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SUMMARY:Annexins II, V, and VI belong to a family of Ca 2ϩ -dependent phospholipid-binding proteins that have been involved mainly in signal transduction, differentiation, membrane trafficking events, or binding to the extracellular matrix, or that might be effective as Ca 2ϩ -channels. They are abundant in the mammalian myocardium and might play a role in ventricular remodeling and altered calcium handling during heart failure. To test this hypothesis, we compared the expression and distribution of these annexins in nonfailing (n ϭ 9) and failing human hearts with idiopathic dilated cardiomyopathy (n ϭ 11). Northern blot and slot blot analysis were used to determine the annexin mRNA levels and Western blots were used to quantify the amounts of annexin proteins. Distribution of annexins was studied by immunohistofluorescence labeling and compared with that of a sarcolemmal marker (Na ϩ /K ϩ -ATPase) and of a myofibrillar protein (␣-actinin). We showed that nonfailing hearts contained a higher amount of annexin VI than of annexin V or II (13.5 Ϯ 1.8, 3.7 Ϯ 0.2, and 2.5 Ϯ 0.5 g/mg protein, respectively). In failing hearts, there was a parallel increase in both mRNA and protein levels of annexin II (146% and 132%, p Ͻ 0.05, respectively) and annexin V (152%, p Ͻ 0.01, 147%, p Ͻ 0.005, respectively); the protein level of annexin VI was also increased (117%, p Ͻ 0.05), whereas the increase of its mRNA level was statistically insignificant. We observed a predominant localization of annexin II in interstitium, and of annexins V and VI in cardiomyocytes at the level of the sarcolemma, T-tubules, and intercalated disks in nonfailing hearts, whereas in failing hearts enlarged interstitium contained all three annexins. Furthermore, annexin V staining at the level of cardiomyocytes almost disappeared. In conclusion, we showed that heart failure is accompanied by marked overexpression of annexins II and V, as well as translocation of annexin V from cardiomyocytes to interstitial tissue. The data suggest that annexins may contribute to ventricular remodeling and annexin V to impaired Ca 2ϩ handling in failing heart. (Lab Invest 2000, 80:123-133).

This study indicated for the first time that annexin A5 was externalized at a very early stage of apoptosis and could have a proapoptotic effect in cardiomyocytes.

Chemically skinned (by treatment with saponin, 40 μg/ml) isolated cardiomyocytes were used to study the intracellular diffusion of ADP and creatine (Cr). Stimulation of respiration was studied in these cardiomyocytes without intact sarcolemma and in isolated heart mitochondrial by addition of ADP and Cr in the presence of 0.2 mM ATP (via mitochondrial creatine kinase reaction: Cr + MgATP = MgADP + PCr). The Michaelis constant (Km) for Cr was similar in both cases, 5.67 ± 0.11 (SD) mM in skinned myocytes and 6.9 ± 0.2 mM in mitochondria, showing that there is no significant restriction to the diffusion of this substrate. However, the apparent Km for external ADP increased from 17.6 ± 1.0 μM for mitochondria to 250 ± 38 μM for skinned cardiomyocytes, showing decreased diffusivity of ADP as a result of binding to cellular structures. In the presence of 25 mM Cr, the Km for ADP for myocytes decreased to 35.6 ± 5.6 μM due to the coupling of the creatine kinase and oxidative phosphorylation reactions. Provision of substrate for the creatine kinase reaction amplified the weak ADP signal in the regulation of respiration. The activity of the mitochondrial creatine kinase was decreased by a factor of two in cardiomyopathic hamsters and human hearts and was associated with a twofold decrease in creatine-stimulated respiration. These data show a potentially key role of mitochondrial creatine kinase in the regulation of cellular respiration and the possible importance of changes in its activity for the functional disturbances of the cardiomyopathic heart. cellular respiration; creatine kinase; oxidative phosphorylati

No abstract

Chemically skinned (by treatment with saponin, 40 μg/ml) isolated cardiomyocytes were used to study the intracellular diffusion of ADP and creatine (Cr). Stimulation of respiration was studied in these cardiomyocytes without intact sarcolemma and in isolated heart mitochondrial by addition of ADP and Cr in the presence of 0.2 mM ATP (via mitochondrial creatine kinase reaction: Cr + MgATP = MgADP + PCr). The Michaelis constant (Km) for Cr was similar in both cases, 5.67 ± 0.11 (SD) mM in skinned myocytes and 6.9 ± 0.2 mM in mitochondria, showing that there is no significant restriction to the diffusion of this substrate. However, the apparent Km for external ADP increased from 17.6 ± 1.0 μM for mitochondria to 250 ± 38 μM for skinned cardiomyocytes, showing decreased diffusivity of ADP as a result of binding to cellular structures. In the presence of 25 mM Cr, the Km for ADP for myocytes decreased to 35.6 ± 5.6 μM due to the coupling of the creatine kinase and oxidative phosphorylation reactions. Provision of substrate for the creatine kinase reaction amplified the weak ADP signal in the regulation of respiration. The activity of the mitochondrial creatine kinase was decreased by a factor of two in cardiomyopathic hamsters and human hearts and was associated with a twofold decrease in creatine-stimulated respiration. These data show a potentially key role of mitochondrial creatine kinase in the regulation of cellular respiration and the possible importance of changes in its activity for the functional disturbances of the cardiomyopathic heart. cellular respiration; creatine kinase; oxidative phosphorylation

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Monceau, Virginie, Yulia Belikova, Gueorgui Kratassiouk, Estelle Robidel, Françoise Russo-Marie, and Daniele Charlemagne. Myocyte apoptosis during acute myocardial infarction in rats is related to early sarcolemmal translocation of annexin A5 in border zone. Am J Physiol Heart Circ Physiol 291: H965-H971, 2006. First published February 24, 2006 doi:10.1152/ajpheart.01053.2005.-Annexin A5 is a Ca 2ϩ -dependent phospholipid binding protein well known for its high phosphatidylserine affinity. In vitro, translocation to sarcolemma and externalization of endogenous annexin A5 in the cardiomyocyte has recently been demonstrated to exert a proapoptotic effect. To determine whether these in vitro findings occurred in vivo, we performed myocardial infarction (MI) and studied the time course of apoptosis and annexin A5 localization (0.5 to 8 h) in the border zone around the infarcted area. This zone that was defined as Evans blue unstained and triphenyltetrazolium chloride (TTC) stained, represented 42.3 Ϯ 5.5% of the area at risk and showed apoptotic characteristics (significant increases in caspase 3 activity 2.3-fold at 0.5 h; P Ͻ 0.05), transferase-mediated dUTP nick-end labelingpositive cardiomyocytes (15.8 Ϯ 0.8% at 8 h), and DNA ladder. When compared with sham-operated rats, we found that in this area, annexin A5 was translocated to the sarcolemma as early as 0.5 h after MI and that translocation increased with time. Moreover, the amount of annexin A5 was unchanged in the border zone and decreased in the infarcted area after 1 h (77.1 Ϯ 4.8%; P Ͻ 0.01 vs. perfused area), suggesting a release in the latter but not in the former. In conclusion, we demonstrated that annexin A5 translocation is an early and rapid event of the whole border zone, likely due to Ca 2ϩ increase. Part of this translocation occurred in areas where apoptosis was later detected and suggests that in vivo as in vitro annexin A5 might be involved in the regulation of early apoptotic events during cardiac pathological situations. myocardial ischemia; terminal deoxyribonucleotide transferase ANNEXINS are a structurally related family of proteins known to bind phospholipids in a Ca 2ϩ