The muscular dystrophy with myositis (mdm) mouse carries a deletion in the muscle protein titin. A previous study found that mdm mice shiver at a lower than expected frequency for their body size, are mostly heterothermic with a narrow thermoneutral zone beginning at 34C, and have reduced active muscle stiffness in vivo compared to their wildtype siblings. Impairment in heat production (shivering thermogenesis) could be due to the N2A deletion in the titin protein leading to a more compliant muscle and a lower shivering frequency. The ability of mdm mice to use nonshivering thermogenesis via Uncoupling Protein 1 in brown adipose tissue to generate heat during cold stress may also be impaired and contributing to their heterothermy. The purpose of this study was to evaluate mechanisms for the inability of mdm mice to maintain homeothermy during cold stress and to further understand their metabolic properties. We hypothesized the inability of mdm mice to maintain homeothermy is due to an impairment in heat production (e.g. shivering thermogenesis and nonshivering thermogenesis) and that metabolic rate will reach a maximum at a higher ambient temperature than in wildtype mice. In a temperature experiment, mice were placed in a mouse cage that used indirect calorimetry to measure metabolic rate at four different temperatures (34C, 29C, 24C, 19C). Nonshivering thermogenesis was stimulated by administering 1.2 mg kg−1 of norepinephrine subcutaneously. In the temperature experiment, there was a significant interaction between genotype and temperature, with significant differences in metabolic rates at the lowest temperatures of 19C and 24C (Two‐Way ANOVA and Tukey HSD, p<0.05. Mdm mice had significantly lower body temperatures than wildtype mice at 29C, 24C, and 19C, and reached their maximum metabolic rate at 24C, suggesting that a temperature between 19–24C is the minimum at which mdm mice are able to sustain thermogenesis. Area under the curve (AUC) using the trapezoidal rule was used to calculate total thermogenic capacity following norepinephrine injection. Mdm mice had a lower overall AUC (Welch's T‐Test, p=0.03) as well as a lower overall time that they increased metabolic rate after norepinephrine injection (Welch's T‐Test, p=0.02). Our results suggest that in addition to a deficiency in shivering frequency, mdm mice also have a reduced capacity for nonshivering thermogenesis. The deficiency in shivering thermogenesis and nonshivering thermogenesis combined likely lead to inability to maintain euthermic body temperatures below 34C. Support or Funding Information NSF IOS‐0742483, NSF IOS‐1025806, and NSF IOS‐1456868, the W. M. Keck Foundation, and the Technology Research Initiative Fund of Northern Arizona University. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remains limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with non-failing or DCM pediatric patient serum activates the fetal gene program (FGP). Here we show that serum treatment with Proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating microRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is up-regulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA sequencing (RNA-Seq), indicates extracellular matrix remodeling and focal adhesion pathways are upregulated in pediatric DCM serum and serum-treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum-treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM-serum treatment. Our results show that serum circulating proteins promote pathological changes in gene expression and cellular stiffness, and circulating miRNAs are protective against pathological changes.

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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: 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.

Introduction: Heart failure (HF) remains a leading cause of death and indication for transplant in single ventricle congenital heart disease (SV). However, little is known regarding the molecular mechanisms leading to HF in SV. The purpose of this study was to characterize mitochondrial function in the myocardium of failing (SVHF) and non-failing (SVNF) SV patients compared to biventricular NF controls (BVNF). Furthermore, we investigated the effect of ex vivo treatment with the phosphodiesterase-5 inhibitor (PDE5i) sildenafil on mitochondrial function. Methods: Freshly explanted ventricular tissue was saponin permeabilized and mitochondrial oxygen consumption was measured sequentially throughout the electron transport system using SUbstrate-Inhibitor-Titration (“SUIT”) protocols and an Oroboros O2k high resolution respirometer. Permeabilized ventricular tissue was treated for 40 min with sildenafil [1μM] prior to measurement of oxygen consumption. A Western blot for PDE5 was performed in isolated mitochondrial proteins from SVHF subjects ± PDE5i. Results: Compared to BVNF (n=15) and SVNF (n=6), SVHF (n=8) hearts have decreased function of Complex I and Complex I and II (A, B), and decreased maximal respiration (C), all of which improve with acute ex vivo treatment with sildenafil in SVHF (SVHF+PDE5i, n=6). Importantly, mitochondrial function is impaired in BVNF+PDE5i (n=5) and SVNF+PDE5i hearts (n=5) (A-C, one-way Anova p<0.05). PDE5 protein is expressed in SVHF mitochondria, but expression is not affected by ex vivo PDE5i treatment (D). Conclusions: Our results indicate that mitochondrial function is impaired in SVHF, PDE5 protein is expressed in SVHF mitochondria, and PDE5i improves mitochondrial function in SVHF, but may be detrimental to mitochondrial function in SVNF and BVNF. Together these data suggest that mitochondrial PDE5 is a potential therapeutic target, but that indiscriminate use of PDE5i in SV patients may not be advisable.

Background Pediatric dilated cardiomyopathy (DCM) is a devastating and poorly understood disease with most clinical treatment paradigms extrapolated from the adult population. Our studies showed that aspects of metabolism and mitochondria function are dysregulated in pediatric hearts. One potential factor which may be implicated in pediatric cardiac dysfunction is the Branched‐Chain Amino Acid Transaminase 1 (BCAT1), an enzyme highly expressed in pediatric non‐failing hearts and down‐regulated in response to DCM‐induced heart failure. While BCAT1’s role in cardiac dysfunction is unclear, in cancer cells and macrophages, BCAT1 is important for mitochondria function. The objective of this study is to investigate the role of BCAT1 in the heart, which may result in age‐specific therapies. Methods We measured metabolite composition of the pediatric DCM and non‐failing (NF) hearts. Activity and level of BCAT1 by enzymatic assay, RT‐qPCR and Western Blot in adult and pediatric and DCM and NF hearts was also measured. Pathologic changes in gene expression and mitochondrial function were assessed in Neonatal Rat Ventricular Myocytes (NRVMs) by RT‐qPCR and Seahorse assay in the presence or absence of BCAT1 inhibitor. Results Analysis of metabolite content showed a dramatic accumulation of branched‐chain amino acids (BCAAs) in pediatric DCM hearts. BCAT1 mRNA and protein levels are increased in pediatric NF hearts compared to adult NF hearts, and decreased in pediatric DCM hearts. In addition, inhibition of BCAT1 in NRVMs results in pathologic gene expression and inhibition of pro‐inflammatory cytokines. Finally, Seahorse assay showed a decrease of mitochondrial respiration in NRVMs treated with BCAT1 inhibitor. Conclusions Decreased levels of BCAT1 are detrimental to the pediatric failing heart. These findings suggest that BCAT1 could be a specific therapeutic target for the pediatric population. Support or Funding Information R01 HL139968 and HL126928

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