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Myocardial leptin transcription in feline hypertrophic cardiomyopathy
Research in Veterinary Science 112 (2017) 105–108
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Myocardial leptin transcription in feline hypertrophic cardiomyopathy Sonja Fonfara a,b,⁎,1, Sarah Kitz c, Udo Hetzel b,c, Anja Kipar b,c a b c
Companion Animal Studies, University of Bristol, Langford House, Langford, Bristol BS40 5DU, United Kingdom Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, 00014, Finland Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 268, 8057 Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 6 December 2016 Received in revised form 8 February 2017 Accepted 10 February 2017 Available online xxxx Keywords: Thrombus Cardiac remodeling Adipokine Heart Cardiomyocyte Atrium
a b s t r a c t Leptin is an adipokine, which is in humans with cardiac disease suspected to be involved in myocardial remodeling and thrombus formation. In cats, however, it is not known whether leptin plays a role in cardiac disease, i.e. hypertrophic cardiomyopathy (HCM) and the presence of an atrial thrombus (AT). The objective of the study was therefore to establish whether leptin is transcribed in the feline myocardium and to compare myocardial leptin mRNA concentrations in cats with HCM with and without AT, and in cats without cardiac diseases. Myocardial samples from 15 cats with HCM (five of these with AT), and 12 cats without cardiac diseases were investigated for leptin mRNA expression using quantitative reverse transcriptase PCR, and the transcription levels were correlated with those obtained for a range of cytokines and remodeling parameters. Leptin mRNA expression was detected in the myocardium in all heart regions, with generally higher concentrations in the atria than in the ventricles. Cats with HCM exhibited higher atria and ventricular leptin transcription than cats without cardiac diseases, but reduced ventricular transcription levels in the presence of AT. A positive correlation between leptin, cytokine and remodeling marker transcription levels was observed. The present study shows that leptin is constitutively transcribed in the feline myocardium. The observed increase in leptin mRNA concentrations in the myocardium from cats with HCM and the reduction when an AT is present suggests varying gene activation in different stages of the disease and a potential involvement of leptin in the feline cardiac remodeling process. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Leptin is a mainly adipocyte-derived adipokine and one of the most important and widely studied factors in the control of energy balance. In humans, elevated circulating levels are observed with obesity and in cardiac disease, independent of body weight, and an association with low-grade inflammation and a pro-thrombotic state has been reported (Feijoo-Bandin et al., 2015; Ghantous et al., 2015; Schafer and Konstantinides, 2011). Leptin has been shown to promote platelet adhesion, activation and aggregation (Elbatarny and Maurice, 2005; Schafer and Konstantinides, 2011). Similarly, obese cats exhibit increased circulating leptin concentrations and an association with a increased clotting tendency was suspected (Appleton et al., 2000; Abbreviations: AT, atrial thrombus; HCM, hypertrophic cardiomyopathy; IL, interleukin; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; TGF, transforming growth factor; TNF, tumor necrosis factor. ⁎ Corresponding author at: Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON, N1G 2W1, Canada. E-mail addresses: [email protected] (S. Fonfara), [email protected] (S. Kitz), [email protected] (U. Hetzel), [email protected] (A. Kipar). 1 Present address: Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.
http://dx.doi.org/10.1016/j.rvsc.2017.02.006 0034-5288/© 2017 Elsevier Ltd. All rights reserved.
Bjornvad et al., 2014; Bjornvad et al., 2012). In the heart of humans, mice and rats, cardiomyocytes, pericardial fat and vascular smooth muscle cells have been shown to produce leptin, and its receptor is abundantly expressed on cardiomyocytes (Feijoo-Bandin et al., 2015; Ghantous et al., 2015). Leptin expression by cardiomyocytes is increased by endothelin-1 and angiotensin II, which are both elevated in cardiac disease. This would suggest a potential paracrine or autocrine role of leptin in the regulation of cardiac function as well as its contribution to pathological processes in the myocardium (Rajapurohitam et al., 2006). Leptin has been shown to stimulate several factors involved in extracellular matrix deposition in vitro, including metalloproteinases and collagen profiles, and may therefore contribute to myocardial remodeling and heart failure (Ghantous et al., 2015). The recently identified presence of a leptin receptor on the mitochondria of cardiomyocytes further suggests that intracellularly synthesized leptin could directly modulate mitochondrial function (Martinez-Abundis et al., 2015). However, whether leptin has damaging or protective effects on the heart is so far not known (Feijoo-Bandin et al., 2015). Some authors report anti-apoptotic and anti-fibrotic and therefore potentially cardioprotective effects of leptin (Feijoo-Bandin et al., 2015; Ghantous et al., 2015). For women, a U-shaped association between serum leptin concentrations and cardiovascular and all-cause mortality has been reported, with increased risk
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of death at both low and high circulating leptin levels (Mishra et al., 2015). Hypertrophic cardiomyopathy (HCM) is the most common cardiac disease in cats (Payne et al., 2015b), and an association with an increased body condition score has been reported (Payne et al., 2015b). The disease is characterized by idiopathic left ventricular hypertrophy accompanied by myocardial fibrosis, cardiomyocyte disorientation and intramural coronary arteriosclerosis (Biasato et al., 2015; Khor et al., 2015). Cardiac thrombus formation and subsequent thromboembolic events are known complications, and are associated with reduced survival (Payne et al., 2015a). Only little is known about potential factors involved in feline cardiac remodeling and thrombus formation. Whether leptin is produced by the feline myocardium and if it might vary in cats with HCM and atrial thrombus (AT) is not known. The objective of the study was therefore to investigate whether leptin is expressed in the feline myocardium and whether its expression is altered in cats with HCM and AT in comparison to cats without cardiac diseases. We hypothesized that HCM and AT is associated with increased myocardial leptin transcription.
2. Material and methods Fifteen cats with HCM and 12 cats without cardiac diseases were included in the study. These cats were patients that had been presented at the Universities of Helsinki and Bristol. HCM was clinically diagnosed by echocardiography, the diagnostic criterion for HCM was an idiopathic diffuse or segmental left ventricular wall thickness of ≥6 mm measured at end-diastole (Fox et al., 1995). Cats without cardiac diseases did not exhibit clinical evidence of cardiac disease, and echocardiography was not routinely carried out. The cats had been euthanized upon owner's request due to poor prognosis, impaired quality of life or financial constraints. Informed consent was obtained from owners prior to inclusion into the study and cats were assigned arbitrary numbers as identifiers. Institutional ethical approval was obtained from the University of Bristol. From each cat, the signalment including breed, sex, and age was recorded. The weight was available for 24 cats (13 cats with HCM, 11 cats with systemic diseases). The heart was removed within 1 h after death and grossly examined. Epicardial fat was removed and myocardial samples from the interventricular septum, right atrium and ventricle, and left atrium and ventricle were collected for RNA extraction and stored in RNA stabilising solution (RNAlater; Ambion, Life Technologies, Paisley, UK) at − 20 °C until analysed. Hearts were subsequently fixed in 10% formalin and samples from the same sites as those for RNA extraction were prepared and routinely paraffin wax embedded for histological examination (Fonfara et al., 2015). Twenty-four cats underwent full necropsy to identify relevant disease conditions, confirm HCM in cats with clinically diagnosed HCM, and exclude cardiac diseases in cats that did not exhibit clinical signs of heart disease. Samples were collected from all major organs and any gross abnormalities, fixed in 10% formalin and routinely paraffin-embedded for histological examination. For three cats that presented without cardiac diseases (one each with nasal polyp, oesophageal stricture, and discus prolapse) owners did not give consent for a full necropsy and only dissection and further examination of the heart was permitted. RNA extraction and real time reverse transcriptase PCR was carried out as reported previously (Fonfara et al., 2015). Primers for feline leptin (forward 5′-GCAGCTTGGCTGACAATTTAAAA-3′, reverse 5′-TCTAGTCTTGGCCCAGCTACCTAT-3′) were designed using Primer Express software (Applied Biosystems, Life Technologies), and BLAST searches performed to confirm gene specificity. Validation of the primer and PCR protocols was carried out as previously described (Fonfara et al., 2015). For the amplification of the cytokines IL-1, IL-2, IL-6, TNF-α, TGF-β, the matrix metalloproteinases (MMP)-2, -3, -9, -13 and the tissue inhibitors of matrix metalloproteinases (TIMP)1, -2, -3 published primer pairs were applied (Fonfara et al., 2015).
Statistical analysis was carried out using SPSS. Leptin, cytokines, MMP and TIMP were log transformed to improve normality and the model assumptions necessary for parametric analysis. Due to there being no difference in transcriptions between left and right atrium (data not shown), results were combined for analysis and are reported as ‘atria’. Similarly, no differences in transcriptions were observed between ventricular regions (interventricular septum, left and right ventricle; data not shown) and results were combined for analysis and are reported as ‘ventricles’. Cats were grouped into cats with HCM without AT (‘HCM without AT’), HCM with AT (‘HCM with AT’) and cats without cardiac diseases (‘non-cardiac’). Cardiac regions, groups and gender were compared using 1-way ANOVA and unpaired t-tests as appropriate. For the investigation of the relationship between leptin and age and the markers investigated ‘HCM without AT’ and ‘HCM with AT’ cats were combined (‘HCM’). Scatter plots were used to explore the association, followed by Pearson correlation test. Results are reported as mean and standard deviation. Statistical significance was defined as p b 0.05. 3. Results Twenty-seven cats were included in the study, ten cats with HCM without AT (‘HCM without AT’), five cats with HCM and AT (‘HCM with AT’), and 12 cats without cardiac diseases (‘non-cardiac’). ‘HCM without AT’ cats had a mean age of 6.5 years (±4.9 years) and a mean weight of 4.6 kg (±1.5 kg). Nine of these cats were male neutered, one was female entire. Four ‘HCM with AT’ cats were male neutered, one was female neutered; they had a mean age of 10.4 years (±4 years) and a mean body weight of 4.6 kg (±1.5 kg). ‘Non-cardiac’ cats had a mean age of 7 years (± 3.8 years) and a mean weight of 4.2 kg (± 1 kg), ten cats were female neutered, one male neutered and one male. Age and weight did not differ between ‘HCM without AT’ and ‘non-cardiac’ cats (p = 0.580 and p = 0.164, respectively); ‘HCM with AT’ cats were significantly older than ‘HCM without AT’ (p = 0.001) and ‘non-cardiac’ cats (p b 0.001). ‘Non-cardiac’ cats were diagnosed with localised disease (i.e. nasal polyp, oesophageal stricture, discus prolapse; n = 5), different forms of lymphoma (n = 3) or age-related nonspecific changes (n = 1) or had been euthanized due to behaviour abnormalities (n = 3) (Fonfara et al., 2015). None of these cats displayed histological myocardial abnormalities. Cats with HCM showed myocardial changes described for the disease (Biasato et al., 2015; Khor et al., 2015). Leptin was transcribed in all samples investigated, with significantly higher leptin mRNA concentrations in atria than in ventricles both in ‘HCM without AT’ cats (p = 0.003) and ‘non-cardiac’ cats (p b 0.001), but not in ‘HCM with AT’ cats (p = 0.083; Table 1). A comparison between the different groups revealed higher leptin mRNA concentrations in atria (p = 0.036) and ventricles (p = 0.001) from ‘HCM without AT’ cats than ‘non-cardiac’ cats (Table 1). ‘HCM with AT’ cats exhibited lower ventricular leptin mRNA concentrations in comparison to ‘HCM without AT’ (p = 0.003) and ‘non-cardiac’ cats (p = 0.039). No difference was detected for atrial leptin transcription comparing ‘HCM with AT’ cats with ‘HCM’ cats (p = 0.156) and ‘non-cardiac’ cats (p = 0.442; Table 1). A weak negative correlation of age and leptin was observed for ‘HCM’ cats (r = −0.352, p = 0.005, Fig. 1), but not for ‘non-cardiac’ cats (r = 0.01, p = 0.942). Furthermore, ‘HCM’ cats showed strong positive correlations between leptin and IL-2 (r = 0.793, p b 0.001; Fig. 2a), Table 1 Relative leptin mRNA concentrations in atria and ventricles from cats with hypertrophic cardiomyopathy (HCM; n = 10), HCM and atrial thrombus (HCM, AT; n = 5) and cats without cardiac diseases (non-cardiac; n = 12).
HCM HCM, AT Non-cardiac
Atria
Ventricles
P
3.1 (±0.53)# 2.4 (±1.2) 2.8 (±0.37)#
2.6 (±0.41)#$ 1.6 (±0.94)*$ 2.2 (±0.42)*#
0.003 0.083 b0.001
#, *, $: significant differences between groups. AT: atrial thrombus, HCM: hypertrophic cardiomyopathy.
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moderate correlations between leptin and IL-1 (r = 0.446, p = 0.001) and TNF-α (r = 0.579, p b 0.001), and a weak correlation between leptin and IL-6 (r = 0.385, p = 0.006).
4. Discussion
Fig. 1. Scatterplot showing a weak negative correlation between relative leptin mRNA expression and age (r = −0.352, p = 0.005).
TGF-β (r = 0.650, p b 0.001; Fig. 2b), MMP-2 (r = 0.697, p b 0.001; Fig. 2c), MMP-3 (r = 0.851, p b 0.001), MMP-13 (r = 0.642, p b 0.001), TIMP2 (r = 0.790, p b 0.001; Fig. 2d), and TIMP-3 (r = 0.637, p b 0.001),
Leptin was transcribed in all samples investigated, indicating that the feline myocardium does produce leptin constitutively, similar to what has been reported for other species (Fonfara et al., 2011; Karmazyn et al., 2013; Purdham et al., 2004). Consistent with our hypothesis, an increase in leptin transcription was observed in cats with HCM, however, cats with HCM and AT exhibited lower ventricular mRNA concentrations in comparison to cats with HCM alone and cats without cardiac disease. This indicates that leptin transcription varies in different stages of the disease and suggests that it might be involved in cardiac remodeling (Karmazyn et al., 2013). The positive correlation of leptin and inflammatory and pro-fibrotic cytokines, MMPs and TIMPs supports an association of leptin with inflammatory, immune modulatory and remodeling parameters in cats, as reported in human medicine. For humans, a controversy about the cardiac effect of leptin exists (Feijoo-Bandin et al., 2015). The inflammatory and pro-thrombotic effects of leptin have been suggested to have a damaging influence on the heart. On the other hand, leptin was suspected to be cardioprotective by stimulating nitric oxide signaling and anti-apoptotic and anti-hypertrophic effects (Feijoo-Bandin et al., 2015). However, a pro-apoptotic influence has
Fig. 2. a: Scatterplot showing a strong positive correlation between relative IL-2 and leptin mRNA expression (r = 0.793, p b 0.001). b: Scatterplot showing a strong positive correlation between relative TGF-β and leptin mRNA expression (r = 0.650, p b 0.001). c: Scatterplot showing a strong positive correlation between relative MMP-2 and leptin mRNA expression (r = 0.697, p b 0.001). d: Scatterplot showing a strong positive correlation between relative TIMP-2 and leptin mRNA expression (r = 0.790, p b 0.001).
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been reported under damaging conditions (Feijoo-Bandin et al., 2015), which were most likely present in the cats with HCM of the current study, considering that these had end-stage disease. Increased leptin transcription and positive correlation with several cytokines in the cats with HCM might reflect ongoing myocardial inflammation and remodeling associated with progression of the disease. Myocardial fibrosis and myofibre disorganisation are well-known pathological findings in feline HCM and an inflammatory cell infiltration has been reported (Biasato et al., 2015; Khor et al., 2015). Alternatively, the inflammatory response and the MMP and TIMP activation could indicate a cardiac remodeling process that results in impaired ventricular function, with a functional reserve sufficient to reduce the risk of thrombus formation in some cats. Consequentially, the lower mRNA concentration of leptin in cats with HCM and AT might be consistent with a different form of progression of the disease, or could be a subsequent event associated with progressed remodeling processes and ventricular dysfunction that results in blood flow abnormalities which promote thrombus formation in the atria. Clinically, AT formation in cats with HCM suggests a more progressed stage of disease and is associated with an increased risk of death (Payne et al., 2015a). The generally higher leptin mRNA concentrations in atria than in ventricles suggest regional differences in myocardial reactivity as has been reported previously for other parameters of inflammation and remodeling (Fonfara et al., 2015). A direct contribution of leptin to thrombus formation, as suggested in people (Elbatarny and Maurice, 2005; Schafer and Konstantinides, 2011), seems unlikely considering the unchanged atrial and reduced ventricular leptin transcription in cats with HCM and AT in comparison to cats with HCM alone and without cardiac diseases. However, it is possible that the results obtained for myocardial transcription are not comparable to results for circulating leptin concentrations. The negative association of leptin and age in cats with HCM might support a potential association of leptin with impaired myocardial reactivity and ability for repair. Increased ventricular stiffness has been reported with increasing age and might contribute to the cardiac remodeling processes associated with HCM (Payne et al., 2013; Schober and Fuentes, 2001). However, this association was weak only and the absence of a negative correlation in the cats without cardiac disease suggests that the disease and not age influences this result. Limitations of the study include the small number of cats, in particular of cats with HCM and AT, which will have limited statistical significance. A body condition score was only recorded for a few individual cats and a muscle condition score was not obtained. Obesity has been reported to increase circulating leptin concentrations in humans and cats, and might have influenced results (Appleton et al., 2000; Bjornvad et al., 2014; Feijoo-Bandin et al., 2015). However, the cats included were not obese. A gender difference was present between the groups. Higher leptin concentrations have been reported for women, however, no sex difference was found in cats (Bjornvad et al., 2014). It is therefore unlikely that the higher mRNA concentrations detected for the cats with HCM, which were primarily male, are due to gender difference. The group of cats without cardiac diseases was inhomogeneous and clinical information was not complete for all cats. Different diseases might influence cardiac leptin concentrations. However, no difference was detected comparing leptin transcription between cats with diseases that might have had an influence on cardiac function and diseases unlikely to have cardiac effects (data not shown). Furthermore, echocardiography was not obtained in these cats, however, gross examination and histology did not identify any cardiac disease. We have investigated myocardial and not circulating leptin concentrations and results are likely to differ.
5. Conclusion The results of this study showed leptin transcription in the myocardium of cats with and without cardiac diseases. The observed increase in leptin transcription in the myocardium of cats with HCM and the reduction in cats with HCM and AT suggests varying gene activation in different stages of the disease and a potential involvement of leptin in feline cardiac remodeling processes. References Appleton, D.J., Rand, J.S., Sunvold, G.D., 2000. Plasma leptin concentrations in cats: reference range, effect of weight gain and relationship with adiposity as measured by dual energy X-ray absorptiometry. J. Feline Med. Surg. 2, 191–199. Biasato, I., Francescone, L., La Rosa, G., Tursi, M., 2015. Anatomopathological staging of feline hypertrophic cardiomyopathy through quantitative evaluation based on morphometric and histopathological data. Res. Vet. Sci. 102, 136–141. Bjornvad, C.R., Wiinberg, B., Kristensen, A.T., 2012. Obesity increases initial rate of fibrin formation during blood coagulation in domestic shorthaired cats. J. Anim. Physiol. Anim. Nutr. (Berl) 96, 834–841. Bjornvad, C.R., Rand, J.S., Tan, H.Y., Jensen, K.S., Rose, F.J., Armstrong, P.J., Whitehead, J.P., 2014. Obesity and sex influence insulin resistance and total and multimer adiponectin levels in adult neutered domestic shorthair client-owned cats. Domest. Anim. Endocrinol. 47, 55–64. Elbatarny, H.S., Maurice, D.H., 2005. Leptin-mediated activation of human platelets: involvement of a leptin receptor and phosphodiesterase 3A-containing cellular signaling complex. Am. J. Physiol. Endocrinol. Metab. 289, E695–E702. Feijoo-Bandin, S., Portoles, M., Rosello-Lleti, E., Rivera, M., Gonzalez-Juanatey, J.R., Lago, F., 2015. 20 years of leptin: role of leptin in cardiomyocyte physiology and physiopathology. Life Sci. 140, 10–18. Fonfara, S., Hetzel, U., Tew, S.R., Dukes-McEwan, J., Cripps, P., Clegg, P.D., 2011. Leptin expression in dogs with cardiac disease and congestive heart failure. J. Vet. Intern. Med. 25, 1017–1024. Fonfara, S., Hetzel, U., Hahn, S., Kipar, A., 2015. Age- and gender-dependent myocardial transcription patterns of cytokines and extracellular matrix remodelling enzymes in cats with non-cardiac diseases. Exp. Gerontol. 72, 117–123. Fox, P.R., Liu, S.K., Maron, B.J., 1995. Echocardiographic assessment of spontaneously occurring feline hypertrophic cardiomyopathy. An animal model of human disease. Circulation 92, 2645–2651. Ghantous, C.M., Azrak, Z., Hanache, S., Abou-Kheir, W., Zeidan, A., 2015. Differential role of leptin and adiponectin in cardiovascular system. Int. J. Endocrinol. 2015, 534320. Karmazyn, M., Gan, X.T., Rajapurohitam, V., 2013. The potential contribution of circulating and locally produced leptin to cardiac hypertrophy and failure. Can. J. Physiol. Pharmacol. 91, 883–888. Khor, K.H., Campbell, F.E., Owen, H., Shiels, I.A., Mills, P.C., 2015. Myocardial collagen deposition and inflammatory cell infiltration in cats with pre-clinical hypertrophic cardiomyopathy. Vet. J. 203, 161–168. Martinez-Abundis, E., Rajapurohitam, V., Gertler, A., Karmazyn, M., 2015. Identification of functional leptin receptors expressed in ventricular mitochondria. Mol. Cell. Biochem. 408, 155–162. Mishra, S., Harris, T.B., Hsueh, W.C., Hue, T., Leak, T.S., Li, R., Mehta, M., Vaisse, C., Sahyoun, N.R., 2015. The association of serum leptin with mortality in older adults. PLoS One 10, e0140763. Payne, J.R., Borgeat, K., Connolly, D.J., Boswood, A., Dennis, S., Wagner, T., Menaut, P., Maerz, I., Evans, D., Simons, V.E., Brodbelt, D.C., Luis Fuentes, V., 2013. Prognostic indicators in cats with hypertrophic cardiomyopathy. J. Vet. Intern. Med. 27, 1427–1436. Payne, J.R., Borgeat, K., Brodbelt, D.C., Connolly, D.J., Luis Fuentes, V., 2015a. Risk factors associated with sudden death vs. congestive heart failure or arterial thromboembolism in cats with hypertrophic cardiomyopathy. J. Vet. Cardiol. 17 (Suppl. 1), S318-328. Payne, J.R., Brodbelt, D.C., Luis Fuentes, V., 2015b. Cardiomyopathy prevalence in 780 apparently healthy cats in rehoming centres (the CatScan study). J. Vet. Cardiol. 17 (Suppl. 1), S244–S257. Purdham, D.M., Zou, M.X., Rajapurohitam, V., Karmazyn, M., 2004. Rat heart is a site of leptin production and action. Am. J. Physiol. Heart Circ. Physiol. 287, H2877–H2884. Rajapurohitam, V., Javadov, S., Purdham, D.M., Kirshenbaum, L.A., Karmazyn, M., 2006. An autocrine role for leptin in mediating the cardiomyocyte hypertrophic effects of angiotensin II and endothelin-1. J. Mol. Cell. Cardiol. 41, 265–274. Schafer, K., Konstantinides, S., 2011. Adipokines and thrombosis. Clin. Exp. Pharmacol. Physiol. 38, 864–871. Schober, K.E., Fuentes, V.L., 2001. Effects of age, body weight, and heart rate on transmitral and pulmonary venous flow in clinically normal dogs. Am. J. Vet. Res. 62, 1447–1454.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Myocardial leptin transcription in feline hypertrophic cardiomyopathy Sonja Fonfara a,b,⁎,1, Sarah Kitz c, Udo Hetzel b,c, Anja Kipar b,c a b c
Companion Animal Studies, University of Bristol, Langford House, Langford, Bristol BS40 5DU, United Kingdom Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, 00014, Finland Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 268, 8057 Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 6 December 2016 Received in revised form 8 February 2017 Accepted 10 February 2017 Available online xxxx Keywords: Thrombus Cardiac remodeling Adipokine Heart Cardiomyocyte Atrium
a b s t r a c t Leptin is an adipokine, which is in humans with cardiac disease suspected to be involved in myocardial remodeling and thrombus formation. In cats, however, it is not known whether leptin plays a role in cardiac disease, i.e. hypertrophic cardiomyopathy (HCM) and the presence of an atrial thrombus (AT). The objective of the study was therefore to establish whether leptin is transcribed in the feline myocardium and to compare myocardial leptin mRNA concentrations in cats with HCM with and without AT, and in cats without cardiac diseases. Myocardial samples from 15 cats with HCM (five of these with AT), and 12 cats without cardiac diseases were investigated for leptin mRNA expression using quantitative reverse transcriptase PCR, and the transcription levels were correlated with those obtained for a range of cytokines and remodeling parameters. Leptin mRNA expression was detected in the myocardium in all heart regions, with generally higher concentrations in the atria than in the ventricles. Cats with HCM exhibited higher atria and ventricular leptin transcription than cats without cardiac diseases, but reduced ventricular transcription levels in the presence of AT. A positive correlation between leptin, cytokine and remodeling marker transcription levels was observed. The present study shows that leptin is constitutively transcribed in the feline myocardium. The observed increase in leptin mRNA concentrations in the myocardium from cats with HCM and the reduction when an AT is present suggests varying gene activation in different stages of the disease and a potential involvement of leptin in the feline cardiac remodeling process. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Leptin is a mainly adipocyte-derived adipokine and one of the most important and widely studied factors in the control of energy balance. In humans, elevated circulating levels are observed with obesity and in cardiac disease, independent of body weight, and an association with low-grade inflammation and a pro-thrombotic state has been reported (Feijoo-Bandin et al., 2015; Ghantous et al., 2015; Schafer and Konstantinides, 2011). Leptin has been shown to promote platelet adhesion, activation and aggregation (Elbatarny and Maurice, 2005; Schafer and Konstantinides, 2011). Similarly, obese cats exhibit increased circulating leptin concentrations and an association with a increased clotting tendency was suspected (Appleton et al., 2000; Abbreviations: AT, atrial thrombus; HCM, hypertrophic cardiomyopathy; IL, interleukin; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; TGF, transforming growth factor; TNF, tumor necrosis factor. ⁎ Corresponding author at: Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON, N1G 2W1, Canada. E-mail addresses: [email protected] (S. Fonfara), [email protected] (S. Kitz), [email protected] (U. Hetzel), [email protected] (A. Kipar). 1 Present address: Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.
http://dx.doi.org/10.1016/j.rvsc.2017.02.006 0034-5288/© 2017 Elsevier Ltd. All rights reserved.
Bjornvad et al., 2014; Bjornvad et al., 2012). In the heart of humans, mice and rats, cardiomyocytes, pericardial fat and vascular smooth muscle cells have been shown to produce leptin, and its receptor is abundantly expressed on cardiomyocytes (Feijoo-Bandin et al., 2015; Ghantous et al., 2015). Leptin expression by cardiomyocytes is increased by endothelin-1 and angiotensin II, which are both elevated in cardiac disease. This would suggest a potential paracrine or autocrine role of leptin in the regulation of cardiac function as well as its contribution to pathological processes in the myocardium (Rajapurohitam et al., 2006). Leptin has been shown to stimulate several factors involved in extracellular matrix deposition in vitro, including metalloproteinases and collagen profiles, and may therefore contribute to myocardial remodeling and heart failure (Ghantous et al., 2015). The recently identified presence of a leptin receptor on the mitochondria of cardiomyocytes further suggests that intracellularly synthesized leptin could directly modulate mitochondrial function (Martinez-Abundis et al., 2015). However, whether leptin has damaging or protective effects on the heart is so far not known (Feijoo-Bandin et al., 2015). Some authors report anti-apoptotic and anti-fibrotic and therefore potentially cardioprotective effects of leptin (Feijoo-Bandin et al., 2015; Ghantous et al., 2015). For women, a U-shaped association between serum leptin concentrations and cardiovascular and all-cause mortality has been reported, with increased risk
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of death at both low and high circulating leptin levels (Mishra et al., 2015). Hypertrophic cardiomyopathy (HCM) is the most common cardiac disease in cats (Payne et al., 2015b), and an association with an increased body condition score has been reported (Payne et al., 2015b). The disease is characterized by idiopathic left ventricular hypertrophy accompanied by myocardial fibrosis, cardiomyocyte disorientation and intramural coronary arteriosclerosis (Biasato et al., 2015; Khor et al., 2015). Cardiac thrombus formation and subsequent thromboembolic events are known complications, and are associated with reduced survival (Payne et al., 2015a). Only little is known about potential factors involved in feline cardiac remodeling and thrombus formation. Whether leptin is produced by the feline myocardium and if it might vary in cats with HCM and atrial thrombus (AT) is not known. The objective of the study was therefore to investigate whether leptin is expressed in the feline myocardium and whether its expression is altered in cats with HCM and AT in comparison to cats without cardiac diseases. We hypothesized that HCM and AT is associated with increased myocardial leptin transcription.
2. Material and methods Fifteen cats with HCM and 12 cats without cardiac diseases were included in the study. These cats were patients that had been presented at the Universities of Helsinki and Bristol. HCM was clinically diagnosed by echocardiography, the diagnostic criterion for HCM was an idiopathic diffuse or segmental left ventricular wall thickness of ≥6 mm measured at end-diastole (Fox et al., 1995). Cats without cardiac diseases did not exhibit clinical evidence of cardiac disease, and echocardiography was not routinely carried out. The cats had been euthanized upon owner's request due to poor prognosis, impaired quality of life or financial constraints. Informed consent was obtained from owners prior to inclusion into the study and cats were assigned arbitrary numbers as identifiers. Institutional ethical approval was obtained from the University of Bristol. From each cat, the signalment including breed, sex, and age was recorded. The weight was available for 24 cats (13 cats with HCM, 11 cats with systemic diseases). The heart was removed within 1 h after death and grossly examined. Epicardial fat was removed and myocardial samples from the interventricular septum, right atrium and ventricle, and left atrium and ventricle were collected for RNA extraction and stored in RNA stabilising solution (RNAlater; Ambion, Life Technologies, Paisley, UK) at − 20 °C until analysed. Hearts were subsequently fixed in 10% formalin and samples from the same sites as those for RNA extraction were prepared and routinely paraffin wax embedded for histological examination (Fonfara et al., 2015). Twenty-four cats underwent full necropsy to identify relevant disease conditions, confirm HCM in cats with clinically diagnosed HCM, and exclude cardiac diseases in cats that did not exhibit clinical signs of heart disease. Samples were collected from all major organs and any gross abnormalities, fixed in 10% formalin and routinely paraffin-embedded for histological examination. For three cats that presented without cardiac diseases (one each with nasal polyp, oesophageal stricture, and discus prolapse) owners did not give consent for a full necropsy and only dissection and further examination of the heart was permitted. RNA extraction and real time reverse transcriptase PCR was carried out as reported previously (Fonfara et al., 2015). Primers for feline leptin (forward 5′-GCAGCTTGGCTGACAATTTAAAA-3′, reverse 5′-TCTAGTCTTGGCCCAGCTACCTAT-3′) were designed using Primer Express software (Applied Biosystems, Life Technologies), and BLAST searches performed to confirm gene specificity. Validation of the primer and PCR protocols was carried out as previously described (Fonfara et al., 2015). For the amplification of the cytokines IL-1, IL-2, IL-6, TNF-α, TGF-β, the matrix metalloproteinases (MMP)-2, -3, -9, -13 and the tissue inhibitors of matrix metalloproteinases (TIMP)1, -2, -3 published primer pairs were applied (Fonfara et al., 2015).
Statistical analysis was carried out using SPSS. Leptin, cytokines, MMP and TIMP were log transformed to improve normality and the model assumptions necessary for parametric analysis. Due to there being no difference in transcriptions between left and right atrium (data not shown), results were combined for analysis and are reported as ‘atria’. Similarly, no differences in transcriptions were observed between ventricular regions (interventricular septum, left and right ventricle; data not shown) and results were combined for analysis and are reported as ‘ventricles’. Cats were grouped into cats with HCM without AT (‘HCM without AT’), HCM with AT (‘HCM with AT’) and cats without cardiac diseases (‘non-cardiac’). Cardiac regions, groups and gender were compared using 1-way ANOVA and unpaired t-tests as appropriate. For the investigation of the relationship between leptin and age and the markers investigated ‘HCM without AT’ and ‘HCM with AT’ cats were combined (‘HCM’). Scatter plots were used to explore the association, followed by Pearson correlation test. Results are reported as mean and standard deviation. Statistical significance was defined as p b 0.05. 3. Results Twenty-seven cats were included in the study, ten cats with HCM without AT (‘HCM without AT’), five cats with HCM and AT (‘HCM with AT’), and 12 cats without cardiac diseases (‘non-cardiac’). ‘HCM without AT’ cats had a mean age of 6.5 years (±4.9 years) and a mean weight of 4.6 kg (±1.5 kg). Nine of these cats were male neutered, one was female entire. Four ‘HCM with AT’ cats were male neutered, one was female neutered; they had a mean age of 10.4 years (±4 years) and a mean body weight of 4.6 kg (±1.5 kg). ‘Non-cardiac’ cats had a mean age of 7 years (± 3.8 years) and a mean weight of 4.2 kg (± 1 kg), ten cats were female neutered, one male neutered and one male. Age and weight did not differ between ‘HCM without AT’ and ‘non-cardiac’ cats (p = 0.580 and p = 0.164, respectively); ‘HCM with AT’ cats were significantly older than ‘HCM without AT’ (p = 0.001) and ‘non-cardiac’ cats (p b 0.001). ‘Non-cardiac’ cats were diagnosed with localised disease (i.e. nasal polyp, oesophageal stricture, discus prolapse; n = 5), different forms of lymphoma (n = 3) or age-related nonspecific changes (n = 1) or had been euthanized due to behaviour abnormalities (n = 3) (Fonfara et al., 2015). None of these cats displayed histological myocardial abnormalities. Cats with HCM showed myocardial changes described for the disease (Biasato et al., 2015; Khor et al., 2015). Leptin was transcribed in all samples investigated, with significantly higher leptin mRNA concentrations in atria than in ventricles both in ‘HCM without AT’ cats (p = 0.003) and ‘non-cardiac’ cats (p b 0.001), but not in ‘HCM with AT’ cats (p = 0.083; Table 1). A comparison between the different groups revealed higher leptin mRNA concentrations in atria (p = 0.036) and ventricles (p = 0.001) from ‘HCM without AT’ cats than ‘non-cardiac’ cats (Table 1). ‘HCM with AT’ cats exhibited lower ventricular leptin mRNA concentrations in comparison to ‘HCM without AT’ (p = 0.003) and ‘non-cardiac’ cats (p = 0.039). No difference was detected for atrial leptin transcription comparing ‘HCM with AT’ cats with ‘HCM’ cats (p = 0.156) and ‘non-cardiac’ cats (p = 0.442; Table 1). A weak negative correlation of age and leptin was observed for ‘HCM’ cats (r = −0.352, p = 0.005, Fig. 1), but not for ‘non-cardiac’ cats (r = 0.01, p = 0.942). Furthermore, ‘HCM’ cats showed strong positive correlations between leptin and IL-2 (r = 0.793, p b 0.001; Fig. 2a), Table 1 Relative leptin mRNA concentrations in atria and ventricles from cats with hypertrophic cardiomyopathy (HCM; n = 10), HCM and atrial thrombus (HCM, AT; n = 5) and cats without cardiac diseases (non-cardiac; n = 12).
HCM HCM, AT Non-cardiac
Atria
Ventricles
P
3.1 (±0.53)# 2.4 (±1.2) 2.8 (±0.37)#
2.6 (±0.41)#$ 1.6 (±0.94)*$ 2.2 (±0.42)*#
0.003 0.083 b0.001
#, *, $: significant differences between groups. AT: atrial thrombus, HCM: hypertrophic cardiomyopathy.
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moderate correlations between leptin and IL-1 (r = 0.446, p = 0.001) and TNF-α (r = 0.579, p b 0.001), and a weak correlation between leptin and IL-6 (r = 0.385, p = 0.006).
4. Discussion
Fig. 1. Scatterplot showing a weak negative correlation between relative leptin mRNA expression and age (r = −0.352, p = 0.005).
TGF-β (r = 0.650, p b 0.001; Fig. 2b), MMP-2 (r = 0.697, p b 0.001; Fig. 2c), MMP-3 (r = 0.851, p b 0.001), MMP-13 (r = 0.642, p b 0.001), TIMP2 (r = 0.790, p b 0.001; Fig. 2d), and TIMP-3 (r = 0.637, p b 0.001),
Leptin was transcribed in all samples investigated, indicating that the feline myocardium does produce leptin constitutively, similar to what has been reported for other species (Fonfara et al., 2011; Karmazyn et al., 2013; Purdham et al., 2004). Consistent with our hypothesis, an increase in leptin transcription was observed in cats with HCM, however, cats with HCM and AT exhibited lower ventricular mRNA concentrations in comparison to cats with HCM alone and cats without cardiac disease. This indicates that leptin transcription varies in different stages of the disease and suggests that it might be involved in cardiac remodeling (Karmazyn et al., 2013). The positive correlation of leptin and inflammatory and pro-fibrotic cytokines, MMPs and TIMPs supports an association of leptin with inflammatory, immune modulatory and remodeling parameters in cats, as reported in human medicine. For humans, a controversy about the cardiac effect of leptin exists (Feijoo-Bandin et al., 2015). The inflammatory and pro-thrombotic effects of leptin have been suggested to have a damaging influence on the heart. On the other hand, leptin was suspected to be cardioprotective by stimulating nitric oxide signaling and anti-apoptotic and anti-hypertrophic effects (Feijoo-Bandin et al., 2015). However, a pro-apoptotic influence has
Fig. 2. a: Scatterplot showing a strong positive correlation between relative IL-2 and leptin mRNA expression (r = 0.793, p b 0.001). b: Scatterplot showing a strong positive correlation between relative TGF-β and leptin mRNA expression (r = 0.650, p b 0.001). c: Scatterplot showing a strong positive correlation between relative MMP-2 and leptin mRNA expression (r = 0.697, p b 0.001). d: Scatterplot showing a strong positive correlation between relative TIMP-2 and leptin mRNA expression (r = 0.790, p b 0.001).
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been reported under damaging conditions (Feijoo-Bandin et al., 2015), which were most likely present in the cats with HCM of the current study, considering that these had end-stage disease. Increased leptin transcription and positive correlation with several cytokines in the cats with HCM might reflect ongoing myocardial inflammation and remodeling associated with progression of the disease. Myocardial fibrosis and myofibre disorganisation are well-known pathological findings in feline HCM and an inflammatory cell infiltration has been reported (Biasato et al., 2015; Khor et al., 2015). Alternatively, the inflammatory response and the MMP and TIMP activation could indicate a cardiac remodeling process that results in impaired ventricular function, with a functional reserve sufficient to reduce the risk of thrombus formation in some cats. Consequentially, the lower mRNA concentration of leptin in cats with HCM and AT might be consistent with a different form of progression of the disease, or could be a subsequent event associated with progressed remodeling processes and ventricular dysfunction that results in blood flow abnormalities which promote thrombus formation in the atria. Clinically, AT formation in cats with HCM suggests a more progressed stage of disease and is associated with an increased risk of death (Payne et al., 2015a). The generally higher leptin mRNA concentrations in atria than in ventricles suggest regional differences in myocardial reactivity as has been reported previously for other parameters of inflammation and remodeling (Fonfara et al., 2015). A direct contribution of leptin to thrombus formation, as suggested in people (Elbatarny and Maurice, 2005; Schafer and Konstantinides, 2011), seems unlikely considering the unchanged atrial and reduced ventricular leptin transcription in cats with HCM and AT in comparison to cats with HCM alone and without cardiac diseases. However, it is possible that the results obtained for myocardial transcription are not comparable to results for circulating leptin concentrations. The negative association of leptin and age in cats with HCM might support a potential association of leptin with impaired myocardial reactivity and ability for repair. Increased ventricular stiffness has been reported with increasing age and might contribute to the cardiac remodeling processes associated with HCM (Payne et al., 2013; Schober and Fuentes, 2001). However, this association was weak only and the absence of a negative correlation in the cats without cardiac disease suggests that the disease and not age influences this result. Limitations of the study include the small number of cats, in particular of cats with HCM and AT, which will have limited statistical significance. A body condition score was only recorded for a few individual cats and a muscle condition score was not obtained. Obesity has been reported to increase circulating leptin concentrations in humans and cats, and might have influenced results (Appleton et al., 2000; Bjornvad et al., 2014; Feijoo-Bandin et al., 2015). However, the cats included were not obese. A gender difference was present between the groups. Higher leptin concentrations have been reported for women, however, no sex difference was found in cats (Bjornvad et al., 2014). It is therefore unlikely that the higher mRNA concentrations detected for the cats with HCM, which were primarily male, are due to gender difference. The group of cats without cardiac diseases was inhomogeneous and clinical information was not complete for all cats. Different diseases might influence cardiac leptin concentrations. However, no difference was detected comparing leptin transcription between cats with diseases that might have had an influence on cardiac function and diseases unlikely to have cardiac effects (data not shown). Furthermore, echocardiography was not obtained in these cats, however, gross examination and histology did not identify any cardiac disease. We have investigated myocardial and not circulating leptin concentrations and results are likely to differ.
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