An in-vitro study on effects of laser activation on dye penetration in human root dentin.

Objective: To evaluate the penetration of a dye in root dentin after activation with different laser wavelengths. Materials: Palatal roots of 38 human molars were enlarged and disinfected. Irrigation activation was performed with an Er:YAG laser: @50 mJ, 15 Hz (Er:YAG); a 9.3 µm CO2 laser: @40% power (CO2); diode lasers 455 nm/970 nm: @0.8 W, 15 Hz (D455, D970) and 808/980 nm: @1 W (D808, D980) and compared to positive control: etching with 35% H3PO4 (POS); negative control: water (NEG) and conventional needle irrigation: NaOCl and ethylenediaminetetraacetic acid (EDTA) (CONV). Methylene blue solution was introduced in the canal and laser-activated or left untouched for 100 s before the roots were dried and cut into horizontal slices. Dye penetration was automatically calculated by color recognition of two samples per root third (n = 8 per group in each coronal, middle and apical root thirds). The presence and absence of a smear layer was checked in two additional samples of the negative and positive control under scanning electron microscopy (SEM). Results: Full-depth infiltration was not achieved in any group. Dye penetration in CONV was significantly less than in Er:YAG, CO2, POS, D455, D970, D808 and similar to NEG and D980 when results of different root thirds were pooled. Conclusion: Laser activation using certain parameters enhanced dye penetration compared to conventional needle irrigation with NaOCl and EDTA (CONV).

Activation of endodontic irrigants using a 9.3 µm CO2 and diode lasers: A laboratory proof of concept model.

PURPOSE: To investigate the differences between irrigant propagation and temperature changes using laser-activated irrigation (LAI) at different settings in an artificial root canal model. METHODS: Using an artificial resin root canal model, irrigant activation was achieved in 19 experimental groups with eight samples each. A 9,300 nm CO2 laser, two diode lasers with different settings (wavelengths 455, 808, 970, and 980 nm) were compared to 2,940 nm Er:YAG laser and traditional needle irrigation. Er:YAG and CO2 laser were activated in the pulpal chamber only, while diode lasers and needles were inserted into the main root canal. Lasers were activated for 5x 20 seconds resulting in 100 seconds of activation or rinsing for each sample. After each activation of 20 seconds, a photo was taken of the side canals and the propagation of the dye was measured with a digital measuring tool after calibration. Further, the temperature of the irrigant was reported after activation of 20 seconds and repeated 5 times. Data were checked for normality and statistically compared. RESULTS: All lasers increased the irrigant propagation compared to conventional irrigation. Significant differences were found between groups regarding propagation and temperature (P< 0.0027). Er:YAG and CO2 laser had similar effects on irrigant propagation in middle and apical located side-canals with specific power parameters and were superior to diode lasers and syringe irrigation. The irrigant's temperature increased significantly with the diode and CO2 lasers. CLINICAL SIGNIFICANCE: Diode lasers and CO2 lasers have not been established for irrigant activation. 9,300 nm CO2 lasers absorb well in water and were shown to introduce vapor bubble formation and streaming in water. Diode lasers are highly accepted in periodontics. The laser light is not absorbed in water but interacts with bacteria as well as soft tissues and contributes therefore to infection control. With a modified laser tip it was however possible to introduce cavitation and streaming in irrigants.

3D-printed TCP-HA scaffolds delivering MicroRNA-302a-3p improve bone regeneration in a mouse calvarial model.

OBJECTIVE: To demonstrate hydroxyapatite nanoparticles modified with cationic functional molecules. 3-aminopropyltriethoxysilane (HA-NPs-APTES) carrying microRNA-302a-3p (miR) in the 3D-printed tricalcium phosphate/Hydroxyapatite (TCP/HA) scaffold can increase healing of the critical-sized bone defect. MATERIALS AND METHODS: 3D-printed TCP/HA were modified with HA-NPs-APTES by two methods (M1, M2). The dispersion of particles was visualized by fluorescent microscopy. Biocompatibility of the scaffolds was tested by alizarin assay. Delivery of miR to the cells and osteogenic gene expression were evaluated by qPCR. After selecting best method (M2), scaffolds, scaffolds+HA-NPs-APTES with or without miR were implanted in 4 mm mouse calvarium defect (n = 4 per group). After 2,4 and 6 weeks, bone regeneration were evaluated by microCT and histology sections. RESULTS: Both M1 and M2 scaffolds were biocompatible with cell adhesion on its surface. M2 scaffold showed significant increase of miR, suggesting successful delivery, resulted in downregulation of its target mRNA COUP-TFII, and upregulation of RUNX2 mRNA. Calvarium defect with M2 scaffold also showed significantly higher BV/TV and higher number of filled spaces at all time points. Histomorphometry demonstrated new bone formed at the center of the HA-NPs-APTES-miR scaffold earlier than controls. CONCLUSION: TCP/HA scaffold modified with HA-NPs-APTES facilitated delivery of miR and enhanced bone regeneration.

Alveolar ridge augmentation with 3D-printed synthetic bone blocks: A clinical case series.

This report documents the clinical and histological outcome of 3D-printed calcium phosphate blocks placed in two-stage procedures to successfully rehabilitate atrophic alveolar ridges. This approach yielded a functionally favorable result. Histological evaluations were performed after healing periods of 6 months and showed ongoing bone regeneration and sprouting capillaries.

Adipose-Derived Stromal Cells within a Gelatin Matrix Acquire Enhanced Regenerative and Angiogenic Properties: A Pre-Clinical Study for Application to Chronic Wounds.

This study evaluates the influence of a gelatin sponge on adipose-derived stromal cells (ASC). Transcriptomic data revealed that, compared to ASC in a monolayer, a cross-linked porcine gelatin sponge strongly influences the transcriptome of ASC. Wound healing genes were massively regulated, notably with the inflammatory and angiogenic factors. Proteomics on conditioned media showed that gelatin also acted as a concentrator and reservoir of the regenerative ASC secretome. This secretome promoted fibroblast survival and epithelialization, and significantly increased the migration and tubular assembly of endothelial cells within fibronectin. ASC in gelatin on a chick chorioallantoic membrane were more connected to vessels than an empty sponge, confirming an increased angiogenesis in vivo. No tumor formation was observed in immunodeficient nude mice to which an ASC gelatin sponge was transplanted subcutaneously. Finally, ASC in a gelatin sponge prepared from outbred rats accelerated closure and re-vascularization of ischemic wounds in the footpads of rats. In conclusion, we provide here preclinical evidence that a cross-linked porcine gelatin sponge is an optimal carrier to concentrate and increase the regenerative activity of ASC, notably angiogenic. This formulation of ASC represents an optimal, convenient and clinically compliant option for the delivery of ASC on ischemic wounds.

Delivery of microRNA-302a-3p by APTES modified hydroxyapatite nanoparticles to promote osteogenic differentiation in vitro.

OBJECTIVE: To demonstrate the miRNA delivery by hydroxyapatite nanoparticles modified with APTES (HA-NPs-APTES) and promote osteogenic gene expression. MATERIALS AND METHODS: Osteosarcoma cells (HOS, MG-63) and primary human mandibular osteoblasts (HmOBs) were co-cultured with HA-NPs-APTES conjugated with miRNA-302a-3p. Resazurin reduction assay was performed to evaluate HA-NPs-APTES biocompatibility. Intracellular uptake was demonstrated by confocal fluorescent and scanning electron microscopy. The miRNA-302a-3p and its mRNA targets expression levels including COUP-TFII and other osteogenic genes were assessed by qPCR on day1 or day5 post-delivery. Calcium deposition induced by the osteogenic gene upregulation was shown by alizarin red staining on day7 and 14 post-delivery. RESULTS: Proliferation of HOS cells treated with HA-NPs-APTES was similar to that of untreated cells. HA-NPs-APTES was visualized in cell cytoplasm within 24 hours. MiRNA-302a-3p level was upregulated in HOS, MG-63 and HmOBs as compared to untreated cells. As a result, COUP-TFII mRNA expression was reduced, followed by an increase of RUNX2 and other osteogenic genes mRNA expression. Calcium deposition induced by HA-NPs-APTES-miR-302a-3p in HmOBs was significantly higher than in untreated cells. CONCLUSION: HA-NPs-APTES may support the delivery of miRNA-302a-3p into bone cells, as assessed by osteogenic gene expression and differentiation improvement once this combination is used on osteoblast cultures.

Identification of Type-H-like Blood Vessels in a Dynamic and Controlled Model of Osteogenesis in Rabbit Calvarium.

Angiogenesis and bone regeneration are closely interconnected processes. Whereas type-H blood vessels are abundantly found in the osteogenic zones during endochondral long bone development, their presence in flat bones' development involving intramembranous mechanisms remains unclear. Here, we hypothesized that type-H-like capillaries that highly express CD31 and Endomucin (EMCN), may be present at sites of intramembranous bone development and participate in the control of osteogenesis. A rabbit model of calvarial bone augmentation was used in which bone growth was controlled over time (2-4 weeks) using a particulate bone scaffold. The model allowed the visualization of the entire spectrum of stages throughout bone growth in the same sample, i.e., active ossification, osteogenic activity, and controlled inflammation. Using systematic mRNA hybridization, the formation of capillaries subpopulations (CD31-EMCN staining) over time was studied and correlated with the presence of osteogenic precursors (Osterix staining). Type-H-like capillaries strongly expressing CD31 and EMCN were identified and described. Their presence increased gradually from the regenerative zone up to the osteogenic zone, at 2 and 4 weeks. Type-H-like capillaries may thus represent the initial vascular support encountered in flat bones' development and which organize osteogenic niches.

Pre-Treat Xenogenic Collagenous Blocks of Bone Substitutes with Saline Facilitate Their Manipulation and Guarantee High Bone Regeneration Rates, Qualitatively and Quantitatively.

Deproteinized bovine bone mineral particles embedded in collagen (DBBM-C) are widely used for bone regenerations with excellent, albeit sometimes variable clinical outcomes. Clinicians usually prepare DBBM-C by mixing with blood. Replacing blood by saline represents an alternative. We investigated if saline treatment could improve DBBM-C i. handling in vitro and ii. biological performances in a rabbit calvarial model. In vitro, DBBM-C blocks soaked in saline or blood were submitted to compression tests. In vivo, four poly ether ether ketone (PEEK)cylinders were placed on 16 rabbit skulls, filled with DBBM-C soaked in blood or saline for 2-4-8-12 weeks before histomorphometry. DBBM-C blocks were fully hydrated after 30 s in saline when 120 s in blood could not hydrate blocks core. Stiffness gradually decreased 2.5-fold after blood soaking whereas a six-fold decrease was measured after 30 s in saline. In vivo, saline treatment allowed 50% more bone regeneration during the first month when compared to blood soaking. This difference was then no longer visible. New bone morphology and maturity were equivalent in both conditions. DBBM-C saline-soaking facilitated its handling and accelerated bone regeneration of highly qualitative tissues when compared to blood treatment. Saline pretreatment thus may increase the clinical predictability of bone augmentation procedures.

Evaluation of the Validity of Digital Optical Microscopy in the Assessment of Marginal Adaptation of Dental Adhesive Interfaces.

Analysis of marginal adaptation of dental adhesive interfaces using scanning electron microscopy has proven to be a powerful nondestructive method to evaluate the quality of adhesion. This methodology is, however, time-consuming and needs expensive equipment. The purpose of this study was to evaluate the possibility and efficiency of using a digital optical microscope (DOM) to perform marginal analysis and to compare it with the scanning electron microscope (SEM) analysis. Fifteen defect-free molars were selected for this study. Class V cavities were prepared and restored with resin composite, and epoxy replicas were obtained from silicone impressions of the restored teeth. Custom-made image analysis software was then used to measure the percentage of the noncontinuous margins (NCM) of each sample. To compare the DOM to the gold standard, SEM, each sample was analyzed 10 times using the DOM and three times using the SEM, by the same experienced operator. The repeatability coefficient and concordance were evaluated, and a Bland and Altman analysis was used for comparison of the two methods of measurements. To validate the DOM analysis method, an ANOVA approach (Gage R R) was used. Repeatability and reproducibility, which are two components of precision to validate the DOM analysis system, were calculated. For this, the same restorations were analyzed by two additional operators three times with the DOM. The duration of each step of the analysis using both methods was also recorded as a secondary outcome. Regarding the repeatability of each method, the Friedman test showed no statistically significant difference within the repetitions of measurements by SEM and DOM (p = 0.523 and p = 0.123, respectively). Moreover, the Bland-Altman analysis revealed a bias of 0.86 and concluded no statistically significant difference between the two methods, DOM and SEM. ANOVA evaluated DOM measurement system variation including the variances of repeatability and reproducibility that represented, respectively, 0.3% and 4% of the variance components. Reproducibility or inter-operator variability represented the principal source of variability with a statistically significant difference (p = 0.024). The time required for analysis with SEM was almost double that of DOM. The use of digital optical microscopy appears to be a valid alternative to the SEM for the analysis of marginal adaptation of dental adhesive interfaces. Further studies to evaluate the effect of training of operators in digital optical microscopy could reveal higher accuracy for this method and inter-operator agreement when experience is gained.

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Translational research on clinically failed zirconia implants.

OBJECTIVES: To provide fractographic analysis of clinically fractured zirconia implants recovered with their cemented crown. To calculate bending moments, corresponding stress and crack onset location on the implant's fracture surface using a mathematical model integrating spatial coordinates of the crown-implant part and occlusal loading obtained from 2D and 3D images. METHODS: 15 fractured zirconia implants parts (11 posterior and 4 anterior) with their all- ceramic crowns still cemented on it were recovered. The implants were first generations from four manufacturers (AXIS Biodental, Z-Systems, Straumann, Swiss Dental Solutions). The time-to-failure varied between 2weeks and 9years. Fractography was performed identifying the failure origin and characteristic surface crack features. From 2D and 3D digital images of the crown-implant part, spatial coordinates anchoring the crown's occlusal contacts with the implant's central axis and reference plane were integrated in a mathematical model spreadsheet. Loads of 500 N in total were selectively distributed over identified occlusal contacts from wear patterns. The resultant bending and torsion moments, corresponding shear, tensile, maximum principal stress and von Mises stress were calculated. The fracture crack onset location on the implant's fracture surface was given by an angular position with respect to an occlusal reference and compared with the location of the fracture origin identified from fractographic analysis. RESULTS: Implants fractured from the periphery of the smaller inner diameter between two threads at the bone-entrance level except for one implant which failed half-way within the bone. The porous coating (AXIS Biodental) and the large grit alumina sandblasting (Z-System) created surface defects directly related to the fracture origin. The model spreadsheet showed how occlusal loading with respect to the implant's central axis affects bending moments and crack onset. Dominant loads distributed on contacts with important wear pattern provided a calculated crack onset location in good agreement with the fractographic findings of the fracture origin. SIGNIFICANCE: Recovered broken zirconia implant parts with their restorative crowns can provide not only information regarding the failure origin using fractography but also knowledge regarding occlusal crown loading with respect to the implant's axis. The mathematical model was helpful in showing how occlusal loading affects the location of the fracture initiation site on clinical zirconia implant fracture cases.

G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block.

Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies "sick sinus" syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Cav1.3 Ca(2+) channels (Cav1.3(-/-)) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K(+) channel (IKACh) could rescue SSS and heart block in Cav1.3(-/-) mice. We found that genetic inactivation of IKACh abolished SSS symptoms in Cav1.3(-/-) mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of IKACh either in Cav1.3(-/-) mice or following selective inhibition of Cav1.3-mediated L-type Ca(2+) (ICa,L) current in vivo. Ablation of IKACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from IKACh suppression achieved by gene therapy or selective pharmacological inhibition.

Cardiac arrhythmia induced by genetic silencing of 'funny' (f) channels is rescued by GIRK4 inactivation.

The mechanisms underlying cardiac automaticity are still incompletely understood and controversial. Here we report the complete conditional and time-controlled silencing of the 'funny' current (If) by expression of a dominant-negative, non-conductive HCN4-channel subunit (hHCN4-AYA). Heart-specific If silencing caused altered [Ca(2+)]i release and Ca(2+) handling in the sinoatrial node, impaired pacemaker activity and symptoms reminiscent of severe human disease of pacemaking. The effects of If silencing critically depended on the activity of the autonomic nervous system. We were able to rescue the failure of impulse generation and conduction by additional genetic deletion of cardiac muscarinic G-protein-activated (GIRK4) channels in If-deficient mice without impairing heartbeat regulation. Our study establishes the role of f-channels in cardiac automaticity and indicates that arrhythmia related to HCN loss-of-function may be managed by pharmacological or genetic inhibition of GIRK4 channels, thus offering a new therapeutic strategy for the treatment of heart rhythm diseases.

The G-protein-gated K+ channel, IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation.

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein-activated K(+) current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4(-/-) mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4(-/-) mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4(-/-) animals. Although the relative extent of heart rate regulation of Girk4(-/-) mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological ß-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4(-/-) animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct ß-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation.

Upregulation of the hyperpolarization-activated current increases pacemaker activity of the sinoatrial node and heart rate during pregnancy in mice.

BACKGROUND: Pregnancy is associated with a faster heart rate (HR), which is a risk factor for arrhythmias. However, the underlying mechanisms for this increased HR are poorly understood. Therefore, this study was performed to gain mechanistic insight into the pregnancy-induced increase in HR. METHODS AND RESULTS: Using surface ECG we observed that pregnant (P) mice have faster HR (531±14 beats per minute [bpm]) compared with nonpregnant (NP) mice (470±27 bpm; P<0.03). Results obtained with Langendorff-perfused hearts showed that this difference persisted in the absence of autonomic nervous innervation (NP, 327±16 bpm; P, 385±18 bpm; P<0.02). Spontaneous action potentials of sinoatrial node cells from pregnant mice exhibited higher automaticity (NP, 292±13 bpm; P, 330±12 bpm; P=0.047) and steeper diastolic depolarization (NP, 0.20±0.03 V/s; P, 0.40±0.06 V/s; P=0.004). Pregnancy increased the density of the hyperpolarization-activated current (If) (at -90mV: NP, -15.2±1.0 pA/pF; P, -28.6±2.9 pA/pF; P=0.0002) in sinoatrial node cells. Voltage dependence of the If activation curve and the intracellular cAMP levels were unchanged in sinoatrial node cells of pregnant mice. However, there was a significant increase in HCN2 channel protein expression with no change in HCN4 expression. Maximal depolarizing shift of the If activation curve induced by isoproterenol was attenuated in pregnancy. This reduced response to isoproterenol may be attributable to the lower cAMP sensitivity of HCN2 isoform compared with that of HCN4. CONCLUSIONS: This study shows that an increase in If current density contributes to the acceleration of sinoatrial node automaticity and explains, in part, the higher HR observed in pregnancy.

Pacemaker activity and ionic currents in mouse atrioventricular node cells.

It is well established that Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of I(Na) by tetrodotoxin (TTX) or of I(Ca,L) by isradipine abolished AVNCs pacemaker activity. TTX-resistant (I(Nar)) and TTX-sensitive (I(Nas)) Na(+) currents were recorded in mouse AVNCs, as well as T-(I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents I(Ca,L) density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (I(f)) and that of the fast component of the delayed rectifier current (I(Kr)) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of I(f) by 3 µM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for I(f) in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K(+) current (I(to)). In contrast, no detectable slow component of the delayed rectifier current (I(Ks)) could be recorded in AVNCs. The lower densities of I(f) and I(Ca,L), as well as higher expression of I(Kr) in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.

Functional roles of Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of mouse atrioventricular cells: insights into the atrioventricular pacemaker mechanism.

The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Ca(v)3.1 (Ca(v)3.1(-/-)), Ca(v)1.3 (Ca(v)1.3(-/-)), channels or both (Ca(v)1.3(-/-)/Ca(v)3.1(-/-)). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Ca(v)3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Ca(v)3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Ca(v)1.3 channels were required for spontaneous automaticity, because Ca(v)1.3(-/-) and Ca(v)1.3(-/-)/Ca(v)3.1(-/-) AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Ca(v)1.3 channels are required for automaticity, Ca(v)3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for ß-adrenergic regulation.

Control of heart rate by cAMP sensitivity of HCN channels.

"Pacemaker" f-channels mediating the hyperpolarization-activated nonselective cation current I(f) are directly regulated by cAMP. Accordingly, the activity of f-channels increases when cellular cAMP levels are elevated (e.g., during sympathetic stimulation) and decreases when they are reduced (e.g., during vagal stimulation). Although these biophysical properties seem to make f-channels ideal molecular targets for heart rate regulation by the autonomic nervous system, the exact contribution of the major I(f)-mediating cardiac isoforms HCN2 and HCN4 to sinoatrial node (SAN) function remains highly controversial. To directly investigate the role of cAMP-dependent regulation of hyperpolarization activated cyclic nucleotide activated (HCN) channels in SAN activity, we generated mice with heart-specific and inducible expression of a human HCN4 mutation (573X) that abolishes the cAMP-dependent regulation of HCN channels. We found that hHCN4-573X expression causes elimination of the cAMP sensitivity of I(f) and decreases the maximum firing rates of SAN pacemaker cells. In conscious mice, hHCN4-573X expression leads to a marked reduction in heart rate at rest and during exercise. Despite the complete loss of cAMP sensitivity of I(f), the relative extent of SAN cell frequency and heart rate regulation are preserved. Our data demonstrate that cAMP-mediated regulation of I(f) determines basal and maximal heart rates but does not play an indispensable role in heart rate adaptation during physical activity. Our data also reveal the pathophysiologic mechanism of hHCN4-573X-linked SAN dysfunction in humans.

Nkx2.5 cell-autonomous gene function is required for the postnatal formation of the peripheral ventricular conduction system.

The ventricular conduction system is responsible for rapid propagation of electrical activity to coordinate ventricular contraction. To investigate the role of the transcription factor Nkx2.5 in the morphogenesis of the ventricular conduction system, we crossed Nkx2.5(+/-) mice with Cx40(eGFP/+) mice in which eGFP expression permits visualization of the His-Purkinje conduction system. Major anatomical and functional disturbances were detected in the His-Purkinje system of adult Nkx2.5(+/-)/Cx40(eGFP/+) mice, including hypoplasia of eGFP-positive Purkinje fibers and the disorganization of the Purkinje fiber network in the ventricular apex. Although the action potential properties of the individual eGFP-positive cells were normal, the deficiency of Purkinje fibers in Nkx2.5 haploinsufficient mice was associated with abnormalities of ventricular electrical activation, including slowed and decremented conduction along the left bundle branch. During embryonic development, eGFP expression in the ventricular trabeculae of Nkx2.5(+/-) hearts was qualitatively normal, with a measurable deficiency in eGFP-positive cells being observed only after birth. Chimeric analyses showed that maximal Nkx2.5 levels are required cell-autonomously. Reduced Nkx2.5 levels are associated with a delay in cell cycle withdrawal in surrounding GFP-negative myocytes. Our results suggest that the formation of the peripheral conduction system is time- and dose-dependent on the transcription factor Nkx2.5 that is cell-autonomously required for the postnatal differentiation of Purkinje fibers.

Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1/alpha1G T-type calcium channels.

The generation of the mammalian heartbeat is a complex and vital function requiring multiple and coordinated ionic channel activities. The functional role of low-voltage activated (LVA) T-type calcium channels in the pacemaker activity of the sinoatrial node (SAN) is, to date, unresolved. Here we show that disruption of the gene coding for CaV3.1/alpha1G T-type calcium channels (cacna1g) abolishes T-type calcium current (I(Ca,T)) in isolated cells from the SAN and the atrioventricular node without affecting the L-type Ca2+ current (I(Ca,L)). By using telemetric electrocardiograms on unrestrained mice and intracardiac recordings, we find that cacna1g inactivation causes bradycardia and delays atrioventricular conduction without affecting the excitability of the right atrium. Consistently, no I(Ca,T) was detected in right atrium myocytes in both wild-type and CaV3.1(-/-) mice. Furthermore, inactivation of cacna1g significantly slowed the intrinsic in vivo heart rate, prolonged the SAN recovery time, and slowed pacemaker activity of individual SAN cells through a reduction of the slope of the diastolic depolarization. Our results demonstrate that CaV3.1/T-type Ca2+ channels contribute to SAN pacemaker activity and atrioventricular conduction.