Electronic health records (EHRs) in clinical research and platform trials: Application of the innovative EHR-based methods developed by EU-PEARL.

OBJECTIVE: Electronic Health Record (EHR) systems are digital platforms in clinical practice used to collect patients' clinical information related to their health status and represents a useful storage of real-world data. EHRs have a potential role in research studies, in particular, in platform trials. Platform trials are innovative trial designs including multiple trial arms (conducted simultaneously and/or sequentially) on different treatments under a single master protocol. However, the use of EHRs in research comes with important challenges such as incompleteness of records and the need to translate trial eligibility criteria into interoperable queries. In this paper, we aim to review and to describe our proposed innovative methods to tackle some of the most important challenges identified. This work is part of the Innovative Medicines Initiative (IMI) EU Patient-cEntric clinicAl tRial pLatforms (EU-PEARL) project's work package 3 (WP3), whose objective is to deliver tools and guidance for EHR-based protocol feasibility assessment, clinical site selection, and patient pre-screening in platform trials, investing in the building of a data-driven clinical network framework that can execute these complex innovative designs for which feasibility assessments are critically important. METHODS: ISO standards and relevant references informed a readiness survey, producing 354 criteria with corresponding questions selected and harmonised through a 7-round scoring process (0-1) in stakeholder meetings, with 85% of consensus being the threshold of acceptance for a criterium/question. ATLAS cohort definition and Cohort Diagnostics were mainly used to create the trial feasibility eligibility (I/E) criteria as executable interoperable queries. RESULTS: The WP3/EU-PEARL group developed a readiness survey (eSurvey) for an efficient selection of clinical sites with suitable EHRs, consisting of yes-or-no questions, and a set-up of interoperable proxy queries using physicians' defined trial criteria. Both actions facilitate recruiting trial participants and alignment between study costs/timelines and data-driven recruitment potential. CONCLUSION: The eSurvey will help create an archive of clinical sites with mature EHR systems suitable to participate in clinical trials/platform trials, and the interoperable proxy queries of trial eligibility criteria will help identify the number of potential participants. Ultimately, these tools will contribute to the production of EHR-based protocol design.

Enhanced sarcolemmal FAT/CD36 content and triacylglycerol storage in cardiac myocytes from obese zucker rats.

In obesity, the development of cardiomyopathy is associated with the accumulation of myocardial triacylglycerols (TAGs), possibly stemming from elevation of myocardial long-chain fatty acid (LCFA) uptake. Because LCFA uptake is regulated by insulin and contractions, we examined in cardiac myocytes from lean and obese Zucker rats the effects of insulin and the contraction-mimetic agent oligomycin on the initial rate of LCFA uptake, subcellular distribution of FAT/CD36, and LCFA metabolism. In cardiac myocytes from obese Zucker rats, under basal conditions, FAT/CD36 was relocated to the sarcolemma at the expense of intracellular stores. In addition, the LCFA uptake rate, LCFA esterification rate into TAGs, and the intracellular unesterified LCFA concentration each were significantly increased. All these metabolic processes were normalized by the FAT/CD36 inhibitor sulfo-N-succinimidyloleate, indicating its antidiabetic potential. In cardiac myocytes isolated from lean rats, in vitro administration of insulin induced the translocation of FAT/CD36 to the sarcolemma and stimulated initial rates of LCFA uptake and TAG esterification. In contrast, in myocytes from obese rats, insulin failed to alter the subcellular localization of FAT/CD36 and the rates of LCFA uptake and TAG esterification. In cardiac myocytes from lean and obese animals, oligomycin stimulated the initial rates of LCFA uptake and oxidation, although oligomycin only induced the translocation of FAT/CD36 to the sarcolemma in lean rats. The present results indicate that in cardiac myocytes from obese Zucker rats, a permanent relocation of FAT/CD36 to the sarcolemma is responsible for myocardial TAG accumulation. Furthermore, in vitro these cardiac myocytes, although sensitive to contraction-like stimulation, were completely insensitive to insulin, as the basal conditions in hyperinsulinemic, obese animals resemble the insulin-stimulated condition in lean littermates.

Ketone bodies disturb fatty acid handling in isolated cardiomyocytes derived from control and diabetic rats.

According to the current paradigm, fatty acid (FA) utilization is increased in the diabetic heart. Since plasma levels of competing substrates such as ketone bodies are increased during diabetes, the effect of those substrates on cardiac FA handling was explored. Cardiomyocytes were isolated from control and streptozotocin-treated diabetic rats and incubated with normal (80 microM) and elevated (160 microM) palmitate concentrations in the absence or presence of ketone bodies, including acetoacetate (AcAc). Comparing control cardiomyocytes under normal conditions (80 microM, no AcAc) with diabetic cardiomyocytes (160 microM, 3 mM AcAc) showed that palmitate uptake was increased from 35.2 +/- 4.8 to 60.2 +/- 14.0 nmol x 3 min(-1) x g wet weight(-1) respectively. Under these conditions, palmitate oxidation rates were comparable (58.9 +/- 23.6 versus 53.2 +/- 18.5 nmol x 30 min(-1) x g wet weight(-1)). However, in the absence of AcAc, palmitate oxidation was significantly enhanced in diabetic cardiomyocytes, indicating that ketone bodies are able to suppress cardiac FA oxidation in diabetes. The concomitantly increased FA uptake in diabetic cells, mainly due to the elevated extracellular FA levels, may be responsible for the accumulation of FA and triacylglycerol, as observed in the diabetic heart in situ.

Critical steps in cellular fatty acid uptake and utilization.

Despite decades of extensive research, the transport routes, mechanisms of uptake and points of flux control of long-chain fatty acids (FA) in mammalian organs are still incompletely understood. In non-fenestratred organs such as heart and skeletal muscle, membrane barriers for blood-borne FA are the luminal and abluminal membranes of endothelial cells, the sarcolemma and the mitochondrial membranes. Transport of FA through the phospholipid bilayer of the cellular membrane is most likely accomplished by diffusion of protonated FA. Evidence is accumulating that membrane-associated proteins, such as plasmalemmal fatty acid-binding protein (FABPpm) and fatty acid translocase (FAT/CD36), either alone or in conjunction with albumin binding protein (ABP), are instrumental in enhancing the delivery of FA to the cellular membrane. Inside the cell, cytoplasmic fatty acid-binding proteins (FABPc) are involved in diffusion of FA from the plasmalemma to the intracellular sites of conversion, such as the mitochondrial outer membrane. After conversion of FA to FACoA, the fatty acyl chain is transported across the mitochondrial inner membrane in a carnitine-mediated fashion. Uptake and utilization of FA by muscle cells are finely tuned, most likely to avoid the intracellular accumulation of FA, as these are cytotoxic at high concentrations. On a short-term basis, net uptake is, among others, regulated by intracellular translocation of FAT from intracellular stores to the sarcolemma and by the concentration gradient of FA across the sarcolemma. The latter implies that, among others, the rate of FA utilization determines the rate of uptake. The rate of utilization is governed by a variety of factors, including malonylCoA, the ratio acetylCoA/CoA and the availability of competing substrates such as glucose, lactate, and ketone bodies. Long-term regulation of uptake and utilization is accomplished by alterations in the rate of expression of genes, encoding for FA-handling proteins. Circumstantial evidence indicates that FA themselves are able to modulate the expression of FA-handling genes via nuclear transcription factors such as peroxisome proliferator-activated receptors (PPARs).

Protein acylation in the cardiac muscle like cell line, H9c2.

Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with 3H-palmitate or 3H-myristate, cells were lysed and proteins precipitated, followed by extensive delipidation. The delipidated proteins were subjected to SDS-PAGE and transferred to nitro-cellulose prior to autoradiography. In addition, TLC was used to separate the various lipid classes. The first aspect we addressed was the extent of protein acylation as a function of time, relative to fatty acid incorporation into various lipid classes. Cells were incubated for 30 min, 1 h and 2 h with 100 microCi palmitate (PA, 2.3 nmol) or 125 microCi myristate (MA, 2.5 nmol). Palmitoylation increased from 0.48 +/- 0.25 to 1.25 +/- 0.56 microCi/mg protein between 30 min to 2 h, while myristoylation increased from 0.25 +/- 0.12 to 0.77 +/- 0.36 microCi/mg protein. Furthermore, delipidated proteins subjected to autoradiography showed that a set of distinct proteins was labelled with 3H-palmitate. Incorporation into phospholipids (PL) increased from 40-60% of the total amount of radio-labelled PA or MA supplied between 30 min and 2 h. Only the FA pool differed between MA and PA, with a higher FA content present after incubations with MA. Second, we investigated palmitoylation and incorporation into cellular lipids as a function of the amount of PA applied. Palmitoylation showed saturation at high PA concentrations. The percentage incorporation of 3H-PA in the various lipids depended on the amount of PA added: a decline in the PL pool with a concomitant increase in the size of the diacylglycerol pool at high PA concentrations. Third, inhibition of palmitoylation by cerulenin and tunicamycin was investigated. While both were able to inhibit palmitoylation, cerulenin also inhibited the incorporation of PA into various lipid classes, indicating differences in inhibitory action.