Apelin and APJ orchestrate complex tissue-specific control of cardiomyocyte hypertrophy and contractility in the hypertrophy-heart failure transition.

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, ß-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 (APJendo-/-) and myocardium (APJmyo-/-). No baseline difference was observed in left ventricular function in APJendo-/-, APJmyo-/-, or control (APJendo+/+, APJmyo+/+) mice. After exposure to transaortic constriction, APJendo-/- mice displayed decreased left ventricular systolic function and increased wall thickness, whereas APJmyo-/- 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. Ca2+ transients did not change with stretch in either APJ-/- or APJ+/+ cardiomyocytes. Application of apelin to APJ+/+ cardiomyocytes resulted in decreased Ca2+ transients. Furthermore, hearts of mice treated with apelin exhibited decreased phosphorylation in cardiac troponin I NH2-terminal residues (Ser22 and Ser23) consistent with increased Ca2+ 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 Ca2+ transients while maintaining contractility through myofilament Ca2+ 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 Ca2+ sensitization, and identifying potential mitigating effects of apelin in APJ stretch-induced hypertrophic signaling.

Load-dependent effects of apelin on murine cardiomyocytes.

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.

Endogenous regulation of cardiovascular function by apelin-APJ.

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.

CCR2-/- knockout mice revascularize normally in response to severe hindlimb ischemia.

OBJECTIVE: Monocyte chemoattractant protein-1 (MCP-1) is reported to stimulate ischemia-induced arteriogenesis (collateral artery development) by recruiting monocytes and macrophages into areas of active arteriogenesis. To determine whether the MCP-1-mediated response occurs through its receptor, CC-chemokine receptor 2 (CCR2), we induced hindlimb ischemia in mice lacking the receptor for MCP-1 (CCR2 -/- ) and measured limb blood flow recovery, collateral artery development, and monocyte and macrophage recruitment. METHODS AND RESULTS: Hindlimb ischemia was induced by excising the left femoral artery in CCR2 -/- and wild-type mice. Hindlimb blood flow recovery, as measured using laser Doppler perfusion imaging, was equivalent in both groups ( P = .78 for foot and P = 0.38 for calf). Collateral artery development, as measured by angiography at postoperative day 14 and 31, likewise did not differ between the 2 groups ( P = .46 and P = .67). Counts of monocytes and macrophages in calf and thigh muscle sections of mice sacrificed on postoperative day 7 revealed that although CCR2 -/- mice recruited 44% fewer monocytes and macrophages to areas of ischemia in the calf, they recruited similar numbers of monocytes and macrophages to areas of active arteriogenesis in the thigh. Intercellular adhesion molecule-1 and MCP-1 mRNA levels were higher in the thigh muscle of CCR2 -/- mice than in wild-type mice (5.5-fold and 42.3-fold induction operated to unoperated vs 2.6-fold and 6.1-fold induction operated to unoperated, respectively). CONCLUSIONS: 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.