The mediator complex subunit Med10 regulates heart valve formation in zebrafish by controlling Tbx2b-mediated Has2 expression and cardiac jelly formation.

In search for novel key regulators of cardiac valve formation, we isolated the zebrafish cardiac valve mutant ping pong (png). We find that an insertional promoter mutation within the zebrafish mediator complex subunit 10 (med10) gene is leading to impaired heart valve formation. Expression of the T-box transcription factor 2b (Tbx2b), known to be essential in cardiac valve development, is severely reduced in png mutant hearts. We demonstrate here that transient reconstitution of Tbx2b expression rescues AV canal development in png mutant zebrafish. By contrast, overexpression of Forkhead box N4 (Foxn4), a known upstream regulator of Tbx2b, is not capable to reconstitute tbx2b expression and heart valve formation in Med10-deficient png mutant hearts. Interestingly, hyaluronan synthase 2 (has2), a known downstream target of Tbx2 and producer of hyaluronan (HA) - a major ECM component of the cardiac jelly and critical for proper heart valve development - is completely absent in ping pong mutant hearts. We propose here a rather unique role of Med10 in orchestrating cardiac valve formation by mediating Foxn4 dependent tbx2b transcription, expression of Has2 and subsequently proper development of the cardiac jelly.

Paxillin and Focal Adhesion Kinase (FAK) Regulate Cardiac Contractility in the Zebrafish Heart.

An orchestrated interplay of adaptor and signaling proteins at mechano-sensitive sites is essential to maintain cardiac contractility and when defective leads to heart failure. We recently showed that Integrin-linked Kinase (ILK), ß-Parvin and PINCH form the IPP-complex to grant tuned Protein Kinase B (PKB) signaling in the heart. Loss of one of the IPP-complex components results in destabilization of the whole complex, defective PKB signaling and finally heart failure. Two components of IPP, ILK and ß-Parvin directly bind to Paxillin; however, the impact of this direct interaction on the maintenance of heart function is not known yet. Here, we show that targeted gene inactivation of Paxillin results in progressive decrease of cardiac contractility and heart failure in zebrafish without affecting IPP-complex stability and PKB phosphorylation. However, we found that Paxillin deficiency leads to the destabilization of its known binding partner Focal Adhesion Kinase (FAK) and vice versa resulting in degradation of Vinculin and thereby heart failure. Our findings highlight an essential role of Paxillin and FAK in controlling cardiac contractility via the recruitment of Vinculin to mechano-sensitive sites in cardiomyocytes.

Developmental alterations in centrosome integrity contribute to the post-mitotic state of mammalian cardiomyocytes.

Mammalian cardiomyocytes become post-mitotic shortly after birth. Understanding how this occurs is highly relevant to cardiac regenerative therapy. Yet, how cardiomyocytes achieve and maintain a post-mitotic state is unknown. Here, we show that cardiomyocyte centrosome integrity is lost shortly after birth. This is coupled with relocalization of various centrosome proteins to the nuclear envelope. Consequently, postnatal cardiomyocytes are unable to undergo ciliogenesis and the nuclear envelope adopts the function as cellular microtubule organizing center. Loss of centrosome integrity is associated with, and can promote, cardiomyocyte G0/G1 cell cycle arrest suggesting that centrosome disassembly is developmentally utilized to achieve the post-mitotic state in mammalian cardiomyocytes. Adult cardiomyocytes of zebrafish and newt, which are able to proliferate, maintain centrosome integrity. Collectively, our data provide a novel mechanism underlying the post-mitotic state of mammalian cardiomyocytes as well as a potential explanation for why zebrafish and newts, but not mammals, can regenerate their heart.

In vivo characterization of human myofibrillar myopathy genes in zebrafish.

Myofibrillar myopathies (MFM) are progressive diseases of human heart and skeletal muscle with a severe impact on life quality and expectancy of affected patients. Although recently several disease genes for myofibrillar myopathies could be identified, today most genetic causes and particularly the associated mechanisms and signaling events that lead from the mutation to the disease phenotype are still mostly unknown. To assess whether the zebrafish is a suitable model system to validate MFM candidate genes using targeted antisense-mediated knock-down strategies, we here specifically inactivated known human MFM disease genes and evaluated the resulting muscular and cardiac phenotypes functionally and structurally. Consistently, targeted ablation of MFM genes in zebrafish led to compromised skeletal muscle function mostly due to myofibrillar degeneration as well as severe heart failure. Similar to what was shown in MFM patients, MFM gene-deficient zebrafish showed pronounced gene-specific phenotypic and structural differences. In summary, our results indicate that the zebrafish is a suitable model to functionally and structurally evaluate novel MFM disease genes in vivo.

Automatic zebrafish heartbeat detection and analysis for zebrafish embryos.

A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual detection of the heart rates of wild-type zebrafish embryos 36-120 h postfertilization and of embryonic hearts with bradycardia and pauses in the cardiac contraction.

The Arabidopsis NIMIN proteins affect NPR1 differentially.

NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) is the central regulator of the pathogen defense reaction systemic acquired resistance (SAR). NPR1 acts by sensing the SAR signal molecule salicylic acid (SA) to induce expression of PATHOGENESIS-RELATED (PR) genes. Mechanistically, NPR1 is the core of a transcription complex interacting with TGA transcription factors and NIM1-INTERACTING (NIMIN) proteins. Arabidopsis NIMIN1 has been shown to suppress NPR1 activity in transgenic plants. The Arabidopsis NIMIN family comprises four structurally related, yet distinct members. Here, we show that NIMIN1, NIMIN2, and NIMIN3 are expressed differentially, and that the encoded proteins affect expression of the SAR marker PR-1 differentially. NIMIN3 is expressed constitutively at a low level, but NIMIN2 and NIMIN1 are both responsive to SA. While NIMIN2 is an immediate early SA-induced and NPR1-independent gene, NIMIN1 is activated after NIMIN2, but clearly before PR-1. Notably, NIMIN1, like PR-1, depends on NPR1. In a transient assay system, NIMIN3 suppresses SA-induced PR-1 expression, albeit to a lesser extent than NIMIN1, whereas NIMIN2 does not negatively affect PR-1 gene activation. Furthermore, although binding to the same domain in the C-terminus, NIMIN1 and NIMIN2 interact differentially with NPR1, thus providing a molecular basis for their opposing effects on NPR1. Together, our data suggest that the Arabidopsis NIMIN proteins are regulators of the SAR response. We propose that NIMINs act in a strictly consecutive and SA-regulated manner on the SA sensor protein NPR1, enabling NPR1 to monitor progressing threat by pathogens and to promote appropriate defense gene activation at distinct stages of SAR. In this scenario, the defense gene PR-1 is repressed at the onset of SAR by SA-induced, yet instable NIMIN1.