Jan Matthes

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.

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)

Disturbed atrio-ventricular conduction and normal contractile function in isolated hearts from Cav1.3-knockout mice

Cardiac L-type calcium channels are formed by two α-subunits, Cav1.2 (α1C) and Cav1.3 (α1D). In contrast to the uniform expression pattern of Cav1.2, Cav1.3 is highly expressed in sino-atrial node (SAN) and atrial tissue, but not in the ventricle. Accordingly, knockout of Cav1.3 (Cav1.3−/−) in mice was shown to lead to a cardiac phenotype characterised by severe bradycardia in vivo and in isolated SAN cells. Cav1.3 may therefore constitute a novel pharmacological target for specific bradycardic agents.RNAse protection assays of murine wild type hearts revealed rather high Cav1.3 levels comparable to Cav1.2, suggesting functional relevance of Cav1.3 outside specialised tissues such as SAN. Due to the lack of specific Cav1.3 blockers, we directly examined the functional role of Cav1.3 using isolated working hearts from adult wild type (WT) and Cav1.3−/− mice. Histological analysis of hearts revealed no pathological changes. Ventricular contractility and inotropic effects of isoproterenol were unaltered in Cav1.3−/− hearts. Severe sinus bradycardia already noted in vivo was accompanied by ventricular extrasystoles. This phenotype was restored to nearly normal values by the cumulative addition of isoproterenol. Electrocardiograms of Cav1.3−/− hearts revealed delayed atrio-ventricular (AV) conduction and a decoupling of heart rate and PR interval duration. Isoproterenol did not improve disturbance of AV conduction.In conclusion, suppression of Cav1.3 does not alter ventricular contractile function, and the decrease in sinus node frequency is counterbalanced by adrenergic stimulation. Importantly, bradyarrhythmia is partly due to an intrinsic AV node dysfunction, which is resistant to adrenergic counterbalance. These findings help to predict the clinical pattern of selective Cav1.3 blockade.

Transgenic simulation of human heart failure-like L-type Ca2+-channels: implications for fibrosis and heart rate in mice

AIMS Cardiac L-type Ca(2+)-currents show distinct alterations in chronic heart failure, including increased single-channel activity and blunted adrenergic stimulation, but minor changes of whole-cell currents. Expression of L-type Ca(2+)-channel beta(2)-subunits is enhanced in human failing hearts. In order to determine whether prolonged alteration of Ca(2+)-channel gating by beta(2)-subunits contributes to heart failure pathogenesis, we generated and characterized transgenic mice with cardiac overexpression of a beta(2a)-subunit or the pore Ca(v)1.2 or both, respectively. METHODS AND RESULTS Four weeks induction of cardiac-specific overexpression of rat beta(2a)-subunits shifted steady-state activation and inactivation of whole-cell currents towards more negative potentials, leading to increased Ca(2+)-current density at more negative test potentials. Activity of single Ca(2+)-channels was increased in myocytes isolated from beta(2a)-transgenic mice. Ca(2+)-current stimulation by 8-Br-cAMP and okadaic acid was blunted in beta(2a)-transgenic myocytes. In vivo investigation revealed hypotension and bradycardia upon Ca(v)1.2-transgene expression but not in mice only overexpressing beta(2a). Double-transgenics showed cardiac arrhythmia. Interstitial fibrosis was aggravated by the beta(2a)-transgene compared with Ca(v)1.2-transgene expression alone. Overt cardiac hypertrophy was not observed in any model. CONCLUSION Cardiac overexpression of a Ca(2+)-channel beta(2a)-subunit alone is sufficient to induce Ca(2+)-channel properties characteristic of chronic human heart failure. beta(2a)-overexpression by itself did not induce cardiac hypertrophy or contractile dysfunction, but aggravated the development of arrhythmia and fibrosis in Ca(v)1.2-transgenic mice.

Functional Adenylyl Cyclase Inhibition in Murine Cardiomyocytes by 2′(3′)-O-(N-Methylanthraniloyl)-Guanosine 5′-[γ-Thio]triphosphate

β1-Adrenergic receptor activation stimulates cardiac L-type Ca2+ channels via adenylyl cyclases (ACs), with AC5 and AC6 being the most important cardiac isoforms. Recently, we have identified 2′(3′)-O-(N-methylanthraniloyl)-guanosine 5′-[γ-thio-]triphosphate (MANT-GTPγS) as a potent competitive AC inhibitor. Intriguingly, MANT-GTPγS inhibits AC5 and -6 more potently than other cyclases. These data prompted us to study the effects of MANT-GTPγS on L-type Ca2+ currents (ICa,L) in ventricular myocytes of wild-type (WT) and AC5-deficient (AC5–/–) mice by whole-cell recordings. In wild-type myocytes, MANT-GTPγS attenuated ICa,L stimulation following isoproterenol application in a concentration-dependent manner (control, +77 ± 13%; 100 nM MANT-GTPγS, +43 ± 6%; 1 μM MANT-GTPγS, +21 ± 9%; p < 0.05). The leftward shift of current-voltage curves was abolished by 1 μM but not by 100 nM MANT-GTPγS. In myocytes from AC5–/– mice, the residual stimulation of ICa,L was not further attenuated by the nucleotide, indicating AC5 to be the major AC isoform mediating acute β-adrenergic stimulation in WT mice. Interestingly, basal ICa,L was lowered by 1 μM but not by 100 nM MANT-GTPγS. The decrease was less pronounced in myocytes from AC5–/– mice compared with wild types (–23 ± 1 versus –40 ± 7%), indicating basal ICa,L to be partly driven by AC5. Collectively, we found a concentration-dependent inhibition of ICa,L by MANT-GTPγS, both under basal conditions and following β-adrenergic stimulation. Comparison of data from wild-type and AC5-deficient mice indicates that AC5 plays a major role in ICa,L activation and that MANT-GTPγS predominantly acts via AC5 inhibition.

Rare Mutations of CACNB2 Found in Autism Spectrum Disease-Affected Families Alter Calcium Channel Function

Autism Spectrum Disorders (ASD) are complex neurodevelopmental diseases clinically defined by dysfunction of social interaction. Dysregulation of cellular calcium homeostasis might be involved in ASD pathogenesis, and genes coding for the L-type calcium channel subunits CaV1.2 (CACNA1C) and CaVβ2 (CACNB2) were recently identified as risk loci for psychiatric diseases. Here, we present three rare missense mutations of CACNB2 (G167S, S197F, and F240L) found in ASD-affected families, two of them described here for the first time (G167S and F240L). All these mutations affect highly conserved regions while being absent in a sample of ethnically matched controls. We suggest the mutations to be of physiological relevance since they modulate whole-cell Ba2+ currents through calcium channels when expressed in a recombinant system (HEK-293 cells). Two mutations displayed significantly decelerated time-dependent inactivation as well as increased sensitivity of voltage-dependent inactivation. In contrast, the third mutation (F240L) showed significantly accelerated time-dependent inactivation. By altering the kinetic parameters, the mutations are reminiscent of the CACNA1C mutation causing Timothy Syndrome, a Mendelian disease presenting with ASD. In conclusion, the results of our first-time biophysical characterization of these three rare CACNB2 missense mutations identified in ASD patients support the hypothesis that calcium channel dysfunction may contribute to autism.

Pharmacoresistant Cav2·3 (E-type/R-type) voltage-gated calcium channels influence heart rate dynamics and may contribute to cardiac impulse conduction

Voltage‐gated Ca2+ channels regulate cardiac automaticity, rhythmicity and excitation–contraction coupling. Whereas L‐type (Cav1·2, Cav1·3) and T‐type (Cav3·1, Cav3·2) channels are widely accepted for their functional relevance in the heart, the role of Cav2·3 Ca2+ channels expressing R‐type currents remains to be elucidated.

Spectrum of Cav1.4 dysfunction in congenital stationary night blindness type 2.

Defective retinal synaptic transmission in patients affected with congenital stationary night blindness type 2 (CSNB2) can result from different dysfunction phenotypes in Cav1.4 L-type calcium channels. Here we investigated two prototypical Cav1.4 variants from either end of the functional spectrum. Using whole-cell and single-channel patch-clamp techniques, we provide analysis of the biophysical characteristics of the point mutation L860P and the C-terminal truncating mutation R1827X. L860P showed a typical loss-of-function phenotype attributed to a reduced number of functional channels expressed at the plasma membrane as implied by gating current and non-stationary noise analyses. This phenotype can be rationalized, because the inserted proline is predicted to break an amphipatic helix close to the transmembrane segment IIIS1 and thus to reduce channel stability and promote misfolding. In fact, L860P was subject to an increased turnover. In contrast, R1827X displayed an apparent gain-of-function phenotype, i.e., due to a hyperpolarizing shift of the IV-curve and increased single-channel activity. However, truncation also resulted in the loss of functional C-terminal modulation and thus unmasked calcium-dependent inactivation. Thus R1827X failed to support continuous calcium influx. Current inactivation curtails the dynamic range of photoreceptors (e.g., when adapting to variation in illumination). Taken together, the analysis of two representative mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches.

Gαi2- and Gαi3-Specific Regulation of Voltage-Dependent L-Type Calcium Channels in Cardiomyocytes

Background Two pertussis toxin sensitive Gi proteins, Gi2 and Gi3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous Gi isoforms are functionally distinct. To test for isoform-specific functions of Gi proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC). Methods Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gαi2 (Gαi2 −/−) or Gαi3 (Gαi3 −/−). mRNA levels of Gαi/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gαi and Cavα1 protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings. Results In cardiac tissue from Gαi2 −/− mice, Gαi3 mRNA and protein expression was upregulated to 187±21% and 567±59%, respectively. In Gαi3 −/− mouse hearts, Gαi2 mRNA (127±5%) and protein (131±10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gαi2 −/− mice was lowered (−7.9±0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (−10.7±0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gαi3 −/− mice (−14.3±0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gαi2 (but not of Gαi3) and following treatment with pertussis toxin in Gαi3 −/−. The pore forming Cavα1 protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Cavα1 and Cavβ2 subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gαi2. Conclusion Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gαi proteins. In particular, loss of Gαi2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.