Rationale: Excess signaling through cardiac G␤␥ subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein-coupled receptor kinase (GRK)2 to agonist-stimulated ␤-adrenergic receptors (␤-ARs) in HF, leading to chronic ␤-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting G␤␥ signaling and its interaction with GRK2 could be of therapeutic value in HF. Objective: We sought to investigate small molecule G␤␥ inhibition in HF. Methods and Results: We recently described novel small molecule G␤␥ inhibitors that selectively block G␤␥-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of G␤␥ and GRK2 in vitro and in HL60 cells. Here, we show they reduced ␤-AR-mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to ␤-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and ␤-myosin heavy chain). Conclusions: These data suggest a promising therapeutic role for small molecule inhibition of pathological G␤␥ signaling in the treatment of HF. (Circ Res. 2010;107:532-539.)Key Words: G proteins Ⅲ adrenergic receptor Ⅲ G protein-coupled receptor kinases Ⅲ cardiomyopathy Ⅲ heart failure Ⅲ cardiomyocyte H eart failure (HF) is a devastating disease with poor prognosis, and remains a leading cause of death worldwide. 1,2 Excess signaling through cardiac G protein G␤␥ subunits is an important component of HF pathophysiology. In particular, they recruit elevated levels of cytosolic G protein-coupled receptor kinase 2 (GRK2) (␤-adrenergic receptor kinase [␤-ARK]1) to agonist-stimulated ␤-ARs in HF, 3 leading to the chronic ␤-AR desensitization, downregulation and pathological signaling that are hallmarks of HF. 4,5 Increasing evidence suggests a critical role for G␤␥-mediated signaling in HF. In particular, GRK2 is significantly upregulated in cardiomyocytes of animal models of HF and human HF patients; this elevates G␤␥-GRK2 interactions and contributes to chronic desensitization of ␤-AR signaling 6,7 ; interestingly, levels of GRK2 appear to correlate with the severity of HF. 6,8 Enhancing G␤␥-GRK2 interaction by cardiac targeted overexpression of GRK2(s) can directly cause HF in experimental animal models 9 ; its genetic ablation has generally proven to be...

G protein-coupled receptors (GPCRs) represent the largest family of membrane receptors and are responsible for regulating a wide variety of physiological processes. This is accomplished via ligand binding to GPCRs, activating associated heterotrimeric G proteins and intracellular signaling pathways. G protein-coupled receptor kinases (GRKs), in concert with β-arrestins, classically desensitize receptor signal transduction, thus preventing hyperactivation of GPCR second messenger cascades. As changes in GRK expression have featured prominently in many cardiovascular pathologies, including heart failure, myocardial infarction, hypertension, and cardiac hypertrophy, GRKs have been intensively studied as potential diagnostic or therapeutic targets. Herein, we review our evolving understanding of the role of GRKs in cardiovascular pathophysiology.

Cardiac myocytes, in the intact heart, are exposed to shear/fluid forces during each cardiac cycle. Here we describe a novel Ca 2+ signalling pathway, generated by 'pressurized flows' (PFs) of solutions, resulting in the activation of slowly developing (∼300 ms) Ca 2+ transients lasting ∼1700 ms at room temperature. Though subsequent PFs (applied some 10-30 s later) produced much smaller or undetectable responses, such transients could be reactivated following caffeineor KCl-induced Ca 2+ releases, suggesting that a small, but replenishable, Ca 2+ pool serves as the source for their activation. PF-triggered Ca 2+ transients could be activated in Ca 2+ -free solutions or in solutions that block voltage-gated Ca 2+ channels, stretch-activated channels (SACs), or the Na + -Ca 2+ exchanger (NCX), using Cd 2+ , Gd 3+ , or Ni 2+ , respectively. PF-triggered Ca 2+ transients were significantly smaller in quiescent than in electrically paced myocytes. Paced Ca 2+ transients activated at the peak of PF-triggered Ca 2+ transients were not significantly smaller than those produced normally, suggesting functionally separate Ca 2+ pools for paced and PF-triggered transients. Suppression of nitric oxide (NO) or IP 3 signalling pathways did not alter the PF-triggered Ca 2+ transients. On the other hand, mitochondrial metabolic uncoupler FCCP, in the presence of oligomycin (to prevent ATP depletion), reversibly suppressed PF-triggered Ca 2+ transients, as did the mitochondrial Ca 2+ uniporter (mCU) blocker, Ru360. Reducing agent DTT and reactive oxygen species (ROS) scavenger tempol, as well as mitochondrial NCX (mNCX) blocker CGP-37157, inhibited PF-triggered Ca 2+ transients. In rhod-2 AM-loaded and permeabilized cells, confocal imaging of mitochondrial Ca 2+ showed a transient increase in Ca 2+ on caffeine exposure and a decrease in mitochondrial Ca 2+ on application of PF pulses of solution. These signals were strongly suppressed by either Na + -free or CGP-37157-containing solutions, implicating mNCX in mediating the Ca 2+ release process. We conclude that subjecting rat cardiac myocytes to pressurized flow pulses of solutions triggers the release of Ca 2+ from a store that appears to access mitochondrial Ca 2+ .

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics. 1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal. 2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals. 3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound. 4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function. 6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment. Begin by securing an isoflurane anesthetized mouse to an animal-handling platform in the supine position. Place a nose cone over the animal's nose and mouth to deliver 0.5-1% isoflurane to maintain the anesthesia. 2. Secure the paws of the mouse to the electrode pads with conducting gel. Ensure appropriate ECG, body temperature at 37°C and check respiratory rate for physiological assessment during imaging. 3. Apply depilatory cream to the chest and upper abdomen of the mouse. 4. After 2 minutes, use wet gauze remove to the cream.1. Once the mouse has been prepared for imaging, tilt the left side of the platform to rotate the animal handling platform 30 degrees about the anterioposterior axis. 2. Orient the transducer in the vertical position and rotate 10 degrees counterclockwise with the notch pointed toward the posterior of the mouse. 3. Next, while in the two-dimensional viewing/video "B-mode", lower the transducer over the left parasternal line until the heart comes into view.Once the pulmonary artery comes into view, collect images and store them. 4. Still in B-mode, move the transducer left or right until the aortic outflow and apex come into view. Some rotation of the probe may be necessary to ensure proper alignment with t...

G-protein-coupled receptor (GPCR)-kinase interacting protein-1 (GIT1) is a multi-function scaffold protein. However, little is known about its physiological role in the heart. Here we sought to identify the cardiac function of GIT1. Global GIT1 knockout (KO) mice were generated and exhibited significant cardiac hypertrophy that progressed to heart failure. Electron microscopy revealed that the hearts of GIT1 KO mice demonstrated significant morphological abnormities in mitochondria, including decreased mitochondrial volume density, cristae density and increased vacuoles. Moreover, mitochondrial biogenesis-related gene peroxisome proliferator-activated receptor γ (PPARγ) co-activator-1α (PGC-1α), PGC-1β, mitochondrial transcription factor A (Tfam) expression, and total mitochondrial DNA were remarkably decreased in hearts of GIT1 KO mice. These animals also had impaired mitochondrial function, as evidenced by reduced ATP production and dissipated mitochondrial membrane potential (Ψm) in adult cardiomyocytes. Concordant with these mitochondrial observations, GIT1 KO mice showed enhanced cardiomyocyte apoptosis and cardiac dysfunction. In conclusion, our findings identify GIT1 as a new regulator of mitochondrial biogenesis and function, which is necessary for postnatal cardiac maturation.

Mammalian enabled (Mena) of the Drosophila enabled/vasodilator-stimulated phosphoprotein gene family is a cytoskeletal protein implicated in actin regulation and cell motility. Cardiac Mena expression is enriched in intercalated discs (ICD), the critical intercellular communication nexus between adjacent muscle cells. We previously identified Mena gene expression to be a key predictor of human and murine heart failure (HF). To determine the in vivo function of Mena in the heart, we assessed Mena protein expression in multiple HF models and characterized the effects of genetic Mena deletion on cardiac structure and function. Immunoblot analysis revealed significant upregulation of Mena protein expression in left ventricle tissue from patients with end-stage HF, calsequestrin-overexpressing mice, and isoproterenol-infused mice. Characterization of the baseline cardiac function of adult Mena knockout mice (Mena(-/-)) via echocardiography demonstrated persistent cardiac dysfunction, including a significant reduction in percent fractional shortening compared with wild-type littermates. Electrocardiogram PR and QRS intervals were significantly prolonged in Mena(-/-) mice, manifested by slowed conduction on optical mapping studies. Ultrastructural analysis of Mena(-/-) hearts revealed disrupted organization and widening of ICD structures, mislocalization of the gap junction protein connexin 43 (Cx43) to the lateral borders of cardiomyoycytes, and increased Cx43 expression. Furthermore, the expression of vinculin (an adherens junction protein) was significantly reduced in Mena(-/-) mice. We report for the first time that genetic ablation of Mena results in cardiac dysfunction, highlighted by diminished contractile performance, disrupted ICD structure, and slowed electrical conduction.

G protein-coupled receptors (GPCRs) are a virtually ubiquitous class of membrane-bound receptors, which functionally couple hormone or neurotransmitter signals to physiological responses. Dysregulation of GPCR signaling contributes to the pathophysiology of a host of cardiovascular disorders. Pharmacological agents targeting GPCRs have been established as therapeutic options for decades. Nevertheless, the persistent burden of cardiovascular diseases necessitates improved treatments. To that end, exciting drug development efforts have begun to focus on novel compounds that discriminately activate particular GPCR signaling pathways.

Mammalian enabled (Mena) is a key regulator of cytoskeletal actin dynamics, which has been implicated in heart failure (HF). We have previously demonstrated that cardiac Mena deletion produced cardiac dysfunction with conduction abnormalities and hypertrophy. Moreover, elevated Mena expression correlates with HF in human and animal models, yet the precise role of Mena in cardiac pathophysiology is unclear. In these studies, we evaluated mice with cardiac myocyte-specific Mena overexpression (TTA/TgTetMena) comparable to that observed in cardiac pathology. We found that the hearts of TTA/TgTetMena mice were functionally and morphologically comparable to wild-type littermates, except for mildly increased heart mass in the transgenic mice. Interestingly, TTA/TgTetMena mice were particularly susceptible to cardiac injury, as these animals experienced pronounced decreases in ejection fraction and fractional shortening as well as heart dilatation and hypertrophy after transverse aortic constriction (TAC). By "turning off" Mena overexpression in TTA/TgTetMena mice either immediately prior to or immediately after TAC surgery, we discovered that normalizing Mena levels eliminated cardiac hypertrophy in TTA/TgTetMena animals but did not preclude post-TAC cardiac functional deterioration. These findings indicate that hearts with increased levels of Mena fare worse when subjected to cardiac injury and suggest that Mena contributes to HF pathophysiology.