In this grant we will use a new stable isotope method called "proteome dynamics" to measure the rate of synthesis of individual mitochondrial proteins encoded on nuclear and mtDNA in the heart. Recent advances by our group in stable isotopic tracer methods and peptide analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables measurement of protein synthesis using deuterium (2H) labeled precursors and assessment of isotopically enriched protein products. Oral administration of "heavy water" (2H2O;a safe, non-radioactive compound) results in rapid steady state labeling of body water and transfer of 2H from 2H2O to 2H-labeled amino acids, which is followed by slow incorporation into proteins dependent upon the rate of synthesis of the specific protein. Assessment of protein dynamics requires measurement of enrichments of either tissue amino acids or total body water, and tryptic peptides, and calculation of the asymptotical number of 2H incorporated into a peptide using specialized software. We propose to use proteome dynamics to compare the synthesis rates for proteins encoded on mtDNA vs. nuclear DNA in the normal heart, in hearts with cardiac hypertrophy and early heart failure, and in hearts with advance heart failure and mitochondrial dysfunction. In addition, we will extend 2H2O tracers to measure the synthesis rate of mtDNA. Experiments will be performed in an established rat model of pressure overload HF that results in mitochondrial dysfunction. We will evaluate our hypotheses in three Specific Aims: 1) Determine if HF decreases the synthesis of proteins on the inner mitochondrial membrane encoded by mtDNA more than those on nuclear DNA. 2) Assess the synthesis rate of mtDNA by following the time course of the incorporation of 2H into mtDNA in the normal and failing heart. 3) Assess effects of HF on the synthesis rate of key cytosolic cardiac proteins.
Heart failure afflicts 6 million Americans, and despite aggressive treatment patient prognosis remains poor. There is growing evidence that part of the underlying problem in the failing heart is defective energy metabolism due to dysfunction mitochondria (the part of the cell that converts food to a form that the heart can use for pumping blood). In this project we will investigate the underlying mechanism for mitochondrial dysfunction in heart failure.
|Shekar, Kadambari Chandra; Li, Ling; Dabkowski, Erinne R et al. (2014) Cardiac mitochondrial proteome dynamics with heavy water reveals stable rate of mitochondrial protein synthesis in heart failure despite decline in mitochondrial oxidative capacity. J Mol Cell Cardiol 75:88-97|
|Kasumov, Takhar; Dabkowski, Erinne R; Shekar, Kadambari Chandra et al. (2013) Assessment of cardiac proteome dynamics with heavy water: slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria. Am J Physiol Heart Circ Physiol 304:H1201-14|