Preserved cardiac function by vinculin enhances glucose oxidation and extends health- and life-span

Abstract

Despite limited regenerative capacity as we age, cardiomyocytes maintain their function in part through compensatory mechanisms, e.g., Vinculin reinforcement of intercalated discs in aged organisms. This mechanism, which is conserved from flies to non-human primates, creates a more crystalline sarcomere lattice that extends lifespan, but systemic connections between the cardiac sarcomere structure and lifespan extension are not apparent. Using the rapidly aging fly system, we found that cardiac-specific Vinculin-overexpression [Vinculin heart-enhanced (VincHE)] increases heart contractility, maximal cardiac mitochondrial respiration, and organismal fitness with age. Systemic metabolism also dramatically changed with age and VincHE; steady state sugar concentrations, as well as aerobic glucose metabolism, increase in VincHE and suggest enhanced energy substrate utilization with increased cardiac performance. When cardiac stress was induced with the complex I inhibitor rotenone, VincHE hearts sustain contractions unlike controls. This work establishes a new link between the cardiac cytoskeleton and systemic glucose utilization and protects mitochondrial function from external stress.

FIG. 1.

Increased climbing ability and significant increases in lifespan are observed in VincHE flies. (a) Rapid Iterative Negative Geotaxis (RING) assay described in detail in the methods was performed weekly over the entire lifespan of each genotype, and the climbing distance over an interval of 5 s was recorded for each fly. n = 250 at the start of the experiment. Data represent mean ± SEM. All negative geotaxis data were analyzed with 2-way-ANOVA with post hoc Bonferroni test. *** p < 0.001. (b) Survival curves for VincHE and controls in the RING assay show median survival indicated by the dashed black line for each genotype. ***p < 0.0001.

FIG. 2.

VincHE hearts exhibit sustained oxygen consumption due to contraction and increased maximal respiration under cardiovascular stress with age. (a) Oxygen consumption rates (OCR) were calculated as the difference between the endogenous (basal) and non-mitochondrial respiration (after addition of rotenone and antimycin A) at the given ages. 12 < n < 20 for all samples in biological triplicates. (b) Stimulated OCR of VincHE and control hearts were measured after the addition of 1 mM epinephrine and corrected for background rates determined with the addition of rotenone and antimycin A. Data represented as mean ± SEM. 12 < n < 20 for all samples in biological triplicates. All respiration data were analyzed with a two-tailed Student's t-test and Mann-Whitney post-test where *p < 0.05. Fly ages are indicated on the x-axis.

FIG. 3.

Whole fly GC-MS metabolic profiling reveals metabolic changes in energy metabolism in VincHE with age. (a) Principal component analysis of biological replicates scaling expression values is plotted for given genotypes (green for VincHE and black for control) and ages (squares for 1 week and triangles for 5 weeks). (b) Heat-map visualization of changes in metabolites identified in whole fly GC-MS analysis normalized to maximal intensity detected across samples and scaled on an expression scale of 1–12 for each genotype and age specified. Metabolites were sorted by ontology and color coded to the left with colors and associated ontology specified at the bottom. Dashed black box highlights sugar ontology. (c) Sucrose, (d) glucose, and (e) melezitose abundance plotted for each genotype at 1 or 5 weeks of adulthood. n = 4 with each five flies per condition. Data were analyzed, and metabolites identified using MetaboliteDetectore software. Statistical significance calculated by 2-way ANOVA with post-hoc Dunn's test. * p < 0.05.

FIG. 4.

VincHE flies exhibit increased ability to metabolize glucose via incorporation into the TCA cycle. Mole percent enrichment (MPE) of [U-¹³C₆]glucose shown for pyruvate, lactate, TCA cycle intermediates, and related amino acids relative to control conditions at 1 week of adulthood. Flies were fasted for 12 h before offering labeled glucose. The metabolic map depicts incorporation of [¹³C]glucose-derived carbons into downstream metabolites. Open circles depict ¹²C, closed circles ¹³C atoms (blue first turn, grey second turn of the TCA cycle). Data represented as mean ± SEM. n = 4 with five flies per condition. Statistical significance calculated by non-parametric Students t-test with Mann Whitney post-test. * p < 0.05.

FIG. 5.

VincHE hearts exhibit increased resistance to mitochondrial stress when subjected to rotenone treatment compared to controls and maintain physiological heart wall velocities. (a) Percent of hearts contracting with fractional shortening >5% were plotted vs time for VincHE and control hearts exposed to mitochondrial stressor 1 μM rotenone. n = 25 for 1 week old adult hearts. Data were analyzed by Log-rank Mantel Cox test. *** p < 0.0001. (b) Heart wall shortening (contraction) velocities were assessed for VincHE hearts in hemolymph or treated with either 0.05% DMSO or 1 μM rotenone in 0.05% DMSO for 30 min (green) and untreated controls (black). All data were analyzed with a one-way ANOVA and Tukey post-test. n > 15. * p < 0.05, *** p < 0.001.