Il processo di apoptosi provocata dalla transizione di permeabilità mitocondriale è un tipo di morte cellulare programmata durante il quale la membrana mitocondriale interna mostra un’aumentata permeabilità con un conseguente influsso osmotico di soluti nella matrice mitocondriale. Questo evento è mediato dal complesso proteico del poro mitocondriale di transizione della permeabilità, una piattaforma multiproteica di cui la composizione non è precisamente stabilita. In due studi precedenti, abbiamo dimostrato che la subunità c del complesso F1FO ATP sintasi ricopre un ruolo fondamentale nell’attività e nella formazione del poro mPTP, prima di tutto dimostrando una forte correlazione tra lo stato funzionale dell’mPTP e l’espressione della subunità c, in particolare tra la modalità a diversi passaggi con cui avviene l’apertura dell’mPTP data dal disassemblaggio dei dimeri di ATP sintasi e da riarrangiamenti conformazionali del c-ring. Studi recenti di cardiologia hanno riportato il ruolo chiave dell’apertura dell’mPTP nella progressione della morte delle cellule del miocardio in seguito a riperfusione, infatti più del 50% dell’area finale dell’infarto è dovuta al danno da ischemia e riperfusione. In questo progetto, abbiamo validato un nuovo approccio farmacologico tramite la scoperta, l’ottimizzazione e gli studi di relazione struttura-attività delle prime piccole molecole inibitrici dell’apertura dell’mPTP basate su uno 1,3,8-triazaspiro[4.5]decane scaffold che colpisce la subunità c. Abbiamo identificato tre potenziali composti che mostrano un’attività inibitoria dell’mPTP aumentata a dosi inferiori ai composti di riferimento, una migliore localizzazione nel compartimento mitocondriale e effetti positivi anche in un modello di MI ex vivo, inoltre non mostrano effetti collaterali né a livello cellulare né a livello mitocondriale preservando il contenuto mitocondriale di ATP nonostante la loro interazione diretta con il complesso dell’ATP sintasi. Sia modelli in vitro che preclinici hanno dimostrato l’effetto deleterio dell’apertura dell’mPTP nei primi minuti dopo la riperfusione, giocando un ruolo chiave nell’accelerazione del processo di danno tissutale, quindi colpire il mPTP può essere una valida strategia adiuvante per contrastare il danno da riperfusione. Studi recenti hanno ipotizzato che una delle proteine componenti il poro dell’mPTP è la subunità c della porzione FO dell’ATP sintasi. I suoi livelli circolanti sono stati associati con endpoints surrogati della riperfusione miocardica in pazienti con infarto miocardico con sopraslivellamento del tratto ST (STEMI). Non sono mai stati correlati dati sui determinanti genetici nella subunità c al danno da riperfusione in pazienti con infarto del miocardio. Complessivamente in questo studio clinico mostriamo come l’attività dell’mPTP misurata nei fibroblasti correla in modo significativo con quella monitorata nei cardiomiociti dello stesso paziente, in questo modo in quelle condizioni in cui le biopsie cardiache sono impossibili da ottenere, la funzione dell’mPTP nei fibroblasti di pazienti STEMI può essere predittiva dell’attività dell’mPTP nei cardiomiociti. Inoltre, per la prima volta abbiamo dimostrato una correlazione tra l’mPTP e il danno da riperfusione dopo infarto in pazienti. In aggiunta, un nuovo polimorfismo è stato trovato nel gene ATP5G1 che codifica per la subunità c in due pazienti STEMI ed è stato associato ad un peggioramento nel danno da riperfusione in vitro.

Mitochondrial permeability transition (MPT)-driven apoptosis is a type of programmed cell death during which the inner mitochondrial membrane (IMM) exhibits increased permeability with a consequent osmotic influx of solutes in the mitochondrial matrix. This event is mediated by the mitochondrial permeability transition pore complex (PTPC), a membrane multiprotein platform composed of pore-forming parts and modulators that contribute to its conformational state and, thus, to its mechanism of action. In two previous studies, we provided experimental evidence that the c subunit of F1FO ATP synthase plays a pivotal role in mitochondrial permeability transition pore (mPTP) activity and mPTP formation, demonstrating first, a strong correlation between the mPTP functional state and c subunit expression; and second, the multi-step nature of the mPTP opening by ATP synthase dimers disassembly and c-ring conformational arrangements. Recent cardiology research studies have reported a key role for mPTP opening in the progression of myocardial cell death secondary to reperfusion. Since up to 50% of the final infarct size is due to ischemia-reperfusion injury, targeting the PTPC could be a valuable adjunct in reducing infarct size. In this first project, we validated a new pharmacological approach as an adjunct to reperfusion in myocardial infarction (MI) treatment and described the discovery, optimization, and structure−activity relationship (SAR) studies of the first small-molecule mPTP opening inhibitors based on a 1,3,8-triazaspiro[4.5]decane scaffold that targets the c subunit of the F1FO ATP synthase complex. We identified three potential compounds with increased mPTP inhibitory activity at low concentrations, a specific localization to the mitochondrial compartment and beneficial effects in an ex vivo model of MI, they did not show off-target effects at the cellular and mitochondrial levels; moreover, the compounds preserved mitochondrial ATP content despite interacting with the ATP synthase complex. In vitro and pre-clinical models have demonstrated the deleterious effects of mitochondrial Permeability Transition Pore (mPTP) opening in the first few minutes upon reperfusion, becoming a key player in the acceleration of the injury process. According to this, targeting mPTP might be a valid adjuvant strategy to counteract the reperfusion damage. Hitherto, no evidences are present in a clinical scenario. Recent studies hypothesized that one of the pore-forming proteins of the mPTP is the FO ATP synthase c subunit. Its circulating levels had been found to be associated with surrogate endpoints of myocardial reperfusion in patients with ST-segment elevation myocardial infarction (STEMI). No data about genetic determinants in c subunit have ever been related to reperfusion injury in patients with MI. Overall in this project, the CROFT clinical study showed how mPTP activity measured in fibroblasts significantly correlate with that monitored in myocytes from the same patient, so in those conditions where cardiac biopsies are impossible to be taken, mPTP function in fibroblasts from STEMI patients may be predictive of the mPTP activity in myocytes. Moreover, for the first time we provided a proof of mPTP correlation to reperfusion injury developed upon MI in patients. In addition, a new polymorphism found in ATP5G1 gene encoding for c subunit in two STEMI patients was responsible for worsening reperfusion injury in vitro.

The role of c subunit of F1FO-ATP synthase in mitochondrial permeability transition pore activity for the treatment of reperfusion injury after myocardial infarction

PEDRIALI, GAIA
2020-02-14T00:00:00+01:00

Abstract

Mitochondrial permeability transition (MPT)-driven apoptosis is a type of programmed cell death during which the inner mitochondrial membrane (IMM) exhibits increased permeability with a consequent osmotic influx of solutes in the mitochondrial matrix. This event is mediated by the mitochondrial permeability transition pore complex (PTPC), a membrane multiprotein platform composed of pore-forming parts and modulators that contribute to its conformational state and, thus, to its mechanism of action. In two previous studies, we provided experimental evidence that the c subunit of F1FO ATP synthase plays a pivotal role in mitochondrial permeability transition pore (mPTP) activity and mPTP formation, demonstrating first, a strong correlation between the mPTP functional state and c subunit expression; and second, the multi-step nature of the mPTP opening by ATP synthase dimers disassembly and c-ring conformational arrangements. Recent cardiology research studies have reported a key role for mPTP opening in the progression of myocardial cell death secondary to reperfusion. Since up to 50% of the final infarct size is due to ischemia-reperfusion injury, targeting the PTPC could be a valuable adjunct in reducing infarct size. In this first project, we validated a new pharmacological approach as an adjunct to reperfusion in myocardial infarction (MI) treatment and described the discovery, optimization, and structure−activity relationship (SAR) studies of the first small-molecule mPTP opening inhibitors based on a 1,3,8-triazaspiro[4.5]decane scaffold that targets the c subunit of the F1FO ATP synthase complex. We identified three potential compounds with increased mPTP inhibitory activity at low concentrations, a specific localization to the mitochondrial compartment and beneficial effects in an ex vivo model of MI, they did not show off-target effects at the cellular and mitochondrial levels; moreover, the compounds preserved mitochondrial ATP content despite interacting with the ATP synthase complex. In vitro and pre-clinical models have demonstrated the deleterious effects of mitochondrial Permeability Transition Pore (mPTP) opening in the first few minutes upon reperfusion, becoming a key player in the acceleration of the injury process. According to this, targeting mPTP might be a valid adjuvant strategy to counteract the reperfusion damage. Hitherto, no evidences are present in a clinical scenario. Recent studies hypothesized that one of the pore-forming proteins of the mPTP is the FO ATP synthase c subunit. Its circulating levels had been found to be associated with surrogate endpoints of myocardial reperfusion in patients with ST-segment elevation myocardial infarction (STEMI). No data about genetic determinants in c subunit have ever been related to reperfusion injury in patients with MI. Overall in this project, the CROFT clinical study showed how mPTP activity measured in fibroblasts significantly correlate with that monitored in myocytes from the same patient, so in those conditions where cardiac biopsies are impossible to be taken, mPTP function in fibroblasts from STEMI patients may be predictive of the mPTP activity in myocytes. Moreover, for the first time we provided a proof of mPTP correlation to reperfusion injury developed upon MI in patients. In addition, a new polymorphism found in ATP5G1 gene encoding for c subunit in two STEMI patients was responsible for worsening reperfusion injury in vitro.
PINTON, Paolo
File in questo prodotto:
File Dimensione Formato  
PhD Thesis Pedriali.pdf

embargo fino al 13/02/2023

Descrizione: Tesi di dottorato Pedriali Gaia
Tipologia: Tesi di dottorato
Dimensione 5.08 MB
Formato Adobe PDF
5.08 MB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11392/2487953
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact