Empagliflozine improves myocardial fibrosis and diastolic function in a non-diabetic porcine model of ischaemic heart failure

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Empagliflozine improves myocardial fibrosis and diastolic function in a non-diabetic porcine model

of ischaemic heart failure.

by Alvaro Garcia Ropero, MD

A thesis submitted to The Department of Medicine, Faculty of Medicine – Universidad Autonoma de Madrid,

for the degree of Doctor of Philosophy, 2020.

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Dr Juan Badimon, Director of the Atherothrombosis Research Unit at Mount Sinai Hospital in New York City, United States, and Dr Jose Tuñon, Chief-physician of the Cardiology Unit at Fundacion Jimenez Diaz Hospital in Madrid, Spain, hereby certify that Dr Alvaro Garcia Ropero, graduated by the Universidad Complutense de Madrid, has conducted the thesis entitled ‘Empagliflozine improves myocardial fibrosis and diastolic function in a non-diabetic porcine model of ischaemic heart failure’ under their supervision.

According to them, this research project is suitable to be presented as a thesis work and it meets the requirements to obtain the degree of Doctor of Philosophy by the

Universidad Autonoma de Madrid.

Yours sincerely,

Dr Juan Badimon Maestro DNI: 38054248-N

Dr Jose Tunon Fernandez DNI: 09355481-R

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Figures, tables and appendices

Figure 1. Representative tracing (A) and curves (B) of right and left ventricular stroke work for each level of atrial pressure ... 21 Figure 2. Left ventricular pressure-volume relationships of rats according to infarct size

... 42 Figure 3. Tracing of intracardiac pressures during cardiac cycle and mitral inflow

correlation ... 53 Figure 4. Invasive analysis of diastolic function ... 59 Figure 5. Calculation of Tau (isovolumetric fall) and dP/dt (isovolumetric rise) ... 63 Figure 6. Conductance (or impedance) catheter (7F) with pigtail for use in human,

schematically showed positioned along the left ventricle axis ... 66 Figure 7. Influence of external forces on PV curve ... 68 Figure 8. Doppler criteria for classification of diastolic function ... 78 Figure 9. Algorithm for diagnosis of left ventricular diastolic dysfunction and

estimation of filling pressures ... 81 Figure 10. Left ventricular filling curves assessed by CMR ... 83 Figure 11. Comparison of renal-glucose filtration and reabsorption process in a non-

diabetic individual (A) and in a diabetic subject (B) ... 85 Figure 12. The anti-inflammatory effects of SGLT-receptor inhibition ... 106 Figure 13. Systemic effects of SGLT2 inhibitors ... 110 Figure 14. Working hypothesis with empagliflozin on a porcine model of heart failure

... 114 Figure 15. Overview of study design ... 130

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Figure 16. Cardiomyocyte perimeter (in µm) ... 134

Figure 17. Cardiomyocyte size ... 134

Figure 18. Myocardial interstitial fibrosis ... 136

Figure 19. Myocardial interstitial fibrosis ... 136

Figure 20. Myocardial extracellular volume assessed by CMR using T1 mapping sequences ... 137

Figure 21. Myocardial hydroxyproline levels ... 137

Figure 22. Myocardial gene expression of TGF-β and stiffer type-1 collagen assessed by PCR ... 138

Figure 23. Myocardial molecular remodeling and nitric oxide-related pathways ... 140

Figure 24. Myocardial oxidative stress assessed by 8-OHdG ... 142

Figure 25. Myocardial oxidative stress assessed by 8-OHdG ... 142

Figure 26. Myocardial oxidative stress assessed by MDA ... 143

Figure 27. Myocardial oxidative stress assessed by SOD ... 143

Figure 28. Sarcomere relaxation assessment ... 145

Figure 29. Echocardiographic assessment of diastolic function ... 147

Figure 30. Longitudinal strain ... 149

Figure 31. Circumferential strain ... 149

Figure 32. Radial strain ... 149

Figure 33. Strain rate during early diastole ... 150

Figure 34. Strain rate during isovolumetric relaxation time ... 150

Figure 35. Strain imaging diastolic index ... 150

Figure 36. Diastolic function assessed by CMR ... 152

Figure 37. Time versus volume CMR curves ... 152

Figure 38. Diastolic function assessed by invasive cardiac catheterization (I) ... 154

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Figure 39. Diastolic function assessed by invasive cardiac catheterization (II) ... 154

Table 1. Pharmacokinetics and Pharmacodynamics of SGLT2 inhibitors ... 92

Table 2. CMR assessment at one-day post-MI induction ... 132

Appendix A. Baseline and one-day post-MI 3D-echocardiography analysis ... 206

Appendix B. Cardiomyocyte hypertrophy (perimeter in µm) ... 207

Appendix C. Myocardial interstitial fibrosis ... 208

Appendix D. Myocardial extracellular volume assessed by CMR using T1 mapping sequences ... 209

Appendix E. Myocardial hydroxyproline levels ... 210

Appendix F. Myocardial gene expression of TGF-β and stiffer type-1 collagen assessed by PCR ... 211

Appendix G. Myocardial oxidative stress ... 212

Appendix H. Echocardiographic assessment of diastolic function ... 213

Appendix I. Echocardiographic assessment of diastolic function: strain ... 214

Appendix J. Diastolic function assessed by CMR ... 215

Appendix K. Diastolic function assessed by invasive cardiac catheterization ... 216

Appendix L. Hemodynamic and analytic data at the end of the study ... 217

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Dedication

To my mother, always there.

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Acknowledgements

I would like to thank Dr Juan J. Badimon and Dr J. Tuñon for their wonderful supervision throughout this journey. They provided much encouragement and patient during the long process of writing this thesis. I would also like to thank all staff involved in this amazing research project, whose hard work and tremendous support made this thesis possible. In addition, my special recognition for all those patients who suffer from heart failure, their families and the professionals involved in their care, for their unconditional willingness to in research project and help others. Many friends, family and colleagues have supported me throughout the process of working on this dissertation. I deeply appreciate all of the encouragement, feedback, humour and

support through the various ups and downs of this process. Finally, I would like to bring the attention over all those animals who are involved in research studies and that make possible the progress of science and hence, saving human lives in many cases.

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Glossary of key terms and acronyms

8-OHdG: 8-hydroxydeoxyguanosine

A: peak atrial Doppler mitral valve flow velocity AII: angiotensin II

AAALAC: Association for Assessment and Accreditation of Laboratory Animal Care ACCF: American College of Cardiology Foundation

ACE: angiotensin-converting enzyme

ACEi: angiotensin-converting enzyme inhibitors AF: atrial fibrillation

AHA: American Heart Association

AMPK: adenosine-monophosphate-protein-activated protein kinase ANP: atrial natriuretic peptide

Ar: atrial systolic reversal

ARB: angiotensin-receptor blockers

ARVC: arrhythmogenic right ventricular cardiomyopathy AT₁: angiotensin II-type 1 receptor

ATP: adenosine triphosphate ATPase: adenosine triphosphatase BB: beta-blockers

BNP: brain natriuretic peptide BP: blood pressure

BSA: body surface area

cAMP: cyclic adenosine monophosphate Ca²⁺: Calcium

CaMKII: Ca2⁺/calmodulin dependent kinase II

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cGMC: cyclic guanosine monophosphate CHD: Congenital heart disease

CMR: cardiac magnetic resonance

COPD: chronic obstructive pulmonary disease CK: creatine kinase

CKD: chronic kidney disease

CRT: cardiac resynchronization therapy CS: circumferential strain

CTGF: connective tissue growth factors CV: cardiovascular

CVD: cardiovascular disease DAPI: diamidino phenylindole DCM: dilated cardiomyopathy DM: diabetes mellitus

DNA: deoxyribonucleic acid DPP-4: Dipeptidyl peptidase 4 DT: deceleration time

E: early Doppler mitral valve flow velocity EAE: European association of echocardiography ECV%: Extracellular volume fraction

EDP: end-diastolic pressure

EDPV: end-diastolic pressure-volume eGFR: estimated glomerular filtration rate EMA: European Medicines Agency EMB: Endomyocardial biopsy ESC: European society of cardiology ERK: extracellular-signal-regulated kinase

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FDA: Food and drug administration F: french

FA: fatty acids

FFV: first filling volume

GLP-1: glucagon-like-peptide 1 GLS: global longitudinal strain GLUT1: glucose transporter 1 GLUT2: glucose transporter 2 HCM: hypertrophic cardiomyopathy

HDL-C: high-density lipoprotein cholesterol HF: heart failure

HFmrEF: HF with mid-range EF (LVEF 40 – 40%) HFpEF: heart failure with preserved ejection fraction HFrEF: heart failure with reduced ejection fraction HTN: hypertension

I/R: ischemia/reperfusion

ICD: implantable cardioverter defibrillator iECV: indexed extracellular volume IHD: ischemic heart disease

IL-6: interleukin 6 IL-10: interleukin 10 i.m.: intramuscular IV: intravenous

IVR: isovolumetric relaxation IVRT: isovolumetric relaxation time K⁺: potassium

KB: ketone bodies

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LA: left atrium

LAD: left anterior descending coronary artery

LAVi: left atrium volume indexed to body surface area LBBB: left bundle branch block

LDH: lactate dehydrogenase

LDL-C: low-density lipoprotein cholesterol LGE: late gadolinium enhancement

LS: longitudinal strain LV: left ventricle

LVEDP: left ventricle end-diastolic pressure LVEDV: left ventricle end-diastolic volume LVEF: left ventricle ejection fraction LVESV: left ventricle end-sistolic volume

MACE: major adverse cardiovascular events (i.e. nonfatal stroke, nonfatal myocardial infarction, and cardiovascular death)

MDA: malondialdehyde MI: myocardial infarction

MIF: myocardial interstitial fibrosis miRNA: micro ribonucleic acid MMP: matrix metalloproteinase MR: mitral regurgitation

MRA: mineralocorticoid receptor antagonists MS: mitral stenosis

Na⁺: sodium

NADPH: nicotinamide adenine dinucleotide phosphate NHE: sodium-hydrogen exchanger

NHE1: sodium-hydrogen exchanger isoform 1

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NHE3: sodium-hydrogen exchanger isoform 3 NO: nitric oxide

NOS: nitric oxide synthase

NOX2: nicotinamide adenine dinucleotide phosphate oxidase 2 NT-proBNP: N-terminal pro-brain natriuretic peptide

NYHA: New York Heart Association OCT: optimal cutting temperature PCP: pulmonary capillary pressure PCR: polymerase chain reaction PCT: proximal convoluted tubule

PCWP: pulmonary capillary wedge pressure PFR: peak filling volume rate

PKC: protein kinase C PKG: protein kinase G PO: orally

PP: protein phosphatase PP1: protein phosphatase 1 PV loop: pressure-volume loop RA: right atrium

RAAS: renin-angiotensin-aldosterone system RCTs: randomized controlled trials

RNA: ribonucleic acid ROS: reactive oxygen species RS: radial strain

RV: right ventricle

SCD: sudden cardiac death SD: standard deviation

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SERCA2a: sarcoplasmic reticulum ATPase SGLT: sodium glucose cotransporters

SGLT1: sodium glucose cotransporter type 1 SGLT2: sodium glucose cotransporter type 2 SI-DI: strain imaging diastolic index

SL: sarcomere length SOD: superoxide dismutase

SRE: Strain rate during early diastole

SRIVR: Strain rate during isovolumetric relaxation SSFP: Steady-state free precession

SV: stroke volume

T1: longitudinal relaxation time T1/2: half-life

T1DM: type 1 diabetes mellitus T2DM: type 2 diabetes mellitus

Tau: time constant of left ventricular isovolumetric relaxation TBARS: Thiobarbituric acid reactive substances

TCA: trichloroacetic acid solution TDI: tissue Doppler imaging TE: echo time

TGF-β: transforming growth factor-β

TGF-β1: transforming growth factor-β subtype 1 TI: inversion time

Tmax: maximum plasma levels

TmG: maximum renal glucose reabsorptive capacity TNFα: tumor necrosis factor alpha

TR: repetition time

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TR: tricuspid regurgitation UTI: urinary tract infection

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Abstract

Introduction: heart failure (HF) is considered the fastest growing cardiovascular condition worldwide. Irrespective of aetiology, the final common response is a neurohormonal activation along with greater cardiac mechanical stress, which ultimately leads to adverse cardiac remodeling and impaired cardiac performance.

Interestingly, the novel antidiabetic therapy, the sodium-glucose cotransporter type 2 inhibitors (SGLT2i), have demonstrated to significantly increase survival rates among patients with type 2 diabetes mellitus (T2DM). These results were mostly achieved by a significant reduction in HF hospitalizations. Their effect seems to be irrespective of their glucose-lowering action, although the underlying mechanisms remain under investigation.

Objectives: to evaluate the hypothesis that empagliflozin, a SGLT2i, may improve myocardial cell performance – specifically diastolic function – by mitigating adverse cardiac remodeling.

Material and methods: ischaemic HF model was created by inducing a myocardial infarction (MI), following 2-h balloon occlusion of the proximal left anterior

descending artery, in nondiabetic pigs. Thereafter, animals (n = 14) were then randomized in a 1:1 ratio to receive either empagliflozin or placebo for 2 months.

Cardiac adverse remodeling was evaluated by histology and molecular techniques (cardiomyocyte size and interstitial fibrosis), upon study termination. In addition, myocardial diastolic function was assessed with invasive and non-invasive techniques, at baseline and at 2 months of follow up.

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Results: despite similar baseline myocardial injury following MI induction, the empagliflozin-treated group showed mitigation of adverse cardiac remodeling at 2- months of follow up (lower cardiomyocyte hypertrophy and lower interstitial fibrosis).

In addition, diastolic function indexes significantly improved in the treated group compared with placebo group.

Conclusion: empagliflozin ameliorates cardiac adverse remodeling in a non-diabetic animal model of ischaemic heart failure. This may translate into an improvement in myocardial diastolic function.

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Background

1. Heart failure: brief history and developed therapies

1.1. First description of heart failure

Andreas Nerlich, a German pathologist, was believed to first described a case of decompensated heart failure (HF) over 3500 years ago [1]. He examined the pulmonary histology of Nebiri, an Egyptian dignitary, and confirmed the presence of pulmonary oedema – likely due to ‘heart failure’. By histochemical staining, he excluded other causes of fluid in the lungs, including infections such as tuberculosis. The remains of this first case of HF are currently housed in the Egyptian museum in Turin, Italy.

Little was known about the pumping function of the heart until 1628, when William Harvey, an English physician, extensively described the cardiovascular (CV) circulation [2]. He also demonstrated that blood stream flows in two different loops and thus, he first described two independent systems, termed pulmonary and systemic circulation. This view provided the basis for a comprehensive approach to the cardiac haemodynamics and HF.

The first allusion in literature to adverse cardiac remodeling was probably made by Lancisi, an Italian clinician who served Pope Innocent XI in the late- 17^th century. He observed that valvular regurgitation leads to cardiac dilation and this, eventually weakens the heart function [3]. Thereafter, various scientists detailed different forms of cardiac enlargement including hypertrophy - both

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concentric and eccentric - as well as maladaptive changes that occur within the failing heart, contributing to acute and chronic heart HF [4].

The turning point occurred in 1918 when Ernest Starling, a British physiologist, published the ‘Law of the Heart’ [5]. He announced that by

increasing end-diastolic volume (EDV), the cardiac performance improved. This statement caused a tremendous impact initially, given the contradiction with previous theories that postulated an adverse relationship between cardiac dilation and myocardial function.

1.2. Initial heart failure therapies

Although the introduction of cardiac catheterization and cardiac surgery in the 1940s and 1960s extraordinarily contributed to characterize several forms of HF, the only available therapies were digitalis and diuretics along with bed rest and fluid restriction [4].

Little research was focused on the heart by then; however, the idea of cardiac contractility gained notorious importance following the description of ventricular function curves, based on Starling´s Law [6]. In 1954 Sarnoff, an American doctor, described that the ventricular function curve shows an initial steep rise, during which 1 cm H2O increase in atrial pressure, may increase the ventricular stroke work as much as 300%; at high filling pressures the curve flattens off to a plateau with no descending limb in the normal heart.

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Figure 1. Representative tracing (A) and curves (B) of right and left ventricular stroke work for each level of atrial pressure [6].

The stroke work for each ventricle in grammeters (a unit of energy equal to 100 gram-centimetres), was calculated according to the following formula:

(𝑀𝑒𝑎𝑛 𝑎𝑟𝑡𝑒𝑟𝑖𝑎𝑙 𝑝𝑟𝑒𝑠𝑠𝑢𝑟𝑒 (𝑐𝑚 𝐻2𝑂) − 𝑀𝑒𝑎𝑛 𝑎𝑟𝑡𝑒𝑟𝑖𝑎𝑙 𝑝𝑟𝑒𝑠𝑠𝑢𝑟𝑒 (𝑐𝑚 𝐻2𝑂)) 𝑥 𝑠𝑡𝑟𝑜𝑘𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 100

Stroke volume (in cm³) was obtained by dividing systemic blood flow per minute by the heart rate.

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Interestingly, a significant alteration in the circulatory state produced different ventricular curves. For instance, following a left main artery occlusion, there was a depression of the left ventricle function curve together with the appearance of a descending limb and subsequent HF. Thus, established the connection between reduced contractility and HF, investigations focused on enhancing this myocardial contractility by developing new drugs such as inotropes. Surprisingly, all clinical trials with inotropes were terminated prematurely due to poor outcomes [7].

In December 1967, Christian Barnard performed the first interhuman (orthotopic) heart transplantation in Cape Town, South Africa. The recipient was a 54-year-old man with and end-stage ischemic cardiomyopathy who received the heart of a girl, hit by a drunk driver [8]. Following an initial excellent recovery within the first days after transplantation, the patient eventually died.

In 1960s, left ventricle (LV) assist devices (LVAD) also emerged as an

alternative to these patients [9]. They underwent tremendous developments and improvements over the last decades, and currently they are considered a crucial therapy for end-stage HF patients.

In the 1970s, the scientific interest was focused on vasodilators [10]. It was believed that by reducing cardiac afterload, there would be an increase in myocardial efficiency, cardiac output and thus, ejection fraction (EF). In addition, intracardiac pressures would be reduced and subsequently, diastolic function along with cardiomyocyte energetics would also improve. Promising results were first observed in the clinical trial V-HeFT I [11], with different vasodilators in patients with HF. However, the initial enhancement of CV

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haemodynamics was not confirmed in the long-term analysis as the trial did not show increased survival rates among treated patients [12–14].

1.3. Neurohormonal blockade drugs

In the 1980s the emerging theory was the existence of neurohormonal system response in untreated HF patients. This idea was based on the fact that nature would provide mammals with survival mechanisms to counterbalance acute cardiovascular disturbances (for instance, excessive bleeding following traumatic event, such as hunt) [4]. The response consists of renin-angiotensin- aldosterone system (RAAS) activation along with catecholamine release by sympathetic nervous system [15]. This response, which may be beneficial in the short-term, exerts deleterious effects if chronically maintained, with myocyte damage and cardiac architectural changes.

This scientific milestone motived the development of what is termed nowadays ‘neurohormonal blockade therapies’, and it changed outstandingly the prognosis of patients with HF. Angiotensin-converting enzyme inhibitors

(ACEi), together with beta-blockers (BB), widely demonstrated to improve survival rates and decrease hospitalization rates in all patients with HF and reduced EF (HFrEF). This benefit was observed regardless of the severity of symptoms [16,17]. Both classes of drugs ameliorate adverse cardiac remodeling and their effect seems to be independent of etiopathogenesis of myocardial dysfunction [4].

Mineralocorticoid receptor antagonists (MRA), such as spironolactone, were launched in 1960 as diuretic medications for treating oedematous

conditions, including aldosteronism and hypertension [18]. Later, Selye

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discovered that spironolactone protected rats from aldosterone-induced cardiac necrosis [19]. However, this pioneering work on aldosterone-induced

inflammation was not clinically considered for years. The most inspiring trial of MRA on HF patients (RALES) was published in 1999, when Pitt et al.

demonstrated a significant 30% reduction in mortality rates [20]. Of note, patient enrolled in this trial were symptomatic, with severe LV systolic dysfunction and spironolactone was added on top of standard therapies, including BB and ACEi, when tolerated. Few years later, angiotensin-receptor blockers (ARB) were incorporated to the therapeutic armamentarium for HF management as an alternative to ACEi. These drugs also showed to significantly reduce adverse cardiac outcomes in patients with reduced LVEF [21,22].

1.4. Devices and heart failure

Cellular damage, disarray and ventricular scarring in HF is a favourable substrate for ventricular arrhythmias [23]. In the late 1960s, Michel Mirowski and Morton Mower described for the first time the use of effective implantable cardioverter defibrillator (ICD), following a successfully delivered shock in a dog with ventricular fibrillation [24,25]. Despite initial disbelieves, ICD was finally recognized as an opportunity to address this form of cardiac death. The first human ICD implantation occurred at the Johns Hopkins Hospital in 1980 and ever since, a number of randomized controlled trials (RCT) supported the use of this device given improvements in survival rates in primary and

secondary prevention of sudden cardiac death (SCD) [26].

Thereafter, the introduction of cardiac resynchronization therapy (CRT) also demonstrated a significant increase in survival rates among selected patient

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with HF and reduced LVEF [27]. By harmonizing the contraction of the

ventricle walls, this revolutionary device enhances cardiac contractility, reverses adverse remodeling and hence, improves LVEF [4].

1.5. Molecular biology and genetics

In 1990 Geisterfer-Lowrance et al. published the first molecular cause of familiar cardiomyopathy, a missense mutation in the cardiac beta-myosin heavy chain gene (MYH7), associated with hypertrophic cardiomyopathy [28]. This led to consider gene therapy (i.e. replacement of faulty genes with correct copies delivered by viral vectors) as an alternative therapy for HF patients [4]. Over the last years, preclinical studies have showed promising results and a realistic opportunity for treating HF patients. Although recent studies in humans did not confirm efficacy of this therapy, they demonstrated the safety of the gene therapy and defined the methods for gene delivery [29].

Micro ribonucleic acid (miRNA) has also emerged as an encouraging treatment for patients with HF. These small non-coding RNA segments regulate gene-transcription and protein synthesis by silencing messenger RNA [4]. They have been found to participate in cardiac development and cardiomyocyte proliferation [30]. In mice, for instance, miRNA stimulated cardiac regeneration and restored cardiac function after myocardial infarction (MI) [31]. In addition, transcriptome analysis of human failing hearts suggested that reactivation of a foetal miRNA pattern may contribute to a gene expression pattern that

resembles foetal heart. This represents a switching of structural proteins from the adult to the foetal isoforms, including a decrease in the fast-shortening- velocity isoform, α-myosin heavy chain (α-MHC), along with an increase in the

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slow-shortening-velocity isoform β-MHC [30]. Ultimately, this leads to decreased myocardial contractility.

Stem cell therapy has also been investigated for HF. Under the hypothesis that implanted stem cells will differentiate in cardiomyocytes, various strategies have been explored, including autologous bone marrow- derived mononuclear cells [32] and mesenchymal stem cells from adipose tissue [33]. However, it seems that the best cell type, among other technical aspects, remain to be determined.

Finally, the latest approach being explored for treating patients with HF is the chemical decellularizing of a human heart, with preservation of its architecture and vascularity. Few studies reported that cardiomyocytes seeded onto this extracellular matrix are capable of form functional connections [34].

Further research will clarify the future of a manufactured human heart.

1.6. Latest developments in heart failure

For nearly 30 years there were no new pharmacological therapies for HF, until the publication of the PARADIGM-HF trial in 2014 [35]. This study

evaluated the efficacy of a dual ARB (valsartan) plus a neprilysin inhibitor (sacubitril) – also termed ‘ARNI’ (angiotensin receptor neprilysin inhibitor). It enrolled symptomatic HF patients with LVEF ≤ 40% and on standard medical therapies. For the primary composite outcome of CV death and HF

hospitalization, the group treated with this combination, valsartan/sacubitril, showed a significant 20% risk reduction compared with the group treated with enalapril alone in.

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Neprilysin is a neutral endopeptidase that degrades various endogenous vasoactive peptides such as natriuretic peptides, bradykinin and adrenomedullin [36–38]. These substances attenuate the neurohormonal activation that accounts in HF patients leading ultimately to vasoconstriction, sodium retention and adverse cardiac remodeling [39]. By inhibiting neprilysin, there is an increase in these peptides and thus, a counterbalance in the neurohormonal response.

The combination of ACEi plus endopeptidase inhibitors is superior to ACEi alone in terms of improving cardiac filling pressures and induced

natriuresis with preservation of renal glomerular filtration [40]. Nonetheless, this combination could be associated with an increased risk of serious angioedema [41].

In symptomatic patients with LVEF ≥ 45%, the PARAGON-HF trial did not show significant differences between ARNI and valsartan alone for the primary composite endpoint of HF hospitalization and CV death [42]. However, it could exert a major impact in long terms outcomes for patients admitted due to acute decompensated HF. In this regard, the PIONEER-HF trial randomized hospitalized patients with LVEF of ≤ 40% to receive either sacubitril-valsartan or enalapril [43]. This study demonstrated that initiation of sacubitril-valsartan whilst on admission was associated with a significant reduction of N-terminal pro-brain natriuretic peptide (NT-proBNP) compared with enalapril alone. In addition, there were no significant differences in safety outcomes, including worsening renal function, hyperkalaemia or symptomatic hypotension.

Other studies have failed to demonstrate benefit in hospitalized patient with HF. For instance, serelaxin (a vasodilator hormone that contributes to CV

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and renal adaptations during pregnancy) did not improved survival rates within this patient with acute HF decompensation compared with placebo [44].

2. Heart failure: definition and terminology

2.1. Definition of heart failure

Since the very detailed description of heart muscle diseases by the German clinician Rudolf Virchow in his publication ‘Die Cellular Pathologie’

in 1858, HF definition has noticeably evolved over the years [45]. In 2016, the European Society of Cardiology (ESC) defined this condition as a clinical syndrome characterized by typical symptoms (breathlessness, ankle swelling, etc.…), that may be accompanied by signs (i.e. elevated jugular venous pressure or pulmonary crackles), and that is caused by a structural and/or functional cardiac abnormality [46]. The ultimate consequence is reduced cardiac output and/or elevated intracardiac pressures. It is worth mentioning that this definition restricts itself to symptomatic stages, as opposite to 2013 guidelines of the American Heart Association, wherein they described different stages of structural heart disease that may or may not be associated with HF symptoms [47].

The most common underlying cause of HF is a myocardial injury;

however, abnormalities affecting cardiac valves, pericardium, endocardium and/or conduction system disease, may also precipitate a HF episode. The

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identification of the primary disorder is crucial for further management and prognosis [46].

2.2. Terminology in heart failure

Heart failure can be classified according to severity of symptoms, presence of cardiac structural abnormalities, LVEEF or clinical course, among others.

2.2.1. Symptom-based classification of patients

The New York Heart Association (NYHA) stratifies HF patients

according to severity of symptoms. It is indeed the most widely used functional classification of such patients. It was formally proposed in 1972 as a general outline to emphasize the ‘whole patient’ and to facilitate communication among physicians of different specialties. It is a subjective assessment of patient´s symptoms and it can change frequently over short periods of time. Nevertheless, this classification has showed to be an independent predictor of mortality [48], and it has been outlined as a general eligibility criteria in many research trials.

According to this, patients are stratified as follows:

- Class I: No limitation of physical activity. Ordinary physical activity does not cause undue fatigue, palpitation, and dyspnoea.

- Class II: Slight limitation of physical activity. Comfortable at rest.

Ordinary physical activity results in fatigue, palpitation, and dyspnoea.

- Class III: Marked limitation of physical activity. Comfortable at rest. Less than ordinary activity causes fatigue, palpitation, or dyspnoea.

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- Class IV: Unable to carry on any physical activity without discomfort.

Symptoms of heart failure at rest. If any physical activity is undertaken, discomfort increases.

2.2.2. Objective assessment of heart failure stage

The American College of Cardiology Foundation (ACCF), together with the American Heart Association (AHA), emphasized the development of a more objective assessment of HF patients based on both risk factors and abnormalities in cardiac structure [47]. Within this classification, stages are progressive and inviolate (i.e. there is no regression to a prior stage once patient moves to higher stage). Of note, it has been demonstrated a reduced 5-year survival when

progression to advanced stage [49]. This classification include:

- Stage A: At high risk for HF but without structural heart disease or symptoms of HF.

- Stage B: Structural heart disease but without signs or symptoms of HF.

- Stage C: Structural heart disease with prior or current symptoms of HF.

- Stage D: Refractory HF requiring specialized interventions.

2.2.3. Classification based on ejection fraction

The LVEF is the classic parameter used to describe HF patients.

According to this, patients are categorized as follows [46]:

• HFrEF: HF with reduced EF (LVEF < 40%)

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• HFpEF: HF with preserved EF (LVEF ≥ 50%)

• HFmrEF: HF with mid-range EF (LVEF 40 – 49%)

Distinction of patients based on LVEF is important in terms of prognosis given the fact that most clinical trials published after 1990 enrolled patients based upon this criterion [46]. These trials undoubtedly showed benefits in patients with HFrEF, by lowering mortality rates. However, these results are still controversial in other HF patients [50]. A cut-off value of 40% of LVEF is questionable as this is a continuous variable that may need to be indexed by additional cofactors such as sex and age [51]. Recent guidelines indicate that the lower normal values for LVEF are 52% in men and 54% in women [52] and thus, values between 41-52% in men, and 41-53% in women, are considered as mildly impairment. However, these patients have not been historically included in HFrEF trial, as they were designed to have high event rates by limiting a sample size to a minimum to obtain significant results in a cost-effectiveness approach [51].

It is also worth mentioning that, due to a significant intra- and interobserver dependency, along with a marked reliance on the imaging

acquisition technique, an large proportion of patients could be reclassified if the measurements were repeated [51]. This variability could be as much as 7% if LVEF was analysed in the same patient, using the same methods and by highly experienced echocardiographer; however, these disparities could be pronounced in clinical practice [51].

When assessing by circulating biomarkers, patients with HF and LVEF >

50% typically show increased levels of proteins that reflect systemic

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inflammation, endothelial injury and myocardial fibrosis. In contrast, patients with HFrEF exhibit higher markers of cardiomyocyte injury, loss and stretch.

Interestingly, patients with LVEF 40 – 50% show a circulating profile that resembles patients with LVEF < 40% [46].

In regards to therapies and HFmrEF, several large randomized controlled trials (RCTs) strongly support the neurohormonal blockade medications for this subgroup of patients. A meta-analysis of 11 RCTs comparing BB versus

placebo, demonstrated a marked reduction in mortality rates in patients with HF and LVEF of 40 – 49% versus those with LVEF higher than 50% [53]. In

addition, this association was comparable to that seen in patients with and LVEF lower than 40%. Moreover, the CHARM-Preserved trial, demonstrated that candesartan significantly reduced CV death rates and HF hospitalizations in patient with LVEF of 40 – 49% but not in those with LVEF ≥ 50%, compared with placebo [54]. Similarly, in the TOPCAT trial, spironolactone showed a positive outcomes with reduced CV deaths and HF hospitalizations, in HF patients with an EF of 45 – 49%, but not for those with an EF ≥ 50%, compared with placebo [55]. And most recently, the prespecified subgroup analysis of the PARAGON-HF trial, showed a significant reduction in CV deaths and HF hospitalizations in HF patients with LVEF of 45 – 57% receiving

sacubitril/valsartan versus valsartan alone, and not in those with LVEF higher than 57% [42].

2.2.4. Other nomenclature in heart failure

According to time course and presentation, chronic HF applies to

patients who have had a previous episode of congestive HF (i.e. with symptoms

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of volume overload). When a patient has been previously treated and their clinical status remains unchanged for at least 1 month, then the term stable could apply. However, when patient deteriorates, then this episode is described as decompensated HF. This clinical deterioration can be either acute or subacute, if progresses gradually.

If there is no history of previous HF episode, terms new-onset or de novo are generally used. Although symptoms and signs may resolve, the underlying cause of cardiac dysfunction may persist and there is a risk of recurrence. On the other side, the aetiology may completely resolve such as acute viral myocarditis, or persist chronically.

According to the recent position statement of the ESC, ‘advanced’,

‘refractory’ and ‘end-stage’ HF are often interchangeable terms and they

identify patients who should be evaluated for advanced HF therapies [56]. They apply to a stage where conventional treatments including pharmacological therapies, devices and conventional surgery, are insufficient to control

symptoms and thus, additional therapies are should be explored. These involve either advanced therapies, such as cardiac transplantation or mechanical

circulatory support, or palliative care (i.e. inotropic infusions, renal replacement therapies to control volume overload and/or end-of-life comfort care) [56].

Many of these terms may be applied to the same patient at different times, depending upon their stage of disease.

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3. Heart failure: epidemiology and aetiology

3.1. Incidence, prevalence and prognosis

Most of the literature on HF epidemiology is derived from developed countries and thus, it should be carefully extrapolated to global population.

Among these countries, however, there seems to be a stabilization of HF prevalence and even possible reduction in trends [45]. This is mainly due to improvements in both primary prevention of CV disease and also, in

management of ischemic heart disease (IHD) [57].

The global incidence of HF ranges from 100 to 900 cases per 100,000 person-years [58]. Between 1950 and 1999, the incidence of HF in the Framingham cohort in the USA declined from 420 to 327 cases per 100,000 person-years in women [59]. Nonetheless, this reduction was not observed in men, whose HF incidence remained at 564 cases per 100,000 person-years.

Additionally, a significant reduction in age- and sex-adjusted HF incidence was observed between 2000 and 2010 in the Olmsted County cohort in the USA, from 315.8 to 219.3 cases per 100,000 person-years [60]. This decline was substantially greater in women (43%) than in men (29%). There are also

disparities on HF incidence amongst different ethnic groups, with higher rates in African-American individuals, intermediate rates in white and Hispanic

individuals, and the lowest rates among Chinese-American subjects [61]. The burden of CV risk factors and also, in socioeconomic disparities, contribute to this differences [61].

Similar reduction in HF incidence was corroborated in Europe. For instance, a Stockholm-based study showed an incidence of 380 new cases per

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100,000 person-years in 2010, with an absolute decline of 90 cases compared with 2006 [62]. However, approximately 80% of global CV disease burden occurs in middle-income and low-income countries [45]. The PURE cohort, which includes patients from high-, middle- and low-income nations, reported an incidence of 271 new HF cases per 100,000 person-years [63]. Interestingly, decompensated HF was the most common diagnosis amongst African patients hospitalized for CV events [64].

The estimated global prevalence of HF was 37.7 million people in 2010 [65]. It is considered the fastest growing CV condition worldwide. In developed countries, the prevalence of HF in adult population is 1-2%, rising over 10%

among patients aged older than 70 years [46]. In this regard, it is worth to mention that one in six patients who present to primary care with dyspnoea on exertion will have unrecognized HF [66]. Although the age adjusted-prevalence of HF is decreasing, the absolute number of patients diagnosed with HF is markedly increasing. For instance, in the USA the projected increase in HF prevalence will be 46% by 2030 (>8 million people) [67]. The primary driver for this escalation is the aging of national population [45].

Approximately 68.7% of global HF causes include one of the following:

IHD, chronic obstructive pulmonary disease (COPD), hypertensive heart disease and rheumatic heart disease [65]. The proportion of patients with HFpEF widely ranges from 22 – 73%, depending on the registries [46]. This population tends to be older, and includes more women, with a strong background of hypertension (HTN) and/or atrial fibrillation (AF).

Numerous prognostic markers of death and/or HF hospitalization have been identified, including demographic data, clinical status of patients,

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neurohormonal biomarkers, myocardial remodeling and genetic testing.

However, none of >115 prognostic models analysed has showed strong accuracy to predict mortality and/or HF hospitalization [68]. In addition, estimation of HF mortality rates alone is challenging given associated conditions such us CAD, which are often regarded as the major cause of death. Most deaths among HF patients are due to CV causes, especially sudden death and worsening HF [46].

In developed countries, survival rates significantly improved in the 1990s. For instance, the Framingham Heart Study reported a decline in 5-year mortality over 10% for the period between 1950 – 1969 compared with the period between 1990 – 1999 [59]. This may be attributed to the developments of the neurohormonal blockade therapies. However, this improvement in prognosis of HF seems to be diminished in the early 2000s [69]. Current studies indicate that one-, five- and ten-year survival rates are approximately 81%, 48% and 26%, respectively, in developed nations [69]. After adjusting to age and sex, fatality rates could be 3.7 times higher in low-income countries, and 2.6 times higher in middle-income countries, compared with high-income nations [63].

The ESC-HF pilot study showed a 12-month hospitalization rate of 44%

for patients admitted due to HF decompensation, and 32% for ambulatory/stable HF patients [70]. These hospitalizations are frequently related to non-CV causes, particularly in patients with HFpEF.

3.2. Aetiology

There is no agreement in single classification system and a wide variety of cardiac conditions, inherited anomalies and systemic diseases may result in HF. There is also a marked overlapping between categories and different

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pathologies can coexist in the same patient. Identification of underlying conditions may offer targeted therapeutic opportunities.

In developed countries, IHD and COPD are especially frequent whereas hypertensive and rheumatic heart disease are more prevalent in developing countries [65].

- Ischemic heart disease: it is the leading cause of death globally,

accounting for 15.7% of all age-standardized deaths [71]. The incidence of MI is higher in Eastern Europe and Central Asia compared with the lower incidence rates observed in developed nations in Asia [72]. As a result of cardiomyocyte hypoxia, the myocardial oxygen supply is insufficient to meet metabolic demands [73]. If prolongs, this situation leads to a myocardial cell death and ultimately, LV dysfunction. The proportion of ischemic HFrEF has gradually decreased in high-income countries through both, primary and secondary prevention strategies; however, this proportion has slightly increased among patients with HFpEF [45]. Ischemic HF can occur quickly following a sudden interruption of coronary circulation, or it can be chronic in the setting of prolonged imbalance between

cardiomyocyte oxygen supply and demand. Different causes of increased myocardial oxygen include abnormal loading conditions, heart rate or contractility; whereas those of decreased cardiomyocyte oxygen supply comprise coronary artery stenosis, thrombosis, spams or endothelial dysfunction [73]. Imaging assessment of myocardial viability is decisive, given the potential reversibility of ischemic HF by revascularization techniques [73].

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- Hypertensive heart disease: high blood pressure may lead to ventricular hypertrophy and increased cardiac mass by exposing cardiomyocytes to mechanical stress along with neurohormones, growth factors and cytokines [74]. As a result, there is a change in the extracellular matrix with greater fibrosis and LV stiffness [75]. The ultimate consequence is an elevation in LV filling pressure that triggers symptoms and HF decompensation [74].

Interestingly, hypertensive HF as been associated with both preserved and reduced EF. The Olmsted County cohort reported a higher percentage of patients with HTN among HFpEF group compared with HFrEF group [60].

Although the underlying mechanism between HTN and reduced LVEF remains challenging, myocardial ischemia due to abnormalities of the intramyocardial coronary vasculature seems crucial [74]. Among individuals with blood pressure (BP) higher than 160/90 mmHg, the

lifetime risk of HF is double that of those with BP lower than 140/90mmhg [76]. Improvements in high BP control have contributed to decline the incidence of hypertensive HF. Of note, optimal control of HTN may reduce the risk of HF as much as by 87% [45].

- Valvular and rheumatic heart disease: although data of valvular heart disease and HF is scarce, rheumatic fever is globally the leading cause of valvular HF [45], with estimated 470,000 new cases and 233,000 deaths per year [77]. However, there are considerable demographic differences. For instance, the incidence of rheumatic fever is >100 cases per 100,000 person- years in Sudan, whereas in developed countries it has fallen below 1 case

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per 100,000 person-years [78]. This is attributable to improvements in social and living conditions, together with wide availability of antibiotic therapy [45]. In developed countries, degenerative disease is the most common cause of valvular disease, with a prevalence that substantially increases with age [79].

- Cardiomyopathies: in developed nations, dilated cardiomyopathy (DCM) has been reported to be the second most common cause of HF, with up to 36% of total cases, and only exceeded by IHD [80]. In clinical practice, a clear distinction between inherited and acquired conditions is often difficult.

Routine genetic testing has a major role in scenarios such hypertrophic cardiomyopathy (HCM) or arrhythmogenic right ventricular

cardiomyopathy (ARVC), since results may exert a crucial role with clinical implications and marked impact on outcomes [46]. On the other side,

inflammatory, infectious and nutritional deficiencies may be the underlying cause of HF in low-income countries. For instance, HIV-related

cardiomyopathy and endomyocardial fibrosis have a significant prevalence in sub-Saharan Africa [45]. Interestingly, Chagas disease is the leading cause of non-ischemic cardiomyopathy in Latin America [81], and over 38% of these patients progress to HF over a 10-year follow-up period [82].

These patients are at higher risk of complications due to conduction disease and malignant arrhythmias, compared with other non-ischemic

cardiomyopathy patients. Antiparasitic treatments, benznidazole and nifurtimox, are recommended in the acute phase. However, their effectiveness remains controversial in patients with establishes Chagas

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cardiomyopathy as they did not demonstrate to reduce cardiac clinical deterioration [83].

- Congenital heart disease (CHD): estimated global prevalence of CHD ranges from 0.4 – 5% of all births [84]. Nearly 25% of these patients will develop HF [85], primarily due to a volume or pressure overload [86].

Genetic testing may also play a decisive role amongst these patients.

Survival rates in children with CHD have dramatically improved over the past decades, mostly due to surgical advances [87]. Adults with CHD are also living longer with an overall median age at death that has increased from 37 years in 2002 to 57 years in 2007 [84]. This impact in cumulative survival rates means that more patients with CHD are reaching the

adulthood and thus, increasing the cohort of patient at risk of developing HF. It is worth to mention that these patients may have adapted to their long-standing limitations and thus, they may not report symptoms despite significant impaired exercise performance when objectively assessed by tests such as six-minute walk test or cardiopulmonary exercise test. In addition, application of general HF classifications, such as NYHA, may underestimate the severity. Interestingly, none of these scoring methods have been validated in predicting outcomes in patients with CHD [87].

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4. Adverse cardiac remodeling and myocardial fibrosis

4.1. Cardiac remodeling: concept and aetiology

The term LV remodeling refers to alterations in global myocardium geometric (shape and size), together with changes in cellular and extracellular composition of the LV, in response to mechanical stress and neurohormonal activation. It includes 2 main aspects: cardiomyocyte injury (hypertrophy, necrosis, apoptosis, amongst others) and myocardial interstitial fibrosis (MIF) [88].

This remodeling contributes to cardiac dysfunction and impaired contractility that ultimately results in HF [89]. The term initially referred to ventricular dilation and myocyte changes following MI in rats [90].

Subsequently, Pfeffer et al. introduced the key concept that defined and quantified cardiac remodeling, a shift of the ventricular end-diastolic pressure- volume (EDPV) relationship [91]. This study showed an increase in LV volumes following MI in rats (reflected by a rightward shift of the EDPV curve). The extent of the dilation was related to the size and the duration of the MI. In addition, this dilation was notably more appreciated at high filling pressures (i.e.

end-diastolic pressure (EDP)), due to higher distension and stretching of both, the scar and the residual normal myocardium.

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Figure 2. Left ventricular pressure-volume relationships of rats according to infarct size [91].

A: small-, B: moderate-, C: large-sized myocardial infarct. Shaded area represents mean value (±2 standard errors) for volume at each level of pressure in rats without infarct. Time in days post-infarction appears above each curve. Note modest rightward shift in low-pressure range for moderate- and large- sized infarcts and leftward shift in high-pressure range for small-sized infarcts during early post- infarction phase. During late post-infarction phase considerable rightward shift in the curve occurred in rats with moderate- and large-sized infarcts such those volumes were double those of rats without infarcts by 106 days post-infarction.

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Proposed causes of cardiac remodeling include: MI, pressure overload (aortic stenosis, HTN…), volume overload (valvular regurgitation), or

myocardial inflammation. Physiologic and thus, non pathological remodeling, can also be seen in athletes [92]. The final proposed pathway is a local increased in different substances such as norepinephrine, angiotensin, aldosterone and cytokines, amongst others. All these changes result in alteration of protein expression that ultimately leads to myocyte hypertrophy and collagen synthesis [92]

4.2. Cardiac remodeling: cellular, extracellular and molecular changes

4.2.1. Cardiomyocyte hypertrophy

They are considered the main cell involved in the remodeling process.

Following and insult, such as MI, myocytes die and the total number declines.

As an initial compensatory mechanism to maintain stroke volume, surviving myocytes become elongated and hypertrophied [92]. Membrane stretching activates cyclic adenosine monophosphate (cAMP) and cation exchange channels, allowing Ca²⁺ and Na⁺ enter the cell. These disturbances may affect transcription process and generate altered proteins [93] that may also influence other signalling pathways. Ultimately, new contractile proteins are synthetized – although often less efficient. The way these cell assembles the new sarcomeres varies. For instance, in volume-overloaded ventricles the new sarcomeres are laid down primarily in series to elongate myocytes (eccentric hypertrophy). The sympathetic nervous system seems to play a decisive role in this process [94]. In

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contrast, a pure pressure overloaded LV increases the number of sarcomeres in a parallel fashion (concentric hypertrophy) [95].

Cardiomyocyte hypertrophy is a reversion to a more foetal phenotype, that includes greater tolerance for low oxygen levels, with abundant glycogen, higher rates of glycolytic metabolism and low levels of adenosine triphosphatase (ATPase) and lactate dehydrogenase [93]. There is also a predominance of β- myosin isozymes and creatinine kinase-MB. Eventually, the fraction of cell occupied by myofibrils increases and there is an imbalance between these ATPase-consuming myofibrils and ATPase-generating mitochondria [96].

4.2.2. Myocardial interstitial fibrosis

Within the heart muscle, a syncytium of cardiomyocytes is tethered by a structural protein network composed primarily of fibrillar type I collagen [97].

There is a turnover of collagen in this extracellular matrix (ECM), by continuous degradation and synthesis. The main role of the ECM is to provide a scaffold for cardiomyocyte array and additionally, to integrate functional and electrical cardiac performance [97]. Alterations in the ECM contribute to develop pathological cardiac remodeling.

Histologically MIF is defined by the diffuse deposition of excess fibrous tissue relative to the cardiomyocyte mass [98]. There are two types of MIF, which may coexist in the same patient:

- Reparative/replacement fibrosis, where MIF replaces small foci of dead cardiomyocytes forming micro-scars.

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- Reactive fibrosis, where MIF accumulates in the perivascular space around intramural coronary arteries.

4.2.3. Mechanisms of myocardial fibrosis

Given the minimal regenerative capacity of the adult mammalian heart, the most extensive fibrotic remodeling occurs following acute cardiac cell death (i.e. MI) [99]. The cardiomyocyte loss is often the trigger event responsible for the MIF. However, several other intrinsic cardiac diseases or systemic factors (such as HTN, aging, metabolic injury, pressure or volume overload) may lead to the same final common pathway, by injuring the myocardium without associated cell death [99]. Within this injured myocardium there is an increase on inflammatory cytokines, reactive oxygen species (ROS) and fibrotic growth factors, such as transforming growth factor-β (TGF-β) and connective tissue growth factors (CTGF). There is also an increased activity of RAAS [98].

Following cardiomyocyte loss and/or injured myocardium, myofibroblast generation occurs. The sources of theses cells are both cardiac resident

fibroblasts, and circulating and resident fibroblast progenitor cells. When activated by inflammatory factors, fibroblasts differentiate into a non- proliferative secretory phenotype, the myofibroblasts [98]. These cells show characteristics of smooth muscle cells, including contractile stress fibres and the expression of α-smooth muscle actin. They also have an extensive endoplasmic reticulum reflecting their active synthesis.

Once activated, myofibroblasts produce and secrete collagen type I and III, along with molecules required to regulate their turnover (also termed,

‘secretome’) [98]. Sequence-specific DNA binding transcription factors,

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together with alterations in non-coding RNAs, are involved in this secreting process. The MIF process is perpetuated by the secretion of autocrine and paracrine substances that control collagen turnover, and favour fibre formation over degradation [98]. In addition, oxidation of specific collagen lysine by enzymes of the lysyl oxidase family is crucial to maintain MIF [98]. As a consequence of this oxidation, collagen fibres resist to degradation by matrix metalloproteinase (MMP).

4.2.4. The role of sympathetic nerve system in cardiac remodeling Hyperadrenergic state, such as the one associated with HF, triggers a non-ischemic type of cardiomyocyte necrosis termed ‘mitochondriocentric signal-transducer-effector pathway’ [97]. Raised catecholamine levels induce intracellular and intramitochondrial Ca²⁺ overloading that prompts oxidative stress and loss of adenosine triphosphate (ATP) synthesis [97]. When the rate of generation of ROS overwhelms the rate of detoxification, the mitochondrial inner membrane permeability-transition-pore opens and enables passive solute entry. As a consequence, there is an osmotic swelling of the mitochondria that degenerates, and ultimately leads to cell necrosis [97].

4.2.5. Inflammatory and immune cells in myocardial fibrosis

Following cardiac cell necrosis, there is increased serum and local concentrations of proteins such as creatine kinase (CK), lactate dehydrogenase (LDH) and myoglobin, among others, that may result in activation of the immune system [97]. For instance, T cells have showed enhanced adhesion for the activated vascular endothelium and further cardiac infiltration within injured

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myocardium compared with healthy subjects [100]. This T cell recruitment was also associated with HF progression due to induced cardiac fibrosis and

hypertrophy. Whereas Th1 cells are involved in acute inflammation response and cytokine secretion, Th1 cells participate in chronic remodeling response and fibroblast activation [101].

During the first days following cardiomyocyte necrosis, circulating inflammatory cells are recruited to the site of injury, guided by a gradient in concentration of chemokines [88]. They participate in the wound-healing response through degrading and removing dead cells by proteolysis and phagocytosis. The Zn²⁺-dependent MMP, activated by ROS, paly a major

function in this process [102]. Macrophages M1 are pro-inflammatory mediators and participate in the acute response post-cardiomyocyte injury. In contrast, macrophages M2 show anti-inflammatory properties and are critical in

wounding healing [88]. On the other hand, mast cells also exert pro-fibrotic and inflammatory functions. Among their released products, histamine, for instance, has been associated with fibroblast stimulation and fibrosis [88].

4.2.6. Renin-angiotensin-aldosterone system and cardiac remodeling During early stages of wound repair, macrophages that express renin and angiotensin-converting enzyme (ACE), produce angiotensin II (AII) at the site of injury [103]. Via its binding to AII-type 1 receptor (AT₁), AII stimulates upregulation of TGF-β₁expression and segregation by inflammatory cells [104].

Furthermore, released TGF-β₁ induces myofibroblasts differentiation and collagen synthesis. In addition, TGF-β₁ suppresses myofibroblast gene

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expression of MMP. The ultimate effect is an altered equilibrium between collagen synthesis/degradation and thus, larger MIF.

4.2.7. Assessment of myocardial interstitial fibrosis

Endomyocardial biopsy (EMB) is the gold standard for MIF diagnosis.

However, due to its limitations, other techniques have been emerged.

- Circulating biomarkers: only 3 have demonstrated an association with histologically proven MIF [98]:

- Serum carboxy-terminal propeptide of procollagen type I (PICP): it is generated during conversion of procollagen type I into collagen type I and released from the heart into the circulation in HF patients [105]. Of note, serum concentrations correlate with severity of both MIF and HF, together with mortality and ventricular arrhythmias [98].

- Serum amino-terminal propeptide of procollagen type III (PIIINP): it is generated from the conversion of procollagen type III into collagen type III. It also correlates with severity and outcomes in HF [98].

- Serum collagen type I telopeptide-to-serum matrix metalloproteinase-1 ratio (CITP-to-MMP-1 ratio): the higher this ratio, the lower the cleavage of this peptide by MMP enzyme and thus, higher resistance of the peptide [98].

Circulating biomarkers may present several limitations since they are not cardiac-specific and their concentration may be influenced by altered

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- Imaging biomarkers: promising results have been demonstrated with cardiac magnetic resonance (CMR). These techniques require the use of gadolinium-based contrast agents, which shorten longitudinal relaxation time (T1) of tissues in which they accumulate, providing a high-intensity signal (white coloured) on T1-weighted imaging [106]. Contrast agents invade extracellular space and are washed out at a slower rate within the injured myocardium. Among these procedures it is worth to mention:

- Late gadolinium enhancement (LGE): it enables to detect focal myocardial fibrosis and it shows prognostic correlation. Nevertheless, this specific CMR sequence cannot detect diffuse cardiac fibrosis [98].

- T1 mapping: is the cornerstone of MIF. These techniques interrogate tissue recovery from longitudinal magnetization following saturation or inversion pre-pulses [106] and they include:

- Native (pre-contrast) T1 values: they reflect the combined

extracellular and intracellular compartments and are measured on a per-segment basis. They are increased in myocardial fibrosis [106].

- Extracellular volume fraction (ECV%): it targets only the extracellular space and it represents the extracellular matrix as a proportion of total LV myocardial volume [106].

- Indexed extracellular volume (iECV): it also targets solely the extracellular space but, contrarily to ECV%, it adjusts for LV myocardial volume (ECV% multiplied by LV myocardial volume) [106].

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Replacement fibrosis correlates with LGE whereas reactive fibrosis can be detected by T1 mapping [106]. It is worth to mention that ECV techniques quantify very precisely diffuse MIF in he absence of myocardial oedema or myocardial infiltrative disease, such as amyloidosis [98]. Due to technique limitation and partial volume effect, T1 mapping is mostly confined to the mid myocardium because pixels straddling the subendocardial or subepicardial border may be contaminated by blood pool or epicardial fat, respectively [98].

4.2.8. Overview of anti-fibrotic therapies for cardiovascular disease There are several treatments that have showed to reduce cardiac fibrosis in animal models. However, some of them have not been tested in humans or they have proved disappointing outcomes in clinical trials.

- RAAS inhibitors: AII interacts with its receptor type I and stimulates fibroblast proliferation and collagen synthesis. Several clinical trials have demonstrated that both ACEi and ARB reduce cardiac fibrosis regardless of their antihypertensive effect [107,108]. In addition, MRA (spironolactone and eplerenone) have showed to reduce cardiac fibrosis in humans

[109,110]. However, the population in these studies is rather small and RAAS inhibitors only demonstrated a modest regression of cardiac fibrosis [111].

- Beta-blockers: they have demonstrated to prevent cardiac fibrosis and improve survival rate in an animal model of diastolic dysfunction [112].

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However, they have showed inconsistent results [113,114] and whether or not beta-blockers attenuate cardiac fibrosis in humans remains unknown.

- Anti TNFα therapies: TNFα plays an important role in cardiac fibrosis.

Nonetheless, few studies have failed to improve CV outcomes in patients with HF including the RENEWAL trial with etanercept [115], and the ATTACH trial with infliximab [116].

- Statins: they exert a potent anti-inflammatory effect. Although rosuvastatin showed positive results reducing cardiac fibrosis in animal models [117], overall the statin’s effect on cardiac fibrosis in human are generally disappointing [111].

- TGF-β inhibitors: alike TNFα, TGF-β also is decisive in activating cardiac fibrosis. Various TGF-β inhibitors including pirfenidone and tranilast have been showed to reduce cardiac fibrosis in animal studies [118]. However, they have not showed consistent results in clinical trials and they have also been associated with significant adverse events such as liver dysfunction [111].

- Other anti-fibrotic therapies: the impact of other anti-fibrotic agents has been evaluated in CV clinical trial. These drugs include bosentan

(endothelin inhibitor), torasemide (loop diuretic), MMP inhibitors, sildenafil and relaxin (vasodilator hormone). Nonetheless, they showed negative or neutral effects on improving cardiac adverse remodeling and CV outcomes.

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5. Left ventricular diastolic function

5.1. Introduction to diastology

In broad terms, one can refer to cardiac diastolic function as two main aspects: myocardial relaxation and chamber stiffness. Its assessment is more challenging as it is modulated by several parameters, including right ventricle (RV) - LV interaction, LA function, pericardial influence, coronary blood flow and cardiomyocyte perfusion [119].

During isovolumetric relaxation (IVR) of cardiac cycle, LV pressure declines rapidly. When intraventricular pressure falls below atrial pressure, a pressure gradient between these two chambers is established and mitral valve opens. Thereafter, LV fills quickly, giving rise to E-velocity (i.e. early diastole).

During diastasis, LV and LA pressures equilibrate and transmitral flow occurs at a low velocity. Finally, atrial contraction causes late diastolic filling, giving rise to A-velocity (i.e. end diastole) [120].

Transmitral pressure gradient represents the driving force for the transmitral flow. As long as this gradient is positive, mitral velocity increases and reaches the peak when pressure gradient is zero. Thereafter, when gradient reverses, mitral flow decelerates [120,121].

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Figure 3. Tracing of intracardiac pressures during cardiac cycle and mitral inflow correlation [121].

Empagliflozine improves myocardial fibrosis and diastolic function in a non-diabetic porcine model of ischaemic heart failure