Abstract
Heart physiology depends on oxidative metabolism that likely requires dynamic and permanent reorganization of mitochondria by fusion and fission. To directly evaluate mitochondrial fusion dynamics in cardiomyocytes (CM), mitochondrial matrix-targeted photoactivatable GFP and DsRed were introduced either in vitro or in vivo by adenovirus and were followed by confocal microscopy. Four conditions were analyzed: 24 to 48 h cultured neonatal and in vitro transduced adult CM, and CM from in vivo infected rat hearts. In the latter case, CM were isolated 7-10 days after infection and were imaged promptly or 24-48 h post harvesting. Neonatal CM mitochondria form a highly connected network, whereas both in vitro and in vivo transformed cultured CM displayed only some connectivity. Impressively, in vivo transduced adult CM that were imaged promptly after harvesting, unveiled a significantly higher connectivity among mitochondria than the 24-48h cultured adult CM. Furthermore, fusion events (f.e./75 square micrometers/min) were almost absent in cultured in vitro transduced CM, meanwhile in vivo transduced and cultured CM showed 0.4±0.2 f.e./min, whereas isolated, freshly-imaged CM displayed 1.4± 0.1 f.e./min. Imaging in perfused whole heart ex vivo, showed considerable mitochondrial continuity and fusion activity in ventricular CM. To study more directly the role of CM's contractile activity in mitochondrial fusion, CM were incubated with Verapamil (10μM), that blocked spontaneous contraction and partially suppressed the fusion activity of mitochondria. Also, mitochondrial fusion activity appeared to be higher after spontaneous contraction or short term field stimulation in isolated freshly-imaged CM. Thus, mitochondria are dynamic in both neonatal and adult CM, but under culture conditions, adult CM lose mitochondrial fusion activity. This might be at least in part, because cardiomyocyte contractile activity is altered in culture and contractions likely provide some factors to support mitochondrial fusion activity.
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