Journal of Molecular and Cellular Cardiology 47 (2009) 423–425
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Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y j m c c
MLP: A stress sensor goes nuclear
The sarcomeric Z-disc is one of the most complex macromolecular structures in biology . Some of its constituents have important structural functions and an increasing number of recent publications point to additional, previously unexpected features. A new view is now emerging, whereby Z-disc proteins are involved as important intra- and intercellular signaling nodes . Translocation of Z-disc proteins to the nucleus and probably to the M-band as well as to other compartments, their interaction with additional signaling molecules and ability to facilitate macromolecular protein complexes are only a few properties to indicate their multi-functionality. In this context it was shown, that I-band proteins such as FHL1  or muscle ankyrin repeat proteins such as CARP  as well as the titin kinase, which is located at the sarcomeric M-band  are probably all involved in cardiac mechanosensation (please see for a review ). Titin contains speciﬁc elastic domains within its I-band region (such as the N2B, N2BA, PEVK and Ig domains) and as such might serve as a sarcomeric length sensor. In contrast, the sarcomeric Z-disc might serve as a tension sensor. A better understanding of these emerging novel physiological roles of Z-disc proteins has been achieved using a variety of genetically altered animal models, such as mouse and zebraﬁsh as well as Drosophila. Even more insights might be provided with the use of reductionist approaches by precisely analyzing one particular protein in the setting of cardiomyocyte cell culture systems, where preload and afterload conditions can be modiﬁed. One of these proteins is muscle LIM protein (MLP, also known as cysteine rich protein 3 (CSRP3)), which has been discovered as a myogenic factor and was ﬁrst described in 1994 . MLP is a LIM only protein, it consists of 194 amino acids which constitute two LIM domains each followed by a glycine rich domain. Soon after its initial description the protein became important for cardiovascular research when it was demonstrated in 1997  that MLP deﬁcient animals developed hypertrophy followed by a dilated cardiomyopathy (DCM) phenotype — at this time the ﬁrst genetically altered mouse model for this devastating disease. Perhaps even more important is that human MLP mutations are able to cause human forms of cardiomyopathy [9– 13]. The precise mechanisms by which MLP mutations cause these diseases are still less clear. In this context it was shown that additional ablation of phospholamban (PLB) in MLP−/− animals, which exhibit normal or enhanced SR-calcium ATPase function , rescues the heart-failure phenotype (i.e. no heart failure in PLB and MLP double knock-out animals) . It was also shown that MLP−/− cardiomyocytes are defective in brain natriuretic peptide (BNP) induction upon mechanical stimulation whereas endothelin or phenylephrine were still able to induce BNP expression in these cells . Based on these, as well as additional data , it was suggested that the primary defect is not located in the downstream pathway for gene induction but in the initial sensing of the stretch stimulus. Thus, MLP was 0022-2828/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2009.07.012
suggested to be part of a macromolecular, cardiac mechanical stretch sensor. In this context, MLP was found to be present in different cellular compartments, including the sarcomeric Z-disc/I-band of different species such as Drosophila , rat [12,17] and mouse [8,18–19], the costamere (where β1-spectrin interacts with MLP) , intercalated disc (where N-RAP interacts with MLP)  and the cytoplasm (where actin interacts with MLP)  as well as the nucleus  (please see Fig. 1). It might be of interest to elucidate, which MLP pool shuttles into the nucleus and/or whether all of these different MLP pools are able to exchange information with the nucleus (into and out of the nucleus?). Recently Geier et al. found MLP mostly cytoplasmic and not at the Z-disc  and challenged as well a disease causing role for the 10T → C (Trp4Arg) MLP variant . However, the largest pedigree presented in this paper is not informative (due to the presence of hypertension in some members) and several individuals were called “not affected”, in contrast to current guidelines . Nevertheless, the paper by Geier et al. highlights the need for more research in this ﬁeld. Previously, Boateng et al. demonstrated quite elegantly that MLP in cardiac myocytes is present in two different molecular forms : the oligomeric form, which is present at the sarcolemma and the cytoskeleton versus the monomeric form, which they found exclusively in the nucleus. The carboxyterminal end of the MLP protein was required for this process, thus it is possible that the carboxyterminal target of the monoclonal MLP antibody used by Geier et al. is protected by oligomerization (“masked epitope”). After myocardial infarction and pressure overload in animals as well as in failing human hearts nuclear MLP levels in the myocardium increased at the expense of non-nuclear MLP. In failing human hearts almost no MLP was detectable outside the nucleus. In their current work Boateng et al. extend on their previous research by providing unequivocal evidence for nucleo-cytoplasmic shuttling of MLP on the basis of the putative nuclear localization signal (NLS) – and also by providing signiﬁcant insight into the functional properties of nuclear MLP  – important evidence which has been missing up to now. They used cell permeable synthetic peptides containing the putative nuclear localization signal (RKYGPG) of MLP and were able to inhibit any further shuttling of this protein, thus clearly documenting functionality of this (up to now only hypothesized) NLS. In addition, inhibition of nuclear translocation prevented the increased protein accumulation usually seen under phenylephrine treatment of isolated cardiomyocytes, thus pointing to an important role of MLP in the control of hypertrophy after agonist stimulation. Interestingly, cyclic strain of myocytes after prior NLS treatment resulted in disarrayed sarcomeres, an observation that has also been made in vivo in cardiomyocyte cytoarchitecture of MLP knock-out mice . Increased protein synthesis and brain natriuretic peptide
Fig. 1. The ﬁgure displays a putative cardiomyocyte and indicates various MLP (CSRP3) localizations. For reasons of simplicity oligomerization of MLP was not taken into account. MLP is probably localized at the sarcomeric Z-disc/I-band, at costameres as well as at intercalated discs and in the cytoplasm. It remains to be elucidated, which MLP molecules translocate into the nucleus (indicated by “?”).
(BNP) expression were also abolished when nuclear shuttling was inhibited during cyclic mechanical strain, suggesting that MLP is required for remodelling of myoﬁlaments and altered gene expression. A direct link between BNP gene expression and MLP has been shown earlier  and additional research in heterozygous MLP knock-out animals after myocardial infarction pointed in the same direction . Although the functional signiﬁcance of nuclear MLP (especially for myocyte hypertrophy following biomechanical stress, gene expression and sarcomere assembly) has been clearly demonstrated in this study, even more questions are now arising. The most important is how a small protein-adaptor molecule like MLP can exert its actions in different cellular compartments including the nucleus. In this regard, the prohypertrophic phosphatase calcineurin was previously identiﬁed as a cytoskeletal target/interaction partner of MLP and it was demonstrated that MLP at the Z-disc is necessary for calcineurin activation in cardiac myocytes [19,25,26]. What are the nuclear targets of MLP in cardiac myocytes? The present study points to an important feed back mechanism between MLP and protein translation — while MLP cannot directly bind DNA, one could speculate that it enhances DNA binding by well characterized stress responsive cardiac transcription factors like GATA4, GATA6, AP1 or serum response factor (SRF). It has in fact been shown previously, that CSRP2, which is closely related to MLP (CSRP3), can bind GATAs and SRF and thereby synergistically enhance gene expression in smooth muscle cells . By linking different transcription factor complexes, LIM proteins of the CSRP family may integrate the activities of multiple nuclear regulatory proteins in order to coordinate gene expression. It is also possible that MLP facilitates nucleo-cytoplasmic shuttling of its non-nuclear target calcineurin, which has been demonstrated to translocate to the nucleus in hypertrophied cardiac myocytes . Research on MLP centered so far on putative cytoskeletal functions of this protein only. Particularly the nuclear localization sequence (NLS), ﬁrst described by Fung et al. several years ago  which is localized within the ﬁrst glycine rich domain, remained elusive. Although the interaction of MLP with different transcription factors such as MyoD, MRF4 and myogenin was shown in skeletal myocytes  and MLP nuclear localization following an increase in biomechanical stress was also observed in
right ventricular murine hypertrophy , the functional signiﬁcance of this NLS remained elusive until the current report by Boateng et al. However, more work is needed to unravel the nuclear versus cytoplasmic targets of MLP. Importantly, MLP is acetylated within its NLS on position K69 , a modiﬁcation thought to affect calcium sensitivity which might potentially be able to affect MLP's nuclear–cytoplasmic shuttling. It might also be interesting to analyze the effects of human mutations on nuclear localization and to analyze their ability to activate a speciﬁc gene program. Particularly the K69R-MLP mutation, found in an individual affected by DCM and endocardial ﬁbroelastosis , is worth to be analyzed. Besides acetylation, additional potentially existing, but not yet well understood posttranslational modiﬁcations such as phosphorylation, sumoylation and/or polyubiquitinylation might well have their effects on MLP function. Last but not least, given the importance of MLP for cardiac integrity and the myocardial stress response, it might be worthwhile to consider MLP, which appears dislocated from its membrane/cytoskeletal localization in failing human myocardium, as a therapeutic target in heart failure. Maybe restoration of non-nuclear MLP in failing myocardium repairs the defects observed in myocardial stress response observed in this disease.
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Sylvia Gunkel1 Ralph Knöll⁎ Working Group on Cardiovascular Molecular Genetics, Heart Centre, Georg August University, Göttingen, Germany. E-mail address: [email protected]
⁎Corresponding author. Working Group on Cardiovascular Molecular Genetics, Heart Centre, University Medical Centre Göttingen, Georg August University, NGFN coordinator, Robert Koch Str. 40, 37075 Göttingen, Germany. Tel.: +49 551 39 5316; fax: +49 (0)551 39 13592. Jörg Heineke1 Denise Hilﬁker-Kleiner Department of Cardiology and Angiology, Rebirth - Cluster of Excellence, Hannover Medical School, Hannover, Germany. 1 Both authors contributed equally. 22 April 2009