Summary dTOR (target of rapamycin) and dFoxo respond to changes in the nutritional environment to induce a broad range of responses in multiple tissue types. Both dTOR and dFoxo have been demonstrated to control the rate of age-related decline in cardiac function. Here, we show that the Eif4e-binding protein (d4eBP) is sufficient to protect long-term cardiac function against age-related decline and that up-regulation of dEif4e is sufficient to recapitulate the effects of high dTOR or insulin signaling. We also provide evidence that d4eBP acts tissue-autonomously and downstream of dTOR and dFoxo in the myocardium, where it enhances cardiac stress resistance and maintains normal heart rate and myogenic rhythm. Another effector of dTOR and insulin signaling, dS6K, may influence cardiac aging nonautonomously through its activity in the insulinproducing cells, possibly by regulating dilp2 expression. Thus, elevating d4eBP activity in cardiac tissue represents an effective organ-specific means for slowing or reversing cardiac functional changes brought about by normal aging.

The epidemic of obesity and diabetes is causing an increased incidence of dyslipidemia-related heart failure. While the primary etiology of lipotoxic cardiomyopathy is an elevation of lipid levels resulting from an imbalance in energy availability and expenditure, increasing evidence suggests a relationship between dysregulation of membrane phospholipid homeostasis and lipid-induced cardiomyopathy. In the present study, we report that the Drosophila easily shocked (eas) mutants that harbor a disturbance in phosphatidylethanolamine (PE) synthesis display tachycardia and defects in cardiac relaxation and are prone to developing cardiac arrest and fibrillation under stress. The eas mutant hearts exhibit elevated concentrations of triglycerides, suggestive of a metabolic, diabetic-like heart phenotype. Moreover, the low PE levels in eas flies mimic the effects of cholesterol deficiency in vertebrates by stimulating the Drosophila sterol regulatory element-binding protein (dSREBP) pathway. Significantly, cardiac-specific elevation of dSREBP signaling adversely affects heart function, reflecting the cardiac eas phenotype, whereas suppressing dSREBP or lipogenic target gene function in eas hearts rescues the cardiac hyperlipidemia and heart function disorders. These findings suggest that dysregulated phospholipid signaling that alters SREBP activity contributes to the progression of impaired heart function in flies and identifies a potential link to lipotoxic cardiac diseases in humans.

Why is Drosophila a good model of cardiac physiology?The fly heart has been an excellent model of cardiovascular development for over a decade. Since the discovery of the homeobox transcription factor tinman (Azpiazu and Frasch 1993, Bodmer 1993, Bodmer et al. 1990) and the recognition that it is conserved in vertebrates [reviewed in (Bodmer 1995, Harvey 1996], more and more evidence has corroborated the idea that much of the regulatory genetic network controlling the specification and differentiation of the heart is conserved from flies to mammals [reviewed in (Bodmer and Frasch 1999, Bodmer et al. 2005, Cripps and Olson 2002, Zaffran and Frasch 2002 ], laying the ground work for molecular models of congenital heart disease in humans [reviewed in (Chien and Olson 2002, Prall et al. 2002, Seidman and Seidman 2002, Olson 2004, Srivastava 2006]. Given the remarkable conservation of molecular and embryological mechanisms underlying cardiogenesis in the animal kingdom, it seems plausible that the genetic control of heart function may also be conserved. Clearly, many proteins that carry out cardiac function, such as ion channels and contractile proteins, are highly conserved (reviewed in Bodmer et al. 2005): contributors to excitation-contraction coupling, such as the ryanodine receptor, SERCA, myosin, troponin, and ion channels likely to be involved in pacemaking, such as Ih/HCN (Monier et al. 2005), are all present in fly cardiomyocytes. Also, plasma membrane invag inations forming T tubules have been observed in the fly's heart, much like in vertebrates, and the mononucleate cardiomyocytes that comprise the heart tube are electrically connected by GAP junctions formed by innexin proteins in invertebrates. Thus, it is conceivable that the way these conserved proteins function within the mature heart to ensure a normal heartbeat has also evolved from a common evolutionary design that was in place prior to the invertebratevertebrate split.In the following we review recent advances in elucidating the genetics of cardiac function and aging in Drosophila and propose that the control of the cardiac physiology and rhythmicity is conserved between in many ways vertebrates and invertebrates. As a consequence, the fly heart is a potentially useful genetic model not only for understanding congenital heart disease that Correspondence: R. Bodmer (rolf@burnham.org) and X. Wu

The neuronal Cdk5 kinase is composed of the catalytic subunit Cdk5 and the activator protein p35 nck5a or its isoform, p39nck5ai . To identify novel p35 nck5a -and p39 nck5ai -binding proteins, fragments of p35 nck5a and p39 nck5ai were utilized in affinity isolation of binding proteins from rat brain homogenates, and the isolated proteins were identified using mass spectrometry. With this approach, the nuclear protein SET was shown to interact with the N-terminal regions of p35 nck5a and p39 nck5ai . Our detailed characterization showed that the SET protein formed a complex with Cdk5/p35 nck5a through its binding to p35 nck5a . The p35 nck5a -interacting region was mapped to a predicted ␣-helix in SET. When cotransfected into COS-7 cells, SET and p35 nck5a displayed overlapping intracellular distribution in the nucleus. The nuclear co-localization was corroborated by immunostaining data of endogenous SET and Cdk5/ p35 nck5a from cultured cortical neurons. Finally, we demonstrated that the activity of Cdk5/p35 nck5a , but not that of Cdk5/p25 nck5a , was enhanced upon binding to the SET protein. The tail region of SET, which is rich in acidic residues, is required for the stimulatory effect on Cdk5/p35 nck5a .Cdk5 is distinct from other cyclin-dependent kinases by virtue of its functions in post-mitotic neurons, but not in proliferating cells. Although Cdk5 is ubiquitously expressed, Cdk5-associated kinase activity has been primarily demonstrated in central nervous system neurons. In such neurons, Cdk5 is associated with p35 nck5a or a p35 nck5a isoform (p39 nck5ai ), two Cdk5 activators with restricted expression in central nervous system neurons (1-3). In a recent report, p35 nck5a was also found in muscle cells at the neuromuscular junction (4). Besides the full-length protein of p35 nck5a , a proteolytic fragment called p25 nck5a exists in central nervous system neurons. The p25 nck5a protein is generated when the N-terminal 98 amino acids are removed from p35 nck5a (1, 5-7). Moreover, p25 nck5a is fully functional in terms of Cdk5 activation (8). In association with p35 nck5a /p25 nck5a and p39 nck5ai , Cdk5 exhibits a variety of functions in neuronal differentiation and neurocytoskeleton dynamics as well as neuronal degeneration and cell death (9 -13).Despite little homology between p35 nck5a and cyclins at the primary sequence level, it was proposed that p35 nck5a forms a core structure similar to that of cyclins to support Cdk5 enzyme activity (14 -16). The minimal region required for Cdk5 binding and activation was localized to a region in the C-terminal half of p35 nck5a as well as in p25 nck5a (16,17). Moreover, Cdk5/ p35 nck5a shows many distinct regulatory properties. Cdk5 is highly activated upon association with p35 nck5a /p25 nck5a , and the activation process is not regulated by the cyclin-dependent kinase-activating kinase Cdk7/cyclin H (8, 16). Moreover, upregulation of Cdk5/p35 nck5a activity was observed when Cdk5 was phosphorylated at Tyr-15 by the cellular tyrosine kinase c-Abl in complex ...

SUMMARY Reactive oxygen species (ROS) can act cell autonomously and in a paracrine manner by diffusing into nearby cells. Here, we reveal a ROS-mediated paracrine signaling mechanism that does not require entry of ROS into target cells. We found that under physiological conditions, nonmyocytic pericardial cells (PCs) of the Drosophila heart contain elevated levels of ROS compared to the neighboring cardiomyocytes (CMs). We show that ROS in PCs act in a paracrine manner to regulate normal cardiac function, not by diffusing into the CMs to exert their function, but by eliciting a downstream D-MKK3-D-p38 MAPK signaling cascade in PCs that acts on the CMs to regulate their function. We find that ROS-D-p38 signaling in PCs during development is also important for establishing normal adult cardiac function. Our results provide evidence for a previously unrecognized role of ROS in mediating PC/CM interactions that significantly modulates heart function.

Cdk5 and its neuronal activator p35 play an important role in neuronal migration and proper development of the brain cortex. We show that p35 binds directly to ␣/␤-tubulin and microtubules. Microtubule polymers but not the ␣/␤-tubulin heterodimer block p35 interaction with Cdk5 and therefore inhibit Cdk5-p35 activity. p25, a neurotoxin-induced and truncated form of p35, does not have tubulin and microtubule binding activities, and Cdk5-p25 is inert to the inhibitory effect of microtubules. p35 displays strong activity in promoting microtubule assembly and inducing formation of microtubule bundles. Furthermore, microtubules stabilized by p35 are resistant to coldinduced disassembly. In cultured cortical neurons, a significant proportion of p35 localizes to microtubules. When microtubules were isolated from rat brain extracts, p35 co-assembled with microtubules, including cold-stable microtubules. Together, these findings suggest that p35 is a microtubule-associated protein that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation.As a distinct member of the CDK family, Cdk5 is activated by a neuron-specific protein p35 or the p39 homologue of p35 in the central nervous system (1). Both Cdk5 and p35 are required for neurite outgrowth (2). Studies in animal models have revealed their crucial involvements in neuronal migration during nervous system development as mice deficient of Cdk5 or p35 display abnormal brain cortex (3, 4). To date, a wide range of evidence has been accumulated indicating that Cdk5-p35 is a multifunctional kinase that acts in the regulation of various neuronal activities, including organization of the microtubule cytoskeleton (1). In living cells, the dynamic properties of microtubules are modulated through a sophisticated mechanism involving microtubule-associated proteins (MAPs), 2 which bind microtubule polymers and promote microtubule polymerization by stabilizing the polymer structure (5). Cdk5 phosphorylates several MAPs including MAP1b, MAP2, tau, and doublecortin, mediating their association with microtubules and their microtubule-stabilizing functions (1, 6, 7).It is poorly understood how Cdk5 activity is regulated. Although p35 shows little apparent sequence homology to cyclins, it resembles the cyclin A structure with distinct features to bind specifically to Cdk5 (8,9). The binding of p35 highly stimulates Cdk5 activity (10). Several proteins, including C42, protein kinase CK2, and three importin family members (importin-␤, importin-5, and importin-7), show inhibitory effects toward Cdk5 activation via binding to p35 (11-13). Under neurotoxic conditions, p35 is transformed into the N-terminally truncated p25 protein, which causes sustained activation and mislocalization of Cdk5 (14 -16). Moreover, p25 deregulation of Cdk5 has been linked to neuronal cell death and pathogenesis of neurodegenerative diseases such as Alzheimer disease (1). In this report, we have identified direct association of p35 with tubulin and microtubules a...

A central feature of obesity-related cardiometabolic diseases is the impaired ability to transition between fatty acid and glucose metabolism. This impairment, referred to as “metabolic inflexibility”, occurs in a number of tissues, including the heart. Although the heart normally prefers to metabolize fatty acids over glucose, the inability to upregulate glucose metabolism under energetically demanding conditions contributes to a pathological state involving energy imbalance, impaired contractility, and post-translational protein modifications. This review discusses pathophysiologic processes that contribute to cardiac metabolic inflexibility and speculates on the potential physiologic origins that lead to the current state of cardiometabolic disease in an obesogenic environment.

The heart has emerged as an important organ in the regulation of systemic lipid homeostasis; however, the underlying mechanism remains poorly understood. Here, we show that Drosophila cardiomyocytes regulate systemic lipid metabolism by producing apolipoprotein B-containing lipoproteins (apoB-lipoproteins), essential lipid carriers that are so far known to be generated only in the fat body. In a Drosophila genetic screen, we discovered that when haplo-insufficient, microsomal triglyceride transfer protein (mtp), required for the biosynthesis of apoB-lipoproteins, suppressed the development of diet-induced obesity. Tissue-specific inhibition of Mtp revealed that whereas knockdown of mtp only in the fat body decreases systemic triglyceride (TG) content on normal food diet (NFD) as expected, knockdown of mtp only in the cardiomyocytes also equally decreases systemic TG content on NFD, suggesting that the cardiomyocyte- and fat body-derived apoB-lipoproteins serve similarly important roles in regulating whole-body lipid metabolism. Unexpectedly, on high fat diet (HFD), knockdown of mtp in the cardiomyocytes, but not in fat body, protects against the gain in systemic TG levels. We further showed that inhibition of the Drosophila apoB homologue, apolipophorin or apoLpp, another gene essential for apoB-lipoprotein biosynthesis, affects systemic TG levels similarly to that of Mtp inhibition in the cardiomyocytes on NFD or HFD. Finally, we determined that HFD differentially alters Mtp and apoLpp expression in the cardiomyocytes versus the fat body, culminating in higher Mtp and apoLpp levels in the cardiomyocytes than in fat body and possibly underlying the predominant role of cardiomyocyte-derived apoB-lipoproteins in lipid metabolic regulation. Our findings reveal a novel and significant function of heart-mediated apoB-lipoproteins in controlling lipid homeostasis.