Augmentation of myocardial If dysregulates calcium homeostasis and causes adverse cardiac remodeling.

HCN channels underlie the depolarizing funny current (If) that contributes importantly to cardiac pacemaking. If is upregulated in failing and infarcted hearts, but its implication in disease mechanisms remained unresolved. We generated transgenic mice (HCN4tg/wt) to assess functional consequences of HCN4 overexpression-mediated If increase in cardiomyocytes to levels observed in human heart failure. HCN4tg/wt animals exhibit a dilated cardiomyopathy phenotype with increased cellular arrhythmogenicity but unchanged heart rate and conduction parameters. If augmentation induces a diastolic Na+ influx shifting the Na+/Ca2+ exchanger equilibrium towards 'reverse mode' leading to increased [Ca2+]i. Changed Ca2+ homeostasis results in significantly higher systolic [Ca2+]i transients and stimulates apoptosis. Pharmacological inhibition of If prevents the rise of [Ca2+]i and protects from ventricular remodeling. Here we report that augmented myocardial If alters intracellular Ca2+ homeostasis leading to structural cardiac changes and increased arrhythmogenicity. Inhibition of myocardial If per se may constitute a therapeutic mechanism to prevent cardiomyopathy.

The symptom complex of familial sinus node dysfunction and myocardial noncompaction is associated with mutations in the HCN4 channel.

BACKGROUND: Inherited arrhythmias were originally considered isolated electrical defects. There is growing evidence that ion channel dysfunction also contributes to myocardial disorders, but genetic overlap has not been reported for sinus node dysfunction (SND) and noncompaction cardiomyopathy (NCCM). OBJECTIVES: The study sought to investigate a familial electromechanical disorder characterized by SND and NCCM, and to identify the underlying genetic basis. METHODS: The index family and a cohort of unrelated probands with sinus bradycardia were examined by electrocardiography, Holter recording, exercise stress test, echocardiography, and/or cardiac magnetic resonance imaging. Targeted next-generation and direct sequencing were used for candidate gene analysis and mutation scanning. Ion channels were expressed in HEK293 cells and studied using patch-clamp recordings. RESULTS: SND and biventricular NCCM were diagnosed in multiple members of a German family. Segregation analysis suggested autosomal-dominant inheritance of the combined phenotype. When looking for potentially disease-causing gene variants with cosegregation, a novel hyperpolarization-activated cyclic nucleotide channel 4 (HCN4)-G482R mutation and a common cysteine and glycine-rich protein 3 (CSRP3)-W4R variant were identified. HCN4-G482R is located in the highly conserved channel pore domain. Mutant subunits were nonfunctional and exerted dominant-negative effects on wild-type current. CSRP3-W4R has previously been linked to dilated and hypertrophic cardiomyopathy, but was also found in healthy subjects. Moreover, different truncation (695X) and missense (P883R) HCN4 mutations segregated with a similar combined phenotype in an additional, unrelated family and a single unrelated proband respectively, which both lacked CSRP3-W4R. CONCLUSIONS: The symptom complex of SND and NCCM is associated with heritable HCN4 defects. The NCCM phenotype may be aggravated by a common CSRP3 variant in one of the families.

The neuromuscular junction: selective remodeling of synaptic regulators at the nerve/muscle interface.

The peripheral synapses between motoneurons and skeletal muscle fibers, the neuromuscular junctions, are ideal to investigate the general principles of synaptogenesis that depend on the interaction of activity-dependent and activity-independent signals. Much has been learned from gene "knock out" mouse models that helped to identify major synaptic regulators. The "knock out" approach, however, may not distinguish between changes arising from the disruption of molecular signaling pathways and changes caused by the absence of synaptic transmission. To circumvent these problems, postsynaptic activity was modulated in mouse models by specifically targeting endplate receptors or the activity of synaptic regulators such as MuSK. Both regulators have multiple functions and acetylcholine receptors are not just signal transducers but regulate the localization and architecture of endplates. The results show that detailed analysis of mouse models will help to understand the complexity in mechanisms that regulate synaptic remodeling.

Novel mouse model reveals distinct activity-dependent and -independent contributions to synapse development.

The balanced action of both pre- and postsynaptic organizers regulates the formation of neuromuscular junctions (NMJ). The precise mechanisms that control the regional specialization of acetylcholine receptor (AChR) aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. Synaptic activity is of central importance and to understand synaptogenesis, it is necessary to distinguish between activity-dependent and activity-independent processes. By engineering a mutated fetal AChR subunit, we used homologous recombination to develop a mouse line that expresses AChR with massively reduced open probability during embryonic development. Through histological and immunochemical methods as well as electrophysiological techniques, we observed that endplate anatomy and distribution are severely aberrant and innervation patterns are completely disrupted. Nonetheless, in the absence of activity AChRs form postsynaptic specializations attracting motor axons and permitting generation of multiple nerve/muscle contacts on individual fibers. This process is not restricted to a specialized central zone of the diaphragm and proceeds throughout embryonic development. Phenotypes can be attributed to separate activity-dependent and -independent pathways. The correct patterning of synaptic connections, prevention of multiple contacts and control of nerve growth require AChR-mediated activity. In contrast, myotube survival and acetylcholine-mediated dispersal of AChRs are maintained even in the absence of AChR-mediated activity. Because mouse models in which acetylcholine is entirely absent do not display similar effects, we conclude that acetylcholine binding to the AChR initiates activity-dependent and activity-independent pathways whereby the AChR modulates formation of the NMJ.

Time lapse in vivo visualization of developmental stabilization of synaptic receptors at neuromuscular junctions.

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.

Differential muscle-driven synaptic remodeling in the neuromuscular junction after denervation.

We used knock-in mice that express green fluorescent protein (GFP)-labeled embryonic-type acetylcholine receptors to investigate postsynaptic responses to denervation of fast-twitch and slow-twitch muscle fibers, and to visualize the integration of newly synthesized GFP-labeled embryonic-type receptors into adult synapses. The embryonic-type receptors are transiently expressed and incorporated into the denervated endplates. They replaced synaptic adult-type receptors in a directed fashion, starting from the endplate's periphery and proceeding to its central regions. The progress of embryonic-type receptor expression with respect to transcriptional control is a transient, short-term activation mechanism. The less pronounced increase in the expression levels of the GFP-labeled receptors revealed a differential shift in the integration and degradation processes that constitute the dynamic equilibrium of the synaptic receptor pool. Therefore, we were able to model the changes in the total receptor load of the neuromuscular endplate following denervation as a function of the abundance of available receptors and the initial receptor load of the endplate.

Transcription profiling of HCN-channel isotypes throughout mouse cardiac development.

Hyperpolarization-activated ion channels, encoded by four mammalian genes (HCN1-4), contribute in an important way to the cardiac pacemaker current I(f). Here, we describe the transcription profiles of the four HCN genes, the NRSF, KCNE2 and Kir2.1 genes from embryonic stage E9.5 dpc to postnatal day 120 in the mouse. Embryonic atrium and ventricle revealed abundant HCN4 transcription but other HCN transcripts were almost absent. Towards birth, HCN4 was downregulated in the atrium and almost vanished from the ventricle. After birth, however, HCN isotype transcription changed remarkably, showing increased levels of HCN1, HCN2 and HCN4 in the atrium and of HCN2 and HCN4 in the ventricle. HCN3 showed highest transcription at early embryonic stages and was hardly detectable thereafter. At postnatal day 10, HCN4 was highest in the sinoatrial node, being twofold higher than HCN1 and fivefold higher than HCN2. In the atrium, HCN4 was similar to HCN1 and sevenfold higher than HCN2. In the ventricle, in contrast, HCN2 was sixfold higher than HCN4, while HCN1 was absent. Subsequently all HCN isotype transcripts declined to lower adult levels, while ratios of HCN isotypes remained stable. In conclusion, substantial changes of HCN isotype transcription throughout cardiac development suggest that a regulated pattern of HCN isotypes is required to establish and ensure a stable heart rhythm. Furthermore, constantly low HCN transcription in adult myocardium may be required to prevent atrial and ventricular arrhythmogenesis.

AChR channel conversion and AChR-adjusted neuronal survival during embryonic development.

We generated knock-in mice that express GFP-labeled embryonic-type acetylcholine receptors (AChR) to follow postsynaptic differentiation and innervation during embryonic development and to visualize the postnatally occurring channel conversion from embryonic- to adult-type AChR. The dynamics of AChRgamma/AChRepsilon conversion at the neuromuscular junction indicates that muscle-specific programs of receptor subunit gene transcription control AChR replacement. While conversion proceeds from peripheral to central regions for individual endplates, it does not occur simultaneously for all endplates. Although GFP-labeled receptors were expressed at reduced levels, the localization of postsynaptic sites and synapse formation was not noticeably altered. However, these alterations correlated with a striking reduction of motoneuron programmed cell death, transient increase of neurite growth and axon branching. This animal model refines the view on reciprocal neuromuscular signaling and suggests that motoneuron survival and axon branching are directly regulated by AChR function to enable optimal innervation and timing of neurally evoked muscle contraction.