Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan

Abstract

Aging is associated with extensive remodeling of the heart, including basement membrane (BM) components that surround cardiomyocytes. Remodeling is thought to impair cardiac mechanotransduction, but the contribution of specific BM components to age-related lateral communication between cardiomyocytes is unclear. Using a genetically tractable, rapidly aging model with sufficient cardiac genetic homology and morphology, e.g. Drosophila melanogaster, we observed differential regulation of BM collagens between laboratory strains, correlating with changes in muscle physiology leading to cardiac dysfunction. Therefore, we sought to understand the extent to which BM proteins modulate contractile function during aging. Cardiac-restricted knockdown of ECM genes Pericardin, Laminin A, and Viking in Drosophila prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of Laminin A expression correlated with an overall preservation of contractile velocity with age and extension of organismal lifespan. Global heterozygous knockdown confirmed these data, which provides new evidence of a direct link between BM homeostasis, contractility, and maintenance of lifespan.

Keywords: Aging; Basement membrane; Drosophila; Extracellular matrix; Laminin A.

Figure 1

Decreased Fractional Shortening in w¹¹¹⁸ Correlates with Decreased Longevity and Increased BM Thickness. (A) Fractional shortening was calculated for each genotype and age (mean ± s.e.m., n=20) and is plotted at 1 and 5 weeks. * p<0.05 (B) Survival curves for indicated wildtypes show median survival indicated by the dashed black line for each genotype, 55 and 35 days for yw (n=215) and w¹¹¹⁸ (n=223), respectively. ***p<0.001. (C) Brightfield image of AFM cantilever (open triangular shape) positioned over midline (center) of the Drosophila heart tube during nanoindentation analysis of Drosophila tri-layered myocardium. Scale bar is 100 µm. (D) Schematic of 3 layered AFM nanoindentation (black circle) into Drosophila heart tube depicting: (i) the 1^st layer indentation of the VM, (ii) the complete compaction of the VM layer and 2^nd layer indentation of the BM, and (iii) the final compaction of the first 2 layers and 3^rd layer indentation of the CM layer. (E) A representative force vs. indentation depth plot in black showing the respective Linearized-Hertz fits for both the shallow (green) VM and deep (red) CM with a BM layer indicated by a linear region shown in blue starting and ending within the brackets. (F) BM stiffness and BM thickness reported as the length over which the BM linear region was fit were measured by AFM at the ventral midline and plotted at indicated ages. All data represented as mean ± s.e.m of >15 flies.*p<0.05 and **p<0.01 for indicated comparisons.

Figure 2

Cardiac Basement Membrane Protein Regulation Differences in wildtype Drosophila. (A) Scatter plot of the MS1 intensity values for each protein. MS1 intensity values (protein abundance) are plotted for w¹¹¹⁸ value on the x-axis and yw value on the y-axis. Colors of points indicate biological function as determined by Software Tool for Rapid Annotation of Proteins (STRAP) analysis and relative percentages in each quadrant are given (ECM - green, Cytoskeleton - blue). Red arrow indicates Pericardin [Prc]. Data represents a mean value of 4 biological replicates of 50 pooled fly hearts for protein analysis. (B) Absolute distance of MS1 intensity values from a zero log₂ fold change or x=y were plotted for each protein in decreasing fashion. 25 proteins with highest calculated distance values are shown in subpanel (grey dashed box) color coded by STRAP analysis (ECM - green, Cytoskeleton - blue) with ECM proteins of interest listed on the x-axis and Laminin A (the cardiac specific laminin subunit within trimer) highlighted with green arrow and Pericardin in red arrow. (C) Representative immunofluorescent images for the indicated genotypes at 1 week for proteins Laminin A (green), Viking (purple), Pericardin (red), and F-actin (white). All fluorescent intensities were normalized to F-actin within region of interest (ROI) selected over bulk of heart tube (top left, white dashed box). Scale bar is 50 µm. (D) Mean pixel intensity for each indicated protein as measured within ROI, normalized to F-actin intensity within ROI, and shown as w¹¹¹⁸ normalized to yw. Mean Factin intensity varied <10% sample to sample between each genotype or with age. n=4 hearts per comparison.

Figure 3

High Speed Image Analysis Reveals Dilated Heart Tube in ECM KD Flies and Prevention of Age-Related Tube Restriction (A) Still ventral images of fly heart from 120 fps video capture used to create motion-mode kymographs. Lines depict where measurements for dimensions (diastole, red, and systole, green) are taken in segment a2. (B) Diastolic heart diameters were quantified at 1 and 5 weeks for indicated genotypes. (C) Systolic heart diameters were quantified at 1 and 5 weeks for indicated genotypes. (D) Fractional Shortening were quantified for indicated genotypes and ages. All data represented as mean ± s.e.m. n>20. All data were analyzed via 2-way-ANOVA with post hoc Bonferroni test. Non-parametric student’s two-tailed t-test was used to assess significance within each genotype with age. *p< 0.05, **p< 0.01, ***p<0.001.

Figure 4

Knockdown of ECM genes Decreases BM Thickness and Decreases LanAKD Cardiac Stiffness. (A) BM thickness reported at midline by AFM for indicated genotypes at 1 week. All KD flies were not statistically different from each other, but ***p<0.001 for comparisons between KD flies and their control. (B) BM stiffness reported in kiloPascals by AFM for indicated genotypes at 1 week. All data reported as mean ± s.e.m of >15 flies. All data analyzed with non-parametric Student’s two-tailed t-test with unequal variances assumption.

Figure 5

ECM KD Leads to Preserved Cardiac Mechanics with Age. (A) To the left, motion-mode images of a heart sequentially loaded by increasing relative viscosity, υ, by increasing Ficoll w/v% in the hemolymph in which the fly hearts are bathed. Arrows indicate where systole and diastole are located in the motion-mode images. To the right, m-mode image expanded to show contraction length (CL) in blue, shortening interval (SI) in green, and lengthening interval (LI) in red used for velocity measurements below. (B) Heart wall shortening (contraction) and lengthening (relaxation) velocities were assessed under viscous load and fit by Hill’s model for each genotype and age. (C) Fractional shortening were measured under viscous load for each indicated genotype and age. Data represented as mean ±s.e.m. n>20. All viscous load data were analyzed with 2-way-ANOVA with post hoc Bonferroni test. Significance indicates load/genotype as source of variance. *p<0.05, **p<0.01, ***p<0.001 (D) Survival curves for indicated genotypes show median survival indicated by the dashed black line for each genotype. ***p<0.0001