J. Kurt Chuprun

A Novel and Efficient Model of Coronary Artery Ligation and Myocardial Infarction in the Mouse

Rationale: Coronary artery ligation to induce myocardial infarction (MI) in mice is typically performed by an invasive and time-consuming approach that requires ventilation and chest opening (classic method), often resulting in extensive tissue damage and high mortality. We developed a novel and rapid surgical method to induce MI that does not require ventilation. Objective: The purpose of this study was to develop and comprehensively describe this method and directly compare it to the classic method. Methods and Results: Male C57/B6 mice were grouped into 4 groups: new method MI (MI-N) or sham (S-N) and classic method MI (MI-C) or sham (S-C). In the new method, heart was manually exposed without intubation through a small incision and MI was induced. In the classic method, MI was induced through a ventilated thoracotomy. Similar groups were used in an ischemia/reperfusion injury model. This novel MI procedure is rapid, with an average procedure time of 1.22±0.05 minutes, whereas the classic method requires 23.2±0.6 minutes per procedure. Surgical mortality was 3% in MI-N and 15.9% in MI-C. The rate of arrhythmia was significantly lower in MI-N. The postsurgical levels of tumor necrosis factor-&agr; and myeloperoxidase were lower in new method, indicating less inflammation. Overall, 28-day post-MI survival rate was 68% with MI-N and 48% with MI-C. Importantly, there was no difference in infarct size or post-MI cardiac function between the methods. Conclusions: This new rapid method of MI in mice represents a more efficient and less damaging model of myocardial ischemic injury compared with the classic method.

Uncovering G protein-coupled receptor kinase-5 as a histone deacetylase kinase in the nucleus of cardiomyocytes

G protein-coupled receptor (GPCR) kinases (GRKs) are critical regulators of cellular signaling and function. In cardiomyocytes, GRK2 and GRK5 are two GRKs important for myocardial regulation, and both have been shown to be up-regulated in the dysfunctional heart. We report that increased levels and activity of GRK5 in failing myocardium may have unique significance due to its nuclear localization, a property not shared by GRK2. We find that transgenic mice with elevated cardiac GRK5 levels have exaggerated hypertrophy and early heart failure compared with control mice after pressure overload. This pathology is not present in cardiac GRK2-overexpressing mice or in mice with overexpression of a mutant GRK5 that is excluded from the nucleus. Nuclear accumulation of GRK5 is enhanced in myocytes after aortic banding in vivo and in vitro in myocytes after increased Gαq activity, the trigger for pressure-overload hypertrophy. GRK5 enhances activation of MEF2 in concert with Gq signals, demonstrating that nuclear localized GRK5 regulates gene transcription via a pathway critically linked to myocardial hypertrophy. Mechanistically, we show that this is due to GRK5 acting, in a non-GPCR manner, as a class II histone deacetylase (HDAC) kinase because it can associate with and phosphorylate the myocyte enhancer factor-2 repressor, HDAC5. Moreover, significant HDAC activity can be found with GRK5 in the heart. Our data show that GRK5 is a nuclear HDAC kinase that plays a key role in maladaptive cardiac hypertrophy apparently independent of any action directly on GPCRs.

Stable Myocardial-Specific AAV6-S100A1 Gene Therapy Results in Chronic Functional Heart Failure Rescue

Background— The incidence of heart failure is ever-growing, and it is urgent to develop improved treatments. An attractive approach is gene therapy; however, the clinical barrier has yet to be broken because of several issues, including the lack of an ideal vector supporting safe and long-term myocardial transgene expression. Methods and Results— Here, we show that the use of a recombinant adeno-associated viral (rAAV6) vector containing a novel cardiac-selective enhancer/promoter element can direct stable cardiac expression of a therapeutic transgene, the calcium (Ca2+)-sensing S100A1, in a rat model of heart failure. The chronic heart failure–rescuing properties of myocardial S100A1 expression, the result of improved sarcoplasmic reticulum Ca2+ handling, included improved contractile function and left ventricular remodeling. Adding to the clinical relevance, long-term S100A1 therapy had unique and additive beneficial effects over &bgr;-adrenergic receptor blockade, a current pharmacological heart failure treatment. Conclusions— These findings demonstrate that stable increased expression of S100A1 in the failing heart can be used for long-term reversal of LV dysfunction and remodeling. Thus, long-term, cardiac-targeted rAAV6-S100A1 gene therapy may be of potential clinical utility in human heart failure.

Level of G protein–Coupled Receptor Kinase-2 Determines Myocardial Ischemia/Reperfusion Injury via Pro- and Anti-Apoptotic Mechanisms

Rationale: Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein–coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function. Objective: A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac &bgr;-adrenergic receptor (&bgr;AR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the &bgr;ARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model. Methods and Results: We demonstrate, using cardiac-specific GRK2 and &bgr;ARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with &bgr;ARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2-overexpressing mice (45.0±2.8% versus 31.3±2.3% in controls) and significantly smaller in &bgr;ARKct mice (16.8±1.3%, P<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via &bgr;ARKct, and these antiapoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of &bgr;2ARs abolished &bgr;ARKct-mediated cardioprotection, suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival. Conclusion: The novel effect of reducing acute ischemic myocardial injury via increased Akt activity and NO production adds significantly to the therapeutic potential of GRK2 inhibition with the &bgr;ARKct not only in chronic HF but also potentially in acute ischemic injury conditions.

Paroxetine is a direct inhibitor of g protein-coupled receptor kinase 2 and increases myocardial contractility.

G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. Herein we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine-Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.

G Protein–Coupled Receptor Kinase 2 Activity Impairs Cardiac Glucose Uptake and Promotes Insulin Resistance After Myocardial Ischemia

Background— Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure. The chronic adrenergic stimulation that accompanies heart failure is a signaling abnormality that leads to the upregulation of G protein–coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part owing to uncoupling of the &bgr;-adrenergic receptor system. In this study, we explored the possibility that enhanced GRK2 expression and activity, as seen during heart failure, can negatively affect cardiac metabolism as part of its pathogenic profile. Methods and Results— Positron emission tomography studies revealed in transgenic mice that cardiac-specific overexpression of GRK2 negatively affected cardiac metabolism by inhibiting glucose uptake and desensitization of insulin signaling, which increases after ischemic injury and precedes heart failure development. Mechanistically, GRK2 interacts with and directly phosphorylates insulin receptor substrate-1 in cardiomyocytes, causing insulin-dependent negative signaling feedback, including inhibition of membrane translocation of the glucose transporter GLUT4. This identifies insulin receptor substrate-1 as a novel nonreceptor target for GRK2 and represents a new pathological mechanism for this kinase in the failing heart. Importantly, inhibition of GRK2 activity prevents postischemic defects in myocardial insulin signaling and improves cardiac metabolism via normalized glucose uptake, which appears to participate in GRK2-targeted prevention of heart failure. Conclusions— Our data provide novel insights into how GRK2 is pathological in the injured heart. Moreover, it appears to be a critical mechanistic link within neurohormonal crosstalk governing cardiac contractile signaling/function through &bgr;-adrenergic receptors and metabolism through the insulin receptor.Background Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure (HF). The chronic adrenergic stimulation that accompanies HF is a signaling abnormality that leads to the up-regulation of G protein-coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part due to uncoupling of the β-adrenergic receptor (βAR) system. In this study we explored the possibility that enhanced GRK2 expression and activity, as seen during HF, can negatively affect cardiac metabolism as part of its pathogenic profile.

Targeted Inhibition of Cardiomyocyte Gi Signaling Enhances Susceptibility to Apoptotic Cell Death in Response to Ischemic Stress

Background— A salient characteristic of dysfunctional myocardium progressing to heart failure is an upregulation of the adenylyl cyclase inhibitory guanine nucleotide (G) protein &agr; subunit, G&agr;i2. It has not been determined conclusively whether increased Gi activity in the heart is beneficial or deleterious in vivo. Gi signaling has been implicated in the mechanism of cardioprotective agents; however, no in vivo evidence exists that any of the G&agr; subunits are cardioprotective. We have created a novel molecular tool to specifically address the role of Gi proteins in normal and dysfunctional myocardium. Methods and Results— We have developed a class-specific Gi inhibitor peptide, GiCT, composed of the region of G&agr;i2 that interacts specifically with G protein–coupled receptors. GiCT inhibits Gi signals specifically in vitro and in vivo, whereas Gs and Gq signals are not affected. In vivo expression of GiCT in transgenic mice effectively causes a “functional knockout” of cardiac G&agr;i2 signaling. Inducible, cardiac-specific GiCT transgenic mice display a baseline phenotype consistent with nontransgenic mice. However, when subjected to ischemia/reperfusion injury, GiCT transgenic mice demonstrate a significant increase in infarct size compared with nontransgenic mice (from 36.9±2.5% to 50.9±4.3%). Mechanistically, this post-ischemia/reperfusion phenotype includes increased myocardial apoptosis and resultant decreased contractile performance. Conclusions— Overall, our results demonstrate the in vivo utility of GiCT to dissect specific mechanisms attributed to Gi signaling in stressed myocardium. Our results with GiCT indicate that upregulation of G&agr;i2 is an adaptive protective response after ischemia to shield myocytes from apoptosis.

Prodeath Signaling of G Protein–Coupled Receptor Kinase 2 in Cardiac Myocytes After Ischemic Stress Occurs Via Extracellular Signal–Regulated Kinase-Dependent Heat Shock Protein 90–Mediated Mitochondrial Targeting

Rationale: GRK2 is abundantly expressed in the heart and its expression and activity is increased in injured or stressed myocardium. This up-regulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes and its inhibition by a peptide comprised of the last 194 amino acids of GRK2 (known as βARKct) is cardioprotective. Objective: The aim of this study was to elucidate the signaling mechanism that accounts for the pro-death signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the βARKct. Methods and Results: Using in vivo mouse models of ischemic injury and also cultured myocytes we found that GRK2 localizes to mitochondria providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced pro-death signaling. Localization of GRK2 to mitochondria was dependent upon phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal-regulated kinases (ERKs), resulting in enhanced GRK2 binding to heat shock protein 90 (Hsp90), which chaperoned GRK2 to mitochondria. Mechanistic studies invivo and invitro showed that ERK regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent pro-death signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca2+-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. Conclusions: We identify GRK2 as a pro-death kinase in the heart acting in a novel manner through mitochondrial localization via ERK regulation.Rationale: G protein–coupled receptor kinase 2 (GRK2) is abundantly expressed in the heart, and its expression and activity are increased in injured or stressed myocardium. This upregulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes, and its inhibition by a peptide comprising the last 194 amino acids of GRK2 (known as carboxyl-terminus of &bgr;-adrenergic receptor kinase [bARKct]) is cardioprotective. Objective: The aim of this study was to elucidate the signaling mechanism that accounts for the prodeath signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the carboxyl-terminus of &bgr;-adrenergic receptor kinase. Methods and Results: Using in vivo mouse models of ischemic injury and also cultured myocytes, we found that GRK2 localizes to mitochondria, providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced prodeath signaling. Localization of GRK2 to mitochondria was dependent on phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal–regulated kinases, resulting in enhanced GRK2 binding to heat shock protein 90, which chaperoned GRK2 to mitochondria. Mechanistic studies in vivo and in vitro showed that extracellular signal–regulated kinase regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent prodeath signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca2+-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. Conclusions: We identify GRK2 as a prodeath kinase in the heart, acting in a novel manner through mitochondrial localization via extracellular signal–regulated kinase regulation.

The Evolving Impact of G Protein-Coupled Receptor Kinases in Cardiac Health and Disease

G protein-coupled receptors (GPCRs) are important regulators of various cellular functions via activation of intracellular signaling events. Active GPCR signaling is shut down by GPCR kinases (GRKs) and subsequent β-arrestin-mediated mechanisms including phosphorylation, internalization, and either receptor degradation or resensitization. The seven-member GRK family varies in their structural composition, cellular localization, function, and mechanism of action (see sect. II). Here, we focus our attention on GRKs in particular canonical and novel roles of the GRKs found in the cardiovascular system (see sects. III and IV). Paramount to overall cardiac function is GPCR-mediated signaling provided by the adrenergic system. Overstimulation of the adrenergic system has been highly implicated in various etiologies of cardiovascular disease including hypertension and heart failure. GRKs acting downstream of heightened adrenergic signaling appear to be key players in cardiac homeostasis and disease progression, and herein we review the current data on GRKs related to cardiac disease and discuss their potential in the development of novel therapeutic strategies in cardiac diseases including heart failure.

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.