Mitochondrial cristae form, function, and organization dependent upon metabolic sources and implication in heart failure

Project Information

MitoFORMSinHF

Grant agreement ID: 101027093

DOI

10.3030/101027093

Project closed

EC signature date 29 March 2021

Start date 1 September 2022

End date 31 August 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 171 473,28

EU contribution

€ 171 473,28

Coordinated by

UNIVERSITA DEGLI STUDI DI PADOVA
Italy

Periodic Reporting for period 1 - MitoFORMSinHF (Mitochondrial cristae form, function, and organization dependent upon metabolic sources and implication in heart failure)

Reporting period: 2022-09-01 to 2024-08-31

Summary of the context and overall objectives of the project

Acute myocardial infarctions (AMIs) are the leading cause of mortality worldwide and lead to the vast majority of heart failure (HF) cases. While recently there has been a decline in AMIs leading to mortality due to advancements in managing the disease, the prevalence of HF is only increasing. Mitochondria are double membrane organelles that synthesize most cellular ATP, but they also mediate death signals by initiating intrinsic apoptosis and necrosis, implicating their role in nearly all pathophysiology including acute myocardial infarction (AMI) and heart failure (HF). Mitochondria are dynamic and the size, shape, and number of mitochondria respond to changes in the cellular environment. Mitochondrial dynamics are regulated by a dynamin related family of proteins that control fusion and fission of mitochondria. At the level of the inner mitochondrial membrane, Optic Atrophy 1 (OPA1), a dynamin-related protein, orchestrates IMM fusion but also has a genetically distinct role in maintaining mitochondrial cristae form and ultrastructure. Opa1-mediated cristae maintenance is implicated in cardiac ischemia-reperfusion injury (I/R) as apoptotic signaling disassemble Opa1 oligomers, allowing cytochrome c to release to the cytosol, inducing intrinsic apoptosis and cell death.

Recent studies indicate metabolism can profoundly influence mitochondrial morphology as starvation increases mitochondrial fusion events and enhances cristae density in an Opa1-dependent manner but it is unclear how specific carbon sources regulate Opa1-mediated cristae maintenance and how this is implicated in acute ischemia reperfusion injury or heart failure. In the MitoFORMsinHF project we hypothesized OPA1 assembly and cristae maintenance are dependent on metabolic substrate utilization and are implicated in I/R damage pathogenesis. The overall objective of the MitoFORMSinHF project assessed if shifting metabolism can prevent pathogenic cristae remodeling and provide cardioprotective benefits during I/R injury. This is important for society as these novel descriptions of unknown mechanisms-of-action of crosstalk between single carbon sources and IMM architecture will be exploited by the scientific community and biotech sector to translate the basic science into potential novel therapeutic targets for I/R injury or HF.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the MitoFORMSinHF project we first solely feed cells single carbon sources and characterized how specific carbon sources contributed to Opa1-dependent mitochondrial morphology using super resolution microscopy. Next, we set out to precisely determine how metabolic sources regulate Opa1-macromolecular assembly by fusing Opa1 to a proximity dependent biotin ligase named TurboID generating an Opa1-TurboID chimeric protein. The Opa1-TurboID fusion protein correctly localizes to the inner mitochondrial membrane, faces the intermembrane space and displays biotinylation activity. Once expressed in Opa1-/- cells, Opa1-TurboID restores mitochondrial ultrastructure and fusion, confirming OPA1-TurboID can vicariate endogenous Opa1 and that Opa1-TurboID is appropriate to use to evaluate how changes in the Opa1 protein interactome. We therefore used Opa1-TurboID to unbiasedly identify by label free proteomics changes in the Opa1 protein interactome by culturing cells stably expressing Opa1-TurboID in different metabolic conditions (glucose, starvation, ketones and fatty acids, amino acids, or in a physiologic like cell culture medium) while inducing biotinylation. Protein lysates were collected, trypsin digested, biotinylated peptides were enriched with streptavidin beads, and peptides were identified with liquid chromatography mass spectrometry. Bioinformatic analysis identified 231 bona fide mitochondrial proteins as candidate Opa1-TurboID interactors and that interactors were uniquely and significantly enriched in distinct metabolic conditions. We also generated a Slc25a12 knockout cell line and subsequent investigation into this cell line unveiled the mitochondrial aspartate-glutamate carrier is a metabolic sensor that orchestrates organelle morphology and cristae maintenance through interactions with Opa1. Last, we also have generated a first-of-its-kind conditional Opa1-TurboID gain-of-function murine model which we have breed to obtain a cardiac specific, tamoxifen inducible Opa1-TurboID gain-of-function mice (Opa1iHTID). In future studies we will subject the Opa1iHTID mouse model to ischemia-reperfusion injury to identify specific protein candidates implicated in Opa1-mediated cristae remodeling in I/R injury and during heart failure.

Overall our results indicate axes exists between fuel availability and Opa1-mediated cristae dynamics pinpointing metabolic enzymes relay individual fuel sources to cristae biogenesis machinery.

The Opa1-TurboID plasmid is being deposited into the addgene plasmid depository. The large-scale proteomics dataset of the results of the discovery based proteomics screen is being deposited onto public repositories of mass spectrometry datasets. Novel genetically modified cell and mouse models will made available to researchers upon request.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Between 2012 and 2019 in Europe the proportion of acute myocardial infarction related deaths per 1,000 individuals declined from 5.0 % to 3.5% both in the entire population and separately in males and females. This increase in the survival rate of acute myocardial infarction-patients is encouraging but patients with injured hearts develop heart failure. The prevalence of heart failure continues to increase concurrently with life expectancy and development of better therapeutics for managing heart failure Today, over 15 million Europeans live with heart failure placing an enormous socioeconomic burden on both the individual and society.

The heart failure patient has an overall lower quality of life displaying symptoms such as shortness of breath, fatigue, dyspnea, and difficulty with walking and climbing stairs. Difficulty performing these daily activities not only reduces work productivity but can lead to depression and social anxiety. Heart failure patients must rely on friends and family to perform daily activities whom spend on average ~20 hours a week caring for heart failure patients which can cut into their worker productivity, place an economic burden on the caretaker, and disrupt the social activities of the caretaker leading to lower quality of life as well. Heart failure is a progressive disease and symptoms get worse. After diagnosis of an myocardial infarction patients lose 1 month of life per year and patients are hospitalized as much as once per year placing a significant burden on the healthcare system. Heart failure patients are hospitalized ~2 million times a year in Europe and cardiovascular diseases place an enormous socioeconomic burden costing 282 billion Euros representing roughly 2% percent of the EU’s GDP. The MitoFORMsinHF project aims to provide novel insights into basic biology that can be translated into therapies that at the patient level will increase overall quality of life and reduce HF associated mortality, at the caregiver level reduce lost time of productivity, and at the societal level decrease human resource and economic burdens on the healthcare system and governmental budgets.

Overview of discovery based proteomics screen