204 J Biocncrg Bionicmbr (2009) 41:195 214 arrangement induce fturctional alterations in nomral cardiac i V FW. 5 pm cells.

Interestingly, even after trypsin treatment mitochondria V _ _ "" are always of granular fonn and still, to some extent, in fixed V. position. Only at much higher trypsin concentration distinct ;= ._`_ ` mitochondria could be completely released into the medium. Q; - A ` _ ? ln no case was the fusion of granular mitochondria into * ` filamentous fomis observed in our study. V V , X Kg With the aim of verification of these findings concerning " ~ * ` ri the absence of mitochondrial fusion in intact cardiomyo- ii log ikq,_`VV cytes under normal conditions, we studied the functional! .V *2,, "' I ' electrical connectivity of mitochondria. in ` i*= ii` glqii g if ,_ .r Wt. i r. Disconnectivity qfmitochondria in adult curdiomyocytes V E. '*"iV K`, To assess the question of whether mitochondrial fusion talces _ at i - ii r. Ii place in adult cardiomyocytes, we tested the functional/ =;. ii L_ ia electrical connectivity Of rilimchondua in isolated adhlt mt Fig. 4 Demonstration of thc abscncc of intcr mitochondrial conneccvrdwmyocytes- Indeed, fusion should lead to inc foinlahoh of tivity in cardiomyocytes Simultaneous image analysis of mitochon amitochondrial reticulum, thus to electrical continuity between dria by MitoTracker *> Green (gyeenV/Zumemenee) and mitochondrial mrreehemrm (Skulachev 1990, 2001; srmnerrev et ar. 2004; ij$imbr? r;V(jV Vj1V;r@}h_TMVV{<(fi <:t/lg~r~L gr dmils Am'rkV Et *1*- *9**; Twig rt 3*- 20%- * i*'i at ri- Qcflfrsrilgc .,?M lrSrLL`r`r%` JZ?mZZ,l `rZr'2r 200l)- lh this siiiilya the nihochondiial lncinhlia-no Potential shows norrrrally cncrgizcd and fully dcpolarizcd ncighboring mito was monitored using very low concentrations ofthe specific chondria (indicated by arrows), which is consistent with the absence

potcntiometric fluorescent probe TMRM, a probe of mito- "i i "i l " ii"iiY * ` " g ihem chondrial membrane potential. Similarly to TMRE and JC-1, this dye is widely used for the assessment of mitochondrial inner membrane potential in various cells using different fluorescent techniques such as fluorimetry, FACS, and in particular confocal microscopy (Zorov et al. 2000, 2004; mitochondria. The latter indicates the existence of rnitochonPetronilli et al. 2001; Floryk and Houstek 1999). In our study, diial neighbours exhibiting different energization status. mitochondrial imaging by MitoTracker Green was per- Notably, these results also confimi that l\/IitoTracker* Green formed simultaneously with imaging of TMRM (Fig. 4). labels all mitochondria independentlyof theirinnermembrane Therefore, we could distinguish mitochondria with high or potential (Presley et al. 2003). Therefore, the rapid, low low membrane potential by the intensity of TMRM fluores- amplitude mitochondrial fluctuations observed above can not cence signal together with the mitochondrial imaging be related to the oscillations of the membrane potential perfomied by MitoTracl<er Green staining. described in some reports (Cortassa ct al. 2004; Hattori etal. For these co-localization studies, cells were preloaded with 2005). Our results point out that cardiac mitochondria are TMRM (50 nM) and with MitoTracker Green (0.2 uM). ln probably not electrically connected, since distinct individual control test experiments, dissipation of membrane potential was mitochondria can be depolarized by photo-oxidative stress. checked after addition ofthe respiratory inhibitor antimycin A This finding confirms that mitochondria within adult (5 uM) in combination with the uncoupler FCCP (4 uM), which cardiomyocytes are electrically unconnected, under normal, always strongly decreased TMRM fluorescence (data not non-pathological conditions. These results will also be

shown). Under conditions of nomially polarized mitochondria, consistent with differential functional properties among incubation of cells with TMRM results in its efficient mitochondria within the network (Collins et al. 2002; Collins accumulation within the organelle and strong fluorescent signal and Bootman 2003; Kuznetsov et al. 2006) but, at the same (red fluorescence). However, under stressful conditions (e.g. by time, being metabolically synchronized during excitation/ photodynamic ROS production during laser irradiation), contraction cycles. It has been shown before (Aon et al. mitochondria may lose their membrane potential due to 2003,2006, 2007) that in cardiomyocytes mitochondria form pemieability transition (opening of permeability transition a regular network where they are connected by chemical pore) (Zorov et al. 2000). In this case, as shown in Fig. 4, messengers through diffusion. Under physiological condithe collapse of membrane potential of distinct mitochondria is tions, diffusion of reactive oxygen species (ROS) is likely to detected as a strong decrease in TMRM fluorescence, be effective in the micrometer range among local neighborrevealing green spots of only lVlitoTracke@ Green labeled ing mitochondria. Q sprrrrger