Fig 1: Cardiac oxygen consumption and key metabolic enzymes in WT and NLRX1-/- mouse hearts. (A) Myocardial oxygen consumption rate (MVO2) and (B) MVO2/RPP at 25 min baseline perfusion (n=14-16 per group); (C) Total pyruvate dehydrogenase (PDH) relative toα-tubulin, and phospho-PDH (p-PDH) in hearts of both genotypes (n=9 per group); (D) Cardiac enzyme activities for hexokinase (HK), citrate synthase (CS), lactate dehydrogenase (LDH) and short chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) (n=4–6 per group). RPP, rate pressure product. Values represent mean ± SD. *P < 0.05 by t test.
Fig 2: Metabolism of isolated beating WT and NLRX1-/- hearts characterized following 35 min perfusion with U-13C6 labeled 5.5 mM glucose or U-13C16 0.4 mM palmitate. (A) Metabolite sequential enrichment in glycolysis (hexose-6-phosphate, PEP, pyruvate), pentose phosphate pathway (erythrose-4-phosphate), pyruvate dehydrogenase (PDH) complex reaction (mtAcCoA) and TCA cycle (citrate, α-ketoglutarate and malate) from 13C glucose. The mitochondrial enrichments reflect the calculated contributions of carbons coming from acetyl CoA in the first turn; (B) Flux contribution (ϕ;), depicted as the ratio of precursor to product enrichment, determined by MIMOSA analysis along central carbon metabolism with each “parent” metabolite giving rise to its respective direct or clustered isotopomer(s); (C) Flux contribution of the 13C palmitate-derived labeled butyrylCoA into acetylCoA or citrate, and acetylCoA into citrate. (D) 13C labeling in PEP and lactate; (E) Flux contribution of the glycolytic intermediate PEP to lactate as proxy for lactate generation; APE(%), atomic percent enrichment (=(labeled isotopomer/total metabolite) * 100); PEP = phosphoenolpyruvate, Values represent mean ± SD (n= 5-10 per group). *P < 0.05, **P < 0.01, ***P < 0.001, by two-way ANOVA followed by LSD test or by t test.
Fig 3: Survival signaling pathways and inflammatory factors at early stage of reperfusion in severe IRI model of WT and NLRX1-/- isolated mouse hearts of protocol 3. (A, B) Representative immunoblots and analysis of total Akt and phospho-Akt; (C, D) Representative immunoblots and analysis of total AMPK and phospho-AMPK; (E, F) Representative immunoblots and analysis of total STAT3 and phospho-STAT3. Values represent mean ± SD. (n=6/5 per group). *P < 0.05 by t test.
Fig 4: The impact of NLRX1 deletion on maintenance of mechanical function during 20 min normoxic perfusion in the isolated hearts of protocols 1 to 5. For all parameters, the change in parameter at T=20 min relative to the value at T= 0 min is depicted. (A) end diastolic pressure (EDP), (B) heart rate (HR), (C) Rate Pressure Product (RPP), (D) maximum contraction rate of left ventricle (+dp/dt), (E) maximum relaxation rate of left ventricle (-dp/dt), and (F) Perfusion pressure (Pperf). DLVP, developed left ventricular pressure. All values represent median ± IQ (n=41 in WT group; n=34 in NLRX1-/- group). *P < 0.05, **P < 0.01 by Mann-Whitney U test.
Fig 5: (A) Perfusion protocols for NLRX1 effects on IR injury, survival kinases at reperfusion and 13C cardiac metabolism. Hearts from WT mice and NLRX1-/- mice were perfused with mixed substrates as described in methods part and subjected to 20 min baseline perfusion. For IR injury, hearts were subjected to 20 or 35 min ischemia followed by 60 min reperfusion. For measuring vital survival proteins, hearts were subjected to 35 min ischemia and 5 min reperfusion. For detecting metabolism, hearts were perfused for 35 min with either U-13C6 glucose or U-13C16 palmitate. (B) immunoblot results showing the presence of NLRX1 in WT hearts, and the absence of NLRX1 in NLRX1-/- hearts.
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