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...