Accumulation of mitochondrial electron transport chain (ETC) defects is a recognized hallmark of the age-associated decline in cardiac bioenergetics; however, the molecular events involved are only poorly understood. In the present work, we hypothesized that age-related ETC deterioration stemmed partly from disassociation of large solid-state macromolecular assemblies termed "supercomplexes". Mitochondrial proteins from young and old rat hearts were separated by Blue Native-PAGE, protein bands analyzed by LC-MALDI-MS/MS, and protein levels quantified by densitometry. Results showed that supercomplexes comprised of various stoichiometries of complexes I, III and IV were observed, and declined significantly (p < 0.05, n = 4) with age. Supercomplexes displaying the highest molecular masses were the most severely affected. Considering that certain diseases (e.g. Barth Syndrome) display similar supercomplex destabilization as our results for aging, the deterioration in ETC supercomplexes may be an important underlying factor for both impaired mitochondrial function and loss of cardiac bioenergetics with age.

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Inflammation results in heightened mitochondrial ceramide levels, which cause electron transport chain dysfunction, elevates reactive oxygen species, and increases apoptosis. As mitochondria in aged hearts also display many of these characteristics, we hypothesized that mitochondrial decay stems partly from an age-related ceramidosis that heretofore has not been recognized for the heart. Intact mitochondria or their purified inner membranes (IMM) were isolated from young (4-6 mo) and old (26-28 mo) rats and analyzed for ceramides by LC-MS/MS. Results showed that ceramide levels increased by 32% with age and three ceramide isoforms, found primarily in the IMM (e.g. C16-, C18-, and C24:1-ceramide), caused this increase. The ceramidosis may stem from enhanced hydrolysis of sphingomyelin, as neutral sphingomyelinase (nSMase) activity doubled with age but with no attendant change in ceramidase activity. Because (R)-α-lipoic acid (LA) improves many parameters of cardiac mitochondrial decay in aging and lowers ceramide levels in vascular endothelial cells, we hypothesized that LA may limit cardiac ceramidosis and thereby improve mitochondrial function. Feeding LA [0.2% wt/wt] to old rats for two weeks prior to mitochondrial isolation reversed the age-associated decline in glutathione levels and concomitantly improved Complex IV activity. This improvement was associated with lower nSMase activity and a remediation in mitochondrial ceramide levels. In summary, LA treatment lowers ceramide levels to that seen in young rat heart mitochondria and restores Complex IV activity which otherwise declines with age.

Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary (IFM) and subsarcolemmal (SSM) mitochondria were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (~10,000 pmols/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (~70 pmols/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16 to 24 carbon units in their acyl side-chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmols/mg protein). Three ceramide isotypes (C 20 -, C 22 -, and C 24 -ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.

Abstract— The effect of external calcium level, calcium ionophore A23187 and red light on the circadian rhythm of Robinia pseudoacacia leaflet movements has been studied. Fifteen minute red light pulses shifted the phase of leaflet rhythmic movement with a phase‐response curve type 0. Maximum advances and delays (about 10 h and 8 h, respectively) were obtained between circadian time (CT) 10 and CT 12 at the end of a subjective day. An almost null effect was obtained at the end of a subjective night. Phytochrome is the photoreceptor involved in phase shifting since this effect of red light is reversed by 5 min of far red light. Two hour pulses of external calcium, applied as CaCl2 (10 mM), and 2 h pulses of calcium ionophore A23187 (10–50 μM) also shifted the phase of leaflet circadian movement and caused the same type of phase‐response curve, with maximum advances and delays at the same time as those produced by red light. Two hour pulses of an external calcium chelator, EGTA (5 mM), and a calcium channel blocker, LaCl3 (10–50 mM), damped the circadian rhythm or did not change the phase when they were applied at lower concentration. These results indicate that phytochrome could control the circadian oscillator, which drives Robinia leaflet movements by increasing the intracellular calcium concentration.