Molecular basis of Period 1 regulation by adrenergic signaling in the heart.

The cardiac circadian clock is responsible for the modulation of different myocardial processes, and its dysregulation has been linked to disease development. How this clock machinery is regulated in the heart remains an open question. Because noradrenaline (NE) can act as a zeitgeber in cardiomyocytes, we tested the hypothesis that adrenergic signaling resets cardiac clock gene expression in vivo. In its anti-phase with Clock and Bmal1, cardiac Per1 abundance increased during the dark phase, concurrent with the rise in heart rate and preceded by an increase in NE levels. Sympathetic denervation altered Bmal1 and Clock amplitude, while Per1 was affected in both amplitude and oscillatory pattern. We next treated mice with a ß-adrenergic receptor (ß-AR) blocker. Strikingly, the ß-AR blockade during the day suppressed the nocturnal increase in Per1 mRNA, without altering Clock or Bmal1. In contrast, activating ß-AR with isoproterenol (ISO) promoted an increase in Per1 expression, demonstrating its responsiveness to adrenergic input. Inhibitors of ERK1/2 and CREB attenuated ISO-induced Per1 expression. Upstream of ERK1/2, PI3Kγ mediated ISO induction of Per1 transcription, while activation of ß2-AR, but not ß1-AR induced increases in ERK1/2 phosphorylation and Per1 expression. Consistent with the ß2-induction of Per1 mRNA, ISO failed to activate ERK1/2 and elevate Per1 in the heart of ß2-AR-/- mice, whereas a ß2-AR antagonist attenuated the nocturnal rise in Per1 expression. Our study established a link between NE/ß2-AR signaling and Per1 oscillation via the PI3Ky-ERK1/2-CREB pathway, providing a new framework for understanding the physiological mechanism involved in resetting cardiac clock genes.

Increased cholinergic activity under conditions of low estrogen leads to adverse cardiac remodeling.

Cholinesterase inhibitors are used in postmenopausal women for the treatment of neurodegenerative diseases. Despite their widespread use in the clinical practice, little is known about the impact of augmented cholinergic signaling on cardiac function under reduced estrogen conditions. To address this gap, we subjected a genetically engineered murine model of systemic vesicular acetylcholine transporter overexpression (Chat-ChR2) to ovariectomy and evaluated cardiac parameters. Left-ventricular function was similar between Chat-ChR2 and wild-type (WT) mice. Following ovariectomy, WT mice showed signs of cardiac hypertrophy. Conversely, ovariectomized (OVX) Chat-ChR2 mice evolved to cardiac dilation and failure. Transcript levels for cardiac stress markers atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were similarly upregulated in WT/OVX and Chat-ChR2/OVX mice. 17ß-Estradiol (E2) treatment normalized cardiac parameters in Chat-ChR2/OVX to the Chat-ChR2/SHAM levels, providing a link between E2 status and the aggravated cardiac response in this model. To investigate the cellular basis underlying the cardiac alterations, ventricular myocytes were isolated and their cellular area and contractility were assessed. Myocytes from WT/OVX mice were wider than WT/SHAM, an indicative of concentric hypertrophy, but their fractional shortening was similar. Conversely, Chat-ChR2/OVX myocytes were elongated and presented contractile dysfunction. E2 treatment again prevented the structural and functional changes in Chat-ChR2/OVX myocytes. We conclude that hypercholinergic mice under reduced estrogen conditions do not develop concentric hypertrophy, a critical compensatory adaptation, evolving toward cardiac dilation and failure. This study emphasizes the importance of understanding the consequences of cholinesterase inhibition, used clinically to treat dementia, for cardiac function in postmenopausal women.

Locus coeruleus noradrenaline depletion and its differential impact on CO2-induced panic and hyperventilation in male and female mice.

CO2 exposure has been used to investigate the panicogenic response in patients with panic disorder. These patients are more sensitive to CO2, and more likely to experience the "false suffocation alarm" which triggers panic attacks. Imbalances in locus coeruleus noradrenergic (LC-NA) neurotransmission are responsible for psychiatric disorders, including panic disorder. These neurons are sensitive to changes in CO2/pH. Therefore, we investigated if LC-NA neurons are differentially activated after severe hypercapnia in mice. Further, we evaluated the participation of LC-NA neurons in ventilatory and panic-like escape responses induced by 20% CO2 in male and female wild type mice and two mouse models of altered LC-NA synthesis. Hypercapnia activates the LC-NA neurons, with males presenting a heightened level of activation. Mutant males lacking or with reduced LC-NA synthesis showed hypoventilation, while animals lacking LC noradrenaline present an increased metabolic rate compared to wild type in normocapnia. When exposed to CO2, males lacking LC noradrenaline showed a lower respiratory frequency compared to control animals. On the other hand, females lacking LC noradrenaline presented a higher tidal volume. Nevertheless, no change in ventilation was observed in either sex. CO2 evoked an active escape response. Mice lacking LC noradrenaline had a blunted jumping response and an increased freezing duration compared to the other groups. They also presented fewer racing episodes compared to wild type animals, but not different from mice with reduced LC noradrenaline. These findings suggest that LC-NA has an important role in ventilatory and panic-like escape responses elicited by CO2 exposure in mice.

RFamide-related Peptide 3 Signaling via Neuropeptide FF Receptor Stimulates Prolactin Secretion in Female Rats.

The RF-amide peptides comprise a family of neuropeptides that includes the kisspeptin (Kp), the natural ligand of kisspeptin receptor (Kiss1r), and the RFamide-related peptide 3 (RFRP-3) that binds preferentially to the neuropeptide FF receptor 1 (Npffr1). Kp stimulates prolactin (PRL) secretion through the inhibition of tuberoinfundibular dopaminergic (TIDA) neurons. Because Kp also has affinity to Npffr1, we investigated the role of Npffr1 in the control of PRL secretion by Kp and RFRP-3. Intracerebroventricular (ICV) injection of Kp increased PRL and LH secretion in ovariectomized, estradiol-treated rats. The unselective Npffr1 antagonist RF9 prevented these responses, whereas the selective antagonist GJ14 altered PRL but not LH levels. The ICV injection of RFRP-3 in ovariectomized, estradiol-treated rats increased PRL secretion, which was associated with a rise in the dopaminergic activity in the median eminence, but had no effect on LH levels. The RFRP-3-induced increase in PRL secretion was prevented by GJ14. Moreover, the estradiol-induced PRL surge in female rats was blunted by GJ14, along with an amplification of the LH surge. Nevertheless, whole-cell patch clamp recordings showed no effect of RFRP-3 on the electrical activity of TIDA neurons in dopamine transporter-Cre recombinase transgenic female mice. We provide evidence that RFRP-3 binds to Npffr1 to stimulate PRL release, which plays a role in the estradiol-induced PRL surge. This effect of RFRP-3 is apparently not mediated by a reduction in the inhibitory tone of TIDA neurons but possibly involves the activation of a hypothalamic PRL-releasing factor.

Norepinephrine modulation of heat dissipation in female rats lacking estrogen.

Postmenopausal hot flushes are caused by lack of estradiol (E2) but their neuroendocrine basis is still poorly understood. Here, we investigated the interrelationship between norepinephrine and hypothalamic neurons, with emphasis on kisspeptin neurons in the arcuate nucleus (ARC), as a regulatory pathway in the vasomotor effects of E2. Ovariectomized (OVX) rats displayed increased tail skin temperature (TST), and this increase was prevented in OVX rats treated with E2 (OVX + E2). Expression of Fos in the hypothalamus and the number of ARC kisspeptin neurons coexpressing Fos were increased in OVX rats. Likewise, brainstem norepinephrine neurons of OVX rats displayed higher Fos immunoreactivity associated with the increase in TST. In the ARC, the density of dopamine-ß-hydroxylase (DBH)-immunoreactive (ir) fibers was not altered by E2 but, importantly, DBH-ir terminals were found in close apposition to kisspeptin cells, revealing norepinephrine inputs to ARC kisspeptin neurons. Intracerebroventricular injection of the α2-adrenergic agonist clonidine (CLO) was used to reduce central norepinephrine release, confirmed by the decreased 3-methoxy-4-hydroxyphenylglycol/norepinephrine ratio in the preoptic area and ARC. Accordingly, CLO treatment in OVX rats reduced ARC Kiss1 mRNA levels and TST to the values of OVX + E2 rats. Conversely, CLO stimulated Kiss1 expression in the anteroventral periventricular nucleus (AVPV) and increased luteinizing hormone secretion. These findings provide evidence that augmented heat dissipation in OVX rats involves the increase in central norepinephrine that modulates hypothalamic areas related to thermoregulation, including ARC kisspeptin neurons. This neuronal network is suppressed by E2 and its imbalance may be implicated in the vasomotor symptoms of postmenopausal hot flushes.

Reduced dopaminergic tone during lactation is permissive to the hypothalamic stimulus for suckling-induced prolactin release.

Dopamine from tuberoinfundibular dopaminergic (TIDA) neurones tonically inhibits prolactin (PRL) secretion. Lactational hyperprolactinaemia is associated with a reduced activity of TIDA neurones. However, it remains controversial whether the suckling-induced PRL surge is driven by an additional decrease in dopamine release or by stimulation from a PRL-releasing factor. In the present study, we further investigated the role of dopamine in the PRL response to suckling. Non-lactating (N-Lac), lactating 4 hour apart from pups (Lac), Lac with pups return and suckling (Lac+S), and post-lactating (P-Lac) rats were evaluated. PRL levels were elevated in Lac rats and increased linearly within 30 minutes of suckling in Lac+S rats. During the rise in PRL levels, dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the median eminence (ME) and neurointermediate lobe of the pituitary did not differ between Lac+S and Lac rats. However, dopamine and DOPAC were equally decreased in Lac and Lac+S compared to N-Lac and P-Lac rats. Suckling, in turn, reduced phosphorylation of tyrosine hydroxylase in the ME of Lac+S. Domperidone and bromocriptine were used to block and activate pituitary dopamine D2 receptors, respectively. Domperidone increased PRL secretion in both N-Lac and Lac rats, and suckling elicited a robust surge of PRL over the high basal levels in domperidone-treated Lac+S rats. Conversely, bromocriptine blocked the PRL response to suckling. The findings obtained in the present study provide evidence that dopamine synthesis and release are tonically reduced during lactation, whereas dopamine is still functional with respect to inhibiting PRL secretion. However, there appears to be no further reduction in dopamine release associated with the suckling-induced rise in PRL. Instead, the lower dopaminergic tone during lactation appears to be required to sensitise the pituitary to a suckling-induced PRL-releasing factor.