Sarah M. Schumacher

Paroxetine-mediated GRK2 inhibition reverses cardiac dysfunction and remodeling after myocardial infarction

GRK2 inhibition with paroxetine improves cardiac function independent of its central nervous system effects. Taking antidepressants to heart Drug repurposing—extending currently Food and Drug Administration (FDA)–approved drugs to treat additional diseases—has both economic and safety advantages over new drug development. The selective serotonin reuptake inhibitor (SSRI) paroxetine, which is used as an antidepressant, has been shown to selectively inhibit G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor kinase 2 (GRK2), which is thought to contribute to heart failure progression. Now Schumacher et al. report that paroxetine can block or even reverse heart damage after myocardial infarction in a mouse model. These affects are separate from its SSRI functions and are further enhanced in the presence of current standard-of-care β-blockers. If these data hold true in humans, paroxetine therapy could be an additional or even additive strategy for treating heart failure. Heart failure (HF) is a disease of epidemic proportion and is associated with exceedingly high health care costs. G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR) kinase 2 (GRK2), which is up-regulated in the failing human heart, appears to play a critical role in HF progression in part because enhanced GRK2 activity promotes dysfunctional adrenergic signaling and myocyte death. Recently, we found that the selective serotonin reuptake inhibitor (SSRI) paroxetine could inhibit GRK2 with selectivity over other GRKs. Wild-type mice were treated for 4 weeks with paroxetine starting at 2 weeks after myocardial infarction (MI). These mice were compared with mice treated with fluoxetine, which does not inhibit GRK2, to control for the SSRI effects of paroxetine. All mice exhibited similar left ventricular (LV) dysfunction before treatment; however, although the control and fluoxetine groups had continued degradation of function, the paroxetine group had considerably improved LV function and structure, and several hallmarks of HF were either inhibited or reversed. Use of genetically engineered mice indicated that paroxetine was working through GRK2 inhibition. The beneficial effects of paroxetine were markedly greater than those of β-blocker therapy, a current standard of care in human HF. These data demonstrate that paroxetine-mediated inhibition of GRK2 improves cardiac function after MI and represents a potential repurposing of this drug, as well as a starting point for innovative small-molecule GRK2 inhibitor development.

MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics

Mitochondrial Ca(2+) Uniporter (MCU)-dependent mitochondrial Ca(2+) uptake is the primary mechanism for increasing matrix Ca(2+) in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired [Ca(2+)]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca(2+)-dependent mitochondrial metabolism.

A peptide of the RGS domain of GRK2 binds and inhibits Gαq to suppress pathological cardiac hypertrophy and dysfunction

A GRK2-derived peptide inhibits pathological cardiac hypertrophy and may prevent heart failure. GRK2 against heart failure Sustained high blood pressure causes the heart walls to thicken to deal with the increased load, a process called cardiac hypertrophy. If left unchecked, cardiac hypertrophy leads to heart failure. A particular part of the kinase and scaffolding protein GRK2 inhibits a G protein that promotes cardiac hypertrophy. Schumacher et al. generated mice that overexpressed a peptide of this inhibitory region of GRK2 in the heart. These mice developed less cardiac hypertrophy and retained greater cardiac function under conditions that cause heart failure. Thus, targeting this peptide to the heart could suppress hypertrophic signaling and prevent heart failure. G protein–coupled receptor (GPCR) kinases (GRKs) play a critical role in cardiac function by regulating GPCR activity. GRK2 suppresses GPCR signaling by phosphorylating and desensitizing active GPCRs, and through protein-protein interactions that uncouple GPCRs from their downstream effectors. Several GRK2 interacting partners, including Gαq, promote maladaptive cardiac hypertrophy, which leads to heart failure, a leading cause of mortality worldwide. The regulator of G protein signaling (RGS) domain of GRK2 interacts with and inhibits Gαq in vitro. We generated TgβARKrgs mice with cardiac-specific expression of the RGS domain of GRK2 and subjected these mice to pressure overload to trigger adaptive changes that lead to heart failure. Unlike their nontransgenic littermate controls, the TgβARKrgs mice exhibited less hypertrophy as indicated by reduced left ventricular wall thickness, decreased expression of genes linked to cardiac hypertrophy, and less adverse structural remodeling. The βARKrgs peptide, but not endogenous GRK2, interacted with Gαq and interfered with signaling through this G protein. These data support the development of GRK2-based therapeutic approaches to prevent hypertrophy and heart failure.

Interleukin 10 Inhibits Bone Marrow Fibroblast Progenitor Cell-Mediated Cardiac Fibrosis in Pressure Overloaded Myocardium

Background: Activated fibroblasts (myofibroblasts) play a critical role in cardiac fibrosis; however, their origin in the diseased heart remains unclear, warranting further investigation. Recent studies suggest the contribution of bone marrow fibroblast progenitor cells (BM-FPCs) in pressure overload–induced cardiac fibrosis. We have previously shown that interleukin-10 (IL10) suppresses pressure overload–induced cardiac fibrosis; however, the role of IL10 in inhibition of BM-FPC–mediated cardiac fibrosis is not known. We hypothesized that IL10 inhibits pressure overload–induced homing of BM-FPCs to the heart and their transdifferentiation to myofibroblasts and thus attenuates cardiac fibrosis. Methods: Pressure overload was induced in wild-type (WT) and IL10 knockout (IL10KO) mice by transverse aortic constriction. To determine the bone marrow origin, chimeric mice were created with enhanced green fluorescent protein WT mice marrow to the IL10KO mice. For mechanistic studies, FPCs were isolated from mouse bone marrow. Results: Pressure overload enhanced BM-FPC mobilization and homing in IL10KO mice compared with WT mice. Furthermore, WT bone marrow (from enhanced green fluorescent protein mice) transplantation in bone marrow–depleted IL10KO mice (IL10KO chimeric mice) reduced transverse aortic constriction–induced BM-FPC mobilization compared with IL10KO mice. Green fluorescent protein costaining with &agr;-smooth muscle actin or collagen 1&agr; in left ventricular tissue sections of IL10KO chimeric mice suggests that myofibroblasts were derived from bone marrow after transverse aortic constriction. Finally, WT bone marrow transplantation in IL10KO mice inhibited transverse aortic constriction–induced cardiac fibrosis and improved heart function. At the molecular level, IL10 treatment significantly inhibited transforming growth factor-&bgr;–induced transdifferentiation and fibrotic signaling in WT BM-FPCs in vitro. Furthermore, fibrosis-associated microRNA (miRNA) expression was highly upregulated in IL10KO-FPCs compared with WT-FPCs. Polymerase chain reaction–based selective miRNA analysis revealed that transforming growth factor-&bgr;–induced enhanced expression of fibrosis-associated miRNAs (miRNA-21, -145, and -208) was significantly inhibited by IL10. Restoration of miRNA-21 levels suppressed the IL10 effects on transforming growth factor-&bgr;–induced fibrotic signaling in BM-FPCs. Conclusions: Our findings suggest that IL10 inhibits BM-FPC homing and transdifferentiation to myofibroblasts in pressure-overloaded myocardium. Mechanistically, we show for the first time that IL10 suppresses Smad–miRNA-21–mediated activation of BM-FPCs and thus modulates cardiac fibrosis.

Noncanonical Roles of G Protein-coupled Receptor Kinases in Cardiovascular Signaling

G protein-coupled receptor kinases (GRKs) are classically known for their role in regulating the activity of the largest known class of membrane receptors, which influence diverse biological processes in every cell type in the human body. As researchers have tried to uncover how this family of kinases, containing only 7 members, achieves selective and coordinated control of receptors, they have uncovered a growing number of noncanonical activities for these kinases. These activities include phosphorylation of nonreceptor targets and kinase-independent molecular interactions. In particular, GRK2, GRK3, and GRK5 are the predominant members expressed in the heart. Their canonical and noncanonical actions within cardiac and other tissues have significant implications for cardiovascular function in healthy animals and for the development and progression of disease. This review summarizes what is currently known regarding the activity of these kinases, and particularly the role of GRK2 and GRK5 in the molecular alterations that occur during heart failure. This review further highlights areas of GRK regulation that remain poorly understood and how they may represent novel targets for therapeutic development.

G protein-coupled receptor kinase 2 promotes cardiac hypertrophy

The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3β), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ). Moreover, inhibition of PI3Kγ as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3Kγ mediated Akt phosphorylation and subsequent inactivation of GSK3β, resulting in enhanced NFAT activity.

Comparative effects of urocortins and stresscopin on cardiac myocyte contractility

RATIONALE There is a current need for the development of new therapies for patients with heart failure. OBJECTIVE We test the effects of members of the corticotropin-releasing factor (CRF) family of peptides on myocyte contractility to validate them as potential heart failure therapeutics. METHODS AND RESULTS Adult feline left ventricular myocytes (AFMs) were isolated and contractility was assessed in the presence and absence of CRF peptides Urocortin 2 (UCN2), Urocortin 3 (UCN3), Stresscopin (SCP), and the β-adrenergic agonist isoproterenol (Iso). An increase in fractional shortening and peak Ca(2+) transient amplitude was seen in the presence of all CRF peptides. A decrease in Ca(2+) decay rate (Tau) was also observed at all concentrations tested. cAMP generation was measured by ELISA in isolated AFMs in response to the CRF peptides and Iso and significant production was seen at all concentrations and time points tested. CONCLUSIONS The CRF family of peptides effectively increases cardiac contractility and should be evaluated as potential novel therapeutics for heart failure patients.

Designer Approaches for G Protein–Coupled Receptor Modulation for Cardiovascular Disease

Summary The new horizon for cardiac therapy may lie beneath the surface, with the downstream mediators of G protein–coupled receptor (GPCR) activity. Targeted approaches have shown that receptor activation may be biased toward signaling through G proteins or through GPCR kinases (GRKs) and β-arrestins, with divergent functional outcomes. In addition to these canonical roles, numerous noncanonical activities of GRKs and β-arrestins have been demonstrated to modulate GPCR signaling at all levels of receptor activation and regulation. Further, research continues to identify novel GRK/effector and β-arrestin/effector complexes with distinct impacts on cardiac function in the normal heart and the diseased heart. Coupled with the identification of once orphan receptors and endogenous ligands with beneficial cardiovascular effects, this expands the repertoire of GPCR targets. Together, this research highlights the potential for focused therapeutic activation of beneficial pathways, with simultaneous exclusion or inhibition of detrimental signaling, and represents a new wave of therapeutic development.

The Challenge of Mentorship

It is hard to pinpoint a particular event or experience that prompted me to get into science in the first place, probably because science has always been an interest and part of my life. I am continually fascinated by the complexity and adaptability of nature and the process of scientific inquiry. It all began with catching bugs, making plant concoctions in my playhouse, and dyeing part of my mom’s new carpet blue with my first chemistry set. Fortunately, I had parents who recognized and fostered this passion from an early age. They allowed a constant stream of insects, reptiles, and amphibians to be caught and observed in my terrarium and each mentored a junior high Science Fair project all the way to the state competition at the University of Illinois. This acceptance and support bolstered my passion for a life of scientific discovery. My career in science officially began during my undergraduate years at Indiana Wesleyan University. As a student in a small liberal arts college with a strong science department, I had opportunities one might not have at a larger state or private school. I had the privilege all 4 years to serve as a laboratory technician and undergraduate student instructor. Initially, this work involved organizing stocks and preparing laboratory supplies. As I progressed in my studies and began my independent research with Dr Burton Webb, my responsibilities …

Science Signaling Podcast for 22 March 2016: Preventing heart failure with GRK2

A GRK2-derived peptide reduces pathological cardiac hypertrophy in mice. Abstract This Podcast features an interview with Sarah Schumacher and Walter Koch, authors of a Research Article that appears in the 22 March 2016 issue of Science Signaling, about a peptide that can inhibit pathological cardiac hypertrophy in mice. Untreated or poorly controlled high blood pressure can cause the walls of the heart to thicken, a condition known as cardiac hypertrophy, which can lead to heart failure. Signaling from G protein–coupled receptors (GPCRs) through the G protein Gαq promotes cardiac hypertrophy. GPCR kinase 2 (GRK2) inhibits Gαq in cultured cells, and the abundance of GRK2 is increased in hypertrophic hearts, suggesting that GRK2 may contribute to heart failure. Schumacher et al. found that expressing a peptide corresponding to the domain of GRK2 that binds to and inhibits Gαq can reduce cardiac hypertrophy in response to pressure overload in mice. Listen to Podcast