Metin Avkiran

Cardiovascular side effects of cancer therapies: a position statement from the Heart Failure Association of the European Society of Cardiology

The reductions in mortality and morbidity being achieved among cancer patients with current therapies represent a major achievement. However, given their mechanisms of action, many anti‐cancer agents may have significant potential for cardiovascular side effects, including the induction of heart failure. The magnitude of this problem remains unclear and is not readily apparent from current clinical trials of emerging targeted agents, which generally under‐represent older patients and those with significant co‐morbidities. The risk of adverse events may also increase when novel agents, which frequently modulate survival pathways, are used in combination with each other or with other conventional cytotoxic chemotherapeutics. The extent to which survival and growth pathways in the tumour cell (which we seek to inhibit) coincide with those in cardiovascular cells (which we seek to preserve) is an open question but one that will become ever more important with the development of new cancer therapies that target intracellular signalling pathways. It remains unclear whether potential cardiovascular problems can be predicted from analyses of such basic signalling mechanisms and what pre‐clinical evaluation should be undertaken. The screening of patients, optimization of therapeutic schemes, monitoring of cardiovascular function during treatment, and the management of cardiovascular side effects are likely to become increasingly important in cancer patients. This paper summarizes the deliberations of a cross‐disciplinary workshop organized by the Heart Failure Association of the European Society of Cardiology (held in Brussels in May 2009), which brought together clinicians working in cardiology and oncology and those involved in basic, translational, and pharmaceutical science.

Na(+)/H(+) exchange inhibitors for cardioprotective therapy: progress, problems and prospects.

Extensive pre-clinical work indicates that inhibition of the sarcolemmal Na(+)/H(+) exchanger (NHE) affords significant protection to myocardium subjected to ischemia and reperfusion, predominantly through reduced intracellular accumulation of Na(+) and consequently Ca(2+). In contrast, recent clinical studies with the NHE inhibitors cariporide and eniporide in patients with evolving myocardial infarction (MI) and those at risk of MI have provided mixed and somewhat contradictory data. The experimental evidence suggests that the key mechanism through which NHE inhibitors afford protection consists in slowing the progression of myocardial injury during ischemia and thereby enhancing myocardial salvage by reperfusion. It follows from this that, to obtain maximum cardioprotective benefit, 1) the NHE inhibitor must be present in jeopardized myocardium, at a concentration sufficient to inhibit NHE activity, before (or as soon as possible after) the onset of ischemia, and 2) ischemia must be terminated by timely reperfusion. Thus, in the GUARDIAN trial, the cardioprotective efficacy of cariporide was limited to the subset of high-risk patients who underwent coronary artery bypass graft surgery, in whom both prerequisites could be readily fulfilled. In contrast, no cardioprotective benefit was observed in the ESCAMI trial, in which eniporide was administered late as an adjunct to reperfusion therapy in patients with evolving MI. Ongoing clinical studies will determine whether NHE inhibition will find therapeutic application in the setting of cardiac surgery, while pre-clinical investigations continue to test the potential of NHE inhibitors in the treatment of other cardiovascular diseases such as heart failure.

Protein Kinase D Is a Novel Mediator of Cardiac Troponin I Phosphorylation and Regulates Myofilament Function

Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown. Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins. In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin. Peptide mass fingerprint analysis of cTnI by liquid chromatography–coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets. Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T. PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD. Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (≈2 mol phosphate/mol cTnI) by both PKD and PKA. To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation. PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension–pCa relationship, indicating reduced myofilament Ca2+ sensitivity. At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics. Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.

Ca2+/Calmodulin-Dependent Protein Kinase IIδ and Protein Kinase D Overexpression Reinforce the Histone Deacetylase 5 Redistribution in Heart Failure

Cardiac hypertrophy and heart failure (HF) are associated with reactivation of fetal cardiac genes, and class II histone deacetylases (HDACs) (eg, HDAC5) have been strongly implicated in this process. We have shown previously that inositol trisphosphate, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and protein kinase (PK)D are involved in HDAC5 phosphorylation and nuclear export in normal adult ventricular myocytes and also that CaMKII&dgr; and inositol trisphosphate receptors are upregulated in HF. Here we tested whether, in our rabbit HF model, nucleocytoplasmic shuttling of HDAC5 was altered either at baseline or in response to endothelin-1, which would indicate HDAC5 phosphorylation and transcription effects. The fusion protein HDAC5–green fluorescent protein (HDAC5-GFP) was more cytosolic in HF myocytes (Fnuc/Fcyto 3.3±0.3 vs 7.2±0.4 in control), and HDAC5 was more phosphorylated. Despite this baseline cytosolic HDAC5 shift, endothelin-1 produced more rapid HDAC5-GFP nuclear export in HF versus control myocytes. We also find that PKD and CaMKII&dgr;C expression and activation state are increased in both rabbit and human HF. Inhibition of either CaMKII or PKD in HF myocytes partially restored the HDAC5-GFP Fnuc/Fcyto toward control, and simultaneous inhibition restored Fnuc/Fcyto to that in control myocytes. Moreover, adenovirus-mediated overexpression of PKD, CaMKII&dgr;B, or CaMKII&dgr;C reduced baseline HDAC5 Fnuc/Fcyto in control myocytes (3.4±0.5, 3.8±0.5, and 5.2±0.5, respectively), approaching that seen in HF. We conclude that chronic upregulation and activation of inositol trisphosphate receptors, CaMKII, and PKD in HF shifts HDAC5 out of the nucleus, derepressing transcription of hypertrophic genes. This may directly contribute to the development and/or maintenance of HF.

Thrombin Activates the Sarcolemmal Na+-H+ Exchanger: Evidence for a Receptor-Mediated Mechanism Involving Protein Kinase C

Thrombin can activate the plasma membrane Na(+)-H+ exchanger in a variety of noncardiac cells. We have studied (1) the effect of thrombin on the activity of the sarcolemmal Na(+)-H+ exchanger in freshly isolated quiescent ventricular myocytes from the adult rat heart and (2) the signaling mechanism(s) underlying any effect. Reverse-transcription polymerase chain reaction analysis revealed thrombin receptor mRNA expression in a myocyte-enriched cell preparation. As an index of Na(+)-H+ exchanger activity, acid efflux rates (JHS) were determined in single myocytes (n = 4 to 11 per group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after two consecutive intracellular acid pulses (induced by transient exposure to 20 mmol/L NH4Cl) in bicarbonate-free medium. At a pHi of 6.9, JH did not change significantly during the second pulse relative to the first in control cells. However, when the second pulse occurred in the presence of 0.2, 1, or 5 U/mL thrombin, JH increased by 30%, 62% (P < .05), and 87% (P < .05), respectively. A hexameric thrombin receptor-activating peptide (SFLLRN) mimicked the effect of thrombin and increased JH by 73% (P < .05) at 25 mumol/L. In contrast, an inactive control peptide (FLLRN) was without effect at 25 mumol/L. In cells pretreated with 100 nmol/L GF109203X or 5 mumol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 mumol/L SFLLRN produced a significant increase in JH. In the presence of 10 mumol/L HOE-694 (a Na(+)-H+ exchanger inhibitor), pHi did not recover after an acid load, even during exposure to 5 U/mL thrombin or 25 mumol/L SFLLRN, confirming that the Na(+)-H+ exchanger was the primary acid efflux mechanism under the conditions used. Neither 5 U/mL thrombin nor 25 mumol/L SFLLRN affected resting pHi and Ca2+ or background acid loading. We conclude that (1) adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation results in increased activity of the sarcolemmal Na(+)-H+ exchanger, and (2) this effect appears to occur through a protein kinase C-mediated mechanism.

Glycogen Synthase Kinase-3 Inactivation Is Not Required for Ischemic Preconditioning or Postconditioning in the Mouse

The inactivation of glycogen synthase kinase-3β (GSK-3β) is proposed as the event integrating protective pathways initiated by preconditioning and other interventions. The inactivation of GSK-3 is thought to decrease the probability of opening of the mitochondrial permeability transition pore. The aim of this study was to verify the role of GSK-3 using a targeted mouse line lacking the critical N-terminal serine within GSK-3β (Ser9) and the highly homologous GSK-3α (Ser21), which when phosphorylated results in kinase inactivation. Postconditioning with 10 cycles of 5 seconds of reperfusion/5 seconds of ischemia and preconditioning with 6 cycles of 4 minutes of ischemia/6 minutes of reperfusion, similarly reduced infarction of the isolated perfused mouse heart in response to 30 minutes of global ischemia and 120 minutes of reperfusion. Preconditioning caused noticeable inactivating phosphorylation of GSK-3. However, both preconditioning and postconditioning still protected hearts of homozygous GSK-3 double knockin mice. Moreover, direct pharmacological inhibition of GSK-3 catalytic activity with structurally diverse inhibitors before or after ischemia failed to recapitulate conditioning protection. Nonetheless, cyclosporin A, a direct mitochondrial permeability transition pore inhibitor, reduced infarction in hearts from both wild-type and homozygous GSK-3 double knockin mice. Furthermore, in adult cardiac myocytes from GSK-3 double knockin mice, insulin exposure was still as effective as cyclosporin A in delaying mitochondrial permeability transition pore opening. Our results, which include a novel genetic approach, suggest that the inhibition of GSK-3 is unlikely to be the key determinant of cardioprotective signaling in either preconditioning or postconditioning in the mouse.

Sarcolemmal Na+/H+ exchanger activity and expression in human ventricular myocardium

OBJECTIVES To determine sarcolemmal Na+/H+ exchanger (NHE) activity and expression in human ventricular myocardium. BACKGROUND Although the sarcolemmal NHE has been implicated in various physiological and pathophysiological phenomena in animal studies, its activity and expression in human myocardium have not been studied. METHODS Ventricular myocardium was obtained from unused donor hearts with acute myocardial dysfunction (n = 5) and recipient hearts with chronic end stage heart failure (n = 11) through a transplantation program. Intracellular pH (pHi) was monitored in enzymatically isolated single ventricular myocytes by microepifluorescence. As the index of sarcolemmal NHE activity, the rate of H+ efflux at a pHi of 6.90 J(H6.9)) was determined after the induction of intracellular acidosis in bicarbonate-free medium. Na+/H+ exchanger isoform 1 (NHE1) expression in ventricular myocardium was determined by immunoblot analysis. RESULTS Human ventricular myocytes exhibited readily detectable sarcolemmal NHE activity after the induction of intracellular acidosis, and this activity was suppressed by the NHE1-selective inhibitor HOE-642 (cariporide) at 1 micromol/L. Sarcolemmal NHE activity of myocytes was significantly greater in recipient hearts (JH6.9 = 1.95+/-0.18 mmol/L/min) than it was in unused donor hearts (J(H6.9 = 1.06+/-0.15 mmol/L/min). In contrast, NHE1 protein was expressed in similar abundance in ventricular myocardium from both recipient and unused donor hearts. CONCLUSIONS Sarcolemmal NHE activity of human ventricular myocytes arises from the NHE1 isoform and is inhibited by HOE-642. Sarcolemmal NHE activity is significantly greater in recipient hearts with chronic end-stage heart failure than it is in unused donor hearts, and this difference is likely to arise from altered posttranslational regulation.

Protection of the ischaemic myocardium by Na+/H+ exchange inhibitors: potential mechanisms of action

nal hypothesis by Lazdunski et al. (21) proposed that increased sarcolemmal NHE activity, arising from the rapid normalisation of extracellular pH and the generation of an outwardly directed H+ gradient upon reperfusion, may play a role in reperfusion injury. Although this hypothesis is supported by the studies which have shown cardioprotective benefit with NHE inhibitors when given from shortly before or early during reperfusion (discussed elsewhere in this issue), it is not consistent with the majority of studies (starting with the original observations of Karmazyn (15)) which have shown superior benefit when the NHE inhibitor is given prior to the onset of or early during ischaemia (5, 12, 17, 18, 25, 35, 37). In this context, it has been suggested that the relative inefficacy of NHE inhibitors when given from shortly before or early during reperfusion in intact hearts may arise from inadequate drug delivery to the site of action (24). However, the recent work of Klein et al. (18) has shown that late administration of an NHE inhibitor by intracoronary infusion does not limit infarct size in pigs in vivo, even when an effective drug concentration is achieved in coronary venous blood prior to the onset of reperfusion. Furthermore, the evidence obtained with NHE inhibitors of the amiloride class, which have been shown to attenuate intracellular Na+ (26, 28, 41) and Ca2+ (26) accumulation during ischaemia in parallel with their cardioprotective effects, suggests that NHE activity is maintained and contributes substantially to the loss of ionic homeostasis during ischaemia, and that this is a key determinant of the overall extent of injury during ischaemia and reperfusion. Nevertheless, the conclusions of these studies have been open to conjecture, on the basis that amiloride and its derivatives can additionally inhibit ion transporting proteins other than the sarcolemmal NHE that may also contribute to the loss of Na+ and Ca2+ homeostasis during ischaemia (10). Therefore, it is important to emphasise recent studies which have shown that the highly specific NHE inhibitor cariporide (previously known as HOE-642 (34)) also attenuBR C 04 Dr. M. Avkiran (Y) Centre for Cardiovascular Biology and Medicine King’s College London The Rayne Institute St Thomas’ Hospital London SE1 7EH, UK E-Mail: metin.avkiran@kcl.ac.uk Received: 23 April 2001 Returned for revision: 8 May 2001 Revision received: 21 May 2001 Accepted: 21 May 200

Protein Kinase D Selectively Targets Cardiac Troponin I and Regulates Myofilament Ca2+ Sensitivity in Ventricular Myocytes

Protein kinase D (PKD) is a serine/threonine kinase with emerging myocardial functions; in skinned adult rat ventricular myocytes (ARVMs), recombinant PKD catalytic domain phosphorylates cardiac troponin I at Ser22/Ser23 and reduces myofilament Ca2+ sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca2+]i transients in intact ARVMs. In myocytes transduced to express wild-type PKD, the heterologously expressed enzyme was activated by endothelin 1 (ET1) (5 nmol/L), as reflected by PKD phosphorylation at Ser744/Ser748 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). The ET1-induced increase in cellular PKD activity was accompanied by increased cardiac troponin I phosphorylation at Ser22/Ser23; this measured approximately 60% of that induced by isoproterenol (10 nmol/L), which activates cAMP-dependent protein kinase (PKA) but not PKD. Phosphorylation of other PKA targets, such as phospholamban at Ser16, phospholemman at Ser68 and cardiac myosin-binding protein C at Ser282, was unaltered. Furthermore, heterologous PKD expression had no effect on isoproterenol-induced phosphorylation of these proteins, or on isoproterenol-induced increases in sarcomere shortening and relaxation rate and [Ca2+]i transient amplitude. In contrast, heterologous PKD expression suppressed the positive inotropic effect of ET1 seen in control cells, without altering ET1-induced increases in relaxation rate and [Ca2+]i transient amplitude. Complementary experiments in “skinned” myocytes confirmed reduced myofilament Ca2+ sensitivity by ET1-induced activation of heterologously expressed PKD. We conclude that increased myocardial PKD activity induces cardiac troponin I phosphorylation at Ser22/Ser23 and reduces myofilament Ca2+ sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.

Roles of Mitogen-Activated Protein Kinases and Protein Kinase C in α1A-Adrenoceptor–Mediated Stimulation of the Sarcolemmal Na+-H+ Exchanger

Activation of the sarcolemmal Na(+)-H(+) exchanger (NHE) has been implicated as a mechanism of inotropic, arrhythmogenic, antiacidotic, and hypertrophic effects of alpha(1)-adrenoceptor (AR) stimulation. Although such regulation of sarcolemmal NHE activity has been shown to be selectively mediated through the alpha(1A)-AR subtype, distal signaling mechanisms remain poorly defined. We investigated the roles of various kinase pathways in alpha(1A)-AR-mediated stimulation of sarcolemmal NHE activity in adult rat ventricular myocytes. As an index of NHE activity, trans-sarcolemmal acid efflux rate (J(H)) was determined through microepifluorescence in single cells, during recovery from intracellular acidosis in bicarbonate-free conditions. Extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase (MAPK), and p90(rsk) activities were indexed on the basis of analysis of their phosphorylation status. In control cells, there was no change in J(H) in response to vehicle. Phenylephrine and A61603, an alpha(1A)-AR subtype-selective agonist, increased J(H), as well as cellular ERK and p90(rsk) activities. Neither agonist affected p38 activity, which was increased with sorbitol. The MAPK kinase inhibitor PD98059 abolished phenylephrine- and A61603-induced increases in J(H) and cellular ERK and p90(rsk) activities. In contrast, the PKC inhibitor GF109203X abolished phenylephrine- and A61603-induced increases in J(H) but failed to prevent the increases in ERK and p90(rsk) activities. Our findings suggest that alpha(1A)-AR-mediated stimulation of sarcolemmal NHE activity in rat ventricular myocytes requires activation of the ERK (but not the p38) pathway of the MAPK cascade and that the ERK-mediated effect may occur via p90(rsk). Activation of PKC is also required for alpha(1A)-AR-mediated NHE stimulation, but such regulation occurs through an ERK-independent pathway.