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Age-dependent Lamin changes induce cardiac dysfunction via dysregulation of cardiac transcriptional programs

Nature Aging volume 3, pages 17–33 (2023)Cite this article

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Abstract

As we age, structural changes contribute to progressive decline in organ function, which in the heart act through poorly characterized mechanisms. Taking advantage of the short lifespan and conserved cardiac proteome of the fruit fly, we found that cardiomyocytes exhibit progressive loss of Lamin C (mammalian Lamin A/C homolog) with age, coincident with decreasing nuclear size and increasing nuclear stiffness. Premature genetic reduction of Lamin C phenocopies aging’s effects on the nucleus and subsequently decreases heart contractility and sarcomere organization. Notably, Lamin C reduction downregulates myogenic transcription factors and cytoskeletal regulators, possibly via reduced chromatin accessibility. Subsequently, we find a role for cardiac transcription factors in regulating adult heart contractility and show that maintenance of Lamin C and cardiac transcription factor expression, prevents age-dependent cardiac decline. Our findings are conserved in aged nonhuman primates and mice, demonstrating that age-dependent nuclear remodeling is a major mechanism contributing to cardiac dysfunction.

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Fig. 1: Age-associated changes in cardiac nuclear morphology and mechanics.
Fig. 2: Natural aging downregulates nuclear envelope proteins.
Fig. 3: LamC, but not LamB, impacts cardiomyocyte aging, heart function and lifespan.
Fig. 4: Chromatin accessibility decreases with age and LamC RNAi at sites of myogenic control.
Fig. 5: LamC loss transcriptionally regulates myogenic transcription factors.
Fig. 6: Myogenic transcription factors maintain heart contractility in aged flies.
Fig. 7: Nuclear remodeling induces adult-onset transcription factor loss, a process conserved in mice and nonhuman primates.

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Data availability

RNA-seq and ATAC-seq data are deposited at Gene Omnibus Express accession codes GSE185967 and GSE185923, respectively. Source data files are provided and all supporting data from this study are available from the corresponding author upon reasonable request.

Code availability

Software to image fly hearts, analyze their contraction and create kymographs (SOHA) is available at http://sohasoftware.com/index.html. Python code to assess lamin distribution is available at http://englea52.github.io/Englerlab/. Any ImageJ macros have been included in the supplementary software file.

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Acknowledgements

The authors thank K. Jepsen of the Institute for Genomic Medicine (UCSD) for technical assistance with sequencing; E. Molina (Sanford Consortium Stem Cell Core) for access and maintenance of the LSM780 microscope; K. Christman, R. Middleton, J. Tuler and K. Birmingham (UCSD) for use and dissection of hearts from the aged mouse model; N. Varki and S. Choudhury of the Mouse Phenotyping Core (UCSD) for assistance with histological preparations; and UCSD undergraduate students S. Skalak, M. Naguib, I. Madan, J. Yun and S. Sharma for assistance with fly husbandry and immunostaining experiments. We thank the Bloomington Drosophila Stock Center (P40OD018537), R. Bodmer, K. Ocorr, J. Grosshans and L. Walrath for providing fly strains. The authors acknowledge funding and equipment support from the NIH (R01AG045428 to A.J.E. and S10OD026929 to UCSD IGM Genomics Center) as well as intramural support from the NIA (to E.G.L. and M.W.). Fellowship support was provided by the American Heart Association (20POST35180048 to N.J.K.), the National Science Foundation (to A.J.W. and P.B.), the ARCS Foundation (to A.J.W.) and the NIH (T32GM008666 to J.D.H.). Work at the Center for Epigenomics was supported in part by the UCSD School of Medicine.

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Authors and Affiliations

  1. Department of Bioengineering, University California San Diego, La Jolla, CA, USA

    Natalie J. Kirkland, Scott H. Skalak, Alexander J. Whitehead, Pranjali Beri & Adam J. Engler

  2. Sanford Consortium for Regenerative Medicine, La Jolla, CA, USA

    Natalie J. Kirkland, Scott H. Skalak, Alexander J. Whitehead, Pranjali Beri & Adam J. Engler

  3. Cell and Molecular Medicine, University California San Diego, La Jolla, CA, USA

    James D. Hocker & Bing Ren

  4. Biomedical Sciences Program, University California San Diego, La Jolla, CA, USA

    James D. Hocker, Bing Ren & Adam J. Engler

  5. Development, Aging and Regeneration Program, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA

    Geo Vogler, Bill Hum & Rolf Bodmer

  6. Laboratory of Cardiovascular Science, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA

    Mingyi Wang & Edward G. Lakatta

  7. Ludwig Institute for Cancer Research, La Jolla, CA, USA

    Bing Ren

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Contributions

N.J.K., R.B. and A.J.E. conceived the project and designed experiments. N.J.K. and S.H.S. performed experiments. B.H. and G.V. developed the HCR protocol for adult fly hearts. N.J.K. analyzed experimental data. P.B. developed the Python Lamin distribution analysis pipeline. N.J.K., A.J.W. and J.D.H. performed ATAC-seq and analyzed sequencing data. B.R. oversaw ATAC-seq assay design and provided technical expertise. G.V. oversaw and provided technical expertise for fly imaging and HCR experiments. M.W. performed macaque husbandry and isolated macaque tissue. E.G.L. managed the macaque colony. The manuscript was written by N.J.K., R.B. and A.J.E. with input from the other authors.

Corresponding author

Correspondence to Adam J. Engler.

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Nature Aging thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Nuclear Dynamics in Cardiac and Skeletal Muscle Cells.

(A) Kaplan–Meier survival curve for yw (gray) and w1118 (black) flies. n = 103 and flies, respectively, were used in the plot. p < 10−3 based on Log-Rank (Mantel-Cox) test between the two strains. (B) Plots of 2D projected area (left) and circularity data (right) for yw flies. n = 129, 108, and 143 for yw flies at 1-, 3-, and 5 weeks, respectively. (C) Cardiomyocyte nuclear area (left) and aspect ratio (right) plotted for w1118 and yw flies as a function of adult age. n = 96, 116, and 141 nuclei for for w1118 flies and n = 129, 108, and 143 for yw flies at 1-, 3-, and 5 weeks of adulthood, respectively. (D) Ventral muscle nuclear area (left), perimeter (center), and aspect ratio (right) from w1118 flies at 1-, 3-, and 5 weeks of adulthood. n = 528, 604, 661 ventral muscle nuclei from w1118 flies at 1-, 3-, and 5 weeks of adulthood. (E) Representative images of the 3D wireframe mesh of cardiomyocyte nuclei from w1118 flies at 1- (top), 3- (middle), and 5 weeks (bottom) of adulthood. Scale bar is 5 μm. *p < 0.05, **p < 10−2, ***p < 10−3, and ****p < 10-4 by one-way ANOVA with Tukey multiple comparisons test. Error bars in all graphs refer to mean + /- SD.

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Extended Data Fig. 2 Natural Aging Downregulates LamC and LamB but does not affect their localization.

(A) MA plot of all genes from 1- and 5-week adult w1118 fly hearts showing log2 fold change (FC) and mean normalized expression counts. Data is shown in black for genes −1.25 < FC < 1.25 (dashed lines) or p-adj > 0.05. Open circles represent genes that do not map to a nuclear ontological term. Green and purple data represent DEGs that are up- or downregulated in 5-week adult flies, respectively. (B) Cellular component and molecular function ontological terms for genes associated with the nuclear envelope GO term, organized by their elimination pruning p-value. (C) Representative images of 1 and 5 week w1118 fly heart nuclei stained for DNA, and LamC (green) and LamB (purple) mRNA transcripts. Scale bar, 5 μm. (D) Left plot shows area percentage occupied by mRNA transcripts per cardiomyocyte in 1- and 5-week-old adult w1118 fly hearts. Right plot normalizes data to mean area at 1 week for each transcript. n = 49, 36, 49, and 36 nuclei from w1118 flies at 1 and 5 weeks for LamC and LamB, respectively. (E) Corrected total nuclear fluorescence (CTNF) of 1, 3, and 5 week yw flies for LamC (top) and LamB (bottom). n = 94, 106, and 133 nuclei for LamC and n = 173, 115, and 90 nuclei for LamB for 1, 3, and 5 week adults, respectively. (F) Image showing a representative nucleus with multiple lines radiating out from its centroid (left) to create line plots that are averaged into a single radial profile of the fluorescent intensity (right). Lower panel, the ratio of edge to center intensity is plotted. n = 72, 63, 73, and 33 nuclei from w1118 flies at 1 and 5 weeks for LamC and LamB, respectively. n = 51, 87, 77, and 42 nuclei from yw flies at 1 and 5 weeks for LamC and LamB, respectively. *p < 0.05, **p < 10−2, ***p < 10−3, and ****p < 10-4 by one-way ANOVA with Tukey multiple comparisons test. Bars in (D) refer to min to max, with median and 25th and 75th interquartile range and error bars in (E-F) refer to mean + /- SD.

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Extended Data Fig. 3 Validation and Morphological and Functional Characterization of LamB and LamC RNAi lines.

Corrected total nuclear fluorescence (CTNF) for cardiomyocytes from (A) LamC RNAi and (B) LamB RNAi fly lines and their respective controls, that is, attp2 and attp40, n = 47, 36, 79, and 69 nuclei from attp2 and LamC RNAi flies at 1 and 4 weeks (left to right). n = 53, 29, 65, and 61 nuclei from attp40 and LamB RNAi flies at 1 and 4 weeks (left to right). (C) Plots quantifying nuclear perimeter (left, n = 111, 96, 46, and 89 nuclei/condition) and aspect ratio (right, n = 113, 97, 46, and 92 nuclei/condition) for LamB, LamC RNAi and their genetic controls. (D) Plots quantifying nuclear area, perimeter, aspect ratio, and circularity (left to right) for LamB and LamC RNAi lines and their genetic control background at 4 weeks. For all plots, n = 136, 101, 86, and 95 nuclei/condition left to right. (E-F) Plots of diastolic and systolic diameters, and shortening velocity determined from SOHA imaging for attp2 control and LamC RNAi flies (E), and attp40 control and LamB RNAi flies (F) at 1 and 4 weeks. Each LamC RNAi plots, n = 21, 23, 25, and 27 nuclei/condition, left to right. Each LamB RNAi plots, n = 31, 21, 26, and 25 nuclei/condition, left to right. For panels A-D, *p < 0.05, **p < 10−2, ***p < 10−3, and ****p < 10-4 from a two-sided unpaired t-test at each time point and RNAi line. For E-F, *p < 0.05, **p < 10−2, ***p < 10−3, and ****p < 10-4 by one-way ANOVA with Tukey multiple comparisons test. Error bars in (A-D) refer to mean + /- SD, and bars in (E, F) refer to min to max, with median and 25th and 75th interquartile range.

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Extended Data Fig. 4 Validation of transgenic fly background, effects of LamB on myogenic transcription factor expression, and LamC heterozygous flies.

(A) Volcano plot and (B) heat map of bulk RNA-seq from surgically dissected attp2 heart tubes. Fold change represents 5-week attp2 fly hearts normalized to 1-week-old hearts and p-adj was computed from quintuplicate. 1,998 differentially expressed genes (DEGs) were assessed from cutoffs of −0.32 > log2(FC) > 0.32 (or −1.25 > FC > 1.25) (dashed lines) from comparisons of 15 fly hearts per replicate; DEGs increasing and decreasing with age are shown in green and purple, respectively. Heat map columns were hierarchically clustered using Euclidean distance and linkage shown by the dendrogram. Heat maps are normalized within each gene/row. (C) 635 of 688 genes were co-regulated DEGs (92.3%) in the w1118 and attp2 control fly hearts, and plotted based on their fold change with age. DEGs were annotated based on their ontological categorization as nuclear (orange), extracellular matrix (ECM; green), or cytoskeletal (blue). A subset of DEGs either did not fit those categories (black/white) or lacked a known ontology (gray). Only 7.7% of all DEGs were dysregulated. (D) Representative images of attp40 control and LamB RNAi flies at 1 and 4 weeks showing H15, Hand and Tin transcription factor mRNA and DAPI. Scale bar is 5 μm. (E) Quantification of the per cell percentage area for each transcript in attp40 control and LamB RNAi flies at 1 and 4 weeks. For H15, n = 93, 87, 85, and 71 cells, left to right. For Hand, n = 111, 96, 105, and 76 cells, left to right. For Tin, n = 105, 118, 102, and 76 cells, left to right. (F) Quantification of cardiomyocyte nuclear area, circularity and LamC corrected total nuclear fluorescence for 1-week-old female control yw/w1118 and heterozygous LamC excision mutants yw/w1118;LamCex187/+ and yw/w1118;LamC296/+ fly hearts and their representative images in (G) showing staining for LamC (green) and DNA (Magenta), scale bare = 5 µm. For (F), n = 29, 26, 25 (CM nuclei, left to right) (H) Quantification of heart parameters: fractional shortening, diastolic diameter and systolic diameter for background control and heterozygous LamC excision mutants. n = 17, 16, 16 (heart tubes, left to right) (I) Representative images of Hand, H15 and Tin mRNA in heterozygous LamC excision mutants, presented with quantification of per cell percentage area for each transcript in (J). n = 52, 39, 46 (CM nuclei, left to right). *p < 0.05, **p < 10−2, ***p < 10−3, and ****p < 10-4 by one-way ANOVA with Tukey multiple comparisons test. Box plots in (E, H, J) refer to min to max, with median and 25th and 75th interquartile range and error bars in (F) refer to mean + /- SD.

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Extended Data Fig. 5 Effects of Adult Myogenic Transcription Factor Loss on of Heart Tube Morphology and Function.

(A) Representative images of A2-A3 heart region visualized by phalloidin (F-Actin) with the indicated transgenes expressed under the control of the Hand-Gal4 promoter. Scale bar is 10 μm. At least three heart tubes were imaged for each condition. (B) Diastolic and (C) systolic diameters of heart tubes from control fly lines (attp40 and attp2) and their corresponding transgenic flies expressing the indicated RNAi. n = 10, 12, 18, 15, 19 and 14 (heart tubes, left to right). (D) CM nuclear area in attp2 control and LamC RNAi hearts subject to regime described in Fig. 6a. n = 29 and 22 (CM Nuclei, left to right). (E-G) Quantification of the per cell, percentage area for Hand, H15 and Tin transcripts upon KD of (E) LamC, (F) Hand (red),Tin (pink) and respective control attp2 (grey), and (G) H15 (blue) and attp40 control (grey), induced as in Fig. 6a. n = 32 and 26 (CM Nuclei/genotype) in (E), n = 20, 23, 28 (CM Nuclei/genotype, left to right) in (F) and n = 43 and 42 (CM Nuclei/genotype) in (G). (H) Schematic of temperature-sensitive transgenic expression where 29 °C enables transgenic expression due to the denaturation of Gal4 transcription factor suppressor, Gal80ts. (I) Fractional shortening of surgically exposed heart tubes at 18 °C and 29 °C for controls (black), and KD of tin (pink), Hand (brown), and H15 (blue) with corresponding diastolic and systolic diameters in (J-M). n = 13, 15, 18, 15, 26, 27, 16, 19, 29, 27, 29, and 21 (heart tubes/transgene/ temperature; left to right as shown in (I)). *p < 0.05, **p < 10−2, ***p < 10−3, and ****p < 10-4 by independent t-test and one-way ANOVA with Kruskal–Wallis test and Dunn’s comparisons test. Box plots show to min to max, with median and 25th and 75th interquartile range and error bars in (D) refer to mean + /- SD.

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Extended Data Fig. 6 Effects of LamC and Myogenic Transcription Factor Overexpression.

For panels A-D and as outlined in Fig. 6a, flies were reared at 18 °C and shifted to 25 °C upon eclosure. Nuclear area (A) and CTNF (B) for hearts overexpressing LacZ (control) and LamC from 1 to 7 weeks. n = 31, 41, 23, 30, 26, and 31 cells (A, left to right) and n = 31, 31, 24, 29, 30, and 40 cells (B, left to right). (C) Diastolic and (D) systolic diameters of LacZ and Hand OE flies at 1, 4, and 7 weeks of adulthood. (E) Representative images (left) for nuclei from flies at 18 °C and 29 °C expressing GFPNLS transgene or LamC OE from HandGal,tubGal80ts;tubGal80ts based upon regime described in S5H. Scale bar is 5 μm. Plots of (F) projected nuclear area, (G) circularity, and (H) and corrected total nuclear fluorescence (CTNF) of LamC protein as a function of temperature and transgene expression. n = 39, 40, 46, and 41 nuclei (left to right). (I) For GFPNLS and LamC OE at 18 °C and 29 °C, representative images of LamC mRNA transcripts. Scale bar is 5 μm. (J) Quantification of LamC transcript area per CM in GFPNLS and LamC OE hearts, at 18 °C and 29 °C. n = 24, 36, 20 and 40 CMs, left to right. Diastolic (K) and systolic (L) diameters, and (M) fractional shortening for GFPNLS and LamC OE at 18 °C and 29 °C. n = 32, 19, 30 and 26 heart tubes, left to right. (N). A plot for each transcription factor is shown and quantifies the per cell, percentage area for each transcript. For all transcripts, n = 50, 56, 49 and 35 cells, left to right. (O) Representative images for tin (pink), Hand (brown), and H15 (blue) transcripts in GFPNLS and LamC OE at 18 °C and 29 °C. Scale bar is 5 μm. *p < 0.05, **p < 10−2, ***p < 10−3, and ****p < 10-4 by two-way ANOVA with Sidaks multiple comparisons test (A-D) and one-way ANOVA with Kruskal-Wallis test and Dunn’s comparisons test (F-N). Error bars in (A, B, F-H) refer to mean + /- SD and all box plots display min to max, with median and 25th and 75th interquartile range.

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Extended Data Fig. 7 Myogenic transcription factor expression in the left ventricles of aged nonhuman primates and mice.

(A) Expression of three housekeeping genes for mice are plotted as a function of age with a linear and associated p-value shown. Data is plotted for raw Cq values. (B) Expression of four transcription factors in mice is shown, normalized to each housekeeper gene. Data normalized to Eef1e1 (black), Rpl4 (medium gray), and ACTB (light gray) are plotted using the left and right y-axes, depending on the axis label colors. P-values for each fit are shown in the upper right corner. (C) Expression of three housekeeping genes for rhesus macaques are plotted as a function of age with a linear and associated p-value shown. Data is plotted for raw Cq values. (D) Expression of four transcription factors in rhesus macaques is shown, normalized to each housekeeper gene. Data normalized to Rpl13a (black) and TUBB2 (light gray) use the left y-axis whereas Rpl32 uses the right y-axis (medium gray). P-values for each fit are shown in the upper right corner.

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Supplementary information

Reporting Summary

Supplementary Table 1

Age-associated transcriptome changes of w1118 flies. Table showing changes in the transcriptome of w1118 flies. Data listed are annotated for common gene name followed by (left to right): gene description, fold change for the comparison of 5-week/1 week adults, log2FC value, P value, Padj, average log2 expression, raw fragments per kilobase of transcript per million mapped reads (FPKM), gene ID and any gene aliases, if available.

Supplementary Table 2

Gene Ontologies of age-associated transcriptome changes of w1118 flies. Table showing GO associated with transcriptome changes in w1118 flies. Data listed are annotated for (left to right): GO identifier, common name, P value, FDR-adjusted P value, Elim pruning P value, number of genes in term, number of genes that are also in this filter or cluster, number of upregulated genes, number of downregulated genes and the negative log10 Elim pruning P value.

Supplementary Table 3

Differentially accessible chromatin regions for age, LamC RNAi and LamB RNAi. This table annotates ATAC-sequencing data for age comparisons in the w1118 background, in the LamB RNAi versus attp40 flies, and in the LamC RNAi versus attp2 flies. In each tab, data annotates (left to right): peak number, chromosome, peak start location, peak end location, peak annotation, detailed peak annotation, distance to transcription start site (TSS), nearest promoter ID, Entrez ID of nearest gene, Unigene ID for nearest gene, nearest Refseq, gene name, gene alias, gene description, gene type, gene concentration, conc_group1, conc_group2, fold change, P value and FDR.

Supplementary Table 4

Analysis of DARs common to aging versus LamC RNAi and aging versus LamB RNAi. This table annotates co-regulated DAR from ATAC-sequencing data for LamB RNAi versus attp40 flies and for LamC RNAi versus attp2 flies. In tabs one (Age_and_LamC_EdgeR) and three (Age_and_LamB_EdgeR), each comparison is denoted with ‘x’ and ‘y’ corresponding to RNAi versus control and 5wk versus 1wk aging flies, respectively, and annotated for (left to right): peak number, chromosome, peak start location, peak end location, peak annotation, detailed peak annotation, distance to TSS, nearest promoter ID, Entrez ID of nearest gene, Unigene ID for nearest gene, nearest Refseq, gene name, gene alias, gene description, gene type, gene concentration, conc_group1, conc_group2, fold change, P value and FDR. In tabs two (age and LamC genes only) and four (age and LamB genes only), data are annotated just for the fold change for each gene associated with the DAR.

Supplementary Table 5

Gene Ontologies for co-downregulated DAR in aging and LamC iR. This table annotates the GO for biological process (BP) and cellular component (CC) for co-downregulated genes based on the closest assigned gene for the peak and associated DAR. Data are annotated (left to right) for: GO complete name, D. melanogaster peak, number of genes in dataset, expected gene number, gene over/under, fold enrichment, raw P value, FDR and −log10 (FDR P value).

Supplementary Table 6

LamC RNAi-mediated transcriptome changes in attp2 fly background. Table showing changes in the transcriptome of LamC RNAi flies at 1 week of adulthood versus their control attp2 background flies. Data plotted are annotated for common gene name followed by (left to right): gene description, fold change for the comparison of LamC/attp2, log2FC value, P value, Padj, average log2 expression, raw FPKM, gene ID and any gene aliases, if available.

Supplementary Table 7

Age-associated transcriptome changes of attp2 flies. Table showing changes in the transcriptome of attp2 flies. Data plotted are annotated for common gene name followed by (left to right): gene description, fold change for the comparison of 5-week-old/1-week-old adults, log2FC, P value, Padj, average log2 expression, raw FPKM, gene ID and any gene aliases, if available.

Supplementary Table 8

Co-regulated transcriptome changes in attp2 versuss w1118 fly background. Table showing DEGs as a function of age between the attp2 and w1118 fly genotypes. Only genes differentially expressed with age in both systems are shown. Data are annotated for common gene symbol followed by (left to right): gene ID, fly base alias, gene description and then in the attp2 fly first and w1118 second, we show the log2FC value with age, P value for the age fold change and Padj value for the age fold change.

Supplementary Table 9

Co-regulated transcriptome changes in LamC RNAi versus attp2 and biological process ontology terms. Mutually significant DEGs from LamC RNAi and aged fly hearts are listed in this table. In the first tab listing co-regulated genes, data are annotated (left to right) by: symbol, gene ID, gene alias, gene description, base mean gene expression with age, log2FC with age, Padj for age fold change, base mean gene expression for LamC RNAi versus attp2, log2FC for LamC RNAi versus attp2, Padj for LamC RNAi versus attp2 fold change, protein type, additional protein description, protein classification and the type of cellular component GO terms associated with the gene, for example, cytoskeleton, nucleus, extracellular matrix, other or unknown. In the second tab, PANTHER biological process GO terms are annotated for co-regulated terms.

Supplementary Table 10

Primer sequences. All forward and reverse primer sequences for mice and rhesus macaques are shown here.

Supplementary Software file 1.

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Kirkland, N.J., Skalak, S.H., Whitehead, A.J. et al. Age-dependent Lamin changes induce cardiac dysfunction via dysregulation of cardiac transcriptional programs. Nat Aging 3, 17–33 (2023). https://doi.org/10.1038/s43587-022-00323-8

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