Phosphodiesterase 11A in brain is enriched in ventral hippocampus and deletion causes psychiatric disease-related phenotypes

Phosphodiesterase 11A (PDE11A) is the most recently identified family of phosphodiesterases (PDEs), the only known enzymes to break down cyclic nucleotides. The tissue expression profile of this dual specificity PDE is controversial, and little is understood of its biological function, particularly in the brain. We seek here to determine if PDE11A is expressed in the brain and to understand its function, using PDE11A^−/− knockout (KO) mice. We show that PDE11A mRNA and protein are largely restricted to hippocampus CA1, subiculum, and the amygdalohippocampal area, with a two- to threefold enrichment in the ventral vs. dorsal hippocampus, equal distribution between cytosolic and membrane fractions, and increasing levels of protein expression from postnatal day 7 through adulthood. Interestingly, PDE11A KO mice show subtle psychiatric-disease–related deficits, including hyperactivity in an open field, increased sensitivity to the glutamate N-methyl-D-aspartate receptor antagonist MK-801, as well as deficits in social behaviors (social odor recognition memory and social avoidance). In addition, PDE11A KO mice show enlarged lateral ventricles and increased activity in CA1 (as per increased Arc mRNA), phenotypes associated with psychiatric disease. The increased sensitivity to MK-801 exhibited by PDE11A KO mice may be explained by the biochemical dysregulation observed around the glutamate α-amino-3-hydroxy-5-methyl-4-isozazolepropionic (AMPA) receptor, including decreased levels of phosphorylated-GluR1 at Ser845 and the prototypical transmembrane AMPA-receptor–associated proteins stargazin (γ2) and γ8. Together, our data provide convincing evidence that PDE11A expression is restricted in the brain but plays a significant role in regulating brain function.     

The PDZ-binding motif of the beta2-adrenoceptor is essential for physiologic signaling and trafficking in cardiac myocytes

beta1- and beta2-adrenergic receptors (AR) regulate cardiac myocyte function through distinct signaling pathways. In addition to regulating cardiac rate and contractility, beta1AR and beta2AR may play different roles in the pathogenesis of heart failure. Studies on neonatal cardiac myocytes from beta1AR and beta2AR knockout mice suggest that subtype-specific signaling is determined by subtype-specific membrane targeting and trafficking. Stimulation of beta2ARs has a biphasic effect on contraction rate, with an initial increase followed by a sustained Gi-dependent decrease. Recent studies show that a PDZ domain-binding motif at the carboxyl terminus of human beta2AR interacts with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, a PDZ-domain-containing protein. The human beta2AR carboxyl terminus also binds to N-ethylmaleimide-sensitive factor, which does not contain a PDZ domain. We found that mutation of the three carboxyl-terminal amino acids in the mouse beta2AR (beta2AR-AAA) disrupts recycling of the receptor after agonist-induced internalization in cardiac myocytes. Nevertheless, stimulation of the beta2AR-AAA produced a greater contraction rate increase than that of the wild-type beta2AR. This enhanced stimulation of contraction rate can be attributed in part to the failure of the beta2AR-AAA to couple to Gi. We also observed that coupling of endogenous, wild-type beta2AR to Gi in beta1AR knockout myocytes is inhibited by treatment with a membrane-permeable peptide representing the beta2AR carboxyl terminus. These studies demonstrate that association of the carboxyl terminus of the beta2AR with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, N-ethylmaleimide-sensitive factor, or some related proteins dictates physiologic signaling specificity and trafficking in cardiac myocytes.     

Mechanisms and time course of beta 1 adrenoceptor desensitisation in mammalian cardiac myocytes

To study the mechanisms and time course of beta1 adrenoceptor desensitisation in mammalian heart tissue neonatal rat cardiac myocytes (greater than 90% pure) were cultured in serum free medium. Cells were exposed to 1 mumol X litre-1 (-)-isoprenaline for 30 min, 4 h, and 16 h. In myocyte membranes mean(SEM)) 125I-iodocyanopindolol binding was 167(46) pmol X litre-1 (n = 5) and did not differ at 30 min, 4 h, or 16 h in control compared with (-)-isoprenaline treated cells. The maximum number of binding sites was 84(32) fmol X mg protein-1 and was unchanged at 30 min, but (-)-isoprenaline stimulated adenylate cyclase activity significantly decreased from 221(62) to 103(37) pmol X mg protein-1 30 min-1. (-)-Isoprenaline competition curves at 30 min showed a significant increase in the proportion of low affinity binding sites from 46% to 62% (n = 5). By 4 h the maximum number of binding sites was significantly decreased by 54%, adenylate cyclase activity remained depressed, and agonist affinity decreased threefold in the (-)-isoprenaline treated cells. At 16 h (-)-isoprenaline treated cells showed alterations similar to the 4 h values in the maximum number of binding sites, adenylate cyclase activity, and affinity for (-)-isoprenaline. (-)-Isoprenaline stimulated adenylate cyclase activity took 72 h to recover after desensitisation. Overnight ultracentrifugation of the cytosol showed a significant 40% increase in beta adrenoceptor density in cells exposed to (-)-isoprenaline for 4 h (n = 5), suggesting receptor internalisation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin signaling in the brain of adult female mice is required for sexual preference

A role for serotonin in male sexual preference was recently uncovered by our finding that male mutant mice lacking serotonin have lost sexual preference. Here we show that female mouse mutants lacking either central serotonergic neurons or serotonin prefer female over male genital odors when given a choice, and displayed increased female–female mounting when presented either with a choice of a male and a female target or only with a female target. Pharmacological manipulations and genetic rescue experiments showed that serotonin is required in adults. Behavioral changes caused by deficient serotonergic signaling were not due to changes in plasma concentrations of sex hormones. We demonstrate that a genetic manipulation reverses sexual preference without involving sex hormones. Our results indicate that serotonin controls sexual preference.     

Simultaneous absence of dopamine D1 and D2 receptor-mediated signaling is lethal in mice

Dopamine (DA) controls a wide variety of physiological functions in the central nervous system as well as in the neuroendocrine and gastrointestinal systems. DA signaling is mediated by five cloned receptors named D1-D5. Knockout mouse models for the five receptors have been generated, and, albeit impaired for some important DA-mediated functions, they are viable and can reproduce. D1 and D2 receptors are the most abundant and widely expressed DA receptors. Cooperative/synergistic effects mediated by these receptors have been suggested, in particular, in the control of motor behaviors. To analyze the extent of such interrelationship, we have generated double D1/D2 receptor mutants. Interestingly, in contrast to single knockouts, we found that concurrent ablation of the D1 and D2 receptors is lethal during the second or third week after birth. This dramatic phenotype is likely to be related to altered feeding behavior and dysfunction of the gastrointestinal system, especially because major anatomical changes were not identified in the brain. Similarly, in the absence of functional D1, heterozygous D2 mutants (D1r(-/-);D2r(+/-)) showed severe growth retardation and did not survive their postweaning period. The analysis of motor behavior in D1r/D2r compound mutants showed that loss of D2-mediated functions reduces motor abilities, whereas the effect of D1r ablation on locomotion strongly depends on the experimental paradigms used. These studies highlight the interrelationship between D1 and D2 receptor-mediated control of motor activity, food intake, and gastrointestinal functions, which has been elusive in the single-gene ablation studies.     

Lymphotoxin beta receptor signaling is required for inflammatory lymphangiogenesis in the thyroid

Infiltration of lymphocytes into the thyroid gland and formation of lymph node-like structures is a hallmark of Hashimoto's thyroiditis. Here we demonstrate that lymphatic vessels are present within these infiltrates. Mice overexpressing the chemokine CCL21 in the thyroid (TGCCL21 mice) developed similar lymphoid infiltrates and lymphatic vessels. TGCCL21 mice lacking mature T and B cells (RAGTGCCL21 mice) did not have cellular infiltrates or increased number of lymphatic vessels compared with controls. Transfer of CD3(+)CD4(+) T cells into RAGTGCCL21 mice promoted the development of LYVE-1(+)podoplanin(+)Prox-1(+) vessels in the thyroid. Genetic deletion of lymphotoxin beta receptor or lymphotoxin alpha abrogated development of lymphatic vessels in the inflamed areas in the thyroid but did not affect development of neighboring lymphatics. These results define a model for the study of inflammatory lymphangiogenesis in the thyroid and implicate lymphotoxin beta receptor signaling in this process.     

Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca current in cardiac myocytes

Endothelial nitric oxide synthase (NOS3) regulates the functional response to β-adrenergic (β-AR) stimulation via modulation of the L-type Ca²⁺ current (I_Ca). However, the NOS3 signaling pathway modulating I_Ca is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I_Ca. Myocytes were isolated from NOS3 knockout (NOS3^−/−) and wildtype (WT) mice. We measured I_Ca (whole-cell voltage-clamp), and simultaneously measured Ca²⁺ transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased β-AR stimulated (isoproterenol, ISO)-I_Ca, and Ca²⁺ transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I_Ca, but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3^−/− myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I_Ca, or Ca²⁺ transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I_Ca, and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I_Ca; YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca²⁺ transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I_Ca and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating β-AR stimulated I_Ca, while limiting effects on cardiac contraction.     

Intracellular ATP is required for mitochondrial apoptotic pathways in isolated hypoxic rat cardiac myocytes

OBJECTIVES: The present study examined the possibility that intracellular ATP levels dictate whether hypoxic cardiac myocytes die by apoptosis or necrosis. BACKGROUND: Although apoptosis and necrosis may appear to be distinct forms of cell death, recent studies suggest that the two may represent different outcomes of a common pathway. In ischemic myocardium, apoptosis appears early, while energy stores are presumably still available, followed only later by necrosis. METHODS: Neonatal rat cardiac myocytes were exposed to continuous hypoxia, during which the intracellular ATP concentration was modulated by varying the glucose content in the medium. The form of cell death was determined at the end of the hypoxic exposure. RESULTS: Under total glucose deprivation, ATP dropped precipitously and cell death occurred exclusively by necrosis as determined by nuclear staining with ethidium homodimer-1 and smearing on DNA agarose gels. However, with increasing glucose concentrations (10, 20, 50, 100 mg/dl) cellular ATP increased correspondingly, and apoptosis progressively replaced necrosis until it became the sole form of cell death, as determined by nuclear morphology, DNA fragmentation on agarose gels, and caspase-3 activation. The data showed a significantly positive correlation between myocyte ATP content and the percentage of apoptotic cells. Hypoxia resulted in lactate production and cellular acidification which stimulates apoptosis. However, acidification-induced apoptosis was also increased in an ATP-dependent fashion. Loss of mitochondrial membrane potential and cytochrome c release from the mitochondria was observed in both the apoptotic and necrotic cells. Furthermore, translocation of Bax from cytosol into mitochondria preceded these events associated with mitochondrial permeability transition. Increased lactate production and a lack of effect by the mitochondrial inhibitor oligomycin indicated that ATP was generated exclusively through glycolysis. CONCLUSIONS: We demonstrate that ATP, generated through glycolysis, is a critical determinant of the form of cell death in hypoxic myocytes, independently of cellular acidification. Our data suggest that necrosis and apoptosis represent different outcomes of the same pathway. In the absence of ATP, necrosis prevails. However, the presence of ATP favors and promotes apoptosis.     

Prefrontal D1 dopamine signaling is required for temporal control

Temporal control, or how organisms guide movements in time to achieve behavioral goals, depends on dopamine signaling. The medial prefrontal cortex controls many goal-directed behaviors and receives dopaminergic input primarily from the midbrain ventral tegmental area. However, this system has never been linked with temporal control. Here, we test the hypothesis that dopaminergic projections from the ventral tegmental area to the prefrontal cortex influence temporal control. Rodents were trained to perform a fixed-interval timing task with an interval of 20 s. We report several results: first, that decreasing dopaminergic neurotransmission using virally mediated RNA interference of tyrosine hydroxylase impaired temporal control, and second that pharmacological disruption of prefrontal D1 dopamine receptors, but not D2 dopamine receptors, impaired temporal control. We then used optogenetics to specifically and selectively manipulate prefrontal neurons expressing D1 dopamine receptors during fixed-interval timing performance. Selective inhibition of D1-expressing prefrontal neurons impaired fixed-interval timing, whereas stimulation made animals more efficient during task performance. These data provide evidence that ventral tegmental dopaminergic projections to the prefrontal cortex influence temporal control via D1 receptors. The results identify a critical circuit for temporal control of behavior that could serve as a target for the treatment of dopaminergic diseases.     

Localization of cardiac L-type Ca(2+) channels to a caveolar macromolecular signaling complex is required for beta(2)-adrenergic regulation

L-type Ca(2+) channels play a critical role in regulating Ca(2+)-dependent signaling in cardiac myocytes, including excitation-contraction coupling; however, the subcellular localization of cardiac L-type Ca(2+) channels and their regulation are incompletely understood. Caveolae are specialized microdomains of the plasmalemma rich in signaling molecules and supported by the structural protein caveolin-3 in muscle. Here we demonstrate that a subpopulation of L-type Ca(2+) channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for beta(2)-adrenergic receptor (AR) regulation of I(Ca,L). Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy detected extensive colocalization of caveolin-3 and the major pore-forming subunit of the L-type Ca channel (Ca(v)1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Ca(v)1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Ca(v)1.2, beta(2)-AR (not beta(1)-AR), G protein alpha(s), adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized beta(2)-AR/Ca(v)1.2 signaling complex, beta(2)-AR stimulation (salbutamol plus atenolol) of I(Ca,L) was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of I(Ca,L) in response to beta(2)-AR activation was eliminated by disruption of caveolae with 10 mM methyl beta-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas beta(1)-AR stimulation (norepinephrine plus prazosin) of I(Ca,L) was not altered. These findings demonstrate that subcellular localization of L-type Ca(2+) channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways.     

Cardiac conduction is required to preserve cardiac chamber morphology

Electrical cardiac forces have been previously hypothesized to play a significant role in cardiac morphogenesis and remodeling. In response to electrical forces, cultured cardiomyocytes rearrange their cytoskeletal structure and modify their gene expression profile. To translate such in vitro data to the intact heart, we used a collection of zebrafish cardiac mutants and transgenics to investigate whether cardiac conduction could influence in vivo cardiac morphogenesis independent of contractile forces. We show that the cardiac mutant dco^s226 develops heart failure and interrupted cardiac morphogenesis following uncoordinated ventricular contraction. Using in vivo optical mapping/calcium imaging, we determined that the dco cardiac phenotype was primarily due to aberrant ventricular conduction. Because cardiac contraction and intracardiac hemodynamic forces can also influence cardiac development, we further analyzed the dco phenotype in noncontractile hearts and observed that disorganized ventricular conduction could affect cardiomyocyte morphology and subsequent heart morphogenesis in the absence of contraction or flow. By positional cloning, we found that dco encodes Gja3/Cx46, a gap junction protein not previously implicated in heart formation or function. Detailed analysis of the mouse Cx46 mutant revealed the presence of cardiac conduction defects frequently associated with human heart failure. Overall, these in vivo studies indicate that cardiac electrical forces are required to preserve cardiac chamber morphology and may act as a key epigenetic factor in cardiac remodeling.     

Hic-5 is required for fetal gene expression and cytoskeletal organization of neonatal cardiac myocytes

Cardiac costameres link the extracellular matrix to the sarcomere at the z-disc and contain proteins such as integrins and other signaling molecules implicated in the regulation of pathological hypertrophy. Paxillin family members, hic-5 and paxillin, are scaffolding proteins associated with the integrin complex that have been shown to mediate numerous protein interactions in other cell types. While paxillin has been described in postnatal heart, hic-5 has not been identified. Our results provide evidence of hic-5 in neonatal cardiac myocytes co-localized with paxillin and α-actinin at the z-discs and the ends of actin filaments. Treatment with the hypertrophic agonist phenylephrine resulted in increased hic-5 expression while having no effect on paxillin levels. To see if increased hic-5 expression was sufficient to induce changes in cytoskeletal organization, hic-5 was overexpressed in myocytes by adenoviral infection. Hic-5 overexpression significantly increased the number of cells with organized cytoskeleton. Using siRNA mediated knockdown, we examined the requirement of hic-5 and paxillin in regulation of phenylephrine induced gene expression and cytoskeletal organization. Our results indicate that hic-5, not paxillin is required for upregulation of ANF and α-skeletal actin genes as well as in cytoskeletal reorganization. Finally, we demonstrated that hic-5 upregulation occurs downstream of MEK1/2-ERK1/2 signaling as inhibition of MEK1/2 using U0126 inhibitor completely inhibited hic-5 upregulation by PE. In a complimentary study, we showed that hic-5 knockdown had no effect on PE induced ERK1/2 phosphorylation. These findings demonstrate a novel role for hic-5 in the regulation of actin cytoskeleton and fetal gene expression.     

The effects of phospholipase A2 on beta adrenoceptor function in isolated cardiac cells

The role of phospholipids in the maintenance of beta-adrenoceptor function was investigated in isolated canine myocytes prepared from eight adult mongrel dogs by using collagenase. The characteristics of beta-adrenoceptors were assessed by determining the number and the affinity of receptors by a radioactive ligand binding assay using 125I-iodocyanopindolol. The increase in cyclic AMP content induced by isoproterenol or forskolin was also determined by radioimmunoassay with or without pretreatment with phospholipase (PLase) A2. The amount of free fatty acids released from isolated myocytes by PLase A2 was measured by high-performance liquid chromatography. PLase induced a significant decrease in the number of beta-adrenoceptors but did not affect their affinity. Although the isoproterenol-stimulated increase in cyclic AMP was significantly inhibited by the pretreatment with PLase A2, the forskolin-stimulated increase was not affected. Responsive accumulation of cyclic AMP to isoproterenol was much more impaired than the decrease in beta-adrenoceptor number. These results indicate that PLase A2 deteriorates the function of the adenylate cyclase system linked-beta-adrenoceptor, and suggest that PLase A2 affects both beta-adrenoceptors and the coupling of beta-adrenoceptors with adenylate cyclase.     

Syndecan 4 is required for endothelial alignment in flow and atheroprotective signaling

Atherosclerotic plaque localization correlates with regions of disturbed flow in which endothelial cells (ECs) align poorly, whereas sustained laminar flow correlates with cell alignment in the direction of flow and resistance to atherosclerosis. We now report that in hypercholesterolemic mice, deletion of syndecan 4 (S4^−/−) drastically increased atherosclerotic plaque burden with the appearance of plaque in normally resistant locations. Strikingly, ECs from the thoracic aortas of S4^−/− mice were poorly aligned in the direction of the flow. Depletion of S4 in human umbilical vein endothelial cells (HUVECs) using shRNA also inhibited flow-induced alignment in vitro, which was rescued by re-expression of S4. This effect was highly specific, as flow activation of VEGF receptor 2 and NF-κB was normal. S4-depleted ECs aligned in cyclic stretch and even elongated under flow, although nondirectionally. EC alignment was previously found to have a causal role in modulating activation of inflammatory versus antiinflammatory pathways by flow. Consistent with these results, S4-depleted HUVECs in long-term laminar flow showed increased activation of proinflammatory NF-κB and decreased induction of antiinflammatory kruppel-like factor (KLF) 2 and KLF4. Thus, S4 plays a critical role in sensing flow direction to promote cell alignment and inhibit atherosclerosis.Syndecan 4 (S4) is a transmembrane heparan sulfate proteoglycan that serves as a coreceptor for extracellular matrix proteins and growth factors (1–3). S4^−/− mice are viable and fertile (4, 5) but show defective wound healing consequent to impaired angiogenesis (6). They also have higher mortality after LPS injection (7) and exhibit defective muscle repair and myofiber organization as a result of inefficient differentiation and migration of muscle satellite cells (8). We and others have also demonstrated that S4 plays a critical role in the control of cell polarity, by controlling Rho GTPase activity (9–11), as well as in planar cell polarity (12). S4 has also been recently identified as a putative mechanosensor (13).Atherosclerosis is an inflammatory disease of large to midsized arteries that is the major cause of illness and death in developed nations and is rapidly increasing in developing nations (14, 15). It is linked to a variety of risk factors including high LDL cholesterol level and triglycerides, diabetes, smoking, hypertension, sedentary lifestyle, and inflammatory mediators. However, atherosclerotic lesions occur selectively in regions of arteries that are subject to disturbances in fluid shear stress (FSS), the frictional force flowing blood exerts on the endothelium. Regions of arteries with lower flow magnitude, flow reversal, and other complex spatial/temporal flow patterns are predisposed to atherosclerosis. Systemic risk factors appear to synergize with local biomechanical factors in the initiation and progression of atherosclerotic lesions (16).The importance of S4 in endothelial biology prompted us to test its role in atherogenesis. Surprisingly, S4 deletion not only drastically increased atherosclerotic plaque burden in hypercholesterolemic mice but also caused plaque to form in regions that are normally resistant to disease. These findings led us to investigate the role of S4 in flow signaling. Our results showed that S4 is specifically required in alignment of endothelial cells (ECs) in flow and suggest that loss of this atheroprotective mechanism leads to increased atherosclerosis in S4^−/− mice.     

Constitutive BDNF/TrkB signaling is required for normal cardiac contraction and relaxation

BDNF and its associated tropomyosin-related kinase receptor B (TrkB) nurture vessels and nerves serving the heart. However, the direct effect of BDNF/TrkB signaling on the myocardium is poorly understood. Here we report that cardiac-specific TrkB knockout mice (TrkB^−/−) display impaired cardiac contraction and relaxation, showing that BDNF/TrkB signaling acts constitutively to sustain in vivo myocardial performance. BDNF enhances normal cardiomyocyte Ca²⁺ cycling, contractility, and relaxation via Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Conversely, failing myocytes, which have increased truncated TrkB lacking tyrosine kinase activity and chronically activated CaMKII, are insensitive to BDNF. Thus, BDNF/TrkB signaling represents a previously unidentified pathway by which the peripheral nervous system directly and tonically influences myocardial function in parallel with β-adrenergic control. Deficits in this system are likely additional contributors to acute and chronic cardiac dysfunction.In the brain, BDNF has a pleiotropic profile, preserving cell viability and function, preventing neuronal degeneration during stress (1), and acting as an antidepressant (2). BDNF is also expressed in various nonneuronal tissues, including smooth and skeletal muscle cells, where it enhances airway smooth muscle cell contractility (3) and acts as a metabolic enhancer (4), respectively.In the mammalian heart, BDNF is essential for organ development because its genetic deletion leads to the thinning of cardiac chambers, microvascular leakage, and ultimately, early death in mice (5). In adult mammals, BDNF governs autonomic transmission to the heart (6) and exerts prominent angiogenic effects (7). Recent evidence also points to the presence of the BDNF receptor, tropomyosin-related kinase receptor B (TrkB), in the myocardium (8). However, the role of myocardial BDNF/TrkB signaling in cardiac physiology and myocardial response to pathologic stress, independent from its well-known trophic actions on blood vessels and autonomic efferent neurons (9), is largely unknown.In neurons, BDNF regulates Ca²⁺ signaling, and calmodulin-dependent protein kinase II (CaMKII) is one of the downstream effectors of the BDNF/TrkB signaling pathway. Because CaMKII is one of the important regulators of excitation–contraction coupling, and because BDNF also enhances smooth muscle contraction by augmenting intracellular Ca²⁺ signaling, we first determined the effects of BDNF on cardiomyocyte contractility, relaxation, and Ca²⁺ dynamics. Then, to establish whether BDNF/TrkB stimulation is relevant in maintaining basal cardiac function in vivo, we generated cardiac-specific TrkB^−/− mice by crossing conditional TrkB^−/− mice with MHC promoter-driven Cre mice and subjected these mice and their WT littermates to pressure–volume analysis. We further investigated which signaling pathway mediates the BDNF cardiac effects, and finally, we tested whether modulation of myocyte function by BDNF is preserved or altered in myocytes from failing hearts.