Age-dependent Lamin remodeling induces cardiac dysfunction via dysregulation of cardiac transcriptional programs

Natalie Kirkland University of California, San Diego Alexander Whitehead University of California, San Diego James Hocker University of California, San Diego Pranjali Beri University of California, San Diego Geo Vogler Sanford Burnham Prebsy Medical Discovery Institute Bill Hum Sanford Burnham Prebsy Medical Discovery Institute Mingyi Wang National Institute on Aging https://orcid.org/0000-0001-6412-369X Edward Lakatta National Institute on Aging Bing Ren University of California, San Diego Rolf Bodmer Sanford Burnham Prebys Medical Discovery Institute Adam Engler (  aengler@ucsd.edu ) University of California, San Diego https://orcid.org/0000-0003-1642-5380


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With aging comes a progressive decline in organ function 1,2 , but age-related decline in heart 33 performance is especially critical as cardiovascular disease is the leading cause of mortality 34 worldwide 3 . Aging results in the progressive loss of structural organization 4,5 , which can limit 35 contractility 1,6 and result in heart failure 7 . High prevalence of age-related cardiac dysfunction 36 may in part be because cardiomyocyte renewal is limited 8 and therefore, maintenance of cardiac 37 function over time must rely on compensatory mechanisms; these are multifaceted but tightly 38 linked to the integrity of key structural elements, e.g., intercalated discs, sarcomeres, and 39 extracellular matrix. Reducing force on cardiomyocytes (CMs) or compensating with transgenic 40 overexpression of intercalated disc proteins can partially reverse heart dysfunction, typically by 41 restoring structural organization and gene expression 6,7 . Since physical forces transduced to the 42 nucleus can impact chromatin organization and induce changes in gene expression 9-11 , nuclear 43 remodeling may similarly be a mechanism of age-associated cardiac dysfunction. via lamina associated domains (LADs) 16 . Along with the perinuclear cytoskeleton 12,13 and 49 chromatin 14,15 , the nuclear lamina regulates nuclear properties, including stiffness, size and 50 shape 17-26 . In mechanically active tissues, Lamin mutations give rise to muscular dystrophy 27,28 51 and cardiomyopathies 29 , which also manifest in premature aging syndromes, e.g., Hutchinson 52 Gilford Progeria (HGPS) 30 . Lamin mutations cause dysmorphic nuclei, epigenetic dysregulation 53 and DNA damage 31-34 . However, changes in nuclear shape, which are conserved from 54 invertebrates 35,36 to humans 37 , have been observed upon aging in the absence of Lamin 55 mutations and accompany loss of heterochromatin 37,38 and accumulation of DNA damage 37 . In 56 some cases, Progerin (truncated Lamin A) has been identified in aging skin 39 and dilated 57 cardiomyopathy 40 in the absence of mutations. Furthermore, Lamins decrease in expression 58 with age in some tissues 41-43 , with loss of Lamin B being a well-known aging marker 42 that may 59 decrease cardiomyocyte regenerative capacity and increase ploidy 44 . Lamin A and C (Lamin A/C, 60 two splice variants of the lmna gene) are the dominant adult cardiac Lamins, and age-associated 61 reduction has been observed in mouse cardiomyocytes 41 , but a role in heart function and cardiac 62 aging is unknown. Insights from Lamin A haploinsufficient mutant mice suggest Lamin reduction 63 is as detrimental to heart function as progerin mutants; mice develop dilated cardiomyopathy via 64 loss of sarcomere-nuclear coupling, show defective nuclear transport and fail to activate 65 compensatory hypertrophic pathways 45 . Thus, age-associated nuclear remodeling could be a 66 major mechanism contributing to organ dysfunction, yet mechanisms contributing to age-67 dependent nuclear remodeling and how it affects tissue function remain elusive. 68 69 To investigate a role for age-dependent nuclear remodeling in regulating heart function, we 70 primarily employ the invertebrate Drosophila melanogaster. Drosophila are rapidly aging, 71 possess a simple but highly conserved heart 46 , and importantly, demonstrate age-depended 72 cardiac decline 5,47 . We identified age-dependent remodeling unique to CM nuclei, which is 73 strongly influenced by an age-dependent reduction of Lamin C (LamC), the fly homologue to 74 mammalian Lamin A/C. Genetic reduction of LamC in young flies phenocopies age-associated 75 nuclear stiffening, decreased heart contractility and sarcomere disorganization, and ultimately 76 shortens lifespan. We show that LamC loss decreases expression of cardiomyocyte transcription 77 factors, as well as cytoskeletal regulators, likely by reducing their chromatin accessibility. 78 8 conditions ( Fig. 5B; Table S9). Mutually conserved genes presented biological process terms 203 related to aging (red, Fig. 5C; Table S9), suggesting that LamC loss creates differential gene 204 expression similar to natural aging. As validation, we also observed terms previously identified 205 from ATAC-Seq, including anatomical structure development and morphogenesis (blue, Fig. 5C), 206 in which CM transcription factors tin and H15 were downregulated (Fig. 5D). HCR validated CM 207 specificity of tin, H15, and Hand and showed that all three were reduced in both aged and LamC 208 RNAi hearts (Fig. 5E). Conversely for LamB RNAi, hearts showed only an aging phenotype and 209 no transgenic effect ( Fig. 5E and S4D do not yet establish if loss of a myogenic program is critical for adult myocyte function. 213 214

Adult-onset myogenic transcription factor loss induces heart dysfunction while LamC 215 preserves heart function 216
The importance of myogenic transcription factors is highlighted by significant sarcomere defects 217 present when any one factor is silenced throughout development (Fig. S5A). This begs the 218 question of whether CM transcription factor loss in adulthood, due to age-associated LamC loss, 219 could influence heart function. To reduce expression only in the adult fly and assess whether 220 their loss phenocopies LamC reduction, flies possessing the temperature sensitive suppressor 221 of Gal4, TubGal80 ts , and heart specific drive Hand-Gal4 were used (Fig. 6A). Within 24 hours of 222 eclosure, adult flies were maintained at the permissive (18 o C) or shifted to the non-permissive 223 temperature (29 o C), and after 2 weeks their heart function assessed. Live heart imaging showed 224 that loss of each transcription factor only in adulthood still caused a significant decrease in 225 fractional shortening compared to control backgrounds which exhibit a slight, but insignificant 226 reduction in fractional shortening due to relative differences in aging between flies maintained at 227  Conversely, we asked if adult-onset LamC overexpression could preserve myogenic 229 factor expression and function with age. When LamC expression is induced at 29 o C, we 230 observed nuclear size was consistent with 18 o C flies, in contrast to GFP overexpression controls 231 that showed an expected age-dependent reduction in nuclear size ( Fig. 6D and S5C). LamC 232 protein levels did not significantly decrease, in contrast to GFP overexpression controls ( Fig. 9 S5D), which corresponded to an increase in LamC transcript levels only at 29 o C for LamC OE 234 hearts (Fig. S5E). Importantly, with additional LamC in older flies, fractional shortening was 235 preserved ( Fig. 6E and S5F), as well as CM specific expression of myogenic transcript factors 236 tin, H15, and Hand ( Fig. 6F-G). Together, our results establish that adult loss of myogenic 237 programs is mediated by age-associated LamC loss and their chromatin remodeling, which 238 subsequently reduces adult cardiomyocyte function (Fig. 7A). 239 240

Non-human Primates 242
Despite physiological differences between tubular and chambered hearts, there is surprising 243 overlap between the Drosophila and human cardiac proteomes 46 . We therefore sought to assess 244 whether similar structural and transcriptional changes are conserved from the fly heart to the 245 mammalian heart 41 . We observed in both mouse and monkey heart sections that nuclear size 246 decreased and circularity increased upon aging, as we found in the fly heart tube ( Fig. 7B-C). 247 Furthermore, immunofluorescence staining of the mouse heart sections confirmed reduction of 248

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The role nuclear remodeling has in heart function during natural aging has thus far been largely 259 unexplored. Here, we demonstrate that CM nuclear remodeling, i.e., age-related loss of nuclear 260 lamins, is intimately linked with tissue-level dysfunction. Genetically inducing nuclear remodeling 261 leads to reduction in heart contractility, sarcomere disorganization and shortens lifespan by 262 mimicking transcriptional changes that occur in natural aging. Our findings suggest that 263 transcriptional misregulation downstream of nuclear remodeling may occur due to altered 264 chromatin accessibility and, strikingly, this represses CM fate transcription factors and 265 sarcomeric structural components. Importantly, we show that preserving "youthful" nuclear 266 properties, e.g., high Lamin expression and nuclear morphology, maintains CM transcription 267 factor expression and heart function. These changes are conserved in both mice and non-human 268 primates further demonstrating nuclear remodeling and myogenic transcriptional programs as 269 potential therapeutic targets for preserving heart function during aging. for progeria-related laminopathies 19,23,33,37 . Rather than increasing in size and dysmorphia, we 274 observe that aging CM nuclei atrophy and become rounder. We also, for the first time to our 275 knowledge, demonstrate that CM nuclei stiffen upon aging in situ, an observation only seen 276 previously in cell culture for progeria cells and only after multiple rounds of passaging 19 . Further 277 supported by our assessment of non-cardiomyocyte ventral muscle nuclei that hypertrophy with 278 age within the heart tube, our findings suggest that cardiomyocytes have specific mechanisms 279 mediating nuclear remodeling. 280 In the context of Drosophila CMs, we sought to understand how nuclear remodeling 281 occurred upon aging and identified that nuclear lamins, LamC and LamB, in addition to nesprin-282 related proteins Klar and Msp300, were downregulated upon aging. Consistent with our data, 283 Lamin B has been previously reported to be downregulated with age 43,51,56 possibly due to its 284 role in senesence 42 , while a functional role for age-associated Lamin A/C reduction has not 285 previously been explored. We found that genetically reducing LamC prematurely was sufficient 286 to induce aging-like nuclear atrophy and increased circularity, but conversely, overexpression 287 was required to change nuclear size in Xenopus and HeLa 25 . While A and B-type Lamins 288 differentially contribute to nuclear mechanics 20 , we observed that reduction of A-type LamC 289 increased CM nuclear stiffness despite cultured cells' nuclei soften with reduced Lamin A/C 290 expression 20,26 . These differences could be accounted for by several hypotheses; First, 291 Drosophila LamB and LamC could have differing functions compared to mammalian 292 counterparts, although in other cell types, there is conservation between Drosophila and human 293 Lamins 57 . Second, it is increasingly apparent that nuclei respond differently in 2D and 3D 294 environments. In 2D cell culture, nuclear wrinkling indicates membrane laxity, whereas in 3D 295 environments 58,59 , wrinkling is dependent on actin filaments intrusion into the perinuclear space 296 and wrinkling infers high membrane tension 58 . Third, cell-or developmental-specific differences 297 may result in alternative mechanics upon Lamin depletion. For  repressive H3K9me3 marks in C. elegans 68 . These conflicting instances, along with our data, 320 suggest that accessibility both globally, and locally for specific loci, could be context specific, 321 and thus our data suggests that in the context of aging, reduced accessibility could be coupled 322 to dysfunction. 323 We show that ultimately, LamC-mediated nuclear remodeling appears to be a conserved 324 process in vertebrates that reduces the expression of cardiomyocyte transcription factors, e.g., 325 Hand/HAND1/2, Tin/NKX2-5 and H15/Tbx20. We observe that Hand specifically is less 12 accessible with aging and LamC reduction. In Drosophila, the highly conserved Tin is an early 327 initiator of cardiogenesis and binds between Hand exons 3 and 4 69 , an intron we observe to have 328 reduced accessibility upon LamC reduction (Fig. 4F). Thus, reduced gene accessibility could 329 further downregulate Hand and downstream myogenic transcription. We predict reduced 330 chromatin accessibility might also account for the reduction of Tin/NKX2-5 and H15/Tbx20 with 331 age across flies, mice, and monkeys. Our findings provide a new Lamin-mediated interpretation 332 for previous observations of reduced NKX2-5 in aged, isolated mouse cardiomyocytes 70 and 333 provides them with a role beyond development. We show in Drosophila that their adult-specific 334 reduction gives rise to a marked reduction in heart function, supported by studies that find an 335 adult-specific role for TBX20 when deleted in mice 71-73 . Consistent with these observations, CM 336 transcription factors are misregulated in remodeling events leading to heart failure 74 , e.g., HAND 337 is downregulated in rodent hypertrophy 75 and in human cardiomyopathy 76 . Therefore, Lamin-338 mediated misregulation of myogenic transcriptional programs likely has a significant impact on 339 mediating heart dysfunction during aging and may precede the development of heart failure. 340 Since preserving LamC, and therefore nuclear morphology, maintained CM transcription factor 341 expression and heart function despite aging in flies, our findings suggest nuclear lamina 342 remodeling is a unique mechanism in age-related organ dysfunction. Furthermore, our work 343 presents several avenues for investigating therapeutic interventions to increase health span into 344 advanced age. 345 346

Drosophila melanogaster 348
Fly stocks were raised in non-crowded conditions on standard fly food medium consisting of 349 yeast, cornstarch and molasses (10% yeast, 12% sugar and 1.5% agar). Flies were raised at 350 25°C except for the temperature sensitive fly crosses (HandGal4, TubGal80ts; TubGal80ts, Fig.  351 6) which were raised at 18°C until eclosure, then 50% of eclosed flies were aged at 29°C and 352 50% at 18°C. Freshly eclosed flies were collected and aged such that day of collection was day 353 1. Flies were transferred to fresh food every 2-3 days. Female flies were used for subsequent 354 heart analysis to ensure consistent heart morphology. The following fly lines were used from the  imaged on a Keyence All-in-One BZ-X Series Fluorescence Microscope, with a 60X objective, 418 1x Zoom, 1µm depth resolution and 1920 x 1440 XY pixel resolution. 419 420 Macaque heart sections were received from the NIA. For staining and imaging, slides were first 421 rehydrated using the following steps: 2 x 10 minutes with Xylene, 100% ethanol, 95% ethanol 422 (in DI water), 70% ethanol and 50% ethanol before rinsing with DI water. Slides were 423 subsequently immersed in PBS with 0.5% Triton X-100 for 30 minutes and incubated with DAPI 424 (Sigma) for 30 minutes, prior to 3 x 5 minute washes with PBST and 3 x 5 minutes with PBS. 425 Slides were prepared using ProLong TM Glass Antifade Mountant (Invitrogen) and imaged as 426 described for mouse heart sections. 427 428

Fly nuclear morphology and intensity analysis 429
For two-dimensional analysis of nuclear morphology, 3D stack images were acquired of the A2-430 A3 region of the heart as described above in the (Methods: Immunofluorescence and Imaging). 431 The A2-A3 heart region possesses 3-4 cardiomyocyte pairs and therefore 6-8 total CM nuclei. 432 Using ImageJ, the CM nuclei were cropped from the larger heart image, within a 22.17 2 µm / 433

Lamin localization 542
A custom python code 81 was modified to assess the intensity of Lamin at radially increasing 543 distances from the center of the nucleus to the periphery for the max projected images also 544 generated for nuclear morphology and intensity analysis ( Figure S2F). The average mean 545 intensity measurement at periphery was then divided by the average mean intensity the center 546 to obtain the fold enrichment of Lamin at the periphery. 547 548

Sarcomere organization 549
Using ImageJ and confocal stack images of actinin stained hearts, the dorsal region of the A2-550 were generated as part of the QC step using RSeQC 88 . DEseq2 was also used to calculate fold 618 changes and p-values and perform optional covariate correction. Clustering of genes for the final 619 heatmap of differentially expressed genes was done using the PAM (Partitioning Around 620 Medoids) method using the fpc R library 89 . Hypergeometric distribution was used to analyze the 621 enrichment of pathways, gene ontology, domain structure, and other ontologies. The topGO R 622 library 90, was used to determine local similarities and dependencies between GO terms in order 623 to perform Elim pruning correction. Several database sources were referenced for enrichment 624 analysis, including Interpro 91 , NCBI 92 MSigDB 93,94 , REACTOME 95 , WikiPathways 96 . Enrichment 625 was calculated relative to a set of background genes relevant for the experiment. Panther was 626 used to assess GO terms for gene lists generated in Rosalind. 627 628

Hybridization Chain Reaction (HCR) 629
Hearts were dissected as previously described 52 to expose the heart in a 2.5mm dish. The hearts 630 were relaxed with 10mM EGTA in oxygenated hemolymph and fixed with 4% formaldehyde in 631 0.1% Tween 20, PBS for 20 minutes. Next, the hearts were washed 2 x 5 minutes with 0.1% 632 Tween 20, PBS. Then on ice, hearts were incubated, each for 5 minutes, with 25%, 50%, 75%, 633 100%, 75%, 50% and finally 25% methanol in PBS. Hearts were then permeabilized for 2 hours 634 at room temperature with 1% Triton 100-X in PBS. A second fixation was repeated at room 635 temperature and samples were washed 2 x 5 minutes with 0.1% Tween, PBS on ice. Then, a 636 50% 0.1% Tween 20, PBS; 50% 5X SSCT (20XSSC, 10% Tween 20, Ultrapure water) solution 637 was used to wash the samples for 5 minutes on ice and replaced by 5X SSCT for a further 5 638 minutes. The cuticle with the heart attached was then trimmed down to a small rectangle and 639 carefully transferred to a 96 well plate well (each containing a maximum of 7 hearts). Within the 640 well, the hearts were incubated with probe hybridization buffer (Molecular Instruments) on ice 641 for 5 minutes, then the plate was transferred to 37 o C for 30 minutes. added with the first 5X SSCT 30-minute wash or stained subsequently for 15 minutes in PBST, 652 followed by 3 x 10-minute PBST washes and 3 x PBS rinses. Samples were prepared and 653 imaged as described above (Methods section: Immunofluorescence and Imaging). 654

655
To quantify RNA expression levels, the processed hearts were imaged as described in the 656 Immunofluorescence and Imaging section and then imported into ImageJ. For Hand, Tinman 657 and H15 quantification, the A2-A3 heart region confocal stack was converted to a max projection, 658 duplicated and then binarized. Using the max projected image as a guide, the cytoplasmic 659 pockets surrounding the CM nuclei were then then traced, the ROI copied to the binary imaged 660 for particle analysis. As the segmentation was imperfect for transcripts very close together and 661 to account for differences in pocket size, the % area covered by the transcripts was used to 662 assess statistical significance in Prism (Graphpad).
As Lamin C and B are expressed in cells other than the CM nuclei, i.e., the ventral muscle nuclei 665 and the cuticle, the narrowest stacks were taken around the nuclear-cytoplasmic pocket to 666 eliminate interfering non-CM transcripts, and then the same analysis was conducted as for H15, 667 Tinman and H15. The macro is as follows: and wildtype vs LamC iR attp2 samples. Samples were read into R Studio using dba(), count 717 densities per peak were calculated using dba.count(), filtering out peaks with <1 read per sample 718 and a summit width of 100 (as recommended by the Diffbind3 vignette). Differential accessibility 719 was calculated using the EdgeR wrapper of dba.analyze(). BED files were generated for each 720 comparison using dba.report() and annotated using HOMER annotatePeaks.pl. Regions were 721 filtered based on a log2 fold change of 0.32 and FDR of ≤ 0.1. Common features between 722 comparisons were isolated using dplyr's inner_join function of the "Nearest.Refseq" column 723 output of HOMER. Plots were generated using ggplot2 and ggrepel packages. Panther was used 724 to assess GO terms for gene lists. 725

Quantitative PCR for Monkey and Mouse left ventricle 727
Total RNA was isolated from mouse and monkey frozen left ventricle sections by first, grinding 728 frozen tissue in a pestle and mortar with liquid nitrogen to ensure samples did not degrade. 729 Ground tissue was transferred to an Eppendorf and resuspended in 600ul of RLT lysis buffer 730 from the RNeasy mini RNA extraction kit (Qiagen). The suspension was then transferred to a 731 QIAshredder column and centrifuged at <10,000 rcf for 5 minutes for further homogenization. 732 The supernatant was collected and total RNA was extracted using the RNAeasy mini RNA 733 extraction kit (Qiagen) as per the protocol. RNA quality was assessed using an Agilent Tape 734 station system. Poly(A) + RNA was reverse transcribed using oligo(dT) reagent of the 735 SuperScript IV First-Strand Synthesis kit (Thermofisher) and cDNA library generated using 736 manufacturers protocol with a final RNase step. RT-qPCR was then performed in triplicate for 737 each sample using SYBR Green PCR Master mix (Thermofisher) and the CFX96 hardware 738 (Biorad). Each gene of interest was normalized to three housekeeping genes 77,78 using the 739 delta CT equation 2 -(AvgCqGOI -AvgCqHK) . Primer sequences are shown in Table S10 and were obtained. For SOHA live heart imaging, >13 hearts were imaged and analyzed. For actinin 751 organization, >14 hearts were analyzed. For lifespan assays, more than 100 flies were recorded. 752 The following statistical significance cut off was applied: n.s. p>0.05, * p<0.05, **p<0.01, 753 ***p<0.01, ****p<0.0001. No tests were conducted to measure statistical power or normality of 754