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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.

Pediatric dilated cardiomyopathy (DCM) is a devastating and poorly understood disease with most clinical treatment paradigms extrapolated from the adult population. Our studies have demonstrated that aspects of metabolism and mitochondrial function are dysregulated in pediatric DCM hearts. Cardiolipin (CL), a unique phospholipid in the inner mitochondrial membrane, is essential for optimal mitochondrial function and was shown to be dysregulated in both the failing adult and pediatric human heart. The objective of this study is to investigate if serum circulating factors from pediatric DCM patients can remodel CL resulting in mitochondrial dysfunction in vitro , similar to what is observed in the failing pediatric heart. Using a novel in vitro model that consists of treating neonatal rat ventricular myocytes (NRVMs) with serum from pediatric DCM patients or from non-failing (NF) healthy controls, mitochondrial respiration was assessed using the Agilent Seahorse, and reactive oxygen species (ROS) was assessed using Electron Paramagnetic Resonance Spectroscopy. Relative mitochondrial DNA (mtDNA) copy number was determined by qPCR and expression of enzymes involved in CL biosynthesis and remodeling were analyzed using RT-qPCR. Mass-spectrometry was used to quantitate total and specific CL species and to investigate the metabolite composition of NRVMs treated with NF or DCM serum. While mitochondrial ROS and mtDNA copy number were not significantly altered, we show that DCM serum decreases mitochondrial function, which is associated with alterations in CL content and composition and the downregulation of enzymes implicated in CL biosynthesis and remodeling. Analysis of metabolite content showed an alteration of pathways involved in fatty acid metabolism, mitochondrial biogenesis and regulation of β-oxidation by the transcription factor PPARα. In conclusion, pediatric DCM serum circulating factors can promote CL remodeling resulting mitochondrial dysfunction in primary cardiomyocytes. These findings suggest that CL could be a novel therapeutic target for this particular population.

Introduction: Despite current standard of care, heart failure (HF) remains a leading cause of death and indication for transplant in the single ventricle congenital heart disease (SV) population. However, little is known regarding the molecular mechanisms underlying remodeling and eventual HF in SV patients. The purpose of this study was to characterize the transcriptional profile of SV myocardium in both failing (SVHF) and non-failing (SVNF) SV patients compared to biventricular NF controls (BVNF). Furthermore, we conducted high resolution respirometry to assess mitochondrial function in each of these populations. Methods: Library prep was performed using the TruSeq Ribo Zero rRNA depletion kit, and 2x150 total RNAseq (Illumina HiSEQ 4000) was performed on age-matched explanted RV myocardium from BVNF (n=4 biventricular donors), SVNF (n=8 SV primary transplants, normal function), and SVHF (n=9 SV systolic HF transplants). Samples were aligned to hg19 and were normalized and annotated using the edgeR pipeline. Significant changes in gene expression were calculated using an FDR adjusted p-value (q Results: RNAseq identified 1,007 differentially expressed genes in SVNF and 2,109 in SVHF myocardium relative to BVNF controls. Transcriptome pathway analysis demonstrated multiple pathways that are similarly dysregulated in SVNF and SVHF, while pathways involved in mitochondrial metabolism and function were significantly dysregulated specifically in the SVHF population. Moreover, mitochondrial oxygen flux was significantly decreased, particularly through complexes I and II, in SVHF relative to BVNF controls. Conclusions: Our results provide new insights into SVHF by identifying unique gene expression changes, including those related to metabolism, and impaired mitochondrial function. Together these data suggest dysregulated metabolic gene expression and mitochondrial dysfunction are phenotypes associated with the failing single ventricle and may serve as potential therapeutic targets for the treatment or prevention of HF in the SV population.

Organization of the mitochondrial electron transport chain (ETC) into a protein “supercomplex” has been shown to be critical for optimal mitochondrial respiration, and is dependent on the phospholipid composition of the inner mitochondrial membrane. A close physical interaction between the ETC supercomplex and the fatty acid beta-oxidation system (FAO, which provides necessary reducing equivalents for ETC) has also been proposed. We have previously demonstrated that content of the primary mitochondrial phospholipid, cardiolipin, is altered in pediatric dilated cardiomyopathy (DCM), with evidence for its dysregulated biosynthesis. We hypothesized that altered cardiolipin content in pediatric DCM is correlated with altered supercomplex-associated ETC activity and mitochondrial fatty acid β-oxidation. A cross-sectional investigation was performed using myocardium from 16 children with DCM and 15 non-failing (NF) controls from the University of Colorado Heart Tissue Bank. Using blue native (BN) -PAGE with in-gel activity staining we demonstrated lower activity of supercomplex-associated complexes I (DCM 80% of NF, P

Background— Remodeling of myocardial phospholipids has been reported in various forms of heart failure for decades, but the mechanism and pathophysiological relevance of this phenomenon have remained unclear. We examined the hypothesis that δ-6 desaturase (D6D), the rate-limiting enzyme in long-chain polyunsaturated fatty acid biosynthesis, mediates the signature pattern of fatty acid redistribution observed in myocardial phospholipids after chronic pressure overload and explored plausible links between this process and disease pathogenesis. Methods and Results— Compositional analysis of phospholipids from hearts explanted from patients with dilated cardiomyopathy revealed elevated polyunsaturated fatty acid product/precursor ratios reflective of D6D hyperactivity, manifesting primarily as lower levels of linoleic acid with reciprocally higher levels of arachidonic and docosahexaenoic acids. This pattern of remodeling was attenuated in failing hearts chronically unloaded with a left ventricular assist device. Chronic inhibition of D6D in vivo reversed similar patterns of myocardial polyunsaturated fatty acid redistribution in rat models of pressure overload and hypertensive heart disease and significantly attenuated cardiac hypertrophy, fibrosis, and contractile dysfunction in both models. D6D inhibition also attenuated myocardial elevations in pathogenic eicosanoid species, lipid peroxidation, and extracellular receptor kinase 1/2 activation; normalized cardiolipin composition in mitochondria; reduced circulating levels of inflammatory cytokines; and elicited model-specific effects on cardiac mitochondrial respiratory efficiency, nuclear factor κ B activation, and caspase activities. Conclusions— These studies demonstrate a pivotal role of essential fatty acid metabolism in myocardial phospholipid remodeling induced by hemodynamic stress and reveal novel links between this phenomenon and the propagation of multiple pathogenic systems involved in maladaptive cardiac remodeling and contractile dysfunction.