Stefan Herzig

Increased Availability and Open Probability of Single L-Type Calcium Channels From Failing Compared With Nonfailing Human Ventricle

BACKGROUND The role of the L-type calcium channel in human heart failure is unclear, on the basis of previous whole-cell recordings. METHODS AND RESULTS We investigated the properties of L-type calcium channels in left ventricular myocytes isolated from nonfailing donor hearts (n= 16 cells) or failing hearts of transplant recipients with dilated (n=9) or ischemic (n=7) cardiomyopathy. The single-channel recording technique was used (70 mmol/L Ba2+). Peak average currents were significantly enhanced in heart failure (38.2+/-9.3 fA) versus nonfailing control hearts (13.2+/-4.5 fA, P=0.02) because of an elevation of channel availability (55.9+/-6.7% versus 26.4+/-5.3%, P=0.001) and open probability within active sweeps (7.36+/-1.51% versus 3.18+/-1.33%, P=0.04). These differences closely resembled the effects of a cAMP-dependent stimulation with 8-Br-cAMP (n= 11). Kinetic analysis of the slow gating shows that channels from failing hearts remain available for a longer time, suggesting a defect in the dephosphorylation. Indeed, the phosphatase inhibitor okadaic acid was unable to stimulate channel activity in myocytes from failing hearts (n=5). Expression of calcium channel subunits was measured by Northern blot analysis. Expression of alpha1c- and beta-subunits was unaltered. Whole-cell current measurements did not reveal an increase of current density in heart failure. CONCLUSIONS Individual L-type calcium channels are fundamentally affected in severe human heart failure. This is probably important for the impairment of cardiac excitation-contraction coupling.

PROBLEM-BASED LEARNING VERSUS LECTURE-BASED LEARNING IN A COURSE OF BASIC PHARMACOLOGY : A CONTROLLED, RANDOMIZED STUDY

Since its first implementation in a medical programme at McMaster University, Canada, problem‐based learning (PBL) has become a well‐established means of teaching and learning medicine. Extensive research has been conducted and a number of strengths of the method are well supported. Several items, however, remain unclear1 although there is evidence that no relevant difference exists in factual knowledge among students from PBL and traditional curricula, a controlled, randomized study has not been conducted to address this issue. The Medical Faculty of the University of Cologne is in the process of integrating elements of PBL into its curriculum. In the spring term of 1997, after seven semesters of experience with PBL supplementing the traditional course of basic pharmacology, we did for the first time use PBL instead of the lecture‐based course (LBL) and conducted a controlled prospective study to determine the effects of this intervention. One‐hundred and twenty‐three students were randomly assigned to either PBL (n = 63), with tutorial groups of up to nine students, or to the traditional, lecture‐based course (n = 60). Analysis of the results of both groups in the examination of basic pharmacology, consisting of multiple‐choice and short‐essay questions, revealed similar scores with a tendency favouring PBL students in the category of short‐essay questions. Hence, it seems clear that PBL does not imply a disadvantage in terms of factual knowledge. Students considered PBL to be an effective learning method and favoured it over the lecture format. Furthermore, students reported positive effects of PBL in terms of use of additional learning resources, inter‐disciplinarity, team work and learning fun.

Functional Properties of a Newly Identified C-terminal Splice Variant of Cav1.3 L-type Ca2+ Channels

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Cav1.3 L-type Ca2+ channels (Cav1.3L) is a major determinant of their voltage- and Ca2+-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Cav1.342A channels that activate at a more negative voltage range and exhibit more pronounced Ca2+-dependent inactivation. Here we describe the discovery of a novel short splice variant (Cav1.343S) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Cav1.342A, still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Cav1.343S also activated at more negative voltages like Cav1.342A but Ca2+-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Cav1.3L. The presence of the proximal C terminus in Cav1.343S channels preserved their modulation by distal C terminus-containing Cav1.3- and Cav1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca2+ influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Cav1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca2+ channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca2+ accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca2+-induced neurodegenerative processes.

Single-channel properties support a potential contribution of hyperpolarization-activated cyclic nucleotide-gated channels and If to cardiac arrhythmias.

Background—The pacemaker current If is present in atrial and ventricular myocytes. However, it remains controversial whether If overexpression in diseased states might play a role for arrhythmogenesis, because first If activation in whole-cell recordings hardly overlapped the diastolic voltage of working myocardium. Methods and Results—To obtain further insight into IHCN and If properties, we provide for the first time detailed single-channel analysis of heterologously expressed hyperpolarization-activated cyclic nucleotide-gated (HCN) isoforms and native human If. HCN subtypes differed significantly in single-channel amplitude, conductance, and activation kinetics. Interestingly, threshold potentials of HCN isoforms were more positive than would have been expected from whole-cell measurements. Single-channel properties of cells cotransfected with HCN2 and HCN4 were distinct from cells expressing HCN2 or HCN4 alone, demonstrating that different HCN isoforms can influence current properties of a single HCN channel complex, thus providing direct functional evidence for HCN heteromerization. Pooled data of homomeric and heteromeric HCN channels and of native If extrapolated from maximum likelihood fits indicated a multistate gating scheme comprising 5 closed- and 4 open-channel states. Single-channel characteristics of If in human atrial myocytes closely resembled those of HCN4 or HCN2+HCN4, supporting the hypothesis that native If channels in atrial myocardium are heteromeric complexes composed of HCN4 and/or HCN2. Most interestingly, half-maximal activation of single-channel atrial If (−68.3±4.9 mV; k=−9.9±1.5; n=8) was well within the diastolic voltage range of human atrial myocardium. Conclusions—These observations support a potential contribution of HCN/If to the arrhythmogenesis of working myocardium under pathological conditions.

Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes.

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

Cardioprotection specific for the G protein Gi2 in chronic adrenergic signaling through β2-adrenoceptors

Two subtypes of β-adrenoceptors, β1 and β2, mediate cardiac catecholamine effects. These two types differ qualitatively, e.g., regarding G protein coupling and calcium channel stimulation. Transgenic mice overexpressing human β2-adrenoceptors survive high-expression levels, unlike mice overexpressing β1-adrenoceptors. We examined the role of inhibitory Gi proteins, known to be activated by β2- but not β1-adrenoceptors, on the chronic effects of human β2-adrenoreceptor overexpression in transgenic mice. These mice were crossbred with mice where Gαi2, a functionally important cardiac Gi α-subunit, was inactivated by targeted gene deletion. Survival of β2-adrenoreceptor transgenic mice was reduced by heterozygous inactivation of Gαi2. Homozygous knockout/β2-adrenoreceptor transgenic mice died within 4 days after birth. Heterozygous knockout/β2-adrenoreceptor transgenic mice developed more pronounced cardiac hypertrophy and earlier heart failure compared with β2-adrenoreceptor transgenic mice. Single calcium-channel activity was strongly suppressed in heterozygous knockout/β2-adrenoreceptor transgenic mice. In cardiomyocytes from these mice, pertussis toxin treatment in vitro fully restored channel activity and enhanced channel activity in cells from homozygous Gαi2 knockout animals. Cardiac Gαi3 protein was increased in all Gαi2 knockout mouse strains. Our results demonstrate that Gαi2 takes an essential protective part in chronic signaling of overexpressed β2-adrenoceptors, leading to prolonged survival and delayed cardiac pathology. However, reduction of calcium-channel activity by β2-adrenoreceptor overexpression is due to a different pertussis-toxin-sensitive pathway, most likely by Gαi3. This result indicates that subtype-specific signaling of β2-adrenoreceptor functionally bifurcates at the level of Gi, leading to different effects depending on the Gα isoform.

Increased Expression of the Auxiliary β2-subunit of Ventricular L-type Ca2+ Channels Leads to Single-Channel Activity Characteristic of Heart Failure

Background Increased activity of single ventricular L-type Ca2+-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary β-subunits as a possible explanation. Methods and Results By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC β-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac β-subunits: Unlike β1 or β3 isoforms, β2a and β2b induce a high-activity channel behavior typical of failing myocytes. In accordance, β2-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac CaV1.2 also reveal increased single-channel activity and sarcolemmal β2 expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing (“Adaptive Phase”), reveal the opposite phenotype, viz : reduced single-channel activity accompanied by lowered β2 expression. Additional evidence for the cause-effect relationship between β2-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive CaV1.2 and inducible β2 cardiac overexpression. Here in non-failing hearts induction of β2-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. Conclusions Our study presents evidence of the pathobiochemical relevance of β2-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Stimulation of protein phosphatases as a mechanism of the muscarinic-receptor-mediated inhibition of cardiac L-type Ca2+ channels

Acetylcholine decreases currents through cardiac L-type Ca2+ channels after stimulation with agents which elevate levels of cyclic adenosine monophosphate, such as isoproterenol, but there is still a controversy over the mechnisms of this muscarinic effect. We tested the hypothesis of whether, after isoproterenol stimulation, protein phosphatases are activated by acetylcholine. Whole-cell currents were recorded from guinea-pig ventricular myocytes. The effect of 10−5 M acetylcholine on currents induced by 10−8 M isoproterenol was studied in the absence or presence of protein phosphatase inhibitors. Three agents reduced the acetylcholine response: okadaic acid (3 or 9 · 10−6 M) and cantharidin (3 · 10−6 M) added to the pipette solution, and bath-applied fluoride (3 mM). In contrast, pipette application of other phosphatase inhibitors, namely the inhibitor PPI2 (1000 U/ml), ciclosporin (10−5 M), or calyculin A (10−6 M) did not significantly diminish the acetylcholine effect. Interestingly, there was no correlation between the effects of the compounds on basal Ca2+ current and their interference with the muscarinic response. An activation of type 2A phosphatases by acetylcholine would explain these findings. Indeed, okadaic acid is 3 orders of magnitude more potent in vitro in its inhibition of this isoform (purified from cardiac myocytes) than is calyculin A, while type-1 phosphatases are inhibited equally. The data support the attractive possibility that stimulation of protein phosphatases is part of the signal transduction cascade of Ca2+ channel inhibition by acetyl-choline.

PACAP induces bradycardia in guinea-pig heart by stimulation of atrial cholinergic neurones.

Based on previous studies which indicated that pituitary adenylate cyclase activating peptide (PACAP) acts as a positive inotropic and chronotropic substance in different species via the cAMP signal transduction pathway, the objective of the present work was to investigate cAMP-regulated myocardial key proteins in response to PACAP in isolated ventricular cells of the guinea pig. Surprisingly, the two molecular forms of PACAP, PACAP(1–27) and PACAP(1–38), showed no effect on intracellular cAMP-levels, L-type Ca2+ channel current or phosphorylation of troponin inhibitor (TnI) and phospholamban (PLB). Additionally, inotropy of isolated guinea-pig ventricular strips was not affected by the neuropeptide.However, in isolated spontaneously beating guinea-pig atria, PACAP(1–27) and PACAP(1–38), but not VIP induced severe bradycardia in a dose-dependent manner. This effect could be prevented by preincubation with the PACAP receptor antagonist PACAP(6–38), by atropine and by ω-conotoxin, a blocker of neuronal N-type Ca2+channels. PACAP stimulates release of [3H]-labelled acetylcholine. Only preparations showing an increase in [3H]acetylcholine release developed bradycardia, indicating a causal relationship between both phenomena. It was concluded that PACAP exerts no influence on guinea-pig ventricular tissue, but induces negative chronotropic effects in isolated guinea-pig atria by stimulation of acetylcholine release from parasympathetic neurons via PACAP type 1 receptors.

Mechanism of Cav1.2 channel modulation by the amino terminus of cardiac β2-subunits

L‐type calcium channels are composed of a pore, α1c (CaV1.2), and accessory β‐ and α2δ‐subunits. The β‐subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five ß2‐subunits isoforms expressed in human heart ( β 2a‐e) on the single L‐type calcium channel current. These splice variants differ only by amino‐terminal length and amino acid composition. Single‐channel modulation by β2‐subunit isoforms was investigated in HEK293 cells expressing the recombinant L‐type ion conducting pore. All β2‐subunits increased open probability, availability, and peak current with a highly consistent rank order (ß2a≈ ß2b>ß2e≈ ß2c>ß2d). We show graded modulation of some transition rates within and between deep‐closed and inactivated states. The extent of modulation correlates strongly with the length of amino‐terminal domains. Two mutant ß2‐subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.—Herzig, S., Khan, I. F. Y., Gründemarin, D., Matthes, J., Ludwig, A., Michels, G., Hoppe, U. C., Chaudhuri, D., Schwartz, A., Yue, D. T., Hullin, R. Mechanism of Cav1.2 channel modulation by the amino terminus of cardiac ß2‐sub‐units. FASEB J. 21, 1527–1538 (2007)