Jinliang Li

An adenylyl cyclase-mAKAPβ signaling complex regulates cAMP levels in cardiac myocytes

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein β (mAKAPβ) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPβ selectively binds type 5 AC in the heart and that mAKAPβ-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPβ-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245–340), and expression of this peptide disrupts endogenous mAKAPβ-AC association. Accordingly, disruption of mAKAPβ-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPβ complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy.

The mAKAPβ Scaffold Regulates Cardiac Myocyte Hypertrophy via Recruitment of Activated Calcineurin

mAKAPbeta is the scaffold for a multimolecular signaling complex in cardiac myocytes that is required for the induction of neonatal myocyte hypertrophy. We now show that the pro-hypertrophic phosphatase calcineurin binds directly to a single site on mAKAPbeta that does not conform to any of the previously reported consensus binding sites. Calcineurin-mAKAPbeta complex formation is increased in the presence of Ca(2+)/calmodulin and in norepinephrine-stimulated primary cardiac myocytes. This binding is of functional significance because myocytes exhibit diminished norepinephrine-stimulated hypertrophy when expressing a mAKAPbeta mutant incapable of binding calcineurin. In addition to calcineurin, the transcription factor NFATc3 also associates with the mAKAPbeta scaffold in myocytes. Calcineurin bound to mAKAPbeta can dephosphorylate NFATc3 in myocytes, and expression of mAKAPbeta is required for NFAT transcriptional activity. Taken together, our results reveal the importance of regulated calcineurin binding to mAKAPbeta for the induction of cardiac myocyte hypertrophy. Furthermore, these data illustrate how scaffold proteins organizing localized signaling complexes provide the molecular architecture for signal transduction networks regulating key cellular processes.

S-nitrosoglutathione reductase–dependent PPARγ denitrosylation participates in MSC-derived adipogenesis and osteogenesis

Bone marrow-derived mesenchymal stem cells (MSCs) are a common precursor of both adipocytes and osteoblasts. While it is appreciated that PPARγ regulates the balance between adipogenesis and osteogenesis, the roles of additional regulators of this process remain controversial. Here, we show that MSCs isolated from mice lacking S-nitrosoglutathione reductase, a denitrosylase that regulates protein S-nitrosylation, exhibited decreased adipogenesis and increased osteoblastogenesis compared with WT MSCs. Consistent with this cellular phenotype, S-nitrosoglutathione reductase-deficient mice were smaller, with reduced fat mass and increased bone formation that was accompanied by elevated bone resorption. WT and S-nitrosoglutathione reductase-deficient MSCs exhibited equivalent PPARγ expression; however, S-nitrosylation of PPARγ was elevated in S-nitrosoglutathione reductase-deficient MSCs, diminishing binding to its downstream target fatty acid-binding protein 4 (FABP4). We further identified Cys 139 of PPARγ as an S-nitrosylation site and demonstrated that S-nitrosylation of PPARγ inhibits its transcriptional activity, suggesting a feedback regulation of PPARγ transcriptional activity by NO-mediated S-nitrosylation. Together, these results reveal that S-nitrosoglutathione reductase-dependent modification of PPARγ alters the balance between adipocyte and osteoblast differentiation and provides checkpoint regulation of the lineage bifurcation of these 2 lineages. Moreover, these findings provide pathophysiological and therapeutic insights regarding MSC participation in adipogenesis and osteogenesis.

Anchored p90 Ribosomal S6 Kinase 3 is Required for Cardiac Myocyte Hypertrophy

Rationale: Cardiac myocyte hypertrophy is the main compensatory response to chronic stress on the heart. p90 ribosomal S6 kinase (RSK) family members are effectors for extracellular signal-regulated kinases that induce myocyte growth. Although increased RSK activity has been observed in stressed myocytes, the functions of individual RSK family members have remained poorly defined, despite being potential therapeutic targets for cardiac disease. Objective: To demonstrate that type 3 RSK (RSK3) is required for cardiac myocyte hypertrophy. Methods and Results: RSK3 contains a unique N-terminal domain that is not conserved in other RSK family members. We show that this domain mediates the regulated binding of RSK3 to the muscle A-kinase anchoring protein scaffold, defining a novel kinase anchoring event. Disruption of both RSK3 expression using RNA interference and RSK3 anchoring using a competing muscle A-kinase anchoring protein peptide inhibited the hypertrophy of cultured myocytes. In vivo, RSK3 gene deletion in the mouse attenuated the concentric myocyte hypertrophy induced by pressure overload and catecholamine infusion. Conclusions: Taken together, these data demonstrate that anchored RSK3 transduces signals that modulate pathologic myocyte growth. Targeting of signaling complexes that contain select kinase isoforms should provide an approach for the specific inhibition of cardiac myocyte hypertrophy and for the development of novel strategies for the prevention and treatment of heart failure.

The Scaffold Protein Muscle A-Kinase Anchoring Protein β Orchestrates Cardiac Myocyte Hypertrophic Signaling Required for the Development of Heart Failure

Background—Cardiac myocyte hypertrophy is regulated by an extensive intracellular signal transduction network. In vitro evidence suggests that the scaffold protein muscle A-kinase anchoring protein &bgr; (mAKAP&bgr;) serves as a nodal organizer of hypertrophic signaling. However, the relevance of mAKAP&bgr; signalosomes to pathological remodeling and heart failure in vivo remains unknown. Methods and Results—Using conditional, cardiac myocyte–specific gene deletion, we now demonstrate that mAKAP&bgr; expression in mice is important for the cardiac hypertrophy induced by pressure overload and catecholamine toxicity. mAKAP&bgr; targeting prevented the development of heart failure associated with long-term transverse aortic constriction, conferring a survival benefit. In contrast to 29% of control mice (n=24), only 6% of mAKAP&bgr; knockout mice (n=31) died in the 16 weeks of pressure overload (P=0.02). Accordingly, mAKAP&bgr; knockout inhibited myocardial apoptosis and the development of interstitial fibrosis, left atrial hypertrophy, and pulmonary edema. This improvement in cardiac status correlated with the attenuated activation of signaling pathways coordinated by the mAKAP&bgr; scaffold, including the decreased phosphorylation of protein kinase D1 and histone deacetylase 4 that we reveal to participate in a new mAKAP signaling module. Furthermore, mAKAP&bgr; knockout inhibited pathological gene expression directed by myocyte-enhancer factor-2 and nuclear factor of activated T-cell transcription factors that associate with the scaffold. Conclusions—mAKAP&bgr; orchestrates signaling that regulates pathological cardiac remodeling in mice. Targeting of the underlying physical architecture of signaling networks, including mAKAP&bgr; signalosome formation, may constitute an effective therapeutic strategy for the prevention and treatment of pathological remodeling and heart failure.

mAKAP-a master scaffold for cardiac remodeling.

Abstract: Cardiac remodeling is regulated by an extensive intracellular signal transduction network. Each of the many signaling pathways in this network contributes uniquely to the control of cellular adaptation. In the last few years, it has become apparent that multimolecular signaling complexes or “signalosomes” are important for fidelity in intracellular signaling and for mediating crosstalk between the different signaling pathways. These complexes integrate upstream signals and control downstream effectors. In the cardiac myocyte, the protein mAKAP&bgr; serves as a scaffold for a large signalosome that is responsive to cAMP, calcium, hypoxia, and mitogen-activated protein kinase signaling. The main function of mAKAP&bgr; signalosomes is to modulate stress-related gene expression regulated by the transcription factors NFATc, MEF2, and HIF-1&agr; and type II histone deacetylases that control pathological cardiac hypertrophy.

The Scaffold Protein mAKAPβ Orchestrates Cardiac Myocyte Hypertrophic Signaling Required for the Development of Heart Failure

Background—Cardiac myocyte hypertrophy is regulated by an extensive intracellular signal transduction network. In vitro evidence suggests that the scaffold protein muscle A-kinase anchoring protein &bgr; (mAKAP&bgr;) serves as a nodal organizer of hypertrophic signaling. However, the relevance of mAKAP&bgr; signalosomes to pathological remodeling and heart failure in vivo remains unknown. Methods and Results—Using conditional, cardiac myocyte–specific gene deletion, we now demonstrate that mAKAP&bgr; expression in mice is important for the cardiac hypertrophy induced by pressure overload and catecholamine toxicity. mAKAP&bgr; targeting prevented the development of heart failure associated with long-term transverse aortic constriction, conferring a survival benefit. In contrast to 29% of control mice (n=24), only 6% of mAKAP&bgr; knockout mice (n=31) died in the 16 weeks of pressure overload (P=0.02). Accordingly, mAKAP&bgr; knockout inhibited myocardial apoptosis and the development of interstitial fibrosis, left atrial hypertrophy, and pulmonary edema. This improvement in cardiac status correlated with the attenuated activation of signaling pathways coordinated by the mAKAP&bgr; scaffold, including the decreased phosphorylation of protein kinase D1 and histone deacetylase 4 that we reveal to participate in a new mAKAP signaling module. Furthermore, mAKAP&bgr; knockout inhibited pathological gene expression directed by myocyte-enhancer factor-2 and nuclear factor of activated T-cell transcription factors that associate with the scaffold. Conclusions—mAKAP&bgr; orchestrates signaling that regulates pathological cardiac remodeling in mice. Targeting of the underlying physical architecture of signaling networks, including mAKAP&bgr; signalosome formation, may constitute an effective therapeutic strategy for the prevention and treatment of pathological remodeling and heart failure.

Regulation of MEF2 transcriptional activity by calcineurin/mAKAP complexes.

The calcium/calmodulin-dependent protein phosphatase calcineurin is required for the induction of transcriptional events that initiate and promote myogenic differentiation. An important effector for calcineurin in striated muscle is the transcription factor myocyte enhancer factor 2 (MEF2). The targeting of the enzyme and substrate to specific intracellular compartments by scaffold proteins often confers specificity in phosphatase activity. We now show that the scaffolding protein mAKAP organizes a calcineurin/MEF2 signaling complex in myocytes, regulating gene transcription. A calcineurin/mAKAP/MEF2 complex can be isolated from C2C12 cells and cardiac myocytes, and the calcineurin/MEF2 association is dependent on mAKAP expression. We have identified a peptide comprising the calcineurin binding domain in mAKAP that can disrupt the binding of the phosphatase to the scaffold in vivo. Dominant interference of calcineurin/mAKAP binding blunts the increase in MEF2 transcriptional activity seen during myoblast differentiation, as well as the expression of endogenous MEF2-target genes. Furthermore, disruption of calcineurin binding to mAKAP in cardiac myocytes inhibits adrenergic-induced cellular hypertrophy. Together these data illustrate the importance of calcineurin anchoring by the mAKAP scaffold for MEF2 regulation.

p90 ribosomal S6 kinase 3 contributes to cardiac insufficiency in α-tropomyosin Glu180Gly transgenic mice

Myocardial interstitial fibrosis is an important contributor to the development of heart failure. Type 3 p90 ribosomal S6 kinase (RSK3) was recently shown to be required for concentric myocyte hypertrophy under in vivo pathological conditions. However, the role of RSK family members in myocardial fibrosis remains uninvestigated. Transgenic expression of α-tropomyosin containing a Glu180Gly mutation (TM180) in mice of a mixed C57BL/6:FVB/N background induces a cardiomyopathy characterized by a small left ventricle, interstitial fibrosis, and diminished systolic and diastolic function. Using this mouse model, we now show that RSK3 is required for the induction of interstitial fibrosis in vivo. TM180 transgenic mice were crossed to RSK3 constitutive knockout (RSK3(-/-)) mice. Although RSK3 knockout did not affect myocyte growth, the decreased cardiac function and mild pulmonary edema associated with the TM180 transgene were attenuated by RSK3 knockout. The improved cardiac function was consistent with reduced interstitial fibrosis in the TM180;RSK3(-/-) mice as shown by histology and gene expression analysis, including the decreased expression of collagens. The specific inhibition of RSK3 should be considered as a potential novel therapeutic strategy for improving cardiac function and the prevention of sudden cardiac death in diseases in which interstitial fibrosis contributes to the development of heart failure.

RSK3: A regulator of pathological cardiac remodeling.

The family of p90 ribosomal S6 kinases (RSKs) are pleiotropic effectors for extracellular signal‐regulated kinase signaling pathways. Recently, RSK3 was shown to be important for pathological remodeling of the heart. Although cardiac myocyte hypertrophy can be compensatory for increased wall stress, in chronic heart diseases, this nonmitotic cell growth is usually associated with interstitial fibrosis, increased cell death, and decreased cardiac function. Although RSK3 is less abundant in the cardiac myocyte than other RSK family members, RSK3 appears to serve a unique role in cardiac myocyte stress responses. A potential mechanism conferring the unique function of RSK3 in the heart is anchoring by the scaffold protein muscle A‐kinase anchoring protein β (mAKAPβ). Recent findings suggest that RSK3 should be considered as a therapeutic target for the prevention of heart failure, a clinical syndrome of major public health significance.