Juliane Stieber

Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2

Hyperpolarization‐activated cation (HCN) channels are believed to be involved in the generation of cardiac pacemaker depolarizations as well as in the control of neuronal excitability and plasticity. The contributions of the four individual HCN channel isoforms (HCN1—4) to these diverse functions are not known. Here we show that HCN2‐deficient mice exhibit spontaneous absence seizures. The thalamocortical relay neurons of these mice displayed a near complete loss of the HCN current, resulting in a pronounced hyperpolarizing shift of the resting membrane potential, an altered response to depolarizing inputs and an increased susceptibility for oscillations. HCN2‐null mice also displayed cardiac sinus dysrhythmia, a reduction of the sinoatrial HCN current and a shift of the maximum diastolic potential to hyperpolarized values. Mice with cardiomyocyte‐ specific deletion of HCN2 displayed the same dysrhythmia as mice lacking HCN2 globally, indicating that the dysrhythmia is indeed caused by sinoatrial dysfunction. Our results define the physiological role of the HCN2 subunit as a major determinant of membrane resting potential that is required for regular cardiac and neuronal rhythmicity.

Ultraflexible vesicles, Transfersomes, have an extremely low pore penetration resistance and transport therapeutic amounts of insulin across the intact mammalian skin.

New vehicles for the non-invasive delivery of agents are introduced. These carriers can transport pharmacological agents, including large polypeptides, through the permeability barriers, such as the intact skin. This capability depends on the self-regulating carrier deformability which exceeds that of the related but not optimized lipid aggregates by several orders of magnitude. Conventional lipid suspensions, such as standard liposomes or mixed lipid micelles, do not mediate a systemic biological effect upon epicutaneous applications. In contrast to this, the properly devised adaptable carriers, when administered on the intact skin, transport therapeutic amounts of biogenic molecules into the body. This process can be nearly as efficient as an injection needle, as seen from the results of experiments in mice and humans with the insulin-carrying vesicles. The carrier-mediated transcutaneous insulin delivery is unlikely to involve shunts, lesions or other types of skin damage. Rather than this, insulin is inferred to be transported into the body between the intact skin cells with a bio-efficiency of at least 50% of the s.c. dose action.

The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart

Hyperpolarization-activated, cyclic nucleotide-gated cation currents, termed If or Ih, are generated by four members of the hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channel family. These currents have been proposed to contribute to several functions including pacemaker activity in heart and brain, control of resting potential, and neuronal plasticity. Transcripts of the HCN4 isoform have been found in cardiomyocytes and neurons, but the physiological role of this channel is unknown. Here we show that HCN4 is essential for the proper function of the developing cardiac conduction system. In wild-type embryos, HCN4 is highly expressed in the cardiac region where the early sinoatrial node develops. Mice lacking HCN4 channels globally, as well as mice with a selective deletion of HCN4 in cardiomyocytes, died between embryonic days 9.5 and 11.5. On average, If in cardiomyocytes from mutant embryos is reduced by 85%. Hearts from HCN4-deficient embryos contracted significantly slower compared with wild type and could not be stimulated by cAMP. In both wild-type and HCN4-/- mice, cardiac cells with “primitive” pacemaker action potentials could be found. However, cardiac cells with “mature” pacemaker potentials, observed in wild-type embryos starting at day 9.0, were not detected in HCN4-deficient embryos. Thus, HCN4 channels are essential for the proper generation of pacemaker potentials in the emerging sinoatrial node.

HCN4 provides a ‘depolarization reserve’ and is not required for heart rate acceleration in mice

Cardiac pacemaking involves a variety of ion channels, but their relative importance is controversial and remains to be determined. Hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels, which underlie the If current of sinoatrial cells, are thought to be key players in cardiac automaticity. In addition, the increase in heart rate following beta‐adrenergic stimulation has been attributed to the cAMP‐mediated enhancement of HCN channel activity. We have now studied mice in which the predominant sinoatrial HCN channel isoform HCN4 was deleted in a temporally controlled manner. Here, we show that deletion of HCN4 in adult mice eliminates most of sinoatrial If and results in a cardiac arrhythmia characterized by recurrent sinus pauses. However, the mutants show no impairment in heart rate acceleration during sympathetic stimulation. Our results reveal that unexpectedly the channel does not play a role for the increase of the heart rate; however, HCN4 is necessary for maintaining a stable cardiac rhythm, especially during the transition from stimulated to basal cardiac states.

IRAG is essential for relaxation of receptor-triggered smooth muscle contraction by cGMP kinase.

Signalling by cGMP‐dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKI‐dependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5‐trisphosphate receptor I (IP3RI)‐associated protein (IRAG), which decreases hormone‐induced IP3‐dependent Ca2+ release. We show now that the targeted deletion of exon 12 of IRAG coding for the N‐terminus of the coiled‐coil domain disrupted in vivo the IRAG–IP3RI interaction and resulted in hypomorphic IRAGΔ12/Δ12 mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol‐ and phenylephrine‐contracted smooth muscle strips from colon and aorta, respectively, of IRAGΔ12/Δ12 mice were not relaxed by cGMP, while cAMP‐mediated relaxation was unperturbed. Norepinephrine‐induced increases in [Ca2+]i were not decreased by cGMP in aortic smooth muscle cells from IRAGΔ12/Δ12 mice. In contrast, cGMP‐induced relaxation of potassium‐induced smooth muscle contraction was not abolished in IRAGΔ12/Δ12 mice. We conclude that cGMP‐dependent relaxation of hormone receptor‐triggered smooth muscle contraction essentially depends on the interaction of cGKI–IRAG with IP3RI.

Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice

Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention.

Pathophysiology of HCN channels

Hyperpolarization-activated cation currents termed If/h are observed in many neurons and cardiac cells. Four genes (HCN1-4) encode the channels underlying these currents. New insights into the pathophysiological significance of HCN channels have been gained recently from analyses of mice engineered to be deficient in HCN genes. Lack of individual subunits results in markedly different phenotypes. Disruption of HCN1 impairs motor learning but enhances spatial learning and memory. Deletion of HCN2 results in absence epilepsy, ataxia, and sinus node dysfunction. Mice lacking HCN4 die during embryonic development and develop no sinoatrial node-like action potentials. In the present review, we summarize the physiology and pathophysiology of HCN channel family members based primarily on information from the transgenic mouse models and on data from human patients carrying defects in HCN4 channels.

Molecular Basis for the Different Activation Kinetics of the Pacemaker Channels HCN2 and HCN4

The pacemaker channels HCN2 and HCN4 have been identified in cardiac sino-atrial node cells. These channels differ considerably in several kinetic properties including the activation time constant (τact), which is fast for HCN2 (144 ms at –140 mV) and slow for HCN4 (461 ms at –140 mV). Here, by analyzing HCN2/4 chimeras and mutants we identified single amino acid residues in transmembrane segments 1 and 2 and the connecting loop between S1 and S2 that are major determinants of this difference. Replacement of leucine 272 in S1 of HCN4 by the corresponding phenylalanine present in HCN2 decreased τact of HCN4 to 149 ms. Conversely, activation of the fast channel HCN2 was decreased 3-fold upon the corresponding mutation of F221L in the S1 segment. Mutation of N291T and T293A in the linker between S1 and S2 of HCN4 shifted τact to 275 ms. While residues 272, 291, and 293 of HCN4 affected the activation speed at basal conditions they had no obvious influence on the cAMP-dependent acceleration of activation kinetics. In contrast, mutation of I308M in S2 of HCN4 abolished the cAMP-dependent decrease in τact. Surprisingly, this mutation also prevented the acceleration of channel activation observed after deletion of the C-terminal cAMP binding site. Taken together our results indicate that the speed of activation of the HCN4 channel is determined by structural elements present in the S1, S1-S2 linker, and the S2 segment.

Programming and Isolation of Highly Pure Physiologically and Pharmacologically Functional Sinus-Nodal Bodies from Pluripotent Stem Cells

Summary Therapeutic approaches for “sick sinus syndrome” rely on electrical pacemakers, which lack hormone responsiveness and bear hazards such as infection and battery failure. These issues may be overcome via “biological pacemakers” derived from pluripotent stem cells (PSCs). Here, we show that forward programming of PSCs with the nodal cell inducer TBX3 plus an additional Myh6-promoter-based antibiotic selection leads to cardiomyocyte aggregates consisting of >80% physiologically and pharmacologically functional pacemaker cells. These induced sinoatrial bodies (iSABs) exhibited highly increased beating rates (300–400 bpm), coming close to those found in mouse hearts, and were able to robustly pace myocardium ex vivo. Our study introduces iSABs as highly pure, functional nodal tissue that is derived from PSCs and may be important for future cell therapies and drug testing in vitro.

HCN channels in the heart: lessons from mouse mutants

Hyperpolarization‐activated cation channels generate the If current in the heart. In the sino‐atrial node (SAN), If is thought to play an essential role in setting the heart rate and mediating its autonomic control. This review focuses on the role of If in pacemaking and non‐pacemaking cardiomyocytes and the resulting therapeutic implications. HCN4 represents the principal isoform underlying sino‐atrial If, but other isoforms may also be of importance. To examine the functional role of cardiac channels, several mouse mutants, most of them targeting HCN4, have been generated by different groups. Unexpectedly, these lines display greatly different and as yet unexplained phenotypes. We provide an overview about these HCN mutants and suggest an interpretation of the functional significance of If in the SAN in light of these studies. HCN channels are also present in ventricular myocytes, and an up‐regulation of If in the hypertrophic and failing heart may contribute to arrhythmogenesis. Inhibition of If by HCN channel blockers is a novel approach in the treatment of cardiac disorders, and ivabradine is approved for treatment of stable angina pectoris. Remarkably, a recent clinical trial assessing this substance in heart failure showed a significantly improved outcome. The mechanism underlying this beneficial effect is not yet clear and might lie beyond heart rate slowing. Thus, the growing knowledge about cardiac HCN channels will undoubtedly promote the development of the promising class of HCN channel blockers.