Significance: Maximum heart rate (mHR) declines with age, contributing to the reduced aerobic capacity of the elderly. A parallel reduction in "intrinsic heart rate" (which is measured during autonomic blockade) underlies the decline in mHR, and indicates that pacemaker function of the sinoatrial node is compromised during aging. In this study we demonstrate that the slower heart rates in the elderly result from depressed spontaneous activity of individual sinoatrial node myocytes (SAMs). Patch-clamp electrophysiology revealed that aged SAMs have slowed AP firing rates, altered AP waveforms, and changed properties of Ca ²⁺ currents and the cardiac "funny current,"I _f . Our findings demonstrate that age-dependent changes ion channel activity in SAMs are a major cause of the decline in mHR during aging.

The sympathetic nervous system increases heart rate by activating β adrenergic receptors and increasing cAMP levels in myocytes in the sinoatrial node. The molecular basis for this response is not well understood; however, the cardiac funny current (If) is thought to be among the end effectors for cAMP signaling in sinoatrial myocytes. If is produced by hyperpolarization-activated cyclic nucleotide–sensitive (HCN4) channels, which can be potentiated by direct binding of cAMP to a conserved cyclic nucleotide binding domain in the C terminus of the channels. β adrenergic regulation of If in the sinoatrial node is thought to occur via this direct binding mechanism, independent of phosphorylation. Here, we have investigated whether the cAMP-activated protein kinase (PKA) can also regulate sinoatrial HCN4 channels. We found that inhibition of PKA significantly reduced the ability of β adrenergic agonists to shift the voltage dependence of If in isolated sinoatrial myocytes from mice. PKA also shifted the voltage dependence of activation to more positive potentials for heterologously expressed HCN4 channels. In vitro phosphorylation assays and mass spectrometry revealed that PKA can directly phosphorylate at least 13 sites on HCN4, including at least three residues in the N terminus and at least 10 in the C terminus. Functional analysis of truncated and alanine-substituted HCN4 channels identified a PKA regulatory site in the distal C terminus of HCN4, which is required for PKA modulation of If. Collectively, these data show that native and expressed HCN4 channels can be regulated by PKA, and raise the possibility that this mechanism could contribute to sympathetic regulation of heart rate.

Serotonin [5-hydroxytryptamine (5-HT)], an important neurotransmitter and a paracrine messenger in the gastrointestinal tract, regulates intestinal secretion by its action primarily on 5-HT₃and 5-HT₄receptors. Recent studies highlight the role of gut microbiota in 5-HT biosynthesis. In this study, we determine whether human-derived gut microbiota affects host secretory response to 5-HT and 5-HT receptor expression. We used proximal colonic mucosa-submucosa preparation from age-matched Swiss Webster germ-free (GF) and humanized (HM; ex-GF colonized with human gut microbiota) mice. 5-HT evoked a significantly greater increase in short-circuit current (Δ I_sc) in GF compared with HM mice. Additionally, 5-HT₃receptor mRNA and protein expression was significantly higher in GF compared with HM mice. Ondansetron, a 5-HT₃receptor antagonist, inhibited 5-HT-evoked Δ I_scin GF mice but not in HM mice. Furthermore, a 5-HT₃receptor-selective agonist, 2-methyl-5-hydroxytryptamine hydrochloride, evoked a significantly higher Δ I_scin GF compared with HM mice. Immunohistochemistry in 5-HT_3A-green fluorescent protein mice localized 5-HT₃receptor expression to enterochromaffin cells in addition to nerve fibers. The significant difference in 5-HT-evoked Δ I_scbetween GF and HM mice persisted in the presence of tetrodotoxin (TTX) but was lost after ondansetron application in the presence of TTX. Application of acetate (10 mM) significantly lowered 5-HT₃receptor mRNA in GF mouse colonoids. We conclude that host secretory response to 5-HT may be modulated by gut microbiota regulation of 5-HT₃receptor expression via acetate production. Epithelial 5-HT₃receptor may function as a mediator of gut microbiota-driven change in intestinal secretion.NEW & NOTEWORTHY We found that gut microbiota alters serotonin (5-HT)-evoked intestinal secretion in a 5-HT₃receptor-dependent mechanism and gut microbiota metabolite acetate alters 5-HT₃receptor expression in colonoids.View this article's corresponding video summary at https://www.youtube.com/watch?v=aOMYJMuLTcw&feature=youtu.be .

Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT₃receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT_3Apromoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR andin situhybridization confirmed the presence of 5-HT_3AmRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT_3A-driven GFP respond to 10 μm5-HT and this response is blocked by 1 μmondansetron, a 5-HT₃antagonist, and mimicked by application of 10 μmm-chlorophenylbiguanide, a 5-HT₃agonist. Pharmacological blockade of 5-HT₃receptorsin vivoor genetic deletion of the 5-HT₃receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT₃receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT₃receptors, accounting for a significant proportion of the neural taste response.SIGNIFICANCE STATEMENTHistorically, serotonin (5-hydroxytryptamine; 5-HT) has been described as a candidate neurotransmitter in the gustatory system and recent studies show that type III taste receptor cells release 5-HT in response to various taste stimuli. In the present study, we demonstrate that a subset of gustatory sensory neurons express functional 5-HT₃receptors that play a significant role in the neurotransmission of taste information from taste buds to nerves. In addition, we show that the anesthetic pentobarbital, widely used in taste nerve recordings, blocks 5-HT₃signaling. Therefore, many conclusions drawn from those data need to be reexamined in light of this anesthetic effect.

The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by “funny” current (I_f) through HCN4 channels (the “Membrane Clock“ hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two (“Coupled Clock”). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of I_f in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.