The vertebrate heart possesses autoregulatory mechanisms enabling it first to sense and then to adapt its force of contraction to continually changing demands. The molecular components of the cardiac mechanical stretch sensor are mostly unknown but of immense medical importance, since dysfunction of this sensing machinery is suspected to be responsible for a significant proportion of human heart failure. In the hearts of the ethylnitros-urea (ENU)-induced, recessive embryonic lethal zebrafish heart failure mutant main squeeze (msq), we find stretch-responsive genes such as atrial natriuretic factor (anf) and vascular endothelial growth factor (vegf) severely down-regulated. We demonstrate through positional cloning that heart failure in msq mutants is due to a mutation in the integrin-linked kinase (ilk) gene. ILK specifically localizes to costameres and sarcomeric Z-discs. The msq mutation (L308P) reduces ILK kinase activity and disrupts binding of ILK to the Z-disc adaptor protein ␤-parvin (Affixin). Accordingly, in msq mutant embryos, heart failure can be suppressed by expression of ILK, and also of a constitutively active form of Protein Kinase B (PKB), and VEGF. Furthermore, antisense-mediated abrogation of zebrafish ␤-parvin phenocopies the msq phenotype. Thus, we provide evidence that the heart uses the Integrin-ILK-␤-parvin network to sense mechanical stretch and respond with increased expression of ANF and VEGF, the latter of which was recently shown to augment cardiac force by increasing the heart's calcium transients.[Keywords: Integrin-linked kinase (ILK); zebrafish; cardiac stretch sensor; ␤-parvin (Affixin)] Supplemental material is available at http://www.genesdev.org.

The FRQ1 gene is essential for growth of budding yeast and encodes a 190-residue, N-myristoylated (myr) calcium-binding protein. Frq1 belongs to the recoverin/frequenin branch of the EF-hand superfamily and regulates a yeast phosphatidylinositol 4-kinase isoform. Conformational changes in Frq1 due to N-myristoylation and Ca(2+) binding were assessed by nuclear magnetic resonance (NMR), fluorescence, and equilibrium Ca(2+)-binding measurements. For this purpose, Frq1 and myr-Frq1 were expressed in and purified from Escherichia coli. At saturation, Frq1 bound three Ca(2+) ions at independent sites, which correspond to the second, third, and fourth EF-hand motifs in the protein. Affinity of the second site (K(d) = 10 microM) was much weaker than that of the third and fourth sites (K(d) = 0.4 microM). Myr-Frq1 bound Ca(2+) with a K(d)app of 3 microM and a positive Hill coefficient (n = 1.25), suggesting that the N-myristoyl group confers some degree of cooperativity in Ca(2+) binding, as seen previously in recoverin. Both the NMR and fluorescence spectra of Frq1 exhibited very large Ca(2+)-dependent differences, indicating major conformational changes induced upon Ca(2+) binding. Nearly complete sequence-specific NMR assignments were obtained for the entire carboxy-terminal domain (residues K100-I190). Assignments were made for 20% of the residues in the amino-terminal domain; unassigned residues exhibited very broad NMR signals, most likely due to Frq1 dimerization. NMR chemical shifts and nuclear Overhauser effect (NOE) patterns of Ca(2+)-bound Frq1 were very similar to those of Ca(2+)-bound recoverin, suggesting that the overall structure of Frq1 resembles that of recoverin. A model of the three-dimensional structure of Ca(2+)-bound Frq1 is presented based on the NMR data and homology to recoverin. N-myristoylation of Frq1 had little or no effect on its NMR and fluorescence spectra, suggesting that the myristoyl moiety does not significantly alter Frq1 structure. Correspondingly, the NMR chemical shifts for the myristoyl group in both Ca(2+)-free and Ca(2+)-bound myr-Frq1 were nearly identical to those of free myristate in solution, indicating that the fatty acyl chain is solvent-exposed and not sequestered within the hydrophobic core of the protein, unlike the myristoyl group in Ca(2+)-free recoverin. Subcellular fractionation experiments showed that both the N-myristoyl group and Ca(2+)-binding contribute to the ability of Frq1 to associate with membranes.

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca 2؉ -bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca 2؉ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices ( In animal cells and yeast (1, 2), phosphoinositides mediate selective recruitment of proteins to membranes (3-6) and serve as precursors for intracellular second messengers (7-9). Phosphoinositide biosynthesis begins with phosphorylation of the myo-inositol headgroup of phosphatidylinositol (PtdIns) 3 at the D-4 position by PtdIns 4-kinase (ATP:1-phosphatidyl-1D-myo-inositol 4-phosphotransferase, EC 2.7.1.67) (10 -12). The first PtdIns 4-kinase to be purified (13), and the corresponding gene cloned (14), was Pik1 from the yeast Saccharomyces cerevisiae. PIK1 is an essential gene required for vesicular trafficking in the late secretory pathway (15, 16), for nuclear functions (17), and possibly cytokinesis (18). Pik1-like isoforms are conserved in metazoans (10,11,19).Yeast frequenin (Frq1), a 22-kDa Ca 2ϩ -binding protein, copurifies with Pik1 and is essential for its optimal activity (20). The site where Frq1 docks on Pik1 was localized to a region (residues 121-174) that lies far upstream of the catalytic domain (residues 792-1066) (21). Mammalian frequenin also interacts with Pik1 (22), and frequenin may regulate PtdIns 4-kinase activity in animal cells (23-25). Ca 2ϩ -dependent activation of PtdIns 4-kinase by frequenin may be especially important in neurons because modulation of phosphoinositide synthesis by intracellular Ca 2ϩ controls exocytosis (26) and is involved in synaptic plasticity (27).Frq1 and other frequenins belong to the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, which includes recoverin and neurocalcin (28 -31). These proteins are small (Յ25 kDa) and characterized by a consensus signal for N-terminal myristoylation and four EF-hand Ca 2ϩ -binding sites (Fig. 1). We have shown previously that, at saturation, Frq1 binds only three Ca 2ϩ (32). Frq1, which is itself essential for the viability of yeast cells (20), associates with membranes in a manner that depends on both the N-myristoyl group and conformational changes induced upon Ca 2ϩ binding, suggesting that Frq1, like other NCS proteins, may possess a Ca 2ϩ -myristoyl switch (32). Indeed, prior work indicated that N-myristoylation of Frq1 is important (but not essential) for stimulating both the catalytic activity (20) and the membrane recruitment of Pik1 (17).Three-dimensional structures for Frq1 and other NCS proteins have been determined by x-ray crystallography (23,(33)(34)(35)(36)(37) and NMR spectroscopy (32, 38 -40). The structure of * This work was su...

The zebrafish (Danio rerio) is an increasingly popular model organism in cardiovascular research. Major insights into cardiac developmental processes have been gained by studies of embryonic zebrafish. However, the utility of zebrafish for modeling adult-onset heart disease has been limited by a lack of robust methods for in vivo evaluation of cardiac function. We established a physiological protocol for underwater zebrafish echocardiography using high frequency ultrasound, and evaluated its reliability in detecting altered cardiac function in two disease models. Serial assessment of cardiac function was performed in wild-type zebrafish aged 3 to 12 months and the effects of anesthetic agents, age, sex and background strain were evaluated. There was a varying extent of bradycardia and ventricular contractile impairment with different anesthetic drugs and doses, with tricaine 0.75 mmol l−1 having a relatively more favorable profile. When compared with males, female fish were larger and had more measurement variability. Although age-related increments in ventricular chamber size were greater in females than males, there were no sex differences when data were normalized to body size. Systolic ventricular function was similar in both sexes at all time points, but differences in diastolic function were evident from 6 months onwards. Wild-type fish of both sexes showed a reliance on atrial contraction for ventricular diastolic filling. Echocardiographic evaluation of adult zebrafish with diphtheria toxin-induced myocarditis or anemia-induced volume overload accurately identified ventricular dilation and altered contraction, with suites of B-mode, ventricular strain, pulsed-wave Doppler and tissue Doppler indices showing concordant changes indicative of myocardial hypocontractility or hypercontractility, respectively. Repeatability, intra-observer and inter-observer correlations for echocardiographic measurements were high. We demonstrate that high frequency echocardiography allows reliable in vivo cardiac assessment in adult zebrafish and make recommendations for optimizing data acquisition and analysis. This enabling technology reveals new insights into zebrafish cardiac physiology and provides an imaging platform for zebrafish-based translational research.

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Recognition that phosphoinositides and inositol phosphates are key regulators of many processes in eukaryotic cells has brought increased attention to the enzymes that regulate the synthesis and turnover of these molecules (reviewed in Refs. 1-3). Of particular interest are the enzymes responsible for producing the various polyphosphoinositides situated on the cytosolic face of cellular membranes, which initiate several different signaling pathways by serving as highly specific recognition determinants for the selective recruitment of proteins to membranes (reviewed in Refs. 4 -7) and as the precursors for several intracellular second messengers (reviewed in Refs. 8 -10). The first committed step in the synthesis of the polyphosphoinositide, phosphatidylinositol 4,5-bisphosphate, is considered to be ATP-dependent phosphorylation of the hydrophilic myo-inositol head group of phosphatidylinositol (PtdIns) 1 at the D-4 position by PtdIns 4-kinase (ATP:1-phosphatidyl-1D-myo-inositol 4-phosphotransferase, EC 2.7.1.67) (reviewed in Refs. 11-13) . The resulting product, PtdIns(4)P, can be phosphorylated on the D-5 position by PtdIns(4)P 5-kinase to generate PtdIns(4,5)P 2 , PtdIns(4,5)P 2 can be phosphorylated on the D-3 position by yet other lipid kinases, and the phosphoinositides so generated can be converted to other species by specific phosphatases and phospholipases (reviewed in Refs. 14 -17).The first PtdIns 4-kinase to be purified to homogeneity from any organism (18), and to have the corresponding gene cloned (19,20), was Pik1 from the yeast Saccharomyces cerevisiae. Thereafter, a second isoform, Stt4, which is the product of a discrete gene, was described (21). Absence of either Pik1 or Stt4 is lethal, and overproduction of each protein cannot compensate for absence of the other, indicating that these enzymes participate in distinct cellular processes and generate discrete pools of PtdIns(4)P that are essential for yeast cell viability. Indeed, subsequent work has shown that, together, Pik1 and Stt4 account for all of the PtdIns(4)P generated in the yeast cell (22) and that Pik1 is required for vesicular trafficking in the late secretory pathway (23, 24) and perhaps for cytokinesis (20), whereas Stt4 plays roles in cell wall integrity, maintenance of vacuole morphology, and aminophospholipid transport from the endoplasmic reticulum to the Golgi (25-27). The presence of Pik1-and Stt4-like isoforms is also conserved in metazoans (11,12,28).We have shown previously that Frq1, a small calcium-bind-

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