Novel insights into the distribution of cardiac HCN channels: an expression study in the mouse heart.

HCN pacemaker channels (I(f) channels) are believed to contribute to important functions in the heart; thus these channels became an attractive target for generating transgenic mouse mutants to elucidate their role in physiological and pathophysiological cardiac conditions. A full understanding of cardiac I(f) and the interpretation of studies using HCN mouse mutants require detailed information about the expression profile of the individual HCN subunits. Here we investigate the cardiac expression pattern of the HCN isoforms at the mRNA as well as at the protein level. The specificity of antibodies used was strictly confirmed by the use of HCN1, HCN2 and HCN4 knockout animals. We find a low, but highly differential HCN expression profile outside the cardiac conduction pathway including left and right atria and ventricles. Additionally HCN distribution was investigated in tissue slices of the sinoatrial node, the atrioventricular node, the bundle of His and the bundle branches. The conduction system was marked by acetylcholine esterase staining. HCN4 was confirmed as the predominant isoform of the primary pacemaker followed by a distinct expression of HCN1. In contrast HCN2 shows only a confined expression to individual pacemaker cells. Immunolabeling of the AV-node reveals also a pronounced specificity for HCN1 and HCN4. Compared to the SN and AVN we found a low but selective expression of HCN4 as the only isoform in the atrioventricular bundle. However in the bundle branches HCN1, HCN4 and also HCN2 show a prominent and selective expression pattern. Our results display a characteristic distribution of individual HCN isoforms in several cardiac compartments and reveal that beside HCN4, HCN1 represents the isoform which is selectively expressed in most parts of the conduction system suggesting a substantial contribution of HCN1 to pacemaking.

Insights into sick sinus syndrome from an inducible mouse model.

AIMS: Sick sinus syndrome is a generalized abnormality of cardiac impulse formation and is responsible for a large proportion of pacemaker implantations. Although the exact aetiology is not known, it is widely accepted that age-dependent degenerative fibrosis of nodal tissue is the most common cause. Despite its importance, an animal model for sick sinus syndrome is lacking. We attempted to generate a mouse model phenocopying the pathohistological changes as well as the characteristic arrhythmic manifestations of this syndrome. METHODS AND RESULTS: We crossed two genetically engineered mouse lines, ROSA-eGFP-DTA and HCN4-KiT-Cre, to achieve an inducible deletion of cells specifically in the cardiac pacemaking and conduction system. This deletion resulted in a degenerative fibrosis of nodal tissue, which accurately reflects the pathohistological findings in human sick sinus syndrome. The extent of the sino-atrial fibrosis could be controlled by varying the dosage of the inducing substance, tamoxifen. A high-dose protocol resulted in the complete ablation of all sino-atrial cells as demonstrated by histochemical analysis and quantitative reverse transcriptase-polymerase chain reaction. The animals developed a variety of arrhythmias, including progressive bradycardia, sinus pauses, supraventricular and ventricular tachycardia and chronotropic incompetence. Remarkably, the complete destruction of the primary pacemaker centre resulted in only a small increase in mortality. CONCLUSION: This study describes the generation and analysis of an inducible mouse model which closely reflects the pathophysiological characteristics of sick sinus syndrome. The model, with the ability to control the extent of nodal cell ablation and fibrosis, offers new insights into sick sinus syndrome and other cardiac conduction diseases.

Spinal inflammatory hyperalgesia is mediated by prostaglandin E receptors of the EP2 subtype.

Blockade of prostaglandin (PG) production by COX inhibitors is the treatment of choice for inflammatory pain but is also prone to severe side effects. Identification of signaling elements downstream of COX inhibition, particularly of PG receptor subtypes responsible for pain sensitization (hyperalgesia), provides a strategy for better-tolerated analgesics. Here, we have identified PGE2 receptors of the EP2 receptor subtype as key signaling elements in spinal inflammatory hyperalgesia. Mice deficient in EP2 receptors (EP2-/- mice) completely lack spinal PGE2-evoked hyperalgesia. After a peripheral inflammatory stimulus, EP2-/- mice exhibit only short-lasting peripheral hyperalgesia but lack a second sustained hyperalgesic phase of spinal origin. Electrophysiological recordings identify diminished synaptic inhibition of excitatory dorsal horn neurons as the dominant source of EP2 receptor-dependent hyperalgesia. Our results thus demonstrate that inflammatory hyperalgesia can be treated by targeting of a single PG receptor subtype and provide a rational basis for new analgesic strategies going beyond COX inhibition.

Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice.

Although glycine is a major inhibitory transmitter in the mammalian CNS, the role of glycinergic neurons in defined neuronal circuits remains ill defined. This is due in part to difficulties in identifying these cells in living slice preparations for electrophysiological recordings and visualizing their axonal projections. To facilitate the morphological and functional analysis of glycinergic neurons, we generated bacterial artificial chromosome (BAC) transgenic mice, which specifically express enhanced green fluorescent protein (EGFP) under the control of the promotor of the glycine transporter (GlyT) 2 gene, which is a reliable marker for glycinergic neurons. Neurons expressing GlyT2-EGFP were intensely fluorescent, and their dendrites and axons could be visualized in great detail. Numerous positive neurons were detected in the spinal cord, brainstem, and cerebellum. The hypothalamus, intralaminar nuclei of the thalamus, and basal forebrain also received a dense GlyT2-EGFP innervation, whereas in the olfactory bulb, striatum, neocortex, hippocampus, and amygdala positive fibers were much less abundant. No GlyT2-EGFP-positive cell bodies were seen in the forebrain. On the subcellular level, GlyT2-EGFP fluorescence was colocalized extensively with glycine immunoreactivity in somata and dendrites and with both glycine and GlyT2 immunoreactivity in axon terminals, as shown by triple staining at all levels of the neuraxis, confirming the selective expression of the transgene in glycinergic neurons. In slice preparations of the spinal cord, no difference between the functional properties of EGFP-positive and negative neurons could be detected, confirming the utility of visually identifying glycinergic neurons to investigate their functional role in electrophysiological studies.