Subcellular Ca2+ distribution with varying Ca2+ load in neonatal cardiac cell culture.
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Recent work in our laboratory has investigated and modeled subcellular calcium compartmentation and Ca2+ movement under steady-state control conditions. This experimental study is directed to the further description and quantitation of cellular calcium compartmentation patterns and movements as correlated with contraction in neonatal rat cardiac myocytes in culture under a variety of calcium loading conditions. Compartmental contents were assessed after incubations in various [Ca2+]o, 0 Na+/1 mM Ca2+, and 10 microM ouabain/1.0 mM Ca2+ test solutions. The cellular components investigated include sarcolemmal bound, sarcoplasmic reticulum (SR), and mitochondrial calcium. The results indicate that 1) sarcolemmal calcium binding is insensitive to changes in [Ca2+]o in the range tested (0.25-6.0 mM) while highly sensitive to changes in [Na+]i; 2) SR is sensitive to both changes in [Ca2+]o and [Na+]i and exhibits a maximum loading capacity of approximately 750 micromol Ca2+/kg dw; 3) in the [Ca2+]o range between 0.25 and 2.0 mM, contractile amplitude is proportional to SR content; 4) the mitochondria comprise a high-capacity calcium-containing compartment that is sensitive to changes in [Ca2+]o but does not reach saturation under the conditions tested (0.25-8.0 mM [Ca2+]o); 5) SR calcium is divided into at least two functionally discrete pools, one of which is available for release to the myofilaments during a normal ICa-triggered contraction and other of which is caffeine releasable but unavailable for release to the myofilaments during a normal triggered release; and 6) mitochondrial calcium serves as a reservoir of calcium capable of replenishing and/or augmenting SR stores with anywhere from 10% to 50% of mitochondrial calcium cycling through SR calcium compartments. (+info)
Differential expression of stress proteins in rat myocardium after free wheel or treadmill run training.
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High-intensity treadmill exercise increases the expression of a cardioprotective, inducible 72-kDa stress protein (SP72) in cardiac muscle. This investigation examined whether voluntary free wheel exercise training would be sufficient to confer a similar response. Male Sprague-Dawley rats were randomly assigned to either treadmill (TM-Tr) or free wheel (FW-Tr) training groups. By the end of the 8-wk training period, TM-Tr animals ran 1 h/day, 5 days/wk up a 10% grade, covering a distance of 8,282 m/wk. FW-Tr rats ran, on average, 5,300 m/wk, with one-third of the animals covering distances similar to those for the TM-Tr group. At the time of death, hearts of trained and caged sedentary control (Sed) animals were divided into left (LV) and right (RV) ventricles. Citrate synthase activity and the relative immunoblot contents of SP72, SP73 (the constitutive isoform of the SP70 family), and a 75-kDa mitochondrial chaperone (SP75) were subsequently determined. LV and RV did not differ on any measure, and SP73, SP75, and citrate synthase were not affected by training. Cardiac SP72 levels were elevated over fourfold in both ventricles of TM-Tr compared with RV of FW-Sed rats. Despite the animals having run a similar total distance, cardiac SP72 content in FW-Tr rats was not different from that in Sed animals. These data indicate that voluntary exercise training is insufficient to elicit an elevation of SP72 in rat heart and suggest that exercise intensity may be a critical factor in evoking the cardioprotective SP72 response. (+info)
The 20 C-terminal amino acid residues of the chloroplast ATP synthase gamma subunit are not essential for activity.
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It has been suggested that the last seven to nine amino acid residues at the C terminus of the gamma subunit of the ATP synthase act as a spindle for rotation of the gamma subunit with respect to the alpha beta subunits during catalysis (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). To test this hypothesis we selectively deleted C-terminal residues from the chloroplast gamma subunit, two at a time starting at the sixth residue from the end and finishing at the 20th residue from the end. The mutant gamma genes were overexpressed in Escherichia coli and assembled with a native alpha3beta3 complex. All the mutant forms of gamma assembled as effectively as the wild-type gamma. Deletion of the terminal 6 residues of gamma resulted in a significant increase (>50%) in the Ca-dependent ATPase activity when compared with the wild-type assembly. The increased activity persisted even after deletion of the C-terminal 14 residues, well beyond the seven residues proposed to form the spindle. Further deletions resulted in a decreased activity to approximately 19% of that of the wild-type enzyme after deleting all 20 C-terminal residues. The results indicate that the tip of the gammaC terminus is not essential for catalysis and raise questions about the role of the C terminus as a spindle for rotation. (+info)
Sarcolemmal versus mitochondrial ATP-sensitive K+ channels and myocardial preconditioning.
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Ischemic preconditioning (IPC) is a phenomenon in which single or multiple brief periods of ischemia have been shown to protect the heart against a more prolonged ischemic insult, the result of which is a marked reduction in myocardial infarct size, severity of stunning, or incidence of cardiac arrhythmias. Although a number of substances and signaling pathways have been proposed to be involved in mediating the cardioprotective effect of IPC, the overwhelming majority of evidence suggests that the ATP-sensitive potassium channel (KATP channel) is an important component of this phenomenon and may serve as the end effector in this process. Initially, it was hypothesized that the surface or sarcolemmal KATP (sarc KATP) channel mediated protection observed after IPC; however, subsequent evidence suggested that the recently identified mitochondrial KATP channel (mito KATP) may be the potassium channel mediating IPC-induced cardioprotection. In this review, evidence will be presented supporting a role for either the sarc KATP or the mito KATP in IPC and potential mechanisms by which opening these channels may produce cardioprotection; additionally, we will address important questions that still need to be investigated to define the role of the sarc or mito KATP channel, or both, in cardiac pathophysiology. (+info)
Pharmacological and histochemical distinctions between molecularly defined sarcolemmal KATP channels and native cardiac mitochondrial KATP channels.
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A variety of direct and indirect techniques have revealed the existence of ATP-sensitive potassium (KATP) channels in the inner membranes of mitochondria. The molecular identity of these mitochondrial KATP (mitoKATP) channels remains unclear. We used a pharmacological approach to distinguish mitoKATP channels from classical, molecularly defined cardiac sarcolemmal KATP (surfaceKATP) channels encoded by the sulfonylurea receptor SUR2A and the pore-forming subunit Kir6.2. SUR2A and Kir6.2 were expressed in human embryonic kidney (HEK)293 cells, and their activities were measured by patch-clamp recordings of membrane current. SurfaceKATP channels are activated potently by 100 microM pinacidil but only weakly by 100 microM diazoxide; in addition, they are blocked by 10 microM glibenclamide, but are insensitive to 500 microM 5-hydroxydecanoate. This pharmacology, which was confirmed with patch-clamp recordings in intact rabbit ventricular myocytes, contrasts with that of mitoKATP channels as indexed by flavoprotein oxidation. MitoKATP channels in myocytes are activated equally by 100 microM diazoxide and 100 microM pinacidil. In contrast to its lack of effect on surfaceKATP channels, 5-hydroxydecanoate is an effective blocker of mitoKATP channels. Glibenclamide's effects on mitoKATP channels are difficult to assess, because it independently activates flavoprotein fluorescence, consistent with a previously described primary uncoupling effect. Confocal imaging of the subcellular distribution of expressed fluorescent Kir6.2 in HEK cells and in myocytes revealed no targeting of mitochondrial membranes. The differences in drug sensitivity and subcellular localization indicate that mitoKATP channels are distinct from surface KATP channels at a molecular level. (+info)
Direct membrane insertion of voltage-dependent anion-selective channel protein catalyzed by mitochondrial Tom20.
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Insertion of newly synthesized proteins into or across the mitochondrial outer membrane is initiated by import receptors at the surface of the organelle. Typically, this interaction directs the precursor protein into a preprotein translocation pore, comprised of Tom40. Here, we show that a prominent beta-barrel channel protein spanning the outer membrane, human voltage- dependent anion-selective channel (VDAC), bypasses the requirement for the Tom40 translocation pore during biogenesis. Insertion of VDAC into the outer membrane is unaffected by plugging the translocation pore with a partially translocated matrix preprotein, and mitochondria containing a temperature-sensitive mutant of Tom40 insert VDAC at the nonpermissive temperature. Synthetic liposomes harboring the cytosolic domain of the human import receptor Tom20 efficiently insert newly synthesized VDAC, resulting in transbilayer transport of ATP. Therefore, Tom20 transforms newly synthesized cytosolic VDAC into a transmembrane channel that is fully integrated into the lipid bilayer. (+info)
Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis.
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BACKGROUND: The importance of free radical homeostasis and apoptosis in normal and diseased hearts and their interrelationships are poorly defined. We tested whether reactive oxygen species can trigger apoptosis in cardiomyocytes, and we explored the underlying pathways. METHODS AND RESULTS: A cell culture model of isolated cardiac cells and different reactive oxygen species (ROS)-generating systems were used. Apoptosis became evident when cardiomyocytes were exposed to either H2O2 or superoxide anion (O2-). Both H2O2- and O2--induced apoptosis of cardiomyocytes were associated with an increase in p53 protein content, whereas protein levels of Bax and Bcl-2 were unaltered. H2O2, but not O2-, induced an increase in the protein content of Bad. Furthermore, H2O2 elicited translocation of Bax and Bad from cytosol to mitochondria, where these factors formed heterodimers with Bcl-2, which was followed by the release of cytochrome c, activation of CPP32, and cleavage of poly(ADP-ribose) polymerase. Interestingly, this pathway was not activated by O2-. Instead, O2- used Mch2alpha to promote the apoptotic pathway, as revealed by the activation of Mch2alpha and the cleavage of its substrate, lamin A. CONCLUSIONS: Taken together, these results indicate that ROS may play an important pathophysiological role in cardiac diseases characterized by apoptotic cell death and suggest that different ROS-induced activations of the apoptotic cell death program in cardiomyocytes involve distinct signaling pathways. (+info)
Declines in mitochondrial respiration during cardiac reperfusion: age-dependent inactivation of alpha-ketoglutarate dehydrogenase.
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We previously reported that cardiac reperfusion results in declines in mitochondrial NADH-linked respiration. The degree of inactivation increased with age and was paralleled by modification of protein by the lipid peroxidation product 4-hydroxy-2-nonenal. To gain insight into potential sites of oxidative damage, the present study was undertaken to identify specific mitochondrial protein(s) inactivated during ischemia and reperfusion and to determine which of these losses in activity are responsible for observed declines in mitochondrial respiration. Using a Langendorff rat heart perfusion protocol, we observed age-dependent inactivation of complex I during ischemia and complex IV and alpha-ketoglutarate dehydrogenase during reperfusion. Although losses in complex I and IV activities were found not to be of sufficient magnitude to cause declines in mitochondrial respiration, an age-related decrease in complex I activity during ischemia may predispose old animals to more severe oxidative damage during reperfusion. It was determined that inactivation of alpha-ketoglutarate dehydrogenase is responsible, in large part, for observed reperfusion-induced declines in NADH-linked respiration. alpha-Ketoglutarate dehydrogenase is highly susceptible to 4-hydroxy-2-nonenal inactivation in vitro. Thus, our results suggest a plausible mechanism for age-dependent, reperfusion-induced declines in mitochondrial function and identify alpha-ketoglutarate dehydrogenase as a likely site of free radical-mediated damage. (+info)