G-Proteins in Growth and Apoptosis: Lessons from the Heart
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Oncogene (2001) 20, 1626 ± 1634 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc G-proteins in growth and apoptosis: lessons from the heart John W Adams1,2 and Joan Heller Brown*,1 1University of California, San Diego, Department of Pharmacology, 9500 Gilman Drive, 0636, La Jolla, CA, California 92093- 0636, USA The acute contractile function of the heart is controlled by for proliferation of adult cardiomyocytes. In light of the eects of released nonepinephrine (NE) on cardiac these considerations, it is not immediately obvious that adrenergic receptors. NE can also act in a more chronic cardiomyocyte growth and cardiomyocyte death would fashion to induce cardiomyocyte growth, characterized by be responses critical to the normal function of the heart. cell enlargement (hypertrophy), increased protein synth- In fact, the ability of cardiomyocytes to undergo esis, alterations in gene expression and addition of hypertrophic growth, which includes an increase in cell sarcomeres. These responses enhance cardiomyocyte size, is an important adaptive response to a wide range of contractile function and thus allow the heart to conditions that require the heart to work more compensate for increased stress. The hypertrophic eects eectively. As described below, adaptive or compensa- of NE are mediated through Gq-coupled a1-adrenergic tory cardiomyocyte hypertrophy appears to be regulated receptors and are mimicked by the actions of other in large part through stimulation of G-protein coupled neurohormones (endothelin, prostaglandin F2a angiotensin receptors (GPCRs). Often, the ability of cardiomyocytes II) that also act on Gq-coupled receptors. Activation of to function at high capacity under increased workload phospholipase C by Gq is necessary for these responses, cannot be sustained and the heart transitions into a and protein kinase C and MAP kinases have also been condition in which ventricular failure develops. One implicated. Gq stimulated cardiac hypertrophy is also event associated with and suspected to be causally evident in transgenic mouse models. In contrast, stimula- related to this transition to heart failure is the apoptotic tion of Gs-coupled b-adrenergic receptors or Gi-coupled death of cardiomyocytes. An intriguing hypothesis is receptors do not directly eect cardiomyocyte hypertro- that prolonged or intensi®ed activation of GPCRs may phy. Apoptosis is also induced by G-protein-coupled be a critical factor tipping the balance between pathways receptor stimulation in cardiomyocytes. Sustained or promoting hypertrophic growth and those leading to excessive activation of either Gq- or Gs-signaling path- apoptotic death of cardiomyocytes. ways results in apoptotic loss of cardiomyocytes both in vitro and in vivo. Apoptosis is associated with decreased ventricular function in the failing heart. Cardiomyocytes In vitro and in vivo models of cardiomyocyte growth provide an ideal model system for understanding the basis for G-protein mediated hypertrophy and apoptosis, and There are no well-established continuous cell lines that the mechanisms responsible for the transition from can be used to study cardiomyocyte development and compensatory to deleterious levels of signaling. This growth. A line of atrial cells (AT-1), immortalized by information may prove critical for designing interventions expression of SV-40 large T-antigen (Delcarpio et al., that prevent the pathophysiological consequences of heart 1991) has been utilized in electrophysiolgical studies failure. Oncogene (2001) 20, 1626 ± 1634. and the HL-1 cardiac muscle cell line has been derived from them (Claycomb et al., 1998). The availability of Keywords: cardiac; Gq; heart failure; hypertrophy; these cells is limited, however, and conditions for cardiomyocyte maintaining their dierentiated properties are stringent. Some investigators have studied embryonic stem cells dierentiated into cardiomyocytes (Minamino et al., Introduction 1999) but this preparation is not routinely useful. Myocytes isolated from the ventricle of adult rat, Cardiomyocytes comprise 70 ± 80% of the mass of the rabbit, and dog heart have been extensively examined adult heart. These cells are terminally dierentiated and to delineate the acute signaling pathways by which precisely designed to rapidly alter their ionic and GPCRs control cell excitability, calcium and excita- contractile function in response to adrenergic (sympa- tion-contraction coupling. However, adult heart cells thetic) and cholinergic (parasympathetic) input. Cardiac are generally dicult to maintain in cell culture and tumors are extremely rare and there is limited evidence substantial changes in their dierentiated properties can occur. Thus adult cardiomyocytes are rarely used to examine control of cell growth. The vast majority of work carried out over the last *Correspondence: JH Brown 2Current address: Arena Pharmaceuticals, Inc., 6166 Nancy Ridge 2 ± 3 decades has utilized primary cultures of neonatal Drive, San Diego, CA 92121, USA rat ventricular myocytes (NRVM) to map the signaling G proteins and cardiac growth JW Adams and JH Brown 1627 molecules and pathways involved in hypertrophic eciency gene delivery has been achieved by infection cardiomyocyte growth. Since little cell division occurs of NRVMs with cDNAs expressed by replication postnatally in cardiac myocytes, increased cardiac mass de®cient recombinant adenoviral vectors. Because is achieved primarily through increases in cardiomyo- virtually all of the cells are infected and high-level cyte cell volume. Hypertrophic growth of myocytes is expression is obtained, biochemical and molecular accompanied by increases in protein synthesis and changes underlying cardiomyocyte hypertrophy and organization of contractile proteins, such as myosin apoptosis can be assessed. light chain-2 (MLC-2) into sarcomeric units. In The in vitro NRVM system has many advantages addition, fetal isoforms of various genes including b- including ease of culture. Importantly, it circumvents myosin heavy chain (MHC) and atrial natriuretic the complexity imposed by interactions of individual factor (ANF) are re-expressed by hypertrophied myocytes with each other, with non-myocytes, with the myocytes (Parker et al., 1990). extracellular matrix and with circulating growth factors. The initial paradigm demonstrating that neurohor- This degree of isolation provides an ideal platform for mones could increase cardiomyocyte size and their the discovery of signaling molecules through which the capacity for protein synthesis documented hypertrophy cardiomyocyte responds to extracellular stimuli or of cultured NRVM in response to norepinephrine (NE) intracellularly expressed mediators. On the other hand, treatment (Simpson et al., 1982; Simpson, 1983). Using the interactions lacking in this in vitro system could play variations of this model, others have demonstrated that a central role in the in vivo control of cardiomyocyte cardiomyocyte hypertrophy occurs in response to growth and death. This dimension is provided by stimulation by a variety of G-protein coupled receptor studies using transgenic gene expression in the myo- agonists, most of which are listed in Table 1. cardium, and/or deletion of putative signaling molecules Investigation of the signaling pathways mediating by gene knockout. The a-MHC promoter has been hypertrophic growth of NRVMs was signi®cantly commonly used to achieve cardiac speci®c gene advanced by the recognition that these primary expression in transgenic mice. For example, signaling cultured cells could be genetically manipulated. Of molecules including G-protein coupled receptors, G- critical importance was the observation that transcrip- protein alpha subunits, and kinases such as MAP tional responses could be assessed using the sensitive kinases and PKC have been selectively expressed in luciferase reporter gene, driven by promoters for the myocardium by transgenesis. Despite the caveats genes upregulated in hypertrophy (e.g. ANF, MLC-2). applicable to transgenic overexpression of signaling Thus even in the face of the relatively low transfection molecules, this paradigm has been invaluable for eciency obtained using CaPO4 or electroporation of evaluating the physiological function of candidate NRVMs, genes encoding molecules of interest could be molecules discovered using the in vitro cell system (see transfected into NRVMs and their eects on regulation Izumo and Shioi, 1998 for review). of hypertrophic responses examined. Proteins, anti- bodies or cDNAs encoding genes of interest have also been microinjected into NRVMs. While this approach G-protein involvement in cardiomyocyte hypertrophy has also provided important information, only re- sponses that can be measured at the single cell level Gq-signaling pathways in cardiomyocyte hypertrophy (e.g. increased immunostaining for ANF protein or altered sarcomere organization visualized by staining Most stimuli that induce myocardial hypertrophy in with phalloidin) can be monitored. Most recently, high NRVMs activate G-protein coupled receptors that Table 1 G-protein coupled receptors regulating cardiomyocyte growth Agonist Receptor G-protein References (selected) Ang II AT1 Gq (Sadoshima and Izumo, 1993b) ET-1 ETA Gq/Gi (Shubeita et al., 1990; Hilal-Dandan et al., 1992; 1997 ET-3 (Tamamori et al., 1996) PGF2a PGF Gq (Adams et al., 1998a) PE a1-AdrR Gq (Bishopric et al., 1987; Chien et al., 1991) LPA Edg (2, 4 or 7 ?) Gq/Gi (Goetzl et al., 2000) Thrombin PAR-1 Gq/Gi (Glembotski