Glutamine uptake and catabolism is required for myofibroblast formation and persistence.

BACKGROUND: Fibrosis and extracellular matrix remodeling are mediated by resident cardiac fibroblasts (CFs). In response to injury, fibroblasts activate, differentiating into specialized synthetic and contractile myofibroblasts producing copious extracellular matrix proteins (e.g., collagens). Myofibroblast persistence in chronic diseases, such as HF, leads to progressive cardiac dysfunction and maladaptive remodeling. We recently reported that an increase in αKG (alpha-ketoglutarate) bioavailability, which contributes to enhanced αKG-dependent lysine demethylase activity and chromatin remodeling, is required for myofibroblast formation. Therefore, we aimed to determine the substrates and metabolic pathways contributing to αKG biosynthesis and their requirement for myofibroblast formation. METHODS: Stable isotope metabolomics identified glutaminolysis as a key metabolic pathway required for αKG biosynthesis and myofibroblast formation, therefore we tested the effects of pharmacologic inhibition (CB-839) or genetic deletion of glutaminase (Gls1-/-) on myofibroblast formation in both murine and human cardiac fibroblasts. We employed immunofluorescence staining, functional gel contraction, western blotting, and bioenergetic assays to determine the myofibroblast phenotype. RESULTS: Carbon tracing indicated enhanced glutaminolysis mediating increased αKG abundance. Pharmacological and genetic inhibition of glutaminolysis prevented myofibroblast formation indicated by a reduction in αSMA+ cells, collagen gel contraction, collagen abundance, and the bioenergetic response. Inhibition of glutaminolysis also prevented TGFß-mediated histone demethylation and supplementation with cell-permeable αKG rescued the myofibroblast phenotype. Importantly, inhibition of glutaminolysis was sufficient to prevent myofibroblast formation in CFs isolated from the human failing heart. CONCLUSIONS: These results define glutaminolysis as necessary for myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat cardiac fibrosis.

Microtubules Increase Diastolic Stiffness in Failing Human Cardiomyocytes and Myocardium.

BACKGROUND: Diastolic dysfunction is a prevalent and therapeutically intractable feature of heart failure (HF). Increasing ventricular compliance can improve diastolic performance, but the viscoelastic forces that resist diastolic filling and become elevated in human HF are poorly defined. Having recently identified posttranslationally detyrosinated microtubules as a source of viscoelasticity in cardiomyocytes, we sought to test whether microtubules contribute meaningful viscoelastic resistance to diastolic stretch in human myocardium. METHODS: Experiments were conducted in isolated human cardiomyocytes and trabeculae. First, slow and rapid (diastolic) stretch was applied to intact cardiomyocytes from nonfailing and HF hearts and viscoelasticity was characterized after interventions targeting microtubules. Next, intact left ventricular trabeculae from HF patient hearts were incubated with colchicine or vehicle and subject to pre- and posttreatment mechanical testing, which consisted of a staircase protocol and rapid stretches from slack length to increasing strains. RESULTS: Viscoelasticity was increased during diastolic stretch of HF cardiomyocytes compared with nonfailing counterparts. Reducing either microtubule density or detyrosination reduced myocyte stiffness, particularly at diastolic strain rates, indicating reduced viscous forces. In myocardial tissue, we found microtubule depolymerization reduced myocardial viscoelasticity, with an effect that decreased with increasing strain. Colchicine reduced viscoelasticity at strains below, but not above, 15%, with a 2-fold reduction in energy dissipation upon microtubule depolymerization. Post hoc subgroup analysis revealed that myocardium from patients with HF with reduced ejection fraction were more fibrotic and elastic than myocardium from patients with HF with preserved ejection fraction, which were relatively more viscous. Colchicine reduced viscoelasticity in both HF with preserved ejection fraction and HF with reduced ejection fraction myocardium. CONCLUSIONS: Failing cardiomyocytes exhibit elevated viscosity and reducing microtubule density or detyrosination lowers viscoelastic resistance to diastolic stretch in human myocytes and myocardium. In failing myocardium, microtubules elevate stiffness over the typical working range of strains and strain rates, but exhibited diminishing effects with increasing length, consistent with an increasing contribution of the extracellular matrix or myofilament proteins at larger excursions. These studies indicate that a stabilized microtubule network provides a viscous impediment to diastolic stretch, particularly in HF.

Myocardial ultrastructure of human heart failure with preserved ejection fraction.

Over half of patients with heart failure have a preserved ejection fraction (>50%, called HFpEF), a syndrome with substantial morbidity/mortality and few effective therapies1. Its dominant comorbidity is now obesity, which worsens disease and prognosis1-3. Myocardial data from patients with morbid obesity and HFpEF show depressed myocyte calcium-stimulated tension4 and disrupted gene expression of mitochondrial and lipid metabolic pathways5,6, abnormalities shared by human HF with a reduced EF but less so in HFpEF without severe obesity. The impact of severe obesity on human HFpEF myocardial ultrastructure remains unexplored. Here we assessed the myocardial ultrastructure in septal biopsies from patients with HFpEF using transmission electron microscopy. We observed sarcomere disruption and sarcolysis, mitochondrial swelling with cristae separation and dissolution and lipid droplet accumulation that was more prominent in the most obese patients with HFpEF and not dependent on comorbid diabetes. Myocardial proteomics revealed associated reduction in fatty acid uptake, processing and oxidation and mitochondrial respiration proteins, particularly in very obese patients with HFpEF.