In this study, changes in proteins of the platelets are described in dogs with CHF. This represents to the author`s knowledge the first report in which changes in the proteins of platelets are reported in dogs with any disease. Platelet proteins may have a role in the pathophysiology of CHF caused by MMVD by regulating several molecular functions, biological processes and signaling systems. Therefore, this study could be a basis for future developments in order to better elucidate the role of platelets in the physiopathology of CHF and other different diseases in the dog, which could lead to new strategies of treatment and management of these conditions.
CHF cases in this study were diagnosed and classified as stage C according to ACVIM heart failure guidelines for dogs (19). The reason why stage C was selected in this study is because it corresponds to symptomatic dogs but without receiving any medication (19). Thereby the possible effects of cardiac drugs (inotropes, diuretics, beta-blockers and ACE inhibitors) on platelet functions were avoided (8).
In this study, a total of 107 platelet proteins were identified, in which 10 were found to be differentially expressed at statistically significant level. Some of the proteins (ApoA-II, GBP, cathepsin D, COX2, CXCL10, PPP1 and CKB) were already identified from healthy dogs in a previous study (18), whereas some proteins such as myotrophin and ApoC-III are described herein by first time in the dog.
Platelet myotrophin activity was decreased in this study, in line with a previous report in humans with CHF in which this protein was decreased in plasma samples (20). Myotrophin is hypertrophy-inducing factor, which acts in the formal arrangement of actin filaments and promotes cardiac muscle hypertrophy (20, 21), and was reported as a serum biomarker showing early activation in CHF (20). Additionally, myotrophin stimulates the transcription factor-kappa B (NF-kB) activity which regulates expression of several genes involved in immune responses, inflammation, proliferation, and apoptosis in ventricular myocytes. Therefore, decreasing platelet myotrophin activity in this study may be a protective response in the pathophysiology of CHF by limiting the NF-kB activity on cardiomyocytes (22), and the initiation process of myocardial hypertrophy in response to volume overload (23).
We have identified three apolipoproteins in this study; ApoC-III, ApoA-II, and clusterin (ApoJ). ApoC-III is a regulator of triglyceride rich lipoproteins which leads to hypertriglyceridemia and then cardiovascular disease (24–26). In line with our results, Roura et al. (27) found a significant increase in serum ApoC-III in people with dilated cardiomyopathy compared to controls. ApoC-III was described as a thrombogenic factor for cardiac patients due to complex interactions with plasma endogenous thrombin formation and coagulation cascade (24), as well as an increase in the accumulation of atherogenic lipoproteins in the vessel wall (26). Therefore, Apo-CIII could alter the homeostatic balance in a pro-coagulant way, and may promote atherothrombotic complications in dogs with CHF (28). Although these complications cannot be detected clinically during diagnostic work-up in dogs, it should be kept in mind that increasing Apo-CIII may be a risk factor for development of intra-myocardial coronary arteriosclerosis and micro-thrombi which were reported in dogs with CHF (2, 5, 6).
ApoA-II is a major component of HDL particles (24, 25), and controls reverse cholesterol transport to inhibit the development of atherosclerosis (29, 30). Antithrombotic effects of ApoA-II and HDL complex were related to inhibition of the coagulation cascade by stimulating endothelial production of nitric oxide and prostaglandins, and stimulation of clot fibrinolysis (31). Low level of ApoA-II was associated with increased severity and worse outcomes in heart failure patients (32). Thus, in this study, increasing platelet ApoA-II levels in dogs may be considered as a host response to be prevented from thrombotic effects and to manage fibrinolytic mechanisms during CHF.
Serum clusterin levels were reported to increase in various conditions such as myocardial infarction, inflammation, apoptosis, and oxidative stress (33, 34). Overall it has a protective effect by preventing endothelial activation (anti-atherosclerotic effect) (33–35) and alleviating angiotensin II - mediated damage of cardiomyocytes, a key mechanism in the pathogenesis and progression of CHF (36). In the present study, increased level of platelet clusterin may be a cytoprotective reaction against progression of CHF. In contrast, that serum clusterin level decreased due to continuous consumption was associated with an unfavorable prognosis in patients with CHF (34).
GBP subunit alpha-11, called as “G protein”, plays a central physiological role in the regulation of cardiac contractility by neurohumoral signals (37, 38). In addition, G proteins modulate the binding of angiotensin-II to adrenal cortex receptors in the homeostatic regulation of the cardiovascular system (39, 40). The increase found in this study agrees with previous reports in humans with cardiomyopathy, where functional activity of G protein increased (41). Upregulation of GNB in dogs with CHF may be considered as an adaptive protective response after myocardial injury (evidenced by increased serum cTnI levels) to prevent myocytes from apoptosis as reported in mice with ischemic stress (42), and regulate cardiac contractility during CHF.
CXCL10, also known as interferon-inducible protein-10 (IP-10), a member of chemokine family, was one of the down-regulated platelet proteins in dogs with CHF in the present study, similar to the results of serum CXCL10 in mice with myocardial infarction (43). After releasing from leukocytes and endothelial cells, CXCL10 binds to its receptor (G protein – coupled receptor; CXCR3), and leads to a range of inflammatory and immune responses (meaning the regulation of leukocyte and lymphocyte traffic to the damaged tissue); key factors in cardiovascular diseases (CVD) such as atherosclerosis, myocardial infarction (43–45), and cardiac remodeling (46). Circulating CXCL10 was found as the best indicators amongst others chemokines for differentiating healthy and heart failure patients (46).
COX enzyme plays important role for mitochondrial oxidative metabolism and ATP synthesis. In this study, COX-II was one of the down-regulated platelet proteins in dogs with CHF. COX functions are affected by several pathological conditions including myocardial ischemia (47) and cardiomyopathy in humans (48). COX deficiency was associated with increased mitochondrial reactive oxygen species (ROS) production and cellular toxicity (47). Increasing oxidative stress lead to pathological changes in COX structure and function, resulting in exacerbating apoptosis in patients with CHF (49). In the light of this information, down-regulated COX-II protein may be resulted from its excessive use in response to increased oxidative stress in the progression of CHF in dogs. Since COX enzyme handles more than 90% of molecular oxygen produced by the mammalian cells and tissue (47), low COX-II levels may be a factor in the emergence of exercise intolerance and/or respiratory stress in dogs with CHF, as reported in humans (49). Platelet COX activity enhancing drugs may have a potential to limit the progression of adverse cardiac remodeling and heart failure, as suggested for septic patients with low COX activity (50).
Similarly to our study, serum levels of cathepsin D were down-regulated in human with CHF (51) and myocardial infarction (52). Cathepsin D plays a role in cardiomyocyte autophagy, which protects against the progression of post-infarction cardiac remodeling (52). In this study, decreased cathepsin D was most probably resulted from its excessive use for myocardial damage contributing to left sided cardiac remodeling in dogs. This may reflect the impaired protective role of cathepsin D (52), meaning less autophagy in these dogs.
Finally, there were other two proteins showing downregulation in CHF dogs, the cytosolic brain type homodimeric - creatine kinase (CKB) and type 1 of serine/threonine phosphatases (PP1). The lower CKB concentrations in dogs with CHF may be contribute to contractile dysfunction resulted from impaired myocardial energy metabolism in these patients (53). PP1 is considered a key regulator of cardiac function, and modulation of its activity may represent a novel therapeutic target in heart failure in humans (54, 55) and dogs.
Regarding on protein-protein interaction, there is a high interaction potential among COX enzymes and between COX and CYTB to regulate oxidative metabolism. CXCL10 stimulates positively G protein-coupled receptor (CXCR3) and CXCR3 is a chemokine receptor that is expressed mainly on effector T cells (56). Therefore, both of them may play an important role in immune-inflammatory reaction such as T cell trafficking and function during CHF in dogs. In this study, reactome and string analysis showed the relationship between the proteins and their roles in hemostasis, signal transduction, immune system, protein metabolism, muscle contraction, apoptosis, extracellular matrix and chromatin organizations, and individual or protein-protein interaction in the formation of cellular responses to external stimuli. Panther pathway analysis of observed platelet proteins support further the roles of chemokine and cytokine mediated inflammatory pathway, and alpha-adrenergic and endothelin signaling pathways in involvement of the pathogenesis of CHF, in agreement with the previous studies of an experimental model of heart failure in swine (16), and humans with heart failure (17).