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. Key Words: myocardial ischemia Ⅲ myocardial ischemia/reperfusion injury Ⅲ cardiac injury Ⅲ cardiac dysfunction Ⅲ mouse model C ardiovascular disease represents the leading cause of morbidity and death in developed countries. Coronary heart disease, which is the single largest cause of cardiovascular disease, is the narrowing of arteries over time caused by atherosclerotic plaques or the acute occlusion of the coronary artery by thrombosis, both of which lead to possible myocardial infarction (MI) and the eventual development of heart failure. 1,2 Protection from coronary heart disease-induced damage of the myocardium during myocardial ischemia/ reperfusion (I/R) injury has been a target of investigation for the development of innovative cardioprotective therapies. [3][4][5][6][7] The increase in the availability of various types of genetically manipulated mice has brought about the need for more efficient ways to induce myocardial damage for both molecular mechanistic studies and potentially therapeutic interventions. Two of the most common models used by researchers are permanent left main descending coronary artery (LCA) occlusion to induce a MI and also temporary coronary artery occlusion to induce I/R injury. 8,9 The I/R model is generally used to examine the short-term consequences of ischemic injury, whereas the MI model is usually used to investigate myocardial changes such as remodeling that occur over an extended period of time. Although a variety of surgical manipulations have been used during the past decade to induce the ischemic event, ligation of the LCA is still the most commonly practiced method. 3,9 -11 However, most investigators still use a method requiring ventilation and wide opening the chest (referred to as the classic method), which can cause extensive tissue damage, high surgical-related death and can also be quite time consuming for most surgeons. [12][13][14][15] Over the last few years, we have developed a new MI approach in mice that does not require ventilation. 11,16 -18 Complete characterization and description of this model has Original Methods and Results:

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.

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 ␤-adrenergic receptor (␤AR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the ␤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 ␤ARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with ␤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 ␤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 ␤ARKct, and these antiapoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of ␤ 2 ARs abolished ␤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 ␤ARKct not only in chronic HF but also potentially in acute ischemic injury conditions. (Circ Res. 2010;107:1140-1149.) Key Words: acute myocardial ischemia Ⅲ ischemia/reperfusion injury Ⅲ cardioprotection Ⅲ G protein-coupled receptor kinase-2 Ⅲ ␤ARKct Ⅲ myocyte apoptosis

Heart failure (HF) is the end stage of many underlying cardiovascular diseases and is among the leading causes of morbidity and mortality in industrialized countries. One of the striking characteristics of HF is the desensitization of G protein-coupled receptor (GPCR) signaling, particularly the β-adrenergic receptor (βAR) system. GPCR desensitization is initiated by phosphorylation by GPCR kinases (GRKs), followed by downregulation and functional uncoupling from their G proteins. In the heart, the major GRK isoforms, GRK2 and GRK5, undergo upregulation due to the heightened sympathetic nervous system activity that is characteristic of HF as catecholamine levels increase in an effort to drive the failing pump. This desensitization leads to the distinctive loss of inotropic reserve and functional capacity of the failing heart. Moreover, GRK2 and GRK5 have an increasing non-GPCR interactome, which may play critical roles in cardiac physiology. In the current review, the canonical GPCR kinase function of GRKs and the novel non-GPCR kinase activity of GRKs, their contribution to the pathogenesis of cardiac hypertrophy and HF, and the possibility of GRKs serving as future drug targets will be discussed.

Heart failure caused by ischemic heart disease is a leading cause of death in the developed world. Treatment is currently centered on regimens involving G protein-coupled receptors (GPCRs) or nitric oxide (NO). These regimens are thought to target distinct molecular pathways. We showed that these pathways were interdependent and converged on the effector GRK2 (GPCR kinase 2) to regulate myocyte survival and function. Ischemic injury coupled to GPCR activation, including GPCR desensitization and myocyte loss, requires GRK2 activation, and we found that cardioprotection mediated by S-nitrosylation and inhibition of GRK2 depended on endothelial nitric oxide synthase (eNOS). Conversely, the cardioprotective effects of NO bioactivity were absent in a knock-in mouse with a form of GRK2 that cannot be S-nitrosylated. Because GRK2 and eNOS inhibit each other, the balance of the activities these enzymes in the myocardium determined the outcome to ischemic injury. Our findings suggest new insights into the mechanism of action of classic drugs used to treat heart failure and new therapeutic approaches to ischemic heart disease.