Metabolic dysregulation in the heart in obesity-associated HFpEF

Abstract

Background: Obesity and hypertension are among the most prevalent comorbidities in heart failure with preserved ejection fraction (HFpEF). In addition to its relationship with hypertension in HFpEF, obesity is also strongly associated with insulin resistance (IR) and type 2 diabetes (T2D). However, the exact cardiac effects underlying this relationship are unknown. We sought to differentiate the cardiac phenotype associated with increased adiposity in the presence or absence of IR in obese HFpEF. We utilized adipose tissue-specific MitoNEET transgenic mice, which develop chronic, metabolically healthy adipose tissue expansion (obese non-insulin resistant, OB-NIR), and compared them with their wild-type, insulin-resistant littermates (OB-IR).

Methods: OB-NIR MitoNEET and OB-IR wildtype mice were fed a high-fat diet for 16 weeks, at which time HFpEF was induced via uninephrectomy, d-aldosterone infusion, and 1.0% sodium chloride drinking water for 4 additional weeks while maintained on the same diet.

Results: OB-NIR HFpEF mice exhibited reduced cardiac fibrosis without changes in hypertrophy. This reduction was accompanied by increased cardiac expression of SIRT3. Upregulation of several downstream mitochondrial targets of SIRT3 was also observed. These included mitochondrial fission protein 1 (Fis1), a critical regulator of mitochondrial dynamics, and the antioxidant enzyme heme oxygenase-1 (Hmox1). In contrast, levels of hydroxy-3-methylglutaryl coenzyme A (CoA) synthase 2 (HMGCS2) were decreased, while both 3-hydroxybutyrate dehydrogenase 1 (Bdh1) and succinyl-CoA:3-ketoacid CoA transferase (Oxct1) were elevated. Furthermore, genes involved in the electron transport chain, such as ubiquinol-cytochrome C reductase hinge protein (Uqcrh, Complex III) and mitochondrially encoded cytochrome c oxidase I (Mt-Co1, Complex IV), were upregulated.

Discussion: Distinct alterations in cardiac mitochondrial function were observed depending on the presence or absence of IR in obese HFpEF mice. These findings suggest that SIRT3 may play a central role in mediating mitochondrial adaptations in the heart and could represent a promising therapeutic target in HFpEF.

Keywords: HFPEF; SIRT3; insulin resistance; mitochondria metabolism; obesity.

Figure 1

Left ventricular cardiomyocyte size and cardiac fibrosis in obese HFpEF mice with and without insulin resistance (A) cardiomyocyte size and (B) representative hematoxylin-eosin staining images and magnification (right panel, 20x). (C) Quantification of cardiac fibrosis using Picrosirius Red staining and (D) representative microscopic images and magnification (right panel, 20x; R.O.I. indicates region of interest). Data are represented as mean ± SEM. Statistical analysis by unpaired t-test for normally distributed data or Mann–Whitney test for non-normally distributed. OB-IR, obese insulin-resistant HFpEF mice; OB-NIR, obese non-insulin-resistant HFpEF mice. N = 5-8 mice/group.

Figure 2

Cardiac remodeling phenotype in obese HFpEF mice with and without insulin resistance. Gene expression of Nppa, Nppb, Col1a, Col3a, N2b and N2ba relative to OB-IR. Data are represented as mean ± SEM. Statistical analysis by unpaired t-test for normally distributed data or Mann–Whitney test for non-normally distributed. Col1a, collagen 1a; Col3a, collagen 3a; Nppa, natriuretic peptide type A, aka atrial natriuretic peptide; Nppb, natriuretic peptide type b, i.e., brain natriuretic peptide; N2b, titin transcript variant N2b; N2ba, titin transcript variant N2ba. OB-IR. OB-IR, obese insulin-resistant HFpEF mice; OB-NIR, obese non-insulin-resistant HFpEF mice. N = 7-9/group.

Figure 3

Left ventricular SIRT3 protein expression and total lysine acetylation in obese HFpEF mice with and without insulin resistance. (A) SIRT3 protein expression and (B) Total lysine acetylation in the left ventricle of obese insulin-resistant HFpEF mice (OB-IR) and obese non-insulin-resistant HFpEF mice (OB-NIR). Data are represented as mean ± SEM. Statistical analysis by unpaired t-test for normally distributed data or Mann–Whitney test for non-normally distributed. SIRT3, sirtuin 3; Total ACK, total lysine acetylation relative to 25KDa Ponceau Red band. N = 8-9 mice/group.

Figure 4

Schematic of SIRT3 mitochondrial targets. Aco1, aconitase 1; Bdh1, 3-hydroxybutyrate dehydrogenase 1; Cat, catalase; ETC, electron transport chain; Fis1, fission protein 1; Gpx1, glutathione peroxidase 1; HMGCS2, hydroxy-3-methylglutary coenzyme A (CoA) synthase 2; Hmox1, heme oxygenase-1; Mfn, mitofusin; Mnsod2, manganese superoxide dismutase 2; Mt-Co1, mitochondrially encoded cytochrome c oxidase I; Ndufa: NADH:ubiquinone oxidoreductase subunit A; Nrf2, nuclear factor erythroid 2-related factor 2; Opa1, optic atrophy 1; Oxct1, succinyl-CoA:3-ketoacid CoA transferase; OXPHOS, oxidative phosphorylation; Ppargc1a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Sdha, succinate dehydrogenase complex flavoprotein subunit A; Slc16a1, solute carrier family 16 member 1; TCA, tricarboxylic acid; Uqcrh, ubiquinol-cytochrome c reductase hinge protein. Created using Servier Medical Art, licensed under CC BY 4.0.

Figure 5

Cardiac gene expression of mitochondrial biogenesis and dynamics regulators in obese HFpEF mice with and without insulin resistance. Data are represented as mean ± SEM. Statistical analysis by unpaired t-test for normally distributed data or Mann–Whitney test for non-normally distributed. Fis1, fission protein; Mfn, mitofusin; OB-IR, obese insulin-resistant HFpEF mice; OB-NIR, obese non-insulin-resistant HFpEF mice; Opa1, optic atrophy 1; Ppargc1a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha. N = 7-9 mice/group.

Figure 6

Cardiac gene expression of mitochondrial redox balance regulators and MnSOD2 acetylation levels at lysine⁶⁸ in obese HFpEF mice with and without insulin resistance. (A) mRNA expression of Cat, Gpx1, Hmox1 and NFr2 in the left ventricle of obese insulin-resistant HFpEF mice (OB-IR) and obese non-insulin-resistant HFpEF mice (OB-NIR). (B) Cardiac MnSOD2 acetylation at lysine 68 (AcK68) relative to total MnSOD2 protein expression in OB-IR and OB-NIR HFpEF mice. Data are represented as mean ± SEM. Statistical analysis by unpaired t-test for normally distributed data or Mann–Whitney test for non-normally distributed. Cat, catalase; Gpx1, glutathione peroxidase 1; Hmox1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2; MNSOD2, manganese superoxide dismutase 2. N = 7-9 mice/group.

Figure 7

HMGCS2 protein and mitochondrial metabolism mRNA expression from the LV of obese HFpEF mice with and without insulin resistance. (A) Cardiac protein expression of HMGCS2 and (B) mRNA relative expression of ketone body metabolism, tricarboxylic acid (TCA) cycle, and electron transport chain related genes in obese insulin-resistant HFpEF mice (OB-IR) and obese non-insulin-resistant HFpEF mice (OB-NIR). Data are represented as mean ± SEM. Statistical analysis by unpaired t-test for normally distributed data or Mann–Whitney test for non-normally distributed. *P < 0.05, **P < 0.01 vs. OB-IR. Aco1, aconitase 1; Bdh1, 3-hydroxybutyrate dehydrogenase 1; HMGCS2, hydroxy-3-methylglutary coenzyme A (CoA) synthase 2; Mt-Co1, mitochondrially encoded cytochrome c oxidase I; Ndufa, NADH:ubiquinone oxidoreductase subunit A; Oxct1, succinyl-CoA:3-ketoacid CoA transferase; Sdha, succinate dehydrogenase complex flavoprotein subunit A; Slc16a1, solute carrier family 16 member 1; Uqcrh, ubiquinol-cytochrome c reductase hinge protein. N = 7-9 mice/group.