Resistin-Like Molecule α Dysregulates Cardiac Bioenergetics in Neonatal Rat Cardiomyocytes

Figures

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  Figure 1. RELMα downregulates genes related to cardiac energy metabolism. (A) Quantitative PCR analyses of acyl-coenzyme A dehydrogenase straight chain (ACADM), acyl-coenzyme A dehydrogenase short chain (ACADS), long-chain acyl-CoA dehydrogenase (LCAD), and very long-chain acyl-CoA dehydrogenase (VLCAD) in total mRNA derived from neonatal rat cardiomyocytes (NRCMs) exposed to RELMα (50 nM). (B) Immunoblotting of ACADM, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and transcription factors PPARα and ERRα in total protein extract derived from NRCMs exposed to RELMα at indicated concentration. (C) Quantitative PCR analyses of peroxisome PGC-1α, PPARα and ERRα in mRNA derived from NRCMs exposed to RELMα (50 nM). (D) Relative quantification for immunoblots of ACADM, PGC-1α, PPARα, and ERRα in total protein extract isolated from NRCMs exposed to RELMα (50 nM). (E) Quantitative PCR analyses of carnitine palmitoyltransferase-1a and 1b (CPT-1a and CPT-1b) and CD36 in total mRNA derived from NRCMs exposed to RELMα (50 nM). (F) Quantitative PCR analyses of pyruvate dehydrogenase 1a (PDH1a), citrate synthase (CS), glucose transporter 1 (GLUT1), pyruvate dehydrogenase…

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  Figure 2. Neonatal rat cardiomyoctyes (NRCMs) challenged with RELMα exhibit a decreased oxygen consumption rate (OCR). (A) Schematic diagram of OCR kinetics. (B) OCR profile in control and RELMα-treated cardiomyocytes. After four baseline measurements, oligomycin (1 μM, ATP-synthase inhibitor), FCCP (2 μM, electron transfer chain accelerator), and a combination of rotenone (0.5 μM, Complex I inhibitor) and antimycin A (0.5 μM, Complex III inhibitor) were injected into the NRCMs at the indicated times for assessment of basal mitochondrial respiration, ATP-linked OCR, proton leak, maximal respiration, and reserve capacity. (C) OCR based basal and maximal respiration in control and RELMα-treated cardiomyocytes. (D) OCR based ATP-linked OCR and proton leak measurement in cultured cardiomyocytes. n = 5 per group. *p < 0.05 vs. control group.

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  Figure 3. Neonatal rat cardiomyoctyes (NRCMs) challenged with RELMα exhibit an increased ability to augment glycolysis. (A) Extracellular acidification rate (ECAR), (B) Proton production rate, and (C) Metabolic profile of the stimulatory effect of RELMα on oxygen consumption rate (OCR) and glycolysis in primary cultured cardiomyocytes. The OCR (y-axis) and ECAR (x-axis) were plotted against one another at the same time. n = 5 per group. *p < 0.05 vs. control group.

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  Figure 4. Neonatal rat cardiomyocytes (NRCMs) challenged with RELMα or Etomoxir alone or in combination exhibit decreased OCR based mitochondrial bioenergetics. (A) Schematic representation of fatty acid oxidation assay with Etomoxir (courtesy Agilent Technologies) in presence of Palmitate-BSA as substrate to primary cultured cardiomyocytes. Cardiomyocytes were treated with either assay media and/or Etomoxir (final concentration 4 μM,) as port A injection. After three baseline measurements, NRCMs were subjected to a final concentration of oligomycin (1 μM), FCCP (2 μM), and a combination of rotenone (0.5 μM) and antimycin A (0.5 μM) as ports B, C, and D injections, respectively, at the indicated times for assessment of basal mitochondrial respiration, maximal respiration, spare respiratory capacity, ATP-linked OCR, and proton leak. (B) Basal respiration, maximal respiration and spare respiratory capacity in cardiomyocytes treated with RELMα with or without Etomoxir. (C) OCR based ATP-linked OCR and proton leak in cardiomyocytes treated with RELMα with or without Etomoxir. n = 3 per group. *, **, ***p < 0.05, 0.01, 0.001 vs. control group. #p < 0.05 vs. RELMα group.

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  Figure 5. RELMα impairs mitochondrial ultrastructure, biogenesis and its replication machinery in NRCMs. (A) Representative electron micrographs taken from NRCMs stimulated with or without recombinant RELMα. Low magnification images (upper) were used to evaluate mitochondrial density, and high magnification images (lower) were used to investigate the ultrastructure of mitochondria. Arrows indicate structurally abnormal mitochondria (black arrow: giant mitochondria, white arrow: abnormal cristae structure, black arrow with dotted tail: small, fragmented mitochondria). Scale bars are 1 μm. (B) Representative electron micrographs showing mitochondrial morphology quantitated as average mitochondrial size (μm2), and (C) mitochondrial density per microscopic field by using imageJ software. (D) Estimation of mitochondrial DNA copy number (mito-DNA/nuclear DNA ratio) by quantitative PCR in NRCMs. (E) Quantitative PCR analyses of mitochondrial transcription factor A (TFAM), mitochondrial topoisomerase I (TOP1MT), mitochondrial DNA polymerase subunit gamma 2 (POLG2), and mitochondrial DNA-directed RNA polymerase (POLRMT) in NRCMs. n = 4–7 per group. *p < 0.05, **p < 0.01 vs. control group.