1 Introduction Cann et al., 1987; Wallace et al., 1999; Wallace, 2005 Dimauro and Davidzon, 2005; Taylor and Turnbull, 2005 Herrnstadt and Howell, 2004 Santoro et al., 2006 Mishmar et al., 2003; Ruiz-Pesini et al., 2004; Wallace, 2005; Montiel-Sosa et al., 2006 Elson et al., 2004; Kivisild et al., 2006; Ruiz-Pesini and Wallace, 2006 Amo and Brand, 2007; Elson et al., 2007 Baudouin et al., 2005 Protti and Singer, 2007 Baudouin et al., 2005 Wallace, 2005 Ruiz-Pesini et al., 1998; Ruiz-Pesini et al., 2000 Ruiz-Pesini et al., 2000 Montiel-Sosa et al., 2006 Pereira et al., 2005; Pereira et al., 2007 Korshunov et al., 1997; Liu, 1997; Lambert and Brand, 2004 Wallace, 2005 Chagnon et al., 1999 Elson et al., 2006 Raule et al., 2007 In the present study, we analyse bioenergetic capacity and coupling efficiency in mitochondria isolated from cytoplasmic hybrids (cybrids) carrying haplogroups H and T with identical nuclear DNA. Furthermore, to investigate the effects of haplogroups H and T on the bioenergetic status of mitochondria at the cellular level, we analyse the respiratory capacities and mitochondrial coupling efficiencies of intact cybrids. 2 Materials and methods 2.1 Subjects Wallace et al., 1999 Torroni et al., 1997 Achilli et al., 2004 Baudouin et al., 2005 Macaulay et al., 1999 Pike 2006 2.2 Generation of cybrid cell lines ρ 0 King and Attardi, 1989 ρ 0 ρ 0 Chomyn, 1996 Laird et al., 1991 2.3 Mitochondrial respiration and membrane potential Amo and Brand, 2007 2 4 2 − 1 Reynafarje et al., 1985 ψ + Brand, 1995 − 1 − 1 − 1 ψ + + p ψ + − 1 2.4 Modular kinetics Amo and Brand, 2007 ψ ψ ψ ψ Brand, 1990 ψ ψ − 1 ψ ψ ψ Brand, 1998 ψ − 1 ψ ψ ψ Brand et al., 1994 2.5 Cell respiration Wu et al., 2007 l 2 2 2 2 4 2 4 d Fig. 4 6 Wu et al., 2007 2.6 Statistics n n Figs. 1–3 n Fig. 4 t P 3 Results 3.1 Modular kinetic analysis of oxidative phosphorylation Kivisild et al., 2006 Wallace et al., 1999 arg thr Kivisild et al., 2006 Fig. 1 Fig. 1 ψ Fig. 1 ψ Fig. 1 ψ ψ Fig. 1 Fig. 1 Fig. 2 Fig. 1 Fig. 1 ψ Fig. 2 ψ Brand et al., 1993 Fig. 1 Fig. 3 Fig. 3 Fig. 3 Fig. 3 In summary, at the mitochondrial level we observed no significant functional differences between mitochondria isolated from cybrids of haplogroup H and haplogroup T. 3.2 Coupling efficiency of respiration in intact cybrid cells Fig. 4 The residual rate in the presence of rotenone and myxothiazol represents non-mitochondrial oxygen consumption. This comprised 16.7 + 2% of basal respiration rate in H-haplotype cybrids and 18.2 + 2.7% in T-haplotype cybrids. These values were not significantly different. Subtraction of this non-mitochondrial respiration allowed assessment of the rates and coupling efficiency of the remaining mitochondrial respiration. Brand, 1990 Fig. 4 Figs. 2 and 3 Wu et al., 2007 Brand, 2005 Fig. 4 Fig. 4 In summary, at the intact cell level we observed no significant functional differences between mitochondria within cybrids of haplogroup H and haplogroup T. 4 Discussion Amo and Brand, 2007 The strengths of our approach are (i) we used a direct empirical measurement, rather than inference from genetic and physiological traits, (ii) we separated the effects of mitochondrial haplogroup from any confounding effects of nuclear DNA background and (iii) we used a simple in vitro system with the powerful modular kinetic approach where the relevant variables could be tightly controlled and manipulated. However, our approach also has some weaknesses. (i) A small effect of mitochondrial haplogroup operating over hours or days might still be too small to be detected by biochemical experiments. (ii) We measured the bioenergetic capacity using isolated mitochondria and cybrid cells in simple defined media, but effects of mitochondrial haplogroups might only emerge under special conditions (for example, they might require triggers such as sepsis or energetic stress or might require particular nuclear genes to be expressed, and so be tissue-specific or suppressed in the lung carcinoma cell line we employed). Our results fail to provide support for the hypothesis that the common European mitochondrial haplogroups H and T have significant effects on bioenergetics that cause the associations with survival after sepsis and sperm motility that have been reported. If such associations stand up to further scrutiny, they presumably operate through more subtle mechanisms than respiratory rates, mitochondrial coupling efficiencies or ATP supply.