Heart failure and adipose tissue dysfunction appear to interact in a bidirectional fashion. Excessive release of lipolytic substances, such as catecholamines, TNF-# and natriuretic peptides, and increased susceptibility of adipocytes to these stimuli may occur in heart failure. The mechanism may contribute to adipose tissue wasting in cardiac cachexia patients. On the other hand, disordered adipose tissue function may predispose to heart failure through release of negatively inotropic substances. Changes in adipokine and fatty acid secretion patterns may be involved. Furthermore, adipose tissue dysfunction is associated with insulin resistance and low grade systemic inflammation, which could, both, worsen heart failure. Although these ideas have been discussed in the literature in recent years, they have not been sufficiently investigated in patients. The overall goal of WP09 is a detailed analysis of adipose tissue dysfunction in heart failure, with emphasis on the accompanying role of comorbidities, e.g. cardiac cachexia, type 2 diabetes, and obesity. The tasks included in both objectives may help to define molecular targets for future treatment directions and may help to better understand proposed treatment options in heart failure (e.g. the use of natriuretic peptides).
Objective 1: Adipokine production in heart failure
Adipokines are secreted products of adipose tissue. During the last years, the identification of adipokines has developed into a new field of biomedical research. However, many of the newly discovered adipokines have not been characterized in terms of their physiological function and their pathophysiological role. Moreover, in many instances findings in animal experiments data have not been confirmed in subsequent human studies. Therefore, our studies will rely on well characterized human biological samples.
In order to determine adipokine production, WP leader MHH will analyze blood and adipose tissue samples that have been obtained by the clinical partners CHAR, WROC, HULL, and ROME during their clinical recruitment activities. Adipose tissue will be sampled from the deep subcutaneous depot on the abdomen by needle biopsy. Thereafter, the specimen will be washed and snap frozen in liquid nitrogen. Frozen samples will be send on dry ice to WP leader MHH.
During the course of the project, patient subgroups and sample size are planned as follows:
Task 1: Isolation of mRNA and determination of gene expression of selected adipokines in adipose tissue by TaqMan-RT-PCR; analyzed genes will include adiponectin, TNFa, resistin, RBP-4, and other adipokines selected from the literature; genes of receptors that mediate lipolysis (a1-, a2-, ß1-, ß2-, ß3-adrenoceptors, NPrA and NPrC, TNF-receptors).
Task 2: isolation of microRNAs and determination of selected microRNA expression in adipose tissue by TaqMan-RT-PCR; microRNAs will be selected on the basis of adipokine mRNA levels and bio-informatic data mining to reveal candidate microRNAs for dysregulated adipokine mRNAs
Task 3: in blood samples, we will determine: markers of oxidative stress and NO metabolism by GC-MS/MS (nitrate, nitrite, ADMA, malondialdehyd); endocannabinoids by GC-MS/MS; adiponectin, TNF-a, and other adipokines in correspondence to the genes that will be measured by ELISA or RIA
Task 4: calculation of the correlation between clinical phenotypes with adipokine levels in the blood, adipokine mRNA and microRNA levels in adipose tissue samples.
The work described in tasks 1-3 will be done by a research fellow (doctoral student from biology, biochemistry or nutrition sciences). Task 4 will be done by Prof. Jordan and Dr. Stefan Engeli.
Objective 2: Adipocyte Lipolysis in Cardiac Cachexia
Enhanced responsiveness of adipocytes to lipolytic stimuli may play an important role in cardiac cachexia. Lipid mobilisation is not only important for the reduction of adipose tissue mass. Indeed, excessive availability of circulating free fatty acids may result in insulin resistance and ectopic deposition of fatty acids/triglycerides. The latter mechanism is known to elicit lipotoxic damage in skeletal muscle, heart, pancreas, and liver. Thus, increased lipolysis may provide a pathophysiological link between heart failure and cachexia. The three predominant lipolytic stimuli in human subjects are catecholamines, TNFa and natriuretic peptides.
The lipolytic activity of these mechanisms has only been defined in lean or obese/diabetic subjects, but not in heart failure patients. In cooperation with clinical partner CHAR, the following patient groups and numbers will be studied:
All patient and control groups will be matched for age, gender, ethnicity, and concomitant medication.
Task 5: subcutaneous adipocytes will be isolated from adipose tissue biopsy samples by collagenase digestion and centrifugation. Then, aliquots of identical numbers of adipocytes will be incubated with different concentrations of isoproterenol, ANP and TNFa for 2 hours. Adipocytes and incubation medium will be frozen and shipped to the WP leader after the experiments.
Task 6: Glycerol as the classical marker of lipolysis will be measured after 2 hours by a photometric assay by the WP leader institution
Task 7: Adipocytes will be further analyzed by the WP lead institution for protein content (required for normalization) and mRNA expression of adreno-, natriuretic peptide and TNF-receptors
The work related to task 5 will be done on-site by researchers/technical assistants from partner CHAR after training provided by Dr. Engeli from WP09. The laboratory work described in tasks 6-7 will be done by a research fellow (doctoral student from biology, biochemistry or nutrition sciences).
Objective 3: Training and SOPs for a common adipokine biobank
All measurements (RT-PCRs, ELISA; mass spectroscopy, photometric measurements) will be done by the WP09 lead institution. However, some of the procedures named above, namely adipose tissue biopsies, adipocyte isolation, and lipolysis assays, have to be performed on site by the different clinical partners. Thus, the provision of adequate biomaterial by partners CHAR, WORC, HULL, and ROME is key for success. To ensure that requirement, providing SOPs and training to other groups from the consortium will be provided by WP09 personnel (Stefan Engeli). Training and SOP development and distribution will be done in association with WP16 (Task 8).