Cardiac pacemaking generation and modulation rely on the coordinated activity of several processes. Although a wealth of evidence indicates a relevant role of the I f ("funny," or pacemaker) current, whose molecular constituents are the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and particularly HCN4, work with mice where Hcn genes were knocked out, or functionally modified, has challenged this view. However, no previous studies used a cardiac-specific promoter to induce HCN4 ablation in adult mice. We report here that, in an inducible and cardiac-specific HCN4 knockout (ciHCN4-KO) mouse model, ablation of HCN4 consistently leads to progressive development of severe bradycardia (∼50% reduction of original rate) and AV block, eventually leading to heart arrest and death in about 5 d. In vitro analysis of sinoatrial node (SAN) myocytes isolated from ciHCN4-KO mice at the mean time of death revealed a strong reduction of both the I f current (by ∼70%) and of the spontaneous rate (by ∼60%). In agreement with functional results, immunofluorescence and Western blot analysis showed reduced expression of HCN4 protein in SAN tissue and cells. In ciHCN4-KO animals, the residual I f was normally sensitive to β-adrenergic receptor (β-AR) modulation, and the permanence of rate response to β-AR stimulation was observed both in vivo and in vitro. Our data show that cardiac HCN4 channels are essential for normal heart impulse generation and conduction in adult mice and support the notion that dysfunctional HCN4 channels can be a direct cause of rhythm disorders. This work contributes to identifying the molecular mechanism responsible for cardiac pacemaking.funny current | heart rate | sinoatrial node | atrioventricular node | chronotropism T he degree of complexity of the processes involved in pacemaking and their individual contributions is still a hotly debated issue (1, 2). Despite this complexity, and granting the fact that perturbation of any participating mechanism can affect rate, there is evidence for a functional specificity of "funny" (f)-channels in mediating generation and physiological control of pacemaker activity.Native f-channels are encoded by four hyperpolarizationactivated, cyclic nucleotide-gated channel genes (Hcn1-4), of which Hcn4 is by far the most highly expressed in the cardiac pacemaker regions of different species (3-6). HCN4 contributes 80% of total HCN mRNA in rabbit and mouse sinoatrial node (SAN), the remaining 20% being a combination of HCN2 and HCN1, with species-dependent relative abundance (4,5,(7)(8)(9). Evidence for the role of f/HCN4 channels in pacemaking relies on several experimental data (10):i) The "funny" (I f ) current and pacemaker activity are correlated; i.e., functional expression of f-channels is restricted to pacemaking regions [SAN, atrioventricular node (AVN), and the ventricular conduction system], demonstrating a commitment to regulation of pacemaker activity. This correlation exists not only in the adult but also throughout development, such that ...

Abstract-Ventricular pacemaker current (I f ) shows distinct voltage dependence as a function of age, activating outside the physiological range in normal adult ventricle, but less negatively in neonatal ventricle. However, heterologously expressed HCN2 and HCN4, the putative molecular correlates of ventricular I f , exhibit only a modest difference in activation voltage. We therefore prepared an adenoviral construct (AdHCN2) of HCN2, the dominant ventricular isoform at either age, and used it to infect neonatal and adult rat ventricular myocytes to investigate the role of maturation on current gating. The expressed current exhibited an 18-mV difference in activation (V 1/2 Ϫ95.9Ϯ1.9 in adult; Ϫ77.6Ϯ1.6 mV in neonate), comparable to the 22-mV difference between native I f in adult and neonatal cultures (V 1/2 Ϫ98.7 versus Ϫ77.0 mV). This did not result from developmental differences in basal cAMP, because saturating cAMP in the pipette caused an equivalent positive shift in both preparations. In the neonate, AdHCN2 caused a significant increase in spontaneous rate compared with control (88Ϯ5 versus 48Ϯ4 bpm). In adult, where HCN2 activates more negatively, the effect was evident only during anodal excitation, requiring significantly less stimulus energy than control (2149Ϯ266 versus 3140Ϯ279 mV ⅐ ms).

Abstract-Lipid rafts are discrete membrane subdomains rich in sphingolipids and cholesterol. In ventricular myocytes a function of caveolae, a type of lipid rafts, is to concentrate in close proximity several proteins of the ␤-adrenergic transduction pathway. We have investigated the subcellular localization of HCN4 channels expressed in HEK cells and studied the effects of such localization on the properties of pacemaker channels in HEK and rabbit sinoatrial (SAN) cells. We used a discontinuous sucrose gradient and Western blot analysis to detect HCN4 proteins in HEK and in SAN cells, and found that HCN4 proteins localize to low-density membrane fractions together with flotillin (HEK) or caveolin-3 (SAN), structural proteins of caveolae. Lipid raft disruption by cell incubation with methyl-␤-cyclodextrin (M␤CD) impaired specific HCN4 localization. It also shifted the midpoint of activation of the HCN4 current in HEK cells and of I f in SAN cells to the positive direction by 11.9 and 10.4 mV, respectively. These latter effects were not due to elevation of basal cyclic nucleotide levels because the cholesterol-depletion treatment did not alter the current response to cyclic nucleotides. In accordance with an increased I f , M␤CD-treated SAN cells showed large increases of diastolic depolarization slope (87%) and rate (58%). We also found that the kinetics of HCN4-and native f-channel deactivation were slower after lipid raft disorganization. In conclusion, our work indicates that pacemaker channels localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties. Key Words: HCN channels Ⅲ pacemaker current Ⅲ lipid rafts Ⅲ caveolin Ⅲ sinoatrial node T he sinoatrial node (SAN) is the region of the heart from which spontaneous action potentials originate and propagate to determine cardiac rhythm. This particular anatomical district is composed by specialized myocytes (pacemaker cells) whose activity is characterized by a slow diastolic depolarization phase at the end of the action potential. The pacemaker (I f ) current plays a key role in the generation of diastolic depolarization. f-Channels open toward the end of the action potential repolarization process and carry an inward current that depolarizes the membrane and drives the membrane potential up to threshold for initiating a new action potential. Although spontaneous activity is an intrinsic property of the heart, independent of innervation, fine modulation of heart rate is achieved through the release of the neurotransmitters norepinephrine (NE) and acetylcholine (ACh) by sympathetic and parasympathetic branches of the autonomic nervous system. It is well established that in cardiac cells NE and ACh, through specific ␤-adrenergic (␤-AR) and muscarinic (mAChR) receptors, modulate intracellular level of cAMP. Direct binding of cAMP to f-channels shifts their activation curve toward more depolarized potentials, thus increasing the steepness of the diastolic depolarization. 1 cAM...

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Our findings demonstrate that although CStC and BMStC share a common stromal phenotype, CStC present cardiovascular-associated features and may represent an important cell source for more efficient cardiac repair.

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltagegated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin ؉ ), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin ؊ ) indicating that interaction with filamin A affects membrane localization. Also, in filamin ؊ cells the gating kinetics of HCN1 were strongly accelerated relative to filamin ؉ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.

Different cardiac stem/progenitor cells have been recently identified in the post-natal heart. We describe here the identification, clonal expansion and characterization of self-renewing progenitors that differ from those previously described for high spontaneous cardiac differentiation. Unique coexpression of endothelial and pericyte markers identify these cells as cardiac mesoangioblasts and allow prospective isolation and clonal expansion from the juvenile mouse ventricle. Cardiac mesoangioblasts express many cardiac transcription factors and spontaneously differentiate into beating cardiomyocytes that assemble mature sarcomeres and express typical cardiac ion channels. Cells similarly isolated from the atrium do not spontaneously differentiate. When injected into the ventricle after coronary artery ligation, cardiac mesoangioblasts efficiently generate new myocardium in the peripheral area of the necrotic zone, as they do when grafted in the embryonic chick heart. These data identify cardiac mesoangioblasts as committed progenitors, downstream of earlier stem/progenitor cells and suitable for the cell therapy of a subset of juvenile cardiac diseases. Cell Death and Differentiation (2008) Several acute or chronic cardiac diseases are characterized by progressive expansion of the left ventricular chamber, with replacement by fibrous deposition in the ventricular wall. One approach proposed for reverse myocardial remodeling is regeneration of cardiac myocytes using stem cells.1 On the basis of distinct cell surface markers such as Sca-1 or c-Kit, different cardiac stem-like cells have been isolated that can restore cardiac function after ischemic injury.2,3 None of these cells shows spontaneous cardiac differentiation and they also differentiate into other tissue types of the heart.2-5 On the other hand, Isl-1 expressing progenitors appear to be committed to cardiac differentiation but still require interactions with other cells for both proliferation and differentiation. 4 It is also becoming clear that a significant part of the beneficial effect that most of these cells exert on the infarcted heart is due to the secretion of factors that increase survival of residual myocardium and/or favor angiogenesis. 6 This was for example the case of embryonic mesoangioblasts whose transplantation resulted in a 50% recovery of cardiac function but whose differentiation into new cardiomyocytes was rare. 7Our recent work on mesoangioblasts isolated from postnatal skeletal muscle, 8,9 indicated that these cells, possibly because of a local commitment, exhibit efficient differentiation into skeletal muscle. On this basis, we isolated mesoangioblast-like cells from different regions of the post-natal mouse heart.Here we describe the isolation, through a specific explant culture method, of self-renewing committed cardiac progenitors from different regions of the juvenile heart. These cells, operationally termed 'cardiac mesoangioblasts', show a unique phenotype and high spontaneous cardiomyocyte differentiation; they can be e...

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