Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts

Abstract

During human heart failure, the balance of cardiac energy use switches from predominantly fatty acids (FAs) to glucose. We hypothesized that this substrate shift was the result of mitochondrial degeneration; therefore, we examined mitochondrial oxidation and ultrastructure in the failing human heart by using respirometry, transmission electron microscopy, and gene expression studies of demographically matched donor and failing human heart left ventricular (LV) tissues. Surprisingly, respiratory capacities for failing LV isolated mitochondria (n = 9) were not significantly diminished compared with donor LV isolated mitochondria (n = 7) for glycolysis (pyruvate + malate)- or FA (palmitoylcarnitine)-derived substrates, and mitochondrial densities, assessed via citrate synthase activity, were consistent between groups. Transmission electron microscopy images also showed no ultrastructural remodeling for failing vs. donor mitochondria; however, the fraction of lipid droplets (LDs) in direct contact with a mitochondrion was reduced, and the average distance between an LD and its nearest neighboring mitochondrion was increased. Analysis of FA processing gene expression between donor and failing LVs revealed 0.64-fold reduced transcript levels for the mitochondrial-LD tether, perilipin 5, in the failing myocardium (P = 0.003). Thus, reduced FA use in heart failure may result from improper delivery, potentially via decreased perilipin 5 expression and mitochondrial-LD tethering, and not from intrinsic mitochondrial dysfunction.-Holzem, K. M., Vinnakota, K. C., Ravikumar, V. K., Madden, E. J., Ewald, G. A., Dikranian, K., Beard, D. A., Efimov, I. R. Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts.

Keywords: electron microscopy; energy substrate; lipid droplet; oxidative respiration; perilipin 5.

Figure 1.

A) Representative image of a donor human heart, with dashed box indicating the mitochondrial isolation region. B) Mitochondrial respiration measurement protocol and example O₂ concentration and flux curves. C) Representative PM and PC O₂ concentration and flux curves for donor and failing hearts. A, aorta; PA, pulmonary artery; RA, right atrium; RV, right ventricle.

Figure 2.

A–C) Average S2 (A), S3 (B), and S4 (C) respirations with PM and PC substrates for donor hearts (n = 7) and failing hearts (n = 9). D, E) S3/4 (D) and S3/2 (E) respiratory control ratios (RCRs) for donor hearts (n = 7) and failing hearts (n = 9). There were no statistically significant differences between donor and failing hearts for respiration rates or RCRs with either substrate. F) CS-specific activities/g muscle tissue were not different between donor hearts (n = 7) and failing hearts (n = 9). Data are expressed as means ± sem.

Figure 3.

A–C) Illustrated schematic for TEM sample preservation. A) A portion of the LV surrounding a coronary artery was dissected and cannulated. Tissue was then perfused with fixative for optimal preservation. A small core of tissue was then excised (B) and stored in fixative (C). D–I) Representative electron micrographs from donor (D–F) and failing (G–I) heart muscle. D, G) Overview images; red and blue boxes outline nonoverlapping regions for donor hearts (E, F) and for failing hearts (H, I). Original magnification, ×3000 (D, G) and ×8000 (E, F, H, I) Scale bar, 1 μm.

Figure 4.

A, B) Representative zoomed-in images of individual mitochondria from 4 separate donor (A) donor and failing (B) hearts. C–F). Frequency distribution LOWESS curves for each individual heart expressed by count (C, D) and by percentage of total (E, F) for mitochondrial area (C, E) and pixel intensity (D, F). G, H) Pooled mitochondrial area (G) and pixel intensity (H), with individual heart data points, show no differences between donor hearts (n = 5) and failing hearts (n = 6). Data are expressed as means ± sem. Scale bar, 500 nm.

Figure 5.

A, B) Representative electron micrographs showing an LD in contact with mitochondria in donor heart (A) and an isolated LD in failing heart (B). Asterisks indicate LDs; arrowheads indicate LD-mitochondrial coupling. C, D) Percent of LDs in direct mitochondrial contact (C) and euclidean distance between LD centroid and nearest mitochondrial centroid (D) for donor hearts (n = 5) and failing hearts (n = 6). E, F) Expression levels for FA processing genes (E) and NPPA and MYH6 control genes (F) in donor LV (n = 26) vs. failing LV (n = 27). Data are expressed as means ± sem. *P < 0.05; **P < 0.01. Scale bar, 500 nm.