MLP (muscle LIM protein) as a stress sensor in the heart

Muscle LIM protein (MLP, also known as cysteine rich protein 3 (CSRP3, CRP3)) is a muscle-specific-expressed LIM-only protein. It consists of 194 amino-acids and has been described initially as a factor involved in myogenesis (Arber et al. Cell 79:221–231, 1994). MLP soon became an important model for experimental cardiology when it was first demonstrated that MLP deficiency leads to myocardial hypertrophy followed by a dilated cardiomyopathy and heart failure phenotype (Arber et al. Cell 88:393–403, 1997). At this time, this was the first genetically altered animal model to develop this devastating disease. Interestingly, MLP was also found to be down-regulated in humans with heart failure (Zolk et al. Circulation 101:2674–2677, 2000) and MLP mutations are able to cause hypertrophic and dilated forms of cardiomyopathy in humans (Bos et al. Mol Genet Metab 88:78–85, 2006; Geier et al. Circulation 107:1390–1395, 2003; Hershberger et al. Clin Transl Sci 1:21–26, 2008; Knöll et al. Cell 111:943–955, 2002; Knöll et al. Circ Res 106:695–704, 2010; Mohapatra et al. Mol Genet Metab 80:207–215, 2003). Although considerable efforts have been undertaken to unravel the underlying molecular mechanisms—how MLP mutations, either in model organisms or in the human setting cause these diseases are still unclear. In contrast, only precise knowledge of the underlying molecular mechanisms will allow the development of novel and innovative therapeutic strategies to combat this otherwise lethal condition. The focus of this review will be on the function of MLP in cardiac mechanosensation and we shall point to possible future directions in MLP research

Inhibition des nukleären Imports von Calcineurin verhindert die Entwicklung myokardialer Hypertrophie

The Calcineurin/NFAT signaling cascade is a crucial transducer of cellular function. It has recently been emerged that in addition to the transcription factor NFAT, the phosphatase Calcineurin is also translocated to the nucleus. Our traditional understanding of Calcineurin activation via sustained high Ca2+-levels was also advanced by recent findings from this working group (AG Ritter), which showed that Calcineurin is activated by proteolysis of the C-terminal autoinhibitory domain. This leads to the constitutive activation and nuclear translocation of Calcineurin. Therefore, Calcineurin is not only responsible for dephosphorylating of NFAT in the cytosol thus enabling its nuclear import, its presence in the nucleus is also significant in ensuring the full transcriptional activity of NFAT. Formation of complexes between transcription factors and DNA regulates the transcriptional process. Therefore, the time that transcription factors remain nuclear is a major determinant of transcriptional activity. The movement of proteins over ~40 kDa into and out of the nucleus is governed by the nuclear pore complex (NPC). Transcription factors and enzymes that regulate the activity of these proteins are shuttled across the nuclear envelope by proteins that recognize nuclear localization signals (NLS) and nuclear export signals (NES) within the amino acid sequence of these transcription factors. In this study, the precise mechanisms of Calcineurin nuclear import and export were identified. Additionally to the nuclear localization sequence (NLS) and the nuclear export sequence (NES) within the sequence of Calcineurin, the respective nuclear cargo proteins, responsible for nuclear import, Importinβ1, and for nuclear export, CRM1, were identified. Inhibition of the Calcineurin/importin interaction by a competitive peptide, called Import Blocking Peptide (IBP), which mimicked the Calcineurin NLS, prevented nuclear entry of Calcineurin. A non-inhibitory control peptide showed no effect. Using this approach, it was able to prevent the development of myocardial hypertrophy. In Angiotensin II stimulated cardiomyocytes, both the transcriptional and the translational level was suppressed. Additionally, cell size and expression of Brain natriuretic peptide (as molecular marker for hypertrophy) were significantly reduced compared untreated controls. IBP worked dose-dependent, but did not affect the Calcineurin phosphatase activity. In conclusion, Calcineurin is not only capable of dephosphorylating NFAT, thus enabling its nuclear import, its presence in the nucleus is also important for full NFAT transcriptional activity. Using IBP to prevent the nuclear import of Calcineurin is a completely new approach to prevent the development of myocardial hypertrophy.Die Calcineurin/NFAT – Signalkaskade spielt eine wichtige Rolle innerhalb der zellulären Funkionalität. Es wurde bereits gezeigt, dass neben NFAT auch die Phosphatase Calcineurin in den Zellkern transportiert wird. Die bisherigen Vorstellungen zur Aktivierung von Calcineurin durch erhöhte intrazelluläre Ca2+-Konzentrationen wurden durch aktuellste Ergebnisse dieser Arbeitsgruppe (AG Ritter) neu diskutiert. Es wurde gezeigt, dass Calcineurin durch gezielte Proteolyse der autoinhibitorischen Domäne konstitutiv aktiviert werden kann und in den Zellkern transportiert wird. Calcineurin ist daher nicht nur verantwortlich für die im Zytosol stattfindende Dephosphorylierung von NFAT, sondern übernimmt auch eine wichtige Funktion im Zellkern. Die nukleäre Lokalisation von Calcineurin ist essentiell für die vollständige transkriptionelle Aktivität von NFAT. Die Komplexbildung zwischen Transkriptionsfaktoren und DNA reguliert die Transkription. Aus diesem Grund spielt die nukleäre Verweildauer diverser Transkriptionsfaktoren eine tragende Rolle. Der Transport von Proteinen (> 40 kDa) in den und aus dem Zellkern erfolgt über nukleäre Porenkomplexe, wobei die zu transportierenden Proteine mittels nukleärer Lokalisationssequenzen (NLS) / nukleärer Exportsequencen (NES) durch die entsprechenden Transportportproteine (Karyopherine) erkannt werden. In dieser Arbeit wurden die genauen Mechanismen des Imports und des Exports von Calcineurin identifiziert. Zusätzlich konnten die NLS/NES von Calcineurin und die entsprechenden Karyopherine, Importinβ1 für den Import bzw. CRM1 für den Export, ermittelt werden. Die Inhibition der Calcineurin/Importin Interaktion durch ein kompetitives Peptid, welches als Import Blocking Peptide (IBP) bezeichnet wurde und der NLS von Calcineurin entspricht, verhinderte den nukleären Import von Calcineurin. Durch diesen Mechanismus war es möglich, die Entwicklung kardialer Hypertrophie zu verhindern. In Angiotensin II stimulierten Kardiomyozyten konnten sowohl die transkriptionelle, als auch die translationelle Aktivität reduziert werden. Des Weiteren wurden das Zellwachstum und die Expression von BNP unterdrückt. Das Blockpeptid wirkte konzentrationsabhängig, beeinflusste aber die Phosphatase-Eigenschaft von Calcineurin nicht. Zusammenfassend lässt sich sagen, dass Calcineurin, nicht nur verantwortlich für die Dephosphorylierung von NFAT ist, sondern die nukleäre Lokalisierung ebenfalls eine entscheidende Rolle für die volle transkriptionelle Aktivität von NFAT spielt. Die Verwendung von IBP, um den nukleären Import von Calcineurin zu verhindern, stellt ein völlig neues Konzept dar, die Entwicklung kardialer Hypertrophie zu unterdrücken

Fibroblast migration after myocardial infarction is regulated by transient SPARC expression

Secreted protein, acidic, and rich in cysteine (SPARC) is thought to regulate cell matrix interaction during wound repair. We hypothesized that SPARC might promote migration via integrin-dependent mechanisms. The present study was designed to clarify the contribution of SPARC in the wound healing process after myocardial infarction (MI). Adult mice received a specific αv integrin inhibitor or vehicle through osmotic mini pumps. Mice of each group were either sham-operated or MI was induced. SPARC expression was investigated 2 days, 7 days, and 1 month after the surgical procedure. For migration assays, a modified Boyden chamber assay was used. A transient increase of SPARC levels was observed, starting at day 2 (2.55±0.21), day 7 (3.72±0.28), and 1 month (1.9±0.16) after MI. After 2 months, SPARC expression dropped back to normal levels compared to sham-operated hearts. Immunofluorescence analysis showed an increase of SPARC in the infarcted area 2 days after MI, a strong increase in the scar area 7 days after MI, and only low levels in the scar area 2 months after MI. Integrin αv inhibition abolished the up-regulation of SPARC. In vitro migration assays demonstrated that fibronectin-stimulated haptotaxis of fibroblasts was modulated by SPARC. This study provides evidence that SPARC is significantly up-regulated in the infarcted region after MI. This up-regulation is dependent on αv integrins. As SPARC is found to regulate fibroblast migration, it appears to play an important role in the injured myocardium with regard to healing and scar formation

Inhibition of nuclear import of calcineurin prevents myocardial hypertrophy.

The time that transcription factors remain nuclear is a major determinant for transcriptional activity. It has recently been demonstrated that the phosphatase calcineurin is translocated to the nucleus with the transcription factor nuclear factor of activated T cells (NF-AT). This study identifies a nuclear localization sequence (NLS) and a nuclear export signal (NES) in the sequence of calcineurin. Furthermore we identified the nuclear cargo protein importinbeta(1) to be responsible for nuclear translocation of calcineurin. Inhibition of the calcineurin/importin interaction by a competitive peptide (KQECKIKYSERV), which mimicked the calcineurin NLS, prevented nuclear entry of calcineurin. A noninhibitory control peptide did not interfere with the calcineurin/importin binding. Using this approach, we were able to prevent the development of myocardial hypertrophy. In angiotensin II-stimulated cardiomyocytes, [(3)H]-leucine incorporation (159%+/-9 versus 111%+/-11; P<0.01) and cell size were suppressed significantly by the NLS peptide compared with a control peptide. The NLS peptide inhibited calcineurin/NF-AT transcriptional activity (227%+/-11 versus 133%+/-8; P<0.01), whereas calcineurin phosphatase activity was unaffected (298%+/-9 versus 270%+/-11; P=NS). We conclude that calcineurin is not only capable of dephosphorylating NF-AT, thus enabling its nuclear import, but the presence of calcineurin in the nucleus is also important for full NF-AT transcriptional activity

Endothelin-1-stimulated InsP₃-induced Ca²+ release is a nexus for hypertrophic signaling in cardiac myocytes

Ca(2+) elevations are fundamental to cardiac physiology-stimulating contraction and regulating the gene transcription that underlies hypertrophy. How Ca(2+) specifically controls gene transcription on the background of the rhythmic Ca(2+) increases required for contraction is not fully understood. Here we identify a hypertrophy-signaling module in cardiac myocytes that explains how Ca(2+) discretely regulates myocyte hypertrophy and contraction. We show that endothelin-1 (ET-1) stimulates InsP(3)-induced Ca(2+) release (IICR) from perinuclear InsP(3)Rs, causing an elevation in nuclear Ca(2+). Significantly, we show that IICR, but not global Ca(2+) elevations associated with myocyte contraction, couple to the calcineurin (CnA)/NFAT pathway to induce hypertrophy. Moreover, we found that activation of the CnA/NFAT pathway and hypertrophy by isoproterenol and BayK8644, which enhance global Ca(2+) fluxes, was also dependent on IICR and nuclear Ca(2+) elevations. The activation of IICR by these activity-enhancing mediators was explained by their ability to stimulate secretion of autocrine/paracrine ET-1

Conditional neuronal nitric oxide synthase overexpression impairs myocardial contractility.

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca(2+)-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [(3)H]-l-arginine to [(3)H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17+/-3% decrease of +dp/dt(max) compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca(2+)ATPase and additionally with L-type Ca(2+)- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I(Ca,L) density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca(2+)-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca(2+)-channels and in turn reduces Ca(2+)-transients which accounts for the negative inotropic effect