Canine chronic valvular heart disease -how does myxomatous degeneration of the mitral valve ultimately lead to congestive heart failure?

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1 Canine chronic valvular heart disease -how does myxomatous degeneration of the mitral valve ultimately lead to congestive heart failure? Anneka Tuomola Licentiate thesis in veterinary medicine Small Animal Diseases, Department of Equine and Small Animal Medicine Faculty of Veterinary Medicine University of Helsinki 2019

2 Tiedekunta - Fakultet Faculty Eläinlääketieteellinen Tiedekunta Osasto - Avdelning Department Kliinisen hevos- ja pieneläinlääketieteen osasto Tekijä - Författare Author Anneka Tuomola Työn nimi - Arbetets titel Title Canine chronic valvular heart disease -how does myxomatous degeneration of the mitral valve ultimately lead to congestive heart failure? Oppiaine - Läroämne Subject Pieneläinsaraudet Työn laji - Arbetets art Level Lisensiaatin tutkielma Tiivistelmä - Referat Abstract Aika - Datum Month and year Huhtikuu 2019 Sivumäärä - Sidoantal Number of pages 67 Koirien krooninen sydänläpän sairaus (CVHD) on rappeumasairaus, jonka pääkohde on mitraaliläppä. Sairaus on yleinen, aiheuttaen 75% sydän- ja verenkiertoelimistön sairauksista. Myksomatoottinen läppärappeuma aiheuttaa läpän vuotoa ja veren regurgitaatiota eteiseen. Regurgitaatiota pystytään kompensoimaan, usein vuosien yli, eikä tämä aiheuta koiralle oireita. Läppärappeuman edetessä regurgitoitu osa iskutilavuudesta kuitenkin kasvaa, ja dekompensaatiopisteen saavuttaminen voi ajankohtaistua. Tässä kirjallisuuskatsauksessa käsittelen sydämmen sisäisiä ja ulkoisia kompensatorisia muutoksia jotka tukevat riittävän keskiverenpaineen ylläpitämistä CVHD:ssä siitä huolimatta, että eteenpäin suuntautuva iskutilavuus on alentunut. Käsittelen myös niiden epäonnistumista. Ulkoisista tekijöistä sympaattinen hermosto ja reniini-angiotensiini-järjestelmä aktivoituvat. Suuri osa tutkielmasta keskittyy verenkierron reniini-angiotensiini-järjestelmän moninaisiin ja pitkälle kantautuviin seurauksiin. Angiotensiini II, sekä sen aktivoimat aldosteroni ja vasopressiini, vaikuttavat antidiureettisesti ja antinatriureettisesti, nostaen verentilavuutta ja näin verenpainetta. Natriureettinen peptidi-järjestelmä vastapainottaa reniini-angiotensiini-järjestelmän vaikutuksia. Sisäisesti, sydän kompensoi rekrytoimalla Frank-Starling mekanismin sekä eksentrisellä hypertrofialla. Sydämen reniini-angiotensiinijärjestelmä sekä bradykiniini ovat eksentrisessa hypertrofiassa osallisena ja niiden vaikutukset ovat yleisesti vastakohtaiset. Kongestiivinen sydämmen vajaatoiminta CVHD:ssä johtuu lähinnä regurgitoitudun verivolyymin kasvusta. Tämä johtaa ensin eteisen laajentumaan, sit eteispaineen kasvuun, ja lopulta keuhkoödeemaan. Tämän tutkielman tarkoitus on kaksitahoinen. Se voi toimia CVHD:n kompensatoristen muutosten katsauksena. Selostan kuitenkin myös miten osalliset hormonit ja kudoshormonit vaikuttavat reseptoreihinsa. Molekulaaristen mekanismien tuntemus toimii aloituskohtana, mikäli taudinkuvaa halutaan muokata lääkkeellisesti. Säätelymekanismit ovat myös samankaltaisia reseptorin sijainnista huolimatta ja näin usein yleisesti sovelettavissa, varsinkin reniini-angiotensiini-järjestelmän kohdalla. Katsauksen tieto voi myös hyödyntää lääkäreitä, sillä ihmisillä on todettu hyvin samankaltainen sairaus. Avainsanat Nyckelord Keywords Sydän, mitraaliläppä, reniini-angiotensiini-järjestelmä, eksentrinen hypertrofia, volyymiylikuormitus, natriureettinen peptidi Säilytyspaikka Förvaringställe Where deposited HELDA Helsingin Yliopiston digitaalinen arkisto Työn johtaja (tiedekunnan professori tai dosentti) ja ohjaaja(t) Instruktör och ledare Director and Supervisor(s) Johtaja: professori Outi Vapaavuori Ohjaajat: ELT Anna Hielm-Björkman

3 Tiedekunta - Fakultet Faculty Faculty of Veterinary Medicine Tekijä - Författare Author Osasto - Avdelning Department Department of Equine and Small Animal Medicine Anneka Tuomola Työn nimi - Arbetets titel Title Canine chronic valvular heart disease -how does myxomatous degeneration of the mitral valve ultimately lead to congestive heart failure? Oppiaine - Läroämne Subject Small Animal Diseases Työn laji - Arbetets art Level Licentiate thesis Tiivistelmä - Referat Abstract Aika - Datum Month and year April 2019 Sivumäärä - Sidoantal Number of pages 67 Canine chronic valvular heart disease (CVHD) is a degenerative disease, primarily affecting the mitral valve apparatus. The disease is common, accounting for about 75% of cardiovascular diseases in the dog. Myxomatous degeneration of the valve causes valvular insufficiency, and regurgitation of blood into the atrium. This is well compensated, often over years, and does not at onset cause outward symptoms in the dog. However, as valvular degeneration continues, and the regurgitant fraction of the total stroke volume grows, a point of decompensation may be reached. In this literature review I discuss the compensatory changes that occur in CVHD, intrinsic and extrinsic, that allow for the maintenance of a satisfactory mean arterial pressure despite the reduction in forward stroke volume, and also, how they eventually fail. Extrinsically, there is activation of the sympathetic nervous system as well as the renin-angiotensin system. A large part of the thesis focuses on the far reaching consequences of circulatory renin-angiotensin activation. The antidiuretic and antinatriuretic effects of angiotensin II and the hormones it activates, namely aldosterone and vasopressin, lead to an increase in blood volume, aiding in blood pressure upkeep. The natriuretic peptide system acts to counterbalance the effects of renin-angiotensin system activation. Intrinsically, the heart compensates by recruitment of the Frank-Starling mechanism, as well as through eccentric hypertrophy. Eccentric hypertrophy involves the cardiac renin-angiotensin system and also bradykinin, with generally opposing effects. In CVHD, congestive heart failure occurs foremost by increase of the regurgitant fraction, that at first leads to atrial enlargement, then to increased atrial pressures, and eventually pulmonary edema. The function of this thesis is twofold. It can act as a reference for the compensatory changes that occur particularly in CVHD. It also attempts to explain general molecular mechanisms of interactions between the hormones and autacoids involved and their receptors. This is a necessary starting point for possible medical modulation of the disease. Also, mechanisms are similar regardless of receptor location, thus the thesis has general applicability, especially with regards to the renin-angiotensin system. The information presented may also benefit the medical community, as humans are afflicted with a very similar disease. Avainsanat Nyckelord Keywords Heart, mitral valve, renin-angiotensin system, eccentric hypertrophy, volume overload, natriuretic peptide Säilytyspaikka Förvaringställe Where deposited publication repository HELDA Työn johtaja (tiedekunnan professori tai dosentti) ja ohjaaja(t) Instruktör och ledare Director and Supervisor(s) Director: professor Outi Vapaavuori Supervisors: ELT Anna Hielm-Björkman

4 TABLE OF CONTENTS 1 Introduction 1 2 Literature review Canine chronic valvular heart disease, what is it? Hemodynamics Hemodynamics, basic concepts Hemodynamics in CVHD Cardiac output and mean arterial pressure Activation of the sympathetic nervous system Activation of the renin-angiotensin system and other hormonal changes Renin Angiotensin converting enzyme Angiotensin II Angiotensin II in the brain Aldosterone Vasopressin Oxytocin and the natriuretic peptides The pathophysiology of eccentric hypertrophy Volume overload, systolic dysfunction and congestive heart failure 38 3 Discussion 41 4 References 44

5 1 INTRODUCTION Canine Chronic Valvular Heart Disease (CVHD) is a degenerative disease, primarily affecting the dog's mitral valve and its tendinous chords. The degeneration leads to valvular prolapse and insufficiency (in the review by Pedersen & Häggström 2000). During systole, a part of the blood volume which should enter the aorta regurgitates into the left atrium instead. When the reduced forward stroke volume cannot be compensated for intrinsically this reduces blood pressure. When registered by various baroreceptors compensatory systemic neuroendocrine changes lead to a rise in blood pressure, sustaining adequate organ perfusion despite the mitral valvular insufficiency (in the review by Häggström et al. 2004). Local neuroendocrine changes also occur, probably early on in the disease (Fujii et al. 2007, in the review by Dillon et al. 2012). Although mitral valvular degeneration is often a rather benign, slowly progressing disease (Borgarelli et al. 2012), not only does the valvular degeneration progress, but the recruited neuroendocrine compensatory mechanisms play a part in disease progression and modification (in the review by Häggström et al. 2004). Neuroendocrine activation leads both to cardiac remodeling indirectly, by increasing blood volume and thus cardiac wall stress, as well as directly, as elevated levels of autacoids and hormones have a direct effect on cardiac remodeling through their cardiac receptors (in the review by Dillon et al. 2012, in the review by McCutcheon & Manga 2018). Mitral valvular degeneration has thus progressed to a disease encompassing the whole heart. Even though cardiac remodeling affects systolic function to some degree (Borgarelli et al. 2007), it is typically the increasing hypervolemia which ultimately leads to pulmonary edema and congestive heart failure (Kittleson & Brown 2003). Not only does CVHD account for about 75% of cardiovascular diseases in dogs (in the review by Häggström et al. 2004, Atkins et al. 2009, in the review by Borgarelli & Häggström 2010), marked degenerative changes of the mitral valves are seen in in more than 50% of dogs post-mortem (in the review by Pedersen & Häggström 2000). While efforts should be, and have been, made in certain breeds to decrease the incidence of myxomatous mitral valve degeneration through breeding schemes (Birkegård et al. 2016), efforts should also be made to understand disease progression since it will remain a prevalent disease for a long time to come. An understanding of how the disease progresses 1

6 can possibly lead to earlier and more targeted medical intervention, and a further deceleration in the progression to congestive heart failure. In this thesis I will review how activation of the neuroendocrine compensatory mechanisms in CVHD lead to cardiac remodeling and ultimately heart failure. 2 LITERATURE REVIEW 2.1 Canine chronic valvular heart disease, what is it? Canine Chronic Valvular Heart Disease (CVHD) is a degenerative disease, primarily of the heart's mitral valve apparatus. In about a third of cases the tricuspid valve is also involved (in the review by Häggström et al. 2004, Atkins et al. 2009). In a few percent of cases the tricuspid valve is the only diseased valve (in the review by Häggström et al. 2004, Garncarz et al. 2013). The semilunar valves are very rarely affected by CVHD (in the review of Häggström et al. 2004). Thus, throughout this thesis, I will discuss the effects of mitral valvular incompetence. Chronic valvular heart disease is known by several names such as degenerative or myxomatous mitral valve disease or endocardiosis (in the review by Häggström et al. 2004). Chronic valvular disease accounts for about 75% of cardiovascular diseases in the dog (in the review by Häggström et al. 2004, Atkins et al. 2009, in the review by Borgarelli & Häggström 2010). It usually affects older small breed dogs such as Dachshunds, Chihuahuas, Poodles, Whippets or Miniature Schnauzers (Mattin et al. 2015). The Cavalier King Charles Spaniel is especially affected, with 90% of the population showing signs of the disease by ten years of age (in the review by Borgarelli & Häggström 2010, in the review of Parker & Kilroy-Glynn 2012). A polygenic inheritance has been suggested for both the Cavalier King Charles Spaniel and the Dachshund (Olsen et al. 1999a, in the review by Häggström et al. 2004). Recently, genome wide association studies have been performed in several breeds identifying various single nucleotide polymorphisms (SNP) associated with CVHD (Madsen et al. 2011, French et al. 2012, Stern et al. 2015, Lee et al. 2019). Based on the results a polygenic mode of inheritance is favored. 2

7 Myxomatous degeneration, as is the case in CVHD, leads to lenghthening and thickening of the valve leaflets and elongation of the chordae tendinae (in the review by Pedersen & Häggström 2000, in the review by Häggström et al. 2004). Thickening is seen primarily distally, at the leaflet tips, even in advanced disease (Han et al. 2010). Degeneration leads to prolapse of the valve leaflets into the atrium, poor coaptation of the leaflets and thus valvular incompetence (Olsen et al. 1999a, in the review by Häggström et al. 2004, in the review by Borgarelli & Häggström 2010). The turbulent blood flow created by regurgitation of blood into the atrium results in the left apical systolic murmur heard upon auscultation (in the review by Häggström et al. 2004). Murmur intensity correlates positively with the regurgitant jet size into the atrium (Olsen et al. 1999a, Ljungvall et al. 2009). Histologically, myxomatous degeneration of the fibrosa layer of the leaflet is characterized by a reduction of connective tissue density leading to its derangement, a reduction in valvular interstitial cell number in myxomatous areas, and an increase in glucosaminoglycans (in the review by Pedersen & Häggström 2000, in the review by Häggström et al. 2004, Han et al. 2010). Even in severely diseased leaflets, distinctly myxomatous areas were confined to the leaflet tips (Han et al. 2010). In scanning electron microscope studies patchy damage to the endothelial lining of both the valves and the chordae tendinae was evident (Corcoran et al. 2004). In human medicine a disease strongly resembling CVHD is termed mitral valve prolapse (MVP), and several researchers have compared the two (Olsen et al. 1999a, in the review by Pedersen & Häggström 2000, in the review by Häggström et al. 2004, Falk et al. 2006, Han et al. 2010, Madsen et al. 2011). It has been suggested that CVHD could act as a model for the human counterpart, due to faster disease progression and a prevalence of CVHD in dogs being roughly ten times greater than MVP in humans (in the review by Pedersen & Häggström 2000). Despite canine CVHD typically progressing faster than the human counterpart, it is still often a disease of slow progression, especially before reaching heart failure. In a study following 256 nonsymptomatic dogs (ACVIM stages B1 and B2, see below) over a median time of 756 days, more than half had not progressed to heart failure. Eight percent 3

8 of dogs had developed heart failure but were alive, and 12% had died from CVHD. Noncardiac related deaths amounted to roughly 15% (Borgarelli et al. 2012). To be able to distinguish dogs with different CVHD severity, researchers have used various forms of disease staging. The staging used by researchers whose work is referenced to in this thesis is tabulated below. The significance of left atrial or cardiac enlargement used in disease staging is discussed foremost in section 2.6. Table 1. Disease staging used by various researchers Type of staging Staging Description Echocardiographic, based on left atrial to aortic root ratio (LA:Ao ) (Kittleson & Brown 2003) Echocardiographic, based on regurgitant jet (RJ) and LA:Ao (Ljungvall et al. 2009) or RJ only (Ljungvall et al. 2011a,b) Based on a modified New York Heart Association (NYHA) classification scheme (Häggström et al. 2000) Based on the International Small Animal Cardiac Health Council (ISACHC) classification scheme (Garncarz et al. 2013) Based on the American College of Veterinary Internal Medicine (ACVIM) Consensus Statement (Atkins et al. 2009) Mild Moderate Severe Mild Moderate Severe Class I Class II Class III Class IV Class I 1a 1b Class 2 Class 3 3a 3b Stage A Stage B LA:Ao LA:Ao LA:Ao Cutoff values not given RJ < 30% (LA:Ao 1.5) RJ 30-50% (LA:Ao < 1.8) RJ > 50% (LA:Ao 1.8) Murmur, no radiologic or echocardiographic evidence of cardiomegaly. Murmur, cardiomegaly, no clinical or radiological evidence of pulmonary congestion or edema. Murmur, cardiomegaly and radiological evidence of pulmonary congestion and edema. Murmur, pulmonary congestion and pulmonary edema, severe dyspnea Asymptomatic No cardiomegaly Cardiomegaly Mild to moderate heart failure Severe heart failure No hospitalization required Hospitalization required At high risk for developing heart disease without current structural disorder of the heart Structural heart disease without clinical signs of heart failure, past or present. 4

9 B1 B2 Stage C Stage D No radiographic or echocardiographic evidence of cardiac enlargement Radiographic or echocardiographic evidence of left-sided cardiac enlargement. Past or present clinical signs of heart failure associated with structural heart disease. End-stage heart failure, refractory to standard therapy 2.2 Hemodynamics As some of the blood which should be entering the aorta regurgitates into the left atrium during ventricular systole, mitral valvular incompetence leads not only to a reduction in forward stroke volume, but also to pressure and volume changes in the cardiac chambers. One way to visualize ventricular pressure and volume changes is with the help of pressure-volume loops, figure Hemodynamics, basic concepts Before discussing the hemodynamic effects of mitral regurgitation an understanding of the cardiac cycle, as illustrated by the pressure-volume loop of the left ventricle, and the key variables that affect stroke volume is paramount. One cardiac cycle comprises one systole, in which ventricles contract, and one diastole, when ventricular relaxation occurs. In the pressure-volume loop of the left ventricle of a healthy heart (figure 1, solid line loop) systole starts just before point B. Ventricular pressure almost instantly rises above that of the atrium, closing the mitral valve at point B. Between points B and C all valves are closed, and the ventricular volume remains constant. Isovolumetric contraction raises ventricular pressure until the pressure opens the aortic valve, at point C; blood flows into the aorta decreasing ventricular volume. At point D, aortic pressure has exceeded that of the left ventricle, and the aortic valve closes. This is also the start of diastole. During isovolumetric relaxation (between points D and A) all valves are again closed. At point A atrial pressure opens the mitral valve allowing 5

10 for ventricular filling during the latter part of diastole, between points A and B. Then, a new cardiac cycle begins (Mitchell & Wang 2014). Figure 1. Pressure-volume loops for the left ventricle of a healthy heart (solid line), a heart with acute mitral regurgitation (dashed line), and a heart with chronic mitral regurgitation (dotted line). The letters A, B, C and D apply to the healthy heart. Point A: opening of the mitral valve. Point B: closing of the mitral valve. Point C: opening of the aortic valve. Point D: closing of the aortic valve. The shape of the pressure volume loops are adapted from human research (Gämperli et al. 2013) and software simulations (Martinez-Legazpi et al. 2017) while the end systolic and end diastolic volumes and peak systolic pressures are obtained from mongrel dogs, average weight 28,6 kg, before, and after 4 days and 4 weeks of experimental mitral regurgitation (Katayama et al. 1988) Stroke volume (SV, equation 1) is the difference between the end diastolic volume (EDV), the volume at point B, and the end systolic volume (ESV), at point D, and is thus the volume of blood pumped by the ventricle in one cardiac cycle (in the review by Hezzell 2018). The stroke volume depends on three components: preload, afterload and inotropy (Vincent 2008). 6

11 SV = EDV ESV eq. 1 The preload is defined as the maximum stretch of the sarcomeres before contraction. Autoregulation (intrinsic regulation) of the heart under normal conditions depends on this stretch; the more the sarcomeres are stretched (within limits), the stronger the ensuing cardiomyocyte contraction. This is known as the Frank Starling Law (Vincent 2008). The stretch of sarcomeres is dependent on the end diastolic pressure (EDP) or the end diastolic volume (EDV) to which preload is often equated (Delicce & Makaryus 2017). Figure 2 shows several Frank-Starling curves. A higher left ventricular EDP leads to a bigger stroke volume. The afterload is the force against which the ventricles must contract to push blood into the circulation (Vincent 2008, Delicce & Makaryus 2017). When the afterload is reduced, for example by reducing the aortic pressure, ventricular contraction can occur more rapidly; a faster ejection velocity during systole lowers the end systolic volume thus increasing the stroke volume. On the other hand increasing afterload will lower the stroke volume. An increase in afterload shifts the Frank-Starling curve to lower stroke volumes for a given EDP, similarly to a decrease in inotropy, discussed below (Delicce & Makaryus 2017). Afterload can also be viewed as the ventricular end systolic wall stress. Various mathematical models for wall stress calculations have been used (in the review by Yin 1981, Katayama et al. 1988, Olivetti et al. 1990, Perry et al. 2002, Pat et al. 2008). The simplest is based on the Law of LaPlace, in which the ventricle is approximated to a sphere (in the review by Yin 1981). This relationship between wall stress ( ), ventricular pressure (P), its radius (r) and wall thickness (h), is typically known as the LaPlace equation in cardiology, equation 2 (Olivetti et al. 1990, in the review by Carabello 2002). = P * r eq. 2 2 h I will return to this equation in section

12 Inotropy, the third parameter affecting stroke volume, refers to a change in contractile strength, that, unlike preload, is indepedent of sarcomere stretch. Several factors affect inotropy, or contractility, the most prominent being autonomic nervous control (Delicce & Makaryus 2017). Cardiac drugs, such as pimobendan, can also act as inotropes (Boswood et al. 2016). Increased contractility leads to more rapid contraction, which again leads to a faster ejection velocity, lower end diastolic volume and higher stroke volume. In the Frank-Starling curves depicted below (figure 2), the effect of increased or decreased inotropy is seen as an upward or downward shift of the curve at given left ventricular EDP. Figure 2: Frank-Starling Curves. As the end diastolic pressure (EDP) increases so does the stroke volume. At maximun sarcomere stretch further increases in the EDP will no longer increase stroke volume. Increased inotropy, such as through sympathetic stimulation, shifts the curve to higher stroke volumes regardless of EDP. Adapted from a book figure (Miller and Adams 2009) Hemodynamics in CVHD The pressure-volume loops of acute and chronic mitral regurgitation, dashed and dotted line loops respectively (figure 1), have distinctly different shapes from the pressurevolume loop of the healthy heart. Because of mitral valvular insufficiency, isovolumetric contraction and relaxation do not occur. This becomes especially apparent during systole, when regurgitation into the atrium causes an appreciable loss of blood volume from the ventricle during what should be the isovolumetric phase (B to C). Thus the left atrium receives blood from both the ventricle and the pulmonary veins during ventricular systole, 8

13 and when this volume is transferred to the ventricle in diastole, the end diastolic volume grows. According to the Frank-Starling Law the ensueing ventricular contraction will be stronger to compensate. The increased stroke volume is seen as a broader pressurevolume loop. Increased stroke volumes compensate for reduced forward stroke volumes inherent to CVHD (Häggström et al. 1996, Borgarelli et al. 2007). It is likely the first compensatory mechanism to be recruited (in the review by Häggström et al. 2004). In chronic mitral regurgitation (figure 1, dotted line loop) not only is the stroke volume even larger, but the whole loop is typically shifted to higher volumes. The neuroendocrine changes that occur to increase blood volume and the cardiac remodeling that occurs to accomodate the larger blood volumes, allowing continued compensation via the Frank- Starling mechanism, are the main focus of the remainder of the thesis. In congestive heart failure exhaustion of the Frank-Starling mechanism can occur (Komamura et al. 1993) Cardiac output and mean arterial pressure Cardiac output (CO), expressed in liters per minute, is the product of heart rate (HR) and stroke volume (eq. 3). Mean arterial pressure (MAP), on the other hand, is the product of cardiac output and systemic vascular resistance (SVR, eq. 4). CO = SV * HR eq.3 MAP = CO * SVR eq. 4 Thus, apart from altering stroke volume as discussed above, mean arterial pressure can be affected by changing heart rate and the systemic vascular resistance. The effect of the autonomic nervous system on retaining MAP despite a reduced forward stroke volume in CVHD is discussed below. 2.3 Activation of the sympathetic nervous system Nagatsu et al. (1994b) showed, in a dog model of severe experimental mitral regurgitation achieved by severing chordae tendinae, that serum catecholamines were significantly increased both one and three month post chordae tendinae rupture. Also, despite the expected drop in cardiac output, pump- and contractile function where maintained in a 9

14 normal or near-normal range in these dogs until onset of acute esmolol induced betablockade, whereon these indices dropped significantly. Thus the authors concluded betaadrenergic support maintained the indices at near-normal levels (Nagatsu et al. 1994). Beta-adrenergic support, an increase in central sympathetic outflow, is due primarily to the deactivation of the arterial baroreceptor reflex. The arterial baroreceptor reflex is a negative feedback loop regulating blood pressure. In short, when blood pressure falls, because of a decrease in forward stroke volume due to CVHD for example, the reduced stretch of the baroreceptors of the aortic arch and carotid sinus decrease the firing frequency of action potentials to the nucleus tractus solitarius (NTS) of the medulla oblongata (in the review by Antunes-Rodriguez et al. 2004, in the review by Kumagai et al. 2012). This also decreases the activity to the caudal ventrolateral medulla (CVLM) via the NTS. Inhibitory GABAergic neuron activity from the CVLM to the rostral ventrolateral medulla (RVLM), a nucleus primarily involved in blood pressure regulation, leads to increased sympathetic outflow to target organs via the intermediolateral cell column (in the review by Guyenet 2006, in the review by Kumagai et al. 2012, in the review by Lohmeier & Iliescu 2015). Typically, sympathetic outflow occurs in parallel to the heart, the kidneys, the resistance arterioles and capacitance veins (except vessels of the skin) as well as the adrenal medulla (in the review by Guyenet 2006, in the review by Kumagai et al. 2012). Via the release from cardiac sympathetic nerve fibers, noradrenaline concentrations rise in the cardiac interstitial fluid. Increased adrenal catecholamines also reach the heart via the circulation. Activation of the Gs-protein coupled 1-adrenergic receptors in cardiomyocytes leads to camp activation of protein kinase A (PKA) and phosphorylation of a number of proteins, amongst them calcium channels, affecting cytosolic calcium concentrations. Since calcium is essential for cardiomyocyte excitation-contraction coupling, increased cytosolic calcium due to sympathetic stimulation leads to increased inotropy as seen in figure 2. Myocyte relaxation is also hastened (in the review by Marks 2003, in the review by Fearnley et al. 2011). Activation of the 1-adrenergic receptors on the sinoatrial node cells leads to increased chronotropy by camp/pka signaling (Behar et al. 2016). Parasympathetic activity would, in turn, decreases camp/pka signaling (Behar et al. 2016). As the autonomic nervous system so heavily influences heart rate 10

15 through sinoatrial node receptors heart rate variability can be used as a measure of autonomic tone (Rasmussen et al. 2012). Several researchers have thus used heart rate variability (HRV) as a means of quantifying changes in autonomic nervous system activity in mitral regurgitation and the congestive heart failure that follows. Typically HRV was compared between dogs in different stages of CVHD and healthy controls (Häggström 1996, Olsen et al. 1999b, Doxey & Boswood 2004, Oliveira et al. 2012, Rasmussen et al. 2011, Rasmussen et al. 2012, Oliveira et al. 2014, Lopez-Alvarez et al. 2014, Pirintr et al. 2017). One research group caused experimental mitral regurgitation in healthy Beagles by severing chordae tendinae, using the dogs pre-surgery as their own controls (Fujii & Wakao 2003). Different time-domain and frequency-domain heart rate variability indices were calculated from ECG or Holter readings. When comparing dogs with congestive heart failure to healthy controls, all researchers found significantly lowered heart rate variability indices in CHF dogs, indicating a dysbalance between the sympathetic (increased) and parasympathetic (decreased) nervous system. A few researchers (Oliveira et al. 2012, Rasmussen et al. 2012, Oliveira et al. 2014, Pirintr et al. 2017) even found one or several significantly lowered HRV indices in stage B of CHVD. Increasing cardiac chronotropy and inotropy, as discussed above, increases cardiac output and mean arterial pressure (equation 3 & 4). Sympathetic activity also leads to vasoconstriction, through an increase in intracellular calcium mediated by the 1- adrenergic receptors, of the resistance arterioles and capacitance veins (Tallaj et al. 2003, in the review by Stupin et al. 2017). Increasing systemic vascular resistance further increases MAP (equation 4). Lastly, by activating the 1-adrenergic receptors of the renal juxtaglomerular cells, camp is once again increased. In the juxtaglomerular cells this increases secretion of renin, ultimately leading, via the renin-angiotensin system, to increased blood volume and systemic vascular resistance and thus increased MAP. Renal blood pressure reduction due to sympathetic stimulation also increases renin secretion, as discussed in more detail below (section 2.4.1). 11

16 2.4 Activation of the renin-angiotensin system and other hormonal changes Classically, the renin-angiotensin system (RAS) is a system primarily concerned with blood pressure control, as well as blood volume and electrolyte homeostasis. When the aspartyl protease renin is secreted from the renal juxtaglomerular cells, for example by sympathetic stimulation as described above, the prohormone angiotensinogen is cleaved to angiotensin I (in the review by Kurtz 2012). Further cleavage to angiotensin II (Ang II), typically by angiotensin converting enzyme (ACE), produces a highly potent blood pressure and blood volume augmenting hormone, through its very diverse effects on the kidneys, the adrenal glands and the brain. Ang II increases blood levels of aldosterone and vasopressin, adding to its effects (in the review by Fyhrquist & Saijonmaa 2008). Ang II also modulates neurons, both short-term, increasing firing rates, and long-term, increasing noradrenaline synthesis (in the review by Sumners et al. 2002). Thus, there is intense cross-talk between the RAS and the nervous system, both centrally and peripherally, adding to the complexity of the RAS and its activation (in the review by Stupin et al. 2017). The actions of Ang II discussed in this thesis are mediated by the AT1 receptor. After the identification of the classical RAS components in the early 1970s, the diversity of the system has grown significantly. Several new enzymes, new biologically active molecules and new receptors have been discovered, some of which balance the actions of Ang II acting on the AT1 receptors. Also, apart from the classical circulatory RAS many tissue specific, and even intracellular RAS have been discovered (in the reviews by Fyhrquist & Saijonmaa 2008 and Karnik et al. 2015). The cardiac RAS is central to cardiac remodeling. Also, it may be upregulated earlier than the classical circulatory RAS and so contibute significantly to the progression of CVHD (Fujii et al. 2007). Before getting into the complexities of the circulating and tissue RAS as it pertains to CVHD, an important first question to answer is at what point in CVHD is renin secretion increased? 12

17 2.4.1 Renin As the sympathetic nervous system, using heart rate variability analysis, was shown to be activated in stage B of CVHD in some studies (section 2.3) one might expect increased plasma renin activity in the early stages of CVHD. Indeed, this often seems to be the case. When comparing healthy Cavalier King Charles Spaniels to those with mitral insufficiency but without radiographic signs of congestive heart failure plasma renin activity was increased (P = 0.03). There was, however, significant overlap in plasma renin activity between control and diseased dogs. Also, there were no significant differences in plasma renin activity depending on whether mitral insufficiency had caused left atrial enlargement or not (Pedersen et al. 1995). In a study on Dachshunds without signs of heart failure, the amount of mitral valve prolapse (none, > 0 mm 1 mm, > 1mm) correlated positively with plasma renin activity, however, no correlation was found with regards to regurgitant jet class (Pedersen et al. 1998). In a separate experiment, where the sodium content of the diet was standardized, and dogs were sampled both lying on their side and while sitting or standing after 10 minutes of walking (in an attempt to increase sympathetic tone) no correlations to either jet class or the amount of mitral valve prolapse could be found. Thus the researchers concluded that the association between increased plasma renin activity and early CVHD could not be reproduced under all study conditions (Pedersen et al. 1998). Also, plasma renin activity needn't be elevated at all; when mild mitral regurgitation was experimentally induced by severing chordae tendinae and circulating RAS components were measured approximately 3 years later, plasma renin activity (and thus no other components of the RAS) wasn't elevated. The dogs in this study all had substantial eccentric hypertrophy yet no heart failure (Fujii et al. 2007). While prorenin, a larger precursor of renin without enzymatic activity, is constitutively secreted, only the juxtaglomerular cells of the juxtaglomerular apparatus are capable of renin secretion (in the review by Kurtz 2012). The juxtaglomerular apparatus is a functional unit comprising the renin secreting juxtaglomerular cells, smooth muscle cells, mesangial cells, endothelial cells, and macula densa interconnected by gap junctions and 13

18 situated in the proximity of the glomerulus. The unit is responsible for controlled renin release into the circulation (in the review by Gomez & Lopez 2009). Renin release is thought to be the rate determining step in the classical RAS (in the review by Kurtz 2012), however if prorenin binds the prorenin receptor, the active site is exposed, allowing enzymatic activity even with the pro-segment attached (in the review by Fyhrquist & Saijonmaa 2008). In the renin producing juxtaglomerular cells, an increase in intracellular camp, for example by 1-adrenergic receptor activation (section 2.3), increases renin release. Calcium, on the other hand, inhibits adenylate cyclase, camp formation and thus renin secretion (in the review by Kurtz 2012, in the review by Peti-Peterdi & Harris 2010). Ang II increases intracellular calcium, creating a negative feedback loop between Ang II and renin. Likewise vasopressin and endothelin increase juxtaglomerular cell intracellular calcium (Seghers et al. 2016). Cyclic GMP can have both an inhibitory or stimulatory role on renin release (Beierwaltes 2006, in the review by Kurtz 2012). Stimulatory roles are discussed below. When atrial natriuretic peptide activates cgmp renin release is typically, but not always, inhibited (Freeman et al. 1985, Kurtz et al. 1986, Kanamori et al. 1995). This is discussed further in section Apart from direct 1-adrenergic receptor activation increased sympathetic activity in CVHD can also, through renal arteriolar vasoconstriction, lead to a reduction in renal perfusion pressure. The existence of a renal baroreceptor was unequivocally proven in a set of experiments performed on mongrel dogs; a reduction in perfusion pressure under constant blood flow led to a graded release of renin (Skinner et al. 1964). Recent mouse studies have shown that mechanical stretch of the juxtaglomerular cells at high perfusion pressures leads to calcium influx and an inhibition of renin release (Seghers et al. 2016). This inhibition would be absent at low perfusion pressures. A third way sympathetic activity may increase renin release in CVHD is by lowering glomerular filtration rate. A decreased filtration rate leads to a decrease in distal tubular salt concentration (in the review by Kurtz 2012) The macula densa cells, situated at the cortical end of the thick ascending limb of the loop of Henle sense changes in salt 14

19 concentration through their apically situated sodium transporters, the sodium hydrogen exchanger type 2 (NHE2) and the furosemide sensitive Na-K-Cl cotransporter 2 (NKCC2) (in the review by Peti-Peterdi & Harris 2010, in the review by Kurtz 2012). A low distal tubular salt concentration leads to macula densa cell shrinkage activating COX- 2 and neural nitric oxide (nnos), producing prostaglandin E2 (PGE2) and nitric oxide respectively. Both substances stimulate renin release; while PGE2 stimulates camp formation by activating adenylate cyclase, nitric oxide, acting through cgmp, limits its breakdown (Beierwaltes 2006, in the review by Peti-Peterdi and Harris 2010). It should be noted that dietary salt restriction also leads to renin secretion by the mechanism above (Kjolby et al. 2005). Thus some of the studies on CVHD and renin secretion discussed above were partly sodium controlled. Chronic stimulation of renin secretion leads to juxtaglomerular cell hypertrophy and recruitment of new afferent arteriolar smooth muscle cells into renin producing cells (in the review by Kurtz 2012, Neubauer et al. 2013). Metaplastic transformation of smooth muscle cells in mice involves endothelial derived (enos) nitric oxide acting on cgmp (Neubauer et al. 2013) Angiotensin converting enzyme Angiotensin converting enzyme (ACE) is a transmembrane protein with an extracellular catalytic domain. Typically ACE is bound to endothelial cells of the pulmonary vasculature, and in the classical RAS most of the conversion of angiotensin I to Ang II occurs here. However, ACE is also bound to other cell types and other vascular beds (in the review by Studdy et al. 1983, in the review by Riordan 2003). Cleavage of the N- terminal extracellular portion of ACE leads to an unbound form, whose activity can be measured in serum (Lucero et al. 2010). Interestingly, in dogs with CVHD but without heart failure, serum ACE activity correlated negatively with the amount of mitral regurgitation (Pedersen 1996, Pedersen et al. 1998, Pedersen & Mow 1998). However, this may not correlate with overall ACE activity. In the heart, ACE is bound not only to endothelial cells but also to fibroblasts (Dell'Italia 15

20 et al. 1995, in the review by Dostal & Baker 1999). Cardiac wall stress, like that caused by volume overload in mitral regurgitation, increases cardiac ACE activity (Dell'Italia et al. 1995, Stewart et al. 2003, Tallaj et al. 2003, Fujii et al. 2007). Measurement of cardiac ACE activity requires removal of the heart, and thus these experiments were performed on dogs whose mitral regurgitation was induced by chordal rupture. Apart from ACE, mast cell chymase has a very important role in intracardiac catalysis of angiotensin I to Ang II. I will return both to the effects of mast cell chymase and to how wall stress affects ACE activity when discussing cardiac hypertrophy (section 2.5). Unlike renin, which is substrate specific, ACE has several substrates apart from angiotensin I. The most important, from a cardiovascular perspective, is bradykinin, which it degrades. In fact, ACE has a stronger affinity for bradykinin than for angiotensin I in dog studies (in the review by Su 2014). Bradykinin primarily acts as a local vasodilator, and when ACE inhibitors are used in the management of congestive heart failure, blood pressure lowering effects are due primarily to increased bradykinin concentrations, not reduced Ang II concentrations (in the review by Su 2014). Bradykinin also reduces myocardial collagen accumulation and is discussed further in section Angiotensin II In the classical RAS, Ang II is produced by cleavage of angiotensinogen, first by renin to angiotensin I, and then by ACE to Ang II as discussed above. Angiotensinogen is produced and directly secreted, foremost by the liver. Regulation is through control of gene expression (in the review by Wu et al. 2011). For example Ang II, infused at physiological levels in dogs, exerts a minor positive feedback effect (Sernia & Reed 1980) by enhancing angiotensinogen mrna stability (in the review by Wu et al. 2011). There are few articles measuring plasma Ang II in CVHD. Most measure plasma renin activity and plasma aldosterone concentration as indicators of classical RAS activation. In some studies plasma aldosterone concentration were significantly increased in mitral regurgitation before CHF (Pedersen et al. 1995, Pedersen 1996, Pedersen et al. 1998), 16

21 suggesting increased circulating Ang II. This will be further discussed in the aldosterone section (section 2.4.4). One study on experimentally induced chronic mild mitral regurgitation showed no increase in any classical RAS components (renin, angiotensin I, Ang II or aldosterone, Fujii et al. 2007), while another where the induced mitral regurgitation was more severe, showed a more than fivefold increase in circulating Ang II concentrations (Tallaj et al. 2003). In a study following 11 biannually examined Cavalier King Charles Spaniels until the start of congestive heart failure, a decrease in Ang II and aldosterone concentrations were noted in early CHF compared to the previous examination half a year earlier. The researchers suggested this may be due to the counterbalancing effects of the atrial natriuretic peptide; the precursor NT-proANP increased with the increase of cardiac size during the study (Häggström et al. 1997). The atrial natriuretic peptides are discussed in section The seemingly conflicting results above may also be explained by a rise, followed by a fall, of the RAS components once a new blood volume able to restore blood pressure is achieved. In one study, cuffs, inflatable so as to constrict either the pulmonary artery or the thoracic inferior vena cava, were surgically placed, and dogs were given two weeks to recover. Hereafter, the cuffs were inflated and a number of variables, amongst them plasma renin activity, aldosterone concentration, hematocrit, blood volume, arterial pressure as well as urinary sodium and water balance were followed daily for 2 weeks. The researchers found that the sudden drop in arterial pressure lead to a huge rise in plasma renin activity and aldosterone concentration as well as salt and water retention, increased thirst, ascites and edema. If a larger blood volume (with ensueing lower hematocrit) was achieved, that allowed for restoration of blood pressure, RAS activation was no longer needed and plasma renin activity and aldosterone concentration dropped (Watkins et al. 1976). Häggström et al. (1997), in the study above, noted that plasma protein concentration and hematocrit were decreased in their Cavalier King Charles Spaniels, suggesting fluid retention. Thus, it is possible that the recent fluid accumulation, causing early CHF in these Cavalier King Charles Spaniels, has temporarily led to a reduction in RAS activation. How volume overload leads to congestive heart failure, is discussed further in section

22 In this section I will discuss how circulatory Ang II augments blood pressure and volume by affecting the vasculature, the kidneys, the adrenal glands and the sympathetic nervous system. In the brain, Ang II modulates pathways concerned with blood pressure and blood volume control. Circulatory Ang II affects the circumventricular organs outside the blood-brain barrier, but there is also ample evidence of Ang II production within the blood-brain barrier, i.e. the central RAS. This is discussed in section The effects of the cardiac RAS are discussed in section 2.5. The AT1 receptor is Gq-protein coupled (in the review by Karnik et al. 2015). Its activation on vascular smooth muscle cells leads to an increase in intracellular calcium and thus vasoconstriction (in the review by Touyz 2003). Also, Ang II may increase expression and binding of the endothelin-1 ETA receptor causing further vasoconstriction (Lin et al. 2014). Plasma endothelin-1 levels have been shown to be augmented in CVHD (Uchide & Saida 2005). In the kidneys Ang II vasoconstriction is more substantial in the efferent arterioles, increasing hydrostatic pressure in the glomerulus and offsetting the reduction in glomerular filtration rate (GFR) caused by the general constriction induced drop in renal blood flow (in the review by Cogan 1990). Angiotensin II affects not only the renal vasculature but also the tubular system. In the proximal convoluted tubule (PCT) Ang II increases the affinity of the Na + / H + exchanger NHE3. The main two counterions absorbed are bicarbonate and chloride; 70% to 90% of NaHCO3 and about 50% of NaCl are absorbed in this section (in the review by Alpern et al. 1995). Higher Ang II concentrations increase NaCl absorption compared with NaHCO3 absorption by mechanisms described in Cogan et al.'s (1990) review paper. Angiotensin II also increases the Na + Cl - cotransporter (NCC), found primarily in the apical membrane of the initial part of the distal convoluted tubule, as well as activating, together with aldosterone, the epithelial Na + channel (ENaC) in the aldosterone-sensitive distal nephron (in the review by Palmer 2014). Increasing Ang II concentrations thus increase sodium chloride absorption in the kidney tubules. Potassium secretion is decreased by inhibiting the renal outer medullary potassium channel (ROMK, in the review by Palmer 2014). Various sodium, potassium and water channels involved in blood volume homeostasis are tabulated below (table 2). 18

23 In the adrenal cortex, Ang II increases aldosterone synthesis by up-regulating AT1 receptor expression, expression of several enzymes required in aldosterone synthesis, as well as increasing the proliferation of stem cells that develop into the zona glomerulosa (in the review by Matsusaka & Ichikawa 1997). In the adrenal medulla, Ang II increases catecholamine release (in the review by Giacchetti et al. 1996). The positive modulation of catecholamine release, both in the adrenal medulla, a modified ganglion, as well as actual ganglia, occurs through AT1 receptor activation on the postganglionic cell body (Kushiku et al. 2001). The short-term increase in firing rates is due to modulation of neuronal potassium and calcium channels while longer-term modulation occurs through upregulation of neuronal tyrosine hydroxylase, a central enzyme in catecholamine synthesis (in the review by Sumners et al. 2002). These effects are underlined in a study where Ang II was infused into the left atrium in both control dogs and those with one month of experimentally induced mitral regurgitation. Heart rate, mean arterial pressure and cardiac interstitial fluid adrenaline and noradrenaline were significantly increased in both groups, heart rate and MAP more so in controls, whereas interstitial fluid adrenaline and noradrenaline concentrations were higher in dogs with mitral regurgitation (Tallaj et al. 2003). The neurons in the central nervous system are modulated similarly (in the review by Sumners et al. 2002). Table 2. Sodium, potassium and water channels involved in blood volume regulation and modulated by Ang II, aldosterone, vasopressin and ANP Tubular segment Proximal convoluted tubule NHE3 Loop of Henle thick ascending limb Distal Convoluted Tubule DCT1 DCT2 Collecting duct Channel Modulation + Ang II, -ANP NKCC2 + Vasopressin, -ANP NCC ROMK ENaC ROMK ENaC ROMK AQP-2 + Ang II, + Aldosterone - Ang II, + Aldosterone + Ang II, + Aldosterone, + Vasopressin, - ANP - Ang II, + Aldosterone + Ang II, + Aldosterone, + Vasopressin, - ANP - Ang II, + Aldosterone + Vasopressin, -ANP 19

24 Angiotensin II in the brain Angiotensin II has important central effects modulating both blood pressure and volume and a fair amount of research has been done on dogs. With present day knowledge on the actions of Ang II in the brain it comes as no surprise that the highest levels of angiotensin (both angiotensin I and Ang II) in the dog's brain were found in the hypothalamus (Fischer-Ferraro et al. 1970). Likewise, angiotensin receptors (in this study the type was not specified) were abundant in the dorsal motor nucleus of the vagus, in the nucleus tractus solitarius (NTS) of the medulla, in certain circumventricular organs, such as the area postrema (AP), the subfornical organ (SFO) and the organum vasculosum laminae terminalis (OVLT), in the median preoptic nucleus of the hypothalamus (MnPO), and in the anterior portions of the pituitary of the dog (Speth et al. 1985). The NTS has been mentioned before, as the medullary nucleus with a central role in blood pressure regulation (section 2.3). The other brain regions, apart from the dorsal motor nucleus of the vagus, will be discussed below. Figure 3 aids in visualization. The sensory circumventricular organs, the SFO and OVLT adjacent to the third brain ventricle (Biancardi & Stern 2016), and the AP, adjacent to the fourth (Otsuka et al. 1986), are key brain regions when it comes to central actions of circulatory Ang II. The circumventricular organs lack a blood-brain barrier, which allows Ang II, which typically doesn't cross this barrier, to reach them. The other regions mentioned above are within the blood-brain barrier and AT1 receptor stimulation typically occurs by centrally produced Ang II (Biancardi & Stern 2016). The existence of a central RAS in the dog has indisputably been proven by not only the high concentrations of angiotensins especially in the hypothalamus, but also by renin-like activity in brain tissue extracts (Fischer- Ferraro et al. 1970, Ganten et al. 1971). The SFO and the OVLT, together with the MnPO, a hypothalamic nucleus, make up the lamina terminalis along the rostral wall of the third ventricle. This area is crucial for central fluid regulation (in the review by Toney & Stocker 2010, Biancardi & Stern 2016). In severing different parts of the lamina terminalis in dogs several researchers noted a significant decrease in drinking (Marson et al. 1985, Thrasher & Keil 1987, Tramposch 20