Drosophila melanogaster genetics provides the advantage of molecularly defined P-element insertions and deletions that span the entire genome. Although Drosophila has been extensively used as a model system to study heart development, it has not been used to dissect the genetics of adult human heart disease because of an inability to phenotype the adult fly heart in vivo. Here we report the development of a strategy to measure cardiac function in awake adult Drosophila that opens the field of Drosophila genetics to the study of human dilated cardiomyopathies. Through the application of optical coherence tomography, we accurately distinguish between normal and abnormal cardiac function based on measurements of internal cardiac chamber dimensions in vivo. Normal Drosophila have a fractional shortening of 87 ؎ 4%, whereas cardiomyopathic flies that contain a mutation in troponin I or tropomyosin show severe impairment of systolic function. To determine whether the fly can be used as a model system to recapitulate human dilated cardiomyopathy, we generated transgenic Drosophila with inducible cardiac expression of a mutant of human ␦-sarcoglycan (␦sg S151A ), which has previously been associated with familial dilated cardiomyopathy. Compared to transgenic flies overexpressing wild-type ␦sg, or the standard laboratory strain w 1118 , Drosophila expressing ␦sg S151A developed marked impairment of systolic function and significantly enlarged cardiac chambers. These data illustrate the utility of Drosophila as a model system to study dilated cardiomyopathy and the applicability of the vast genetic resources available in Drosophila to systematically study the genetic mechanisms responsible for human cardiac disease.optical coherence tomography ͉ cardiomyopathy

Herein we present multiple lines of evidence which demonstrate that depletion of internal calcium stores is both necessary and sufficient for the activation of calcium-independent phospholipase A 2 during arginine vasopressin (

Calcium-independent phospholipase A 2 (iPLA 2 ) is the major phospholipase A 2 activity in many cell types, and at least one isoform of this enzyme class is physically and functionally coupled to calmodulin (CaM) in a reversible calcium-dependent fashion. To identify the domain in recombinant iPLA 2 ␤ (riPLA 2 ␤) underlying this interaction, multiple techniques were employed. First, we identified calcium-activated CaM induced alterations in the kinetics of proteolytic fragment generation during limited trypsinolysis (i.e. CaM footprinting). Tryptic digests of riPLA 2 ␤ (83 kDa) in the presence of EGTA alone, Ca ؉2 alone, or EGTA and CaM together resulted in the production of a major 68-kDa protein whose kinetic rate of formation was specifically attenuated in incubations containing CaM and Ca ؉2 together. Western blotting utilizing antibodies directed against either the N-or C-terminal regions of riPLA 2 ␤ indicated the specific protection of riPLA 2 ␤ by calcium-activated CaM at a cleavage site Ϸ15 kDa from the C terminus. Moreover, calcium-activated calmodulin increased the kinetic rate of tryptic cleavage near the active site of riPLA 2 ␤. Second, functional characterization of products from these partial tryptic digests demonstrated that Ϸ90% of the 68-kDa riPLA 2 ␤ tryptic product (i.e. lacking the 15-kDa C-terminus) did not bind to a CaM affinity matrix in the presence of Ca 2؉ , although >95% of the noncleaved riPLA 2 ␤ as well as a 40-kDa C-terminal peptide bound tightly under these conditions. Third, when purified riPLA 2 ␤ was subjected to exhaustive trypsinolysis followed by ternary complex CaM affinity chromatography, a unique tryptic peptide ( 694 AWSEM-VGIQYFR 705 ) within the 15-kDa C-terminal fragment was identified by RP-HPLC, which bound to CaM-agarose in the presence but not the absence of calcium ion. Fourth, fluorescence energy transfer experiments demonstrated that this peptide (694 -705) bound to dansylcalmodulin in a calcium-dependent fashion. Collectively, these results identify multiple contact points in the 15-kDa C terminus as being the major but not necessarily the only binding site responsible for the calciumdependent regulation of iPLA 2 ␤ by CaM.The phospholipase A 2 -catalyzed release of arachidonic acid from its phospholipid storage depots is a critical component of intra-and intercellular signal transduction. In most noncirculating cells (e.g. cardiac myocytes, pancreatic islet ␤-cells, hippocampal neurons, and vascular smooth muscle cells), calciumindependent phospholipase A 2 (iPLA 2 ) 1 is the major but not the only phospholipase A 2 activity present (1-6). Multiple lines of experimental evidence implicate iPLA 2 as an important mediator of arachidonic acid release in several cell types including: 1) the inhibition of the large majority of AVP-induced arachidonic acid release in A-10 smooth muscle cells by 1-2 M BEL (7); 2) the attenuation of the release of arachidonic acid in lipopolysaccharide-stimulated macrophages by either BEL or antisense DNA targeted to iPLA 2 ␤ (8, 9...

Herein we demonstrate the calcium-dependent regulation of myocardial phospholipase A2 activity, which is mediated by a cytosolic protein constituent that can be chromatographically resolved from, and subsequently reconstituted with, purified myocardial phospholipase A2. Purification of this protein by sequential column chromatographies revealed an 18-kDa doublet, which was identified as calmodulin by Western blotting, calcium-dependent precipitation with W-7 agarose beads, and reconstitution of calcium-mediated phospholipase A2 inhibition with authentic homogeneous calmodulin. Calcium-induced calmodulin-mediated inhibition of myocardial phospholipase A2 was titrated by physiologic increments of calcium ion (Kd approximately 200 nM). Moreover, ternary complex affinity chromatography with calmodulin-Sepharose demonstrated that inhibition of myocardial phospholipase A2 activity by calmodulin resulted from the direct interaction of calmodulin with the myocardial phospholipase A2 catalytic complex. Exposure of cultured A-10 muscle cells to three structurally disparate calmodulin antagonists (W-7, trifluoperazine, and calmidazolium) resulted in the robust release of arachidonic acid, which was entirely ablated by pretreatment of cells with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2-H-tetrahydropyran-2-one. Collectively, this study identifies a novel mechanism whereby latent phospholipase A2 activity can be released from tonic inhibition by alterations in the interactions between the phospholipase A2 catalytic complex, calcium ion, and the intracellular calcium transducer, calmodulin.

The beta-adrenergic receptor (betaAR) signaling system is one of the most powerful regulators of cardiac function and a key regulator of Ca(2+) homeostasis. We investigated the role of betaAR stimulation in augmenting cardiac function and its role in the activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII) using various betaAR knockouts (KO) including beta(1)ARKO, beta(2)ARKO, and beta(1)/beta(2)AR double-KO (DKO) mice. We employed a murine model of left anterior descending coronary artery ligation to examine the differential contributions of specific betaAR subtypes in the activation of CaMKII in vivo in failing myocardium. Cardiac inotropy, chronotropy, and CaMKII activity following short-term isoproterenol stimulation were significantly attenuated in beta(1)ARKO and DKO compared with either the beta(2)ARKO or wild-type (WT) mice, indicating that beta(1)ARs are required for catecholamine-induced increases in contractility and CaMKII activity. Eight weeks after myocardial infarction (MI), beta(1)ARKO and DKO mice showed a significant attenuation in fractional shortening compared with either the beta(2)ARKO or WT mice. CaMKII activity after MI was significantly increased only in the beta(2)ARKO and WT hearts and not in the beta(1)ARKO and DKO hearts. The border zone of the infarct in the beta(2)ARKO and WT hearts demonstrated significantly increased apoptosis by TUNEL staining compared with the beta(1)ARKO and DKO hearts. Taken together, these data show that cardiac function and CaMKII activity are mediated almost exclusively by the beta(1)AR. Moreover, it appears that beta(1)AR signaling is detrimental to cardiac function following MI, possibly through activation of CaMKII.