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UNIVERSIDAD AUTÓNOMA DE MADRID

PROGRAMA DE DOCTORADO DE BIOCIENCIAS MOLECULARES

ROLE OF NUCLEOTIDE-BINDING OLIGOMERIZATION DOMAIN TYPE I (NOD1) IN HUMAN AND

EXPERIMENTAL HEART FAILURE.

Tesis Doctoral Almudena Val Blasco

Madrid, 2018

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Departamento de Bioquímica Facultad de Medicina

Universidad Autónoma de Madrid

ROLE OF NUCLEOTIDE-BINDING OLIGOMERIZATION DOMAIN TYPE 1 (NOD1) IN HUMAN AND

EXPERIMENTAL HEART FAILURE.

Memoria de Tesis Doctoral presentada por:

Almudena Val Blasco Madrid, 2018

Licenciada en Bioquímica, para optar al grado de Doctora por la Universidad Autónoma de Madrid

Dirigida por:

Dra. María Fernández Velasco

Realizada en el Instituto de Investigación Hospital

Universitario La Paz, IdiPAZ, en Madrid.

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La directora Dra. María Fernández Velasco certifica que Almudena Val Blasco, licenciada en Bioquímica por la Universidad de Zaragoza, ha realizado el trabajo de investigación titulado: “Role of Nucleotide-binding oligomerization domain type I (NOD1) in human and experimental heart failure” bajo su dirección en el Instituto de Investigación Hospital Universitario La Paz, para optar al grado de Doctor por la Universidad Autónoma de Madrid.

Revisado el presente trabajo, expresa su conformidad para la presentación del mismo considerando que reúne los requisitos necesarios para ser sometido a discusión ante el Tribunal correspondiente.

Firmado, para que conste a todos los efectos, en Madrid a 24 de septiembre de 2018.

Fdo. María Fernández Velasco Directora de Tesis

Investigadora Miguel Servet II, IdiPAZ

VºBº Tutor Lisardo Boscá Gomar Investigador Científico, CSIC

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La ciencia y la vida cotidiana no pueden y no deben ser separadas. La ciencia, para mí, proporciona una explicación parcial a la vida. En la medida en que está basada en hechos, experiencia y experimentos.

Rosalind Franklin

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AGRADECIMIENTOS

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AGRADECIMIENTOS

Han pasado cuatro años desde que llegué a Madrid llena de esperanza y alegría por lo que iba a ser una nueva etapa en mi carrera profesional. Todavía recuerdo el día en el que hice la entrevista en el despacho de la Dra. Carmen Delgado para un puesto de estudiante en su laboratorio para realizar mi trabajo fin de master. La Dra. María Fernández Velasco estaba con ella y me dijeron que si quería trabajar con ellas tendría que ir al laboratorio del Instituto de Investigación del Hospital La Paz. Yo les dije que no había ningún problema, y así fue como empecé en el Grupo de Fisiopatología Cardiaca.

En este trabajo ha participado mucha gente, gente del Instituto de Investigaciones Biomédicas Alberto Sols, del Instituto de Investigación del Hospital Universitario La Paz, del Complejo Hospitalario Universitario de A Coruña y del Hospital Universitario 12 de Octubre. Todos ellos han puesto su granito de arena apoyándome en este trabajo. Por eso me gustaría dar las gracias a algunas personas que han sido parte de estos maravillosos tres años en Madrid para realizar mi Tesis Doctoral.

A mi directora de Tesis, la Dra. María Fernández Velasco, por confiar en mí y darme la oportunidad de trabajar en su laboratorio y de realizar esta Tesis Doctoral y por ser un ejemplo a seguir no solo profesionalmente sino también personalmente. Por lo mucho que me ha enseñado, sin sus muchos y muy buenos consejos, su crítica constructiva, su constante apoyo y su inmensa paciencia yo no hubiera sido capaz de llegar hasta aquí.

Al Dr. Lisardo Boscá, mi tutor de Tesis, por ayudarme durante estos años y por apoyarme durante todo este proceso.

A la Dra. Carmen Delgado y la Dra. Gema Ruiz-Hurtado, por su apoyo y sus buenos consejos. Gracias por mostrarme vuestro cariño y acogerme como parte de vuestro equipo. Vuestra opinión siempre es clave.

A mis compañeros de Doctorado, José Alberto Navarro García y María Tamayo, por ser los mejores compañeros de viaje. Gracias a los dos por tantísimas horas discutiendo, hablando y riendo. Aunque no estemos en el mismo laboratorio, la distancia no es una excusa para formar un buen equipo.

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A Laura Martín Nunes, por su asistencia técnica y también por los cafés. Qué hubiera sido de mí si ella no hubiera estado allí para ayudarnos con el aislamiento de los cardiomiocitos.

A el laboratorio B.11, especialmente a la Dra. Patricia Prieto, a Rafael Iñigo, a la Dra. Rocío Brea y a Verónica Terrón por “su ayuda de última hora” con los reactivos y los cafés. Patri, gracias también por todo lo que he aprendido de ti, cultivos celulares, PCRs, inmunoprecipitación, etc.

A la Dra. María José G.M. Piedras, por ser un encanto y por su apoyo. Gracias a ella las técnicas quirúrgicas fueron todo un éxito.

A Ana y a todos los estudiantes que han pasado por el laboratorio, porque me han ayudado con el trabajo diario y he aprendido mucho de ellos.

Al Servicio de Microscopía Óptica (Lucía Sanchez, Diego, Mónica, Lucía, Arancha y María) y Confocal y al Servicio de Medicina Comparativa (Cristina y Marta) en el IIB, por su apoyo técnico y su paciencia.

Al Servicio de Cirugía Experimental del IdiPAZ, en especial a la Dra. Carlota Largo, por su apoyo y diligencia durante estos años.

Al Laboratorio de Imagen del IdiPAZ, en especial a la Dra. María Teresa Vallejo por ser tan buena persona y por su ayuda con la inmunohistoquímica.

A mis amigos de aquí y a los de toda la vida, porque ellos siguen a mi lado, preguntándome sobre mi Tesis incluso sin saber realmente lo que estoy haciendo, y diciéndome en los momentos de desesperación que yo puedo.

Y por supuesto, quiero dedicar esta Tesis Doctoral a mi familia, especialmente a mis padres y hermana, los pilares de mi vida sin los cuales no hubiera sido capaz de caminar este camino. Ellos son los verdaderos responsables de que yo esté aquí. Su amor incondicional y apoyo me dan fuerza.

Gracias a todos vosotros y a muchos otros que he conocido durante este tiempo, finalmente, ¡aquí está mi Tesis Doctoral!

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RESUMEN

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La insuficiencia cardiaca (IC) es un síndrome complejo que actualmente se ha relacionado con una respuesta exacerbada del sistema inmune innato y con un remodelado cardiaco deletéreo. Nucleotide-binding oligomerization domain 1 (NOD1) es un receptor del sistema inmune innato cuya activación selectiva induce disfunción cardiaca alterando el manejo del Ca2+ intracelular cardiaco de manera similar a lo que ocurre en la IC. En base a estos antecedentes, nuestra hipótesis consistió en analizar si NOD1 participa en las alteraciones del dinamismo del Ca2+ intracelular asociadas a la IC.

Nuestros resultados mostraron que NOD1 estaba sobre-expresado en el miocardio insuficiente humano y de ratón inducido por infarto de miocardio (wt-PMI). Para estudiar el papel que juega NOD1 en esta patología, se indujo IC en un modelo de ratón knock-out para NOD1 (Nod1-/-). Los ratones Nod1-/--PMI tenían mejor función cardiaca y menor remodelado estructural comparado con los wt-PMI. Esta mejoría se relacionó con la prevención de las alteraciones del manejo del Ca2+ intracelular cardiaco asociadas a la IC.

Además, el bloqueo farmacológico de NOD1 también previno las alteraciones del dinamismo del Ca2+ intracelular en los ratones wt-PMI. A continuación, evaluamos si la supresión de NOD1 modulaba la regulación β-adrenérgica del manejo del Ca2+ intracelular en la IC en ratones wt y Nod1-/- sanos, así como en ratones wt-PMI y Nod1-/--PMI. La administración de isoproterenol (10-8M) indujo efectos similares en los animales wt y Nod1-/- sanos, sin embargo, esta agonista β-adrenérgico mostró una mejoría significativa del manejo del Ca2+ tanto sistólico como diastólico en las células Nod1-/--PMI comparado con las células wt-PMI.

Finalmente, analizamos la vía de señalización de NOD1 en fibroblastos cardiacos obtenidos de un modelo de ratón de diabetes tipo 2 (db). Nuestros resultados mostraron que tanto la vía de señalización de NOD1 como NF-κB estaban aumentados en los corazones db. Los ratones db tratados con un agonista de NOD1 (C12-iE-DAP, 5mg/kg, 2 semanas) presentaban mayor fibrosis y este efecto se prevenía con el co-tratamiento de C12-iE-DAP y un inhibidor de NF-κB (BAY-11-7082, 2,5mg/kg, 2 semanas).

Los resultados obtenidos en la presente Tesis Doctoral señalan a NOD1 como una nueva diana para el tratamiento de enfermedades cardiovasculares con un aumento de la respuesta pro-inflamatoria como la IC o la cardiomiopatía diabética.

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ABSTRACT

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Heart failure (HF) is a complex syndrome associated with maladaptive innate immune system response that leads to deleterious cardiac remodelling. Nucleotide-binding oligomerization domain type 1 (NOD1) is an innate immune receptor involved in several cardiovascular diseases. Since selective activation of NOD1 induces cardiac dysfunction by impairing Ca2+ handling in a similar way to that occurring during HF, we hypothesized that NOD1 participates in Ca2+ mishandling associated with HF progression.

First, NOD1 was found over-expressed in human failing myocardium. To study the involvement of this mediator in HF progression we evaluated the NOD1 signaling pathway in a mice model of HF induced by myocardial infarction (PMI) in wild type (wt-PMI). The NOD1 pathway was up-regulated in murine failing myocardium. Next, we determined whether the absence of NOD1 impairs cardiac damage linked to HF in a mice model (Nod1-/--PMI). Compared to wt-PMI, hearts from Nod1-/--PMI mice showed less cardiac dysfunction and attenuated structural remodelling. Ameliorated cardiac function in Nod1-

/--PMI mice was linked to a prevention of Ca2+ dynamics impairment associated with HF.

Pharmacological blockade of NOD1 also prevented Ca2+ mishandling in wt-PMI mice.

Then, we evaluated whether the absence of NOD1 regulates the β-adrenergic modulation of Ca2+ signaling in HF. Ca2+ dynamics were examined in cardiomyocytes from wt and Nod1-/- healthy mice, and from wt-PMI and Nod1-/--PMI mice before and after isoproterenol perfusion (10-8M). Isoproterenol administration induced similar effects in wt and Nod1-/- healthy cells but induced a significant improvement in Ca2+ dynamics in Nod1-/--PMI compared to wt-PMI cells.

Finally, we analysed NOD1 pathway in cardiac fibroblasts obtained from a type 2 diabetic mice model (db). The results showed that NOD1 and NF-κB signalling were up-regulated in the db cardiac fibroblasts. Supporting these data, cardiac fibrosis was increased in db mice treated with a NOD1 agonist (C12-iE-DAP, 5mg/kg, 2 weeks) compared to vehicle treated db mice. Moreover, the effect of C12-iE-DAP was prevented in db mice co-treated with a NF- κB inhibitor (BAY-11-7082, 2,5mg/kg, 2 weeks).

The results obtained in this Doctoral Thesis point out NOD1 as a new target for cardiovascular diseases with a pro-inflammatory background such as HF or diabetic cardiomyopathy.

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INDEX

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CONTENT:

INTRODUCTION

………..……….……….…. 1

1. HEART FAILURE ………..………. 3

2. STRUCTURAL REMODELLING IN HEART FAILURE ………..…. 3

3. CALCIUM MISHANDLING IN HEART FAILURE ………..……….. 5

3.1. EXCITATION-CONTRACTION COUPLING ………...….. 5

3.1.1. KEY PARTNERS IN EXCITATION-CONTRACTION COUPLING ……….……….…...… 6

3.1.1.1. L-type calcium channel (LTCC) ………...………..….………. 6

3.1.1.2. Ryanodine receptor 2 (RyR2) ……….…….…..……….. 7

3.1.1.3. Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) ……….………. 9

3.1.1.4. Na+/Ca2+ exchanger (NCX) ……….……….…….….. 9

3.2. IMPAIRMENT OF EXCITATION CONTRACTION COUPLING IN HEART FAILURE ……... 10

3.2.1. INVOLVEMENT OF VOLTAGE-DEPENDENT Ca2+ CURRENT TYPE L (ICaL) IN HEART FAILURE ………..………..….…. 10

3.2.2. RYANODINE RECEPTOR 2 (RyR2) DYSFUNCTION IN HEART FAILURE ………..….. 10

3.2.3. SARCOPLASMIC RETICULUM Ca2+ ATPase 2a (SERCA2a) DYSFUNCTION IN HEART FAILURE ………....….. 12

3.2.4. Na+/Ca2+ EXCHANGER (NCX) MISHANDLING IN HEART FAILURE ……….. 13

4. ROLE OF β-ADRENERGIC SYSTEM IN HEART FAILURE ……… 13

4.1. β-ADRENERGIC SIGNALLING IN THE HEART……….…… 14

4.1.1. β-ADRENERGIC RESPONSE IMPAIRMENT IN HEART FAILURE ……….. 14

5. INNATE IMMUNE SYSTEM AND CARDIOVASCULAR DISEASES ……….. 16

5.1. PATTERN RECOGNITION RECEPTORS (PRRs) ……….. 16

5.2. NOD-LIKE RECEPTORS (NLRs) INVOLVEMENT IN CARDIOVASCULAR DISEASES ……. 17

6. NUCLEOTIDE-BINDING OLIGOMERIZATION DOMAIN TYPE 1 (NOD1) ………. 17

6.1. INVOLVEMENT OF NOD1 IN THE PATHOLOGY….……….……… 18

6.1.1. ROLE OF NOD1 IN VASCULAR DISEASE ……….……… 19

6.1.2. ROLE OF NOD1 IN ATHEROSCLEROSIS ……….………. 20

6.1.3. ROLE OF NOD1 IN DIABETIC CARDIOMYOPATHY ………. 20

6.1.4. ROLE OF NOD1 IN CARDIAC Ca2+ MISHANDLING ………..………… 21

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Role of NOD1 in human and experimental heart failure.

OBJECTIVES

... 23

RESULTS

……….…….. 27

1. ROLE OF NOD1 IN HEART FAILURE PREGRESSION VIA REGULATION OF Ca2+ HANDLING ……… 29

1.1. SUPPLEMENTAL MATERIAL ……..………. 43

2. DEFICIENCY OF NOD1 IMPROVES THE β-ADRENERGIC MODULATION OF Ca2+ HANDLING IN A MOUSE MODEL OF HEART FAILURE ……….…. 59

2.1. SUPPLEMENTAL MATERIAL ……..………..…….. 71

3. NOD1 ACTIVATION IN CARDIAC FIBROBLASTS INDUCED MYOCARDIAL FIBROSIS IN A MURINE MODEL OF TYPE 2 DIABETES ………..….…… 73

3.1. SUPPLEMENTAL MATERIAL ………..………. 87

DISCUSSION

………..……….. 91

1. ROLE OF NOD1 IN HEART FAILURE PREGRESSION VIA REGULATION OF Ca2+ HANDLING ………. 93

2. DEFICIENCY OF NOD1 IMPROVES THE β-ADRENERGIC MODULATION OF Ca2+ HANDLING IN A MOUSE MODEL OF HEART FAILURE ……….…….….. 98

3. NOD1 ACTIVATION IN CARDIAC FIBROBLASTS INDUCED MYOCARDIAL FIBROSIS IN A MURINE MODEL OF TYPE 2 DIABETES ……….………..……..…. 100

4. CLINICAL RELEVANCE ………..………. 101

CONCLUSIONS

………... 103

CONCLUSIONES

……….……..…. 107

BIBLIOGRAPHY

……….. 111

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KEY ABBREVIATIONS

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KEY ABBREVIATIONS

[Ca2+]i Intracellular Ca2+ concentration

[Ca2+]SR Free Ca2+ concentration in the sarcoplasmic reticulum AAV1 Adenovirus 1

AC Adenylyl cyclase AR Adrenergic receptor

BAY BAY-11-7082, (E)-3-(4-Methylphenylsulfonyl)-2-propenenitrile CaMKII Calmodulin kinase II

CARD Caspase recruitment domain CICR Calcium-induced calcium-release CLR C-type lectin receptor

COX2 Cyclooxygenase type 2 CVD Cardiovascular disease DAD Delayed afterdepolarization

DAMP Danger-associated molecular pattern

db Diabetic mice model with a mutation in one of the leptin receptor gene EC Excitation-contraction

FKBP12.6 FK506-binding protein 12.6 Gs protein Stimulating G protein Gi protein Inhibiting G protein HF Heart failure

HRC Histidine-rich calcium-binding protein I/R Ischaemia/reperfusion

ICaL Voltage-gated L-type calcium current iE-DAP D-glutamyl-meso-diaminopilemic acid

IKK IκB kinase

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IL1β Interleukin 1β IL6 Interleukin 6

INa/Ca Sodium/calcium exchange current IRF5 Interferon regulatory factor 5 IκBα Inhibitor κBα

JMK c-Jun N-terminal kinase LRR Leucine-rich repeat LTCC L-type calcium channel

MAPK Mitogen-activated protein kinase MI Myocardial infraction

NATCH Nucleotide-binding domain with ATPase activity NCX Sodium/calcium exchanger

NF-κB Nuclear factor κB NLR NOD-like receptor NLRA NOD-like receptor A NLRB NOD-like receptor B NLRC NOD-like receptor C

NLRP NATCH, LRR and PYD-domain protein containing protein NOD Nucleotide oligomerization domain

NOD1 Nucleotide-binding oligomerization domain type 1

Nod1-/- NOD1 knock-out gene

NOS2 Nitric oxide synthase type 2

p38 Protein 38

PAMP Pathogen-associated molecular pattern PKA Protein kinase A

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KEY ABBREVIATIONS

PKC Protein kinase C

PLN Phospholamban

PMI Post-myocardial infarction

PP Phosphatase

PRR Pattern recognition receptor

PYD Pyrin domain

RIG Retinoic acid-inducible gene

RIPK2 Receptor-interacting serine/threonine-protein kinase type 2 RLR RIG-like receptor

RyR Ryanodine receptor

S100A Ca2+-binding protein S100A

SERCA Sarcoplasmic reticulum calcium adenosine tri-phosphatase SR Sarcoplasmic reticulum

TAK1 TGFβ activation kinase TBK1 TANK-binding kinase 1 TGF-β Tumour growth factor-β TLR Toll-like receptor

TNFα Tumour necrosis factor-α T-tubule Transverse tubule

wt Wild-type

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1

INTRODUCTION

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3

INTRODUCTION

1. HEART FAILURE:

Nowadays, cardiovascular diseases (CVDs) are the leading cause of death in developed countries and constitute an expensive cost burden (1-3). One of the most prevalent CVD is heart failure (HF). HF is a complex syndrome that afflicts more than 15 million people in Europe (1-3).

HF occurs when the heart is unable to maintain cardiac output at normal filling pressures.

Its aetiology is diverse and it can be caused by ischaemia, stroke, mechanical stress, genetic diseases, diabetes or atherosclerosis among others (4). HF occurs in many cases as a consequence of a previous disease. The most common cause of HF in the western world is myocardial infarction (MI) (5). Furthermore, metabolic diseases that involve an impairment of glucose or lipids metabolism, such as diabetes or atherosclerosis, can also induce HF (6).

HF is commonly associated with mechanical stress inducing cardiac remodelling, neuro- hormonal activation, Ca2+ mishandling, structural changes, and increased inflammatory response among others (Figure 1).

2. STRUCTURAL REMODELLING IN HEART FAILURE

The extracellular matrix of the heart is the structural basis in which cardiac cells are supported and it is actively involved in the transduction of mechanical signals to cardiac

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cells. The extracellular matrix is mainly composed by fibroblasts and collagen. Fibroblasts are found throughout the cardiac tissue, surrounding myocytes and bridging spaces between cardiac tissue layers. Cardiac fibroblasts mediate mechanical stress via integrins, ion channels, and second messenger responses (7). Direct interactions between cardiomyocytes and fibroblasts are crucial for the electrical conduction into the heart (8).

Fibroblasts play numerous roles in shaping cardiac structure and function, as well as in cardiac development and remodelling.

It has been demonstrated that several CVDs with a pro-inflammatory background, are also associated with cardiac fibrosis. In this way, HF or diabetic cardiomyopathy are characterized by augmented myocardial fibrosis. After a cardiac injury, fibroblasts express increased levels of proinflammatory cytokines and chemokines, including interleukine-1β (IL-1β), interleukine-6 (IL-6), and tumour necrosis factor-α (TNF-α). These increased expressions lead to enhanced proliferation of cardiac fibroblasts and the transition to a myofibroblast phenotype that are involve in several CVDs (9, 10) (Figure 2).

Increased levels of extracellular matrix proteins, such as collagen or fibronectin, are associated with left ventricular stiffness and diastolic dysfunction in HF (11, 12). In fact, fibroblasts accumulation and excessive deposition of extracellular matrix proteins can cause cardiac dysfunction by impairing cell contractility (9). Importantly, patients with

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5

INTRODUCTION

diabetic cardiomyopathy harbour myocardial hypertrophy and cardiac fibrosis that cause left ventricle stiffness and reduced left ventricle ejection fraction (13).

Furthermore, fibrosis is able to disrupt the electrical coupling between fibroblasts and cardiomyocytes that is related to certain cardiac arrhythmias (9). In this regard, cardiac fibrosis is often related to re-entry arrhythmias that can occur around a fixed anatomic obstacle or in a tissue whose properties permit the maintenance of reentrant circuits.

Cardiac fibrosis constitutes an abnormal grid that modifies the conduction and the refractoriness of the cardiac tissue, promoting reentry arrhythmias (14).

3. CALCIUM MISHANDLING IN HEART FAILURE:

HF is associated with excitation-contraction (EC) coupling impairment that triggers cardiac dysfunction and cardiac arrhythmias. EC coupling is the process by which an electrical stimulus reach the cell and provokes Ca2+ influx that leads to cell contraction. In the heart, EC coupling is based in a process called Ca2+-induced Ca2+-release (CICR). CICR was first described in skinned skeletal muscle fibres in the 1970s (15, 16) and Fabiato and Fabiato characterized CICR in cardiac myocytes (17, 18) as detailed below.

3.1. EXCITATION-CONTRACTION COUPLING:

The second messenger Ca2+ is essential in cardiac electrical activity and is the direct activator of the myofilaments, thus inducing contraction (19). During the plateau phase of the cardiac action potential, an inward Ca2+ current (ICaL) enters the cell through L-type depolarization-activated Ca2+ channels (LTCC). These channels are localised mainly in the T-tubules. The Ca2+ influx through the LTCCs stimulates the ryanodine receptors (RyR2s) which are localized in the dyad regions of the sarcoplasmic reticulum (SR). RyR2s are SR Ca2+ channels that permit the extrusion of Ca2+ from the SR to the cytosol. Thus, the increase of the intracellular concentration of Ca2+ ([Ca2+]i) in the cytoplasm stimulates the troponin C of the myofilaments to produce cardiac contraction (Figure 3).

After cell contraction, Ca2+ must disassociate from the myofilaments and [Ca2+]i must return to diastolic levels, taking place cardiac relaxation. There are two principal mechanisms by which Ca2+ is removed from the cytosol: a) the Ca2+ released by RyR2 goes back to the SR through the SR Ca2+ ATPase 2a pump (SERCA2a); and b) the Ca2+ influx

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through LTCCs must be extruded by the Na+/Ca2+ exchanger (NCX). There are other minority mechanisms that contribute to Ca2+ removal such as the Ca2+-ATPase pump of the plasmatic membrane and the Ca2+ mitochondrial uniporter (19) (Figure 3).

3.1.1. KEY PARTNERS IN EXCITATION-CONTRACTION COUPLING:

3.1.1.1. L-Type Ca2+ channel (LTCC):

The L-type Ca2+ channels (LTCCs) are expressed mainly on the T-tubules of cardiomyocytes, and are activated by the depolarization of the sarcolemma (20, 21).

LTCCs have a main role during the plateau phase of the action potential by allowing Ca2+

influx for cardiac EC coupling (19). The inactivation of these ion channels is both Ca2+ and voltage dependent (20, 21). LTCCs are in close proximity to RyR2 channels forming a dyad.

The close proximity between LTCCs and RyR2 is crucial to develop an adequate Ca2+

induced Ca2+ release process.

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7

INTRODUCTION

3.1.1.2. Ryanodine Receptor 2 (RyR2):

The RyR2 is a large ionic channel with a molecular mass exceeding 2.2MDa. They form homotetramers of ≈5000 residue subunits (22) (Figure 4A). Current data of high- resolution optical microscopy show that cardiac RyR2 forms clusters within the Ca2+

release units (23-25). These RyR2 clusters are mainly located throughout the dyad in the SR membrane, but there are RyR2 outside the clusters, known as rogue RyR2s, whose presence has been confirmed in corbular SR regions non-associated to the T-tubule structure (26).

RyR2s have an overall shape of a mushroom, with a large cap located in the cytosol, and the stem containing the transmembrane area and the intraluminal portion (Figure 4C).

Small molecules or proteins can bind to the cytosolic cap, especially in the central rim.

These ligands interfere with channel opening, by showing differential affinity for the open or closed state of the cytosolic cap. RyR2 contains many targets for multiple kinases that are contained in a flexible loop between two tandem repeats in the cytoplasmic side of RyR2 (22, 27) (Figure 4B).

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RyR2s can be opened when [Ca2+]i reaches a certain threshold into the dyad or when the SR-free Ca2+ ([Ca2+]SR) is over the physiological store levels (28). RyR2 regulation depends on several mechanisms including: a) direct free Ca2+ interactions, both cytosolic and luminal; b) accessory cytoplasmic regulatory proteins, such as, FKBP12.6; and c) SR luminal proteins, for instance, calsequestrin, triadin, and junctin (29).

The RyR2 also forms a complex with two major protein kinases (PKA and CaMKII) and three phosphatases (PP1, PP2, and PP2B) indicating the importance of RyR2 phosphorylation (30). In turn, all these phosphatases are modulated by the action of phosphodiesterases, between them the phosphodiesterase 2, 3, and 4 are the most abundant in the heart and it is well established that they have different roles in the cardiac EC coupling regulation (31, 32).

So far, three functionally relevant RyR2 phosphorylation sites have been identified in mice, including Ser-2808 which can be phosphorylated by PKA and CaMKII, Ser-2030 that is a specific PKA phosphorylation site, and Ser-2814 which is CaMKII phosphorylated (33- 36). Physiological phosphorylation of RyR2 increases the opening of the channel and this has been suggested to contribute to the positive inotropic effects of β-adrenergic stimulation.

Between the processes involved in the termination of Ca2+ released via RyR2 there has been postulated: 1) Ca2+-dependent inactivation/adaptation of RyR2, that involves both cytoplasmic and luminal-SR Ca2+ and proteins whose activity over RyR2 is Ca2+ dependent;

2) the spontaneous decay of RyR2 activity due to stochastic attrition; and 3) the depletion of SR-Ca2+ stores that induces the RyR2 inactivation (37). A suitable inactivation of RyR2s can minimize the inappropriate SR Ca2+ release events between heartbeats (19).

During the refractory period, small SR Ca2+ release can promote a decrease in the luminal Ca2+ levels. There are different forms of diastolic Ca2+ release: invisible Ca2+ leak form by Ca2+ quarks, Ca2+ sparks, Ca2+ waves, or spontaneous Ca2+ transients (38). Ca2+ sparks have a physiological role maintaining the SR-Ca2+ stores balanced between systole and diastole.

But in some CVDs, where Ca2+ handling proteins regulation are misbalanced, diastolic Ca2+

leak can be enhanced promoting cardiac dysfunction and arrhythmias (39).

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INTRODUCTION

3.1.1.3. Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a):

SERCA2a is predominantly expressed in the heart. SERCA2a is distributed close to the T- tubules and longitudinally and transversely in the SR throughout the cardiac myocytes (40).

To transfer Ca2+ ions into the SR, SERCA2a proteins use two specialized domains: E1 and E2. E1 has a high affinity for Ca2+ and its binding site is exposed towards the sarcoplasm, while E2 has a lower affinity for Ca2+ and is hanging towards the lumen of the SR. Once the cardiomyocytes relax, Ca2+ ions are released from troponin C and bind to the binding site of E1, preceded by the binding of ATP (41).

SERCA2a functions as a Ca2+ pump into the SR lumen to restore the steady state of the [Ca2+]SR and so, cardiac relaxation occurs. Although SERCA2a interacts with a wide array of proteins (including HRC, PP1; calreticulin, S100A, and sarcolipin), phospholamban (PLN) is the most important regulator of the pump activity (42). At the unphosphorylated state, PLN inhibits SERCA2a activity by lowering the affinity of the pump for Ca2+. Phosphorylation of PLN at Ser-16 by PKA relieves this inhibition, increasing the pumping rate. SERCA2a activity can also be increased via PLN phosphorylation by CaMKII at Thr-17.

This post-transcriptional modification has been suggested to be responsible of the changes in the SR Ca2+ uptake. In addition to the PLN regulation, SERCA2a is highly sensitive to changes in the metabolic status of the cytosol, including the ATP/ADP ratio, pH and the redox potential (43, 44).

3.1.1.4. Na+/Ca2+ exchanger (NCX):

The NCX is the main route for Ca2+ extrusion from the cardiomyocyte. NCX plays an important role restoring cytosolic Ca2+ levels, and thus the extent of cardiomyocyte relaxation.

The NCX exchanges one molecule of Ca2+ for three molecules of Na+ (INa/Ca) (45) and, although NCX predominantly extrudes Ca2+, it may also work in reverse mode resulting in Ca2+ influx. The driving force which determines NCX direction and function is the electrochemical gradient, namely membrane potential and transmembrane gradients of Ca2+ and Na+ (46).

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A mechanism of NCX regulation is the small inhibitory protein phospholemman. PKA and PKC phosphorylation modulate its inhibitory effect increasing NCX inhibition (47). During PKA and PKC phosphorylation, phospholemman therefore increases contractility, by inhibiting NCX in the forward form and increasing [Ca2+]i.

3.2. IMPAIRMENT OF EXCITATION-CONTRACTION COUPLING IN HEART FAILURE:

Heart failure (HF) is characterized by depressed contractility of cardiomyocytes that contribute to reduced left ventricular contraction during systole. The diminished cardiac contractility in HF is associated with an impairment in the EC-coupling. Disruptions in EC coupling are well documented in almost all types of HF (48). The most common changes of EC coupling associated with HF are: a) reduced systolic SR-Ca2+ release through RyR2, b) decreased reuptake of Ca2+ into SR, c) increased Ca2+ extrusion through NCX, and d) increased diastolic SR Ca2+ leak. All of these alterations contribute to reduce SR Ca2+ load, limiting the amount the SR Ca2+ needed to produce regular myocyte contractions (Figure 5).

3.2.1. INVOLVEMENT OF VOLTAGE-DEPENDENT Ca2+ CURRENT TYPE L (ICaL) IN HEART FAILURE:

The potential role of ICaL in HF is controversial. A number of studies related to HF have found relatively unaltered ICa density in ventricular myocyte (20, 49, 50). However, some have suggested that there may be some reduction in the number of Ca2+ channels, but with higher activity per channel (49, 51). On the other hand, other studies have reported that the ICaL amplitude is increased in failing myocytes while their density remains similar to healthy myocytes (52). These discrepancies might be due to differences in the experimental approaches or in the animal models.

3.2.2. RYANODINE RECEPTOR 2 (RyR2) DYSFUNCTION IN HEART FAILURE:

During HF the activity of RyR2 is significantly increased. RyR2 hyperactivity can induce an increase in the diastolic Ca2+ leak, which implies spontaneous openings of RyR2s in the refractory period diminishing the SR-Ca2+ load (Figure 5).

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Several studies have reported increased Ca2+ leak during diastole in experimental and human failing hearts (43, 46, 53). Diastolic Ca2+ leak is linked to RyR2s hyperactivity which can be related to several factors, such as increased modulatory proteins activity both luminal and cytosolic, higher Ca2+ sensitivity, or elevated posttranscriptional modifications such as phosphorylation or oxidation. RyR2 hyper-phosphorylation has been described to increase the open probability of the channel (54-56). Specific sites in the RyR2 are hyper- phosphorylated by PKA (S2808 and 2830, in mice) and by CaMKII (S2808 and S2814, in mice) during HF. There is general agreement that the CaMKII phosphorylation of RyR2 activates the RyR2 channel to open favouring Ca2+ leak and delayed-afterdepolarizations (DADs) (57-68), although some authors describe PKA phosphorylation of RyR2 as the main mechanism of diastolic Ca2+ leak (69-74). These differences need to be clarify with new approaches given that PKA and CaMKII play important roles in the regulation of EC coupling in the heart.

Despite the fact that RyR2 normally shuts off almost completely during diastole, there is still a finite level of SR Ca2+ leak. The most-known examples of diastole localized

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spontaneous SR Ca2+ release events are Ca2+ sparks recorded by confocal microscopy (75).

Ca2+ spark frequency relates to the finite open probability of individual cardiac RyR2 clusters. During Ca2+ sparks, the amount of local Ca2+ released is high because multiple RyR2 channels within a junction are activated by CICR.

Nevertheless, there are conflicting results regarding the occurrence of these events in HF (68). Ruiz-Hurtado et al. showed in a mice model of HF that Ca2+ sparks were less frequent in the failing cardiomyocytes, although more Ca2+ waves were observed (39). Heinzel et al. demonstrated that in HF there is a subcellular dys-synchrony related to the structural and functional detrimental remodeling of the cardiomyocyte that implies inefficient coupled released, triggering Ca2+ sparks and waves (76). This controversy could be due to different HF models, experimental protocols and approaches, or the level of cardiac remodelling.

Ca2+ leak is mediated by several clusters that can propagate to adjacent RyR2 clusters promoting Ca2+ waves or spontaneous Ca2+ transients (37, 77). In addition, the higher SR Ca2+ leak on HF may increase the likelihood of triggered arrhythmogenic events propagating as Ca2+ waves, which activates a transient inward current that causes a DAD triggering arrthythmias (39, 68, 78, 79).

3.2.3. SARCOPLASMIC RETICULUM Ca2+ ATPase 2a (SERCA2a) DYSFUNCTION IN HEART FAILURE.

In human HF, SR-Ca2+ load is reduced, in part because the expression of SERCA2a is decreased, compromising the Ca2+ reuptake into the SR. SERCA2a activity is modulated by PLN who, in turn, is regulated by PKA and CaMKII phosphorylation. As SERCA2a is unable to reuptake all the Ca2+ from relaxation, NCX expression levels are increased as a compensatory mechanism to extrude the intracellular Ca2+ excess in order to maintain [Ca2+]i.

Both diminished SERCA2a function and augmented NCX activity tend to reduce Ca2+

content in the SR, limiting SR Ca2+ release through RyR2, decreasing the systolic Ca2+

release and impairing cardiomyocyte contractility in HF (19). SERCA2a gene therapy is currently under evaluation in clinical trials for new HF treatments (80-84). Some authors have described that the introduction of SERCA2a into isolated cardiomyocytes from HF

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INTRODUCTION

patients and in experimental models results in the improvement of myocardial contractility (85, 86).

However, there are discrepancies about the beneficial role of targeting SERCA2a at the clinical practice. For instance, the clinical trial CUPID2, which targeted by gene transfer AAV1/SERCA2a, failed to improve clinical outcomes of patients with HF (87). An explanation could be that the efficiency of gene transduction in CUPID2 might have been compromised by the lower number of empty capsids. New study perspectives increasing the drug dose are required. It would be also important to characterise serum effects from the target patient population, given that the biological potency of drugs used for gene transfer could have different potential effect on each patient (87).

3.2.4. Na+/Ca2+ EXCHANGER (NCX) MISHANDLING IN HEART FAILURE:

As previously mentioned, NCX constitutes an essential mechanism involved in controlling [Ca2+]i content (88). NCX upregulation is a common feature of both human and animal failing hearts. As such changes often occur simultaneously with SERCA2a downregulation, a marked increase in NCX/SERCA2a ratio is commonly reported, and it has been implicated in both cardiac dysfunction and arrhythmogenesis (89). Chronic up-regulation of NCX results in maladaptive cardiac remodeling since NCX does not restore SR Ca2+ stores.

As NCX is an electrogenic ion channel, and under pathological conditions, the more Ca2+

is extruded from the cardiomyocyte, the more Na+ enters. This Na+ influx can depolarize the cardiomyocyte membrane generating new action potentials which lead to pro- arrhythmogenic events (78, 90).

Increased diastolic SR Ca2+ release is related to augmented NCX activity in HF, and NCX activity will cause a greater Na+ inward transient current which will produce larger depolarizations and thus promoting DADs.

4. ROLE OF β-ADRENERGIC SYSTEM IN HEART FAILURE:

Heart rate and contractility are regulated by the autonomic nervous system, hormones and other factors. The autonomic nervous system englobes the sympathetic and the parasympathetic system.

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The sympathetic nervous system has two types of adrenergic receptors (AR): α and β. In the cardiovascular system there are β1, β2, β3, α1, and α2 adrenergic receptors. β1 adrenergic receptors are expressed ubiquitously in the heart and their activation increases heart rate and contractility. β2 adrenergic receptors are expressed in cardiac and vascular muscle, and in the coronary circulation and their activation leads to vasodilatation. α1 and α2 adrenergic receptors are expressed in vascular smooth muscle and their activation elicits vasoconstriction (91). α1 and β3 play a minor role in the heart (92).

4.1. β-ADRENERGIC SIGNALLING IN THE HEART:

Once a specific β-AR is activated, cellular changes are initiated by the activation of one or more heterotrimeric G proteins. Depending on the β-AR, the G signalling could be stimulating (Gs proteins), or inhibiting (Gi proteins). Gs proteins activate adenylyl cyclase (AC) that reacts with adenosine-triphosphate (ATP) to form cyclic adenosine- monophosphate (cAMP), which modulates the activity of several essential proteins for cardiac function. cAMP prompts the activation of protein kinase A (PKA) which has a key function in the regulation of important ion channels involved in Ca2+ dynamics, including LTCCs, RyRs, PLN, troponin I, and myosin-binding proteins. cAMP could also bind directly to some ion channels increasing the heart rate. Gi proteins decreases cAMP levels, activates mitogen-activated protein kinases, and helps to regulate the receptor signalling (93) (Figure 6).

4.1.1. β-ADRENERGIC RESPONSE IMPAIRMENT IN HEART FAILURE:

It is well-known that alterations in β-AR responses play a key role in HF progression. During progressive HF, biochemical defects arise in the β-AR signalling pathway. In patients who develop HF, β1-ARs are downregulated in myocardial membranes and the functional coupling of the remaining receptors to the Gs-AC system is decreased. In addition, levels of Gi signalling proteins are increased. Taken together, these changes serve to desensitize the β-adrenergic pathway and result in cardiac dysfunction.

In short term, β-AR activation contributes to maintain the physiologic Ca2+ dynamics in failing hearts. However, chronic β-AR activation during HF is maladaptive and results in Ca2+ handling dysregulation that together with cellular effects, promote the progression

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INTRODUCTION

to myocardial failure (93-96). Whereas physiological sympathetic stimulation of the heart through β-ARs increases CICR and SR Ca2+ uptake, chronic activation of β-AR stimulation in HF has deleterious consequences in long term in cardiac Ca2+ handling. As β-ARs signalling pathway activates PKA and CaMKII, they phosphorylate key cardiac Ca2+

handling proteins that alter their function and disrupts cardiac contractility, promoting arrhythmogenic events and cardiac dysfunction (97) (Figure 6).

A common clinical practice is to administer β-AR blockers as they are proved to improve the myocardial molecular phenotype and reduce mortality (98-100). In fact, β-blockers therapy is recommended in the American and European guidelines for patients with ischaemic heart disease and concomitant left ventricular dysfunction (101, 102).

Nevertheless, whereas short-term clinical trials, such as the METOCARD-CNIC, EARLY- BAMI and BEAT-AMI trials, have reported reduced mortality in patients, additional large- scale clinical trials are needed to evaluate the role of β-blockers therapy in long-term HF patients (103, 104).

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5. INNATE IMMUNE SYSTEM AND CARDIOVASCULAR DISEASES:

Classically, it was described that the innate immune system discriminated between self and non-self, but in 2002 Matzinger et al. postulated that the innate immune system is able to react to “danger signals” rather than self or non-self. Thus, there are two types of activating signals, the exogenous “danger signals” called pathogen-associated molecular patterns (PAMPs) and the endogenous “danger signals” referred as danger-associated molecular patterns (DAMPs). PAMPs are highly conserved motifs in microbial pathogens, such as lipopolysaccharide and peptidoglycan of bacteria. DAMPs are proteins, cytokines, chemokines, and other molecules precedent from over-stressed and injured cells (105).

Both PAMPs and DAMPs can activate the innate immune system through pattern recognition receptors (PRRs) and trigger innate and adaptive immune responses.

5.1. PATTERN RECOGNITION RECEPTORS (PRRs):

PAMPs/DAMPs in turn are recognized by pattern recognition receptors (PRR), which may be membrane bound or cytoplasmic. The binding of a PAMP/DAMP to a PRR, in response to danger signals, leads to activation of different transcription factors through complex signalling pathways, producing substances to response against cell damage (106). There are currently four known families of PRRs: the transmembrane protein families of toll-like receptors (TLR) and C-type lectin receptors (CLR), and the cytoplasmic protein families such as nucleotide oligomerization domain (NOD)-like receptors (NLR), and retinoic acid- inducible gene (RIG)-like receptors (RLR) (107). Besides, cells of the innate immune system, PRR are also present in “non-professional” immune cells including cardiomyocytes and fibroblasts.

NLRs are cytosolic sensors of intracellular DAMPs and PAMPs that include four subfamilies, NLRA, NLRB/NAIP, NLRC, and NLRP. The activated NLRC subgroup members NOD1/2 induce TAK1-IKK-NFκB, TBK1-IRF5 or JMK and p38 signalling events (108).

The human NLR family has 22 members, with most sharing a conserved tripartite structure consisting of an N-terminal caspase recruitment domain (CARD) or pyrin domain (PYD), a central nucleotide-binding domain with NTPase activity (NACHT), and a C-terminal leucine-rich repeat (LRR) domain which mediates intracellular ligand sensing (109).

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INTRODUCTION

NLRs regulate a wide range of molecular functions including NFκB signalling, RLR signalling, autophagy, major histocompatibility complex gene regulation, reproduction and development (110).

5.2. NOD-LIKE RECEPTORS (NLRs) INVOLVEMENT IN CARDIOVASCULAR DISEASES:

The short-term innate immune responses are adaptive and protective for the impaired heart, whereas a long-lasting and exaggerated activation likely counteracts the beneficial effects and contributes to maladaptive tissue damage (111, 112). Thus, chronic activation of proinflammatory cytokines and ongoing inflammation associated with HF development hamper to restore the myocardium homeostasis (113).

In CVDs, NLRs have been demonstrated to be involved in the context of myocardial injury.

The formation of NLRP3 inflammasome was found to be induced by myocardial ischemia, and play an important role in adverse cardiac remodelling in a model of severe ischemic damage (114). In fact, blocking the NLRP3 inflammasome limits infarct size after myocardial I/R injury in an experimental mice model (115, 116). In this line, other NLR, nucleotide-binding oligomerization domain 1 (NOD1), was demonstrated to induce cardiac dysfunction concomitantly with cardiac fibrosis and apoptosis in the mice hearts (117).

6. NUCLEOTIDE-BINDING OLIGOMERIZATION DOMAIN TYPE 1 (NOD1):

Nod1 encodes an intracellular scaffolding protein consisting of a caspase activation and recruitment domain (CARD), a nucleotide-binding oligomerization domain (NOD), and multiple leucine-rich repeats (LRRs). NOD1 resides in an inhibited monomeric state in the cytosol, and upon ligand recognition, it undergoes conformational changes that promote its activation. Once activated, the protein self-oligomerizes and recruits receptor- interacting serine/threonine-protein kinase 2 (RIPK2) through homotypic CARD-CARD interactions (118). RIPK2 then mediates the recruitment and activation of the serine/threonine kinase TAK1 which, in turn, activates the IKK complex and MAPK

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pathway. IKK then phosphorylates IκBα that allows the translocation of NFκB to the nucleus and modulates the expression of downstream target genes (119) (Figure 7).

NOD1 detects D-glutamyl-meso-diaminopilemic acid (iE-DAP), which is a dipeptide that is present in a peptidoglycan that is primarily found in Gram-negative bacteria but also in certain groups of Gram-positive bacteria. However, NOD1 signal transduction can also be stimulated in the absence of direct cellular infection by a bacterial pathogen. Keestra- Gounder et al. demonstrated that pro-inflammatory responses induced by endoplasmic reticulum stress are mediated through NOD1/NOD2 pathway (120), suggesting a potential role of NOD1 in inflammatory diseases associated with endoplasmic reticulum stress.

6.1. INVOLVEMENT OF NOD1 IN THE PATHOLOGY:

NOD1 has been confirmed to be a key player in the development of gut pathologies such as Chron’s and Blau diseases. In addition to this, NOD1 sense bacterial pathogens, but also play a role in viral and parasitic infections, and sense endoplasmic reticulum stress. This finding correlates with the broad implication of endoplasmic reticulum stress with inflammation in several inflammatory pathologies, including diabetes or HF (121, 122).

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INTRODUCTION

Whereas NOD1 is involved in many human pathologies including diabetes or cancer, its role and relevance in the cardiovascular system are less understood (123-125).

NOD1 has been proved to play a key role in vascular collapse and septic shock since Moreno et al. demonstrated that vascular smooth muscle cells express active NOD1 receptors that trigger inflammatory responses upon agonist activation (126, 127).

Fernandez-Velasco et al. first confirmed that NOD1 receptors are expressed in both cardiomyocytes and cardiac fibroblasts. Activation of NOD1 receptors induce apoptotic pathways in isolated adult murine cardiomyocytes and promote activation of pro-fibrotic mediators (such as TGF-β pathway) in cardiac fibroblasts. Furthermore, administration of the specific agonist C12-iEDAP in mice induced cardiac dysfunction characterized by decreased ejection fraction, increased cardiac fibrosis and enhanced cardiomyocyte apoptosis (117).

Recently, a study has proved that activation of NOD1 aggravates cardiac damage and inflammation in I/R mice model. Specifically, Yang et al. demonstrated that activation of NOD1 with diaminopimelic acid (DAP) (a synthetic activator of NOD1) significantly aggravates cardiac I/R injury, enhanced cardiomyocyte apoptosis and inflammation (128).

6.1.1. ROLE OF NOD1 IN VASCULAR DISEASE:

Given the fact that the endothelium represents the first barrier against blood borne bacteria PAMPs, NOD1 represents a critical receptor in sensing pathogens. Nishio et al.

documented that chronic administration of selective NOD1 ligands in mice induced coronary arteritis, vascular inflammation and valvulitis similar to the one seen in the acute phase of Kawasaki disease (129). Indeed, CD11c+ macrophages were identified in acute coronary arteritis induced by NOD1 administration (130). These studies establish a clear pathogenic link between NOD1 activation and vascular inflammation.

In this regard, Moreno et al. revealed that NOD1 is selectively expressed in vascular smooth muscle and the activation of NOD1 leads to increased expression of NOS2 and COX2 (126). Moreover, specific NOD1 ligands were able to activate inflammatory responses on intact human vessels, establishing the relevance of the endothelium to NOD1 activation (127). Hence, NOD1 stimulation induces a significant number of pro- inflammatory genes in endothelial cells (127), pointing to the NOD1 signalling pathway as a potential vascular target in the development of new therapies.

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6.1.2. ROLE OF NOD1 IN ATHEROSCLEROSIS:

Atherosclerosis is a chronic inflammatory process where immune cells are key mediators from the earliest fatty streaks to late-stage complex plaques. A number of recent reports support the notion that innate immune receptors, NLRs to be precise, might be involved in this inflammatory process (131, 132). Kanno et al. demonstrated for the first time that long-term oral administration of a synthetic NOD1 ligand accelerated the development and progression of atherosclerosis in Apoe-/- mice. Additionally, the complete loss of this receptor significantly decreased the size of atherosclerotic lesions, providing evidence of a relationship between NOD1 and atherosclerosis progression (133).

6.1.3. ROLE OF NOD1 IN DIABETIC CARDIOMYOPATHY:

Metabolic problems are linked to chronic inflammation. Schertzer et al. demonstrated that the ablation of NOD1 attenuates high fat diet-induced insulin resistance and hepatic lipid accumulation with a reduced inflammation in both adipose and liver tissue, positioning NOD1 as a link between the immune and metabolic disorders (123).

Diabetic patients often suffer cardiovascular complications, such as increased susceptibility to ventricular arrhythmias, cardiac dysfunction, and sudden cardiac death.

Diabetes is associated with a sterile inflammation, driving to TLR2 and NLRP3 activation in cardiac macrophages (134, 135). The main mechanism underlying cardiac arrhythmias in diabetic individuals is still unknown, but several studies have proved that sterile inflammation triggered by hyperglycaemia can be a possible pathophysiological mechanism. In fact, several groups have associated IL-1β with electrical changes in the heart, such as longer action potentials and long QT syndrome (136-139).

In this line, Prieto et al. demonstrated that NOD1 is up-regulated in cardiac tissue from a diabetes mellitus type 2 animal model and from diabetic patients, contributing to chronic grade inflammation in the heart (125). Cardiomyocytes isolated from diabetic mice showed increased activation of NOD1 together with a greater activation of pro- inflammatory mediators. In fact, synthetic NOD1 activation increased the number of apoptotic cells in heart tissue compared to vehicle-treated animals (125). Hence, NOD1 activation is shown as a new partner of cardiac damage in diabetic cardiomyopathy.

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INTRODUCTION

6.1.4. ROLE OF NOD1 IN CARDIAC Ca2+ MISHANDLING:

Cardiac dysfunction is a hallmark of several cardiovascular diseases, including HF and diabetic cardiomyopathy. Detrimental changes in HF are associated with cardiac dysfunction, being Ca2+ mishandling a major player.

Inflammation is now recognized as a main contributor to different manifestations of cardiovascular diseases. NOD1 is a key factor that has been associated with some diseases with a detrimental cardiovascular outcome.

In this regard, Delgado et al. demonstrated that NOD1 activation promoted a significant decrease in cardiac function mice. Isolated cardiomyocytes from iE-DAP-treated mice showed a diminished ICaL density, depressed Ca2+ transients and a slower time decay of [Ca2+]i transients, which correlated with the reduced expression of SERCA. Furthermore, NOD1-deficient mice treated with iE-DAP, failed to modify the cardiac EC coupling parameters (140). Indeed, systolic Ca2+ mishandling induced by NOD1 activation was accompanied of diastolic release events such as increasing Ca2+ sparks frequency. This changes in the intracellular Ca2+ dynamics are common hallmarks of several cardiovascular diseases, such as HF.

Considering these studies previously mentioned, NOD1 emerges as an important regulator of the Ca2+ handling in the cardiac EC coupling, and provide a new research line for Ca2+ handling impairment in specific CVDs, such as HF.

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OBJECTIVES

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OBJECTIVES

Based in the background previously explained in the Introduction, we hypothesised that NOD1 can play a role in the detrimental cardiac remodelling, heart dysfunction, and Ca2+

mishandling associated with HF progression. Thus, the proposed objectives for the present Doctoral Thesis were the following:

1. To analyse the expression of NOD1 and its signalling pathway in myocardium obtained from individuals with HF.

2. To determine the expression of NOD1 and its signalling pathway in hearts from a mice model with HF.

3. To evaluate whether the absence of NOD1 or its pharmacological blockade determine cardiac dysfunction, structural remodelling, and Ca2+ mishandling in an experimental mice model of HF.

4. To determine whether the genetic deletion of NOD1 modulates ventricular arrhythmias and pro-arrhythmogenic Ca2+ events in an experimental mice model of HF.

5. To study whether the absence of NOD1 modulates the β-adrenergic regulation of Ca2+ handling in a mice model of HF.

6. To study whether NOD1 activation plays a role in the development of cardiac fibrosis in an experimental mice model of type 2 diabetes.

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RESULTS

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RESULTS

1. ROLE OF NOD1 IN HEART FAILURE PROGRESSION VIA REGULATION OF Ca

2+

HANDLING.

Recent studies point that the inflammatory response could be a key partner in heart failure (HF). Specifically, some innate immune system mediators can play a major role in this cardiovascular disease (CVD). NOD1 (nucleotide-binding oligomerization domain type 1) is a subtype of innate immune receptor that it has been recently related to certain CVDs. Some studies have demonstrated that the selective activation of NOD1 induces cardiac dysfunction and impairs cellular Ca2+ handling, common alterations in HF. In the present study we analyse the role that NOD1 plays in human failing hearts and in an experimental mice model of HF.

First, we studied NOD1 expression in human failing myocardium and cardiac tissue from mice with HF induced by myocardial infarction. The NOD1 pathway was up-regulated in human and murine failing myocardium. Next, we determined whether the absence of NOD1 in a Nod1-/- mice (Nod1-/--PMI) impairs cardiac damage linked to HF. Compared to wt-PMI, hearts from Nod1-/--PMI mice showed better cardiac function and attenuated structural remodelling. Then, we performed cardiomyocyte isolation of the different experimental groups to study the intracellular Ca2+ movements. wt-PMI cardiomyocytes showed typical HF characteristics, such as smaller and longer [Ca2+]i transients, low SR Ca2+

load induced by a down-regulation of the SR Ca2+-ATPase expression, and augmented expression of the Na+/Ca2+ exchanger. Ameliorated cardiac function in Nod1-/--PMI mice was associated with a prevention of Ca2+ dynamics impairment linked to HF. Regarding these systolic alterations we studied whether the diastolic Ca2+ leak could explain this Ca2+

mishandling. Increased diastolic Ca2+ release in wt-PMI cardiomyocytes was related to over-phosphorylation of ryanodine receptor 2 (RyR2). This aberrant diastolic Ca2+ release was prevented in Nod1-/--PMI cardiomyocytes together with a normalization of the phosphorylation of RyR2.

Then, electrocardiography assessment was performed under β-adrenergic stimulation.

Compared with wt-PMI mice, Nod1-/--PMI mice showed significantly lower ventricular arrhythmia occurrence and mortality induced by the administration of a β-adrenergic agonist, isoproterenol.

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Finally, intracellular Ca2+ handling and the RyR2 phosphorylation were analysed in cardiomyocytes perfused with isoproterenol. The improvement of Ca2+ handling under β- adrenergic stimulation in Nod1-/--PMI cells were associated with lower aberrant systolic Ca2+ release and with a prevention of the over-phosphorylation of RyR2. Lately, wt-PMI mice were treated with a pharmacological NOD1 inhibitor (Nodinitib-1, 5 µmol/L) three times weekly for 6 weeks. Pharmacological blockage of NOD1 also prevented Ca2+

mishandling associated with HF.

In conclusion, this study points to NOD1 as a new regulator of the Ca2+ release in HF, and it could provide the basis for new therapeutic strategies development in cardiovascular diseases, such as HF.

In the present study I performed, the isolation of cardiomyocytes, and functional (electrocardiography), biochemical (Western Blot), and imaging (confocal microscopy) experiments. I also analysed the data from biochemical analysis and Ca2+ handling experiments.

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RESULTS

2. DEFICIENCY OF NOD1 IMPROVES β-ADRENERGIC MODULATION OF Ca

2+

HANDLING IN A MOUSE MODEL OF HEART FAILURE

Nucleotide-binding oligomerization domain 1 (NOD1) is functional in the heart, both in cardiac fibroblasts and cardiomyocytes. In previous studies we have shown that NOD1 is up-regulated in failing human and mice cardiac tissue. Genetic deletion of NOD1 or its pharmacological blockade prevents cardiac dysfunction and deleterious remodelling in failing hearts, largely by preventing heart failure (HF)-induced Ca2+ mishandling.

Chronic HF is characterised by sustained β-adrenergic stimulation which is associated with an increased risk of triggered arrhythmias and cardiac dysfunction. Desensitization of β1- adrenergic receptor pathway in HF leads to intracellular Ca2+ cycling and contractility impairment. In this regard we studied whether NOD1 modulates the β-adrenergic regulation of cardiac Ca2+ handling in a mice model of HF.

We isolated cardiomyocytes from wild type (wt) and knock-out NOD1 (Nod1-/-) healthy mice and from post-myocardial infarction wt (wt-PMI) and Nod1-/- (Nod1-/--PMI) mice and we examined Ca2+ dynamics before and after isoproterenol (10-8M) perfusion in cardiomyocytes isolated from all groups. Using confocal microscopy, we assessed whether the depressed systolic Ca2+ release, reduced sarcoplasmic reticulum (SR)-Ca2+ load and increased SR-Ca2+ leak linked to HF progression is modulated by β-adrenergic stimulation in failing wt-PMI and Nod1-/--PMI mice. Isoproterenol administration promoted an increase in the amplitude of [Ca2+]i transients and SR-Ca2+load in both groups; however, the increase was limited in wt-PMI and marked in Nod1-/--PMI myocytes. Under β- adrenergic stimulation, Nod1-/--PMI myocytes showed an improvement in cell contractility compared with wt-PMI myocytes. mRNA levels of β1 and β2 adrenergic receptors were significantly higher in Nod1-/--PMI hearts vs wt-PMI hearts.

Additionally, the diastolic Ca2+ leak was evaluated and an increase in Ca2+ waves occurrence induced by isoproterenol were found in wt-PMI cardiomyocytes compared to the Nod1-/--PMI cells. This data suggests that the absence of NOD1 prevented the diastolic Ca2+ release induced by β-adrenergic stimulation.

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By contrast, healthy wt cardiomyocytes pre-treated with a selective agonist of NOD1, C12- iE-DAP (20 µmol/mL), and perfused with isoproterenol showed diminished [Ca2+]i

transients amplitude, cell contraction, and SR-Ca2+ load compared with vehicle-treated cells. C12-iE-DAP-treated cells also presented increased diastolic Ca2+ leak under β- adrenergic stimulation. The selectivity of C12-iE-DAP on Ca2+ handling was validated by pre-treatment with the inactive analogue of NOD1, iE-Lys.

Briefly, NOD1 deficiency improves β-adrenergic regulation of Ca2+ handling in a HF mice model by recovering systolic Ca2+ release, cell contractility, and by preventing the increase in diastolic Ca2+ release.

In the present study I developed intracellular Ca2+ recordings, cardiomyocytes isolation and PCR experiments. In addition, I also analysed the Ca2+ recording data and write a part of the scientific publication.

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