**5. Exploring fly heart function to understand CHD-related cardiomyopathies**

By definition, congenital heart disease refers to the presence of structural heart and large vessel defects at the time of birth. Many of these can be repaired by surgical intervention, but depending on the severity of the defect, patients may require life-long medical followups to monitor cardiac performance and to detect signs of functional decline. Furthermore, late onset complications either due to persistent impact of structural defects or related to the applied intervention are often linked to lethal arrhythmias or progressive congestive heart failure. Such secondary complications might not be caused directly by CHD genes, but due to a maladaptive response of the cardiac tissue. In the light of these considerations it is therefore necessary to understand how cardiac tissues respond to these interventions and which other genes might contribute to arrhythmias and cardiomyopathies. The *Drosophila* model has helped to identify a number of novel genes and pathways required to maintain myofibrillar organization and overall heart structure and function. Because similarities between the *Drosophila* and murine heart are found both at the molecular and functional level it is therefore likely that new risk factors of cardiac disease can be identified using *Drosophila*. The cardiac proteasomes of the mouse and the fly have been shown to be

*Drosophila* Model of Congenital Heart Diseases 147

heterozygous *Cdc42;Nkx2.5* mouse hearts also revealed an impaired heart function when compared to single heterozygous animals, again indicating that this genetic link between *Cdc42* and *tinman*/*Nkx2*.5 is conserved. Such complex *in vivo* screens to unravel genetic interactions in higher eukaryotes are currently only feasible in the fly model organism. With respect to understanding the genetics of heart development and disease, *Drosophila* is the simplest genetic model with a heart (Bier and Bodmer, 2004). Moreover, from a systems biology point of view, the fly is a perfect model organism to rapidly test genetic interactions that are predicted from networks based on genetic information, bioinformatics and the integration of other data obtained from many different model organisms and patients (for the fly, such data can be accessed through DroID, the *Drosophila* interactions database, see

The knowledge of molecular mechanisms underlying important biological processes gained from *Drosophila* has been successfully extended to studies of human diseases especially in the field of neural degenerative diseases (Bilen and Bonini, 2005; Marsh and Thompson, 2006). Recent studies in flies have been directed towards understanding more complex and multifactorial diseases such as heart disease. In this section we specifically demonstrate how *Drosophila* can be used as a model to elucidate the molecular mechanisms of CHD and cardiomyopathies. As we mentioned above, both anatomical and molecular features of *Drosophila* heart development (as outlined in Figure 1) and aspects of adult structure and function (see Figure 2) are similar to those observed in the human heart, making *Drosophila* a useful model system with the advantage of a much simpler genetic and tissue

Primary cardiomyopathies are contractile disorders of the myocardium. The majority of cases of cardiomyopathy are classified into two disease types: hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). HCM, defined as a hypertrophic ventricle with myofibrillar disarrays, can sometimes lead to sudden death in young subjects, but many cases of HCM maintain stable hemodynamics until late stages. DCM, defined as dilated ventricle with systolic dysfunction, also shows myofibrillar disarrays and clinically exhibits refractory arrhythmias and severe heart failure. In both disease types genetic causes have been found. The first case of a male-sibling DCM with X-linked inheritance had a mutation in *dystrophin* (*dys*), a gene that plays an important role in the anchoring of muscle cells (Towbin et al., 1993). Mutations in *dys* are a cause of Duchenne-type (null function of *dys*) and Becker-type (hypomorphic function of *dys*) muscular dystrophy, whose clinical entity is characterized by progressive muscle weakness and degeneration of muscle fibers (Koenig et al., 1988). In both types late-onset cardiac dysfunction is frequently observed, and improvement of heart function is an important therapeutic target for improving life prognosis. As in humans, fly Dys is associated with the plasma membrane at the sarcomeric Z-line and is already present during early embryogenesis of the *Drosophila* heart (Taghli-Lamallem et al., 2008). In *dys* deficiency flies the myofibrillar structure of the heart cells is disorganized in that myofibrils are not tightly packed and appear sparse. This phenotype worsens with age, consistent with the late onset of cardiac dysfunction in muscular dystrophy patients. Real-time imaging of heart movements using high-speed digital video

Yu et al., 2008; Murali et al., 2010).

organization.

**6. A genetic model for heart diseases** 

comparable with respect to their overall composition (Cammarato et al., 2011), and functional analysis revealed further evidence of the conserved cardiac characteristics of the fly heart (e.g. Ocorr et al., 2007a; Buechling et al., 2009; Choma et al., 2010). In particular, the contractile and electrical properties have been investigated in the *Drosophila* heart and found to be remarkably similar in fundamental aspects, such as the contribution of ion channels to heart contraction or the effects of mutations of genes of the myofibrillar apparatus (K. Ocorr, unpublished and Lalevée et al., 2006; Wolf et al., 2006; Ocorr et al., 2007b; Cammarato et al., 2008b; Mery et al., 2008). In addition to determining cardiac parameters in vivo and in situ (see below and Fig. 2), electrical properties can be measured using the fluorescent Ca2+ sensor GCaMP (Nakai et al., 2001), where Ca2+-transients are monitored *in vivo* (Lin et al., 2011).

These techniques have set the stage for using the *Drosophila* model to identify new cardiac genes involved in CHD and cardiac disease. In a recent screen for genes affecting *Drosophila* heart function under stress conditions, components of the CCR4/NOT complex, which is a regulator of gene transcription and mRNA degradation, have been shown to play a pivotal role in maintaining heart function in flies, but also in mice and possibly humans (Neely et al., 2010). In this particular study, hearts in mutant flies were functionally analyzed as semiintact preparations (using the methods described in Ocorr et al., 2007b; 2009; Vogler and Ocorr, 2009) and subsequently analyzed for structural defects (as described in Alayari et al., 2009; Fink et al., 2009b). These methods allow the assessment of numerous fly heart parameters (such as diastolic and systolic diameters and intervals, intrinsic heart rate of the denervated heart and estimates on the degree of arrhythmias; see Fig. 2). For CCR4/NOT complex mutants, it was shown that these flies have hallmarks of dilated cardiomyopathy. Remarkably, *not3+/-* heterozygous mice are haploinsufficient and exhibit less resistance to cardiac stress, indicating that this pathway is required for maintaining proper heart function. Administration of HDAC inhibitors ameliorates these phenotypes, indicating that changes in chromatin remodeling are likely to play a major role. Lastly, the authors showed that a singlenucleotide polymorphism (SNP) near the human NOT3 genes is associated with prolonged long QT intervals. Together, these data show that the cardiac role of the CCR4/NOT complex is highly conserved, and that *Drosophila* indeed is useful for identifying novel genes and pathways involved in cardiac disease. Due to the broad role of the CCR4/NOT complex in regulating both, gene transcription and posttranslational modification, it remains unclear by which mechanisms and genes the cardiac phenotypes become manifest. Further analysis of the CCR4/NOT complex is therefore required, and the fly heart is likely to provide further insights. Importantly, this approach showed that RNAi-mediated genetic screening is a promising approach to identify new cardiac risk genes in humans.

In *Drosophila*, as well as in higher organisms, transheterozygous mutations can unravel a hidden link between two genes (genetic interaction). *Drosophila* is well suited for such experimental screening approaches since most genes are not duplicated, thus interactions are less likely to be masked by compensatory mechanisms. In a recent study, this particular strength of the *Drosophila* model has been successfully exploited to identify a novel link between the cardiac transcription factor *Nkx2.5*/*tinman* and the small GTPase *Cdc42* (Qian et al., 2011). In the aforementioned study, flies that were double heterozygous for *Cdc42* and *tinman* showed altered cardiac function and also showed structural defects, something not observed in the single heterozygous animals. The subsequent analysis of double

comparable with respect to their overall composition (Cammarato et al., 2011), and functional analysis revealed further evidence of the conserved cardiac characteristics of the fly heart (e.g. Ocorr et al., 2007a; Buechling et al., 2009; Choma et al., 2010). In particular, the contractile and electrical properties have been investigated in the *Drosophila* heart and found to be remarkably similar in fundamental aspects, such as the contribution of ion channels to heart contraction or the effects of mutations of genes of the myofibrillar apparatus (K. Ocorr, unpublished and Lalevée et al., 2006; Wolf et al., 2006; Ocorr et al., 2007b; Cammarato et al., 2008b; Mery et al., 2008). In addition to determining cardiac parameters in vivo and in situ (see below and Fig. 2), electrical properties can be measured using the fluorescent Ca2+ sensor GCaMP (Nakai et al., 2001), where Ca2+-transients are monitored *in vivo* (Lin et al.,

These techniques have set the stage for using the *Drosophila* model to identify new cardiac genes involved in CHD and cardiac disease. In a recent screen for genes affecting *Drosophila* heart function under stress conditions, components of the CCR4/NOT complex, which is a regulator of gene transcription and mRNA degradation, have been shown to play a pivotal role in maintaining heart function in flies, but also in mice and possibly humans (Neely et al., 2010). In this particular study, hearts in mutant flies were functionally analyzed as semiintact preparations (using the methods described in Ocorr et al., 2007b; 2009; Vogler and Ocorr, 2009) and subsequently analyzed for structural defects (as described in Alayari et al., 2009; Fink et al., 2009b). These methods allow the assessment of numerous fly heart parameters (such as diastolic and systolic diameters and intervals, intrinsic heart rate of the denervated heart and estimates on the degree of arrhythmias; see Fig. 2). For CCR4/NOT complex mutants, it was shown that these flies have hallmarks of dilated cardiomyopathy. Remarkably, *not3+/-* heterozygous mice are haploinsufficient and exhibit less resistance to cardiac stress, indicating that this pathway is required for maintaining proper heart function. Administration of HDAC inhibitors ameliorates these phenotypes, indicating that changes in chromatin remodeling are likely to play a major role. Lastly, the authors showed that a singlenucleotide polymorphism (SNP) near the human NOT3 genes is associated with prolonged long QT intervals. Together, these data show that the cardiac role of the CCR4/NOT complex is highly conserved, and that *Drosophila* indeed is useful for identifying novel genes and pathways involved in cardiac disease. Due to the broad role of the CCR4/NOT complex in regulating both, gene transcription and posttranslational modification, it remains unclear by which mechanisms and genes the cardiac phenotypes become manifest. Further analysis of the CCR4/NOT complex is therefore required, and the fly heart is likely to provide further insights. Importantly, this approach showed that RNAi-mediated genetic screening is a

promising approach to identify new cardiac risk genes in humans.

In *Drosophila*, as well as in higher organisms, transheterozygous mutations can unravel a hidden link between two genes (genetic interaction). *Drosophila* is well suited for such experimental screening approaches since most genes are not duplicated, thus interactions are less likely to be masked by compensatory mechanisms. In a recent study, this particular strength of the *Drosophila* model has been successfully exploited to identify a novel link between the cardiac transcription factor *Nkx2.5*/*tinman* and the small GTPase *Cdc42* (Qian et al., 2011). In the aforementioned study, flies that were double heterozygous for *Cdc42* and *tinman* showed altered cardiac function and also showed structural defects, something not observed in the single heterozygous animals. The subsequent analysis of double

2011).

heterozygous *Cdc42;Nkx2.5* mouse hearts also revealed an impaired heart function when compared to single heterozygous animals, again indicating that this genetic link between *Cdc42* and *tinman*/*Nkx2*.5 is conserved. Such complex *in vivo* screens to unravel genetic interactions in higher eukaryotes are currently only feasible in the fly model organism. With respect to understanding the genetics of heart development and disease, *Drosophila* is the simplest genetic model with a heart (Bier and Bodmer, 2004). Moreover, from a systems biology point of view, the fly is a perfect model organism to rapidly test genetic interactions that are predicted from networks based on genetic information, bioinformatics and the integration of other data obtained from many different model organisms and patients (for the fly, such data can be accessed through DroID, the *Drosophila* interactions database, see Yu et al., 2008; Murali et al., 2010).
