Peter H. Sugden

“Stress-Responsive” Mitogen-Activated Protein Kinases (c-Jun N-Terminal Kinases and p38 Mitogen-Activated Protein Kinases) in the Myocardium

The best-characterized subfamilies of the mitogen-activated protein kinase (MAPK) superfamily are the extracellularly responsive kinases (ERKs) and the two “stress-responsive” MAPK subfamilies, namely, the c-Jun N-terminal kinases (JNKs) and the p38-MAPKs.1 2 3 4 5 As yet, no single nomenclature has been determined, and the synonyms currently in use are summarized in Table 1⇓. The ERK cascade is the most thoroughly studied of the MAPK cascades, and it is activated principally by G protein–coupled receptor (GPCR) agonists in cardiac myocytes. We have reviewed this topic recently,6 and we will not discuss it in any depth here. The regulation of the JNK and p38-MAPK cascades in the myocardium (Figure 1⇓) forms the principal subject of this review. Figure 1. A scheme for the activation of stress-responsive MAPK cascades. Exposure of cultured cardiac myocytes and whole hearts to stresses or GPCR agonists leads to activation of the JNKs and p38-MAPKs. Cellular stresses induce reorganization of the cytoskeleton and activation of the small G proteins, Rac and Cdc42. GPCR agonists activate the small G protein, Ras, through a PKC-dependent mechanism. Ras is recognized to participate in the activation of the ERK cascade and, either directly or indirectly, may activate the stress-responsive MAPKs (JNKs and p38-MAPKs). The JNKs are activated by MKK4 and MKK7, whereas p38-MAPKs are activated by MKK3 and MKK6 (see Table 2⇓). The upstream activators (the MKKKs) of the stress-responsive MKKs have not been clearly defined but possibly include MEKKs (MAPK or ERK kinase kinases), mixed lineage kinases (MLKs), and/or p21-activated kinases (PAKs). Substrates for the stress-responsive MAPKs include transcription factors, which regulate the transcriptional changes, and MAPKAPK2, which phosphorylates Hsp25/27 and may thereby confer cytoprotection. CHOP indicates C/EBP homologous protein; MEF2C myocyte enhancer factor 2C. View this table: Table 1. Mitogen-Activated Protein Kinase (MAPK) Subfamilies An ever-increasing number of isoforms of …

REGULATION OF THE ERK SUBGROUP OF MAP KINASE CASCADES THROUGH G PROTEIN-COUPLED RECEPTORS

The extracellularly-responsive kinase (ERK) subfamily of mitogen-activated protein kinases (MAPKs) has been implicated in the regulation of cell growth and differentiation. Activation of ERKs involves a two-step protein kinase cascade lying upstream from ERK, in which the Raf family are the MAPK kinase kinases and the MEK1/MEK2 isoforms are the MAPK kinases. The linear sequence of Raf --> MEK --> ERK constitutes the ERK cascade. Although the ERK cascade is activated through growth factor-regulated receptor protein tyrosine kinases, they are also modulated through G protein-coupled receptors (GPCRs). All four G protein subfamilies (Gq/11 Gi/o, Gs and G12/13) influence the activation state of ERKs. In this review, we describe the ERK cascade and characteristics of its activation through GPCRs. We also discuss the identity of the intervening steps that may couple agonist binding at GPCRs to activation of the ERK cascade.

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.

Phenotyping hypertrophy: eschew obfuscation.

Everyone thinks they know what “cardiac hypertrophy” is: a reactive increase in cardiac size/myocardial mass in response to hemodynamic stress that, in humans, predisposes to early death.1 Yet, the term “hypertrophy” has become one of the most misused and inaccurate terms in the cardiovascular basic science literature because of its nonspecificity and, as typically used, lack of mechanistic implication. “Hypertrophy” (noun and verb), derived from Greek hyper (above, more than normal) and trophe (nutrition), is defined as “the enlargement or overgrowth of an organ or part due to an increase in size of its constitute cells.”2 The normal heart is “normal,” and hypertrophy is, by definition, “not normal.” Therefore, normal maturational development at the organ level is not “hypertrophy” (verb) and does not result in cardiac “hypertrophy” (noun), although the cells do “hypertrophy” (verb). (Perhaps the term “eutrophy” is more appropriate to describe maturational development.) Likewise, in many genetic in vivo experimental models, the term “cardiac hypertrophy” has too often been loosely applied to any observed cardiac enlargement, frequently with such modifiers as “physiological” or “pathological.”3 Herein, we reflect on the appropriate meanings of terms and criteria that can be used to more accurately describe cardiac enlargement and myocardial growth, with the anticipation that rigorous mechanistic description of such phenotypes will result in a more coherent appreciation of the parallel and redundant processes that result in “myocardial hypertrophy.” The widespread use of the term “hypertrophy” to describe one or a few specific pathophysiological conditions is a holdover from the earliest scientific work on cardiac response to stress, in which cardiac enlargement was largely assumed to be the result of increased cardiomyocyte size and was qualitatively predictable based on the nature of the provocative stimulus.4 Hence, morphogenic changes in the heart were classified by the nature of …

Regulation of Bcl-2 Family Proteins During Development and in Response to Oxidative Stress in Cardiac Myocytes: Association With Changes in Mitochondrial Membrane Potential

Cardiac myocyte apoptosis is potentially important in many cardiac disorders. In other cells, Bcl-2 family proteins and mitochondrial dysfunction are probably key regulators of the apoptotic response. In the present study, we characterized the regulation of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) Bcl-2 family proteins in the rat heart during development and in oxidative stress-induced apoptosis. Bcl-2 and Bcl-xL were expressed at high levels in the neonate, and their expression was sustained during development. In contrast, although Bad and Bax were present at high levels in neonatal hearts, they were barely detectable in adult hearts. We confirmed that H(2)O(2) induced cardiac myocyte cell death, stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours), caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8 hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL were associated with the mitochondria, but Bad and Bax were predominantly present in a crude cytosolic fraction. Exposure of myocytes to H(2)O(2) stimulated rapid translocation of Bad (<5 minutes) to the mitochondria. This was followed by the subsequent degradation of Bad and Bcl-2 (from approximately 30 minutes). The levels of the mitochondrial membrane marker cytochrome oxidase remained unchanged. H(2)O(2) also induced translocation of cytochrome c from the mitochondria to the cytosol within 15 to 30 minutes, which was indicative of mitochondrial dysfunction. Myocytes exposed to H(2)O(2) showed an early loss of mitochondrial membrane potential (assessed by fluorescence-activated cell sorter analysis) from 15 to 30 minutes, which was partially restored by approximately 1 hour. However, a subsequent irreversible loss of mitochondrial membrane potential occurred that correlated with cell death. These data suggest that the regulation of Bcl-2 and mitochondrial function are important factors in oxidative stress-induced cardiac myocyte apoptosis.

The p38‐MAPK inhibitor, SB203580, inhibits cardiac stress‐activated protein kinases/c‐Jun N‐terminal kinases (SAPKs/JNKs)

SB203580 is a recognised inhibitor of p38‐MAPKs. Here, we investigated the effects of SB203580 on cardiac SAPKs/JNKs. The IC50 for inhibition of p38‐MAPK stimulation of MAPKAPK2 was approximately 0.07 μM, whereas that for total SAPK/JNK activity was 3–10 μM. SB203580 did not inhibit immunoprecipitated JNK1 isoforms. Three peaks of SAPK/JNK activity were separated by anion exchange chromatography, eluting in the isocratic wash (44 kDa), and at 0.08 M (46 and 52 kDa) and 0.15 M NaCl (54 kDa). SB203580 (10 μM) completely inhibited the 0.15 M NaCl activity and partially inhibited the 0.08 M NaCl activity. Since JNK1 antibodies immunoprecipitate the 46 kDa activity, this indicates that SB203580 selectively inhibits 52 and 54 kDa SAPKs/JNKs.

Depletion of Mitogen-Activated Protein Kinase Using an Antisense Oligodeoxynucleotide Approach Downregulates the Phenylephrine-Induced Hypertrophic Response in Rat Cardiac Myocytes

An antisense oligodeoxynucleotide (ODN) approach was used to investigate whether mitogen-activated protein kinase (MAPK) is necessary for the hypertrophic response in cardiac myocytes. A phosphorothioate-protected 17-mer directed against the initiation of translation sites of the p42 and p44 MAPK isoform mRNAs was introduced into cultured cardiac myocytes by liposomal transfection. At an antisense ODN concentration of 0.2 mumol/L, p42 MAPK protein was reduced by 82% (immunoblot) after 48 hours, and p42 and p44 MAPK activities were reduced by 44% and 60%, respectively. The same concentration of anti-MAPK ODN inhibited development of the morphological features of hypertrophy (sarcomerogenesis, increased cell size) in myocytes exposed to phenylephrine. Phenylephrine-induced activation of the atrial natriuretic factor (ANF) promoter (measured by the activity of a transfected ANF promoter/luciferase reporter gene) and induction of ANF mRNA (measured by RNase protection assay) were also attenuated. We conclude that MAPK is important for the development of the hypertrophic phenotype in this model of hypertrophy.

Insulin-like Growth Factor-I Rapidly Activates Multiple Signal Transduction Pathways in Cultured Rat Cardiac Myocytes

In response to insulin-like growth factor-I (IGF-I), neonatal rat cardiac myocytes exhibit a hypertrophic response. The elucidation of the IGF-I signal transduction system in these cells remains unknown. We show here that cardiac myocytes present a single class of high affinity receptors (12,446 ± 3,669 binding sites/cell) with a dissociation constant of 0.36 ± 0.10 nm. Two different β-subunits of IGF-I receptor were detected, and their autophosphorylation was followed by increases in the phosphotyrosine content of extracellular signal-regulated kinases (ERKs), insulin receptor substrate 1, phospholipase C-γ1, and phosphatidylinositol 3-kinase. IGF-I transiently activates c-Raf in cultured neonatal cardiac myocytes, whereas A-raf is activated much less than c-Raf. Two peaks of ERK activity (ERK1 and ERK2) were resolved in cardiac myocytes treated with IGF-I by fast protein liquid chromatography, both being stimulated by IGF-I (with EC50values for the stimulation of ERK1 and ERK2 by IGF-I of 0.10 and 0.12 nm, respectively). Maximal activation of ERK2 (12-fold) and ERK1 (8.3-fold) activities was attained after a 5-min exposure to IGF-I. Maximal activation of p90 S6 kinase by IGF-I was achieved after 10 min, and then the activity decreased slowly. Interestingly, IGF-I stimulates incorporation of [3H]phenylalanine (1.6-fold) without any effect on [3H]thymidine incorporation. These data suggest that IGF-I activates multiple signal transduction pathways in cardiac myocytes some of which may be relevant to the hypertrophic response of the heart.

Signaling in Myocardial Hypertrophy Life After Calcineurin

Cardiac (ventricular) hypertrophy is an important adaptive response in vivo that (at least in the shorter term) allows the organism to maintain or increase its cardiac output. Global ventricular hypertrophy is a recognized response to increased pressure or volume work (reviewed in Reference 11 ), with increased myofibrillogenesis and sarcomere deposition being cardinal features. Although global hypertrophy is clinically important, probably the most significant form of cardiac hypertrophy in terms of patient numbers is the localized hypertrophy of the ventricular wall that may follow loss of myocardium after a survivable myocardial infarct. In the early stages, both global and localized hypertrophy may resemble the readily reversible, nonfibrotic “physiological” hypertrophy that develops after repeated endurance exercise. In the longer term, beneficial, “compensated” hypertrophy may decay into maladaptive “decompensated” hypertrophy and heart failure (reviewed in References 2 and 32 3 ) with diminished coronary flow reserve and increased risk of lethal arrhythmias. Although some aspects of the maladapted state are probably intrinsic to the myocyte [eg, prolongation of the action potential and Ca2+ transient (reviewed in References 4 and 54 5 )], other factors (increased fibrosis and mismatch between O2 supply and demand) are also probably involved in decompensation. The predominating view is that mammalian ventricular myocytes lose their capacity for cell division during the perinatal period and are thus terminally differentiated cells, although this is still a matter of some dispute (reviewed in References 6 and 76 7 ). In contrast, other cells in the heart (endothelial cells, fibroblasts, and smooth muscle cells) retain their mitotic capacity. Although ventricular myocytes are not the only cell type involved in the overall hypertrophic response, much of the ventricular enlargement or remodeling is attributable to their hypertrophy. The identities of signaling pathways that couple the demand for increased …

Hypertrophic Agonists Stimulate the Activities of the Protein Kinases c-Raf and A-Raf in Cultured Ventricular Myocytes

We detected expression of two Raf isoforms, c-Raf and A-Raf, in neonatal rat heart. Both isoforms phosphorylated, activated, and formed complexes with mitogen-activated protein kinase kinase 1 in vitro. However, these isoforms were differentially activated by hypertrophic stimuli such as peptide growth factors, endothelin-1 (ET1), or 12-O-tetradecanoylphorbol-13-acetate (TPA) that activate the mitogen-activated protein kinase cascade. Exposure of cultured ventricular myocytes to acidic fibroblast growth factor activated c-Raf but not A-Raf. In contrast, TPA produced a sustained activation of A-Raf and only transiently activated c-Raf. ET1 transiently activated both isoforms. TPA and ET1 were the most potent activators of c-Raf and A-Raf. Both utilized protein kinase C-dependent pathways, but stimulation by ET1 was also partially sensitive to pertussis toxin pretreatment. c-Raf was inhibited by activation of cAMP-dependent protein kinase although A-Raf was less affected. Fetal calf serum, phenylephrine, and carbachol were less potent activators of c-Raf and A-Raf. These results demonstrate that A-Raf and c-Raf are differentially regulated and that A-Raf may be an important mediator of mitogen-activated protein kinase cascade activation when cAMP is elevated.