Slide 1

Good afternoon. Thank you Mr Chairman, Ladies and Gentlemen. It’s my pleasure to be here. So I want to express my gratitude to the organisers for giving me the opportunity to share with you our current views on the role of salt intake on the heart in hypertensive patients.

Slide 2

I think there is no doubt that an excess of sodium intake is related to an increase in blood pressure. It is also true and we accept easily that increased blood pressure is a main mechanism leading to left ventricular hypertrophy in hypertensive patients. But there are a number of studies to my mind the best proof coming from the European project on genes and hypertension demonstrating also that an excess of sodium intake is a major determinant of left ventricular hypertrophy independently of blood pressure. My task today will be to provide you with data suggesting and even supporting that an excess of sodium intake is also related to a very common fact in hypertensive patients with LVH, in other words left ventricular remodelling and dysfunction.

Slide 3

In fact, a number of structural abnormalities develop in the hypertensive heart leading to the histological picture known as myocardial remodelling. Some of these changes are related to the parenchymal compartment of the myocardium and some of the other changes are related to the intramyocardial vessels perfusing the myocardium.
I would like to focus my attention on this particular structural change, the presence of an increased number of collagen fibres within the interstitium and around the intramyocardial vessels of the myocardium leading to interstitial and perivascular fibrosis.

Slide 4

Myocardial fibrosis is a continuum along the clinical history of hypertensive heart disease. Here you can see data coming from a study from our group demonstrating that the amount of collagen fibres which are localised in the heart increases when hypertensive heart disease evolves from no clinically detected revelations to clinically detected LVH and finally, to clinically present heart failure. Most of this collagen material corresponds to collagen type I. As you can see in the figures represented in the bottom part of the slide more than 90% of collagen fibres, which are abnormally localised within the hypertensive heart correspond to collagen type I fibres.

Slide 5

The question is that this lesion means a high price to pay for the heart. Myocardial fibrosis is critically involved in the development of heart failure in hypertensive patients namely in hypertensive elderly women. In fact, fibrosis alters diastolic function. In very advanced cases fibrosis may also compromise systolic function and always fibrosis facilitates the compromise of cardiac function through facilitation of ventricular arrhythmias and through facilitation of intramyocardial ischemia.
So, this is an important lesion to be considered in all hypertensive patients.

Slide 6

The question is how does myocardial fibrosis develop in the hypertensive heart? Myocardial fibrosis is the main consequence of the loss of the physiological equilibrium existing between the synthesis and the degradation of fibrillar collagen molecules,  namely collagen type I molecules. The loss of this equilibrium is zero or --- to the mechanical loading imposed on the heart by increased systemic blood pressure to the susceptibility to this mechanical overload imposed by gender. Males are more prone to fibrosis than females. We know actually that a number of cytokines or growth factors, for instance, TGF β modulate myocardial response to mechanical overload. There are genetic variations leading mostly on the renin-angiotensin system genes that also determine the final response of the myocardium. There are a number of hormones and vasoactive substances, this is the case for aldosterone and angiotensin II, that also contribute in a very specific way to the final accumulation of collagen tissue within the heart. We know now that the amount of sodium present in the diet may also contribute in a different way to the amount of collagen which is localised within the hypertensive heart.

Slide 7

Let me present a very small, brief data from our own group demonstrating that in fact, the amount of sodium present within the diet may determine the final extent of  myocardial fibrosis in hypertensive patients.
In collaboration with Ed Frohlich in New Orleans and his group we developed this study some years ago that was published last year. We took normotensive Wistar Kyoto rats and spontaneously hypertensive rats and we gave these rats from week 8 of age to week 16 either low sodium diet or high sodium diet. When this experimental period was finished we performed antemortem studies or transthoracic echocardiography and systemic and left ventricular haemodynamic assessments and after the animals were sacrificed we took the heart to perform a number of histomorphological and chemical studies. What we found was the following. In terms of systemic haemodynamics you can see that blood pressure both mean arterial blood pressure and pulse pressure was increased after sodium salt loading both in the normotensive strain of rats and in the hypertensive strain of rats.
The absolute relative increase in pulse pressure and mean arterial pressure was quite similar in the two strains of animals. However, the accompanying increase in left ventricular mass, the hypertrophic response of the heart which was present in the two strains was more marked in the hypertensive strain, even more in this strain the hypertrophic response adapted a classical concentric geometric pattern.

Slide 8

So, despite a similar increase in blood pressure, the increase in hypertrophy, the growth response was higher in the hypertensive animal. So, supporting the notion that the hypertensive phenotype determines the left ventricular growth response to sodium load. But what was really more amazing was the response in terms of fibrosis. We look at fibrosis by measuring the collagen volume fraction in the left ventricle, in other words the fraction of myocardium occupied by collagen tissue and also by measuring left ventricular hydroxyproline concentration as a marker of collagen tissue. What you can see here in the left part of the slide is the response of these two parameters to sodium load in Wistar-Kyoto animals and in the hypertensive animals on the right side.

Slide 9

After sodium loading, after salt loading you can see that no changes in these two parameters reflecting fibrosis were observed in a normotensive strain of animals. Whereas, the two parameters increased significantly in those hypertensive animals after being submitted to salt loading.

Slide 10

You can see one representative image of these changes in the two strains. You can see here the microscopic view of the left ventricular myocardium of the Wistar-Kyoto rat under control conditions, low sodium intake and normotensive Wistar-Kyoto rat under high salt intake conditions. You can see that there is a huge increase in the amount of collagen tissue but this is not significant, what is significant is the huge accumulation of big masses of fibrotic tissue within the hypertensive myocardium under conditions of high salt intake. In other words, it’s the hypertensive phenotype which finally determines the fibrotic response of the myocardium to increase salt intake.

Slide 11

As I said before, the price to pay for this myocardial accumulation of collagen tissue is high. Looking at parameters assessing systolic function, diastolic function and coronary flow reserve we were able to observe that whereas, no changes were observed in left ventricular systolic function in hypertensive salt loaded rats, left ventricular relaxation was really impaired in those salt loaded rats which developed fibrosis and very importantly, coronary flow reserve was also decreased significantly in those hypertensive rats under salt loading conditions. So, the hypertensive phenotype determines myocardial fibrosis and this is associated to the impairment in left ventricular diastolic filling and the ability of the intramyocardial vasculature to perfuse the heart in conditions of stress.

Slide 12

The question is, which are the mediators responsible for this particular fibrotic response of the hypertensive myocardium to sodium load? It is not just the increase in blood pressure, remember that the increase in blood pressure was similar in hypertensive and normotensive animals submitted to sodium load.
In accordance with this we found in hypertensive animals that the increase in collagen volume fraction, the increase in myocardial fibrosis was observed not just in the free wall of the left ventricle which is submitted to mechanical overload imposed by high blood pressure but was observed also in other cardiac locations which are not exposed to mechanical overload. So the mechanical component, the haemodynamic component appears not to be a major determinant of myocardial fibrosis in this particular model.

Slide 13

So we need to move to the non-haemodynamic mediator of myocardial fibrosis. In this setting it is actually considered that the imbalance between the synthesis and degradation of collagen fibres which is responsible for hypertensive myocardial fibrosis reflects the influence on cardiac fibroblasts and myofibroblasts of a number of profibrotic factors that are predominant of the anti-fibrotic factors currently existing within the heart. At the top of those profibrotic factors potentially involved in this sequence of events is angiotensin II. In fact, a number of studies recently published by ourselves suggest that angiotensin II acting through the AT1 receptor located in cardiac fibroblasts and myofibroblasts triggers a signalling pathway leading to a number of different profibrotic responses. On one hand the synthesis and secretion of an increased number of collagen precursors. Second the inhibition of those mechanisms leading to the degradation of collagen fibres and finally, angiotensin II acting through the AT1 receptor has been demonstrated as also inducing a proliferation of fibroblasts. But the important question is that these mechanism can be upregulated in conditions of salt loading.

Slide 14

In fact, as demonstrated some years ago, the increase in salt intake induces cardiac angiotensin converting enzyme in spontaneously hypertensive rats and maybe more importantly, the dietary salt loading has been also shown that induces the AT1 receptor at a cardiac level in Dahl salt-sensitive rats. So it appears very attractive the possibility that salt loading is inducing myocardial fibrosis in hypertensive animals through the activation of the local renin-angiotensin system.

Slide 15

To prove this possibility we developed a second study and this study has already been submitted for publication. In this study we used spontaneously hypertensive rats and we used a similar protocol to the first one. Those animals treated with low sodium diet, animals treated with high sodium diet and hypertensive animals treated with high sodium diet plus a low dose of an angiotensin receptor blocker. We repeated the same experimental procedure and we performed similar tests as performed in the first study. What we found is presented here. First, mean arterial pressure, pulse pressure that were highly elevated in salt loaded rats as compared to non-salt loaded rats were not significantly reduced by the low dose of the ARB used in this study. So, the ARB-treated animals remained hypertensive, such hypertensive --- untreated animals at the end of the study.

Slide 16

Despite this you can see here that the low dose of ARB was able to partially prevent the increase in left ventricular hypertrophy observed in the untreated animals. Again reinforcing the partial role of sodium load in the global left ventricular response of this animals. But the important finding of this study is that a low dose of an angiotensin receptor blocker which does not prevent the increase in blood pressure was able to completely prevent the development of myocardial fibrosis in these hypertensive animals.

Slide 17

You can see here that the collagen volume fraction that was increased in the hypertensive animals submitted to a high salt diet was completely prevented in those animals submitted to the same diet but treated with a low dose of an angiotensin receptor blocker.

Slide 18

So, this is clearly shown in these figures. You can see here that the huge perivascular and interstitial fibrosis present in hypertensive rats submitted to a high salt diet was very reduced, almost completely prevented in those animals treated with a low dose of an ARB which was not able I repeat to prevent the increase in blood pressure.

Slide 19

So this experimental data clearly suggests that the activation of the local renin-angiotensin system in conditions of a high salt diet maybe responsible in a great manner for the fibrotic response of the myocardium which is observed in this particular strain, in this particular model of arterial hypertension. The question is how does this data translate to the clinical arena? We have analysed the possibility that a salt diet may also induce myocardial fibrosis in hypertensive patients. We have used a non-invasive approach. Several years ago we developed this biochemical approach to collagen type I metabolism within the heart. This approach is based on the fact that when the procollagen precursors are transformed into the final collagen molecules which will form the final fibre, some peptides are released which are secreted into the blood stream where they can be easily measured by using ELISA or specific radioimmunoassays. The same is true when these fibres are degraded by matrix metalloproteinases. Another peptide is released which is again secreted into the blood stream. In conditions of non-advanced liver failure, or non-advanced kidney disease the measurement in blood of these peptides can give us an indirect idea of the balance between the synthesis and the degradation of the small molecules.

Slide 20

A number of studies that we have published and namely in Circulation allow us to say that these peptides present in peripheral blood come mainly from the blood not from other tissues organ sources. For instance, you can see here that we found some years ago that this peptide, which is produced when collagen type I molecules are formed, this peptide reflecting synthesis presents a significant correlation with the amount of collagen fibres, namely collagen type I fibres present within the hypertensive heart.

Slide 21

We took advantage of this approach to look if there is some kind of relation between collagen type I metabolism and sodium intake in hypertensive patients. I analysed this data taking into account this presentation, these are hot data. What I’m presenting here is when we analyse this peptide the serum concentration of these peptides reflecting collagen type I synthesis against urinary sodium as a measure of sodium intake in non-treated hypertensive patients, you can see that those patients, those values of abnormally high PICP and those values of abnormally high sodium intake collect, aggregate most of our hypertensive population. In other words there is an association between increased synthesis of collagen type I as assessed by these measures and an increased sodium intake as assessed by these measures in non-treated hypertensive patients.

Slide 22

When we look at the balance between the synthesis and the degradation, we can see here that this balance is more altered, the more higher is the sodium intake. By classifying our patients in three urinary sodium tertiles you can see here that those patients in the third tertile with a higher sodium intake are those patients in which the ratio between this peptide reflecting synthesis and this peptide reflecting degradation is the higher one. So, the higher is the imbalance, the higher is the sodium intake.

Slide 23

So, Mr Chairman, Ladies and Gentlemen I would like to conclude by proposing you that in the first stima that I have presented it is possible to say that accumulating experimental and emerging clinical data suggests that in fact, an excess of sodium intake maybe directly linked to those structural changes leading to remodelling of the hypertensive myocardium and as a consequence to its functional deterioration and that this linkage can be exerted through the activation of local mediators of the fibrogenic response such as the renin-angiotensin aldosterone system.

Slide 24

I will finish by two slides in which I reflect my acknowledgement to those colleagues in Pamplona and New Orleans responsible for the experimental studies.

Slide 25

and those colleagues in Pamplona and San Sebastian very close to Pamplona responsible for the clinical studies that I have presented you. Thank you very much for your attention.

Slide 26

Chairman: Thank you. This presentation is open for discussion. Tillman.

Question: Xavier, you showed that WKY animals despite a similar increase in blood pressure in response to high salt intake had a similar increase in blood pressure but did not develop collagen overexpression and fibrosis in the heart. So, what do you think is it, by which mechanisms hypertension predisposes the heart to respond in this particular fashion to high salt intake, whereas the normal heart is protected against this apparently?

Dr Diez: Yes, cardiac fibroblasts submitted to mechanical stress produce a huge amount of collagen tissue. This is true for cardiac fibroblasts coming from a hypertensive animal not from a normotensive animal. Second, the expression of angiotensin converting enzyme and the density of AT1 receptors present within the hypertensive myocardium, as compared to the normotensive myocardium, are significantly higher. So, despite blood pressure, beyond blood pressure the fibrogenic machinery; cardiac fibroblasts and its biosynthetic pathways are more prone to produce collagen in the hypertensive myocardium than in the normotensive myocardium. So, the hypertensive requirement is through the left ventricular myocardiocyte growth response but not for the non-cardiomyocyte growth response, non-cardiomyocyte synthetic response in the hypertensive myocardium. I think that we can separate in a very clinical way which are cardiomyocyte responses from non-cardiomyocyte responses.

Chairman: Professor Ritz.

Question: Doctor Diez do we have information on reactive oxygen species in salt loaded, normo and hypertensive animals? That would be a logical candidate in addition of course, to the undoubted role of activated renin-angiotensin system.

Dr Diez: Maybe as you know we work in this field. When I say angiotensin II interacting through the AT1 receptor triggers a signalling pathway leading to the activation of the synthetic machinery, very upstream in this signalling pathway is the activation of NADPH oxidase, which produces superoxide which is really the trigger of the signalling pathway leading to marked kinase activation, to this marked activation that at the end executes the fibrogenic response. So in fact, there is no fibrogenic response induced by angiotensin II without oxygen species ----. So the oxygen species appears to be a critical necessary requirement for the fibrogenic response to angiotensin II and many other fibrogenic factors.

Chairman: Next question.

Question: The question is concerning the effects of the sodium, an increase of sodium intake on the insulin resistance that you can see in the case of hypertension also in your rats. Did you measure the insulin and see that it is like metabolic syndrome that is occurring with high salt intake?

Dr Diez: Well, the spontaneously hypertensive rat is a very bad model for metabolic changes and is representative of the metabolic syndrome that we see in humans but anyway unfortunately we haven’t looked at this part of the story. Thank you for the suggestion.

Chairman: Last question. Any more? Ok then I think we will conclude the session. I thank very much the four speakers for very interesting presentations and the brave audience who have stayed until now in the room. Thank you very much.