M. van Bilsen

Release of fatty acid-binding protein from isolated rat heart subjected to ischemia and reperfusion or to the calcium paradox

The release of cardiac fatty acid-binding protein (cFABP) and of fatty acids from isolated rat hearts was measured during both reperfusion following 60 min of ischemia and the calcium paradox (readmission of Ca2+ after a period of Ca2+-free perfusion). Total cFABP release was much more pronounced after Ca2+ readmission (over 50% of tissue content) than during post-ischemic reperfusion (on average, 3% of tissue content), but in both cases, it closely paralleled the release of lactate dehydrogenase. Only minor amounts of long-chain fatty acids, if any, were released from the heart. These observations are challenging the idea that cFABP plays a fatty acid-buffering role under the pathophysiological conditions studied.

Lipid alterations in isolated, working rat hearts during ischemia and reperfusion: its relation to myocardial damage.

Disturbances in lipid metabolism may play an important role in the onset of irreversible myocardial damage. To investigate the effect of ischemia and reperfusion on lipid homeostasis and to delineate its possible consequences for myocardial damage, Krebs-Henseleit-perfused, working rat hearts were subjected to various periods of no-flow ischemia (10 to 90 minutes) with or without 30 minutes of reperfusion. During ischemia, the rise in nonesterified fatty acids (NEFAs) was preceded by the accumulation of substantial amounts of glycerol, indicating the presence of an active triacylglycerol-NEFA cycle. The subsequent rise in NEFAs (from 0.25 to 1.64 μmol/g dry residue wt after 90 minutes [means]) coincided with the reduction of ATP to values lower than 10 μmol/g dry wt and the rise of AMP, a potent inhibitor of acyl-coenzyme A synthetase, to values exceeding 2 μmol/g dry wt, making the latter compound a good candidate to hamper the turnover of endogenous lipids during prolonged ischemia. Reperfusion resulted in an additional rise in NEFAs (up to 4.1 μmol/g dry residue wt after 60 minutes of ischemia). Neither ischemia nor reperfusion resulted in significant decreases in the tissue content of triacylglycerols and the various phospholipids. During reperfusion recovery of stroke volume was still adequate at tissue NEFA levels thought to be incompatible with normal mitochondrial function.37 A positive correlation (r=0.81) was found between NEFA content of reperfused hearts and cumulative release of lactate dehydrogenase during reperfusion. Accordingly it is concluded that 1) reperfusion results in additional changes in myocardial lipid homeostasis, 2) the accumulating NEFAs are compartmentalized, possibly at the cellular level, and 3) the accumulation of NEFAs is a sensitive marker for myocardial cell damage.

Connective tissue growth factor and cardiac fibrosis

Cardiac fibrosis is a major pathogenic factor in a variety of cardiovascular diseases and refers to an excessive deposition of extracellular matrix components in the heart, which leads to cardiac dysfunction and eventually overt heart failure. Evidence is accumulating for a crucial role of connective tissue growth factor (CTGF) in fibrotic processes in several tissues including the heart. CTGF orchestrates the actions of important local factors evoking cardiac fibrosis. The central role of CTGF as a matricellular protein modulating the fibrotic process in cardiac remodelling makes it a possible biomarker for cardiac fibrosis and a potential candidate for therapeutic intervention to mitigate fibrosis in the heart.

Transcriptional regulation of metabolic processes: implications for cardiac metabolism.

Abstract Under normal conditions the oxidation of fatty acids and glucose covers, respectively, approximately 65% and 30% of the energy demand of the beating heart. Evidence is accumulating that various pathophysiological conditions are associated with overt changes in cardiac energy metabolism. For instance, in diabetes cardiac energy conversion relies even more on fatty acid than on glucose oxidation. In contrast, during cardiac hypertrophy the opposite takes place, i.e. the utilization of carbohydrates increases at the expense of lipids. The mechanisms responsible for and the significance of these metabolic adaptations are largely unknown. A growing body of evidence indicates that these metabolic adaptations are brought about, at least in part, through adjustments in the rate of transcription of genes encoding proteins involved in substrate transport and metabolism. There are reasons to believe that the transcriptional regulation of these ”metabolic genes” is subject to modulation by metabolites per se, i.e. by oxygen, glucose and fatty acids. In this review the concept of metabolic remodelling as an important facet of cardiac adaptation to chronic pathophysiological conditions is introduced and the putative roles of metabolites in transcriptional regulation in the heart are considered.

Accumulation of arachidonic acid in ischemic/reperfused cardiac tissue: possible causes and consequences

Under physiological conditions, the content of unesterified arachidonic acid in cardiac tissue is very low. The bulk of arachidonic acid is present in the membrane phospholipid pool. Incorporation of arachidonic acid into phospholipids (reacylation) and liberation of this fatty acid from the phospholipid pool (deacylation) are controlled by a set of finely tuned enzymes, including lysophospholipid acyltransferase and phospholipase A2. At present, at least three subtypes of phospholipase A2 have been identified in cardiac structures, i.e., a low molecular mass group II phospholipase A2, a cytoplasmic high molecular mass phospholipase A2 and a plasmalogen-specific phospholipase A2. Cessation of flow to the heart (ischemia) gives rise to net degradation of membrane phospholipids accompanied by accumulation of fatty acids, including (unesterified) arachidonic acid. Restoration of flow to the previously ischemic cells results in a continued accumulation of fatty acids. The mechanism(s) underlying net phospholipid degradation in ischemic/reperfused myocardial tissue is (are) incompletely understood. Impaired reacylation, enhanced hydrolysis of phospholipids, or a combination of both may be responsible for the phenomena observed. Elevated tissue levels of arachidonic acid may exert both direct and indirect effects on the affected myocardium and healthy cardiac cells adjacent to the injured cardiomyocytes. Indirect effects might be evoked by arachidonic acid metabolites, i.e., eicosanoids. Arachidonic acid may directly influence ion channel activity, substrate metabolism and signal transduction, thereby affecting the functional characteristics of the ischemic/reperfused myocardium.

Impaired cardiac functional reserve in type 2 diabetic db/db mice is associated with metabolic, but not structural, remodelling.

Aim:  To identify the initial alterations in myocardial tissue associated with the early signs of diabetic cardiac haemodynamic dysfunction, we monitored changes in cardiac function, structural remodelling and gene expression in hearts of type 2 diabetic db/db mice.

Peroxisome proliferator-activated receptors and inflammation: take it to heart.

Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors acting as key regulators of lipid metabolism as well as modulators of inflammation. The role of PPARα and PPARγ in cardiac ischaemia‐reperfusion injury, infarct healing and hypertrophy is the subject of intense research. Due to the later development of PPARδ‐specific ligands, the role of this PPAR isoform in cardiac disease remains to be established. Although many studies point to salutatory effects of PPAR ligands in cardiac disease, the exact molecular mechanism is still largely unsolved. Both the metabolic (via transactivation) and the more recently discovered anti‐inflammatory (via transrepression) effects of PPARs are likely to play a role. In this review the reported, and sometimes contradictory, effects of PPAR ligands on ischaemia‐reperfusion, infarct healing and cardiac hypertrophy are critically evaluated. In particular the role of inflammation in these disease processes, the ability of PPARs to interfere with pro‐inflammatory processes, and the mechanisms of transrepression are discussed. Currently, the significance of PPARs as therapeutic targets in cardiovascular disease is receiving widespread attention. Accordingly, detailed understanding of the mechanisms controlling the activity of these nuclear hormone receptors is essential.

Long-term effects of fatty acids on cell viability and gene expression of neonatal cardiac myocytes

Abstract Fatty acids are the most important source of energy for the adult heart. However, cardiac substrate preference changes during development and alters in pathophysiological states. Fatty acids have also been shown to be involved in signal transduction pathways, thereby affecting gene expression in various cell systems. In the present paper the significance of changes in substrate preference and the potential role of fatty acids in signal transduction in the cardiomyocyte are briefly reviewed. Furthermore, the development of a cellular model system, useful in exploring the long-term effects of fatty acids on the normal and hypertrophic cardiomyocyte, is described. Some aspects of this model system are illustrated by showing the effects of different fatty acid species on cell viability and the effects of fatty acids on the expression of heart type fatty acid-binding protein (H-FABP), a 15 kDa protein thought to be involved in intracellular trafficking of fatty acids. To this end primary cultures of rat neonatal ventricular myocytes were kept in defined medium containing various (combinations of) substrates for up to 48 h. First, the effects of prolonged exposure to different fatty acid species, complexed to BSA, on cell viability were investigated. Exposure of the cells to saturated fatty acids (C16:0 or C18:0), but not mono-unsaturated (C16:1 or C18:1) fatty acids, resulted in cell death, as evidenced by the release of intracellular proteins like lactate dehydrogenase. The detrimental effects of saturated fatty acids were nullified by the co-addition of mono-unsaturated fatty acids. Accordingly, the combination of C16:0 C18:1 was used to examine the effects of fatty acids on the expression of H-FABP. Therefore, the cells were incubated with either (i) glucose only, (ii) fatty acids only, or (iii) glucose plus fatty acids. Incubation with fatty acids (with or without glucose) resulted in a nearly four-fold increase of the H-FABP mRNA level. Similarly, at the protein level the cellular H - FABP LDH ratio increased almost two-fold. In hypertrophic cardiomyocytes (stimulated with the α1-adrenergic agonist phenylephrine) the stimulatory effect of fatty acids on H-FABP expression was mitigated. These findings strongly suggest that fatty acids are able to modulate gene expression in the context of the cardiac muscle cell.

Cytochrome P450, peroxisome proliferation, and cytoplasmic fatty acid-binding protein content in liver, heart and kidney of the diabetic rat

Diabetes mellitus generally results in an increased systemic fatty acid mobilization which can be associated with an increase in mitochondrial and peroxisomal β-oxidation of fatty acids in selected tissues. The latter is usually accompanied by a concomitant increase in the tissue content of cytoplasmic fatty acid-binding protein (FABP) which functions in the intracellular translocation of fatty acids. It was previously found that in liver clofibrate-induced proliferation of peroxisomes and increase in FABP expression each are dependent on the induction by cytochrome P4504A1 -mediated (CYP4A1) formation of dicarboxylic acids. We studied whether peroxisome proliferation and an increase of FABP contents in liver, heart and kidney of streptozotocin-induced diabetic rats are also accompanied by an increase of CYP4A1 activity, as this would indicate a possible regulatory role for dicarboxylic acids in peroxisome proliferation and FABP induction in diabetic organs other than liver. In livers of the diabetic rat, a concomitant increase was observed of the activities of CYP4A1 and the peroxisomal key enzyme fatty acyl-CoA oxidase (FACO) and of the FABP content. In the diabetic heart FACO activity and FABSP content also increased, but there was no induction of CYP4A1 activity. Conversely, in diabetic kidney there was no increase in FACO activity nor FABP content in spite of a marked induction of CYP4A1 activity. It is concluded that streptozotocin-induced diabetes leads to increased peroxisome proliferation and increased levels of FABP in both liver and heart, which only in liver is accompanied by an induction of the cytochrome P450 system. Consequently, it is not likely that dicarboxylic acids are involved in the induction of peroxisome proliferation in the heart.

Accumulation of lipids and lipid-intermediates in the heart during ischaemia

The content of non-esterified fatty acids (NEFA) and their CoA and carnitine esters is low in normoxic cardiac tissue. The majority of fatty acids is esterified in the triacylglycerol and phosphoglyceride pool. During myocardial ischaemia beta-oxidation of fatty acids is inhibited. In addition, turnover of the esterified fatty acid pools is most likely disturbed. Accumulation of hydroxy fatty acids, acylCoA and acylcarnitine rapidly occurs after the onset of ischaemia. The accumulation of NEFA is a slower process. In addition to extracellular sources, NEFA originate also from intracellular lipid pools, most likely from phosphoglycerides. Although it has been suggested that activation of phospholipase A2 occurs in ischaemic tissue, the mechanism underlying the enhanced degradation of phosphoglycerides ist still incompletely understood.