HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/11/1300 03-0520fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SPITKOVSKY, D. Articles by WIESNER, R. J. Search for Related Content PubMed PubMed Citation Articles by SPITKOVSKY, D. Articles by WIESNER, R. J.

Activity of complex III of the mitochondrial electron transport chain is essential for early heart muscle cell differentiation

DIMITRY SPITKOVSKY^*, PHILIPP SASSE^,1, EUGEN KOLOSSOV^,1, CORNELIA BÖTTINGER, BERND K. FLEISCHMANN, JÜRGEN HESCHELER and RUDOLF J. WIESNER^*,2

^* Institute of Vegetative Physiology,
Institute of Neurophysiology, Center of Physiology and Pathophysiology, The University of Köln,
Institute of Physiology, University of Bonn, and
Axiogenesis AG, Germany

²Correspondence: Institute of Vegetative Physiology, Robert-Koch-Str. 39, 50931 Köln, Germany. E-mail: Rudolf.wiesner{at}uni-koln.de

SPECIFIC AIMS

The aim of this work was to study whether mitochondrial electron transport chain (ETC) activity is necessary for differentiation of heart muscle cells, since this is accompanied by proliferation of mitochondria. To approach this question, embryonic stem (ES) cells, in which expression of the enhanced green fluorescent protein (EGFP) gene is under control of the -myosin heavy chain (-MHC) promoter, were used to label differentiated cardiomyocytes as fluorescent, spontaneously contracting cells within embroid bodies (EB).

PRINCIPAL FINDINGS

1. Mitochondrial proteins increase only at a late stage in mouse heart development
In the atrium, mitochondrial marker proteins began to rise around E17.5, and there was another marked increase when comparing E19.5 with adult atrium. No significant differences were seen in ventricle samples for E16.5 and adult for cytochrome c oxidase subunits, but a notable increase was found in E19.5 vs. the adult stage. Transcription activators PGC-1 and TFAM, involved in stimulating expression of nuclear encoded mitochondrial genes and mtDNA, respectively, were increased in adult compared with prenatal tissues. Thus, an enhanced requirement for mitochondrial activity seems to occur rather late during embryonic heart development.

2. ETC activity is not necessary for function in ES cell-derived cardiomyocytes within EBs, but complex III is indispensable for cardiomyocyte development
Surprisingly, addition of antimycin A or KCN to EBs containing spontaneously beating cardiomyocytes (blockers of ETC complexes III and IV) did not depress contractile activity. Thus, ATP production by oxidative mitochondrial metabolism is not necessary for either contractile function of early cardiomyocytes or maintenance of their stage of differentiation.

When TTFA, a specific blocker of ETC complex II (Fig. 1 B) or KCN (Fig. 1C ), was applied directly upon transfer of EBs to suspension culture, cardiomyocytes developed into spontaneously beating areas. However, no cardiomyocyte differentiation was detected when antimycin A was added at this early time point (Fig. 1D ). Thus, a normal function of complex III of the ETC seems to be essential for cardiomyocyte differentiation whereas activity of complexes II and IV apparently is not required; the effect was reversible if antimycin A was removed.

View larger version (88K): [in this window] [in a new window]   Figure 1. Appearance of green fluorescent, contracting cardiomyocytes in mouse ES cell-derived embryoid bodies under control conditions (A) or after addition of mitochondrial electron transport chain complex II inhibitor TTFA (B), complex IV inhibitor KCN (C) or complex III inhibitor antimycin A (D). At 2 days, EBs were transferred from hanging drop to suspension culture; at 7 days they were plated into individual wells and incubated for another 6 days. Stippled boxes in the ruler indicate the time of incubation with the different inhibitors. Each panel is representative for 24 individual EBs that had been inspected.

Cultivation of EBs for a total of 12 days after the hanging drop incubation in the presence of antimycin A showed that removal of the blocker on the last day of the experimental period was sufficient to enable the appearance of some EGFP-expressing cardiomyocytes. Thus, the block of differentiation is not due to general toxicity of the drug but presumably to interference with some specific signal(s) starting the cardiomyocyte differentiation program. This result shows that precursors are kept in a state of competence for cardiomyocyte differentiation in the presence of antimycin A, and immediately resume the differentiation program when the block is removed. When antimycin A was applied at different days after transfer from the hanging drop incubation into suspension culture, the results clearly demonstrated that cardiomyocyte precursors had passed a critical stage within 4 days. If antimycin A was applied before day 4 in suspension culture, differentiation into cardiomyocytes was not observed. When applied at later stages, however, it could not block cardiomyocyte differentiation and beating areas developed normally.

Incubation of EBs on day 4 with the Ca²⁺ ionophore ionomycin for 2 h rescued cardiomyocyte development to some extent whereas incubation on day 5 was ineffective. Analysis of the expression of transcriptions factors MEF2C, GATA4 and Nkx2.5, centrally involved in heart muscle cell differentiation, by semiquantitative RT/PCR showed this was not influenced by antimycin A or ionomycin.

3. Complex III of the ETC is necessary for spontaneous Ca²⁺ oscillations in early mouse cardiomyocytes
Antimycin A completely blocked the rhythmic Ca²⁺ spiking typical for embryonic heart muscle cells (Fig. 2 A, n=4) shown by others to be important for cardiomyocyte differentiation in EBs, whereas KCN had no obvious effect (Fig. 2B , n=17). As expected, both antimycin A (n=10) and KCN (n=17) depolarized the inner mitochondrial membrane to approximately the same extent, but less than the protonophore CCCP (n=2) (Fig. 2C ). Thus, complex III of the ETC seems to be essential for Ca²⁺ oscillations whereas activity of complex IV appears to be dispensable.

View larger version (33K): [in this window] [in a new window]   Figure 2. Effect of the mitochondrial ETC inhibitors antimycin A (A) and KCN (B) on spontaneous Ca2+ oscillations in a single mouse embryonic cardiomyocyte analyzed by ratiometric measurement of FURA 2-AM fluorescence (n=5). C) Effect of antimycin A (n=10), KCN (n=17), and the mitochondrial uncoupler CCCP (n=2) on the mitochondrial inner membrane potential of a single mouse embryonic cardiomyocyte analyzed by TMRE fluorescence. Representative experiments are shown.

CONCLUSIONS AND SIGNIFICANCE

In the present study, we show that a functioning complex III of the mitochondrial ETC is necessary for early heart muscle cell differentiation that is not due to its obvious role in oxidative metabolism and thus ATP generation, as may have been expected. During development, however, mitochondrial mass increases in cardiomyocytes only at rather late stages. On the other hand, it has been shown that even at early stages mitochondrial function is necessary for cardiomyogenesis, since ablation of the ETC in mice through early inactivation of the gene for mitochondrial transcription factor A (TFAM) completely inhibited heart development. TFAM is necessary for transcription and replication of mtDNA, which encodes a few, but essential, protein subunits of complex I, III, IV, and V of the oxidative phosphorylation system. Homozygous tfam –/– animals showed severe growth retardation and died in utero between E8.5 and E10.5; recognizable cardiac structures were absent.

The most obvious explanation for these findings and ours may be that ATP production by anaerobic pathways is insufficient to maintain organ function, even in the very early embryo, thus not allowing normal development. However, the absence of cardiac structures in the tfam –/– embryos already precluded our findings, namely, that mitochondrial ETC activity is necessary for heart development but independent of its energy-generating function. Even more interesting, it is not ETC activity in general, but complex III function, that is a prerequisite for cardiomyocyte differentiation whereas function of complexes II and IV is dispensable. Complex III is a major site of superoxide radical formation; thus, its inhibition could be detrimental to developing cardiomyocytes. However, in our hands antimycin A incubation of HeLa cells elicits only very small increases in reactive oxygen species; on the contrary, our group had shown that endogenous ROS-production is necessary and exogenously added H₂O₂ is even stimulating for cardiomyocyte development in EBs.

Another possible explanation for the specific requirement for a functioning complex III is to prevent the buildup of a highly reduced ubiquinol pool, which may inhibit the adequate synthesis of pyrimidine nucleotides via dihydroorotate dehydrogenase. Indeed, it was recently shown that the lack of ubiquinon after ablation of the murine homologue of the C. elegans clk-1 gene leads to embryonic lethality around E9.5, without largely affecting mitochondrial respiratory activity. Thus, complex III of the mitochondrial ETC indeed seems to have a function(s) in development, which is clearly separable from ATP generation.

The transcription factor myocyte enhancer factor MEF2C, among others, is known to be indispensable for early cardiogenesis. It has been shown that absence of Ca²⁺ spiking in early ES cell-derived cardiomyocytes lacking the Ca²⁺ handling protein calreticulin (crt) leads to failure of translocation of MEF2C into the nucleus, a lack of myofibrillogenesis and a block of cardiac differentiation. A normal phenotype could be rescued by ionomycin in such crt –/– cells, triggering an increase in cytosolic Ca²⁺. On the contrary, the developmental block could be mimicked by chelating Ca²⁺ in wild-type cardiomyocytes. Intriguingly, these authors using the same protocol we used here showed that cardiomyocytes seemed to undergo a normal commitment up to 4 days after transfer from hanging drop to suspension culture, but then obviously needed a Ca²⁺ signal to achieve full differentiation. Mitochondria have been shown to interact with the endoplasmic/sarcoplasmic reticulum during Ca²⁺ spiking in neonatal rat cardiomyocytes involving IP3-sensitive Ca release sites. We found in the present study that 1) early cardiomyocytes were sensitive to antimycin A only up to day 2 + 4, 2) antimycin A inhibited Ca²⁺ spiking in early heart cells, and 3) the antimycin A block could also be relieved by a ionomycin pulse on day 4. Expression of key factors necessary for cardiomyocyte differentiation was unchanged. Therefore, we suggest that it is complex III of the mitochondrial ETC that is necessary for autonomous Ca²⁺ spiking, together with the endoplasmic/sarcoplasmic reticulum. This rise in cytosolic Ca²⁺, on the other hand, is indispensable for committing precursor cells to the cardiac differentiation program via Ca²⁺-calmodulin-kinase-dependent phosphorylation of MEF2C, which is then translocated into the nucleus.

View larger version (30K): [in this window] [in a new window]   Figure 3. Schematic diagram outlining the indispensable role of complex III of the mitochondrial electron transport chain (ETC) for a critical checkpoint in cardiomyocyte differentiation, which may be identical to its essential role in Ca2+ oscillations, necessary for Ca-calmodulin-kinase (CAMK) -dependent developmental pathways as nuclear translocation of phosphorylated MEF2C.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0520fje; doi: 10.1096/fj.03-0520fje

This article has been cited by other articles:

				F. Sharifpanah, M. Wartenberg, M. Hannig, H.-M. Piper, and H. Sauer Peroxisome Proliferator-Activated Receptor {alpha} Agonists Enhance Cardiomyogenesis of Mouse ES Cells by Utilization of a Reactive Oxygen Species-Dependent Mechanism Stem Cells, January 1, 2008; 26(1): 64 - 71. [Abstract] [Full Text] [PDF]

				J. M. Facucho-Oliveira, J. Alderson, E. C. Spikings, S. Egginton, and J. C. St. John Mitochondrial DNA replication during differentiation of murine embryonic stem cells J. Cell Sci., November 15, 2007; 120(22): 4025 - 4034. [Abstract] [Full Text] [PDF]

				P. Sasse, J. Zhang, L. Cleemann, M. Morad, J. Hescheler, and B. K. Fleischmann Intracellular Ca2+ Oscillations, a Potential Pacemaking Mechanism in Early Embryonic Heart Cells J. Gen. Physiol., July 30, 2007; 130(2): 133 - 144. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/11/1300 03-0520fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SPITKOVSKY, D. Articles by WIESNER, R. J. Search for Related Content PubMed PubMed Citation Articles by SPITKOVSKY, D. Articles by WIESNER, R. J.