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An increase in cardiac afterload typically produces concentric hypertrophy characterized by an increase in cardiomyocyte width, while volume overload or exercise results in eccentric growth characterized by cellular elongation and addition of sarcomeres in series. The signaling pathways that control eccentric versus concentric heart growth are not well understood.
To determine the role of extracellular signal-regulated kinases 1/2 in regulating the cardiac hypertrophic response.
Here we used mice lacking all ERK1/2 protein in the heart (Erk1−/− Erk2fl/fl-Cre) and mice expressing activated Mek1 in the heart to induce ERK1/2 signaling, as well as mechanistic experiments in cultured myocytes to assess cellular growth characteristics associated with this signaling pathway. While genetic deletion of all ERK1/2 from the mouse heart did not block the cardiac hypertrophic response per se, meaning that the heart still increased in weight with both aging and pathologic stress stimulation, it did dramatically alter how the heart grew. For example, adult myocytes from hearts of Erk1−/− Erk2fl/fl-Cre mice showed preferential eccentric growth (lengthening) while myocytes from Mek1 transgenic hearts showed concentric growth (width increase). Isolated adult myocytes acutely inhibited for ERK1/2 signaling by adenoviral gene transfer showed spontaneous lengthening while infection with an activated Mek1 adenovirus promoted constitutive ERK1/2 signaling and increased myocyte thickness. A similar effect was observed in engineered heart tissue under cyclical stretching, where ERK1/2 inhibition led to preferential lengthening.
Taken together these data demonstrate that the ERK1/2 signaling pathway uniquely regulates the balance between eccentric and concentric growth of the heart.
We studied mice lacking all ERK1/2 protein in the heart and mice expressing activated Mek1 in the heart to evaluate the role of the ERK 1/2 signaling cascade in regulating the cardiac hypertrophic response. While activation of the ERK pathway induced concentric hypertrophy, inhibition of this pathway resulted in eccentric hypertrophy. Using cardiomyocytes isolated from these mouse models, and using ex vivo culture models we show that these effects were mediated directly by ERK signaling. Greater ERK1/2 signaling directly programmed myocyte thickening while inhibition of ERK1/2 signaling promoted myocyte lengthening. Thus, this study sheds light on the molecular mechanisms that are partially responsible for the differential hypertrophic response of the heart to an increase in preload versus afterload.
The myocardium undergoes cellular and ventricular chamber remodeling and/or hypertrophy as a means of augmenting or maintaining cardiac output in response to increased workload or pathologic insults. Cardiac hypertrophy is an independent risk factor for heart failure, arrhythmias, sudden cardiac death and for cardiovascular morbidity and mortality.1, 2 The fundamental response of the myocardium to an increase in afterload is termed concentric hypertrophy that consists of an increase in ventricular wall thickness without chamber enlargement.3 In contrast, conditions that increase preload, including significant valvular regurgitation and volume overload, promote chamber dilatation with no increase or even a decrease in left ventricular wall thickness, a process involving eccentric hypertrophy.
The concentric and eccentric hypertrophy of the entire myocardium is mainly the result of different modes of cardiomyocyte cell growth.4 In eccentric hypertrophy there is preferential assembly of contractile-protein units in series, leading to a relatively greater increase in the length than the width of myocytes. Concentric hypertrophy results from an assembly of contractile-protein units in parallel with a relative increase in the width of individual cardiac myocytes.5, 6 Concentric and eccentric growth likely result from orchestrated activation of specific intracellular signaling pathways, although the identity and mechanisms whereby these signaling pathways differentially regulate myocyte growth are not currently known.
The mitogen-activated protein kinases (MAPKs) have been implicated as focal mediators of cardiac hypertrophy in both cell culture and genetically modified mouse models.7 For example, the extracellular signal-regulated kinases 1/2 (ERK1/2) branch of the greater MAPK signaling cascade appears to induce a unique form of concentric cardiac hypertrophy in transgenic mice due to expression of activated MAPK kinase 1 (Mek1).8 Moreover, dozens of studies conducted in cultured cardiac myocytes have suggested that ERK1/2 signaling is necessary for promoting hypertrophic growth.9 Signaling through the ERK1/2 cascade is classically initiated at the cell membrane by activation of the small G protein Ras that then recruits the MAP3K Raf-1 to the plasma membrane where it is activated.10 ERK1/2 also become activated in cardiac myocytes in response to many stimuli such as G protein-coupled receptor agonists, receptor tyrosine kinase agonists, cytokines, reactive oxygen species, and stretch.7 These various stimuli initiate MAP3Ks, which then phosphorylate and activate the dual-specificity kinases MEK1 and MEK2 (MAP2Ks) that serve as dedicated kinases for ERK1/2 phosphorylation and activation.11
Interestingly, while transgenic mice over-expressing an activated form of Mek1 develop concentric hypertrophy, inhibition of ERK1/2 by overexpression of dual specificity phosphatase 6 (DUSP6), which inactivates ERK1/2 in the heart, did not reduce cardiac hypertrophy following multiple pathologic stimuli.12 These results suggest that while activation of MEK1-ERK1/2 signaling can program cardiac hypertrophy, ERK1/2 signaling may not be needed for cardiac hypertrophy induced by pathologic stimuli. These seemingly contradictory findings may be due to the type of cardiac growth that results from ERK1/2 activation or inactivation, such as concentric versus eccentric cardiac growth. Indeed, here we show that ERK1/2 activation specifically leads to concentric cardiac growth at the level of the cardiac myocyte, while inhibition of ERK1/2 preferentially permits eccentric growth. Through the combined use of Erk1 and Erk2 null mice, activated Mek1 expressing transgenic mice, and adult cardiomyocytes in culture, we show that ERK1/2 regulate the ability of myocytes to grow either in width (concentric) or in length (eccentric). Thus, the MEK1-ERK1/2 pathway may be the first identified signaling pathway capable of specifically directing the mode of cardiomyocyte hypertrophy.
Erk1−/− animals have been described before.13 Erk2-loxP (fl) mice were recently described.14 The Nkx2.5-Cre knock-in mice,15 and transgenic mice in which a nuclear-localizing Cre cDNA was placed under the control of the mouse α-myosin heavy chain (α-MHC) promoter were also described previously.16 Echocardiography for assessment of ventricular size and function was performed in 2% isoflurane anesthetized mice.16
Generation of protein samples from tissue, along with Western blotting and chemifluorescent detection, were described previously.8 All antibodies were obtained from Cell Signaling Biotechnology (Beverly, MA).
Hearts were collected at the indicated times, fixed in 10% formalin-containing PBS, and embedded in paraffin. Serial 5-μm heart sections were cut and stained with hematoxylin and eosin or Masson trichrome.
Invasive hemodynamics in the closed-chest mouse was performed with a 1.4F Millar catheter (Millar Instruments, Houston, Tex), as previously described.17
Adult ventricular myocytes were isolated as previously described.18 For histology or immunofluorescence cells were fixed and stained with antibodies for sarcomeric α-actinin (sigma). Contractility was assessed as described previously.19
A full listing of the methods and materials is given in the online supplement.
Previous studies have shown that Erk1-null mice have normal cardiac structure and function,12 although it was not possible to examine Erk2-null mice because of embryonic lethality.14, 20 To circumvent this problem and to unequivocally determine the necessary function of both ERK1/2 in the heart as hypertrophic mediators, we crossed Erk1−/− mice with Erk2fl/fl targeted mice and a cardiac Cre-recombinase expressing mouse line. We used both Nkx2.5-Cre knock-in mice and transgenicmice expressing Cre under the control of the mouse αMHC promoter.16 Western blotting showed absence of ERK1 protein (upper band) and very efficient depletion of ERK2 protein (lower band) in the same mutant hearts with either the Nkx2.5-Cre or αMHC-Cre lines (Figure 1A). No other changes were observed in ERK5, p38, or c-Jun N-terminal kinase (JNK1/2) protein levels or phosphorylation, although MEK1/2 were hyperphosphorylated with ablation of Erk1/2, highlighting a negative feedback mechanism21 (Figure 1A).
While mice of the genotype Cre, Erk1−/− Cre, Erk2fl/fl Cre, and Erk1−/− Erk2fl/fl had normal life spans, Erk1−/− Erk2fl/fl-Cre (both αMHC-Cre and Nkx2.5-Cre) began dying after 60 days of age and none survived beyond 7 months (Figure 1B and not shown). Interestingly, surviving Erk1−/− Erk2fl/fl-Cre mice showed spontaneous cardiac hypertrophy as defined by an increase in heart weight normalized to body weight (Figure 1C and 1D). This increase in heart weight also correlated with induction of the hypertrophic gene program, such as increased mRNA for Nppa, Nppb, Acta1, and Myh7 (Figure 1E). Histological analysis of these hearts showed ventricular dilation by 60–90 days of age with areas of mild fibrosis as assessed with Massons trichrome staining (Figure 1F and 1G and Online Figure IB and ID). However, analysis of cardiac histological sections and isolated cardiomyocytes did not show changes in myofiber organization or sarcomeric structure (Online Figure I, E and F). Analysis of apoptosis by TUNEL staining in these null hearts did not show increased cell death at baseline in 60–90 day old mice (Online Figure I, A and C). Thus, loss of Erk1/2 from the heart induces pathology and a form of hypertrophy without increased cell death.
Careful assessment of cardiac ventricular chamber dimensions showed dilation beginning even at 30 days of age in Erk1−/− Erk2fl/fl-Nkx-Cre mice, which became more pronounced by 60 and 90 days with both Cre lines (Figure 2A). Corresponding with ventricular dilation, cardiac ventricular performance as measured by echocardiography was significantly and progressively reduced at 30, 60, and 90 days of age in Erk1−/− Erk2fl/fl-Cre mice (both Nkx2.5-Cre and αMHC-Cre lines) (Figure 2B). Analysis of cardiac contractility with a Millar catheter also showed a severe reduction in function in Erk1−/− Erk2fl/fl-Nkx-Cre mice at 60 days of age at baseline and with increasing dosages of dobutamine (Figure 2C). However, Erk1−/− Erk2fl/fl-Nkx-Cre mice were not in overt heart failure at the age of 60 days because left ventricular end diastolic filling pressures were not elevated, nor was lung edema observed (Figure 2D and not shown). Myocytes isolated from hearts of Erk1−/− Erk2fl/fl-Nkx-Cre mice even showed enhanced contractility when compared to control cardiomyocytes (Online Figure II). However, with just 30 more days of age (90 days) double null mice developed overt failure with significantly reduced cardiac function and pulmonary edema as demonstrated by increased lung weight/body weight ratio (Figure 2E). Although resting electrocardiogram recordings were normal in the Erk1−/− Erk2fl/fl-Nkx-Cre mice (not shown), there may be some contribution of arrhythmia to the lethality as these mice aged and dilatory growth became extreme. Thus, we believe that overt heart failure is the primary cause of lethality in Erk1−/− Erk2fl/fl-Cre mice, as also witnessed by panting and reduced mobility with aging (Online movie).
While Erk1−/− Erk2fl/fl-Cre mice showed spontaneous cardiac hypertrophy with aging, it was still formally possible that loss of ERK1/2 from the heart might attenuate stress-induced cardiac hypertrophy given the countless studies that have suggested a necessary role for this signaling pathway in transducing growth of cultured cardiomyocytes. To address this issue we first subjected 45 day-old Erk1−/− Erk2fl/fl-Nkx-Cre mice to 14 days of angiotensin II−/−phenylephrine (AngII/PE) infusion to induce pathologic hypertrophy. Erk1−/− Erk2fl/fl control mice showed a significant increase in cardiac hypertrophy after 14 days of infusion, which was similar to the percent increase in heart weight observed in Erk1−/− Erk2fl/fl-Nkx-Cre mice after infusion (Figure 3A). However, Erk1−/− Erk2fl/fl-Nkx-Cre mice showed a greater increase in left ventricular end diastolic dimension (LVEDD) and a greater decrease in fractional shortening (FS) compared with control mice after 14 days of AngII/PE infusion (Figure 3B and 3C).
We also attempted to perform transverse aortic constriction (TAC) on Erk1−/− Erk2fl/fl-Nkx-Cre mice to assess their hypertrophic potential with this more potent stimulus, but were unsuccessful as all succumbed to rapid lethality given their compromised state. However, we were able to perform TAC on Erk1−/−αMHC-Cre mice, Erk2fl/fl-αMHC-Cre mice, and αMHC-Cre control mice for 2 weeks beginning at 60 days of age. Western blotting confirmed deletion of ERK1 protein in Erk1−/−αMHC-Cre mice and ERK2 protein in Erk2fl/fl-αMHC-Cre mice (Online Figure III, A). While all groups showed the same heart weights, ventricular chamber dimensions and FS under sham conditions, Erk2fl/fl-αMHC-Cre mice showed significantly more cardiac hypertrophy after TAC, a greater reduction in FS, but no change in LVEDD (Figure 3D, 3E, and 3F and Online Figure III, B-D). These results again suggest that ERK1/2 are not required for cardiac hypertrophy, and if anything, a loss of ERK1/2 from the heart produced slightly more growth with pathologic stimulation.
The loss of ERK1/2 also did not prevent physiologic exercise induced hypertrophy (Online Figure IV, A-C), a stimulus that itself tends to induce eccentric heart growth. Indeed, Erk1−/−Erk2fl/fl-Nkx-Cre mice actually developed significantly more cardiac hypertrophy with swimming exercise compared with controls, which was associated with greater chamber dilation and an even greater increase in myocyte length without an increase in myocyte thickness (Online Figure IV, AD).
Hearts from Erk1−/− Erk2fl/fl-Cre mice showed robust dilation and loss of whole organ function even though isolated myocytes from these hearts were not functionally compromised. This suggested that loss of ERK1/2 from the heart induced failure due to extreme ventricular remodeling, dilation and increased wall tension, but not due to myocyte dysfunction as is typically observed in most other forms of heart failure. To further understand the cellular mechanism underlying these observations we first isolated adult myocytes from hearts of Erk1−/− Erk2fl/fl-Nkx-Cre mice and compared them with myocytes from transgenic mice expressing activated (a) MEK1 (greater ERK1/2 activation). Remarkably, adult myocytes from hearts of Erk1−/− Erk2fl/fl-Nkx-Cre mice were significantly longer while aMEK1 myocytes were wider and shorter (Figure 4A and 4B). Indeed, the length-width ratios between these 2 genotypes was even more pronounced, suggesting that ERK1/2 activation promotes concentric myocyte growth, while loss of ERK1/2 results in a default program of eccentric growth (Figure 4C). This growth relationship was even more pronounced after AngII/PE stimulation, where myocytes from hearts of Erk1−/−;Erk2fl/fl-Nkx-Cre mice showed an even greater increase in cell length, although some increase in width was observed (Figure 4D and 4E). Similarly, as discussed above, swimming exercise caused significantly greater elongation of Erk1−/− Erk2fl/fl-Nkx-Cre cardiomyocytes compared with controls, without an increase in width (Online Figure IV, D).
To investigate the mechanism of cellular growth associated with ERK1/2 activity in adult myocytes we performed 2 additional assays. First, we isolated adult rat cardiomyocytes and infected them with recombinant adenoviruses encoding β-galactosidase (control) or dominant negative (dn) Mek1 and Dusp6 together to achieve complete inhibition of ERK1/2 signaling over 3 days in serum-free cultures (Figure 5A). Reciprocally, we infected myocytes with a recombinant adenovirus encoding aMEK1 to induce constitutive ERK1/2 signaling over 3 days (Figure 5A). Myocytes were then immunostained for α-actinin and measured for length and width (Figure 5B). Remarkably, inhibition of ERK1/2 produced longer myocytes while activation of ERK1/2 produced wider myocytes, with even greater differences in length-width ratios (Figure 5C and 5D). While this was a static cell culture based model system, it nonetheless again suggested that ERK1/2 were regulating the intrinsic growth response of the myocyte to discriminate between length and width. As a control, co-infection of Ad-Dusp6 with Ad-aMEK1 fully inhibited ERK1/2 phosphorylation, showing that Dusp6 dominates over aMek1 (Online Figure V, A). More importantly, Ad-aMek1 induced cardiomyocyte concentric growth was fully blocked by Ad-Dusp6 co-infection, conclusively indicating that aMek1 only functions through ERK1/2 in programming growth and that aMek1 has no function on its own apart from ERK1/2 (Online Figure V, B and C).
As a second, potentially more “physiologic” assay, we employed engineered heart tissues (EHTs) wherein neonatal rat cardiomyocytes are reconstituted in an anisotropic myocardial syncytium under defined mechanical load.22 After 7 days in culture, EHTs were infected with a combination of adenoviral vectors encoding dnMek1 and Dusp6 to inhibit ERK1/2 signaling or a control GFP encoding virus (Figure 5E). The EHTs were subsequently transferred to a stretch device and submitted to unidirectional cyclic stretch for an additional 5 days. Unlike the in vivo situation, EHTs are not affected by confounding compensatory mechanisms such as neurohumoral stimulation. In addition, mechanical load on the cardiomyocytes in EHTs are similar between groups and any alteration in morphology is only associated with an intrinsic change in the cardiomyocyte growth program. On culture day 12, EHTs were dispersed with a collagenase mixture and cells were fixed and stained with sarcomeric α-actinin to assess cell dimensions (Figure 5F). In accordance with our observations in vivo, the ex vivo inhibition of ERK1/2 signaling in the EHT system also resulted in a significant increase in cell length and a significant reduction in cell width (Figure 5F and 5G). These results again demonstrate the ability of the ERK signaling cascade to directly modulate cell size, independently of effects on contractility.
Our results suggest a model whereby ERK1/2 signaling is necessary to prevent eccentric cardiomyocyte growth. Indeed, Erk1/2 double null ventricular myocytes lengthen and become slightly thinner with aging and pathologic stimulation to cause whole organ eccentric growth. Antithetically, activation of ERK1/2 with aMek1 preferentially programmed concentric cardiac growth while at the same time partially inhibiting eccentric growth. These results suggest a refined hypothesis whereby ERK1/2 are not technically necessary for whole organ hypertrophy, as measured by heart weight normalized to body weight, but are necessary for promoting a properly coordinated growth response that also allows myocyte thickening and addition of sarcomeres at the periphery (Figure 6). Thus, ERK1/2 signaling is necessary for facilitating a select type of cardiac growth in vivo, concentric hypertrophy, while at the same time preventing eccentric growth and addition of sarcomeres in series (Figure 6).
The model of growth proposed above is also consistent with previous work in which we inhibited the ERK1/2 pathway in vivo using a transgene to overexpress Dusp6 in the heart.12 Dusp6 transgenic mice showed complete loss of ERK1/2 phosphorylation and activity, although the cardiac hypertrophic response was not reduced following TAC or agonist infusion.12 In fact, Dusp6 transgenic mice subjected to long-term TAC showed even larger increases in heart weights compared with control mice, an increase that was associated with chamber dilation suggesting greater eccentric growth. Thus, while we had not previously measured cellular lengths and widths in Dusp6 transgenic hearts, their increased propensity towards dilation with pathologic stimulation suggests a similar mechanism in play, whereby loss of ERK1/2 signaling promotes cardiomyocyte lengthening.
To our knowledge the MEK1-ERK1/2 signaling pathway appears to be unique in its ability to regulate both the length and width decision of a myocyte with aging or stress stimulation. Previously, activation of the MEK5-ERK5 signaling pathway was reported to produce eccentric cardiac hypertrophy by lengthening myocytes, although the reciprocal relationship of increased width was not examined in Mek5 or Erk5 gene-deleted mice.23 Moreover, many transgenic mouse models of cardiomyopathy and heart failure lead to spontaneous dilation with myocyte lengthening, such as with myocyte enhancer factor-2 (MEF2) overexpression.24–26 The data we present with the MEK1-ERK1/2 pathway are unique because activation of this pathway leads to myocyte thickening, while loss leads to myocyte lengthening without cellular dysfunction, suggesting it is a primary effect. Moreover, growth of adult myocytes in culture over 3 days showed the exact same relationship, and even myocytes from beating and cyclically stretched EHTs showed the same effect. Since nearly all other transgenic models of heart failure appear to cause secondary dilation, we believe that the MEK1 effect in driving concentric hypertrophy is highly unique and suggestive a true biologic signaling pathway whose role is to program myocyte thickening.
Ventricular dilation can result from either myocardial cell elongation through an addition of contractile-protein units in series, or it can also result from myocyte slipage and changes in wall architecture.27 Either event, or a mixture therein, changes the geometry of the ventricle by increasing the internal radius and thinning of the free wall, resulting in greater wall tension and secondary neuroendocrine stress signaling.28 In fact, according to Laplace’s law, our echocardiographic measurements in hearts from Erk1−/− Erk2fl/fl-Cre mice suggest an almost doubled load at the end of diastole as compared with control mice. This significantly increased load could explain the reduced FS observed in hearts from Erk1−/− Erk2fl/fl-Cre mice, leading to secondary induction of hypertrophic marker gene expression, fibrosis, and mild desensitization to β-adrenergic receptor agonists. Indeed, volume overload induced eccentric hypertrophy due to mitral regurgitation, can also lead to mechanical dysfunction.29 However, this reduction in whole organ function and secondary disease manifestations are unlikely the result of a primary defect in myocyte contractility in the absence of ERK1/2, as adult myocytes from these hearts showed even better functional performance in isolation. Such results from Erk1/2 deleted adult myocytes are in stark contrast to the observed slowing of contraction in unloaded myocytes from other models of heart failure.30 Such a dramatic increase in series sarcomeres without matching concentric growth of sarcomeres on the periphery of the fibers likely promotes whole organ dysfunction through dilation and dramatically increased wall tension with less myocyte-myocyte connectivity.
Concentric hypertrophy usually results from pressure overload, while eccentric hypertrophy usually results from situations of volume overload or forms of exercise. However, almost all hypertrophic stimuli appear to activate and induce phosphorylation of ERK1/2.7 Together these observations may suggest two divergent points of view. The more simplistic view suggests that the ERK pathway is activated in all types of hypertrophy to program concentric growth of myocytes. Therefore, in pressure overload the MEK1-ERK1/2 pathway would work with other signaling pathways to add sarcomeres in parallel and increase the width of the cells, while in volume overload situations the ERK pathway would work against the machinery that drives the cell towards elongation. Alternatively, it is possible that volume overload does not lead to continual recruitment and activation of ERK1/2 in the myocyte, or more provocatively, could even lead to mild inhibition of ERK1/2 to permit preferential addition of sarcomeres in series. Unfortunately, the relationship between types of cardiac growth (concentric vs eccentric) and the associated profile of ERK1/2 activation has not been carefully mapped in vivo at multiple time points, although Toischer et al recently reported ERK1/2 activation at 24 hrs of pressure overload hypertrophy (with concentric myocyte growth) but not with an eccentric hypertrophy promoting shunting procedure.31
The precise intracellular targets that are phosphorylated by ERK1/2 to mediate concentric growth and suppress eccentric growth have not been identified. In theory, ERK1/2 might phosphorylate one or more regulatory proteins that control sarcomeric assembly, such that ERK1/2 activation inhibits the construction of series units while assembly of sarcomeres in parallel is enhanced. In addition to affecting assembly of sarcomeres, ERK1/2 might regulate the site of new protein synthesis within the sarcomeres, at either the periphery of a fiber area or within the middle of a fiber to produce lengthening (Figure 6). In addition to acute regulation of sarcomerogenesis, ERK1/2 activation might affect the type of hypertrophy through a transcriptional mechanism that secondarily affects how myocytes grow. ERK1/2 might also impact other signaling pathways that control other aspects of cardiomyocyte growth, although analysis AKT, GSK3β, JAK/STAT and calcineurin did not reveal differences between control, Mek1 transgenic or Erk1−/− Erk2fl/fl-Cre mice (Online Figure VI).
Interestingly, autophosphorylation of ERK1/2 on Thr188, which directs ERK1/2 to phosphorylate nuclear targets, did not affect the growth response32 suggesting that MEK1-ERK1/2 signaling within the cytoplasm may be more germane in controlling the type of growth that occurs. Indeed, ERK1/2 are known to directly phosphorylate the cytoplasmic ribosomoal proteins p90 ribosomal S6 kinase (p90RSK) and p70S6K that could control protein synthesis.33 Clearly, additional studies are needed to identify the ERK1/2 effectors in hypertrophy and study them through gain- and loss- of function approaches in vivo to assess their roles in concentric and eccentric hypertrophic growth.
Sources of funding
This work was supported by grants from the National Institutes of Health (J.D.M.), the Fondation Leducq (Heart failure network grant to J.D.M), and the Howard Hughes Medical Institute (J.D.M.). I.K. was supported by a long term fellowship from the Human Frontiers Science Program. W.H.Z. is supported by the German Research Foundation (DFG ZI708/7-1, 8-1, 10-1) and the Federal Ministry of Education and Research (BMBF).