Portal de Periódicos da CAPES

Converging Pathways and Principles in Heart Development and Disease

2002; Cell Press; Volume: 110; Issue: 2 Linguagem: Inglês

10.1016/s0092-8674(02)00834-6

1097-4172

Kenneth R. Chien, Eric N. Olson,

Congenital Heart Disease Studies

"One country, two systems." —Deng Xiaoping, referring to the reunification of Hong Kong and China. New York Times, January 2, 1985 As noted by a former Chairman, the term "one country-two systems" was initially coined to describe a new experimental paradigm for normalized relations between Hong Kong and China, two territories that had long been united, but had evolved along separate paths during the Twentieth Century. Over the past 50 years, studies of the cardiovascular system have also evolved along distinct pathways, largely based upon the territorial boundaries of modern developmental biology and clinical physiology. The developmental viewpoint has been underpinned by genetics, and a reductionist approach toward mechanism, with molecular biology as its primary tool, and the smallest biological unit being individual cell types. The clinical cardiology viewpoint has been based on physiology, and an integrative approach toward mechanism, with interventional/device/imaging technologies as the primary tools, and the smallest biological unit being the intact organ. In short, studies of the cardiovascular system have traditionally been approached by citizens of "two" very different scientific cultures and "countries," each with their own societies, uniforms, icons, and customs. If the recent 67th Quantitative Symposium of Cold Spring Harbor Laboratory on "The Cardiovascular System" is any guide, this era of "one system-two countries" is coming to a close. During this symposium, converging pathways and perspectives of cardiovascular development and physiology were presented, driven by post-genomic tools and technology, a new generation of scientists and physicians trained to work at the interface of developmental biology, genetics, physiology, and human disease, and an integrative approach to biology. This review highlights some of the central themes presented by delegates of both "countries" that provided new insights into developmental principles of human cardiovascular physiology and disease. Zebrafish genetics is beginning to uncover genes and pathways for cardiac development at a breathtaking pace, forging unsuspected connections with human cardiovascular physiology and disease. The translucent nature of the organism has facilitated large scale mutagenesis screens for diverse cardiovascular phenotypes (for a review, see Shin and Fishman 2002Shin J.T Fishman M.C From Zebrafish to Humans Modular Medical Models.Annu. Rev. Genom. Genet. 2002; in pressGoogle Scholar), while the density of the zebrafish genetic map and ongoing genome project have led to an ability to clone the corresponding genes within a relatively short time span (3–6 months). The system appears to be particularly valuable for identifying new genes and pathways in vasculogenesis. gridlock, a gene that encodes a member of the Hairy/Enhancer of Split family of related bHLH proteins, acts as a molecular switch for the distinct identity of arteries and veins by repressing the venous cell fate in pre-angioblasts, which is thereby permissive for artery formation. The zebrafish mutant phenotype resembles aspects of human aortic coarctation (Zhong et al. 2001Zhong T.P Childs S Leu J.P Fishman M.C Gridlock signalling pathway fashions the first embryonic artery.Nature. 2001; 414: 216-220Crossref PubMed Scopus (438) Google Scholar). The ability of gridlock to govern the decision between arterial and venous cell fates is a reflection of its activity as a transcriptional target of the Notch signaling pathway, long known to dictate mutually exclusive cell fates in diverse cell types. Surprisingly, knockout mice for the gridlock ortholog Hey2 do not show aortic coarctation, but frequently succumb to severe neonatal cardiomyopathy. A combined loss of Hey1 and Hey2, however, leads to vascular defects similar to gridlock, suggesting that these genes have undergone partial sub- or neofunctionalization during vertebrate evolution (M. Gessler, University of Wuerzburg). Two new genes that lie in an identical pathway for the endothelial-myocardial driven cues for valvulogenesis (Walsh and Stainier 2001Walsh E.C Stainier D.Y UDP-glucose dehydrogenase required for cardiac valve formation in zebrafish.Science. 2001; 293: 1670-1673Crossref Scopus (231) Google Scholar) (D. Stainier, UCSF) were presented, as well as a new transgenic strain harboring a fli-1 GFP construct that allows in vivo visualization of the entire vasculature. This strain should facilitate subsequent modifier screens utilizing existing mutants with interesting vascular phenotypes (Weinstein, NIH). In this regard, over 700 mutants that affect discrete steps of zebrafish vessel formation and/or maturation have been isolated (H. Habeck, Exelixis, Germany). Mark Fishman (MGH) presented an elegant case for the zebrafish as a system to unravel the "logic" of disease pathogenesis. Toward this end, a compelling argument can be made that a parallel effort on physiological characterization of this model organism is now warranted to allow direct connections between novel genes and clinically relevant functional endpoints. Clearly, studies in zebrafish are demonstrating that the border between developmental biology and disease is dissolving as shared pathways for prenatal morphogenic steps and postnatal physiological events continue to be uncovered. In 1994, the first report of therapeutic angiogenesis based on the single administration of one angiogenic factor, VEGF, appeared (Takeshita et al. 1994Takeshita S Zheng L.P Brogi E Kearney M Pu L.Q Bunting S Ferrara N Symes J.F Isner J.M Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model.J. Clin. Invest. 1994; 93: 662-670Crossref PubMed Scopus (969) Google Scholar). This result pointed to the promise of therapeutic angiogenesis as a new molecular therapy for chronic coronary and peripheral arterial disease. Although therapeutic angiogenesis continues to be a laudable goal, its experimental validation remains an open question, and the clinical efficacy of therapeutic angiogenesis remains unclear. If the recent papers presented at CSH are any measure, it appears as if early studies examining the role of single angiogenic factors as therapeutic strategies may have severely underestimated the biological complexity of vasculogenesis. One of the obligatory triggers for angiogenesis is VEGF, a factor that has been shown to be both necessary and sufficient for angiogenesis in vivo. However, recent studies of a host of additional angiogenic factors, including angiopoietins-1 and -2 (Yancopoulos et al. 2000Yancopoulos G.D Davis S Gale N.W Rudge J.S Wiegand S.J Holash J Vascular-specific growth factors and blood vessel formation.Nature. 2000; 407: 242-248Crossref PubMed Scopus (3154) Google Scholar) (Regeneron), suggest that additional components may be required to form fully mature vessels that contain pericytes, smooth muscle cells, and distinct endothelial subtypes. Further, VEGF can specifically induce expression of the synergistic factor angiopoietin-2 in muscle cells by an intracellular pathway, but not when administered exogenously. This suggests that VEGF gene and protein delivery might elicit different biological effects as therapeutic angiogenesis strategies (H. Blau, Stanford). Intriguingly, it now appears that there may be organ-specific angiogenic programs that are based on tissue-specific angiogenic factors, exemplified by EG-VEGF, an angiogenic factor that is expressed in steroidogenic glands such as ovary, adrenal, and testes, and acts selectively on endothelial cells of such endocrine organs through a distinct class of G-protein-coupled receptor pathways (N. Ferrara, Genentech) (LeCouter et al. 2001LeCouter J Kowalski J Foster J Hass P Zhang Z Dillard-Telm L Frantz G Rangell L DeGuzman L Keller G.A et al.Identification of an angiogenic mitogen selective for endocrine gland endothelium.Nature. 2001; 412: 877-884Crossref PubMed Scopus (452) Google Scholar). Accordingly, it may be over-simplistic to expect that the administration of a single gene would be sufficient to promote therapeutic coronary or peripheral arteriogenesis, without first identifying the unique combination of core and tissue-specific angiogenic programs required in the target organ of interest. This may be particularly important for therapeutic coronary arteriogenesis, as the formation of the coronary arterial system in the fetal heart is now known to require concerted developmental cues derived from four discrete lineages: endothelial, myocardial, pro-epicardial, and neural crest lineages (Figure 1). Growing muscular coronary arteries that are built to withstand the intrinsic biomechanical stress of each heartbeat may first require a better definition of the developmental cues and secreted factors required for normal coronary arteriogenesis. The ability to isolate primed coronary arterial progenitor cells could represent a valid alternative strategy that could complement existing approaches, particularly if these can be effectively isolated from adult stem cell populations, and the factors that are required for their recruitment and self-renewal identified. Direct validation of anti-angiogenic strategies as independent chemotherapeutic regimens for broad classes of human cancers has also been elusive. As judged by the recent data presented at CSH, there not only appear to be distinct molecular pathways for vasculogenesis in specific organ systems, but the existence of multiple, parallel pathways for tumor angiogenesis (R. Hynes, MIT; R. Benezra, Sloan-Kettering), supporting the view that the elimination of a single angiogenic factor may not independently lead to the loss of solid tissue tumors. Recent studies of spontaneous tumors suggest that tumors can escape the blockade in single pathways in part via selection for alternative pathways (Benezra 2001Benezra R Role of Id proteins in embryonic and tumor angiogenesis.Trends Cardiovasc. Med. 2001; 11: 237-241Abstract Full Text Full Text PDF Scopus (49) Google Scholar). It may become possible to tailor the anti-angiogenic strategy for specific tumor types based upon tissue and tumor-specific angiogenic pathways, as well as the synergistic effects of current chemotherapeutic regimens. In this regard, kinase inhibitor treatment of a genetically engineered mouse model of pancreatic islet cancer implicated PDGF receptor signaling in tumor vessel-associated pericytes as crucial for maintenance and continuing angiogenesis of tumor vasculature (Bergers, UCSF). Additional results from that model demonstrated the benefits of combinatorial anti-angiogenic therapies utilizing protease inhibitors and low dose, "metronomic" chemotherapy (Bergers). The clinical link between neural crest lineages and cardiac development was established by the characterization of children harboring a characteristic subset of congenital heart defects that were invariably accompanied by neural-crest-related defects. These children harbor microdeletions in chromosome 22 and a minimal region has been established by careful genotype-phenotype correlations in large global patient populations. Attempts to refine the disease interval and to identify the specific gene(s) responsible for the defects have been elusive at the clinical level, but recent studies in mice have shown that a single gene, TBX 1, is likely to be the most important gene within this region to account for a subset of the neural crest-related cardiac congenital defects (Lindsay et al. 2001Lindsay E.A Vitelli F Su H Morishima M Huynh T Pramparo T Jurecic V Ogunrinu G Sutherland H.F Scambler P.J et al.Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice.Nature. 2001; 410: 97-101Crossref PubMed Scopus (764) Google Scholar, Merscher et al. 2001Merscher S Funke B Epstein J.A Heyer J Puech A Lu M.M Xavier R.J Demay M.B Russell R.G Factor S et al.TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome.Cell. 2001; 104: 619-629Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar, Jerome and Papaioannou 2001Jerome L.A Papaioannou V.E DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1.Nat. Genet. 2001; 27: 286-291Crossref PubMed Scopus (802) Google Scholar). However, to date, no patients with TBX1 mutations have been found that display the full spectrum of cardiac neural crest defects, suggesting that other genes in this minimal region may contribute to the human phenotype. In this regard, TBX1-deficient mice only display a subset of the neural crest defects found in DiGeorge children (aortic arch anomalies), again providing support for DiGeorge as a contiguous gene syndrome. Given the number of genes in the minimal critical region of chromosome 22, and the existence of non-overlapping deletions that result in the same phenotype, it is likely that positional effects and long-range chromatin interactions contribute to the difficulty in pin-pointing a single gene as the culprit in all aspects of this disease. The question arises as to whether the requirement for TBX1 actually is localized within neural crest lineages per se, or whether it reflects non-cell-autonomous effects. Surprisingly, a growing body of evidence now supports the concept that the TBX1 requirement is located outside of neural crest lineages. Thus, the cardiac morphogenic defects associated with DiGeorge syndrome do not reflect a primary effect on neural crest formation, proliferation, or survival, but rather result from the loss of guidance cues from the pharyngeal endoderm that are required for neural crest migration into the aortic arch region (A. Baldini, Baylor). This view has been independently supported by studies of the regulatory programs that control TBX1 expression in the mouse (D. Srivastava, UTSW). Fgf8 may mediate the Tbx1 non-cell-autonomous effects on neural crest cells, as heterozygous deletion of the Fgf8 gene enhances the Tbx1 haploinsufficiency phenotype in mice (Vitelli et al. 2002Vitelli F Taddei I Morishima M Meyers E Lindsay E.A Baldini A A genetic link between Tbx1 and Fibroblast Growth Factor signaling.Development. 2002; in pressGoogle Scholar), a point supported by two other independent laboratories (Frank et al. 2002Frank D Fotheringham L.K Brewer J.A Muglia L.J Tristani-Firouzi M Capecchi M.R Moon A.M An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome.Development. 2002; in pressGoogle Scholar, Abu-Issa et al. 2002Abu-Issa R Smyth G Smoak I Yamamura K.-I Meyers E Fgf8 is Required For Pharyngeal Arch and Cardiovascular Development in the Mouse.Development. 2002; in pressGoogle Scholar). This secondary TBX1 effect on neural crest migration might imply that an additional "hit" within neural crest lineages acts as a modifier to enhance the phenotype, which might reflect actions of other genes within or adjacent to the DiGeorge minimal region. Interestingly, 90% of individuals with neural crest-related defects display hemizygous deletions of CDC 45, a cell cycle regulator which falls within the DiGeorge minimal region, although mice that are heterozygous for a CDC 45 null allele do not display cardiac defects (Yoshida et al. 2001Yoshida K Kuo F George E.L Sharpe A.H Dutta A Requirement of CDC45 for postimplantation mouse development.Mol. Cell. Biol. 2001; 21: 4598-4603Crossref Scopus (28) Google Scholar). Accordingly, it will be of interest to generate compound mutant mice which harbor a complete deficiency of TBX1 and partial deficiencies in other contiguous genes, such as CDC 45, that lie within the DiGeorge minimal region. Such an approach has the potential to identify key modifiers of cardiac congenital heart defects. Hypertension is a complex trait that is influenced by multiple organs, including the brain, heart, kidney, adrenals, and vascular endothelium. Recent studies of rare forms of familial hypertension are beginning to reveal the first genetic pathways for rare familial forms of human hypertension. By collecting families from around the globe which have severe, early onset hypertension, mutations in a number of new human hypertensive genes including those encoding the epithelial sodium channel (ENAC), the mineralocorticoid receptor, and a renal WNK kinase have been identified (Lifton et al. 2001Lifton R.P Gharavi A.G Geller D.S Molecular mechanisms of human hypertension.Cell. 2001; 104: 545-556Abstract Full Text Full Text PDF PubMed Scopus (1306) Google Scholar). Remarkably, in addition to these hypertensive genes, another set of human mutations has been found that lowers blood pressure. All of these genes have their primary effect in renal cell lineages, pointing to the central role of the kidney in the onset of human hypertension. As noted by Rick Lifton (Yale), while it is clear that blood pressure can be controlled by a number of different agents that work outside of these renal pathways, the question arises as to whether the design of specific antagonists to these specific renal targets might have long-term beneficial effects that go beyond effects on the control of blood pressure per se, but extend to chronic effects of hypertension on end-organ diseases of the heart, brain, or the kidney itself. The recent unsuspected major therapeutic benefit of mineralocorticoid antagonists in heart failure raises interesting questions about the potential role of this nuclear hormone receptor in heart muscle cells. Accordingly, it will be interesting to cross mice that harbor cardiac-restricted mutations of this gene into well-characterized mouse models of cardiomyopathy and heart failure. In short, the discovery of these new pathways responsible for rare forms of human hypertension is beginning to provide novel insights and therapeutic targets for common forms of the disease. The identification of a number of quantitative trait loci (QTLs) in spontaneous in-bred hypertensive rat strains (Jacob and Kwitek 2002Jacob H.J Kwitek A.E Rat genetics attaching physiology and pharmacology to the genome.Nat. Rev. Genet. 2002; 3: 33-42Crossref Scopus (208) Google Scholar), coupled with the ongoing advances in the rat genome project, suggest that additional insight into genetic pathways for the control of hypertension will be forthcoming from experimental model systems, which should allow a direct comparison in studies of more common forms of hypertension in human populations. Since the discovery of master regulators of skeletal muscle development, such as MyoD and myogenin, there has been a push to identify corresponding regulators of cardiac cell fate. Such factors would have obvious potential in converting non-cardiac cells to the cardiac lineage as an approach for repair and regeneration of the damaged adult myocardium. However, it appears that forming a cardiac muscle cell is not so simple. While several cardiac-restricted transcription factors have been identified, none have been found to possess the ability to confer cardiac identity on their own. However, combinations of factors such as the zinc finger protein GATA4, the homodomain proteins Nkx2.5 and Tbx5 and the MADS box protein serum response factor (SRF) are able to activate some cardiac genes in transfected cells. Tim Mohun (NIMR, UK) reported that ectopic expression of GATA4 in injected frog animal caps was capable of inducing the formation of beating cardiomyocytes. Similar findings have been made with GATA5 in zebrafish (D. Stainier, UCSF) (Reiter et al. 1999Reiter J.F Alexander J Rodaway A Yelon D Patient R Holder N Stainier D.Y Gata5 is required for the development of the heart and endoderm in zebrafish.Genes Dev. 1999; 13: 2983-2995Crossref PubMed Scopus (337) Google Scholar). Since these GATA factors are not restricted to the heart, and alone cannot activate endogenous cardiac genes in transfection assays, they must act in conjunction with other transcription factors and signaling systems in the context of the frog or fish embryo to initiate cardiogenesis in a subset of cells. The concept that specific sets of extracellular signals must be interpreted in a cell-specific manner to generate the cardiac phenotype is consistent with studies in Drosophila, in which overlapping gradients of cardiogenic signals act in conjunction with cell autonomous transcription factors to specify the identity of cardiac cells (M. Frasch, Mt. Sinai). SRF is required for the opposing processes of cell proliferation and myogenesis. Myocardin, a novel cardiac-restricted transcription factor, has recently been shown to stimulate transcription of SRF-dependent muscle genes through its association with SRF (Wang et al. 2001Wang D Chang P.S Wang Z Sutherland L Richardson J.A Small E Krieg P.A Olson E.N Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor.Cell. 2001; 105: 851-862Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). Injection of myocardin mRNA into frog embryos was reported to be sufficient to induce ectopic cardiac gene expression (E. Olson, UTSW, and P. Krieg, University of Arizona). Conversely, dominant negative mutants of myocardin can prevent heart formation in frog embryos. It will be of interest to identify additional cofactors for myocardin and to investigate its potential roles in cardiac gene expression during later stages of development and disease. In addition, the growing database of putative cardiac transcriptional regulators suggests new insights in the combinatorial pathways that control the gene program may be on the horizon (J. Epstein, University of Pennsylvania, and E. Olson, UTSW). A zebrafish mutant, liebeskummer, with an excess of cardiomyocytes was also reported (M. Fishman, MGH). The mutation was shown to activate reptin, an ATP-dependent helicase that acts through an as yet unknown mechanism to control growth of the ventricles. Ventricular growth in the embryo is dependent on signals from the overlying epicardium. Andrew Lassar (Harvard) presented evidence that epicardial signaling requires Epo and retinoic acid, which act through parallel pathways to stimulate myocardial growth by inducing a cardiac myocyte mitogen in the epicardial cells. Neuregulin signaling from the endocardium to the myocardium was also shown by Richard Harvey (UNSW, AU) to play a critical role in embryonic growth and trabeculation of the ventricular chambers. The adult myocardium responds to extrinsic forms of stress such as hypertension, myocardial infarction, and pressure-overload by a hypertrophic growth response (for a review, see Chien 1999Chien K.R Stress pathways and heart failure.Cell. 1999; 98: 555-558Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Inherited mutations in components of the sarcomere and cytoskeleton also result in hypertrophic cardiomyopathy. Hypertrophy in response to pathologic stimuli is accompanied by activation of a fetal gene program, which results in maladaptive changes in contractility and calcium handling (Figure 2). Growth of the heart during normal postnatal development and in response to exercise also occurs through hypertrophy. A key issue in the field is to decipher the pathways that control pathologic and physiologic hypertrophy, such that the former might be inhibited and the latter augmented pharmacologically. Changes in intracellular calcium handling have been implicated as a trigger for cardiac hypertrophy, and numerous calcium-dependent and calcium-independent (Gq, RAS, PI3K, p38) (for review, see Hoshijima and Chien 2002Hoshijima M Chien K.R Mixed signals in heart failure cancer rules.J. Clin. Invest. 2002; 109: 849-855Crossref PubMed Scopus (139) Google Scholar) signaling systems have been shown to be necessary and sufficient to drive multiple features of cardiac growth. The notion that aberrant calcium handling plays a central role in the hypertrophic program has been supported by several observations: (1) hypertrophic cardiomyocytes exhibit alterations in calcium sensitivity and handling, some of which can be corrected by enhanced activity of the SR Ca-ATPase through inhibition of its inhibitor phospholamban (Minamisawa et al. 1999Minamisawa S Hoshijima M Chu G Ward C.A Frank K Gu Y Martone M.E Wang Y Ross Jr., J Kranias E.G et al.Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy.Cell. 1999; 99: 313-322Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, Hoshijima et al. 2002Hoshijima M Ikeda Y Iwanaga Y Minimisawa S Li X Yusu G Wang L Wilson J Wang Y Ross J.J Chien K.R Chronic Inhibition of Heart Failure Progression by a pseudophosphorylated Mutant of Phospholamban via Cardiac rAAV Gene Delivery.Nat. Med. 2002; 8: 864-871Crossref PubMed Scopus (307) Google Scholar); (2) numerous calcium-sensitive signaling pathways are activated in hypertrophic cardiomyocytes (Molkentin et al. 1998Molkentin J.D Lu J.R Antos C.L Markham B Richardson J Robbins J Grant S.R Olson E.N A calcineurin-dependent transcriptional pathway for cardiac hypertrophy.Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2138) Google Scholar) (for a review, see Frey et al. 2000Frey N McKinsey T.A Olson E.N Decoding calcium signals involved in cardiac growth and function.Nat. Med. 2000; 6: 1221-1227Crossref PubMed Scopus (279) Google Scholar); (3) forced activation of calcium-sensitive signaling pathways is sufficient to induce myocyte hypertrophy in vivo and in vitro (for review, see Leinwand 2001Leinwand L.A Calcineurin inhibition and cardiac hypertrophy a matter of balance.Proc. Natl. Acad. Sci. USA. 2001; 98: 2947-2949Crossref Scopus (42) Google Scholar). Thus, it is reasonable to assume that calcium signaling plays a key role in many forms of hypertrophy, but several other calcium-independent pathways are also likely to mediate key aspects of the hypertrophic response, as well (for a review, see Sugden 2001Sugden P.H Signalling pathways in cardiac myocyte hypertrophy.Ann. Med. 2001; 33: 611-622Google Scholar). The involvement of calcium in hypertrophic signaling raises many obvious questions. For example, where does the calcium come from? Given the extreme fluctuations in intracellular calcium levels that accompany each cycle of contraction and relaxation, is there a specific pool that controls hypertrophy? If so, how is it compartmentalized or insulated from other calcium in the sarcoplasm? While calcineurin, CaMK, and MAPK have each been shown to be sufficient, and in some cases necessary, for hypertrophy, each of these enzymes is activated by different concentrations and waveforms of calcium. Do each of these calcium-sensitive effectors get activated by different hypertrophic signals? How are hypertrophic signaling pathways interconnected, and how do they intersect with the calcium cycling machinery? Mice engineered to express a mutant form of α-MHC with a codon 403 mutation that mimics a common human mutation develop hypertrophic cardiomyopathy and exhibit abnormal calcium homeostasis, such that the mutant sarcomere requires a greater amount of calcium than the wild-type sarcomere to generate the same level of force (Fatkin et al. 2000Fatkin D McConnell B.K Mudd J.O Semsarian C Moskowitz I.G Schoen F.J Giewat M Seidman C.E Seidman J.G An abnormal Ca(2+) response in mutant sarcomere protein-mediated familial hypertrophic cardiomyopathy.J. Clin. Invest. 2000; 106: 1351-1359Crossref PubMed Scopus (171) Google Scholar). SR calcium levels are also reduced in these mutants, suggesting that the mutant sarcomere sequesters a higher level of calcium than wild-type, with resulting perturbation in intracellular calcium homeostasis. This notion is supported by the finding that the L-type calcium channel blocker diltiazem restores SR calcium levels and normal contractility and prevents hypertrophy in the mutant heart. Jon Seidman (Harvard) presented evidence suggesting that sarcomere mutations may activate different hypertrophic signaling pathways from pressure overload. However, the molecular mechanisms whereby sarcomere mutations result in hypertrophic cardiomyopathy remain unknown. Another major question in the field is to determine how hypertrophic signals are transmitted to the nucleus, with resulting reprogramming of cardiac gene expression. The MEF2 transcription factor appears to be a critical target for hypertrophic signals, as revealed with a transgenic mouse line that harbors a lacZ reporter under control of MEF2 binding sites (Naya et al. 1999Naya F.J Wu C Richardson J.A Overbeek P Olson E.N Transcriptional activity of MEF2 during mouse embryogenesis monitored with a MEF2-dependent transgene.Development. 1999; 126: 2045-2052Crossref Google Scholar). Using this mouse, Olson and coworkers showed that the transcriptional activity of MEF2 is activated through a post-translational mechanism in response to hypertrophic signals. Activation of MEF2 appears to be mediated by the signal-dependent dissociation of class II histone deacetylases (HDACs), which are normally tethered to MEF2, resulting in repression of MEF2 target genes (McKinsey et al. 2000McKinsey T.A Zhang C.L Lu J Olson E.N Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation.Nature. 2000; 408: 106-111Crossref PubMed Scopus (829) Google Scholar). Stress signals lead to the phosphorylation of HDACs by an as yet unidentified kinase. Consistent with this model, signal-resistant mutants of HDACs act as irreversible repressors of hypertrophy, and HDAC knockout mice are sensitized to hypertrophic signals. By studying the effects of exercise on expression of the MEF2-lacZ transgene, Leslie Leinwand (University of Colorado) found that physiologic signals do not stimulate MEF2 activity in the heart. Intriguingly, cardiac MEF2 activity is dramatically induced in male, but not fem