Molecular signalling in cardiovascular biology: from molecules to man

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MOLECULAR MEDICINE TODAY, SEPTEMBER 2000 (VOL. 6)

Molecular signalling in cardiovascular biology: from molecules to man International Symposium Organized by the Centre for Cardiovascular Biology and Medicine, King’s College, London King’s College, London, UK, 31 May 2000 Joanne Layland and Ajay M. Shah

This symposium, organized by the Centre for Cardiovascular Biology and Medicine (King’s College, London, UK), promoted a collaborative and multidisciplinary approach to the study of cardiovascular biology, ultimately aimed at improving the morbidity and mortality of cardiovascular disease. An international faculty presented recent research findings, indicating how discoveries at the molecular level might ultimately be translated into clinical therapies.

Cardiovascular function Synchronous contraction of cardiac cells in the ventricles of the heart (systole) provides the power required to pump blood around the circulatory system. Conversely, relaxation of ventricular cells (diastole) permits the ventricles to fill with blood between beats. Many complex processes operate to ensure that cardiac performance is finely tuned to match changes in circulatory demand. In addition, the energy requirements of the heart itself have to be matched with its blood supply through the coronary arteries. Disruption of any of these processes leads to the inability of the heart to pump blood at a rate required by the metabolizing tissues: heart failure. Understanding the molecular and cellular mechanisms underlying normal and abnormal cardiovascular function is therefore an essential pre-requisite for the development of effective clinical therapies against cardiovascular disease.

Intracellular signalling in cardiac cells Cardiac muscle contraction is initiated by a transient increase in intracellular Ca21 (the ‘Ca21 transient’) triggered by depolarization of the sarcolemmal and transverse tubule membranes during the cardiac action potential. The Ca21 transient results both from sarcolemmal Ca21 342

influx, primarily through L-type Ca21 channels generating the Ca21 current (ICa), and from Ca21 released from the sarcoplasmic reticulum (SR) in response to the ICa trigger. By binding to the regulatory myofilament protein, troponin-C, Ca21 promotes cross-bridge cycling between interdigitating actin and myosin filaments, ultimately leading to muscle shortening and/or force generation. Subsequent removal of Ca21 from the cytoplasm, by active transport back into the SR and by extrusion across the sarcolemma, promotes Ca21 dissociation from the myofilaments, permitting relaxation.

‘Slip-mode conductance’: a novel route for excitation–contraction coupling Jon Lederer (University of Maryland, Baltimore, USA) presented evidence that SR Ca21 release can be triggered by Ca21 influx through promiscuous Na1 channels – a radical phenomenon termed ‘slip-mode conductance’. ‘Ca21 sparks’ represent the elementary units of SR Ca21 release and are thought to result from the opening of a single SR Ca21 release channel (ryanodine receptor, RyR). Ca21 sparks can be visualized in cardiac myocytes by confocal microscopy of cells loaded with the fluorescent dye fluo-3. Following depolarization, the local increase in intracellular Ca21 generated by sarcolemmal Ca21 influx is thought to activate nearby RyR to produce a localized Ca21 spark. Summation of these local Ca21 release events produces the Ca21 transient that initiates contraction. It has recently become apparent that under conditions of b-adrenergic activation, Ca21 transients can be elicited even when all Ltype Ca21 channels are blocked. This has fuelled speculation that a voltage-activated SR Ca21 release mechanism, similar to that responsible for

skeletal muscle activation, could contribute to cardiac muscle excitation–contraction coupling. However, Lederer showed that these Ca21 transients involved Ca21 influx via Na1 channels. This ‘slip-mode conductance’ is thought to occur through changes in Na1 channel selectivity resulting from protein kinase A (PKA)induced phosphorylation. Slip-mode conductance occurs during simulated cardiac action potentials and can account for as much as 20–30 % of the Ca21 transient during b-adrenergic stimulation. Interestingly, it is also activated by clinically relevant concentrations of digitalis compounds (frequently used as inotropes in the treatment of heart failure), albeit by a different mechanism of altered Na1 channel selectivity, not requiring PKA. These controversial findings provoked speculation that activation of slip-mode conductance by digitalis could be beneficial in the treatment of ischaemia, when conventional excitation–contraction coupling is depressed.

Cardiac Na1–H1 exchange: regulation and role in heart disease The sarcolemmal Na1–H1 exchanger (NHE) represents a major acid-extrusion pathway in cardiac cells, H1 exchanged for Na1 with 1:1 stoichiometry. Metin Avkiran (King’s College) presented work examining the roles of protein kinase C (PKC) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) in NHE regulation. ERK1/2 is believed to activate a ribosomal S6 kinase which might stimulate NHE by phosphorylation of the regulatory domain of the exchanger. Furthermore, it appears that PKC-mediated regulation of NHE is dependent on the activity of another protein kinase (PKD), which regulates NHE activity in an inhibitory

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MOLECULAR MEDICINE TODAY, SEPTEMBER 2000 (VOL. 6)

manner. Understanding the mechanisms regulating NHE is of clinical significance because activation of NHE is implicated in the progression of certain cardiac pathologies, including hypertrophy and ischaemia-reperfusion injury. Acidosis during ischaemia promotes H1 extrusion by NHE resulting in the accumulation of Na1. This might promote Ca21 influx via Na1–Ca21 exchange, leading to Ca21 overload; thus, generating the arrhythmias and myocyte damage characteristic of reperfusion injury. Indeed, selective inhibitors of the NHE – for example, HOE-642 (cariporide) and EMD-96785 (eniporide) – have been shown to be cardioprotective against experimental ischaemia-reperfusion injury. These findings are currently also being assessed in clinical trials – for example, the GUARDIAN study and the ESCAMI study – which might reveal the full therapeutic potential of NHE inhibition.

Cardiac b2-adrenoceptors: intracellular signalling and physiological relevance Catecholamines act via cardiac adrenoceptors to increase both the strength and the frequency of the heart beat. Cardiac adrenoceptors are classified into 2 types, a and b, with further subtypes within each group (for example, b1 and b2). Agonist-bound b-adrenoceptors interact with stimulatory G proteins (Gs) to trigger a signalling cascade that results in the phosphorylation of regulatory proteins in the sarcolemma, SR and myofilaments and ultimately produces the positive inotropic and lusitropic (accelerated relaxation) effects of b-stimulation. Ed Lakatta (National Institute of Aging, Baltimore, USA) reported that although b1-adrenoceptor signalling follows this classical cascade, b2-adrenoceptor signalling is much more localized and associated with phosphorylation of the sarcolemmal proteins only. It is postulated that b2-receptors are coupled both to inhibitory (Gi) and stimulatory (Gs) G proteins; the Gi proteins antagonizing the effects of Gs. Lakatta also presented evidence that, on exposure to isoprenaline (a non-selective b-agonist), b2-Gi coupling confers properties of cell survival whereas b1-Gs coupling is associated with substantial apoptosis. This could have significant clinical implications for therapy with b-blockers (b1 or b2) in patients with heart failure.

Non-contractile sarcomeric proteins Studies in muscle function have tended to concentrate predominantly on proteins involved directly in muscle contraction, for instance, actin, myosin and the tropomyosin complex. Mathias Gautel (Max-Planck-Institute for Molecular Physiology, Dortmund, Germany) presented his work on an array of other

sarcomeric proteins, notably titin and obscurin. The enormous titin molecule (2.5–3 3 106 Da) is particularly complex and is of great importance in the organization of sarcomeric structure. However, the discovery that titin also contains domains for protein kinase and G-protein binding suggests that titin might also have a more dynamic function as a target for intracellular signalling, transducing mechanical signals by virtue of physical interactions with other proteins.

Cardiovascular development, repair and remodelling Genetic screens in zebrafish: a model system to investigate congenital heart defects Mark Fishman (Massachusetts General Hospital and Harvard Medical School, Boston, USA) presented a fascinating insight into the use of zebrafish as a model organism for genetic research into developmental abnormalities of the cardiovascular system. Zebra fish represent a good model for genetic manipulation because they can be bred in large numbers and, as the early stage zebrafish embryo is able to survive by gaseous diffusion across its body surface, cardiovascular abnormalities that would be lethal in mammals can be studied. Randomly induced mutations in zebrafish embryos have produced cardiovascular abnormalities that serve as models for clinically relevant conditions including hypertrophic cardiomyopathy, hypertension, arrhythmia and heart block. The transparency of the zebrafish embryo lets researchers see how abnormalities in cardiac pumping are translated into inefficient blood circulation. Knowledge of the zebra-fish genome is advanced enough to permit the cloning of genes found to be critical in cardiovascular development. These genes are likely to have human homologues, and could be relevant in understanding and treating human congenital heart malformations. The full clinical implications of this exciting field of research are likely to be realized once the sequencing of the entire human genome is completed.

Neoangiogenesis during ischaemia: beneficial or deleterious? Tissues subjected to hypoxia/ischaemia release an angiogenic factor known as vascular endothelial growth factor (VEGF) that triggers the compensatory formation of new blood vessels, so improving perfusion. The secretion of VEGF in the heart has been associated with the formation of collaterals to improve coronary circulation, and fuelled hopes that induction of VEGF could be used in the treatment of coronary artery disease.

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However, Eli Keshet (The Hebrew University, Jerusalem, Israel) warned that clinical trials might be premature because there is still a very limited understanding of the regulation of angiogenesis by VEGF. To illustrate this scepticism, Keshet described recent experiments using a transgenic mouse model in which VEGF expression was selectively induced in the myocardium at different stages of cardiovascular development. These experiments revealed that the timing of VEGF induction is crucial and that, at certain developmental stages, induction of VEGF can actually be lethal by promoting vascular expansion at the expense of myocardium.

Tumor necrosis factor a in heart failure: friend or foe ? Tumor necrosis factor a (TNF-a) is a proinflammatory cytokine, released in response to a variety of infectious or inflammatory stimuli, that plays an important role in host defence. However, recent evidence has suggested a direct relationship between the level of cardiac TNF-a expression and the severity of cardiac hypertrophy and heart failure. Nonfailing human heart does not express TNF-a, whereas endstage failing human hearts express significant amounts of the protein. Furthermore, increasing the level of TNF-a in experimental animals, either by infusion or by overexpression in transgenic mice, is associated with left ventricular dilatation and dysfunction. Virtually all nucleated cells can produce TNF-a, but cardiac myocytes might be a particularly significant source in the context of heart failure. In isolated cardiac myocytes, TNF-a application has a negative inotropic effect that can be reversed by TNF-a receptor antagonism. Douglas Mann (Baylor College of Medicine and VA Medical Centre, Houston, USA) described how the above research, from his laboratory and others, has progressed to clinical trials, examining the efficacy of TNF-a antagonism in patients with severe heart failure. The TNF-a antagonist, etanercept (Enbrel) targets the extracellular domains of the type 2 TNF-a receptor. Results of the early Phase I clinical trials have proved promising, demonstrating that etanercept administration, at least in the short term, ameliorated left ventricular function, reduced left ventricular size and improved quality of life. Further long-term clinical trials examining the effectiveness of TNF-a antagonism (for example, the RENAISSANCE study) are currently in progress and are scheduled for completion by 2002. The important issue was raised as to whether anti-cytokine therapy might be deleterious in patients with serious infections, such as septic shock, where cytokines might be beneficial in promoting repair 343

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and remodeling. It is clear that cytokines, though protective at low doses, can become injurious at higher doses by promoting apoptosis and aggravating the pathophysiology of heart failure.

Concluding remarks This stimulating symposium attracted many diverse research groups, united by the common aim of understanding the molecular and cellular processes that regulate cardiovascular biology. Despite the fact that cardiovascular disease is

MOLECULAR MEDICINE TODAY, SEPTEMBER 2000 (VOL. 6)

Joanne Layland* PhD Post-doctoral research associate

one of the major causes of mortality worldwide, there are still many gaps in our knowledge of cardiovascular physiology and pathophysiology. Presentations at this symposium emphasized the value of the multidisciplinary approach, illustrating how findings at the cellular level have been reiterated in experimental animals and are ultimately being applied in the clinical setting. The therapeutic potential of the research presented here should become apparent in the next few years.

Ajay M. Shah MD, FRCP, FESC Professor of Cardiology Department of Cardiology, GKT School of Medicine, Bessemer Road, London, UK SE5 9PJ. Tel: 144 020 7346 4023 Fax. 144 020 7346 4771 *e-mail: [email protected]

Have tumor cells learnt from microorganisms how to fool the immune system? Escape from immune surveillance of tumors and microorganisms: emerging mechanisms and shared strategies Naples, Italy, 23–27 March 2000 Rolf Kiessling, Graham Pawelec, Raymond M. Welsh, J. Dave Barry and Soldano Ferrone The increasing application of immunotherapy for the treatment of malignant diseases has convincingly shown that only a minority of patients with cancer benefit from immunotherapy. These disappointing findings reflect, at least in part, the ability of malignant cells to escape from immune destruction. To shed new light on this topic, the escape mechanisms used by tumor cells were compared to those utilized by microorganisms in a course held under the auspices of the Scuola Superiore d’Immunologia Ruggero Ceppellini (Naples, Italy). Tumor cells are less sophisticated than are microorganisms in escaping the host immune attack. Therefore bringing together experts in these two areas of ‘escapology’ was expected to stimulate the design of new strategies to defeat tumor cell escape in a clinical setting.

Antigenic variation Micro-organisms have honed their escape mechanisms during their entire evolution, whereas tumor cells have done so only during the “micro-evolution” of somatic cell growth. This difference is strikingly illustrated by the direct or indirect involvement of as much as 25% of the genome of Trypanosoma in strategies to 344

escape the host immune system. Extracellular bacteria that live in the bloodstream and tissue fluids, including Neisseria and Borrelia and protozoan parasites, such as the African trypanosomes, use mainly changes in molecules on their surface to avoid host immune attack (J. Dave Barry, University of Glasgow, Glasgow, UK; Sven Bergstrom, Umea Universitat, Umea, Sweden). Some are armed with dense coats, composed of functionally inert molecules, which shield their surfaces from immune effector mechanisms. This strategy is very effective as antibodies are the main weapon used by the host to combat these extracellular infections. Antigenic variation of the pathogen that involves a fraction of its infecting population switches the surface coat molecule to an immunologically unrelated one. As a result, the mutated sub-population is not recognized by the antibody population(s) elicited by the original infecting population. Antigenic variation of the pathogen, which involves a fraction of the infecting population, changes the surface coat molecule and results in the loss of recognition by the antibody population(s) elicited by the original pathogen. Antigenic variation can occur repeatedly,

leading to chronic infection with many relapsing peaks of infection in the blood. Furthermore, antigenic variation can be used by intracellular pathogens. For instance, HIV-1 might change the three loops that protect the invariant chemokine receptor-binding region of its gp120 envelope protein as the virus seeks new cells to invade. Antigenic variation might also affect the molecules with which some intracellular parasites, such as the malaria parasite Plasmodium, decorate their host cells to enhance their growth or survival. Loss of epitopes under selective pressure has been described in tumor and viral antigens. This mechanism is utilized by herpes viruses to maintain persistent infection in the host. Even in a more genetically stable virus, such as EBV (Epstein–Barr virus), mutations have been described in human leukocyte antigen (HLA)-A11-restricted EBNA-4 epitopes in EBV isolates from South East Asia, where HLA-A11 allospecificity is expressed in .50% of the population (Maria Grazia Masucci, Karolinska Institutet, Stockholm, Sweden).

MHC class I and TAA evasion Many viruses have also evolved multiple mechanisms to repress the major histocompatibility

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