Adiponectin – stratification biomarker in diastolic cardiac dysfunction

Abstract This study does not propose to elucidate how adiponectin secretion is regulated, but how its adiponectin concentration is an indicator of heart disease. About adiponectin, it is not known whether it is functionally an enzyme, or very likely a cytokine/chemokine/hormone, secreted by fat cells/adipocytes in the abdomen. Abdominal fat secretes 67 hormones, and all of which cause disease. For example, adiponectin generates diabetes and ischaemic heart disease via dyslipidemia. Based on clinical symptoms, electrocardiographic and echocardiographic parameters, a group of 208 patients with diastolic cardiac dysfunction with or without preserved systolic function, developed on a background of painful chronic ischaemic heart disease, stable angina on exertion, was constituted. The serum levels of adiponectin, total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides were measured. Using the identified values, it was appreciated whether adiponectin correlates with the type of any of the two conditions, so that it can be recognised as a diagnostic and risk stratification marker.


Introduction
Having a complex etiopathogeny, but predominantly coronary atherosclerosis, heart failure is not only one of the major causes of death, but also one of the conditions that claim high economic costs [1][2][3][4] .
Among the multifunctional hormone-type peptides, which are synthesised almost exclusively by white adipose tissue cells, there is also adiponectin, with the physiological role of regulating metabolic homeostasis and a protective effect on the cardiovascular system [5][6][7][8][9][10][11][12][13] . Studies on animals, as well as on humans, have shown that by reducing its plasma level, the processes induced by oxidative stress at the level of cardiomyocytes and vascular wall cells are augmented [14][15][16][17][18] .
Although some studies question the implication of adiponectin in atherogenesis due to slow turnover, the research results of the last two decades unanimously support that the reduction of the circulating adiponectin level as a consequence of the pathogenic mechanisms developed both at the level of the coronary wall and directly at the level of the myocardial fibre contributes etiopathogenically to the onset and evolution of diastolic cardiac dysfunction 12,[19][20][21][22][23] .
These arguments are based on the studies of Kumada M. and Osaka from the Coronary Artery Disease Study Group, claiming that men with hypo adiponectinemia (<4.0 microg/mL) had a 2fold increase in CAD prevalence, independent of well-known risk factors 24 . Pathogenically, it is admitted that at the level of the coronary wall, hypoadiponectinemia generates arterial dysfunction, inducing pro-inflammatory effects such as: inhibiting the activation of endothelial cells, adhesion of monocytes to endothelial cells and accelerating the transformation of macrophages into foam cells [24][25][26][27] .
At the same time, a low level of adiponectin correlates with a decrease in coronary dilatation induced by NO-synthetase activity deficiency 22,24,[28][29][30] . Actions are the direct expression of synthesis changes in in the vascular endothelium [31][32][33] . In addition to the anti-atherogenic, anti-inflammatory and vasodilation actions of adiponectin, the specialised literature unanimously recognises the antioxidant, anti-apoptotic and anti-thrombotic effects 12,13,22,28,34 .

Objectives
In the literature, it is almost unanimously accepted that the serum level of adiponectin correlates with the intensity of the protective anti-inflammatory, anti-apoptotic and vasodilation effects at arterial endothelium level. In the circumstances of a reduced serum concentration of adiponectin, the vascular pro atherogenic effects, including at the coronary level, become dominant. The induction of atherosclerosis at the level of the vessels of the heart produces, indirectly, a disturbance of the metabolic homeostasis of the myocardium, leading to the development of secondary consequences at this level. Synergistically, but directly, the reduced synthesis of adiponectin at the level of the myocardial fibre also acts to disturb the properties of the heart. In the same way, but directly, the reduced synthesis of adiponectin from myocardial fibres also acts upon the disturbance of the properties of the heart. Therefore, heart hypoadiponectinemy becomes an independent pathogenic risk factor contributing to the development of diastolic cardiac dysfunction.
The objectives aimed at in this study are: a. identifying a possible correlation between the serum concentration of LDL-cholesterol, the main serum lipoprotein involved in atherogenesis, and the level of triglycerides, on the one hand, and the level of circulating adiponectin, on the other. b. whether adiponectin level can be a disease marker, useful for risk stratification in diastolic cardiac dysfunction with or without ejection fraction reduction.

Material and methods
The present study appoints as a prospective one following ethical principles derived from the Declaration of Helsinki (Committee on Human Investigation). The study comprises of a group of 208 patients with cardiac dysfunction on a background of 2 painful chronic ischaemic heart diseases divided into 2 subgroups, one with preserved ejection fraction (FE ! 50%) and another with a decrease of the ejection fraction (FE < 50%) ( Table 1).
In forming the group, the major concern was to ensure a good homogeneity, by excluding smokers or patients with comorbidities, such as obesity, hypertension, diabetes etc.
Since obesity is recognised as an atherogenic risk factor, both as a consequence of increased level of coexisting pro atherogenic lipid fractions, and through the secretion by adipocytes of some atherogenic hormone/adipokine peptides, we enrolled only patients with normal BMI (Body Mass Index) or with at most a deviation of plus 5% in the group 11,[35][36][37][38][39][40] . Since specially designed studies have demonstrated the existence of a reduced level of adiponectin in hypertensives, only normotensive patients were enrolled 28,40,41 . Also, the study group consisted only of non-smoking patients. This is because the effect of smoking on the secretion of adiponectin has been demonstrated, as well as detected over 12 h post-smoking 42,43 . No patients with diabetes were included into the group [44][45][46] .
Through the elaborated investigation protocol, the patients underwent an electrocardiogram (ECG) and echocardiogram, which on the one hand allowed us to exclude from the group patients with other heart diseases, and on the other hand to evaluate the ejection fraction 47,48 .
Physiologically, knowing that plasma adiponectin levels have circadian variations, the measurement was determined from venous blood collected between 8 and 9 am, when it is assumed that adiponectin secretion reaches its maximum value. The dosage of the serum adiponectin concentration was performed via the ELISA method, from venous blood, collected in the morning, from fasting patients. Until the time of testing, although it was the same day, the collected sample was stored in a refrigerator at À30 C.
From the collected blood samples, the lipid fractions required for the study were also determined: total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides.
Since the circulating level of adiponectin varies according to sex, in the study, we decided to work with the average of the values determined on the groups 1 . Depending on the sex of the patients, we used their average values as normal plasma levels: 7.5 mg/L for men and 9 mg/L for women.
Regardless of gender, we considered for the entire group as a normal value of the serum adiponectin concentration the range defined by the previously mentioned average serum values (7.5-9.5 mg/L), and as an indicator of the risk existence, the limits: <7.5 mg/L ¼ increased risk, and !9.5 mg/L ¼ low risk.

a. Correlation of serum adiponectin concentration with circulating lipid fractions involved in atherogenesis.
As a function of the value of the ejection fraction, the prevalence of cases presenting serum increases in total cholesterol, its fractions and triglycerides is presented in Table 2. The number of patients with reduced serum adiponectin concentration is also presented.

Discussions
Among the adipokines that act on the cardiovascular system such as tumour necrosis factor a (TNFa), leptin, plasmogen activator inhibitor-1, adipocyte fatty acid binding protein, lipocalin-2, monocyte chemotactic protein 1 and resistin, we selected adiponectin for study 19,49,50 . This peptide-hormone is secreted by both adipose tissue cells and cardiomyocytes 32 .
We justify the selection by the fact that all circulating oligomers of adiponectin [trimer, hexamer, globular and high-molecularweight-multimer (HMWM)] at normal serum concentrations are modulators-inducers of protective effects (anti-inflammatory and anti-apoptotic) at the level of the arterial wall, but also of cardiomyocytes 11,40,51-53 ( Figure 1).
In our study we measured the serum level of both the lipoproteins involved in atherosclerosis and of adiponectin, because they can act coadjuvantically in the development of atherosclerosis (when lipid fractions such as LDL-cholesterol and triglycerides increase in the blood and the serum level of adiponectin decreases). In an aggravating sense, the hypoadiponectinemia at the myocardial level develops cardiomyopathy that adds up, pathogenically, to chronic ischaemic cardiopathy induced by atherosclerosis of the coronary vascular system. Etiopathogenically, the mechanisms contribute to disrupting the functional properties of the heart, allowing, among other conditions, the development of diastolic cardiac dysfunction.
Among the circulating lipoprotein fractions, we analysed only the possible correlation of the prevalence of LDL-hypercholesterolaemia and hypertriglyceridaemia with that of hypoadiponectinemia (Table 2). Thus: a. In the studied group, the number of patients who presented circulating values of adiponectin below the lower limit of normal (157 patients ¼ 75.5%) correlates in 3 =4 of the   b. The number of cases in the group with hypoadiponectinemia is identical to that of increased triglyceridemia, which allows us to draw another conclusion: the existence of a synergy of pro-atherosclerotic action of the two serum compounds. c. Much higher than the prevalence of hypertriglyceridaemia is that of cases with a high level of LDL-cholesterol (Table 2). For clinical practice, the correlation of 9 cases with hypoadiponectinemia out of 10 with LDL-hypercholesterolaemia becomes useful, indirectly indicating the way of variation of the two compounds, a way that can become useful therapeutically. d. The correlation of the prevalence of the serum concentration of hypertriglycerides with the low concentration of adiponectin is almost superposable for patients regardless of the value of the ejection fraction. However, in the case of patients with DD and reduced ejection fraction, the values are 50% higher than those with DD and PRESYF (Table 1). e. In the subgroup of patients with DD and reduced systolic flow there is a concordance of reduction in the values of systolic and serum adiponectin concentration as well. The last two analytical findings (d and e) allow us to argue that the reduction of adiponectinemia can be accepted as a risk marker, allowing the stratification. f. The data of the study highlights the dispersion of the values of the elevated circulating lipid fractions consistent with that of the reduction of the serum level of adiponectin, which allows their analysis not only individually, but also concomitantly as a complex parameter. The collective analysis increases the safety and usefulness of the information for the clinical practice of evaluating the diagnosis, but also the evolution/risk in ischaemic heart disease. This may constitute a possible multimarker in cardiovascular diseases, in particular chronic ischaemic heart disease generating diastolic cardiac dysfunction. g. In the specialised medical literature, similar studies have been carried out, but for different cardiovascular conditions, and the reported results are contradictory. It is not necessary to explain some results that do not correspond to ours, results that are from the only two published/identified articles. It is a new field of research about which nothing is known. It is known that a researcher does not take his or another's results for granted, but puts them in contrast, as a new problem that must be studied, in depth (that is, new, more in-depth studies are needed to contribute to knowledge in the field). This is also one of our conclusions.

Conclusions
Adiponectin has a cardiovascular protective role, both at the level of vessels and myocardium. The present paper can be interpreted as an effort to understand the mechanisms of atherosclerosis development, including the one at the coronary level and also the deterioration of the contractile function of the heart. This is because this study analyzes for the first time the evolution of serum adiponectin levels in chronic ischaemic heart disease responsible for the evolution of diastolic cardiac dysfunction. The conclusions of the study lead to the idea of the need to carry out more studies with large number of participants, which in the eventuality of confirming our results become of real benefit to medical practice. This is because the quantification of adiponectin level in the condition of patients with dyslipidemia allows a screening of the coexistence of diastolic cardiac dysfunction, and in dynamics the peptide values allow assessment of the evolution of the disease and/or of the therapy with hypolipidemic medication.
Statistically, the degree of correlation between the increase in the serum concentration of LDL-cholesterol and triglycerides with the reduction of that of adiponectin allows us to admit that the low values of adiponectin can be a potential diagnostic biomarker and a severity indicator of diastolic dysfunction, indirectly suggesting the degree of deterioration of the ejection fraction.