Introduction: Heart failure (HF) remains a leading cause of death and indication for heart transplant in children with single ventricle congenital heart disease (SV). The purpose of this study was to evaluate mitochondrial function in circulating peripheral blood mononuclear cells (PBMCs) from failing pediatric SV patients compared to age-matched nonfailing controls (NF). Prior data obtained at the time of cardiac transplant shows associations between SV PBMC mitochondrial function and ex-vivo high-resolution respirometry analysis conducted on permeabilized myocardial fibers, suggesting that PBMCs may be surrogates for myocardial mitochondrial function. Hypothesis: SV patients will have impaired mitochondrial respiratory capacity in circulating PBMCs. Methods: Subjects were recruited from Children’s Hospital Colorado. SV patients ages 0-18 years were included. Healthy children 0-18 years of age with normal biventricular cardiac function were included as a control group. PBMCs were isolated from whole blood (EDTA) using density gradient centrifugation in Histopaque Accuspin tubes. Oxygen consumption rates (OCR) of intact PBMCs were measured using the Seahorse XFe Bioanalyzer (Agilent). OCR is measured under 4 different conditions (Figure 1): baseline, after addition of oligomycin, after addition of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and after addition of antimycin A and rotenone. Results: Baseline respiratory capacity, coupling efficiency, and mitochondrial oxygen flux were decreased in failing SV PBMCs relative to NF controls (Figure 2). Conclusions: Our results demonstrate impaired mitochondrial respiratory capacity in circulating PBMCs of pediatric SV patients relative to healthy age-matched controls. Combined with our ex vivo data showing decreased mitochondrial function in failing SV myocardium, our findings suggests PBMCs may serve as a viable non-invasive biomarker of myocardial respiratory capacity.
Introduction: Heart failure (HF) remains the leading cause of death and indication for transplant in single ventricle congenital heart disease (SV). Phosphodiesterase-5 inhibitors (PDE5i) are commonly used for the treatment of SVHF, with the primary target being the pulmonary vasculature. We have previously demonstrated that the failing SV heart is characterized by increased PDE5 activity and impaired mitochondrial function. We hypothesize PDE5i-mediated deacetylation of mitochondrial proteins via activation of sirtuin-3 (SIRT3) promotes enhanced mitochondrial bioenergetics. Methods: Mitochondrial bioenergetics were assessed using an Oroboros O2k high resolution respirometer on freshly explanted permeabilized myocardial tissue from 12 biventricular non-failing controls (BVNF), 21 SVHF, and SVHF hearts treated with PDE5i (n=14) or honokiol (a SIRT3 activator, n=5) for 40 minutes. We examined cardiac lysine acetylation in 8 explanted BVNF and 11 SVHF hearts and mitochondrial lysine acetylation in 2 SVHF samples +/-PDE5i via western blot. Statistical analysis was performed using unpaired Mann-Whitney tests between 2 groups and a Welch ANOVA with post-hoc Dunnett’s T3 multiple comparisons test for 3 group comparisons. Results: Mitochondrial function is impaired in SVHF compared to BVNF, but is rescued by treatment with PDE5i (A) and the SIRT3 activator honokiol (B). SVHF myocardial proteins are hyperacetylated compared to BVNF (representative blot, C, p=0.02). Treatment with PDE5i promotes deacetylation of mitochondrial proteins in failing SV hearts (representative blot, D). Conclusions: Mitochondrial bioenergetics may represent a novel therapeutic target for SVHF. Our data shows that failing SV hearts are typified by impaired mitochondrial function and protein hyperacetylation, and suggests that PDE5i improves mitochondrial function in SVHF through SIRT3 mediated deacetylation of mitochondrial proteins.
Introduction: While heart failure (HF) remains a leading cause of death and indication for transplant in single ventricle heart disease (SV) patients, the molecular mechanisms associated with the progression to HF are poorly understood. The purpose of this study is to investigate if serum circulating factors from failing SV patients remodel cardiomyocyte metabolism, as is seen in the failing SV heart. Additionally, we investigated the effect of sildenafil on cardiomyocyte metabolism in vitro. Methods: We used a novel in vitro model that consists of primary cardiomyocytes (NRVMs) treated with serum from failing SV patients (SVHF) or bi-ventricular non-failing (BVNF) controls, +/- sildenafil. Mass spectroscopy was used to quantitate mitochondrial cardiolipin and investigate the metabolite composition of serum-treated NRVMs. Mitochondrial respiration was assessed using the Seahorse Bioanalyzer (Agilent), and reactive oxygen species (ROS) were quantified using the Amplex Red (Thermo Fisher). Enzyme and gene expression were analyzed using RT-qPCR and western blot, and relative mtDNA copy numbers were determined by qPCR. Results: While relative mitochondrial copy number was not significantly altered, failing SV serum decreases NRVM mitochondrial function (Fig. 1A-C), which is associated with decreased mitochondrial cardiolipin, increased reactive oxygen species (Fig. 1D), and altered expression of enzymes implicated in lipid and metabolic remodeling. Importantly, many of these changes are abrogated by sildenafil (Fig 1A-D). Conclusions: Together these data suggest that SV serum circulating factors promote lipid and metabolic remodeling in cardiomyocytes and that mitochondria represent a novel therapeutic target of sildenafil in the failing SV heart. Elucidation of the molecular mechanisms involved in modulation of cardiac energy metabolism in SV will be important to improve cardiac function and enhance transplant-free SV survival.
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