Identifying sex similarities and differences in structure and function of the sinoatrial node in the mouse heart.

Background: The sinoatrial node (SN) generates the heart rate (HR). Its spontaneous activity is regulated by a complex interplay between the modulation by the autonomic nervous system (ANS) and intrinsic factors including ion channels in SN cells. However, the systemic and intrinsic regulatory mechanisms are still poorly understood.

LRMP inhibits cAMP potentiation of HCN4 channels by disrupting intramolecular signal transduction.

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here, we identify the domains of LRMP essential for this regulation, show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating, and demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity.

LRMP inhibits cAMP potentiation of HCN4 channels by disrupting intramolecular signal transduction.

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4 but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here we identify the domains of LRMP essential for regulation.

The funny current If is essential for the fight-or-flight response in cardiac pacemaker cells.

The sympathetic nervous system fight-or-flight response is characterized by a rapid increase in heart rate, which is mediated by an increase in the spontaneous action potential (AP) firing rate of pacemaker cells in the sinoatrial node. Sympathetic neurons stimulate sinoatrial myocytes (SAMs) by activating β adrenergic receptors (βARs) and increasing cAMP. The funny current (If) is among the cAMP-sensitive currents in SAMs.

Mechanisms and significance of tissue-specific MICU regulation of the mitochondrial calcium uniporter complex.

Mitochondrial Ca uptake, mediated by the mitochondrial Ca uniporter, regulates oxidative phosphorylation, apoptosis, and intracellular Ca signaling. Previous studies suggest that non-neuronal uniporters are exclusively regulated by a MICU1-MICU2 heterodimer. Here, we show that skeletal-muscle and kidney uniporters also complex with a MICU1-MICU1 homodimer and that human/mouse cardiac uniporters are largely devoid of MICUs.

Cardiomyocyte-Specific Long Noncoding RNA Regulates Alternative Splicing of the Triadin Gene in the Heart.

Background: Abnormalities in Ca homeostasis are associated with cardiac arrhythmias and heart failure. Triadin plays an important role in Ca homeostasis in cardiomyocytes. Alternative splicing of a single gene produces multiple triadin isoforms.

Regulation of HCN Channels by Protein Interactions.

Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels are key regulators of subthreshold membrane potentials in excitable cells. The four mammalian HCN channel isoforms, HCN1-HCN4, are expressed throughout the body, where they contribute to diverse physiological processes including cardiac pacemaking, sleep-wakefulness cycles, memory, and somatic sensation. While all HCN channel isoforms produce currents when expressed by themselves, an emerging list of interacting proteins shape HCN channel excitability to influence the physiologically relevant output.

Bidirectional flow of the funny current (I) during the pacemaking cycle in murine sinoatrial node myocytes.

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (I) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify I during the cardiac cycle in mouse SAMs.

Intracellular Na Modulates Pacemaking Activity in Murine Sinoatrial Node Myocytes: An Analysis.

: The mechanisms underlying dysfunction in the sinoatrial node (SAN), the heart's primary pacemaker, are incompletely understood. Electrical and Ca-handling remodeling have been implicated in SAN dysfunction associated with heart failure, aging, and diabetes. Cardiomyocyte [Na] is also elevated in these diseases, where it contributes to arrhythmogenesis.

Isoform-specific regulation of HCN4 channels by a family of endoplasmic reticulum proteins.

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins.

Cardiac Pacemaker Activity and Aging.

A progressive decline in maximum heart rate (mHR) is a fundamental aspect of aging in humans and other mammals. This decrease in mHR is independent of gender, fitness, and lifestyle, affecting in equal measure women and men, athletes and couch potatoes, spinach eaters and fast food enthusiasts. Importantly, the decline in mHR is the major determinant of the age-dependent decline in aerobic capacity that ultimately limits functional independence for many older individuals.

Exploiting natural regulation.

Using a short peptide to regulate the activity of HCN ion channels illustrates how physiological modulators could inspire new drugs.

Mammalian γ2 AMPK regulates intrinsic heart rate.

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αβγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia).

Phosphodiesterases 3 and 4 Differentially Regulate the Funny Current, I, in Mouse Sinoatrial Node Myocytes.

Cardiac pacemaking, at rest and during the sympathetic fight-or-flight response, depends on cAMP (3',5'-cyclic adenosine monophosphate) signaling in sinoatrial node myocytes (SAMs). The cardiac "funny current" (I) is among the cAMP-sensitive effectors that drive pacemaking in SAMs. I is produced by hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels.

ParamAP: Standardized Parameterization of Sinoatrial Node Myocyte Action Potentials.

Sinoatrial node myocytes act as cardiac pacemaker cells by generating spontaneous action potentials (APs). Much information is encoded in sinoatrial AP waveforms, but both the analysis and the comparison of AP parameters between studies is hindered by the lack of standardized parameter definitions and the absence of automated analysis tools. Here we introduce ParamAP, a standalone cross-platform computational tool that uses a template-free detection algorithm to automatically identify and parameterize APs from text input files.

Cyclic AMP reverses the effects of aging on pacemaker activity and If in sinoatrial node myocytes.

Aerobic capacity decreases with age, in part because of an age-dependent decline in maximum heart rate (mHR) and a reduction in the intrinsic pacemaker activity of the sinoatrial node of the heart. Isolated sinoatrial node myocytes (SAMs) from aged mice have slower spontaneous action potential (AP) firing rates and a hyperpolarizing shift in the voltage dependence of activation of the "funny current," I Cyclic AMP (cAMP) is a critical modulator of both AP firing rate and I in SAMs. Here, we test the ability of endogenous and exogenous cAMP to overcome age-dependent changes in acutely isolated murine SAMs.

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice.

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive.

High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.

Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. However, current approaches are inefficient. Here we demonstrate that pro-fibrotic signalling potently antagonizes cardiac reprogramming.

Culture and adenoviral infection of sinoatrial node myocytes from adult mice.

Pacemaker myocytes in the sinoatrial node of the heart initiate each heartbeat by firing spontaneous action potentials. However, the molecular processes that underlie pacemaking are incompletely understood, in part because of our limited ability to manipulate protein expression within the native cellular context of sinoatrial node myocytes (SAMs). Here we describe a new method for the culture of fully differentiated SAMs from adult mice, and we demonstrate that robust expression of introduced proteins can be achieved within 24-48 h in vitro via adenoviral gene transfer.

Complete atrial-specific knockout of sodium-calcium exchange eliminates sinoatrial node pacemaker activity.

The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by "funny" current (If) 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.

Depressed pacemaker activity of sinoatrial node myocytes contributes to the age-dependent decline in maximum heart rate.

An inexorable decline in maximum heart rate (mHR) progressively limits human aerobic capacity with advancing age. This decrease in mHR results from an age-dependent reduction in "intrinsic heart rate" (iHR), which is measured during autonomic blockade. The reduced iHR indicates, by definition, that pacemaker function of the sinoatrial node is compromised during aging.

PKA-independent activation of I(f) by cAMP in mouse sinoatrial myocytes.

Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN4) channels produce the "funny current," I(f), which contributes to spontaneous pacemaking in sinoatrial myocytes (SAMs). The C-terminus of HCN channels inhibits voltage-dependent gating, and cAMP binding relieves this "autoinhibition." We previously showed 1) that autoinhibition in HCN4 can be relieved in the absence of cAMP in some cellular contexts and 2) that PKA is required for β adrenergic receptor (βAR) signaling to HCN4 in SAMs.

Cellular context and multiple channel domains determine cAMP sensitivity of HCN4 channels: ligand-independent relief of autoinhibition in HCN4.

Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels produce the I(f) and I(h) currents, which are critical for cardiac pacemaking and neuronal excitability, respectively. HCN channels are modulated by cyclic AMP (cAMP), which binds to a conserved cyclic nucleotide-binding domain (CNBD) in the C terminus. The unliganded CNBD has been shown to inhibit voltage-dependent gating of HCNs, and cAMP binding relieves this "autoinhibition," causing a depolarizing shift in the voltage dependence of activation.

Myristoylated peptides potentiate the funny current (I(f)) in sinoatrial myocytes.

The funny current, I(f), in sinoatrial myocytes is thought to contribute to the sympathetic fight-or-flight increase in heart rate. I(f) is produced by hyperpolarization-activated cyclic nucleotide sensitive-4 (HCN4) channels, and it is widely believed that sympathetic regulation of I(f) occurs via direct binding of cAMP to HCN4, independent of phosphorylation. However, we have recently shown that Protein Kinase A (PKA) activity is required for sympathetic regulation of I(f) and that PKA can directly phosphorylate HCN4.