Hormones and neurotransmitters may mediate common responses through receptors that couple to the same class of heterotrimeric guanine nucleotide-binding (G) protein. For example, several receptors that couple to Gq class proteins can induce cardiomyocyte hypertrophy. Class-specific inhibition of Gq-mediated signaling was produced in the hearts of transgenic mice by targeted expression of a carboxyl-terminal peptide of the alpha subunit Galphaq. When pressure overload was surgically induced, the transgenic mice developed significantly less ventricular hypertrophy than control animals. The data demonstrate the role of myocardial Gq in the initiation of myocardial hypertrophy and indicate a possible strategy for preventing pathophysiological signaling by simultaneously blocking multiple receptors coupled to Gq.

-Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.

Background and objectives: Several novel urinary biomarkers have shown promise in the early detection and diagnostic evaluation of acute kidney injury (AKI). Clinicians have limited tools to determine which patients will progress to more severe forms of AKI at the time of serum creatinine increase. The diagnostic and prognostic utility of novel and traditional AKI biomarkers was evaluated during a prospective study of 123 adults undergoing cardiac surgery.Design, setting, participants, & measurements: Urinary neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CyC), kidney injury molecule-1 (KIM-1), hepatocyte growth factor (HGF), -glutathione-S-transferase (-GST), ␣-GST, and fractional excretions of sodium and urea were all measured at preoperative baseline, postoperatively, and at the time of the initial clinical diagnosis of AKI. Receiver operator characteristic curves were generated and the areas under the curve (AUCs) were compared.Results: Forty-six (37.4%) subjects developed AKI Network stage 1 AKI; 9 (7.3%) of whom progressed to stage 3. Preoperative KIM-1 and ␣-GST were able to predict the future development of stage 1 and stage 3 AKI. Urine CyC at intensive care unit (ICU) arrival best detected early stage 1 AKI (AUC ‫؍‬ 0.70, P < 0.001); the 6-hour ICU NGAL (AUC ‫؍‬ 0.88; P < 0.001) best detected early stage 3 AKI. -GST best predicted the progression to stage 3 AKI at the time of creatinine increase (AUC ‫؍‬ 0.86; P ‫؍‬ 0.002).Conclusion: Urinary biomarkers may improve the ability to detect early AKI and determine the clinical prognosis of AKI at the time of diagnosis.

Transgenic mice were generatedwith cardiacspecific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked ,I-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin 11-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the 13-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype. action between residues within /ARK's carboxyl terminus and the dissociated, membrane-anchored fry subunits of G proteins (11,12). Unlike PARK1, GRKS does not undergo agonist-dependent translocation from cytosol to membrane, but rather is constitutively membrane-bound (10). Previous in vitro reconstitution and intact cell experiments have demonstrated agonist-dependent phosphorylation of many G protein-coupled receptors by various GRKs, including f31-ARs, the predominant subtype in the heart (13-15). Since the myocardial 13-AR system is so critical in normal and pathophysiologic states (8, 9), we sought to determine the extent and specificity to which targeted cardiac overexpression of GRK5 would desensitize G protein-coupled receptors in vivo. Furthermore, since myocardial function is greatly influenced by anesthetic agents, studies were also performed in chronically instrumented transgenic mice in the awake, conscious state.A diverse family of receptors activate G protein-coupled signal transduction pathways to elicit multiple biologic functions. Two G protein-coupled receptors that are closely involved in the regulation of cardiac function and growth in response to neurohormonal and mechanical stimuli are the ,13-adrenergic receptor (X31-AR) and the angiotensin II (AnglI) receptor (1, 2). These receptors, when activated by agonists, stimulate the heterotrimeric G proteins Gs and Gq, respectively, leading to the intracellular accumulation of second messengers; notably, cAMP from f31-AR-Gs coupling and inositol trisphosphate and diacylglycerol from AngII-Gq coupling (3, 4). With persistent stimulation by agonist, many G protein signal transduction systems demonstrate diminished responsiveness referred to as desensitization. Two classes of serine/threonine kinases can regulate receptors through rapid receptor phosphorylation; the second messenger-activated protein kinases, such as the cAMP-dependent protein kinase and protein kinase C (5), and the G protein-coupled receptor kinases (GRKs), which phosphorylate activated receptors, leading to homologous desensitization (5). Currently there are six known members of the emerging ...

Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring ␤-adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular ␤-adrenergic signaling defects including down-regulation of myocardial ␤-adrenergic receptors (␤-ARs), functional ␤-AR uncoupling, and an upregulation of the ␤-AR kinase (␤ARK1). Adenoviral-mediated gene transfer of the human ␤ 2 -AR or an inhibitor of ␤ARK1 to these failing myocytes led to the restoration of ␤-AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of ␤ARK1 activity in the heart.

Background-Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1␣ (also called ␣-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. Methods and Results-In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1. By developing a TPM1-specific antibody, we found that the TPM1 protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1 in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1. Incorporation of increased levels of TPM1 protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1 compared with TPM1␣. Conclusions-This functional analysis of TPM1 provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients. (Circulation. 2010;121:410-418.)Key Words: cardiomyopathy Ⅲ contractility Ⅲ heart failure Ⅲ myocardial contraction T he heart adapts to different challenges presented by an array of mechanical, hormonal, and nutritional signals in the process of maintaining its circulatory function. Isoform switching of sarcomeric proteins is 1 mode the heart uses to adapt to those challenges, along with alterations in the relative abundance and phosphorylation status of contractile and regulatory proteins. 1 These changes in isoform expression and phosphorylation status also play an essential role during cardiac development and in response to cardiac hypertrophy and heart failure (HF). Although sarcomeric protein isoforms are subject to developmental regulation, cardiomyopathy and HF primarily elicit changes in thick filament protein isoforms. 2 The only thin filament protein to change isoform expression in the failing human heart is troponin T. 3,4 Furthermore, altered phosphorylation of troponin I, myosin binding protein C, and other sarcomeric proteins has dramatic effects on cardiac function in the failing human myocardium. 5 Editorial see p 351 Clinical Perspective on p 418To understand the specific role of another thin filament protein, tropomyosin (TM), in the normal and the pathological heart, it is essential to define the TM isoform expression profile. Tropomyosins comprise a family of actin-binding proteins encoded by 4 different genes (TPM1, TPM2, TPM3, and TPM4). Each gene uses alternative splicing, alternative promoters, and differential processing to encode multiple striated muscle, smooth muscle, and cytoskeletal transcripts. For example, the TPM1...

We studied the effect of alterations in the level of myocardial ␤-adrenergic receptor kinase (␤ARK1) in two types of genetically altered mice. The first group is heterozygous for ␤ARK1 gene ablation, ␤ARK1(؉/؊), and the second is not only heterozygous for ␤ARK1 gene ablation but is also transgenic for cardiac-specific overexpression of a ␤ARK1 COOH-terminal inhibitor peptide, ␤ARK1(؉/؊)/␤ARKct. In contrast to the embryonic lethal phenotype of the homozygous ␤ARK1 knockout (Jaber, M., Koch, W. J., Rockman, H. A., Smith, B., Bond, R. A., Sulik, K., Ross, J., Jr., Lefkowitz, R. J., Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974 -12979), ␤ARK1(؉/؊) mice develop normally. Cardiac catheterization was performed in mice and showed a stepwise increase in contractile function in the ␤ARK1(؉/؊) and ␤ARK1(؉/؊)/␤ARKct mice with the greatest level observed in the ␤ARK1(؉/؊)/␤ARKct animals. Contractile parameters were measured in adult myocytes isolated from both groups of gene-targeted animals. A significantly greater increase in percent cell shortening and rate of cell shortening following isoproterenol stimulation was observed in the ␤ARK1(؉/؊) and ␤ARK1(؉/؊)/␤ARKct myocytes compared with wildtype cells, indicating a progressive increase in intrinsic contractility. These data demonstrate that contractile function can be modulated by the level of ␤ARK1 activity. This has important implications in disease states such as heart failure (in which ␤ARK1 activity is increased) and suggests that ␤ARK1 should be considered as a therapeutic target in this situation. Even partial inhibition of ␤ARK1 activity enhances ␤-adrenergic receptor signaling leading to improved functional catecholamine responsiveness.One of the most important mechanisms for rapidly regulating ␤-adrenergic receptor (␤AR) 1 function is agonist-stimulated receptor phosphorylation by G protein-coupled receptor kinases (GRKs) resulting in decreased sensitivity to further catecholamine stimulation (1). GRKs phosphorylate only agonistoccupied receptors leading to homologous desensitization (1, 2). The ␤-adrenergic receptor kinase (␤ARK1) is a member of a family of at least 6 GRKs, which phosphorylate and regulate a wide variety of receptors that couple to heterotrimeric G proteins (3, 4). When ␤ARs or other G protein-coupled receptors are activated by agonist, heterotrimeric G proteins dissociate into G ␣ and G ␤␥ subunits, and the G ␤␥ subunit complex, which is membrane anchored by a lipid group (geranylgeranyl), can target ␤ARK1 to the membrane through a direct physical interaction that facilitates phosphorylation of activated receptors (5, 6).Using a transgenic based strategy for cardiac-specific overexpression of either ␤ARK1 or a peptide inhibitor of ␤ARK1 (␤ARKct), we have recently shown that in vivo, myocardial ␤ 1 -adrenergic and angiotensin II receptors are targets for ␤ARK1 mediated desensitization (7, 8). The ␤ARK1 inhibitor utilized is a peptide containing the carboxyl-terminal 194 amino acids of ␤ARK1, which competes with end...

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