Research ArticleCARDIOVASCULAR AGING

Vinculin network–mediated cytoskeletal remodeling regulates contractile function in the aging heart

See allHide authors and affiliations

Science Translational Medicine  17 Jun 2015:
Vol. 7, Issue 292, pp. 292ra99
DOI: 10.1126/scitranslmed.aaa5843
  • Fig. 1. Comparison of cardiac proteomes of ventricles from adult and aged simians and rats to identify cardiac aging biomarkers.

    (A) Experimental design to analyze LV free wall (dashed white box) from intact hearts from aged and adult simians and rats. (B) Scatter plot of the ratio of aged to adult spectral counts. Each point represents the ratio for a single protein, and colors indicate cell component category (left) or biological function (right) into which each protein falls, as determined by STRAP analysis. (C) IPA for sarcomeric and cytoskeletal proteins yielded network interactions. Protein expression determined by spectral counting for rats is denoted by filled symbols, while simian protein expression is an outlined symbol. Color coding of expression is shown below with a legend for shapes and interactions. Vinculin (VCL) and vinculin-specific interactions are highlighted by bold, blue lines within the network. (D) Network of cardiomyopathy-associated proteins that are up-regulated with age in monkeys. Annotations obtained using IPA and OMIM are available in table S9. Note that uncolored nodes represent protein groups or complexes, not individual or redundant molecules.

  • Fig. 2. Cytoskeletal remodeling in adult and aged rats with ventricular hypertrophy but preserved fractional shortening.

    (A) Western blot for vinculin from LV free wall and quantification of normalized blot intensity [vinculin/GAPDH (glyceraldehyde-3-phosphate dehydrogenase)] for each age cohort. Shown are three technical replicates of six pooled samples at the indicated ages. P value determined by unpaired, nonparametric t test. (B) LV echocardiograms displaying diastolic (Dia) and systolic (Sys) chamber widths for the indicated ages. (C) LV end diastolic and systolic diameters (LVIDd and LVIDs, respectively) and fractional shortening were measured from kymographs in (B) for the indicated ages. (D) Staining for vinculin, Cnx43, actin, and nuclei [DAPI (4′,6-diamidino-2-phenylindole)] in the rat ventricular myocardium. Dashed boxes indicate the magnified areas below. Filled arrowheads indicate IDs; open arrowheads indicate transverse junctions. Scale bars, 10 μm. (E) Vinculin expression with age at both IDs (Cnx43+ pixels) and lateral junctions (Cnx43 pixels) as quantified using a method outlined in fig. S4. In (C) and (E), *P < 0.05, **P < 0.01, or otherwise indicated, by Wilcoxon rank sum test.

  • Fig. 3. Age- and genotype-associated structural remodeling in the Drosophila heart.

    (A) Ventral cartoon and confocal microscopy view of the Drosophila head (Hd), thorax (Thrx), and abdomen (Abd). Abdomen contains the heart (Hrt; red), whose major contractile compartment is the conical chamber (CC). Image modified from (30). Filled arrowheads indicate the cell-cell junctions or IDs along the middle axis of the heart. Illustration of heart during diastole versus systole. Arrowheads indicate the region of the heart measured in (B). Inset illustration shows an orthogonal view through the heart indicating the bilateral myocytes with components of interest highlighted. (B) Images of hearts captured from a 120-fps movie used for physiological assessments. Lines indicate where diastolic and systolic dimensions and phases were measured unless otherwise noted. (C) Heart diameters for the indicated Drosophila genotypes and ages (yw, yellow-white; w1118, white) normalized to the genotype-specific 1-week diameter. Data are normalized averages ± SEM (n > 20). *P < 0.05, ***P < 0.001, unpaired, nonparametric t tests.

  • Fig. 4. Age-associated heart stiffening correlates with cortical actin cytoskeletal remodeling in flies.

    (A) AFM cantilever positioned above the Drosophila heart. Insets are illustrations of a cantilever over the heart (top) and spherical indenter (bottom). (B) βPS1 localizes within hearts at IDs (filled arrowheads) and lateral junctions (open arrowheads) along the length of the fly heart. AFM indentation locations are indicated by red dots and their distance from ventral midline (0, 15, or 30 μm). (C) Cardiac stiffness as a function of distance from ventral midline in yw and w1118 flies (average ± SEM, n > 20). *P < 0.05, **P < 0.01 for 5-week versus 1-week at respective distance, using nonparametric t tests. Green overlay indicates ventral midline data. (D) Change in cardiac stiffness for 5-week yw after indicated pharmacological treatment. Data are individual flies with mean change in stiffness from EGTA ± SD (n = 10 per treatment). *P < 0.05, **P < 0.01, versus mean stiffness in EGTA using a repeated-measures one-way analysis of variance (ANOVA). (E) Ratio of aged (5-week) to adult (1-week) gene expression in the heart (n = 3 biological replicates of 10 pooled hearts per age and genotype). (F) Immunohistochemistry of hearts from 1- and 5-week flies showing vinculin or β1 integrin expression. Plots indicate fluorescence intensity from a line drawn within the box in each image for vinculin (green) or β1 integrin (red). Filled arrowheads indicate ID; open arrowheads, costameres.

  • Fig. 5. Cytoskeletal remodeling correlates with the preservation of genotype-specific basal cardiomyocyte shortening during aging.

    (A) Motion-mode (M-mode) kymographs of fly hearts indicating where time of shortening phase (TS, green), lengthening phase (TL, red), and heart width change (ΔL) are measured. Purple lines indicate the half ΔL or movement of one side of the heart wall. (B) M-mode images of a heart sequentially placed under hemodynamic or viscous loading with relative viscosities (ν) indicated. (C and D) Shortening (C) and lengthening (D) velocities were calculated from the difference in heart width between systole and diastole and time interval (shortening = 2ΔL/TS; lengthening = 2ΔL/TL). Data are average velocities ± SEM for the indicated genotypes and ages as a function of hemolymph viscosity (n > 29 for each age, genotype, and load). Shortening velocity is fit with Hill’s equation for muscle, whereas lengthening velocity is fit with a general log function for illustrative purposes. *P < 0.05 and ***P < 0.001 using two-way ANOVA, where asterisks indicate P values for difference in velocity as a function of viscosity and age, respectively (that is, viscosity/age). N.S., not significant.

  • Fig. 6. Cardiac-specific cytoskeletal reinforcement results in increased cardiac contractile function and extends Drosophila life span.

    (A) Illustration of crosses used to generate control and transgenic lines. (B) Immunohistochemistry of βPS1 and vinculin in 1-week-old genotypes. Plots indicate fluorescence intensity from line drawn within box in each image for vinculin (green) or βPS1 (red). Filled arrowheads indicate ID; open arrowheads, costameres. (C) Cardiac stiffness as a function of distance from the ventral midline. Data are averages ± SEM (n > 20). (D) Change in cardiac stiffness from EGTA after indicated pharmacologic treatment. Data are means ± SD (n = 10). Ventral midline indicated by green overlay. (E) At the indicated position, genotypes in (A) were assessed for heart diameters and fractional shortening. Data are individual flies with mean indicated. In (C) to (E), *P < 0.05, **P < 0.01, compared to control by one-way ANOVA. (F) Heart wall velocities and fractional shortening assessed under viscous load and fit by Hill’s model (shortening velocity) or a general log fit (lengthening velocity, fractional shortening, for illustration purposes). Data are means ± SEM (n > 29). *P < 0.05, **P < 0.01, ***P < 0.001, N.S. (not significant) comparing indicated genotype and control (black), two-way ANOVA. (G) Survival curves for indicated genotypes (n > 100). Gray lines indicate time in days to which 50% of starting population survived. **P < 0.01, ***P < 10−6 for population over time versus control, two-way ANOVA. P = 0.025 for MhcKD + VincHE versus MhcKD (two-way ANOVA).

  • Fig. 7. A proposed role for cytoskeletal regulation of myofilament order and contracility in the aging myocyte.

    (A) Representative transmission electron microscopy (TEM) micrographs of myofibril cross sections of 1-week control (black) and VincHE (green) flies. (B) Interfilament spacing analysis as outlined in fig. S14. Inset image illustrates how distances were measured. Data are histograms indicating distribution of interfilament spacing with Gaussian fits, n = 480 (control) and n = 670 (VincHE) thick filaments. (C) Top: Adult cardiac myocytes (fly, rodent, monkey, and human) have defined cell-cell and cell-matrix junctions, referred to as IDs and costameres, respectively. Costameres couple Z discs to the membrane and cross-link the cortical actin cytoskeleton. The terminal sarcomere ends at the ID, rather than a Z disc, which is populated by actin-based adherens junctions as well as desmosomal and gap junctions. Middle: Vinculin binds to integrin and cadherin complexes and can facilitate remodeling of the actin superstructure by nucleating and bundling F-actin via its tail domain. Bottom: Vinculin is overexpressed with age. We propose that this results in reinforcement of the cortical cytoskeleton via cell junctions, which in turn may have propagating mechanical effects through the myofilament lattice that supports or enhances its crystalline order. Thus, the cortical cytoskeleton can act as an extrasarcomeric regulator of myocyte structure and contractile function. Simplified schematic shown. Proteins indicated by gene name.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/292/292ra99/DC1

    Materials and Methods

    Fig. S1. STRAP analysis of gene ontology and biological function for simian and murine proteomes.

    Fig. S2. Partial interaction map of upstream vinculin regulators.

    Fig. S3. Biometric comparison of adult and aged rats.

    Fig. S4. Analysis of vinculin localization in rat and fly myocytes.

    Fig. S5. Vinculin structure and homology across monkey, rat, and fly.

    Fig. S6. Indentation of isolated adult and aged rat cardiomyocytes and intact fly hearts.

    Fig. S7. Effects of heart tube preparation on its function.

    Fig. S8. Characterization of diastolic diameter, cardiac stiffness, and vinculin expression in the wCS Drosophila genotype.

    Fig. S9. Fitting shortening velocities with Hill’s muscle model.

    Fig. S10. Generation of the UAS-Mhc RNAi;UAS-Vinc line.

    Fig. S11. Quantification of genetic perturbations in Drosophila hearts.

    Fig. S12. Change in VincHE stiffness with cytoskeletal perturbation.

    Fig. S13. Transgenic fly heart rate, period, and rate variance.

    Fig. S14. Analysis of interfilament spacing in TEM images from the Drosophila heart.

    Table S1. Peptides detected by mass spectroscopy for adult and aged rhesus monkey left ventricles.

    Table S2. Peptides detected by mass spectroscopy for adult and aged rat left ventricles.

    Table S3. Proteomic analysis for adult and aged rhesus monkey left ventricles.

    Table S4. Proteomic analysis for adult and aged rat left ventricles.

    Table S5. STRAP annotation of the cellular compartments of proteins detected in both rat and monkey proteomes.

    Table S6. STRAP annotation of biological functions for rat and monkey.

    Table S7. IPA of bio-function expression for rat.

    Table S8. IPA of tox-function expression for rat.

    Table S9. IPA and OMIM annotation of age–up-regulated proteins associated with cardiac function.

    Table S10. IPA of upstream regulators of age-related proteins identified in rat and simian.

    Table S11. Expression of candidate actin-binding molecules in Drosophila hearts using qPCR.

    Table S12. Fitting shortening velocities with Hill’s muscle model.

    Table S13. qPCR primers.

    Reference (47)

  • Supplementary Material for:

    Vinculin network–mediated cytoskeletal remodeling regulates contractile function in the aging heart

    Gaurav Kaushik, Alice Spenlehauer, Ayla O. Sessions, Adriana S. Trujillo, Alexander Fuhrmann, Zongming Fu, Vidya Venkatraman, Danielle Pohl, Jeremy Tuler, Mingyi Wang, Edward G. Lakatta, Karen Ocorr, Rolf Bodmer, Sanford I. Bernstein, Jennifer E. Van Eyk, Anthony Cammarato,* Adam J. Engler*

    *Corresponding author. E-mail: acammar3{at}jhmi.edu (A.C.); aengler{at}ucsd.edu (A.J.E.)

    Published 17 June 2015, Sci. Transl. Med. 7, 292ra99 (2015)
    DOI: 10.1126/scitranslmed.aaa5843

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. STRAP analysis of gene ontology and biological function for simian and murine proteomes.
    • Fig. S2. Partial interaction map of upstream vinculin regulators.
    • Fig. S3. Biometric comparison of adult and aged rats.
    • Fig. S4. Analysis of vinculin localization in rat and fly myocytes.
    • Fig. S5. Vinculin structure and homology across monkey, rat, and fly.
    • Fig. S6. Indentation of isolated adult and aged rat cardiomyocytes and intact fly hearts.
    • Fig. S7. Effects of heart tube preparation on its function.
    • Fig. S8. Characterization of diastolic diameter, cardiac stiffness, and vinculin expression in the wCS Drosophila genotype.
    • Fig. S9. Fitting shortening velocities with Hill’s muscle model.
    • Fig. S10. Generation of the UAS-Mhc RNAi;UAS-Vinc line.
    • Fig. S11. Quantification of genetic perturbations in Drosophila hearts.
    • Fig. S12. Change in VincHE stiffness with cytoskeletal perturbation.
    • Fig. S13. Transgenic fly heart rate, period, and rate variance.
    • Fig. S14. Analysis of interfilament spacing in TEM images from the Drosophila heart.
    • Legends for tables S1 to S13
    • Reference (47)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Peptides detected by mass spectroscopy for adult and aged rhesus monkey left ventricles.
    • Table S2 (Microsoft Excel format). Peptides detected by mass spectroscopy for adult and aged rat left ventricles.
    • Table S3 (Microsoft Excel format). Proteomic analysis for adult and aged rhesus monkey left ventricles.
    • Table S4 (Microsoft Excel format). Proteomic analysis for adult and aged rat left ventricles.
    • Table S5 (Microsoft Excel format). STRAP annotation of the cellular compartments of proteins detected in both rat and monkey proteomes.
    • Table S6 (Microsoft Excel format). STRAP annotation of biological functions for rat and monkey.
    • Table S7 (Microsoft Excel format). IPA of bio-function expression for rat.
    • Table S8 (Microsoft Excel format). IPA of tox-function expression for rat.
    • Table S9 (Microsoft Excel format). IPA and OMIM annotation of age–upregulated proteins associated with cardiac function.
    • Table S10 (Microsoft Excel format). IPA of upstream regulators of age-related proteins identified in rat and simian.
    • Table S11 (Microsoft Excel format). Expression of candidate actin-binding molecules in Drosophila hearts using qPCR.
    • Table S12 (Microsoft Excel format). Fitting shortening velocities with Hill’s muscle model.
    • Table S13 (Microsoft Excel format). qPCR primers.

    [Download Tables S1 to S13]

Stay Connected to Science Translational Medicine

Navigate This Article