Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demonstrate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ.

The db/db mouse is a well-established model of diabetes. Previous reports have documented contractile dysfunction (i.e., cardiomyopathy) in these animals, although the extant literature provides limited insights into cardiac structure and function as they change over time. To better elucidate the natural history of cardiomyopathy in db/db mice, we performed cardiac magnetic resonance (CMR) scans on these animals. CMR imaging was conducted with a 4.7-T magnet on female db/db mice and control db/ϩ littermates at 5, 9, 13, 17, and 22 wk of age. Gated gradient echo sequences were used to obtain cineographic short-axis slices from apex to base. From these images left ventricular (LV) mass (LVM), wall thickness, end-diastolic volume (LVEDV), and ejection fraction (LVEF) were determined. 18 F]FDG metabolic imaging showed a 40% decrease in glucose uptake in db/db mice. Furthermore, contractile dysfunction was observed in 15-wk db/db mice undergoing pressure-volume loops. Finally, real-time quantitative PCR revealed an age-dependent recapitulation of the fetal gene program, consistent with a myopathic process. In summary, as assessed by CMR, db/db mice develop characteristic structural and functional changes consistent with cardiomyopathy. diabetes mellitus; insulin resistance; heart failure; metabolism CONGESTIVE HEART FAILURE (CHF) is a significant yet often underappreciated complication of diabetes mellitus (25). While atherosclerotic coronary artery disease is highly prevalent and likely responsible for CHF in many diabetic patients, findings from several large-scale heart failure clinical trials reveal a 16 -20% prevalence of diabetes in patients with nonischemic cardiomyopathy (9). Moreover, an analysis of hospital discharge data showed a 27% prevalence of diabetes in patients discharged with idiopathic cardiomyopathy, compared with 18% for control subjects (13). Collectively these data suggest an alternative mechanism for CHF in diabetics-one independent of the effects of epicardial coronary disease. However, studies investigating this unique form of "diabetic cardiomyopathy" (51) have failed to establish a unifying mechanistic basis for this phenomenon.The C57BL/KLS-lepr db /lepr db (db/db) mouse, which has a mutation in the leptin receptor, is a well-established animal model of Type 2 diabetes mellitus (19). Leptin resistance results in hyperphagia and weight gain from birth. Homozygous db/db mice become noticeably obese by 3-4 wk of age and develop hyperglycemia at 4 -8 wk. Serum insulin levels increase as early as 10 -14 days, peak at 6 -8 wk, then decrease precipitously afterward (although db/db mice continue to be hyperinsulinemic throughout life). This drop, which is believed to be secondary to pancreatic islet cell dysfunction, further exacerbates the hyperglycemia.In addition to these characteristic phenotypic changes, db/db mice also develop cardiomyopathy. Metabolic experiments using cultured db/db cardiomyocytes have shown impaired glucose oxidation as early as 6 wk of age (1). Echocardiographic studie...

Blood flow recovery, arteriogenesis, and monocyte and macrophage recruitment to the thigh was normal in CCR2 -/- mice. However, monocyte and macrophage recruitment to the ischemic calf was diminished in CCR2 -/- mice. Our results show that MCP-1 signaling through CCR2 is not required for physiologic arteriogenesis in response to severe hindlimb ischemia. ICAM-1 upregulation may substitute for MCP-1 signaling through CCR2 in order to allow normal arteriogenesis in CCR2 -/- mice.

The G protein-coupled receptor APJ is a promising therapeutic target for heart failure. Constitutive deletion of APJ in the mouse is protective against the hypertrophy-heart failure transition via elimination of ligand-independent, β-arrestin-dependent stretch transduction. However, the cellular origin of this stretch transduction and the details of its interaction with apelin signaling remain unknown. We generated mice with conditional elimination of APJ in the endothelium (APJ) and myocardium (APJ). No baseline difference was observed in left ventricular function in APJ, APJ, or control (APJ, APJ) mice. After exposure to transaortic constriction, APJ mice displayed decreased left ventricular systolic function and increased wall thickness, whereas APJ mice were protected. At the cellular level, carbon fiber stretch of freshly isolated single cardiomyocytes demonstrated decreased contractile responses to stretch in APJ cardiomyocytes compared with APJ cardiomyocytes. Ca transients did not change with stretch in either APJ or APJ cardiomyocytes. Application of apelin to APJ cardiomyocytes resulted in decreased Ca transients. Furthermore, hearts of mice treated with apelin exhibited decreased phosphorylation in cardiac troponin I NH-terminal residues (Ser and Ser) consistent with increased Ca sensitivity. These data establish that APJ stretch transduction is mediated specifically by myocardial APJ, that APJ is necessary for stretch-induced increases in contractility, and that apelin opposes APJ's stretch-mediated hypertrophy signaling by lowering Ca transients while maintaining contractility through myofilament Ca sensitization. These findings underscore apelin's unique potential as a therapeutic agent that can simultaneously support cardiac function and protect against the hypertrophy-heart failure transition. NEW & NOTEWORTHY These data address fundamental gaps in our understanding of apelin-APJ signaling in heart failure by localizing APJ's ligand-independent stretch sensing to the myocardium, identifying a novel mechanism of apelin-APJ inotropy via myofilament Ca sensitization, and identifying potential mitigating effects of apelin in APJ stretch-induced hypertrophic signaling.

The apelin peptide is described as one of the most potent inotropic agents, produced endogenously in a wide range of cells, including cardiomyocytes. Despite positive effects on cardiac contractility in multicellular preparations, as well as indications of cardio-protective actions in several diseases, its effects and mechanisms of action at the cellular level are incompletely understood.Here, we report apelin effects on dynamic mechanical characteristics of single ventricular cardiomyocytes, isolated from mouse models (control, apelin-deficient [Apelin-KO], apelin-receptor KO mouse [APJ-KO]), and rat. Dynamic changes in maximal velocity of cell shortening and relaxation were monitored. In addition, more traditional indicators of inotropic effects, such as maximum shortening (in mechanically unloaded cells) or peak force development (in auxotonic contracting cells, preloaded using the carbon fibre technique) were studied.The key finding is that, using Apelin-KO cardiomyocytes exposed to different preloads with the 2-carbon fibre technique, we observe a lowering of the slope of the end-diastolic stress-length relation in response to 10 nM apelin, an effect that is preload-dependent. This suggests a positive lusitropic effect of apelin, which could explain earlier counter-intuitive findings on an apelin-induced increase in contractility occurring without matching rise in oxygen consumption.

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Here we demonstrate a method that allows light-induced activation of voltagegated ion channels and the concurrent imaging of membrane potential changes using voltage-sensitive dyes. This light-induced voltage clamp (LIVC) method uses photostimulation through channelrhodopsin-2 (ChR2) to activate voltagegated ion channels. ChR2 allows light to be immediately transduced into a depolarizing ionic current, which in turn causes voltage-gated ion channels to open. In our system we coexpressed ChR2 either with the voltage-gated potassium channels hERG or hKv1.5 in cell lines and in Xenopus oocytes. In electrophysiological experiments we show that light-induced depolarization through ChR2 sufficed to activate hERG as well as hKv1.5 channels. We were further able to optically monitor the light-induced membrane de-and hyperpolarizations on a millisecond timescale with the voltage-sensitive RH421 and Annine6. The fluorescence readout reflected the dose-response relationships of the hERG blocker Terfenadine and the hKv1.5 inhibitor DPO-1 obtained from patch-clamp measurements. LIVC represents a solely optical technology with remote activation of the target voltage-gated ion channels by the delivery of a flash of blue light and simultaneous detection of their activity employing voltage-sensitive dyes. It combines the high-throughput of optical methods with the high-content of patch clamp concerning high temporal resolution, membrane potential control and repetitive stimulation.