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

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 organization.

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

*Drosophila* Model of Congenital Heart Diseases 149

to perform. Reverse genetics may be able to compensate for these disadvantages of clinical studies, and especially in the fly system the availability of genetic tools for the entire genome is very useful for a systematic approach to test gene functions. For example in the fly, the *MHC* is encoded by a single gene, thus the analysis of specific mutations in this gene can inform us how alterations in myosin structure directly contribute to alterations in

Both, dystrophin and myosin-related cardiomyopathies are caused by a dysfunction from within cardiomyocytes, and therefore are not necessarily linked to defects in heart formation, which are the basis of CHD. But can the fly model be used to investigate CHD, even though it lacks higher-order structures, such as looping, septation, and chamber formation? There are many cases of CHD where cardiomyocyte function is still far from normal, regardless of the success of corrective surgical procedures. This suggests that those cases could have primary defects within cardiomyocytes in addition to the overall morphological defects in the heart's architecture. Investigations of these questions may also benefit from the cardiomyopathy models in *Drosophila* mentioned above. Taking advantage of the *Drosophila* model we recently performed a study using data obtained from a patient with hypoplastic left heart syndrome (HLHS). HLHS is the most severe type of left-sided heart defect, and occurs in 2-4% of all infants born with congenital heart disease (Loffredo, 2000). We found that this patient had a balanced chromosomal translocation whose breakpoint is in close proximity to a member of the kinesin family (Akasaka, Grosfeld, et al., unpubl.). Heart-specific over expression of kinesin in the fly model disrupts the contractile muscle structure and reduces the quantity myofibrils. Those phenotypes resemble what is observed in micrographs of heart tissue from HLHS patients; cardiomyocytes with scant cytoplasm and myofibrillar disarray (Bohlmeyer et al., 2003). Therefore, despite the differences in the fly heart's gross anatomy this system can provide insights into CHD pathogenesis and this information can be applied to the development of both preventive

To understand the complex etiology and genetics of congenital heart disease, synergistic efforts from all fields of medical and biological sciences are required. For many decades, the invertebrate model organism *Drosophila* has provided exciting new insights into the genetics, development and function of multi-cellular organisms. In this review, we have highlighted some of the recent advances and findings gained from a *Drosophila* model for CHD. Despite its evolutionary distance from vertebrates there is a remarkable conservation of genetics and function. The development of technologies such as time-lapse analysis of heart formation and optical techniques to study function suggest that further studies using this system will provide insights into fundamental cellular mechanisms underlying heart function and disease. The fly has been shown to be a useful model that is able to complement the shortcomings of other model systems. Its simpler genetic architecture allows researchers to dissect the basic networks involved in organ formation and by extension to gain insights into the genetics underlying CHD and cardiac diseases in the same way that the *Drosophila* model has advanced our understanding of human genetics

function and the pathophysiological consequences.

and therapeutic strategies in the future.

**7. Conclusions** 

and embryonic development.

recording system allows a detailed analysis of the heart's performance and pathology, with quantitative measurements of heart period, rhythmicity, size, and fractional shortening (an index of contractility, Akasaka and Ocorr, 2009; Fink et al., 2009a). Using this methodology, it was observed that *dys* mutants exhibited a significantly wider diastolic (80-90µm) and systolic (60µm) diameter compared to laboratory wild-type strains (diastolic diameter 60µM, systolic diameter 40µm), suggesting that the *dys* mutant produces a dilated, cardiomegalytype phenotype. In addition, fractional shortening in the mutants is reduced to 25-30% (compared to 35-40% in wild type). Those features are reminiscent of DCM in humans and a Duchenne-type mouse model (*mdx* mouse) (Quinlan et al., 2004; Wehling-Henricks et al., 2005). Interestingly, a short C-terminal form of human *dys* (Dp116, Judge et al., 2006) rescued the DCM phenotype of *dys* mutant flies (Taghli-Lamallem et al., 2008), but this micro-*dys* could not improve skeletal muscle function in the *mdx* mice model. Because this isoform is incorporated into the dystrophin glycoprotein complex (DGC) but is not capable of binding to the actin skeleton, successful DGC formation may be a critical characteristic. Failure to form this complex may then lead to the observed pathogenesis in the heart, which potentially may be due to dysfunction in force transmission and/or impairment of the signal transduction through DGC. This study is just one example of the use of *Drosophila* as a model for comprehensive human cardiac disease, and which may also allow testing the potential of therapeutic strategies such as the introduction of micro-*dys* to the heart.

Molecular and genetic examinations of cardiomyopathy populations have produced data indicating that mutations in sarcomere-related proteins are involved in the cardiomyopathy phenotype (Hershberger and Siegfried, 2011; Seidman and Seidman, 2011). Myosin is a molecular motor composed of two myosin heavy chains (MHC) and four light chains. This hexameric myosin is a major component of the thick filament and allows them to slide along the thin actin filaments in an ATP-dependent manner. Two mutant alleles of myosin, D45 (A261T) and Mhc5 (G200D), have missense mutations occurring close to the ATP catalytic site, and it was postulated that those amino acid substitutions would affect ATPase activity (Kronert et al., 1999). In fact, ATPase activities of both D45 and Mhc5 mutant myosin were depressed compared to wild-type myosin; however, *in vivo* motility of F-actin on a myosin coated slide showed a reduced velocity for D45 myosin to almost half of that of wild-type and an increased velocity in Mhc5 myosin to about 115% of wild-type (Cammarato et al., 2008a). Interestingly, these myosin mutants showed different pathologies in the heart. Compared to wild-type, D45 mutant hearts are dilated exhibiting an increased systolic and diastolic diameter, whereas Mhc5 mutants appear restricted showing a decreased diameter only during diastolic phase (Cammarato et al., 2008a). The depressed motor function and dilation in D45 myosin is evocative of DCM in humans, whereas the increased motor function and reduced diastolic function in Mhc5 is similar to human restricted cardiomyopathy (RCM, Cammarato et al., 2008a), a rare type of cardiomyopathy in which decreased myocardium elasticity affects the ventricular blood filling during the diastolic phase. Unlike the fly D45 and Mhc5 pathogenesis, biochemical and structural investigations in vertebrates are not always able to reveal how mutations contribute to cardiac pathologies. Instead, the role of a particular gene is primarily obtained from the phenotype and/or symptoms of a patient carrying a mutation in this gene. However, even this clinical approach requires costly and labor-intensive efforts in order to first identify these patients. In addition, clinical studies often require supplemental tests, which are sometimes difficult

recording system allows a detailed analysis of the heart's performance and pathology, with quantitative measurements of heart period, rhythmicity, size, and fractional shortening (an index of contractility, Akasaka and Ocorr, 2009; Fink et al., 2009a). Using this methodology, it was observed that *dys* mutants exhibited a significantly wider diastolic (80-90µm) and systolic (60µm) diameter compared to laboratory wild-type strains (diastolic diameter 60µM, systolic diameter 40µm), suggesting that the *dys* mutant produces a dilated, cardiomegalytype phenotype. In addition, fractional shortening in the mutants is reduced to 25-30% (compared to 35-40% in wild type). Those features are reminiscent of DCM in humans and a Duchenne-type mouse model (*mdx* mouse) (Quinlan et al., 2004; Wehling-Henricks et al., 2005). Interestingly, a short C-terminal form of human *dys* (Dp116, Judge et al., 2006) rescued the DCM phenotype of *dys* mutant flies (Taghli-Lamallem et al., 2008), but this micro-*dys* could not improve skeletal muscle function in the *mdx* mice model. Because this isoform is incorporated into the dystrophin glycoprotein complex (DGC) but is not capable of binding to the actin skeleton, successful DGC formation may be a critical characteristic. Failure to form this complex may then lead to the observed pathogenesis in the heart, which potentially may be due to dysfunction in force transmission and/or impairment of the signal transduction through DGC. This study is just one example of the use of *Drosophila* as a model for comprehensive human cardiac disease, and which may also allow testing the

potential of therapeutic strategies such as the introduction of micro-*dys* to the heart.

Molecular and genetic examinations of cardiomyopathy populations have produced data indicating that mutations in sarcomere-related proteins are involved in the cardiomyopathy phenotype (Hershberger and Siegfried, 2011; Seidman and Seidman, 2011). Myosin is a molecular motor composed of two myosin heavy chains (MHC) and four light chains. This hexameric myosin is a major component of the thick filament and allows them to slide along the thin actin filaments in an ATP-dependent manner. Two mutant alleles of myosin, D45 (A261T) and Mhc5 (G200D), have missense mutations occurring close to the ATP catalytic site, and it was postulated that those amino acid substitutions would affect ATPase activity (Kronert et al., 1999). In fact, ATPase activities of both D45 and Mhc5 mutant myosin were depressed compared to wild-type myosin; however, *in vivo* motility of F-actin on a myosin coated slide showed a reduced velocity for D45 myosin to almost half of that of wild-type and an increased velocity in Mhc5 myosin to about 115% of wild-type (Cammarato et al., 2008a). Interestingly, these myosin mutants showed different pathologies in the heart. Compared to wild-type, D45 mutant hearts are dilated exhibiting an increased systolic and diastolic diameter, whereas Mhc5 mutants appear restricted showing a decreased diameter only during diastolic phase (Cammarato et al., 2008a). The depressed motor function and dilation in D45 myosin is evocative of DCM in humans, whereas the increased motor function and reduced diastolic function in Mhc5 is similar to human restricted cardiomyopathy (RCM, Cammarato et al., 2008a), a rare type of cardiomyopathy in which decreased myocardium elasticity affects the ventricular blood filling during the diastolic phase. Unlike the fly D45 and Mhc5 pathogenesis, biochemical and structural investigations in vertebrates are not always able to reveal how mutations contribute to cardiac pathologies. Instead, the role of a particular gene is primarily obtained from the phenotype and/or symptoms of a patient carrying a mutation in this gene. However, even this clinical approach requires costly and labor-intensive efforts in order to first identify these patients. In addition, clinical studies often require supplemental tests, which are sometimes difficult to perform. Reverse genetics may be able to compensate for these disadvantages of clinical studies, and especially in the fly system the availability of genetic tools for the entire genome is very useful for a systematic approach to test gene functions. For example in the fly, the *MHC* is encoded by a single gene, thus the analysis of specific mutations in this gene can inform us how alterations in myosin structure directly contribute to alterations in function and the pathophysiological consequences.

Both, dystrophin and myosin-related cardiomyopathies are caused by a dysfunction from within cardiomyocytes, and therefore are not necessarily linked to defects in heart formation, which are the basis of CHD. But can the fly model be used to investigate CHD, even though it lacks higher-order structures, such as looping, septation, and chamber formation? There are many cases of CHD where cardiomyocyte function is still far from normal, regardless of the success of corrective surgical procedures. This suggests that those cases could have primary defects within cardiomyocytes in addition to the overall morphological defects in the heart's architecture. Investigations of these questions may also benefit from the cardiomyopathy models in *Drosophila* mentioned above. Taking advantage of the *Drosophila* model we recently performed a study using data obtained from a patient with hypoplastic left heart syndrome (HLHS). HLHS is the most severe type of left-sided heart defect, and occurs in 2-4% of all infants born with congenital heart disease (Loffredo, 2000). We found that this patient had a balanced chromosomal translocation whose breakpoint is in close proximity to a member of the kinesin family (Akasaka, Grosfeld, et al., unpubl.). Heart-specific over expression of kinesin in the fly model disrupts the contractile muscle structure and reduces the quantity myofibrils. Those phenotypes resemble what is observed in micrographs of heart tissue from HLHS patients; cardiomyocytes with scant cytoplasm and myofibrillar disarray (Bohlmeyer et al., 2003). Therefore, despite the differences in the fly heart's gross anatomy this system can provide insights into CHD pathogenesis and this information can be applied to the development of both preventive and therapeutic strategies in the future.
