Decline in cardiac pump function is a hallmark of aging where mitochondrial decay is an important underlying cause. Although certainly multifactorial in nature, both dysfunction of the machinery involved in the chemiosmotic process of energy transduction and lower capacity to maintain fatty acid-driven respiration are identified as intrinsic factors of mitochondrial decay in the aged myocardium. Age-associated destabilization of electron transport supercomplexes as a potential factor of mitochondrial decay in the rat heart. Defective operation of the electron transport chain (ETC) constitutes a key mechanism involved in the age-associated loss of mitochondrial energy metabolism. Nevertheless, the molecular events underlying inefficient electron flux that ultimately leads to higher superoxide appearance and impaired respiration are not fully known. As recent biophysical evidence shows that the ETC may form large macromolecular assemblies (i.e. supercomplexes) that disintegrate in certain pathologies (e.g. heart failure or Barth syndrome) reminiscent of aging, we investigated the hypothesis that alterations in supercomplexes are partly responsible for the age-related loss of cardiac ETC function. In this dissertation, age-associated changes in supercomplex organization and stability were investigated in subsarcolemmal (SSM) and interfibrillary (IFM) mitochondria isolated from cardiac tissue from young (3-5 months) and old (24-28 months) male Fischer 344 rats. Blue native-PAGE (BN-PAGE) analysis of digitonin-solubilized mitochondrial membranes coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to investigate supercomplex organization. Results show that both SSM and IFM display supercomplexes comprised of various stoichiometries of complexes I, III and IV (never complex II), which typically organize as high mass (1500-2300 kDa) assemblies containing up to four copies of complex IV (i.e. I���III���IV[subscript N]-type supercomplexes). Interestingly, analysis of IFM proteins showed that, in general, supercomplex levels declined by up to 15 % (p < 0.05) with age; however, different degrees of supercomplex deterioration were observed, depending on the particular supercomplex investigated. Supercomplexes of the highest molecular weights (i.e. 1900-2300 kDa), which were also composed of the most complex stoichiometries (i.e. I1III2IVN, N ��� 2), were primarily lost with age. In particular, I���III���IV���, I���III���IV��� and I���III���IV��� supercomplexes were found to decline by 13% (p < 0.05), 30% (p < 0.05) and 45% (p < 0.05), respectively, on an age basis. Therefore, the age-associated loss of supercomplexes in IFM stems from destabilization of the assemblies that comprise several copies of complex IV, which could partially limit proper electron transfer to O��� for its reduction, affecting mitochondrial respiratory capacity. In contrast to IFM, the aging defects of SSM supercomplexes appeared to be confined to the assembly comprised of only one copy of complex IV (I���III���IV���, 1700 kDa) (37% loss; p = 0.06), while the higher molecular weight supercomplex sub-types that were most affected in IFM (i.e. I���III���IV[subscript N], N ��� 2) were not significantly altered with age. Thus, the results from this dissertation indicate that mitochondria from different subcellular locations in the myocyte show different degrees of supercomplex destabilization in the aging rat heart. The more robust supercomplex deficits noted for IFM fit well with previous observations that electron transport characteristics of this subpopulation are more adversely affected with age than SSM. Although the underlying factor(s) of supercomplex deterioration are not fully known, the hypothesis that age-related alterations of certain constituents of the IMM (e.g. cardiolipin) may be important factors of supercomplex destabilization in cardiac mitochondria was investigated in this dissertation. To this end, LC-MS/MS characterization of supercomplex proteins and HPLC analysis of cardiolipin were used as approaches to elucidate potential factor(s) of supercomplex destabilization in the aging rat heart. Age-related alterations of cardiolipin levels and its acyl-chain content showed a strong parallel to the age-associated destabilization of supercomplexes. Specifically, cardiolipin levels declined by 10% (p < 0.05) in IFM, the mitochondrial subpopulation displaying the highest degree of supercomplex deterioration. In addition, the content of (18:2)���-cardiolipin, the predominant species in the heart, was found to decline by 50% (p < 0.05) on average in both populations of cardiac mitochondria. Therefore, the data presented in this dissertation indicate that changes in cardiolipin may be at least one of the factors involved in supercomplex destabilization in the aging heart. Age-related decline in carnitine palmitoyltransferase I (CPT1) activity as a mitochondrial lesion that limits fatty acid catabolism in the rat heart. Loss of fatty acid utilization, another intrinsic factor of mitochondrial decay in the aged myocardium, has been associated with age-related alterations in the activity of carnitine palmitoyltransferase 1 (CPT1), the rate-controlling enzyme for overall fatty acid ��-oxidation. Nevertheless, the exact molecular mechanism involved in the age-related loss of fatty acid-driven bioenergetics is not fully understood. In this dissertation, it was also investigated whether the aging lesion for fatty oxidation lies in a particular mitochondrial subpopulation or more generally results from cardiac decrements in L-carnitine levels. In order to clarify the role of each one of these factors, the effect of long-term dietary supplementation with the L-carnitine analogue, acetyl-L-carnitine (ALCAR), was also investigated. Results show that aging selectively decreases CPT1 activity in IFM by reducing enzyme catalytic efficiency for palmitoyl-CoA. IFM displayed a 28% (p < 0.05) loss of CPT1 activity, which correlated with a decline (41%, p < 0.05) in palmitoyl-CoA-driven state 3 respiration. Interestingly, SSM had preserved enzyme function and efficiently utilized palmitate. Analysis of IFM CPT1 kinetics showed both diminished V[subscript max] and K[subscript m] (60% and 49% respectively, p < 0.05) when palmitoyl-CoA was the substrate. However, no age-related changes in enzyme kinetics were evident with respect to L-carnitine. ALCAR supplementation restored CPT1 activity in heart IFM, but not apparently through remediation of L-carnitine levels. Rather, ALCAR influenced enzyme activity over time, potentially by modulating conditions in the aging heart that ultimately affect palmitoyl-CoA binding and CPT1 kinetics. In conclusion, this dissertation presents a characterization of age-associated alterations in the macromolecular organization of the IMM components that could partly explain the loss of mitochondrial oxidative capacity that affects the aging heart. In addition, the characterization of an age-related lesion of the controlling enzyme for ��-oxidation is presented as another important factor that limits mitochondrial function and energy metabolism in cardiac mitochondria. / Graduation date: 2013

Artificial divergent selection of chickens for high and low body weight at 8 weeks of age has produced two lines: the high (HWS) and low (LWS) body weight chicken lines. In addition to the difference in body weight, the lines show extreme differences in feeding behaviour and body composition. The aim of this study was to uncover the genetic and molecular factors that contribute to and determine these differences, especially regarding body energy regulation and appetite. In papers I and II, genome-wide gene expression in a brain sample containing hypothalamus and in dissected hypothalamus was analysed using DNA microarray and qRT-PCR. We found that levels of differential expression were generally moderate, which was consistent with the idea that polygenic factors were involved in the establishment of the chicken lines. Genes associated with neural plasticity, lipid metabolism and body energy regulation were differentially expressed. This result indicated that the neural systems regulating feeding behaviour and body weight were altered in the chicken lines. However, genes that were involved in the central melanocortin system were not systematically differentially expressed. Interestingly, the biggest differences in expression between the lines found in endogenous retrovirus sequences of the ALV subgroup E. Thus, in paper III, we characterized the number of integrations, the expression of ALVE retroviral elements and their effects on body weight. A significant correlation between low body weight and high ALVE expression was observed in female F9 birds from an HWS x LWS advanced intercross line. This implied that ev-loci contributing to increased ALVE expression levels were genetically linked to loci influencing the low body weight of the pullets. In paper IV, the carnitine palmitoyltransferase-1b gene (CPT1B), which was highly differentially expressed in the hypothalami, was investigated. We mapped chicken CPT1B to the distal tip of chromosome 1p. The levels of CPT1B mRNA in the HWS line were higher in the hypothalamus and lower in muscle than in the LWS line. This pattern of differential expression indicates that this gene could contribute to the remarkable phenotypic differences between HWS and LWS chickens. However, comparison with quantitative trait loci data showed that the expression of CPT1B is a trans effect, rather than a direct causative locus. In conclusion, the data suggested that the long-term selection for body weight resulted in differential gene expression in the brains of the selected chicken lines. These results may have relevance for the poultry industry and will also contribute to increasing knowledge about human diseases such as obesity and anorexia.

No description available.