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.

Bioavailability, biodistribution, and CNS toxicity of clinical-grade parvovirus H1 after intravenous and intracerebral injection in rats.

The autonomous parvovirus H1 (H1PV) is transmitted in rodent populations. The natural host is the rat, in which H1PV infection is pathogenic only in fetuses and newborns. H1PV infection of human cancer cells leads to strong oncolytic effects in preclinical models. In preparation for a clinical trial of H1PV injection in patients with malignant brain tumors, H1PV had to be prepared to Good Manufacturing Practice standards, including extensive toxicology testing in rats. Because the trial involves direct intracerebral injection of H1PV into the tumor and around the resection cavity, possible toxicity to CNS tissue had to be investigated. In addition, quantitative blood levels and the tissue distribution of H1PV after single intracerebral or intravenous injection were measured. Direct injection of H1PV into rat brain at 3 dose levels (maximum, 7.96 × 107 pfu) did not cause any macroscopic or histologic pathology. Furthermore, H1PV infection of the brain did not alter central or autonomous nervous system function. H1PV DNA was detected in almost all organs at 6 h, 48 h, and 14 d after intravenous and intracerebral injection, with the highest levels in liver and spleen. H1PV concentrations in most organs were similar after intravenous and intracerebral injection, indicating high permeability of the blood-brain barrier for this small virus. The current results demonstrate wide organ distribution of H1PV after intravenous or intracerebral injection, confirm that H1PV is nonpathogenic in adult rats even after direct injection into the brain, and form the basis for the ongoing ParvOryx01 clinical trial.

Pathology, organ distribution, and immune response after single and repeated intravenous injection of rats with clinical-grade parvovirus H1.

Parvovirus H1 (H1PV) is an autonomous parvovirus that is transmitted in rodent populations. Its natural host is rats. H1PV infection is nonpathogenic except in rat and hamster fetuses and newborns. H1PV infection of human cancer cells caused strong oncolytic effects in preclinical models. For a clinical trial of H1PV in patients with brain tumors, clinical-grade H1PV was produced according to Good Manufacturing Practices. This report focuses on results obtained after a single high-dose intravenous injection of highly purified H1PV in 30 rats and multiple (n = 17) intravenous injections at 3 dose levels in 223 rats. In both studies, no virus-related mortality or macroscopic organ changes related to H1PV occurred. Histopathology after multiple virus injections revealed minimal diffuse bile duct hyperplasia in livers of animals of the highest dose group and germinal center development in spleens of animals from the high-dose group. Liver changes were reversible within a 2-wk recovery period after the last injection. Hematology, blood chemistry, and coagulation analyses did not reveal significant toxicologic changes due to H1PV. Virus injection stimulated the production of IgG antibodies but did not alter mononuclear cell function or induce cytokine release. PCR analysis showed dose-dependent levels of viral genomes in all organs tested. The virus was excreted primarily through feces. These data provide important information regarding H1PV infection in its natural host. Due to the confirmation of the favorable safety profile of H1PV in a permissive animal model, a phase I/IIa clinical trial of H1PV in brain tumor patients could be initiated.

New method for sperm evaluation by 3-dimensional laser scanning microscopy in different laboratory animal species.

Sperm analysis is one of the end points in reproductive toxicology studies. Different methods for quantitative sperm analysis have been described. For qualitative morphological sperm analysis, either such techniques or smears of sperm and histological sperm staging are in use. Any of these methods provides morphological results on a light microscopy level. Laser scanning microscopy is a technique using a focused laser for scanning an object. The Olympus 3D Laser Scanning Microscope LEXT OLS4000 with optional possibilities of differential interference contrast provides a microscopic method for visualizing microasperities, which are far beyond the resolving power of a typical light or laser microscope. This technique was applied to sperm of mice, rats, rabbits, and cynomolgus monkeys at magnifications up to ×17 090. The obtained images are comparable to those of a scanning electron microscope under relatively low-power magnifications. Measurements on sperm parameters were taken by an integrated image analysis software tool. Abnormalities were easily detectable.

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.

How to optimize the benefits of computer assisted sperm analysis in experimental toxicology.

Exposure at the working place to various substances, that may affect semen quality is possible and should be investigated in detail. One appropriate method for this is computer-assisted sperm analysis (CASA) which offers multiple benefits in comparison to manual evaluation. However, several pitfalls exist, which make the evaluation of data obtained from CASA difficult to interpret. In the present commentary, we focus on these problems, show some examples, and try to define minimum standards which should be taken into consideration whenever working with computer-assisted sperm analysis.

Early ion-channel remodeling and arrhythmias precede hypertrophy in a mouse model of complete atrioventricular block.

Complete atrioventricular block (CAVB) and related ventricular bradycardia are known to induce ventricular hypertrophy and arrhythmias. Different animal models of CAVB have been established with the most common being the dog model. Related studies were mainly focused on the consequences on the main repolarizing currents in these species, i.e. IKr and IKs, with a limited time point kinetics post-AVB. In order to explore at a genomic scale the electrical remodeling induced by AVB and its chronology, we have developed a novel model of CAVB in the mouse using a radiofrequency-mediated ablation procedure. We investigated transcriptional changes in ion channels and contractile proteins in the left ventricles as a function of time (12h, 1, 2 and 5 days after CAVB), using high-throughput real-time RT-PCR. ECG in conscious and anesthetized mice, left ventricular pressure recordings and patch-clamp were used for characterization of this new mouse model. As expected, CAVB was associated with a lengthening of the QT interval. Moreover, polymorphic ventricular tachycardia was recorded in 6/9 freely-moving mice during the first 24h post-ablation. Remarkably, myocardial hypertrophy was only evident 48 h post-ablation and was associated with increased heart weight and altered expression of contractile proteins. During the first 24 hours post-CAVB, genes encoding ion channel subunits were either up-regulated (such as Nav1.5, +74%) or down-regulated (Kv4.2, -43%; KChIP2, -47%; Navß1, -31%; Cx43, -29%). Consistent with the transient alteration of Kv4.2 expression, I(to) was reduced at day 1, but restored at day 5. In conclusion, CAVB induces two waves of molecular remodeling: an early one (≤24 h) leading to arrhythmias, a later one related to hypertrophy. These results provide new molecular basis for ventricular tachycardia induced by AV block.

Physicochemical and toxicological characterization of a new generic iron sucrose preparation.

Intravenous iron preparations are key components in the management of anaemia of various etiologies. These iron-carbohydrate complexes permit safe systemic delivery of iron, whilst protecting from the potential toxic effects of over-saturation. This in turn permits efficient haematopoiesis following erythropoietin administration. Since the rate of release of iron is dependent upon the structure of this iron-carbohydrate complex, it is essential to ensure that an intravenous iron preparation is well characterized and its properties documented. This report describes physicochemical and toxicological studies into a new iron sucrose generic preparation, "Iron Sucrose Azad (ISA)", using the original iron sucrose product as reference. It could be demonstrated that the specifications and physicochemical characteristics of ISA reflect those of the reference product. Furthermore, in a rat model previously shown to identify possible toxicological effects of "unsimilar" iron sucrose preparations, ISA was found to have the same properties as the reference product, with both being well tolerated.

Variable Na(v)1.5 protein expression from the wild-type allele correlates with the penetrance of cardiac conduction disease in the Scn5a(+/-) mouse model.

BACKGROUND: Loss-of-function mutations in SCN5A, the gene encoding Na(v)1.5 Na+ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a(+/-) mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A. METHODOLOGY/PRINCIPAL FINDINGS: Based on ECG, 10-week-old Scn5a(+/-) mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS< or = 18 ms; QRS in wild-type littermates: 10-18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na+ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a(+/-) mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a(+/-) mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A-mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a(+/-) mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a(+/-) mice had similar Na(v)1.5 mRNA but higher Na(v)1.5 protein expression, and moderately larger I(Na) current than severely affected Scn5a(+/-) mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a(+/-) mice than in mildly affected ones. CONCLUSIONS: Scn5a(+/-) mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a(+/-) mice, phenotype severity correlates with wild-type Na(v)1.5 protein expression.

Biological pacemaker engineered by nonviral gene transfer in a mouse model of complete atrioventricular block.

We hypothesized that a nonviral gene delivery of the hyperpolarization-activated HCN2 channel combined with the beta(2)-adrenergic receptor (ADRB2) would generate a functional pacemaker in a mouse model of complete atrioventricular block (CAVB) induced by radiofrequency ablation of the His bundle. Plasmids encoding HCN2 and ADRB2 mixed with tetronic 304, a poloxamine block copolymer, were injected in the left ventricular free wall (HCN2-ADRB2 mice). Sham mice received a noncoding plasmid. CAVB was induced 5 days later. Ventricular escape rhythms in HCN2-ADRB2 mice were significantly faster than in sham mice at day 15 after ablation and later. In HCN2-ADRB2 mice, QRS complexes were larger than in sham mice and characterized by abnormal axes. Immunostaining of GFP-HCN2 fusion protein showed an expression of HCN2 channel in left ventricular myocardium for at least 45 days after injection. In the mouse, CAVB induces progressive hypertrophy and heart failure leading to 50% mortality after 110 days. HCN2-ADRB2 mice survived 3 weeks longer than sham mice. Finally, beta-adrenergic input increased ventricular escape rhythms significantly more in HCN2-ADRB2 mice than in sham mice. In conclusion, nonviral gene transfer can produce a functional cardiac biological pacemaker regulated by sympathetic input, which improves life expectancy in a mouse model of CAVB.

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.

Chronic heart rate reduction remodels ion channel transcripts in the mouse sinoatrial node but not in the ventricle.

We investigated the effects of chronic and moderate heart rate (HR) reduction on ion channel expression in the mouse sinoatrial node (SAN) and ventricle. Ten-week-old male C57BL/6 mice were treated twice daily with either vehicle or ivabradine at 5 mg/kg given orally during 3 wk. The effects of HR reduction on cardiac electrical activity were investigated in anesthetized mice with serial ECGs and in freely moving mice with telemetric recordings. With the use of high-throughput real-time RT-PCR, the expression of 68 ion channel subunits was evaluated in the SAN and ventricle at the end of the treatment period. In conscious mice, ivabradine induced a mean 16% HR reduction over a 24-h period that was sustained over the 3-wk administration. Other ECG parameters were not modified. Two-way hierarchical clustering analysis of gene expression revealed a separation of ventricles from SANs but no discrimination between treated and untreated ventricles, indicating that HR reduction per se induced limited remodeling in this tissue. In contrast, SAN samples clustered in two groups depending on the treatment. In the SAN from ivabradine-treated mice, the expression of nine ion channel subunits, including Navbeta1 (-25%), Cav3.1 (-29%), Kir6.1 (-28%), Kvbeta2 (-41%), and Kvbeta3 (-30%), was significantly decreased. Eight genes were significantly upregulated, including K+ channel alpha-subunits (Kv1.1, +30%; Kir2.1, +29%; Kir3.1, +41%), hyperpolarization-activated cation channels (HCN2, +24%; HCN4, +52%), and connexin 43 (+26%). We conclude that reducing HR induces a complex remodeling of ion channel expression in the SAN but has little impact on ion channel transcripts in the ventricle.

Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a.

We have examined sino-atrial node (SAN) function in hearts from adult mice with heterozygous targeted disruption of the Scn5a gene to clarify the role of Scn5a-encoded cardiac Na+ channels in normal SAN function and the mechanism(s) by which reduced Na+ channel function might cause sinus node dysfunction. Scn5a+/- mice showed depressed heart rates and occasional sino-atrial (SA) block. Their isolated peripheral SAN pacemaker cells showed a reduced Na+ channel expression and slowed intrinsic pacemaker rates. Wild-type (WT) and Scn5a+/- SAN preparations exhibited similar activation patterns but with significantly slower SA conduction and frequent sino-atrial conduction block in Scn5a+/- SAN preparations. Furthermore, isolated WT and Scn5a+/- SAN cells demonstrated differing correlations between cycle length, maximum upstroke velocity and action potential amplitude, and cell size. Small myocytes showed similar, but large myocytes reduced pacemaker rates, implicating the larger peripheral SAN cells in the reduced pacemaker rate that was observed in Scn5a+/- myocytes. These findings were successfully reproduced in a model that implicated i(Na) directly in action potential propagation through the SAN and from SAN to atria, and in modifying heart rate through a coupling of SAN and atrial cells. Functional alterations in the SAN following heterozygous-targeted disruption of Scn5a thus closely resemble those observed in clinical sinus node dysfunction. The findings accordingly provide a basis for understanding of the role of cardiac-type Na+ channels in normal SAN function and the pathophysiology of sinus node dysfunction and suggest new potential targets for its clinical management.

Mouse model of SCN5A-linked hereditary Lenègre's disease: age-related conduction slowing and myocardial fibrosis.

BACKGROUND: We have previously linked hereditary progressive cardiac conduction defect (hereditary Lenègre's disease) to a loss-of-function mutation in the gene encoding the main cardiac Na+ channel, SCN5A. In the present study, we investigated heterozygous Scn5a-knockout mice (Scn5a+/- mice) as a model for hereditary Lenègre's disease. METHODS AND RESULTS: In Scn5a+/- mice, surface ECG recordings showed age-related lengthening of the P-wave and PR- and QRS-interval duration, coinciding with previous observations in patients with Lenègre's disease. Old but not young Scn5a+/- mice showed extensive fibrosis of their ventricular myocardium, a feature not seen in wild-type animals. In old Scn5a+/- mice, fibrosis was accompanied by heterogeneous expression of connexin 43 and upregulation of hypertrophic markers, including beta-MHC and skeletal alpha-actin. Global connexin 43 expression as assessed with Western blots was similar to wild-type mice. Decreased connexin 40 expression was seen in the atria. Using pangenomic microarrays and real-time PCR, we identified in Scn5a+/- mice an age-related upregulation of genes encoding Atf3 and Egr1 transcription factors. Echocardiography and hemodynamic investigations demonstrated conserved cardiac function with aging and lack of ventricular hypertrophy. CONCLUSIONS: We conclude that Scn5a+/- mice convincingly recapitulate the Lenègre's disease phenotype, including progressive impairment with aging of atrial and ventricular conduction associated with myocardial rearrangements and fibrosis. Our work provides the first demonstration that a monogenic ion channel defect can progressively lead to myocardial structural anomalies.