Molecular Pathways for Cardiac Hypertrophy and Heart Failure Progression

S E C T I O N 3

Cardiac Muscle Diseases

chapter1 5

Molecular Pathways for Cardiac Hypertrophy and Heart Failure Progression Masahiko Hoshijima Susumu Minamisawa Hideo Yasukawa Kenneth R. Chien

Heart failure is emerging as a leading cause of human morbidity and mortality and is predicted to reach epidemic proportions in the early 21st century.1 Unfortunately, current therapy for heart failure is largely based on a complex regimen of drugs that are primarily targeted at symptoms of the disease rather than the fundamental processes that drive disease progression.2–4 One of the major difficulties has been the complexity of the disease, which is chronic, progressive, and propelled by a combination of environmental stimuli and genetic susceptibility.5,6 Studies of rare familial forms of heart failure have documented a trend toward incomplete penetrance with a highly variable genotype-phenotype correlation, suggesting the presence of strong modifiers and associated genetic background effects.7,8 At the same time, diverse environmental stimuli, including postinfarction injury, cardiotoxic side effects of chemotherapeutic agents, viral infection, and chronic exposure to volume and pressure overload can all serve to initiate and modify disease progression. Although decreases in cardiac contractility and relaxation are conserved features of many forms of heart failure, these acute physiologic end points do not always correlate with long-term mortality, again underscoring the complex nature of the heart failure phenotype. Heart failure is a growing, unmet clinical problem, representing both a major scientific opportunity and a clinical challenge for cardiovascular physicians and scientists alike. Many laboratories now view heart failure as the “cancer” of the cardiovascular system, because it is a classic, multifactorial, polygenic, and chronic disease that progresses to a terminal stage. Intriguingly, a growing body of work now suggests another important parallel between the biology of cancer and the biology of heart failure. The massive, abnormal growth in cardiac muscle that accompanies heart failure shares several biologic principles and molecular pathways with cancer biology. The biologic precepts of cell growth, apoptosis, and cell survival are as important in the onset of heart failure as they are in tumor progression, and the molecular signals for cell proliferation

and cardiac myocyte hypertrophy are highly conserved. Both diseases are inexorable and progressive in their nature, and a “multihit” hypothesis applies for both cancer and heart failure progression, largely based on the interplay between genetic susceptibility and environmental stimuli. This view, coupled with the integration of multiple experimental approaches including in vivo somatic gene transfer, bioinformatics, and computational biology, is beginning to provide new insights into the disease process. Insights from genetically based studies in mice and humans are now suggesting new therapeutic targets and strategies for intervening in the disease. With the completion of the human and mouse genome databases and physical mapping of annotated genes that provide a road map, great strides in unraveling the logic of heart failure progression can be made. This chapter highlights several advances in the understanding of the molecular pathways that drive the onset of cardiac hypertrophy and heart failure; these advances are forming the basis for a new wave of biologically targeted therapy.

BIPHASIC TEMPORAL SIGNALING CASCADES DIRECTLY ACTIVATED BY MECHANICAL STRESS: POSITIVE AND NEGATIVE REGULATORS Mechanical stress is one of the most important stimuli for triggering the initial steps toward heart failure. A diverse group of biochemical signals can be rapidly activated during in vivo pressure overload; many can be activated within a few minutes of exposure to dynamic or passive stretch that accompanies increases in aortic pressure or volume overload.9–12 To date, the precise identity of the molecular machinery that initially senses cardiac mechanical stress has remained largely unknown. A series of studies have indicated that a subset of ion-channel or ion exchangers, such as a stretch-activated channel or a sodium-proton exchanger, are regulated by deformation of 273

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the plasma membrane and are found in cardiomyocytes exposed to mechanical or osmotic load.13,14 These mechanical stress-related changes in ionic current have rarely been linked to the onset of hypertrophy in individual cardiomyocytes.15 However, several transmembrane signaling pathways, including MAP kinases, Janus kinase (JAK)/signal transducers and activators of transcription (STAT), and phosphoinositide 3-kinase (PI3K)/Akt (protein kinase B) cascades, have been suggested to connect mechanical load to the onset of cardiac hypertrophy.16–19 Following mechanical stretch of cultured cardiomyocytes, most of these cardiac signaling kinase cascades are activated, in part via the cell adhesion complex,which include integrins and integrin-associated proteins that are linked to the extracellular matrix (ECM).20,21 Cardiac conditional knockout mice of the β1 integrin gene display a blunted MAP kinase activation following in vivo pressure overload.22 In addition, ablation of melusin, an integrin-binding protein, has resulted in blunted response to pressure-overload stimulation to activate Akt/glycogen synthase kinase-3β (GSK3β) signaling and to induce cardiac hypertrophy.23 However, further investigation will be necessary to determine the precise role of the ECM-integrin-cytoskeleton complex in cardiac mechanical stress sensing. In addition, hormonal stimuli, such as adrenergic agonists, angiotensin, endothelin, and gp130 cytokine receptor agonists also acti-

vate these signaling kinases, thereby triggering hypertrophic responses in vitro and in vivo.16–19 Many of these insights in the signaling pathways for cardiac hypertrophy have originated from studies in cultured neonatal rat cardiomyocytes in vitro, followed by studies of normal and genetically engineered mouse models that have been exposed to the pressure overload that accompanies in vivo aortic banding. The acute response to pressure-overload peaks within hours and includes the activation of ERK1/2, JNK, p38, Akt, and gp130/STAT and the induction of early stress-response genes, such as the suppressor of cytokine signaling-3 (SOCS3) (Fig. 15-1,Table 15-1). This early phase response is followed by a hypertrophic response, which starts within 48 hours after aortic banding and reaches a peak within 7 days.This later hypertrophic response is clearly marked by an increase in the left ventricle to body weight ratio and the induction of a series of embryonic gene markers such as ANF, skeletal actin, and β-myosin heavy chain.9,31,32 Interestingly, there is a second peak of activation for many of signaling cascade kinases in this subacute hypertrophic response phase,10,11 suggesting that there may be phasic and discrete roles for a subset of signaling pathways that respond to mechanical stress. The double peak of early mechanical stress responses implicates the presence of inhibitory regulators that act

FIGURE 15-1. Mechanical stress-induced transmembrane signaling and integrated phenotypes of a failing heart. The activation profiles of various signaling cascades and time-dependent changes in phenotypic features of heart failure are illustrated based on our previous studies10 and unpublished observations. Lower panels show biphasic induction of representative stress-responding marker genes and upregulation of left ventricular to body weight (LV/BW) ratio after transverse thoracic aortic constriction in normal adult mice. (Modified from Yasukawa H, Hoshijima M, Gu Y, et al: Suppressor of cytokine signaling-e is a biomechanical stress-inducible gene that suppresses gp130-mediated cardiac myocyte hypertrophy and survival pathways. J Clin Invest 2001;108:1459–1467 and Hoshijima M, Chien KR: Mixed signals in heart failure: Cancer rules. J Clin Invest 2002;109:849–855.)

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TABLE 15-1 MECHANICAL STRESS-RELATED CHANGES IN GENE EXPRESSION DURING HEART FAILURE PROGRESSION IN HUMAN AND EXPERIMENTAL STUDIES Acute (<24 hr) Up c-fos c-jun junB egr-1 nur77 BNP SOCS3

Hypertrophic–Failing (Days-Weeks-Years) Up Secreted proteins ANF, lipocortin I, ET-1 HB-EGF,TGFβ1, BNP Osteoblast specific factor 2 Cytoskeletal proteins αMHC, βMHC MLC1a/v, MLC2a MLC2v, tropomyosin Troponin C, Myomesin Smooth muscle α–actin Skeletal α–actin α–cardiac actin FHL1(HCM), sarcosin Desmin, gelsolin Extracellular matrix Fibulin, fibronectin Laminin, collagen Ion-channels/carriers Na+/Ca2+ exchanger, Kv1.4 Voltage dependent anion channel-1

Down Metabolism/translational proteins Ubiquitin, pyruvate dehydrogenase α NADH ubiquinone oxidoreductase Creatine kinase, Myoglobin Superoxide dismutase 2 Aldose reductase, EF-1a, EF-2, IF-4AII 28S, 60S ribosomal L3 Signaling Gsα, βARK, adenylyl cyclase VII A-kinase, C-kinase inhibitor-1, ILK Rap1B, SOCS3, Id-1, GATA-4 SP1/3, PGD/D2 synthase Others Heat shock 70kD proteins 1, 6, 8 Quaking protein, CARP

Cytoskeletal proteins FHL1 (failing heart) Nonsarcomeric MLC2 Ion-channels/carriers L-type Ca2+ channel SERCA2 Phospholamban Kv4.2, 4.3 Kv1.5 KChIP2 Signaling β1-AR, type-A like Ephrin receptor Others α1-Antichymotrypsin αB-Crystallin Plasminogen activator inhibitor-1 TIM17 Connexin43

Modified from Hoshijima M, Chien KR: Mixed signals in heart failure: Cancer rules. J Clin Invest 2002;109:849–855, and references 24 to 30.

as a negative feedback loop to prevent the continuous activation of any single pathway. Naturally, there must be negative regulators of the response that leads to attenuation of the signal so that the acute response is properly terminated and an orderly compensatory hypertrophic response ensues, as opposed to unbridled reactive growth, which is often seen in mouse models of cardiac hypertrophy induced by cardiac overexpression of signaling molecules.19 A number of studies have recently identified negative feedback regulation of these intracellular signaling pathways. SOCS3 provides an example of a biomechanical stress-inducible negative regulator, which acts to inhibit the gp130 stress-inducible pathway for myocyte survival.10,33 Within minutes of aortic constriction, either by the action of gp130 ligands (e.g., cardiotrophin-1 and LIF) or via unknown mechanisms of direct activation, JAKs phosphorylate a transcriptional regulator STAT3 leading to the translocation of STAT3 into the nucleus, which directly activates the expression of genes that can confer myocyte hypertrophy34,35 and survival (e.g., BCL-x),36–38 and SOCS3.10 SOCS3 is induced in the myocardium within 1 hour of aortic banding, peaking for several hours and suppressing JAK kinase activity by selectively binding to gp130. By inhibiting JAKs, SOCS3 negatively regulates stress-inducible gp130 activation (Fig. 15-2). It is likely that this negative regulatory circuit prevents hyperstimulation of this pathway that is induced by sustained expression of gp130-coupled cytokines, which may have independent pathologic effects on cardiac function. In this manner, the delicate balance between the activation of gp130/JAK signaling and the induction of its negative feedback regulator SOCS3 might be important in the transition between car-

diac hypertrophy and failure.5,10 Disrupting the balance between this positive and negative regulatory loop by chronic exposure to gp130 ligands might lead to secondary effects that impair cardiac function. A number of other novel negative regulators of cardiac signaling have recently been described. Recent studies have identified myocyte-enriched calcineurin-interacting protein-1 (MCIP1), a protein that inhibits cardiac hypertrophy by attenuating calcineurin activity.39 The MCIP1 gene contains NFAT (nuclear factors of activated T cells) binding motifs that mediate its upregulation by calcineurin signals, thereby suggesting that MCIP1 participates in the negative feedback loop of calcineurin signaling in the myocardium.40 A separate negative feedback regulatory system has been found for cardiac MAP kinase signaling: forced expression of MAP kinase phosphatase-1 (MKP-1) has been shown to negatively regulate the cardiac hypertrophic response by downregulating the three branches of MAP kinase cascade (p38s, JNK1/2, and ERK1/2).41 Given this complex temporal pattern of signaling pathways that are activated following in vivo pressure overload, it is not surprising that studies of the effects of the overexpression of diverse components of cardiac signaling pathways have been shown to result in cardiac hypertrophy or failure. It will become increasingly important to examine the temporal sequence of activation of these signaling pathways and to connect them to specific phenotypic features that are associated with discrete physiologic end points associated with the disease, such as defects in contractility, action potential, arrhythmogenesis, and so forth. A more meticulous gene switching in model animals42 and high-efficiency somatic gene delivery systems have become available toward this end.43,44

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FIGURE 15-2. PI3K/Akt signaling in cardiomyocytes. Activation of variety of downstream molecules leads to hypertrophic and survival effects in cardiomyocytes.

HYPERTROPHY RESPONSE: QUANTITATIVE EFFECTS OF INCREASING TRANSLATIONAL EFFICIENCY AND QUALITATIVE EFFECTS ON INDUCING AN EMBRYONIC GENE PROGRAM PI3K/Akt Pathway Cardiac hypertrophy can be broken down into two central phenotypic end points: (1) quantitative effects on increasing the level of sarcomeric and other constitutive proteins and their transcripts and (2) qualitative changes in the cardiac gene program that lead to a return to the fetal phenotype. Interestingly, both of these phenotypic effects can be found during growth stimulation of diverse differentiated cell types. In this regard, insulin-like growth factor-1 (IGF-1) is a potent stimulus for both cardiac45,46 and skeletal muscle hypertrophy47 in vivo. Recent studies have clearly identified that IGF-1 increases translational efficiency of preexisting mRNAs, which underlies the hypertrophic response of skeletal muscle hypertrophy.48–50 A number of independent approaches have convincingly shown a central role of Akt in enhancing translational efficiency by phosphorylating and thereby inhibiting GSK3.48,49,51 Skeletal muscle atrophy leads to a decrease in the Akt/mTOR (mammalian target of rapamycin) pathway, and IGF-1 unexpectedly acts via Akt to antagonize calcineurin signaling during skeletal myotube hypertrophy. Taken together, the activation of the Akt/mTOR pathway and its downstream targets, p70S6K and PHAS-1/4E-BP1,3,52 appear to play a central role in skeletal muscle and perhaps cardiac muscle hypertrophy and atrophy (Fig. 15-2).53 IGF-1 activates PI3K,54,55 thereby activating Akt and phosphoinositide-dependent

kinase 1 (PDK-1), which interact with each other and cooperatively activate downstream signaling.52,56,57 The tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN) antagonizes this pathway by facilitating dephosphorylation of PtdIns(3,4,5)P3 (phosphatidylinositol-3,4,5-triphosphate),58,59 During in vivo pressure overload, an increase in translational efficiency has also been documented, and myocardial Akt is particularly activated during the subacute hypertrophic response phase.10 Taken together, a similar role for Akt signaling in heart failure-related myocardial remodeling is likely, and activation of Akt is noted in the failing human heart.60 Transgenic mouse models of cardiac IGF-1,61 PI3K,62 and Akt overexpression63–65 have been reported to induce cardiac hypertrophy and/or hyperplasia, whereas cardiac overexpression of GSK3β suppresses pressure overload or β-adrenergic stimulation-dependent hypertrophy.53,66 In addition, in vitro overexpression of catalytically inactive mutant of PTEN induces cardiomyocyte hypertrophy,67 and muscle-specific ablation of PTEN enhances in vivo mouse cardiac hypertrophy.68 Taken together, PI3K/Akt and their downstream signaling appear to be an important in vivo determinant of cardiac size. Recently, further complexity of P13K signaling was documented. PI3Kα is linked to receptor tyrosine kinase pathways that regulate cardiac myocyte size, and PI3Kγ, which binds to G-protein βγ subunits,69 negatively regulates cardiomyocyte contractility through β2-adrenergic receptor/cAMP-dependent signaling.68

Calcium Signaling Numerous studies in cardiac muscle now support an important role for calcium signaling in the activation of a hypertrophic response. Earlier, activation of calmodulin

MOLECULAR PATHWAYS FOR CARDIAC HYPERTROPHY AND HEART FAILURE PROGRESSION

(CaM)-dependent kinase (CaMK) was reported to be involved in the regulation of a fetal gene program that is coactivated during cardiac hypertrophy.70 CaMK activity is elevated in failing human hearts.71 Subsequently, a series of studies using transgenic mouse models documented that cardiac overexpression of calcineurin, a calcium-CaM dependent protein phosphatase, can induce massive cardiac hypertrophy.72 A family of transcriptional factors NFATs are located in the downstream of calcineurin signaling.73 In addition, the cardiac overexpression of CaMKIV, a neural CaMK isotype, results in a concentric form of cardiac hypertrophy.74 Myocyte enhancer factor-2 (MEF2), a MADS-box transcription factor highly expressed in myocytes, is a putative downstream target molecule of the CaMK pathway, and dual activation of CaMK and calcineurin-dependent pathways by calcium has been proposed to regulate cardiac hypertrophy.19,73 In this regard, MEF2 associates with NFAT and GATA to facilitate transcriptional activation of hypertrophy-responsive genes75 (Fig. 15-3). From studies in skeletal muscle and neural tissue, the molecular mechanism of CaMK-dependent MEF2 regulation has been proposed as follows76: activated CaMK phosphorylates class II histone deacetylases (HDACs) (4, 5, 7, and 9: abundant in striated muscle and brain) or perhaps MEF2-interacting transcriptional facator-2 (MITR), which is a splicing variant of HDAC and lacks the catalytic domain. Phosphorylated HDACs or MITR binds to an intracellular chaperone protein 14-3-3, and this interaction exposes a nuclear export sequence (NES) on HDACs, which leads to CRM1-dependent translocation of HDAC/14-3-3 complex to the cytoplasm. On the other hand, MITR phosphorylation leads to the redistribution of the MITR/14-3-3 complex in the nucleus

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because MITR lacks the NES. The dislocation of HDACs or MITR releases MEF2–interaction-dependent suppression of muscle gene activation. However, this CaMK/HDAC/MEF2 activation sequence has not yet been proved in cardiomyocytes. Interestingly, in cardiomyocytes the CaMK phosphorylation sites of HDAC/MITR seems to be catalyzed by other kinases, because the HDAC phosphorylation activity in cardiac extracts from calcineurin-transgenic mice or mice with cardiac hypertrophy induced by pressure-overload is insensitive to chemical CaMK inhibitors and EGTA, a divalent cation chelator.77 So far, the molecular identity of the putative cardiac HDAC kinase(s) remains unclear. On the other hand, p38 phosphorylates and directly activates MEF2.78 It also antagonizes nuclear translocalization of NFAT.79 In addition, GSK3 negatively regulates GATA480 and NFAT.66 As described, the transcriptional factor complex, including MEF2, NFAT, and GATA, is localized in the downstream of a web of multiple kinase cascades. Pharmacologic inhibition of calcineurin using cyclosporin A or FK506 has provided mixed results for their antihypertrophic effect in various experimental models.81–83 However, more compelling studies, using cardiac specific expression of dominant negative forms of calcineurin84 and endogenous calcineurin inhibitors,32,85 have confirmed a pivotal role for the calcineurin pathway in certain in vitro and in vivo assay systems. Subsequent studies in calcineurin Aβ-deficient mice revealed that changes in heart weight to tibia length ratio are diminished following acute pressure overload or chronic angiotensin II/isoproterenol infusion.86 Herein, the calcineurin Aβ form dominantly contributes to cardiac protein phosphatase 2B activity.In addition,targeted disruption of NFATc3,but not

FIGURE 15-3. Activation of the embryonic gene program in the hypertrophic heart via diverse membrane-nucleus signaling pathways. The signaling meshwork includes Gq, small GTP-biding proteins, MAP kinases, and calcium-calcineurin/CaM. The well-characterized ANF promoter lesion is shown in the lower panel with positioning of respective transactivators, such as MEF2,Tb×5, Nk×2.5, and GATA4.

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NTATc4, was shown to attenuate both pressure-overload and calcineurin–overexpression-mediated mouse cardiac hypertrophy.87 On the other hand, calcineurin inhibition aggravated cardiac dysfunction in a hypertrophic cardiomyopathy (HCM) model carrying a point mutation of the myosin heavy chain gene,88 which corresponds to the human inherited form of cardiomyopathy89 and points out the need to further probe the clinical significance of calcineurin signaling in cardiac diseases. The recent notion that the constitutive activation of CaMKIIdelta(B), the major cardiac CaMK isotype, results in a dilated form of cardiomyopathy90 suggests another level of complexity regarding calcium-dependent signaling in the regulation of cardiac hypertrophy. The compartmental source for calcium that triggers biochemical calcium signaling has not been identified. It is uncertain whether the pool of calcium that activates components of the hypertrophic response is derived from the beatby-beat transient increase in free cytoplasmic calcium that underlies excitation-contraction coupling91 or whether this reflects a specific intracellular compartmentalization (such as specialized endoplasmic reticulum,92,93 myofilaments,94 or nuclear membrane) of calcium. Mice harboring a targeted disruption of calreticulin display embryonic cardiomyopathy, and the calreticulin-deficient myocytes show reduced NFAT translocation into the nucleus, suggesting the possibility that calcium mobilization from the distinctive cardiac cytoplasmic membrane structure where calreticulin localizes may activate calcineurin-dependent nuclear signaling.95 Another possibility is distinctive calciumdependent signaling pathways that dictate the particular frequency or concentration range of intracellular calcium oscillation.96–98 The pCa of calcineurin activation is also one order of magnitude lower than that required for CaMK.99,100 Thus, it appears that a cohesive, integrated view of the role of calcium signaling in hypertrophy is beginning to appear, although an extensive amount of work will be required. In addition, it will be of interest to determine how this calcium signaling is integrated into other calcium-independent signaling pathways for hypertrophy.

Gq Pathways A growing body of clinical and scientific evidence supports the viewpoint that Gq/G11 pathways are one of the most proximal steps in the hypertrophic response.18 By microinjection in cultured neonatal cardiomyocytes, the Gq protein was initially shown to be critical to the induction of morphologic hypertrophy and ANF expression.101 Subsequently, studies using either in vitro overexpression of Gq via an adenovirus vector or in vivo overexpression of Gq in transgenic mice resulted in cardiomyocyte hypertrophy.102 Excessive expression of Gq provokes cardiomyocyte apoptosis and associated cardiac dysfunction in both experimental systems. Herein, induction of a Bcl-2 family proapoptotic mitochondriatargeted protein Nix/Bni3L by Gq overexpression may play a cytopathic role via cytochrome c release and caspase-3 activation.103 The overexpression of the inhibitory peptide for Gq suppressed in vivo pressure-overload-

induced cardiac hypertrophy in mice,104,105 which was confirmed by a subsequent study using mice that harbor a double cardiac-restricted ablation of both Gq and G11.106 The downstream signaling of Gq/G11 activation is diverse and includes activations of small molecular GTP binding proteins, such as ras, rho, and rac; multiple MAP kinases; rho kinase; phosphatidyl inositol (PtdIns) hydrolysis; PKC; and CaMK16–19 (Fig. 15-3). The extent of crosstalk between calcium signaling, PI3K/Akt, ECMadhesion complex, and Gq/G11 pathways and their differential role in triggering downstream phenotypic features of cardiac hypertrophy are currently unclear. Other membrane G-proteins, including G12/G13, are also involved in ligand-dependent cardiac hypertrophy.107 A new Gqdependent pathway has recently been uncovered, which involves the Gq-dependent transactivation of EGF receptor through cleavage of heparin-binding EGF via the extracellular metalloprotease 12 (ADAM12).108 Further investigation will be needed to confirm that this intracellular or intercellular transactivation of tyrosine kinase receptor is indeed downstream of Gq-dependent signaling in the myocardium.

Protein Kinase C (PKC) Signaling PKC was originally identified in bovine brain extracts as a calcium/phospholipid-dependent protein kinase, and its diverse physiologic functions include regulation of cell proliferation, differentiation, motility, and intermediary metabolism.109 The in vitro effect of activated PKC on cardiomyocyte hypertrophy was noted relatively early (using direct PKC-activating agents; e.g., phorbol esters or diacylglycerols110) and was later confirmed in in vivo cardiac overexpression studies of various isoforms of PKC in transgenic mice.111–114 A combination of biochemical and molecular cloning strategies have now identified more than 12 members of the PKC family,which are categorized into four groups based on structural similarities: classic (cPKC), novel (nPKC), atypical (aPKC), and related genes.115 In cardiomyocytes, cPKCα, nPKCδ, and nPKCε are relatively abundant and consistently identified; aPKCζ and aPLCλ/ι are also detectable. The presence of cPKCβ1/2 in the heart has been inconsistent between laboratories.17,116–118 A two-step model of PKC activation has been proposed. PDK-1 accesses to the activation loop of PKCs for conformational changes associated with phosphorylation,119 which is sufficient for the activation of aPKCs, whereas the primed cPKCs need subsequent coupling with PtdIns hydrolysis.50,115 Gq-coupled agonists can lead to the translocation of nPKCδ and nPKCε to the cardiac membrane fraction from the cytoplasm,120–122 and these PKC isoforms display crosstalk and partly constitute a molecular complex with MAP kinases.123,124 Perhaps through c-Raf dependent ERK1/2 activation,125 nPKCε has been implicated in both cardiac hypertrophy and cardioprotective effects, whereas nPKCδ is known to be proapoptotic115 and may be linked to JNK and p38 pathways.126 Overexpression of an nPKCε-activating peptide ψεRACK1 in mice results in mild cardiac hypertrophy,127 whereas an nPKCε-inhibitory peptide εV1 accelerates induction of a heart failure phenotype in Gq-transgenic mice.128 Intriguingly, nPKCε-null mice, which have normal

MOLECULAR PATHWAYS FOR CARDIAC HYPERTROPHY AND HEART FAILURE PROGRESSION

basal cardiac function, are blunted for sphingosine1-phosphate and ganglioside GM-1 mediated cardiac protection against ischemia-reperfusion injury.129 The cardioprotective action of nPKCε may be related to its partial colocalization with MAP kinases near cardiac mitochondria,130 which induces phosphorylation and inactivation of a proapoptotic protein Bad.130,131 On the other hand, earlier immunohistologic studies using isotype-specific antibodies have documented that nPKCε can rapidly translocate from the cytosol to the Z-disc accompanied by kinase activation,132 thereby raising the possibility that nPKCε is directly involved in remodeling of the cardiac cytoskeletal architecture or Z-disc related mechanical stress sensing machinery (see later).Two possible groups of proteins may recruit nPKCε to the Z-disc related structure: (1) β′COP, a selective binding protein (RACK) for activated nPKCε colocalizes in cardiomyocytes with crosstriation133 and (2) some PDZ-Lim domain proteins including ENH (Enigma-homolog)134,135 and ZASP/Cypher/ Oracle136–138 are known to interact with PKC134,136 and colocalize at the Z-disc.135,136

gp130 Signaling The first link between gp130 signaling and cardiac hypertrophy arose via studies of a novel cytokine that was capable of activating an increase in cardiomyocyte cell size via an expression cloning approach.34 A mouse embryonic stem cell cDNA library was screened for its activity to induce rat neonatal cardiomyocyte hypertrophy. This cytokine was named cardiotrophin-1 (CT-1) and was characterized as a member of the gp130 receptor cytokine family peptides.34,35 Separately, an inflammatory peptide IL-6, another gp130 cytokine, was documented to induce in vivo and in vitro cardiomyocyte hypertrophy by associating with the soluble form of the sIL-6 receptor.139 The gp130 peptide is a single transmembrane domain peptide that forms heterodimer complexes with various α subunit ligand-binding peptides. A LIF-binding subunit β (LIFR)-gp130 heterodimer was identified as CT-1 receptor on the surface of cardiomyocytes using a neutralizing antibody against LIFR.35 Dimerized gp130 cytokine receptor activates JAKs, which then induce phosphorylation of a tyrosine residue of gp130, followed by the phosphorylation of the family of STATs. Phosphorylated and active forms of STATs result in homodimerization or heterodimerization and are translocated into the nucleus, leading to target gene expression.140 Cardiac overexpression of STAT3 induces mild hypertrophy141 and also promotes cardiac hypervascularization, which might be linked to VEGF induction in the myocardium.142 Other kinase signaling cascades, including ERK1/2,143 MEK5/ERK5,144 and Akt,145 have also been linked to gp130 activation. In an in vitro cultured rat neonatal cardiomyocyte system, CT-1/LIF-treated cardiomyocytes showed unique polymorphic features of cardiomyocyte hypertrophy,146 which are distinct from Gq-coupling receptor agonists but often are observed in the myocytes isolated from the heart with eccentric hypertrophy. Accordingly, CT-1 induces longitudinal growth of myofilaments rather than lateral thickening of actin-myosin bundles. Protein and

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RNA synthesis were also enhanced, Several characteristic markers for the embryonic gene program were reactivated: myofilaments were newly formed and cell surface area was enlarged by CT-1 treatment in cultured cardiomyocytes. The molecular mechanisms that lead to this distinct assembly of sarcomeres in series is not well understood at present but may include MEK5/ERK5 signaling144; Rho/Rho kinase dependent nonsarcomeric myosin light chain phosphorylation;147 and/or formation of nascent Z-discs with segregation of α-Actinin and other associated proteins such as ALP, a Z-disc PDZ-Lim protein.148

MAP Kinase Signaling Cytoplasmic MAP kinase, MAP kinase kinase (MKK), and MKK kinase signaling cascades are activated by diverse receptor-linked membrane signaling proteins including Gq and are known to have extensive crosstalk and interaction with many of the other previously mentioned signaling cascades.17,124 This intricate signaling meshwork includes three major nodal points, including ERK1/2, JNK1/2, and p38s (Fig. 15-3). Despite effort to determine the physiologic significance and precise downstream cardiac targets that are selectively triggered by these MAPK signaling molecules, a complete understanding of their exact role in cardiac hypertrophy and failure has been elusive because of their complexity and the inherent difficulty in distinguishing between primary and secondary effects. Endogenous activation of many of these molecules induce cardiac hypertrophy and cytoprotective effects, and enhanced activation results in toxicity with apoptotic or necrotic cell death in many cases. Synthetic small molecule kinase inhibitors are often less selective, and overexpression of dominant negative forms of kinases are uncertain for their specificity for molecular compartmentalization; however, more sophisticated genetic approaches, such as conditional gene ablation, are starting to shed light on this problem. Recent studies suggest that the MEK1/2-ERK1/2 cascade might be a central player in this kinase cascade meshwork and primarily induces a concentric form of cardiac hypertrophy and cardioprotection.124 Cardiac-restricted MEK1 transgenic mice have mild concentric form of hypertrophy with enhanced left-ventricular contractility.149 A significant ERK1/2 downstream nuclear target in the cardiomyocyte may be GATA4, which interacts with other cardiac specific transcriptional factors to regulate muscle gene expression150 (see later). In contrast, cardiac p38 activation is primarily cytopathic in vivo,151 which is partly due to its direct proapoptotic effects and also partly due to reduction in myofilament calcium sensitivity.152 TAK1, a p38 upstream kinase, also promotes cardiac hypertrophy with subsequent heart failure in vivo.153 JNK signaling was originally related to cardiomyocyte hypertrophy in vitro154; however, the significance of JNK signaling in in vivo cardiac hypertrophy was less clear in subsequent studies. MEKK1, a JNK upstream regulating kinase, was genetically ablated and has been tested in two in vivo mouse systems. Under conditions in which JNK activation was blunted by MEKK1 ablation, pressure-overload-induced in vivo

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hypertrophy was not affected, although there was mild induction of cytopathic changes in the myocardium.155 On the other hand, cardiomyopathic changes in Gq transgenic mice were largely diminished by MEKK1 ablation.156 Part of these MEKK1/JNK signaling effects have been linked to JNK-dependent phosphorylation of a gapjunction molecule, connexin43.157 Interestingly, the significance of respective MAP kinase cascade activation has been studied in humans tissue, demonstrating the in vivo activation of JNK1/2 and p38. On the other hand, levels of activated ERK1/2 were found unchanged in the failing heart in these studies.60 Although these studies in human tissue are supportive, they have inherent difficulties related to individual variability, difficulty in obtaining sufficient control specimens, and hazards in extrapolating from a one-time measurement at the end stage of the disease to the pathways that trigger the initial steps of the disease-related pathways.

PATHWAYS FOR SURROGATE FAILING HEART PHENOTYPES

versus diastolic dysfunction, and outflow tract obstruction, leading to three clinical subtypes: HCM, dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM).168,169 Genotyping of human cardiomyopathy was initiated with the surprising discovery of mutations in the β-Myosin heavy chain gene in HCM cases in large pedigrees170; this was subsequently expanded to include other sarcomeric gene mutations in human HCM patients, including myosin binding protein-C,171,172 myosin light chains,173 cardiac actin,174 troponin-T,171,175 troponin-I,176 α-Tropomyosin,171,175 and titin.177 On the other hand, parallel analyses of candidate genes for DCM were more difficult, because of the relatively small pedigrees, incomplete penetrance, and phenotypic diversity. Eventually, studies in genetically engineered mice revealed an initial role for cytoskeletal defects in DCM via studies of mutant mice that are deficient in the cardiac Z-disc–associated cytoskeletal muscle-specific LIM protein (MLP)178 (Fig. 15-4). Later, a missense mutation of MLP was identified in a subset of DCM patients who reside in northern Europe.179 Different MLP mutations were also linked to familial cases of HCM.180 Extensive links between other cytoskeletal proteins and familial forms of human DCM were subsequently established.6,168,169 Based on these early findings, a generalized concept was formulated that suggested that sarcomeric mutations lead to HCM, whereas cytoskeletal mutations result in DCM, thereby reflecting specific defects in the hardwiring within cardiac muscle cells that govern these two distinct phenotypes. Thus, the phenotypic diversity of familial cardiomyopathies appeared to be primarily driven by the disease genotype. However, recent experimental and clinical studies suggest a more complex genotype-phenotype relationship of cardiomyopathies168,169 (Table 15-2). It now appears that mutations in a given sarcomeric gene can lead to a spectrum of cardiomyopathic phenotypes, often overlapping between the clinical subsets of DCM, HCM, and RCM. Although there is little doubt that the disease genotype plays a critical role in initiating the cardiomyopathic process, the ultimate clinical phenotype seems to represent the integrated effect of multiple interacting factors.This view is supported by a host of circumstantial evidence, including the poor penetrance of many cardiomyopathic genotypes; the influence of hemodynamic stress on disease progression; the secondary effects of the loss of cardiac myocyte survival and subsequent replacement fibrosis; strong modifying effects of calcium cycling and calcium signaling; and clear evidence of genetic background effects, which is also apparent in gene-targeted mouse models of cardiomyopathy.5,204

Genotype-Phenotype Relationships in Cardiomyopathy

Chamber Dilation: Diverse Cytoskeletal Pathways

The initial precepts that guided the understanding of human cardiomyopathies were based on the presumption that the primary determinant of the clinical phenotype is the molecular genotype. This clinical viewpoint is supported by noninvasive analyses, such as echocardiography, that can quantitatively assess differences in chamber volume, wall thickness, hypertrophy, systolic

Changes in organ size, shape, and histologic phenotype are central features of cardiomyopathy and related heart failure. Chronic increases in ventricular volume and filling pressure can lead to dilation of the cardiac chamber and subsequent thinning of the ventricular wall in the advancement of heart failure, which results in large increases in diastolic and systolic wall stress in both the

Transcriptional Regulation of Cardiac Genes The activation of a fetal gene program is a highly conserved feature of the hypertrophic response and has been extensively studied as a means to identify pathways that might be involved in the in vivo response. Although the initial steps of the induction of embryonic genes are clearly reversible, chronic changes in the cardiac gene program may trigger pathologic changes in the myocardium that can be associated with irreversible dysfunction. Much of this work has focused on the inducibility of ANF gene, which is reactivated in ventricular muscle early during the hypertrophic response (Fig. 15-3). A host of cardiac transcription factors, including NKX 2.5,158 a zinc-finger protein GATA4,159,160 NFATs,72 TBX5,161,162 dHAND,163 CBP/p300,163,164 MEF2,74 and another MADS box protein serum response factor (SRF),160 work together to control the expression of ANF and a panel of downstream target genes in the embryonic gene program. The recent discovery of a new series of cardiac restricted transcriptional cofactors such as Myocardin165 and HOP166,167 have suggested the combinatorial control of the cardiac gene program in response to defined upstream signals. As mentioned previously, a dual enzyme system composed of histone acetyltransferases (HATs) and HDACs is associated with this transcriptional complex and serves as an epigenetic control point of the embryonic cardiac program.76,163,164

FIGURE 15-4. Lim domain proteins and cardiomyopathy. Cardiac-enriched Lim domain proteins are categorized based on their domain structure. MLP-null mice were initially reported to display DCM phenotypes, followed by the identification of human MLP mutations linked to cardiomyopathy. Later, cardiomyopathic phenotypes were also noted in a series of experimental model animals in which Lim domain proteins are ablated.

TABLE 15-2 MOLECULAR DEFECTS LINKED TO HUMAN CARDIOMYOPATHIES DEFECTS IN HUMANS Genomic Defects

HCM

DCM

Missense171,181,182 Missense173 Missense173 Missense174 Missense/deletion171,175 Missense176 Missense171,175 Missense/deletion171,172

Missense185

Missense177

Missense/deletion188 Missense179

RCM

Sarcomere Myosin heavy chain Myosin essential light chain Myosin regulatory light chain Cardiac actin Troponin-T Troponin-I α-tropomyosin Myosin binding protein-C

Missense186 Deletion185 Missense203 Missense187

Titin/titin-related protein Titin Telethonin (T-cap) Z-disk-associated proteins Missense179

MLP Sarcolemmal cytoskeleton

Deletion189–191 Deletion/duplication192 Missense193 Missense194 Deletion195,196

Dystrophin β-Sarcoglycan δ-Sarcoglycan α-Dystrobrevin Metavinculin Intermediate filaments

Missense197,198 Missense199

Desmin Lamin A/C Miscellaneous Mitochondrial respiratory chain G4.5 (tafazzin) Phospholamban

Nonsense183/ promoter184

Missense200 Nonsense201 Missense/nonsense183–202

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right and left ventricular chambers. Based on the discovery of a cytoskeletal network of DCM-related genes in both experimental and clinical studies, distinct classes of cytoskeletal defects have been uncovered that include components of the macromolecular titin complex, Z-disc and intercalated disc proteins, components of the dystrophin/dystroglycan/sarcoglycan complex, and mutations in the nuclear and intermediate filaments (Table 15-2). These diverse cytoskeletal defects have been proposed to lead to chamber dilation via multiple potential mechanisms, such as abnormalities in force transmission,178,186 force generation,185 sarcolemmal integrity,205 sarcomeric organization/assembly defects,144 and intercalated disc stability.206 In addition to these structural roles of the cardiac cytoskeleton, selective cytoskeletal mutations and associated defects may exert their effects on heart failure via specific effects on intracellular signaling pathways triggered by biomechanical or hormonal stimuli to promote cardiac remodeling.179 MLP was demonstrated to directly bind to telethonin (T-cap), a 19-kd muscle-specific titin-interacting protein, and MLP deficiency causes T-cap dislocation from Z-disc structure. Physiologic analysis of MLP-deficient myocardium, before the gross development of DCM, has revealed a selective defect in the cardiac mechanical stress sensor, which is related to abnormalities in the titin-related complex179 (Fig. 15-5). MLP is downregulated at the protein level in the myocardium of both sporadic DCM and ischemic cardiomyopathy patients.207 In this manner, cardiac cytoskeletal defects in mechanical stress sensing might also trigger chamber dilation in acquired forms of heart failure, extending the observations obtained in rare forms of DCM to more common forms of heart failure. The recent association of cytoskeletal components with distinct cardiac channels (min K)208 and the identification of a mutation of ankyrin-B (a membrane cytoskeletal protein and an associated protein of the sarcolemmal sodium-potassium ATPase) in long QT syndrome type 4209 have further raised the possibility of divergent roles of cytoskeletal components in the direct regulation of cardiac channels involved in the control of cardiac repolarization and/or excitation–contraction coupling. An expanding number of novel musclespecific cytoskeletal components have been found localized in the cardiac Z-disc, thereby implying that the Z-disc may play a specialized function beyond its known role as a structural element210 (Fig. 15-6). At the Z-disc, αactinins are organized into a series of antiparallel dimers that crosslink polymerized actin filaments (thin filaments). The Z-disc also anchors titin, a 2- to 3-Md muscle-specific protein. Titin contains two α-actinin binding domains at its N-terminus and spans the length of each half-sarcomere, reaching to the M line in the middle of the A-band. T-cap interacts with titin at the Z-disc. As mentioned previously, a series of signaling molecules including several PKC isoforms, are known to translocate to Z-disc after activation; accordingly, Z-disc proteins with the PDZ-Lim structure including ENH134,135 and ZASP/Cypher/Oracle136–138 and the calcineurin-binding proteins Calsarcin/FATZ211–213 may serve as mechanicalto-biologic signal transducers.

FIGURE 15-5. MLP/T-cap/titin complex and the cardiac stretch sensor. At peak diastole, as ventricular filling pressure rises, titin elastic segments are reshaped, generating diastolic wall stress. The change in intrinsic elasticity functions to trigger the mechanical stress sensor. In DCM (e.g., the MLP-null mouse heart), ventricular chamber dilation and wall thinning act to enhance diastolic wall stress; the increased wall stress destabilizes the function of the titin/Z-disc complex (e.g., in the MLP-null mouse heart,T-cap, which is mechanically stabilized by MLP, gradually dislocates from Z-disc); the titin/Z-disc related mechanical stress sensor fails in translating mechanical stress to the cytoplasmic signaling cascade. The diastolic wall stress reduction appears to be critical for the prevention and rescue of heart failure progression by stabilizing titin/Z-disc complex. For example, in the MLP/phospholamban double knockout (MLP/PLN double KO) mouse heart, Z-disc abnormality, which is apparent in MLP-null myocardium, is genetically rescued.179 (Modified from Knoll R, Hoshijima M, Hoffman HM, et al: The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell 2002;111:943–955.)

Excitation-Contraction Coupling in Heart Failure Progression: A Common Pathologic Endpoint Associated with Defects in Calcium Cycling During the course of cardiac chamber dilation, a progressive decrease in cardiac contractility and relaxation ensues, which ultimately leads to chronic heart failure. The precise link between chamber dilation and cardiac dysfunction is not completely clear, because there are numerous defects that have been associated with the

MOLECULAR PATHWAYS FOR CARDIAC HYPERTROPHY AND HEART FAILURE PROGRESSION

283

FIGURE 15-6. Cardiac cytoskeletal proteins and cardiomyopathy. Representative cytoskeletal proteins constituting cardiac Z-disc and connecting myofilaments to the costamere are illustrated. (Modified from Chien KR: Genomic circuits and the integrative biology of cardiac diseases. Nature 2000;407:227–232.) *Proteins linked to human and experimental cardiomyopathies.

impairment of cardiac contractile function in heart failure. However, recent studies point to the central role of defects in calcium cycling in heart failure progression. These defects arise from a decrease in the activity of the SR calcium ATPase type 2 (SERCA2) that regulates diastolic and systolic function via the resequestration of calcium into the SR lumen.214–216 During the onset of heart failure, there is a decrease in the content of SERCA2 and its activity relative to the unrestrained inhibitory effect of phospholamban (PLN), an endogenous SERCA2 inhibitory peptide.215 In the normal heart, the inhibitory activity of PLN is under the control of the β-adrenergic receptor (β-AR)-adenylyl cyclase-cAMP dependent PKA pathway in which phosphorylation of the S16 residue of PLN leads to an inactivation of its suppressor effect on SERCA2,217, 218 (Fig. 15-7). In human and experimental models of heart failure, PLN is hypophosphorylated,219–221 which most likely reflects desensitization of the β-adrenergic pathway resulting from the increase in the activity of the β-adrenergic receptor kinase (βARK: GRK2) in the failing heart.222 Recently, a PLN mutation (R9CPLN) was found in patients with familial DCM, which blocks the PKA-dependent site-specific phosphorylation in both R9CPLN and co-oligomerized wild-type PLNs.202 Together with the experimental finding that cardiac restricted R9CPLN overexpression leads to progressive heart fail-

ure,202 SR calcium uptake suppression appears to be a critical step in the progression of heart failure. Although clinical and experimental studies document that simply enhancing cAMP during heart failure can lead to cardiac injury and an associated increase in mortality,223–225 genetic complementation studies and somatic gene transfer in heart failure model animals have suggested that it is possible to markedly improve cardiac diastolic and systolic function independent of effects on cAMP levels via directly enhancing SR calcium cycling. The clear therapeutic effects of β-adrenergic receptor blockade, even at advanced stages of heart failure, suggests that any new therapy will have to be compatible with chronic β-blocker therapy.226 Percutaneous or intramuscular delivery of an adenovirus vector (Adeno) carrying β2-AR (Adeno/β2-AR) delivery results in enhanced contractility in normal rabbit or cardiomyopathic hamsters.227,228 βAR kinase inhibitor peptide (βARKct)-transgenic overexpression partially rescues cardiomyopathic phenotypes of MLP-null mice229 and R403Q mutant α-myosin heavy chain (αMHC) transgenic mice.230 Adeno/βARKct also improves contractility and delays heart failure development in postmyocardial infarction rabbits.231,232 Adenylyl cyclase type VI (AC-type VI) transgenic overexpression rescues the heart failure phenotype in Gq cardiomyopathic mice,233 and percutaneous

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FIGURE 15-7. Regulatory pathways and key molecules in the control of SR calcium cycling. The G-protein coupling receptor (GPCR)/cAMP/PKA axis is the main regulating system and serves as a pharmacologic and genetic target at multiple steps in the cascade to improve cardiac contractility and relaxation in heart failure. (Modified from Hoshijima M, Chien KR: Mixed signals in heart failure: Cancer rules. J Clin Invest 2002;109:849–855.)

Adeno/AC-type VI delivery enhances cardiac contractility in normal pigs.234 SERCA2 transgenic mice have enhanced cardiac function,235 and SERCA2 transgenic overexpression partially rescues MLP-null cardiac dysfunction,236 whereas Adeno/SERCA2 gene delivery improves cardiac contractility and prognosis of postaortic-banded rats.237,238 PLN ablation induces a hyperkinetic phenotype in the normal mouse heart239 and dominantly complements cardiomyopathic changes in the MLP-null animals240 The genetic rescue effect of PLN ablation might be modeldependent, because it is not obvious in Gq transgenic mice (an apoptotic heart failure model) and homozygous myosin binding protein-C mutant animals (a neonatalonset progressive ventricular dilation model)241; however, molecular intervention to target PLN-SERCA2 interaction demonstrates clear beneficial effects, which are indeed chronic.44 Enhancement of isolated cardiomyocytes from endstage heart failure patients is also promoted by suppression of PLN expression.242 Diastolic leakage of calcium from SR through a SR calcium release channel (ryanodine receptor: RyR) has been linked to the PKA-dependent hyper-phosphorylation of RyR and subsequent dissociation of FKBP12.6, a stabilizing protein of RyR from the RyR/FKBP12.6 complex243; enhanced diastolic SR calcium leakage was later confirmed in FKBP12.6-null mice.244 Thus, long-term beneficial effects of β-blocker therapy may be partly attributed

to the stabilization of the RyR/FKBP12.6 complex.245,246 The evidence that human RyR mutations247–249 can lead to polymorphic ventricular tachycardia and associated sudden death250 further supports this possibility. The in vitro transfection of Adeno/FKBP12.6 in cultured cardiomyocyte increases contractility,251 suggesting that RyR stabilization may also improve cardiac contractile in the failing hearts.

Molecular Substrates for Arrhythmogenesis in Heart Failure: Acquired Defects in Conduction Lineages, Intercellular Coupling, Repolarization, and Action Potential Duration (APD) One of the major causes of mortality in heart failure is the onset of life-threatening malignant arrhythmias. The incidence of cardiac sudden death and associated cardiac rhythm abnormalities correlates with the severity of dysfunction, but the mechanism that serves as the substrate for this markedly increased susceptibility is unclear. In addition to the abnormal delayed inward depolarization current that is partly associated with diastolic SR calcium leak (see previous discussion), a growing body of electrophysiologic, genetic, experimental, and clinical data is supporting the concept that hetero-

MOLECULAR PATHWAYS FOR CARDIAC HYPERTROPHY AND HEART FAILURE PROGRESSION

geneity of APD throughout the myocardium of the failing heart may partly account for the arrhythmogenic substrate that creates susceptibility for reentrant ventricular tachyarrrhythmias.252,253 Much of this insight has been gleaned from genetically based forms of arrhythmias in clinical and animal model systems. Several distinct classes of mutations have been shown to be associated with cardiac arrhythmogenesis. Studies of long QT syndrome, an autosomal dominant disease associated with an increased incidence of cardiac sudden death, have indicated that the disease is due to mutations in channel pore-forming proteins and interacting proteins of iontransporters that can lead to prolongation of the QT interval and associated APD prolongation.209,252 A second class of arrhythmogenic defects has recently been uncovered, which are related to abnormalities in cardiac intercellular conduction that is associated with the loss of cardiac connexins.157,254,255 The third class of mutations associated with malfunctions in the conduction system and cardiac sudden death have been discovered in human patients and from studies using genetically engineered mouse models. The mutations occur because of defects in cardiac transcription factors that control muscle gene expression in both ventricular muscle and conduction system cell lineages.162,256,257 Recent studies have further implicated a glycogen-storage abnormality associated with HCM that contributes to progressive conduction defects within the conduction system itself.258–260 Because it is unlikely that somatic mutations account for most cases of cardiac sudden death associated with progressive heart failure, the generalizable mechanisms that accompany arrhythmogenesis in this acquired form

FIGURE 15-8. Ion transporter abnormalities and the substrate for malignant ventricular arrhythmia in KChIP2-null (KChIP-/-) myocardium. The absence of KChIP2 blocks the translocation of Kv4.2/4.3 pore-forming subunits of membrane potassium channels,leading to a loss of the cardiac Ito current. The transmural gradient of Ito current, which is normally high in the outer layer, is lost in the KChIP2-null myocardium,resulting in the heterogeneity of repolarization. This repolarization heterogeneity can create multiple reentry circuits with unidirectional conduction block, which provide a substrate for malignant ventricular arrhythmia. (Modified from Kuo H, Cheng CF, Clark RB, et al: A defect in the Kv Channel-interacting protein 2 (KCHIP2) gene leads to a complete loss of I (to) and confers susceptibility to ventricular tachycardia. Cell 2001;107:801–813.)

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of the disease warrant investigation. There are several conserved electrophysiologic features of human heart failure, which include elongation of APD, enhanced inward current associated with sarcolemmal sodiumcalcium exchanger activity, and the loss of a transmural gradient of phase 1 Ito potassium current.214 The mechanisms that contribute to the loss of the Ito gradient are unclear, and the relative importance of changes in this specific current versus the multitude of other changes in the electrophysiologic phenotype of the failing heart is unknown. Recent studies have clearly identified that the loss of Ito can independently increase the dispersion of repolarization and confer marked susceptibility to lethal forms of ventricular tachycardia.261 These observations are based on the discovery of a cytoplasmic accessory protein (KChIP2) that binds to the Kv4.2 and 4.3 channel proteins and that is markedly downregulated early during pressure-overload hypertrophy.261 This negative regulation mimics the reactivation of the fetal gene program, because the KChIP2 gene is not expressed in the fetal heart until the late embryonic stage and is postnatally activated in the adult heart. Interestingly, KChIP2 quantitatively regulates the Ito current, because heterozygotes for the mutant allele display a precise 50% decrease in current density. Mice that lack KChIP2 can display a loss of the normal Ito current gradient across the ventricular wall and sustained polymorphic ventricular tachycardia with a single, extra stimulus in programmed stimulation studies261 (Fig. 15-8). Because human and canine hearts express the KChIP2 gene in a gradient (epicardium greater than endocardium) that matches with the distribution of Ito current (the Kv4.2 and 4.3 genes are uniformly expressed throughout the

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ventricular wall262,263), it is likely that the KChIP2 gene quantitatively regulates the Ito current in the human heart. Therefore, it will be interesting to determine whether there is a downregulation of KChIP2 gene expression in the failing human heart that might account for the loss of the Ito gradient in human heart failure. If such is the case, then defects in KChIP2 may represent a new class of genetic modifiers that can confer risk for both inheritable and acquired forms of ventricular arrhythmias. Because the expression of KChIP2 is downregulated during cardiac remodeling,261 strategies to maintain Ito by promoting the level of KChIP2 expression in the failing heart may represent a new therapeutic approach for combating the prevalent lethal arrhythmias associated with heart failure. The roles of other channel accessory proteins that may be important in the regulation of other cardiac currents and how they confer susceptibility to arrhythmogenesis in the setting of heart failure should be investigated.

TOWARD NEW THERAPEUTIC PATHWAYS Genomic databases, which provide a universal language that can be used to identify the conserved functions of cardiovascular genes in multiple species and organ systems, are ushering in a new era of integrative biology.6 For example, numerous parallels exist between cardiac muscle and neuromuscular disorders (e.g., excitability, conduction, terminal differentiation, and innervation). Unsuspected connections between neural and cardiac developmental gene programs have been uncovered in gene-targeted mice, and conserved myocyte and neuronal cell survival factors have been discovered, which suggest the existence of other factors. Thus, major opportunities for understanding cardiac diseases may lie in working at the interface of cardiovascular science and neuroscience. To evaluate the real clinical potential of disease pathways found in mouse models, parallel studies in larger animals will be necessary. The difficulty of germ-line gene manipulation in larger animals suggests the value of new strategies using peptide inhibitors, neutralizing antibodies, and somatic gene transfer with smart vectors that allow long-term expression. The many quantitative end points for complex in vivo cardiac phenotypes should empower the next frontier that lies at the boundaries of genomic databases, physiology, and human disease. We are entering a renaissance of integrative biology of the intact organism, and the heart may again play a key role in defining the circuitry for complex in vivo physiologic traits.

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EDITOR’S CHOICE Intravenous nesiritide vs nitroglycerin for treatment of decompensated congestive heart failure: A randomized controlled trial. Jama 2002;287:1531–1540. BNP finds a home as a therapy for acute, decompensated heart failure. Chien KR, Ross J Jr, Hoshijima M: Calcium and heart failure:The cycle game. Nat Med 2003;9:508–509. Promoting calcium cycling via phospholamban inhibition is new therapeutic target to halt heart failure progression. del Monte F, Hajjar RJ:Targeting calcium cycling proteins in heart failure through gene transfer. J Physiol 2003;546:49–61. Overview of gene therapy strategies for heart failure via promting calcium cycling.

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Haghighi K, Kolokathis F, Pater L, et al: Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human. J Clin Invest 2003;111:869–876. Human mutation results in a truncated phospholamban peptide and is associated with genetically base dilated cardiomyopathy, contains cytoplasmic inhibitory domain but also is unstable, may represent a mixed genetic effect of poison peptide and hypomorphic allele. Hajjar RJ, MacRae CA: Adrenergic-receptor polymorphisms and heart failure. N Engl J Med 2002;347:1196–1199. Nice review of the association of human variants of the beta-adrenergic receptor and heart failure. MacLennan DH, Kranias EG: Phospholamban: A crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol 2003;4:566–577. Excellent review of emerging topic of clinical importance. Maisel AS, Krishnaswamy P, Nowak RM, et al: Rapid measurement of Btype natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med 2002;347:161–167. BNP as a biomarker for heart failure; rapidly finding a place as standard diagnostic test in the ER and elsewhere. Olson EN, Schneider MD: Sizing up the heart: Development redux in disease. Genes Dev 2003;17:1937–1956. Thought leaders provide excellent overview of complex field. Prasad SV, Perrino C, Rockman HA: Role of phosphoinositide 3-kinase in cardiac function and heart failure. Trends Cardiovasc Med 2003;13:206–212. Pl-3 pathways may represent a new signaling pathway to regulate cardiac contractility.

Schmitt JP, Kamisago M, Asahi M, et al: Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban. Science 2003;299:1410–1413. Seminal paper provides first clear genetic evidence that the augmentation of phospholamban inhibitory activity is sufficient by itself to cause human dilated cardiomyopathy and heart failure; human validation of earlier studies in mice that suggested that phospholamban is a valid therapeutic target for heart failure. Small KM, Wagoner LE, Levin AM, et al: Synergistic polymorphisms of beta1-and alpha2C-adrenergic receptors and the risk of congestive heart failure. N Engl J Med 2002;347:1135–1142. Genetic susceptibility to heart failure based on variants of adrenergic receptors. Wehrens XH, Marks AR: Myocardial disease in failing hearts: Defective excitation-contraction coupling. Cold Spring Harb Symp Quant Biol 2002;67:533–541. Leader in the molecular biology of ryanodine receptors provides nice overview of large body of work. Wencker D, Chandra M, Nguyen K, et al:A mechanistic role for cardiac myocyte apoptosis in heart failure. J Clin Invest 2003;111:1497–1504. Makes the case for a pivotal role of apoptosis in heart failure progression. Yussman MG, Toyokawa T, Odley A, et al: Mitochondrial death protein Nix is induced in cardiac hypertrophy and triggers apoptotic cardiomyopathy. Nat Med 2002;8:725–730. New pathway for cell death in heart muscle cells could play important role in heart failure progression during chronic stimulation of receptors linked to Gq pathways.