Keshava Rajagopal

Teaching old receptors new tricks: biasing seven-transmembrane receptors

Seven-transmembrane receptors (7TMRs; also known as G protein-coupled receptors) are the largest class of receptors in the human genome and are common targets for therapeutics. Originally identified as mediators of 7TMR desensitization, β-arrestins (arrestin 2 and arrestin 3) are now recognized as true adaptor proteins that transduce signals to multiple effector pathways. Signalling that is mediated by β-arrestins has distinct biochemical and functional consequences from those mediated by G proteins, and several biased ligands and receptors have been identified that preferentially signal through either G protein- or β-arrestin-mediated pathways. These ligands are not only useful tools for investigating the biochemistry of 7TMR signalling, they also have the potential to be developed into new classes of therapeutics.

New Roles for β-Arrestins in Cell Signaling: Not Just for Seven-Transmembrane Receptors

beta-arrestins, originally discovered as molecules that bind to and desensitize the activated and phosphorylated form of the G protein-coupled beta2-adrenergic receptor (beta2-AR), have recently emerged as multifunctional adaptor/scaffold proteins that dynamically assemble a wide range of multiprotein complexes in response to stimulation of most seven-transmembrane receptors (7TMRs). These complexes mediate receptor signaling, trafficking, and degradation. Moreover, beta-arrestins are increasingly found to perform analogous functions for receptors from structurally diverse classes, including atypical 7TMRs such as frizzled and smoothened, the nicotinic cholinergic receptors, receptor tyrosine kinases, and cytokine receptors, thereby regulating a growing list of cellular processes such as chemotaxis, apoptosis, and metastasis.

β-Arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes

The G protein-coupled receptor kinases (GRKs) and β-arrestins, families of molecules essential to the desensitization of G protein-dependent signaling via seven-transmembrane receptors (7TMRs), have been recently shown to also transduce G protein-independent signals from receptors. However, the physiologic consequences of this G protein-independent, GRK/β-arrestin-dependent signaling are largely unknown. Here, we establish that GRK/β-arrestin-mediated signal transduction via the angiotensin II (ANG) type 1A receptor (AT1AR) results in positive inotropic and lusitropic effects in isolated adult mouse cardiomyocytes. We used the “biased” AT1AR agonist [Sar1, Ile4, Ile8]-angiotensin II (SII), which is unable to stimulate Gαq-mediated signaling, but which has previously been shown to promote β-arrestin interaction with the AT1AR. Cardiomyocytes from WT, but not AT1AR-deficient knockout (KO) mice, exhibited positive inotropic and lusitropic responses to both ANG and SII. Responses of WT cardiomyocytes to ANG were dramatically reduced by protein kinase C (PKC) inhibition, whereas those to SII were unaffected. In contrast, cardiomyocytes from β-arrestin2 KO and GRK6 KO mice failed to respond to SII, but displayed preserved responses to ANG. Cardiomyocytes from GRK2 heterozygous knockout mice (GRK2+/−) exhibited augmented responses to SII in comparison to ANG, whereas those from GRK5 KO mice did not differ from those from WT mice. These findings indicate the existence of independent Gαq/PKC- and GRK6/β-arrestin2-dependent mechanisms by which stimulation of the AT1AR can modulate cardiomyocyte function, and which can be differentially activated by selective receptor ligands. Such ligands may have potential as a novel class of therapeutic agents.

Marginal Cardiac Allografts Do Not Have Increased Primary Graft Dysfunction in Alternate List Transplantation

Background— Clinical success with modern heart transplantation (HT) has led to the development of an alternate list (AL) HT strategy, matching marginal cardiac allografts with recipients who do not meet standard criteria for HT. Marginal allografts may be at an increased risk for primary graft dysfunction (PGD), the leading cause of early mortality after HT.1 The incidence of PGD in AL HT relative to standard list (SL) HT has not been evaluated, and may contribute to the greater mortality associated with AL HT.2 The objective of this study was to determine the incidence of and predictors for PGD. Methods and Results— A retrospective analysis was performed on 260 consecutive adult patients undergoing either SL HT (n=207) or AL HT (n=53) at our institution from 1/2000 to 1/2005. PGD was defined by requirement for mechanical circulatory support immediately post-HT or more broadly as the need for either mechanical support or high-dose inotrope (epinephrine ≥0.07 &mgr;g/kg/min). Donor hearts allocated to AL recipients were turned down for SL HT for reasons that included coronary disease, left ventricular dysfunction or hypertrophy, and high-dose inotropic requirement. AL HT recipients were significantly older, with a higher proportion of diabetes mellitus and ischemic cardiomyopathy. Both groups experienced a similar incidence of significant rejection, but overall mortality was higher in the AL HT group.2 The incidence of PGD did not differ between AL and SL HT recipients. Pre-transplant VAD and prolonged total ischemic times (≥4.5hours) were independent predictors of PGD. Conclusion— Select marginal donor hearts used in AL HT do not have an increased incidence of PGD. Pre-transplant VAD and prolonged ischemic times are more important determinants of PGD. These data support continued aggressive utilization of marginal donor hearts in AL HT.

When 7 transmembrane receptors are not G protein–coupled receptors

Classically, 7 transmembrane receptors transduce extracellular signals by coupling to heterotrimeric G proteins, although recent in vitro studies have clearly demonstrated that they can also signal via G protein-independent mechanisms. However, the physiologic consequences of this unconventional signaling, particularly in vivo, have not been explored. In this issue of the JCI, Zhai et al. demonstrate in vivo effects of G protein-independent signaling by the angiotensin II type 1 receptor (AT1R) (see the related article beginning on page 3045). In studies of the mouse heart, they compare the physiologic and biochemical consequences of transgenic cardiac-specific overexpression of a mutant AT1R incapable of G protein coupling with those of a wild-type receptor. Their results not only provide the first glimpse of the physiologic effects of this newly appreciated mode of signaling but also provide important and previously unappreciated clues as to the underlying molecular mechanisms.

Independent β-Arrestin2 and Gq/Protein Kinase Cζ Pathways for ERK Stimulated by Angiotensin Type 1A Receptors in Vascular Smooth Muscle Cells Converge on Transactivation of the Epidermal Growth Factor Receptor

Recent studies in receptor-transfected cell lines have demonstrated that extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A receptor and other G protein-coupled receptors can be mediated by both G protein-dependent and β-arrestin-dependent mechanisms. However, few studies have explored these mechanisms in primary cultured cells expressing endogenous levels of receptors. Accordingly, here we utilized the β-arrestin biased agonist for the angiotensin type 1A receptor, SII-angiotensin (SII), and RNA interference techniques to investigate angiotensin II (ANG)-activated β-arrestin-mediated mitogenic signaling pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA synthesis via the ERK activation cascade. Even though SII cannot induce calcium influx (G protein activation) after receptor stimulation, it does cause ERK activation, although less robustly than ANG. Activation by both ligands is diminished by depletion of β-arrestin2 by small interfering RNA, although the effect is more complete with SII. ERK activation at early time points but not later time points is strongly inhibited by those protein kinase C inhibitors that can block protein kinase Cζ. Moreover, ANG- and SII-mediated ERK activation require transactivation of the epidermal growth factor receptor via metalloprotease 2/9 and Src kinase. β-Arrestin2 facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845 on the EGFR, a known site for Src phosphorylation. These studies delineate a convergent mechanism by which G protein-dependent and β-arrestin-dependent pathways can independently mediate ERK-dependent transactivation of the EGFR in vascular smooth muscle cells thus controlling cellular proliferative responses.

Left Ventricular Assist Device Destination Therapy Versus Extended Criteria Cardiac Transplant

BACKGROUND End-stage heart failure is a growing problem associated with a high mortality using conventional medical care. Although cardiac transplantation is an excellent treatment option, it is a limited resource and most patients are ineligible for cardiac transplantation using standard listing criteria. Increasingly, newer surgical options for these patients include the usage of marginal donor organs in an extended criteria-alternate list heart transplant program (EC-AL), or left ventricular assist devices as destination therapy (DT-LVAD). The purpose of this study was to compare baseline characteristics and outcomes after EC-AL versus DT-LVAD. METHODS From March 2000 to August 2008, 153 consecutive patients who had been turned down for standard heart transplantation underwent either EC-AL or DT-LVAD. The most common reasons for standard heart transplant ineligibility for both groups were advanced age, diabetes mellitus with end-organ dysfunction, and significant renal insufficiency. Patients in the alternate list program received a donor organ that had been turned down by all other centers for standard list recipients. The most common reasons for donor heart refusal were decreased left ventricular function, left ventricle hypertrophy, or coronary artery disease. Outcomes for both groups were retrospectively reviewed after Institutional Review Board permission was obtained. Comparisons were also made between patients that had been matched by propensity score analysis. RESULTS In all, 93 patients underwent EC-AL, and 60 underwent DT-LVAD. Baseline preoperative characteristics of both groups were similar except that 87% of DT-LVAD patients (52 of 60) required preoperative mechanical or inotropic support whereas only 51% of EC-AL patients (47 of 93) required support (p<0.0001). Thirty-day operative mortality and 1-year survival were 2.5% and 82.2% for EC-AL and 6.7% and 77.5% DT-LVAD, respectively (p=0.2411 and p=0.5036). Overall survival at 3 years was better for EC-AL versus DT-LVAD. The DT-LVAD patients had improved survival compared with the Randomized Evaluation of Mechanical Assistance for the Treatment of Congestive Heart Failure (REMATCH) trial DT-LVAD cohort, a historical control. These findings were corroborated in the analysis of propensity-matched patients. CONCLUSIONS Preoperatively, the DT-LVAD cohort was more unstable, with greater need for inotropes or mechanical support. Despite this, perioperative and 1-year mortality was similar for the two groups. Three-year survival was better for EC-AL. The DT-LVAD patient survival was better than that of the REMATCH DT-LVAD cohort.

The use of lung donors older than 55 years: a review of the United Network of Organ Sharing database.

BACKGROUND Current lung transplantation guidelines stipulate that the ideal donor is aged younger than 55 years, but several institutions have reported that outcomes using donors aged 55 years and older are comparable with those of younger donors. METHODS We retrospectively reviewed the United Network for Organ Sharing (UNOS) database to identify all adult lung transplants between 2000 and 2010 in the United States. Patients were stratified by donor age 18 to 34 (reference), 35 to 54, 55 to 64, and ≥ 65 years. Primary outcomes included survival at 30 days and at 1, 3, and 5 years and rates of bronchiolitis obliterans syndrome (BOS). Survival was assessed using the Kaplan-Meier method. Risk factors for mortality were identified by multivariable Cox and logistic regression. RESULTS We identified 10,666 recipients with median follow-up of 3 years (range, 0-10 years). Older donors were more likely to have died of cardiovascular or cerebrovascular causes, but there were no differences in recipient diagnosis, lung allocation score, or incidence of BOS as a function of donor age. The use of donors aged 55 to 64 years was not a risk factor for mortality at 1 year (odds ratio, 1.1; p = 0.304) or 3 years (odds ratio, 0.923; p = 0.571) compared with the reference group; however, use of donors aged > 65 years was associated with increased mortality at both time points (odds ratio, 2.8 and 2.4, p < 0.02). CONCLUSIONS Outcomes after lung transplantation using donors aged 55 to 64 years were similar to those observed with donors meeting conventional age criteria. Donors aged ≥ 65 years, however, were associated with decreased intermediate-term survival, although there was no increased risk of BOS for this group.

Venovenous Extracorporeal Membrane Oxygenation as a Bridge to Lung Transplantation: Successful Transplantation After 155 Days of Support

We present a case of a woman with acute respiratory distress syndrome and irrecoverable lung function that was successfully bridged to lung transplantation after 155 consecutive days of venovenous extracorporeal membrane oxygenation.

Infectious Complications in Extended Criteria Heart Transplantation

BACKGROUND We have previously shown that extended criteria heart transplant recipient mortality is higher than standard list mortality, but this is not associated with an increased incidence of either primary graft dysfunction or acute rejection. We hypothesized that other adverse outcomes, principally determined by recipient characteristics, occur at a higher rate in extended criteria recipients. METHODS A retrospective review of adult heart transplant recipients was conducted at Duke University Medical Center between January 2000 and July 2007. Infectious complications considered risk factors for recipient mortality were identified. In addition, the incidence of these complications was compared between standard and alternate list recipients. RESULTS Infectious complications, including pneumonia, bacteremia and sepsis, were significant predictors of overall mortality (pneumonia hazard ratio 4.2 [95% CI 2.5 to 7.0], bacteremia hazard ratio 3.0 [95% CI 1.9 to 4.9], sepsis hazard ratio 6.0 [95% CI 3.6 to 10.2]). In addition, pneumonia occurred at a significantly higher rate in extended criteria (EC) than in standard list (SL) patients (27% vs 13%, p = 0.005), and bacteremia and sepsis demonstrated a trend toward higher occurrence in EC patients (36% vs 25%, p = 0.076, and 15% vs 8%, p = 0.114, respectively). In contrast, severe acute cellular rejection (ISHLT Grade >/=3A) was not a predictor of mortality, and had a similar incidence in both groups. Finally, although overall survival among patients in the SL group was not influenced by the occurrence of a major infectious complication, survival in the extended criteria group was significantly impacted by major infectious complications (p < 0.001). CONCLUSIONS Infectious complications may account for decreased survival in extended criteria heart transplant recipients.