Part 1. Aim. Diabetic cardiomyopathy (DCM) is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD). Mechanisms that can restore cardiac relaxation (lusitropic effect) improving intracellular Ca2+ dynamics, represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime is a NaK ATPase (NKA) inhibitor with the property of accelerating Ca2+ re-uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation. The project aims to characterize Istaroxime effects at a concentration mostly unaffecting NKA to isolate its effects dependent on SERCA2a only in a model of mild diabetes (type 1). Methods and results. Streptozotocin (STZ) treated rats were evaluated at 9 weeks after STZ injection in comparison to control (CTR) ones. SERCA2a-dependent Istaroxime effects were evaluated in cell-free system and in isolated left ventricular (LV) myocytes. STZ animals showed reduced SERCA2a protein level and activity and increased monomeric PLN/SERCA2a ratio. Intracellular Ca2+ handling and electrical activity were evaluated in isolated ventricular myocytes. In STZ myocytes, SERCA downregulation caused 1) increased diastolic Ca2+, 2) reduction in SR Ca2+ content and Ca2+ transient amplitude following control of membrane potential, 3) slower SR reloading process under Na/Ca exchanger (NCX) inhibition, 4) unchanged SR stability and Ca2+ sparks rate. Action potentials (APs) were significantly prolonged, resulting in an increased short-term variability (STV) of APD. Istaroxime (100 nM) significantly stimulated SERCA2a activity and reverted STZ-induced effects by 1) reducing diastolic Ca2+, 2) increasing Ca2+ transient amplitude and SR Ca2+ content, and 3) accelerating SR Ca2+ reuptake in STZ group. Moreover, Istaroxime, by stimulating SERCA2a, partially restored Ca2+ sparks characteristics and significantly accelerated Ca2+ sparks decay. Conclusions. SERCA2a stimulation by Istaroxime restores STZ-induced intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Part 2. Aim. Heart automaticity is generated in the sino-atrial node (SAN) by a functional interplay between ion channels of the plasma membrane and intracellular ryanodine receptor (RyR)-dependent Ca2+ release. SAN cells are characterized by the expression of voltage-gated L-type Cav1.3 and T-type Cav3.1 Ca2+ (Cav) channels in addition to L-type Cav1.2 channels, which are ubiquitously expressed in the heart. To investigate the significance of Cav expression for heart automaticity we used mutant mice carrying individual or concomitant genetic ablation of Cav1.3 and Cav3.1. Methods and results. Cav ablation additively reduced heart rate in mice. ECG recordings of intact Cav1.3-/-/Cav3.1-/- hearts showed atrioventricular rhythm dissociation and predominantly junctional, rather than SAN driven rhythmicity. Optical mapping of automaticity showed disruption of primary automaticity in Cav1.3-/-/Cav3.1-/- SAN and a shift of the leading pacemaker sites outside the SAN area. We also investigated the role of hyperpolarization-activated f-(HCN) channels, and TTX-sensitive Na+ (Nav) channels in residual automaticity of mutant mice. Concomitant pharmacologic inhibition of f-HCN and TTX-sensitive Nav channels slowed atrial automaticity in wild-type and Cav3.1-/-, while arrested it in 4/6 of Cav1.3-/-, 3/6 of Cav1.3-/-/Cav3.1-/-. Same results were confirmed in isolated Cav1.3-/-/Cav3.1-/- SAN pacemaker cells. Conclusions. Cav1.3 and Cav3.1 Ca2+ channels deletion disrupts normal heart automaticity by inducing bradycardia and altering cardiac conduction. Moreover, in the concomitant absence of Cav1.3 and Cav3.1 channels, f-HCN channels and TTX-sensitive Nav channels are the predominant mechanisms sustaining pacemaker activity.  

Parte 1. La cardiomiopatia diabetica (DCM) è una malattia caratterizzata da una precoce disfunzione diastolica (DD). I meccanismi che possono ripristinare il rilassamento cardiaco, migliorando la dinamica intracellulare di Ca2+, rappresentano un promettente approccio terapeutico per le malattie cardiovascolari associate alla DD. Istaroxime è un inibitore di NaK-ATPase (NKA) e stimolatore del recupero di Ca2+ nel reticolo sarcoplasmatico (SR) attraverso la pompa del SR per il Ca2+ (SERCA2a). Il progetto mira a caratterizzare gli effetti di Istaroxime a una concentrazione che minimamente influenza NKA per isolare i suoi effetti dipendenti da SERCA2a in un modello di diabete di tipo 1. I ratti trattati con streptozotocina (STZ) sono stati valutati a 9 settimane dopo l'iniezione di STZ rispetto ai controlli (CTR). I ratti STZ hanno mostrato una riduzione del livello e dell'attività della proteina SERCA2a e un aumento del rapporto PLNmonomerico/SERCA2a. Le dinamiche del Ca2+ intracellulare e l'attività elettrica sono state valutate in miociti ventricolari isolati. Nei miociti STZ, la downregulation di SERCA ha causato 1) aumento di Ca2+ diastolico 2) riduzione del contenuto di Ca2+ nel SR e dell’ampiezza del transiente di Ca2+, 3) ricarica del SR più lenta con inibito lo scambiatore Na/Ca, 4) stabilità del SR e numero di sparks di Ca2+ invariati. I potenziali d'azione (AP) sono stati significativamente prolungati, con conseguente aumento della short-term variability (STV) dell'APD. Istaroxime (100 nM) ha stimolato in modo significativo l'attività di SERCA2a ripristinando gli effetti indotti da STZ 1) riducendo Ca2+ diastolico, 2) aumentando l’ampiezza del transiente e il contenuto del SR di Ca2+ e 3) accelerando il recupero di Ca2+ nel SR nel gruppo STZ. Inoltre, Istaroxime, stimolando SERCA2a, ha parzialmente ripristinato le caratteristiche delle sparks di Ca2+. La stimolazione di SERCA2a da parte di Istaroxime ripristina le anomalie delle dinamiche del Ca2+ intracellulare indotte da STZ. Pertanto, la stimolazione SERCA2a può essere considerata un promettente approccio terapeutico per il trattamento della DD. Parte 2. L'automaticità cardiaca è generata nel nodo seno-atriale (SAN) attraverso i canali ionici della membrana plasmatica e il rilascio di Ca2+ dipendente dal recettore intracellulare della rianodina (RyR). Le cellule del SAN sono caratterizzate dall'espressione dei canali Cav1.3 di tipo L e Cav3.1 di tipo T (Cav). Per studiare il significato dell'espressione di Cav nell'automaticità cardiaca abbiamo usato topi mutanti con delezione genetica individuale o concomitante di Cav1.3 e Cav3.1. La delezione del CaV ha ridotto in modo additivo la frequenza cardiaca nei topi. Le registrazioni ECG dei cuori intatti di Cav1.3-/-/Cav3.1-/- hanno mostrato una dissociazione del ritmo atrioventricolare e una ritmicità prevalentemente giunzionale, anziché del SAN. La mappatura ottica dell'automatismo ha mostrato l'interruzione dell'automaticità primaria in SAN Cav1.3- -/Cav3.1-/- e uno spostamento dei principali siti di pacemaker al di fuori dell'area SAN. Abbiamo anche studiato il ruolo dei canali f- (HCN) attivati dall'iperpolarizzazione e dei canali Na+ (Nav) sensibili al TTX nell'automaticità residua dei topi mutanti. L'inibizione farmacologica concomitante dei canali Nav sensibili a f-HCN e TTX ha rallentato l'automaticità atriale in wild-type e Cav3.1- -, mentre è stata arrestata in alcuni Cav1.3-/- e Cav1.3- /-/Cav3.1- -. Gli stessi risultati sono stati confermati in cellule di pacemaker SAN Cav1.3-/-/Cav3.1-/- isolate. L'eliminazione dei canali Cav1.3 e Cav3.1 interrompe la normale automaticità cardiaca inducendo bradicardia e alterando la conduzione cardiaca. Inoltre, con la doppia delezione dei Cav1.3 e Cav3.1, i canali f-HCN e i canali Nav sensibili al TTX sono i meccanismi predominanti a sostegno dell'attività del pacemaker.

(2020). Role of SERCA stimulation and voltage-dependent Ca2+ channels in improving Ca2+ handling and sustaining heart automaticity. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2020).

Role of SERCA stimulation and voltage-dependent Ca2+ channels in improving Ca2+ handling and sustaining heart automaticity

TORRE, ELEONORA
2020

Abstract

Part 1. Aim. Diabetic cardiomyopathy (DCM) is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD). Mechanisms that can restore cardiac relaxation (lusitropic effect) improving intracellular Ca2+ dynamics, represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime is a NaK ATPase (NKA) inhibitor with the property of accelerating Ca2+ re-uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation. The project aims to characterize Istaroxime effects at a concentration mostly unaffecting NKA to isolate its effects dependent on SERCA2a only in a model of mild diabetes (type 1). Methods and results. Streptozotocin (STZ) treated rats were evaluated at 9 weeks after STZ injection in comparison to control (CTR) ones. SERCA2a-dependent Istaroxime effects were evaluated in cell-free system and in isolated left ventricular (LV) myocytes. STZ animals showed reduced SERCA2a protein level and activity and increased monomeric PLN/SERCA2a ratio. Intracellular Ca2+ handling and electrical activity were evaluated in isolated ventricular myocytes. In STZ myocytes, SERCA downregulation caused 1) increased diastolic Ca2+, 2) reduction in SR Ca2+ content and Ca2+ transient amplitude following control of membrane potential, 3) slower SR reloading process under Na/Ca exchanger (NCX) inhibition, 4) unchanged SR stability and Ca2+ sparks rate. Action potentials (APs) were significantly prolonged, resulting in an increased short-term variability (STV) of APD. Istaroxime (100 nM) significantly stimulated SERCA2a activity and reverted STZ-induced effects by 1) reducing diastolic Ca2+, 2) increasing Ca2+ transient amplitude and SR Ca2+ content, and 3) accelerating SR Ca2+ reuptake in STZ group. Moreover, Istaroxime, by stimulating SERCA2a, partially restored Ca2+ sparks characteristics and significantly accelerated Ca2+ sparks decay. Conclusions. SERCA2a stimulation by Istaroxime restores STZ-induced intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Part 2. Aim. Heart automaticity is generated in the sino-atrial node (SAN) by a functional interplay between ion channels of the plasma membrane and intracellular ryanodine receptor (RyR)-dependent Ca2+ release. SAN cells are characterized by the expression of voltage-gated L-type Cav1.3 and T-type Cav3.1 Ca2+ (Cav) channels in addition to L-type Cav1.2 channels, which are ubiquitously expressed in the heart. To investigate the significance of Cav expression for heart automaticity we used mutant mice carrying individual or concomitant genetic ablation of Cav1.3 and Cav3.1. Methods and results. Cav ablation additively reduced heart rate in mice. ECG recordings of intact Cav1.3-/-/Cav3.1-/- hearts showed atrioventricular rhythm dissociation and predominantly junctional, rather than SAN driven rhythmicity. Optical mapping of automaticity showed disruption of primary automaticity in Cav1.3-/-/Cav3.1-/- SAN and a shift of the leading pacemaker sites outside the SAN area. We also investigated the role of hyperpolarization-activated f-(HCN) channels, and TTX-sensitive Na+ (Nav) channels in residual automaticity of mutant mice. Concomitant pharmacologic inhibition of f-HCN and TTX-sensitive Nav channels slowed atrial automaticity in wild-type and Cav3.1-/-, while arrested it in 4/6 of Cav1.3-/-, 3/6 of Cav1.3-/-/Cav3.1-/-. Same results were confirmed in isolated Cav1.3-/-/Cav3.1-/- SAN pacemaker cells. Conclusions. Cav1.3 and Cav3.1 Ca2+ channels deletion disrupts normal heart automaticity by inducing bradycardia and altering cardiac conduction. Moreover, in the concomitant absence of Cav1.3 and Cav3.1 channels, f-HCN channels and TTX-sensitive Nav channels are the predominant mechanisms sustaining pacemaker activity.  
ROCCHETTI, MARCELLA
MANGONI, MATTEO
SERCA2; Fosfolambano; Diabete di tipo 1; Pacemaker; Canali del Calcio
SERCA2; Phopholamban; Type 1Diabetes; Pacemaker; Canali del Calcio
BIO/09 - FISIOLOGIA
English
31-gen-2020
BIOLOGIA E BIOTECNOLOGIE
32
2018/2019
open
(2020). Role of SERCA stimulation and voltage-dependent Ca2+ channels in improving Ca2+ handling and sustaining heart automaticity. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2020).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/261917
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