Dennis Rottlaender

Regulation of the Human Cardiac Mitochondrial Ca2+ Uptake by 2 Different Voltage-Gated Ca2+ Channels

Background— Impairment of intracellular Ca2+ homeostasis and mitochondrial function has been implicated in the development of cardiomyopathy. Mitochondrial Ca2+ uptake is thought to be mediated by the Ca2+ uniporter (MCU) and a thus far speculative non-MCU pathway. However, the identity and properties of these pathways are a matter of intense debate, and possible functional alterations in diseased states have remained elusive. Methods and Results— By patch clamping the inner membrane of mitochondria from nonfailing and failing human hearts, we have identified 2 previously unknown Ca2+-selective channels, referred to as mCa1 and mCa2. Both channels are voltage dependent but differ significantly in gating parameters. Compared with mCa2 channels, mCa1 channels exhibit a higher single-channel amplitude, shorter openings, a lower open probability, and 3 to 5 subconductance states. Similar to the MCU, mCa1 is inhibited by 200 nmol/L ruthenium 360, whereas mCa2 is insensitive to 200 nmol/L ruthenium 360 and reduced only by very high concentrations (10 &mgr;mol/L). Both mitochondrial Ca2+ channels are unaffected by blockers of other possibly Ca2+-conducting mitochondrial pores but were activated by spermine (1 mmol/L). Notably, activity of mCa1 and mCa2 channels is decreased in failing compared with nonfailing heart conditions, making them less effective for Ca2+ uptake and likely Ca2+-induced metabolism. Conclusions— Thus, we conclude that the human mitochondrial Ca2+ uptake is mediated by these 2 distinct Ca2+ channels, which are functionally impaired in heart failure. Current properties reveal that the mCa1 channel underlies the human MCU and that the mCa2 channel is responsible for the ruthenium red–insensitive/low-sensitivity non-MCU–type mitochondrial Ca2+ uptake.

Connexin 43 acts as a cytoprotective mediator of signal transduction by stimulating mitochondrial KATP channels in mouse cardiomyocytes

Potassium (K+) channels in the inner mitochondrial membrane influence cell function and survival. Increasing evidence indicates that multiple signaling pathways and pharmacological actions converge on mitochondrial ATP-sensitive K+ (mitoKATP) channels and PKC to confer cytoprotection against necrotic and apoptotic cell injury. However, the molecular structure of mitoKATP channels remains unresolved, and the mitochondrial phosphoprotein(s) that mediate cytoprotection by PKC remain to be determined. As mice deficient in the main sarcolemmal gap junction protein connexin 43 (Cx43) lack this cytoprotection, we set out to investigate a possible link among mitochondrial Cx43, mitoKATP channel function, and PKC activation. By patch-clamping the inner membrane of subsarcolemmal murine cardiac mitochondria, we found that genetic Cx43 deficiency, pharmacological connexin inhibition by carbenoxolone, and Cx43 blockade by the mimetic peptide 43GAP27 each substantially reduced diazoxide-mediated stimulation of mitoKATP channels. Suppression of mitochondrial Cx43 inhibited mitoKATP channel activation by PKC. MitoKATP channels of interfibrillar mitochondria, which do not contain any detectable Cx43, were insensitive to both PKC activation and diazoxide, further demonstrating the role of Cx43 in mitoKATP channel stimulation and the compartmentation of mitochondria in cell signaling. Our results define a role for mitochondrial Cx43 in protecting cardiac cells from death and provide a link between cytoprotective stimuli and mitoKATP channel opening, making Cx43 an attractive therapeutic target for protection against cell injury.

Clinical Impact of Atrial Fibrillation in Patients with Pulmonary Hypertension

Background Pulmonary hypertension (PH) is associated with progressive impairment of right ventricular function, reduced exercise capacity and a poor prognosis. Little is known about the prevalence, clinical manifestation and impact of atrial fibrillation (AF) on cardiac function in PH. Methods In a four year single-centre retrospective analysis 225 patients with confirmed PH of various origins were enrolled to investigate the prevalence of AF, and to assess the clinical manifestation, 6-minute walk distance, NT-proBNP levels, echocardiographic parameters and hemodynamics obtained by right heart catheterization in PH with AF. Results AF was prevalent in 31.1%. In patients with PH and AF, parameters of clinical deterioration (NYHA/WHO functional class, 6-minute walk distance, NT-proBNP levels) and renal function were significantly compromised compared to patients with PH and sinus rhythm (SR). In the total PH cohort and in PH not related to left heart disease occurrence of AF was associated with an increase of right atrial pressure (RAP) and right atrial dilatation. While no direct association was found between pulmonary artery pressure (PAP) and AF in these patients, right ventricular function was reduced in AF, indicating more advanced disease. In PH due to left heart failure the prevalence of AF was particularly high (57.7% vs. 23.1% in other forms of PH). In this subgroup, left atrial dilatation, increase of pulmonary capillary wedge pressure, PAP and RAP were more pronounced in AF than in SR, suggesting that more marked backward failure led to AF in this setting. Conclusion PH is associated with increased prevalence of AF. Occurrence of AF in PH indicates clinical deterioration and more advanced disease.

Glycogen synthase kinase 3β transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K+ channels

Despite compelling evidence supporting key roles for glycogen synthase kinase 3β (GSK3β), mitochondrial adenosine triphosphate-sensitive K+ (mitoKATP) channels, and mitochondrial connexin 43 (Cx43) in cytoprotection, it is not clear how these signaling modules are linked mechanistically. By patch-clamping the inner membrane of murine cardiac mitochondria, we found that inhibition of GSK3β activated mitoKATP. PKC activation and protein phosphatase 2a inhibition increased the open probability of mitoKATP channels through GSK3β, and this GSK3β signal was mediated via mitochondrial Cx43. Moreover, (i) PKC-induced phosphorylation of mitochondrial Cx43 was reduced in GSK3β-S9A mice; (ii) Cx43 and GSK3β proteins associated in mitochondria; and (iii) SB216763-mediated reduction of infarct size was abolished in Cx43 KO mice in vivo, consistent with the notion that GSK3β inhibition results in mitoKATP opening via mitochondrial Cx43. We therefore directly targeted mitochondrial Cx43 by the Cx43 C-terminal binding peptide RRNYRRNY for cardioprotection, circumventing further upstream pathways. RRNYRRNY activated mitoKATP channels via Cx43. We directly recorded mitochondrial Cx43 channels that were activated by RRNYRRNY and blocked by the Cx43 mimetic peptide 43GAP27. RRNYRRNY rendered isolated cardiomyocytes in vitro and the heart in vivo resistant to ischemia/reperfusion injury, indicating that mitochondrial Cx43- and/or mitoKATP-mediated reduction of infarct size was not undermined by RRNYRRNY-related opening of sarcolemmal Cx43 channels. Our results demonstrate that GSK3β transfers cytoprotective signaling through mitochondrial Cx43 onto mitoKATP channels and that Cx43 functions as a channel in mitochondria, being an attractive target for drug treatment against cardiomyocyte injury.

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.

Effects of KCNE2 on HCN isoforms: distinct modulation of membrane expression and single channel properties.

Hyperpolarization-activated cation (HCN) channels give rise to an inward current with similar but not identical characteristics compared with the pacemaker current (I(f)), suggesting that HCN channel function is modulated by regulatory beta-subunits in native tissue. KCNE2 has been proposed to serve as a beta-subunit of HCN channels; however, available data remain contradictory. To further clarify this situation, we therefore analyzed the effect of KCNE2 on whole cell currents, single channel properties, and membrane protein expression of all cardiac HCN isoforms in the CHO cell system. On the whole cell level, current densities of all HCN isoforms were significantly increased by KCNE2 without altering voltage dependence or current reversal. While these results correlated well with the KCNE2-mediated 2.2-fold and 1.6-fold increases of membrane protein levels of HCN2 and HCN4, respectively, no effect of KCNE2 on HCN1 expression was obtained. All HCN subtypes displayed faster activation kinetics upon coexpression with KCNE2. Most importantly, for the first time, we demonstrated modulation of single channel function by KCNE2, thus supporting direct functional interaction with HCN subunits. In the presence of KCNE2, the single channel amplitudes and conductance of HCN1, HCN2, and HCN4 were significantly increased versus control recordings. Mean open time was significantly increased in cells coexpressing HCN2 + KCNE2, whereas it was unaffected in HCN1 + KCNE2 cotransfected cells and reduced in HCN4 + KCNE2 cotransfected cells compared with the respective HCN subunits alone. Thus, we demonstrate KCNE2-mediated distinct effects on HCN membrane expression and direct functional modulation of HCN isoforms, further supporting that KCNE2 surves as a regulatory beta-subunit of HCN channels.

Retraction for Rottlaender et al., "Glycogen synthase kinase 3β transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K+ channels".

MEDICAL SCIENCES Retraction for “Glycogen synthase kinase 3β transfers cytoprotective signaling through connexin 43 onto mitochondrial ATPsensitive K channels,” by Dennis Rottlaender, Kerstin Boengler, Martin Wolny, Astrid Schwaiger, Lukas J. Motloch, Michel Ovize, Rainer Schulz, Gerd Heusch, and Uta C. Hoppe, which appeared in issue 5, January 31, 2012, of Proc Natl Acad Sci USA (109: E242– E251; first published January 11, 2012; 10.1073/pnas.1107479109). The authors wish to note the following: “In the course of intense investigations, the first author (D.R.) has admitted that he has committed intentional, systematic manipulation of the electrophysiological data collected in Cologne. Of note, the manipulation of data does not affect Western blots and infarct size data collected in Essen, Giessen, and Cologne, cell volume data collected in Essen, cell viability data collected in Salzburg and Cologne, or transgenic mice contributed from Essen and Lyon. Accordingly, we wish to retract the paper.”