Martin Wolny

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.

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+.

Introduction The possible role of UCP2 in modulating mitochondrial Ca2+-uptake (mCa2+-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial. Methods Thus, we analyzed mCa2+-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca2+-transients from mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2-/- mice. Results Isolated mitochondria showed a Ru360 sensitive mCa2+-uptake, which was significantly decreased in UCP2-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca2+-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2-/- cardiomyocytes (Ca2+)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca2+)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca2+-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa2+-uptake in WT mitoplasts and (Ca2+)m of cardiomyocytes leading to an increase of (Ca2+)c while no ATP dependent effect was observed in UCP2-/-. Conclusion Our results indicate regulatory effects of UCP2 on mCa2+-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.

Distinct regulation of cardiac If current via thyroid receptors alpha1 and beta1

Thyroid hormone (TH) markedly modulates cardiovascular function and heart rate. The pacemaker current If and encoding hyperpolarization-activated cation (HCN) genes have been identified as TH targets. To analyze the specific contribution and functional significance of thyroid receptor isoforms responsible for HCN gene transactivation, we generated transgenic neonatal rat cardiomyocytes with adenovirus-mediated overexpression of the thyroid receptors alpha1 (TRα1) and beta1 (TRβ1), and analyzed native If current and expression levels of the underlying molecular components HCN2 and HCN4. Initial results revealed that spontaneous beating activity was higher in TRα1- and lower in TRβ1-expressing cardiomyocytes. This was associated with accelerated depolarization velocity and abbreviated action potential duration in cells overexpressing TRα1, while TRβ1 suppressed phase 4 depolarization and prolonged action potentials. Consistently, TRα1-infected myocytes exhibited larger If current densities along with increased HCN2 and HCN4 mRNA and protein levels. In contrast, HCN2 gene expression was not significantly affected by TRβ1. TRβ1 exclusively suppressed HCN4 transcription. T3 application led to significant effects only in controls and TRα1-infected cardiomyocytes; whereas, no ligand-dependent actions were observed in TRβ1-expressing neonatal cardiomyocytes. Our results demonstrate that TRα1 and TRβ1 divergently regulate cardiac pacing activity. TH-induced positive chronotropic effects are likely to be mediated by TRα1 through enhanced expression of If pacemaker current and its underlying genes.

UCP2 Modulates Cardioprotective Effects of Ru360 in Isolated Cardiomyocytes during Ischemia

Introduction: Ruthenium 360 (Ru360) has been shown to induce cardioprotective mechanisms in perfused hearts. The agent is a specific blocker of the main cardiac mitochondrial uptake mechanism, the mitochondrial calcium uniporter (MCU). UCP2, a mitochondrial membrane protein, which influences cardiac ROS formation was reported to interact with the MCU. Methods: To prove whether Ru360 affects ischemic cell injury on the singular cell level, cell viability (CV) in isolated cardiomyocytes from wild type mice (WT) was measured in a model of pelleting hypoxia (PH). To explore a possible influence of UCP2 on cellular survival, as well as on Ru360 function, cardiomyocytes from UCP2−/− mice were investigated. Results: During PH, Ru360 significantly improved CV in WT cardiomyocytes (Control 26.32% ± 1.58% vs. PH 13.60% ± 1.20% vs. PH+Ru360 19.98% ± 0.98%, n = 6; p < 0.05). No differences in the rate of apoptosis were observed in UCP2−/− vs. WT. In UCP2−/− cardiomyocytes, Ru360 reduced the rate of cell death. However, the effect was less pronounced compared to WT cardiomyocytes. Conclusion: Ru360 significantly reduces hypoxic cell injury by preventing single cell apoptosis in WT cardiomyoctes. UCP2 does not affect cell survival in hypoxic cardiomyocytes, but it might modulate cardioprotective effects of Ru360 during ischemia.

UCP3 Regulates Single-Channel Activity of the Cardiac mCa1.

Mitochondrial Ca2+ uptake (mCa2+ uptake) is thought to be mediated by the mitochondrial Ca2+ uniporter (MCU). UCP2 and UCP3 belong to a superfamily of mitochondrial ion transporters. Both proteins are expressed in the inner mitochondrial membrane of the heart. Recently, UCP2 was reported to modulate the function of the cardiac MCU related channel mCa1. However, the possible role of UCP3 in modulating cardiac mCa2+ uptake via the MCU remains inconclusive. To understand the role of UCP3, we analyzed cardiac mCa1 single-channel activity in mitoplast-attached single-channel recordings from isolated murine cardiac mitoplasts, from adult wild-type controls (WT), and from UCP3 knockout mice (UCP3–/–). Single-channel registrations in UCP3−/− confirmed a murine voltage-gated Ca2+ channel, i.e., mCa1, which was inhibited by Ru360. Compared to WT, mCa1 in UCP3−/− revealed similar single-channel characteristics. However, in UCP3−/− the channel exhibited decreased single-channel activity, which was insensitive to adenosine triphosphate (ATP) inhibition. Our results suggest that beyond UCP2, UCP3 also exhibits regulatory effects on cardiac mCa1/MCU function. Furthermore, we speculate that UCP3 might modulate previously described inhibitory effects of ATP on mCa1/MCU activity as well.

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.”