In heart failure (HF), the multi-layered mitochondrial quality control system, involving mitochondrial chaperones and proteases, selective mitophagy and vesicular ejection of dysfunctional mitochondria becomes impaired. This leads to the accumulation of damaged mitochondria that are enriched in reactive oxygen species, the loss of membrane potential and the activation of cardiomyocyte death pathways. This accelerates disease progression, ultimately resulting in end-stage HF. However, the structural and molecular characteristics of these defects in human end-stage HF remain poorly defined. When advanced HF continues to cause severe symptoms despite optimal drug therapy, heart transplantation or LVAD (left ventricular assist device) implantation is indicated. Our aim is to identify and characterize the ultrastructure of mitochondria and defects in the mitochondrial quality control pathways in myocardial tissue samples obtained from explanted hearts and the site of LVAD implantation. Understanding these mechanisms may reveal novel therapeutic strategies to preserve mitochondrial integrity and prevent progression to terminal HF.
For this purpose, we collected human myocardial tissue samples from the site of LVAD implantation at left ventricular apex and from explanted hearts from heart transplantation (HTX). Those samples were processed using a standard five-fold contrasting for transmission electron microscopy (TEM), to investigate both mitochondrial ultrastructure and mitochondrial clearance pattern. We then used the software IMOD to establish a mapping for a quantitative analysis of the established clearance pattern compared to intramitochondrial lysis. To identify the matrix chaperones, proteases, and lipases involved in this process, Tokuyasu cryosectioning was used. The primary antibodies were first validated using immunohistochemistry and then linked with a secondary gold antibody for immuno-EM.
EM images of patients with late- and end-stage HF exhibited the typical clearance pattern. Both LVAD and HTX samples showed lysosomes, vacuolar degradation and mitophagy. But despite the presence of all these clearance pathways, mitochondrial clearance appeared impaired, with large accumulations of damaged mitochondria and large autophagosomes containing multiple mitochondria. Furthermore, we observed mitochondria that showed selective lysis of the mitochondrial inner membrane and matrix, while the outer membrane remained intact with no signs of swelling. This phenomenon has not been previously described. Our quantitative analysis revealed that this intramitochondrial lysis occurs more frequently than the established clearance pattern. Immuno-EM images provided insight into the presence and precise localisation of the intramitochondrial matrix proteases and chaperones.
Despite the presence of lysosomes, vacuolar degradation, and mitophagy, mitochondrial clearance in end-stage heart failure appeared impaired, with accumulation of damaged mitochondria and large autophagosomes. We identified a previously undescribed selective lysis of the inner membrane and matrix while the outer membrane remained intact. This intramitochondrial degradation occurred more frequently than canonical clearance, indicating a profound dysregulation of mitochondrial quality control.
