Gene expression and ultra-structural evidence for metabolic derangement in the primary mitral regurgitation heart

Abstract

Aims: Chronic neurohormonal activation and haemodynamic load cause derangement in the utilization of the myocardial substrate. In this study, we test the hypothesis that the primary mitral regurgitation (PMR) heart shows an altered metabolic gene profile and cardiac ultra-structure consistent with decreased fatty acid and glucose metabolism despite a left ventricular ejection fraction (LVEF) > 60%.

Methods and results: Metabolic gene expression in right atrial (RA), left atrial (LA), and left ventricular (LV) biopsies from donor hearts (n = 10) and from patients with moderate-to-severe PMR (n = 11) at surgery showed decreased mRNA glucose transporter type 4 (GLUT4), GLUT1, and insulin receptor substrate 2 and increased mRNA hexokinase 2, O-linked N-acetylglucosamine transferase, and O-linked N-acetylglucosaminyl transferase, rate-limiting steps in the hexosamine biosynthetic pathway. Pericardial fluid levels of neuropeptide Y were four-fold higher than simultaneous plasma, indicative of increased sympathetic drive. Quantitative transmission electron microscopy showed glycogen accumulation, glycophagy, increased lipid droplets (LDs), and mitochondrial cristae lysis. These findings are associated with increased mRNA for glycogen synthase kinase 3β, decreased carnitine palmitoyl transferase 2, and fatty acid synthase in PMR vs. normals. Cardiac magnetic resonance and positron emission tomography for 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) uptake showed decreased LV [¹⁸F]FDG uptake and increased plasma haemoglobin A1C, free fatty acids, and mitochondrial damage-associated molecular patterns in a separate cohort of patients with stable moderate PMR with an LVEF > 60% (n = 8) vs. normal controls (n = 8).

Conclusion: The PMR heart has a global ultra-structural and metabolic gene expression pattern of decreased glucose uptake along with increased glycogen and LDs. Further studies must determine whether this presentation is an adaptation or maladaptation in the PMR heart in the clinical evaluation of PMR.

Keywords: Lipid and glucose metabolism; Mitochondria dysfunction; Primary mitral regurgitation; [18F]FDG-PET/CMR.

Graphical Abstract

Chronic β-adrenergic drive causes: (i) insulin receptor inhibition, decreased glucose transporter 4, and glucose (red) uptake; (ii) increased free fatty acids and lipid droplets; (iii) increased hexosamine biosynthetic pathway/O-linked N-acetylglucosaminyl and glycogen accumulation; and (iv) mitochondrial damage, increased mitochondrial damage-associated molecular patterns and inflammation, and cardiac dysfunction.

Figure 1

Lipid metabolism. (A and B) Lipid droplets in primary mitral regurgitation cardiac tissues. (A) Representative transmission electron microscopy image of the left ventricle, right atrium, and left atrium from human cardiac tissue biopsies. The arrows show lipid droplets. (B) Total lipid droplets number in one field (magnification, ×5000) were assessed double blindly. Data were inferred from 10 to 15 fields for each patient (n = 6–10). (D–N) Gene expression of lipid metabolism from the hearts of patients with normal and primary mitral regurgitation. Data normalized to 18S and expressed as a fold change from normal (n= 8–11 subjects/group). Data presented as mean ± standard error of the mean. P-values indicated on graph. Multiple comparison adjustment was performed when comparing more than three groups. CPT, carnitine palmitoyltransferase 1 and 2; FASN, fatty acid synthase; LCAD, long-chain acyl-CoA.

Figure 2

Plasma markers of adrenergic drive and lipid derangement in patients with primary mitral regurgitation. (A) Neuropeptide Y, (B) free fatty acids, (C) apolipoprotein A1, and (D and E) mitochondrial damage-associated molecular patterns from the ND1 and ND6 regions, respectively, were measured in both plasma and pericardial fluid of patients with primary mitral regurgitation (n = 11). P-values indicated on the graph.

Figure 3

Mitochondrial damage in patients with primary mitral regurgitation. (A and B) Quantitative transmission electron microscopy analysis of mitochondrial ultra-structure in primary mitral regurgitation hearts biopsies. (A) Representative transmission electron microscopy image of the left ventricle, left atrium, and right atrium (zoom areas are boxed), and the arrows show disrupted mitochondrial cristae. (B) Cristae integrity: normal cristae (=1) and complete disorganized cristae (=3). Cristae integrity assessment was performed double blindly using the scoring level. Data were inferred from 10 fields for each heart region from 6 to 10 patients with primary mitral regurgitation. (C and D) Plasma mitochondrial DNA damage-associated molecular pattern levels of patients with normal and primary mitral regurgitation from the ND1 and ND6 regions, respectively. (E) Glucose-6-phosphate dehydrogenase–dependent NADH production/min in the plasma of patients with normal and severe primary mitral regurgitation plasma (n = 8–16). Data are presented as mean ± standard error.

Figure 4

Glucose metabolism in patients with primary mitral regurgitation. (A) Representative transmission electron microscopy analysis of glycogen accumulation in primary mitral regurgitation heart biopsies (left ventricle, left atrial, and right atrial) (zoom areas are boxed, arrows show glycogen). Data were inferred from 10 fields for each heart region from 6 to 10 patients with primary mitral regurgitation. (B) Glycogen accumulation presented as % area of total field (magnification, ×5000). Data are presented as mean ± standard error. (C–T) Gene expression of glucose metabolism from the hearts of patients with normal and primary mitral regurgitation. Data were normalized to 18S and expressed as a fold change from normal hearts. The sample size for each measurement was n = 8–11 subjects/group. Data are presented as mean ± standard error.

Figure 5

Glucose metabolism in patients with primary mitral regurgitation. (A–I) Gene expression of glucose metabolism from the hearts of patients with normal and primary mitral regurgitation hearts. Data were normalized to 18S and expressed as a fold change from normal hearts. The sample size for each measurement was n = 8–11.

Figure 6

Inflammation in the left ventricle midwall in primary mitral regurgitation. (A and B) T2 heat map in the mid left ventricle of patients with normal and primary mitral regurgitation.

Figure 7

Myocardial glucose uptake. (A) Representative 2-deoxy-2-[¹⁸F]fluoro-D-glucose-positron emission tomography/MR myocardial uptake in patients with normal and primary mitral regurgitation, (focal point with high intensity= high uptake) and (low intensity = low uptake). (B–G) Seventeen segment analysis Max and mean myocardial standard uptake value (Max SUV and mean SUV) in patients with normal and primary mitral regurgitation, normalized by liver (B and C), blood (D and E), and uncorrected (F and G).