under the effect of Des mutations. Furthermore, the positive autophagy contribution to reverse cardiac remodeling was demonstrated in myocardial samples from patients with chronic HF after device-based therapy. Overall, this study highlights the significance of autophagy for muscle tissue homeostasis and is a fundamental basis for future research aimed at therapeutic targeting of distinct autophagy steps.