| Biography: Dr. Rui-Ping Xiao has been working in the Laboratory of Cardiovascular Science since February 1990. She was trained as a physiologist and molecular pharmacologist at Tong-Ji Medical University, China, and at the University of Maryland, where she received her M.D. and Ph.D., respectively. Her main scientific focus has been related to G protein-coupled receptors (GPCRs)-mediated transmembrane signal transduction in the cardiovascular system. In addition, considerable efforts have been put on cardiac aging and heart failure associated changes in GPCR signaling. The breadth of our work covers three intertwined programs: (1) Identification and characterization of cardiovascular disease-related genes; (2) b-adrenergic receptor subtype signaling in cardiovascular system; and (3) Modulation of cardiac excitation-contraction coupling by p38 MAPK or Ca/calmodulin-dependent protein kinase II (CaMKII) in normal and failing hearts. Most studies are designed to integrate information gleaned from genetic manipulations, including gene transfer by adenoviruses, transgenic and gene targeted animal models, in conjunction with electrophysiologic, confocal imaging and cell biological techniques. The mechanistic and interdisciplinary nature of our research has made the past few years particularly fruitful. |
| Overview: The section's focus is on elucidating signal transduction mechanisms for G protein-coupled- receptors, e.g., a and b-adrenergic and opioid receptors and their subtypes in the heart. The interaction of signals emanating from stimulation of these with other receptor-mediated signaling pathways are also investigated. Studies are designed to integrate information gleaned from genetic manipulations, including gene transfer by adenoviruses, transgenic and gene targeted animal models, in conjunction with electrophysiologic, confocal imaging and cell biological techniques to probe novel intracellular regulatory mechanisms. In addition, considerable efforts have been put on cardiac aging and heart failure associated changes in G-protein coupled receptor signaling to understand the pathogenic mechanisms and develop new therapeutic strategies for the treatment of human heart failure. |
| Identification and Characterization of Cardiovascular Proliferative Diseases-related Genes: Vascular proliferative disorders, including atherosclerosis, restenosis after balloon angioplasty, and coronary arteriosclerosis, are the most common causes of severe cardiovascular diseases such as myocardial infarction, ischemic heart failure, and strokes. Neointimal VSMC proliferation constitutes an important etiological factor in vascular proliferative disorders. However, the molecular mechanisms governing VSMC proliferation are largely unknown. Thus, identifying genetic modifiers of VSMC proliferation remains as a major focus in cardiovascular biology and medicine. |
| In order to identify genes involved in VSMC proliferation, we analyzed the gene expression profile of spontaneously hypertensive rat (SHR) VSMCs versus that of Wistar Kyoto rats (WKY) VSMCs using a differential display technique and identified a novel gene. We referred to the cDNA fragment highly expressed in WKY but weakly in SHR as hyperplasia suppressor gene (HSG) (accession number: U41803). The partial (~ 0.35 kb) cDNA identified from differential display was cloned into pGEM-T plasmid vector and sequenced. Using cDNA library screening and 5' RACE reaction, we then cloned the full-length cDNA, consisting of 4151 bp before a poly(A) tail. Sequence analysis revealed an open reading frame encoding a protein of 757 amino acids. |
| We have demonstrated that the expression of rat HSG (rHSG) is markedly downregulated in hyper-proliferative SHR VSMCs and growth factor-stimulated WKY VSMCs. Overexpression of rHSG overtly suppresses serum-stimulated VSMC proliferation, and attenuates balloon injury-induced neointimal formation by 90%, thereby preventing balloon angioplasty-associated restenosis in rat carotid arteries. The rHSG-induced growth suppression is mediated by cell cycle arrest in G0/G1 phases due to inhibition of the extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling cascade. |
| Furthermore, our preliminary studies have shown that adenoviral gene transfer of the human homolog (hHSG) has a potent anti-proliferative effect in a variety of cancer cell lines, including breast cancer cell lines MCF-7 and BM-1, a leukemia cancer cell line U937, a colon cancer cell line LoVo, and a hepatoma cell line Bel 7402, and that the anti-proliferative effect of hHSG is even more potent than that induced by overexpression of p53 (a well established cancer suppressor). Thus, rHSH functions as a powerful cell proliferation suppressor, and that downregulation or inactivation of rHSG leads to vascular proliferative disorders and might be also involved in the pathogenesis of a variety of cancers. |
| Dual Coupling of Cardiac b2-Adrenergic Receptor to Gs and Gi Proteins: G protein-coupled receptors (GPCRs) constitute the largest class of cell surface signaling molecules in eukaryotes and in some prokaryotes. By activating their cognate heterotrimeric guanosine triphosphate (GTP) binding proteins (G proteins), GPCRs transduce stimulatory or inhibitory signals for a wide array of endogenous hormones and neurotransmitters, and ambient physical and chemical stimuli, as well as exogenous therapeutic reagents. b-adrenergic receptors (bARs) are archetypical members of the GPCR superfamily. There are, at least, both b1AR and b2AR present in heart muscle cells. Whereas both bAR subtypes stimulate the classic Gs-adenylyl cyclase-cAMP-protein kinase A (PKA) signaling cascade, b2AR can activate bifurcated signaling pathways through Gs and Gi proteins. Because of their distinct G protein coupling, these bAR subtypes fulfill distinct, sometimes even opposite, physiological and pathological roles. Specifically, in the heart, whereas b1AR-generated cAMP signal can broadcast throughout the cell, the b2AR-stimulated cAMP signal is spatially and functionally compartmentalized to subsurface membrane microdomains by the concurrent Gi activation, thus selectively affecting plasma membrane effectors (such as L-type Ca2+ channels) and bypassing cytoplasmic regulatory proteins (such as phospholamban and myofilaments). Of potentially greater importance, the b2AR-to-Gi pathway also delivers a powerful cardiac protective signal. As a consequence, b1AR and b2AR exhibit opposing effects on heart cell survival: b1AR activation can promote programmed heart cell death (apoptosis); in sharp contrast, b2AR activation can protect heart cells from a wide range of assaulting factors, including enhanced b1AR stimulation, hypoxia, and reactive oxygen species. The b2AR survival pathway sequentially involves Gi, Gb, phosphoinositide 3-kinase (PI3K), and Akt. Furthermore, in vivo overexpression of b1AR, but not b2AR, induces heart muscle cell hypertrophy and heart failure in transgenic mouse models. Furthermore, we have shown that sustained b1AR stimulation promotes cardiac myocyte apoptosis by activation of Ca2+/calmodulin kinase II (CaMKII), independently of PKA signaling. Taken together, the differential G protein coupling, to a large extent, accounts for the distinctly different physiological and pathological roles in the heart for b2AR versus those of b1AR. The opposite effects of b1AR and b2AR on the fate of cardiomyocytes also reveal the rationale for selective b1AR blockade with concurrent b2AR activation as a novel therapy to treat chronic heart failure. |
| In chronically failing heart, the b2AR/Gi coupling is exaggerated. The enhanced Gi signaling underlies the heart failure-associated dysfunction of b2AR. Based on the dual G coupling of b2AR, we conceptualize that receptor ligands may selectively activate a subset(s) of the post-receptor signaling pathways. By screening a variety of b2AR ligands, we have identified one ligand (fenoterol) that selectively activates Gs, bypassing the Gi signaling. Strikingly, fenoterol is able to restore the markedly depressed b2AR contractile response in two experimental chronic heart failure models. Our most recent studies provide compelling evidence that stimulation of b1AR, but not b2AR, induces cardiac apoptosis. The anti-apoptotic effect of b2AR stimulation in cardiac myocytes is mediated by Gi-Gg subunits-PI3 kinase-Akt signaling pathway. These studies not only reveal the diversity and specificity of b-AR subtype and G protein interactions, but also provide new insights for understanding the co-existence and different functional roles of b1AR and b2AR in healthy and failing hearts. |
| Modulation of Cardiac Excitation-contraction Coupling by p38 MAPK: MAPK superfamily is one of the most important signal transduction systems conserved in all eukaryotes. There are three major subgroups identified, including the extracellular signal regulated kinase (ERK1/2), p38 MAPK and c-jun-NH2terminal kinase (JNK). p38 MAPK is one of the most ancient signaling molecules involved in multiple cellular processes, including cell proliferation, cell growth and cell death. |
| In the heart, activation of p38 MAPK has been observed in pressure-overload or ischemia/infarction induced cardiac hypertrophy and heart failure in humans and animal models. In cultured cardiac myocytes, activation of p38 MAPK induces myocyte hypertrophy and apoptosis, and is also implicated in the preconditioning process and ischemia/reperfusion injury. Increasing evidence suggests that inhibition of p38 MAPK is able to improve cardiac contractility in ischemia/reperfusion-injured hearts. |
| The specific goal of this research program is to determine whether p38-MAPK activation modulates cardiac myocyte excitation-contraction coupling and if so, to explore the possible underlying mechanisms. We have examined the possible effects of p38-MAPK activation or inhibition on cardiac contractility at the single cell level, and verified the conclusion obtained from single myocyte experiments by in vivo studies in transgenic mice overexpressing activated mutants of p38 MAPK upstream kinases. In addition, we have examined the potential interaction between bAR and p38 MAPK signaling pathways in regulating cardiac contractility, and the pathophysiological relevance of p38 activation in ischemic contractile dysfunction and cardiomyocyte injury. |
| Our in vivo and in vitro studies have demonstrated, for the first time, that inhibition of p38 MAPK leads to a positive inotropic effect, whereas enhanced p38 MAPK activation inhibits myocyte contractility and negates bAR/PKA-mediated positive inotropic effect. Furthermore, we have shown that inhibition of ischemia-induced, intracellular acidosis-mediated activation of p38 MAPK not only protects myocytes against ischemic death but also reverses ischemic contractile dysfunction. These findings reveal a novel function of p38 MAPK, and provide new insights for a better understanding of the coincidence of enhanced p38 MAPK signaling and cardiac contractile dysfunction under certain pathophysiological conditions, such as cardiac ischemic/reperfusion injury and chronic heart failure. |
| Roles of Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) in Regulating Cardiac Pacemaker Activity and Excitation-Contraction Coupling: The human heart faithfully supplies blood to the body by beating more than 3 billion times in a lifetime. The sinoatrial (SA) node possesses automaticity and serves as the primary physiological pacemaker of the heart. Our recent studies have shown that SA node pacemaker activity is critically dependent on Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated positive feedback regulation of the L-type Ca2+ current (ICa,L). In freshly dissociated rabbit single SA node cells, specific CaMKII inhibitors, a peptide CaMKII inhibitor or KN-93 (0.1 - 3.0 �M), but not its inactive analog KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 3 �M KN-93 or 10 �M CaMKII peptide inhibitor completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP via modulating properties of ICa,L inactivation and local Ca2+ is critically involved in this process. |
| In addition to its regulatory effect on cardiac pacemaker activity, CaMKII plays an essential role in heart rate- or stimulation frequency-dependent augmentation of cardiac contractility and acceleration of relaxation. We have shown that CaMKII-mediated phosphorylation of PLB at Thr17 is augmented in response to increasing pacing frequency in the absence of increase in PKA-dependent phosphorylation of PLB at Ser16 or phosphorylation of SR Ca2+-ATPase (SECAR2a). Our results challenged the well-established sequential model for PLB phosphorylation at Ser16 and Thr17, and led to a new model in which dual site PLB phosphorylation occurs independently with a synergistic effect of PKA and CaMKII signaling on Thr17 phosphorylation. Moreover, CaMKII-mediated phosphorylation of PLB-Thr17 plays a crucial role in the positive cardiac contraction/relaxation-frequency relationship. The frequency-encoded PLB-Thr17 phosphorylation may represent a previously unrecognized feedback mechanism: elevated intracellular Ca2+ regulates its own reuptake into SR, whereas PKA-mediated Ser16 phosphorylation is subjected to tight sympathetic regulation. Interplay between bAR stimulation and heart rate in inducing dual site PLB phosphorylation ensures proper cardiac contractility and relaxation, particularly during stress or exercise. |
