Increasing autophagy and blocking Nrf2 suppress laminopathy‐induced age‐dependent cardiac dysfunction and shortened lifespan

Summary Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age‐dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A‐type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age‐dependent heart defects are not well understood. To address this issue, we modeled human disease‐causing mutations in the Drosophila melanogaster Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1 redox sensing pathway was activated, mitochondria exhibited abnormal morphology, and the autophagy cargo receptor Ref2(P)/p62 was upregulated. Genetic analyses demonstrated that simultaneous over‐expression of the autophagy kinase Atg1 gene and an RNAi against CncC eliminated the cytoplasmic protein aggregates, restored cardiac function, and lengthened lifespan. These data suggest that simultaneously increasing rates of autophagy and blocking the Nrf2/Keap1 pathway are a potential therapeutic strategy for cardiac laminopathies.

delay the onset and/or prevent these cardiac defects during aging, a greater understanding of the molecular basis of the pathology is needed.
The human LMNA gene encodes the developmentally regulated and nearly ubiquitously expressed A-type lamins, Lamin A and C, which are produced by alternate splicing (Burke & Stewart, 2013).
Genetically tractable model organisms have been used to understand the functions of lamins. Mice lacking A-type lamins have severe cardiac and skeletal muscle defects, in addition to a shortened lifespan (Ramos et al., 2012;Zhang, Kieckhaefer & Cao, 2013). Furthermore, studies on the mouse models demonstrated that the cardiac defects and shortened lifespan can be partially reversed by treatment with rapamycin and temsirolimus (a derivative of rapamycin) (Choi et al., 2012;Ramos et al., 2012). Studies in Drosophila demonstrated that mutant lamins, modeled after those that cause human disease, lead to cytoplasmic aggregation of nuclear envelope proteins and loss of redox homeostasis in larval body wall muscles (Dialynas et al., 2012(Dialynas et al., , 2015. Consistent with these findings, human muscle biopsy tissues showed both cytoplasmic aggregation of nuclear envelope (NE) proteins and activation of the Nrf2/Keap-1 signaling pathway. Thus, these models have phenotypes similar to the human disease condition.
Here, we developed a Drosophila model of cardiac laminopathies.
Mutations in the human LMNA that cause dilated cardiomyopathy with conduction defects are often point mutations resulting in amino acid substitutions in residues conserved among species. We modeled these mutations in the Drosophila Lamin C gene (hereafter referred as LamC) and assayed for effects on the fruit fly heart. Mechanisms of cardiac development and function are shared between Drosophila and humans (Diop & Bodmer, 2015;Melkani et al., 2013;Zhu et al., 2017). Furthermore, Drosophila has successfully been used to identify the genetic basis of cardiac deterioration that arises due to aging and metabolic dysregulation (Diop & Bodmer, 2015;Gill, Le, Melkani & Panda, 2015;Melkani et al., 2013).
The cardiolaminopathy Drosophila models exhibited age-dependent decline in cardiac function that resulted in a shortened adult lifespan. Defects were observed in the nucleus, cytoplasm, and mitochondria of cardiomyocytes. In addition, adults showed an agedependent increase in triglycerides. Many of these abnormal features are common in human cardiac laminopathies (Captur et al., 2017).
The Drosophila models allowed for genetic tests of suppression and identified new potential therapeutic targets for individuals with cardiolaminopathy and other types of laminopathies.

| Mutant LamC caused age-dependent cardiac defects and a shortened adult lifespan
To determine the mechanistic basis of cardiolaminopathies, we expressed wild-type and mutant Drosophila LamC transgenes (referred to as R205W and G489V hereafter) in the heart (Figure 1a).
These amino acid substitutions are analogous to human Lamin A/C R190W and G449V, respectively. Mutations in LMNA that give rise to Lamin A/C R190W are associated with progressive cardiac defect (including conduction defects) and reduced cardiac performance (Arbustini et al., 2002;Heller et al., 2017;Hermida-Prieto et al., 2004). Mutations in LMNA that give rise to Lamin A/C G449V cause congenital muscular dystrophy, which is characterized by skeletal muscle defects in childhood and age-dependent dilated cardiomyopathy (Dialynas et al., 2015). Cardiac-specific expression of Drosophila LamC was obtained using the Gal4/UAS system with a Hand-Gal4 driver (Han & Olson, 2005;Melkani et al., 2013). Expression of mutant LamC, but not wild-type, caused adult cardiac defects in flies possessing an otherwise wild-type genetic background (Figure 1b, c).
Defective cardiac function was measured and quantitated according to established protocols (Gill et al., 2015;Melkani et al., 2013). M-mode analysis was performed in which a specified region of one-pixel width along the image of the heart was selected from each movie frame and aligned horizontally, generating a montage that portrays the movement of the heart walls over time (Figure 1b). Hearts from 3-week-old female flies expressing R205W and G489V showed significant dilation and restriction, respectively. Consistent with our finding, mutations in human LMNA result in dominant dilated, hypertrophic, and idiopathic cardiomyopathy (Arbustini et al., 2002;Heller et al., 2017;Marian, 2017). Furthermore, expression of mutant LamC resulted in dysrhythmic beating patterns when compared to hearts from age-matched controls expressing wild-type LamC and the Hand-Gal4 driver alone (Figure 1b).
Expression of the mutant LamC resulted in defective ostia, noncontractile region(s) of the heart, and loss of heartbeat, an indicator of conduction defects (Figure 1c), which are observed in human LMNA patients (Arbustini et al., 2002;Brayson & Shanahan, 2017;Malhotra & Mason, 2009;Wolf et al., 2008). In addition to severe cardiac defects, heart-specific expression of R205W and G489V had a drastic impact on the lifespan for both female and male adults compared to controls (Figure 1d, e). The half-life of females expressing R205W and G489V was 21 and 39 days, respectively, compared to 58 days for females expressing wild-type LamC (Figure 1d). Similarly, the half-life of males expressing R205 and G489V was 18 and 38 days, respectively, compared to 56 days for males expressing wild-type LamC (Figure 1e). Western analysis showed that similar levels of wild-type and mutant LamC were expressed by the Hand-Gal4 driver (Fig. S1a). Transgenic flies expressing wild-type LamC had less than twofold higher level of LamC compared with nontransgenic controls. Importantly, the slightly elevated level of wild-type LamC did not produce cardiac defects (Figure 1b, c). In contrast, the mutant versions of LamC expressed at levels similar to the exogenous wild-type LamC produced obvious cardiac defects (Figure 1b, c). Thus, the cardiac defects were caused by mutant LamC and not increased total amounts of LamC. Furthermore, cardiac-specific expression of mutant LamC did not result in noncardiac muscle defects as measured by adult flight (Fig. S1b). Therefore, the phenotypes were restricted to the muscle tissue in which the mutant LamC heart diameters were significantly enlarged and reduced at one and three weeks of age compared to age-matched controls, respectively.
The cardiac dilation and restriction reduced heart contractility (Figure 2e, f), which was further reflected by decreased fractional F I G U R E 1 Drosophila Lamin C domain structure and effects of mutant LamC on cardiac function and lifespan. (a) Lamins contain a conserved structure with an N-terminal head domain, coiled-coil rod domain, and tail domain possessing an Ig-fold. Amino acid substitutions in the rod and Ig-fold domains used in this study are indicated. Numbering for Drosophila Lamin C is in black; the corresponding human diseasecausing amino acid substitution is in red. (b) M-mode recordings (5-s time periods) of dissected hearts from 3-week-old female Hand-Gal4/+, wild-type and mutant Lamin C (LamC) (R205W and G489V). M-mode analysis revealed that R205W-expressing hearts showed significant dilation and arrhythmias, whereas G489V-expressing hearts showed restricted morphology and arrhythmias. Double-headed arrows in the Mmode traces indicate diastolic diameter (DD) and systolic diameter (SD) between the walls of the heart. (c) Summary of the qualitative cardiac defects from 3-week-old male and female adult controls (Hand/+ and wild-type LamC) and mutants (R205W and G489V) showing the percent of flies exhibiting defective ostia, one or more noncontractile regions (conductive defect), and nonbeating hearts. (d and e) Cardiac-specific expression of mutant LamC (R205W and G489V) resulted in a reduction in lifespan compared to controls expressing Hand/+ and wild-type LamC controls (p < .001). Graphs indicating the percent survival for female adults (n = 150 for each group) versus age days posteclosion shortening ( Figure 2g). Similar defects were observed in adult males (data not shown). Thus, these data demonstrate that heart-specific expression of mutant LamC caused severe and progressive contractility-related cardiac phenotypes. The myofibrillar disorganization and LamC aggregation increased with age ( Fig. S2a-c). Quantitation of LamC aggregates showed that the relative area occupied by the aggregates per total area surveyed was greater in hearts expressing the mutant LamC versus those expressing wild-type LamC (Fig. S2c). Furthermore, the relative area occupied by aggregates increased with age (Fig. S2c). Immunostaining of hearts expressing wild-type LamC with antibodies to Otefin (Ote) [a Drosophila orthologue of the human inner nuclear membrane LEM domain protein emerin (Barton, Lovander, Pinto & Geyer, 2016)] showed localization to the nuclear envelope as anticipated ( Figure 3c). In contrast, hearts expressing mutant LamC showed greater amounts of F I G U R E 2 Mutant LamC caused progressive cardiac physiological dysfunction. One (1W) and three-week (3W)-old female adults (n = 44-71 per genotype) expressing Hand-Gal4/+, wildtype LamC, R205W, and G489V were assayed for the heart period (a), arrhythmia index (b), diastolic and systolic intervals (c and d), diastolic and systolic parameters (e and f), and fractional shortening (g). Minimal differences were observed between the Hand/+ and wild-type LamC for the cardiac parameters measured. In contrast, significant differences were observed for all parameters (a-g) between the mutant LamC (R205W and G489V) and age-matched controls. Data are shown as average AE s.e.m.s; statistical significance was determined using one-way ANOVA and Tukey's post hoc test. For all parameters, statistical significance is denoted as follows: *p < .05; **p < .01; ***p < .001; NS = not significant cytoplasmic aggregation of Ote (Figures 3c and S2c). Thus, mutant LamC caused myofibril disorganization, nuclear blebbing, and cytoplasmic aggregation of nuclear envelope proteins.
To determine the effects of LamC aggregation in cardiac tissue at the ultrastructural level, we employed transmission electron microscopy (TEM) (Figure 4a). TEM micrographs of a transverse section through the dorsal vessel of 3-week-old adults were prepared according to published procedures (Melkani et al., 2013). Adults expressing wild-type LamC showed cardiomyocytes with characteristic discontinuous Z-disks ("Z"). In contrast, similar aged adults expressing mutant F I G U R E 3 Mutant LamC caused myofibrillar disorganization, nuclear blebbing, and cytoplasmic aggregation of LamC and Otefin (orthologue of human Emerin). (a) Immunofluorescence of 3-week-old hearts stained with antibodies to LamC (red), phalloidin (F-actin, green), and DAPI (DNA, blue). R205W and G489V caused disorganization of actin-containing myofibrils that was not seen upon expression of wild-type LamC (upper panels). CF and LF represent circumferential and longitudinal fibers, respectively. Asterisks represent myofibrillar disorganization (upper panels). Hearts expressing R205W and G489V that were stained with DAPI and anti-LamC antibodies showed enlarged nuclei and cytoplasmic aggregation of LamC (arrows, lower panels). High magnification images are shown as insets. (b) Merged images of hearts expressing wild-type LamC, R205W, and G489V stained with anti-LamC antibodies (red) and DAPI (blue) showed cytoplasmic LamC aggregates (arrows) (c) Merged images of hearts expressing wild-type LamC, R205W, and G489V stained with DAPI (DNA), anti-LamC antibodies (red), and antibodies that recognize Otefin (yellow) showed cytoplasmic aggregates (arrows) F I G U R E 4 Cardiac-specific expression of mutant LamC caused ultrastructural defects, upregulation of Ref(2)P, and altered adipose tissue homeostasis. (a) TEM micrographs of transverse sections through the heart of 3-week-old adults expressing wild-type LamC showed contractile cardiomyocytes with characteristic Z-disks (Z). Note that only minor myofibrillar degeneration was observed (black asterisk). In contrast, hearts expressing R205W and G489V showed substantial myofibrillar degeneration (black asterisks) and poorly organized Z-disks (Z). The lumen (L) is a hemolymph-containing compartment surrounded by contractile cardiomyocytes. In 3-week-old control hearts, intact membrane-bound nuclei were detected. In contrast, hearts expressing R205W showed nuclear material outside of membrane-bound nuclei (white asterisks). White arrows indicate the nuclear envelope. No intact nuclei were observed in 3-week-old hearts expressing G489V; however, membrane-bound (black arrow) electron-sparse structures were detected, which may constitute degenerated nuclei and/or vacuoles. Scale bars are 500 nm. (b) Western analysis of protein extracts from dissected hearts of 3-week-old female females expressing wild-type and mutant LamC stained with antibodies that recognize Ref(2)P and histone H2B (loading control). (c) Representative antibody "dot blot" of protein extract from dissected hearts and stained with antibodies that recognize Ref(2)P and histone H2B. (d) Quantification of Ref(2)P staining, normalized to levels of histone H2B, in 3-week-old female flies hearts expressing R205W and G489V compared to same age flies expressing wild-type LamC (**p < .01, n = 3 independent samples). (e) Cardiomyocytes of 3-week-old adults were stained with DAPI (blue) and antibodies to Ref (2) Abnormal cytoplasmic protein aggregation causes nuclear enrichment of the mammalian nuclear factor erythroid-related factor 2 (Nrf2) in a mouse model of mutant aB-crystallin-induced cardiomyopathy (Kannan et al., 2013;Rajasekaran et al., 2011). Nrf2 and its cytoplasmic binding partner Keap1 function in cellular detoxification and are conserved in Drosophila (Deng & Kerppola, 2013. In a Drosophila model of noncardiac muscle laminopathies, Cap-and-collar C (CncC) [the orthologue of mammalian nuclear erythroid 2-related factor 2 (Nrf2)] redox transcriptional regulator (Deng & Kerppola, 2013 accumulated in myonuclei and activated cellular detoxification genes (Dialynas et al., 2015). To determine whether CncC accumulated in this cardiac model of laminopathies, hearts expressing wild-type and mutant LamC were stained with antibody to CncC (Deng & Kerppola, 2013. Hearts expressing wild-type LamC Nuclear CncC (Nrf2) is a characteristic of redox imbalance (Deng & Kerppola, 2013. To determine the redox status of the hearts, reduced (GSH) and oxidized (GSSH) glutathione were measured (Anderson, 1985;Dialynas et al., 2015). The absolute values of both did not show differences between flies expressing wild-type LamC and G489V (Fig. S4a); however, the ratio of GSH:GSSG was lower in 3-week-old adults expressing G489V compared to 3-weekold adults expressing wild-type LamC, suggesting an oxidative redox imbalance at this age (Fig. S4a). and G489V, relative to controls (Figures 4f and S4b, upper panels).

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These cytological findings were supported by quantitative analysis of total triglycerides in adults. One-day-old adults with cardiac-specific expression of wild-type LamC and G489V showed no difference in triglyceride levels (Fig. S4b, lower panel). In contrast, 2-to 5-week-old adults showed significant increases in triglyceride levels. These data demonstrate an age-dependent increase in total triglycerides. | 7 of 14 F I G U R E 5 Cardiac-specific expression of Atg1 and knockdown of CncC suppressed the abnormal cardiac, fat, and aging phenotypes caused by mutant LamC. Atg1 OE in hearts of 3-week-old adults (n = 30-70 per genotype) expressing G489V suppressed the heart period defects (a), cardiac dysrhythmia (b), and enhanced cardiac performance as represented by fractional shortening (c). In contrast, expression of Atg1 DN in hearts of 3-week-old adults expressing G489V enhanced the cardiac period and cardiac dysrhythmia and caused further deterioration of the heart (a-c). Cardiac-specific expression of a CncC RNAi under the same conditions enhanced cardiac performance and suppressed heart period and cardiac dysrhythmia. An RNAi against GFP showed no effect on these cardiac parameters (a-c). These genetic modifiers had little to no effect on the physiology of hearts expressing wild-type LamC (a-c). (d) Suppression of cardiac defects correlated with a reduction in cytoplasmic LamC aggregates. Cardiac-specific expression of Atg1 and CncC RNAi suppressed the cytoplasmic aggregates, whereas Atg1 DN and GFP RNAi did not. The relative area of LamC aggregates/total area surveyed in confocal images was plotted. (e) Representative confocal images of the hearts stained with phalloidin (green), antibody against LamC (red), and DAPI (blue) showed a reduction in LamC protein aggregates. Cardiac-specific of expression of CncC RNAi and Atg1 OE suppressed cytoplasmic LamC aggregates (arrows) and myofibrillar disorganization (*) in 3-week-old adults. Cardiac-specific expression of Atg1 DN resulted in increased LamC aggregates and further deterioration of the organization of the actin-containing myofibrils organization. Contractile circumferential fiber (CF) was missing; however, noncontractile longitudinal fibers (LF) were retained. (f) These genetic manipulations had little to no effect in hearts expressing wild-type LamC (g) Cardiac-specific Atg1 OE lowered the levels of total triglycerides. In contrast, Atg1 DN and RNAi against CncC showed no effect on triglyceride levels. Numbers correspond to the numbered genotypes in panels (a-c). (h) Representative confocal images of adipose tissue from adults with cardiac-specific expression of G489V and Atg1 OE stained with Nile Red showed a reduction in lipid droplets compared to the control expressing G489V alone. (i) Atg1 OE lengthened lifespan of adults with cardiac-specific expression of G489V. The lifespan of adults (150-250 female per genotype) was determined for adults of the indicated genotypes. Atg1 OE suppressed the G489V-induced shortened lifespan, whereas Atg1 DN and a CncC RNAi did not alter lifespan. The effect of these genetic modulators on lifespan of flies with cardiacspecific expression of wild-type LamC was used as a control. Statistical significance in A-D, G, and F is denoted as follows: *p < .05; **p < .01; ***p < .001; NS = not significant G489V, suggesting that nuclear enrichment of CncC contributes to the cardiac pathology.

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To determine the cytological changes that account for the genetic suppression of the heart defects, confocal microscopy was used to image cardiac cells. Cardiac-specific KD of CncC in hearts expressing G489V reduced cytoplasmic LamC aggregates, restored nuclear morphology, and promoted organization of actin-containing myofibrils (Figure 5d- To determine the generalizability of these findings, we performed similar genetic tests using flies that express the R205W transgene. Atg1 OE suppressed the cardiac dysfunction (Fig. S7a-c), cytoplasmic aggregates (Fig. S7d), and shortened lifespan (Fig. S7e). Collectively, these results are similar to the suppression observed for flies expressing the G489V transgene. In contrast, cardiac-specific KD of CncC did not suppress mutant phenotypes caused by R205W ( Fig. S7a-e). These data suggest that different mutant versions of To determine whether the suppression of cardiac dysfunction observed with the double treatment suppressed the cardiac cellular phenotypes, hearts were stained with antibodies to LamC. There was an absence of cytoplasmic LamC in the suppressed hearts. In addition, the nuclear morphological defects, myofibrillar disorganization, and nuclear enrichment of CncC were suppressed (Figure 6d and S9a, c). In contrast, simultaneous cardiac-specific expression of Atg1 DN and a CncC RNAi did not suppress LamC cytoplasmic aggregates, nuclear defects, and myofibrillar disorganization (Figure 6d). In addition, enrichment of CncC was enhanced (S9D). In controls expressing wild-type LamC, the double treatment caused minor myofibrillar disorganization, but had no apparent effect on LamC localization and nuclear morphology (Figure 6f). Collectively, these findings demonstrate that upregulation of autophagy and blocking the CncC/Keap1 pathway ameliorates the cardiac cellular defects caused by mutant LamC.
Simultaneous manipulation of autophagy and the CncC/Keap1 pathway was also tested for effects on adipose tissue homeostasis. BHIDE ET AL.

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The double treatment reduced total triglyceride levels in adults expressing G489V (Fig. S9e). In contrast, simultaneous expression of Atg1 DN and CncC RNAi did not alter the elevated triglyceride levels ( Fig. S9e). Furthermore, these genetic combinations did not significantly change triglyceride levels in the LamC background (Fig. S9e).
We reason that the suppressive effect of the double treatment on triglycerides is likely through increased autophagy as a similar effect was observed with Atg1 OE alone.
We next determined whether restoration of cardiac function and maintenance of adipose tissue homeostasis impacted the lifespan of G489V-expressing adults (Figure 6h). The double treatment completely restored lifespan (Figure 6h). In contrast, simultaneous F I G U R E 6 An interplay between autophagy and CncC/Keap1 suppressed cardiac dysfunction caused by mutant LamC. (a-c) The cardiac function of 3-week-old flies (n = 33-70 for each genotype) expressing wild-type and mutant LamC was compared. Hearts expressing G489V, Atg1 OE, and RNAi against CncC showed a reduction in lower heart period (a), cardiac dysrhythmia (b), and enhanced cardiac performance as represented as fractional shortening (c) compared to hearts expressing G489V alone. Expression of G489V in combination with Atg1 DN and a CncC RNAi (KD) resulted in enhancement of cardiac period and dysrhythmia and increased deterioration compared with hearts expressing G489V alone. (d) Atg1 OE and CncC RNAi suppressed the LamC cytoplasmic aggregates. Quantification of the cytoplasmic aggregates was performed by taking the relative average area of the aggregates per the total area of the confocal images surveyed. (e-f) Representative confocal images of hearts from 3-week-old adults stained with phalloidin (green), antibodies against LamC (red), and DAPI (blue) showed that simultaneous OE of Atg1 and a CncC RNAi resulted in nearly complete suppression of cytoplasmic LamC aggregation (represented by arrows) and myofibrillar disorganization (represented by *) caused by G489V. In contrast, simultaneous expression of Atg1 DN and a CncC RNAi enhanced the disorganization of the actin-containing myofibrils, with contractile circumferential fibers (CF) completely disorganized. The impact of this genetic combination resulted in subtle myofibrillar disorganization in the wild-type LamC background, but did not alter nuclear morphology and LamC nuclear envelope localization. (g) Simultaneous cardiac-specific expression of Atg1 and a CncC RNAi suppressed all of the cardiac parameters analyzed. (h) Simultaneous cardiac-specific Atg1 OE with a CncC RNAi completely suppressed the G489V-induced shortening of lifespan (150-250 adults were assayed per genotype). Expression of the Atg1 DN in combination with a CncC RNAi did not rescue the shortened lifespan caused by G489V. Statistical significance in A-D is denoted as follows: *p < .05; **p < .01; ***p < 0.001; NS = not significant expression of Atg1 DN and an RNAi against CncC caused further reduction in the lifespan compared to flies expressing G489V alone ( Figure 6h). As a control, the double treatment had little effect on the lifespan of adults with cardiac-specific expression of wild-type LamC. However, cardiac-specific expression of Atg1 DN, CncC RNAi, and wild-type LamC reduced lifespan compared to LamC alone.
Taken together, these findings demonstrated that simultaneous over-expression of Atg1 in combination with expression of CncC RNAi suppressed cytoplasmic aggregation of LamC, restored cardiac contractility, and improved lifespan.
We have generated Drosophila melanogaster models of age-dependent cardiac dysfunction. In these models, mutations synonymous with those causing disease in humans were introduced into Droso- F I G U R E 7 Model for the interactions between the autophagy and CncC/Keap1 signaling pathway in mutant lamin-induced cardiac disease. Cellular and metabolic stress is triggered by the abnormal aggregation of nuclear envelope proteins in the cytoplasm (red and yellow circles with irregular margins), leading to increased levels of Ref (2) Cardiac-specific expression of mutant LamC altered CncC subcellular localization (Fig. S3). Previously, Drosophila larval body wall muscles expressing G489V were shown to experience reductive stress, an atypical redox state characterized by high levels of reduced glutathione and NADPH, and upregulation CncC target genes (Dialynas et al., 2015). Cardiac-specific CncC RNAi in the wildtype LamC background did not produce major cardiac defects (Figures 5 and S5). Consistent with this, Nrf2 deficiency in mice does not compromise cardiac and skeletal muscle performance (Kannan et al., 2013;Rajasekaran et al., 2011). Cardiac-specific CncC RNAi suppressed G489V-induced cardiac dysfunction and reduced cytoplasmic LamC aggregation, but not R205W-induced defects (Figures 5, S5-S6, S7). However, cardiac-specific RNAi against CncC did not affect G489V-induced adipose tissue accumulation and lifespan shortening. Similar to the nuclear enrichment of CncC in hearts expressing G489V (Fig. S3), human muscle biopsy tissue from an individual with a point mutation in the LMNA gene that results in G449V (analogous to Drosophila G489V) showed nuclear enrichment of Nrf2 (Dialynas et al., 2015). Disruption of Nrf2/Keap1 signaling has also been reported for Hutchinson-Gilford progeria, an earlyonset aging disease caused by mutations in LMNA (Kubben et al., 2016). In this case, however, the thickened nuclear lamina traps Nrf2 at the nuclear envelope that results in a failure to activate Nrf2 target genes, leading to oxidative stress (Kubben et al., 2016). In our studies, we observed CncC nuclear enrichment; however, a redox imbalance was not readily observed at the three-time points investigated (Fig. S4b). This might indicate that there is a window of time in disease progression in which redox imbalance occurs and that mechanisms are in place to re-establish homeostasis.
It has been postulated that there is cross-talk between autophagy and Nrf2/Keap1 signaling (Jain et al., 2010;Stepkowski & Kruszewski, 2011). We tested for this by manipulating autophagy and CncC ( Our findings support a model whereby autophagy and Nrf2 signaling are central to cardiac health (Figure 7). We propose that cytoplasmic aggregation of LamC increases levels of Ref(2)P (p62), which competitively binds to Keap1 (Dialynas et al., 2015;Jain et al., 2010), resulting in CncC (Nrf2) translocation to the nucleus. Inside the nucleus, Nrf2 regulates genes involved in detoxification (Dialynas et al., 2015;Jain et al., 2010). Continued expression of antioxidant genes results in the disruption of redox homeostasis, defective mitochondria, and dysregulation of energy homeostasis/energy sensor such as AMPK and its downstream targets. Simultaneously, upregulation of Ref(2)P (p62) causes inhibition of autophagy via activation of TOR, which leads to the inactivation of AMPK (Mihaylova & Shaw, 2011). AMPK inactivation in combination with activation of the TOR pathway causes cellular and metabolic stress that leads to cardiomyopathy. In support of our model, transcriptomics data from muscle tissue of an individual with muscular dystrophy expressing Lamin A/C G449V (analogous to Drosophila G489V) showed (1) upregulation of transcripts from Nrf2 target genes, (2) upregulation of genes encoding subunits of the mTOR complex, and (3)