The fruit fly, Drosophila melanogaster, is an excellent model system that has a vast set of molecular tools and mutants to dissect the genetic pathways that are responsible for the normal and abnormal cardiac function. While the majority of studies have focused on heart development in the Drosophila embryo, attention has recently focused on the structure and function of the adult fly heart as a model of human heart failure. Here we review strategies to identify novel genes and pathways that cause or modify dilated cardiomyopathy in adult Drosophila.

Rationale: Cardiac muscle adapts to increase workload by altering cardiomyocyte size and function resulting in cardiac hypertrophy. G protein-coupled receptor signaling is known to govern the hypertrophic response through the regulation of ion channel activity and downstream signaling in failing cardiomyocytes. Objective: Transient receptor potential canonical (TRPC) channels are G protein-coupled receptor operated channels previously implicated in cardiac hypertrophy. Our objective of this study is to better understand how TRPC channels influence cardiomyocyte calcium signaling. Methods and Results: Here, we used whole cell patch clamp of adult cardiomyocytes to show upregulation of a nonselective cation current reminiscent of TRPC channels subjected to pressure overload. This TRPC current corresponds to the increased TRPC channel expression noted in hearts of mice subjected to pressure overload. Importantly, we show that mice lacking TRPC1 channels are missing this putative TRPC current. Moreover, Trpc1(-/-) mice fail to manifest evidence of maladaptive cardiac hypertrophy and maintain preserved cardiac function when subjected to hemodynamic stress and neurohormonal excess. In addition, we provide a mechanistic basis for the protection conferred to Trpc1(-/-) mice as mechanosensitive signaling through calcineurin/NFAT, mTOR and Akt is altered in Trpc1(-/-) mice. Conclusions: From these studies, we suggest that TRPC1 channels are critical for the adaptation to biomechanical stress and TRPC dysregulation leads to maladaptive cardiac hypertrophy and failure.

The Calsequestrin (Csq) transgenic mouse model of cardiomyopathy exhibits wide variation in phenotypic progression dependent on genetic background. Seven heart failure modifier (Hrtfm) loci modify disease progression and outcome. Here we report Tnni3k (cardiac Troponin I-interacting kinase) as the gene underlying Hrtfm2. Strains with the more susceptible phenotype exhibit high transcript levels while less susceptible strains show dramatically reduced transcript levels. This decrease is caused by an intronic SNP in low-transcript strains that activates a cryptic splice site leading to a frameshifted transcript, followed by nonsense-mediated decay of message and an absence of detectable protein. A transgenic animal overexpressing human TNNI3K alone exhibits no cardiac phenotype. However, TNNI3K/Csq double transgenics display severely impaired systolic function and reduced survival, indicating that TNNI3K expression modifies disease progression. TNNI3K expression also accelerates disease progression in a pressure-overload model of heart failure. These combined data demonstrate that Tnni3k plays a critical role in the modulation of different forms of heart disease, and this protein may provide a novel target for therapeutic intervention.

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 beta1ARKO, beta2ARKO and beta1/beta2AR 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 ISO stimulation was significant attenuated in beta1ARKO and DKO compared to either the beta2ARKO or WT mice indicating that beta1ARs are required for catecholamine induced increases in contractility and CaMKll activity. Eight weeks post myocardial infarction (MI), beta1ARKO and DKO mice showed a significant attenuation in fractional shortening compared to either the beta2ARKO or WT mice. CaMKII activity after MI was significantly increased only in the beta2ARKO and WT hearts and not in the beta1ARKO and DKO hearts. The border zone of the infarct in the beta2ARKO and WT hearts demonstrated significantly increased apoptosis by TUNEL staining compared to the beta1ARKO and DKO hearts. Taken together, these data show that cardiac function and CaMKII activity is mediated almost exclusively by the beta1AR. Moreover, it appears that beta1AR signaling is detrimental to cardiac function following myocardial infarction, possibly through activation of CaMKII. Key words: Ca2+/calmodulin-dependent kinase II,-adrenergic receptors, heart failure, apoptosis.

AIMS: Cardiac hypertrophy is associated with a reduction in the contractile response to beta-adrenergic stimulation, and with re-expression of foetal genes such as beta-myosin heavy chain (MHC). However, whether these two markers of pathology develop concordantly in the same individual cells or independently in different cells is not known. METHODS AND RESULTS: To answer this question, we examined the beta-adrenergic response of individual beta-MHC expressing and non-expressing myocytes from hypertrophic hearts, using a previously generated mouse model (YFP/beta-MHC) in which a yellow fluorescent protein (YFP) is fused to the native beta-MHC protein allowing easy identification of beta-MHC expressing cells. Yellow fluorescent protein/beta-MHC mice were submitted to 4 weeks of transverse aortic constriction (TAC), and the contractile parameters of isolated individual myocytes in response to the beta-adrenergic agonist isoproterenol were assessed. Our results demonstrate that the decrease in isoproterenol-induced cell shortening that develops in TAC hearts occurs only in those hypertrophic myocytes that re-express beta-MHC. Hypertrophic myocytes that do not express beta-MHC have contractility indices indistinguishable from non-TAC controls. CONCLUSION: These data show that the reduction of beta-adrenergic response occurs only in subsets, rather than in all myocytes, and is coincident with re-expression of beta-MHC.

beta1-adrenergic receptor (beta1AR) stimulation confers cardioprotection via beta-arrestin-dependent transactivation of epidermal growth factor receptors (EGFRs), however the precise mechanism for this salutary process is unknown. We tested the hypothesis that the beta1AR and EGFR form a complex that differentially directs intracellular signaling pathways. beta1AR stimulation and EGF ligand can each induce equivalent EGFR phosphorylation, internalization and downstream activation of ERK1/2, but only EGF ligand causes translocation of activated ERK to the nucleus, whereas beta1AR-stimulated/EGFR-transactivated ERK is restricted to the cytoplasm. beta1AR and EGFR are shown to interact as a receptor complex both in cell culture and endogenously in human heart, an interaction that is selective and undergoes dynamic regulation by ligand stimulation. While catecholamine stimulation mediates the retention of beta1AR-EGFR interaction throughout receptor internalization, direct EGF ligand stimulation initiates the internalization of EGFR alone. Continued interaction of beta1AR with EGFR following activation is dependent upon C-terminal tail GRK phosphorylation sites of the beta1AR and recruitment of beta-arrestin. These data reveal a new signaling paradigm in which beta-arrestin is required for the maintenance of a beta1AR-EGFR interaction that can direct cytosolic targeting of ERK in response to catecholamine stimulation.

Despite substantial evidence that nitric oxide (NO) and/or endogenous S-nitrosothiols (SNOs) exert protective effects in a variety of cardiovascular diseases, the molecular details are largely unknown. Here we show that following left coronary artery ligation, mice with a targeted deletion of the S-nitrosoglutathione reductase gene (GSNOR(-/-)) have reduced myocardial infarct size, preserved ventricular systolic and diastolic function, and maintained tissue oxygenation. These profound physiological effects are associated with increases in myocardial capillary density and S-nitrosylation of the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha) under normoxic conditions. We further show that S-nitrosylated HIF-1alpha binds to the vascular endothelial growth factor (VEGF) gene, thus identifying a role for GSNO in angiogenesis and myocardial protection. These results suggest innovative approaches to modulate angiogenesis and preserve cardiac function.

Tafazzin is a conserved mitochondrial protein that is required to maintain normal content and composition of cardiolipin. We used electron tomography to investigate the effect of tafazzin deletion on mitochondrial structure and found that cellular differentiation plays a crucial role in the manifestation of abnormalities. This conclusion was reached by comparing differentiated cardiomyocytes with embryonic stem cells from mouse and by comparing different tissues from Drosophila melanogaster. The data suggest that tafazzin deficiency affects cardiolipin in all mitochondria, but significant alterations of the ultrastructure, such as remodeling and aggregation of inner membranes, will only occur after specific differentiation.

beta-arrestin1 and beta-arrestin2 were initially identified by sequence homology to visual arrestins and by their ability to bind to and inactivate signaling of the beta-2-adrenergic receptor in a process known as desensitization. While the role of beta-arrestins in desensitization has been known for some time, more recent evidence has revealed that beta-arrestins are multifunctional scaffolding proteins that are involved in numerous aspects of G protein-coupled receptor (GPCR) signaling. Interestingly, exciting new data shows that beta-arrestins can mediate signaling in their own right independent of classical second messenger mediated signaling, and that this beta-arrestin-mediated signaling may be cardioprotective. Identifying novel ligands for GPCRs that can block G protein-mediated signaling while simultaneously promoting beta-arrestin-mediated signaling could provide powerful new therapies for cardiac disease.