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Decreased myocardial injury and improved contractility after administration of a peptide derived against the alpha-interacting domain of the L-type calcium channel.

Viola HM, Jordan MC, Roos KP, Hool LC - J Am Heart Assoc (2014)

Bottom Line: Activation of the channel also alters mitochondrial function.In search of the mechanism for the effect, we found that intracellular calcium ([Ca(2+)]i, Fura-2), superoxide production (dihydroethidium fluorescence), mitochondrial membrane potential (Ψm, JC-1 fluorescence), reduced nicotinamide adenine dinucleotide production, and flavoprotein oxidation (autofluorescence) are decreased after application of AID-TAT peptide.Application of AID-TAT peptide significantly decreased infarct size and supported contractility up to 12 weeks postcoronary artery occlusion as a result of a decrease in metabolic demand during reperfusion.

View Article: PubMed Central - PubMed

Affiliation: School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, WA, Australia (H.M.V., L.C.H.).

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Effect of AID‐TAT peptide on calcium influx in vitro. A, Representative traces of ratiometric Fura‐2 fluorescence recorded in a myocyte before and after 5 minutes of exposure to 40 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase and a myocyte before and after 5 minutes of exposure to 0 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase. Vertical arrows indicate when drugs were added. B, Mean±SEM of changes in ratiometric Fura‐2 fluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, and 10 μmol/L of nisoldipine (Nisol) as indicated. C, Representative traces of ratiometric Fura‐2 fluorescence recorded in myocytes before and after 5 minutes of exposure to 40 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase in the presence of either 1 or 10 μmol/L of AID(S)‐TAT or AID‐TAT peptide as indicated. Vertical arrows indicate when drugs were added. D, Mean±SEM of changes in ratiometric Fura‐2 fluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, 1 or 10 μmol/L of AID(S)‐TAT or AID‐TAT peptide, and 10 μmol/L of nisoldipine (Nisol) as indicated. Statistical significance was determined using the Kruskal‐Wallis test followed by Dunn's multiple comparison test. Inset above: mean±SEM of changes in ratiometric Fura‐2 fluorescence for myocytes before and 30 and 60 minutes after exposure to 10 μmol/L of AID‐TAT peptide as indicated. AID indicates alpha‐interacting domain; TAT, transactivator of transcription.
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fig03: Effect of AID‐TAT peptide on calcium influx in vitro. A, Representative traces of ratiometric Fura‐2 fluorescence recorded in a myocyte before and after 5 minutes of exposure to 40 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase and a myocyte before and after 5 minutes of exposure to 0 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase. Vertical arrows indicate when drugs were added. B, Mean±SEM of changes in ratiometric Fura‐2 fluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, and 10 μmol/L of nisoldipine (Nisol) as indicated. C, Representative traces of ratiometric Fura‐2 fluorescence recorded in myocytes before and after 5 minutes of exposure to 40 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase in the presence of either 1 or 10 μmol/L of AID(S)‐TAT or AID‐TAT peptide as indicated. Vertical arrows indicate when drugs were added. D, Mean±SEM of changes in ratiometric Fura‐2 fluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, 1 or 10 μmol/L of AID(S)‐TAT or AID‐TAT peptide, and 10 μmol/L of nisoldipine (Nisol) as indicated. Statistical significance was determined using the Kruskal‐Wallis test followed by Dunn's multiple comparison test. Inset above: mean±SEM of changes in ratiometric Fura‐2 fluorescence for myocytes before and 30 and 60 minutes after exposure to 10 μmol/L of AID‐TAT peptide as indicated. AID indicates alpha‐interacting domain; TAT, transactivator of transcription.

Mentions: Because AID‐TAT peptide slows the inactivation rate of ICa‐L current,19–20 we examined the effect of the peptide on changes in [Ca2+]i after activation of the channel. We and others have shown previously that application of H2O2 can directly increase ICa‐L current.4,10–11 As little as 5 minutes of exposure to 30 to 50 μmol/L of H2O2 can cause further release of superoxide from the mitochondria without inducing apoptosis or necrosis and is sufficient to increase protein synthesis, consistent with the development of myocyte hypertrophy.10,12 ROS‐induced ROS‐release is proposed to occur during reperfusion and can lead to mitochondrial permeability transition pore opening and arrhythmia.27–29 We mimicked the OS associated with reperfusion by applying 40 μmol/L of H2O2 to ventricular myocytes for 5 minutes, followed by 10 U/mL of CAT to degrade the H2O2. Consistent with previous studies,10 addition of H2O2 resulted in a small, but significant, increase in [Ca2+]i assessed as changes in Fura‐2 that could be attenuated with addition of the ICa‐L antagonist, nisoldipine (Figure 3A and 3B). Application of 1 μmol/L of AID‐TAT peptide had no effect on the increase in Fura‐2 signal induced by 40 μmol/L of H2O2. However, the addition of 10 μmol/L of AID‐TAT peptide attenuated the increase in Fura‐2 signal induced by H2O2 by ≈75% (Figure 3C and 3D). Application of AID‐TAT peptide did not alter basal Fura‐2 signal, indicating that the peptide did not prevent calcium influx through the alpha subunit of the channel (Figure 3D, inset right). These data suggest that 10 μmol/L, but not 1 μmol/L, of AID‐TAT peptide can alter ICa‐L‐activated calcium influx in myocytes.


Decreased myocardial injury and improved contractility after administration of a peptide derived against the alpha-interacting domain of the L-type calcium channel.

Viola HM, Jordan MC, Roos KP, Hool LC - J Am Heart Assoc (2014)

Effect of AID‐TAT peptide on calcium influx in vitro. A, Representative traces of ratiometric Fura‐2 fluorescence recorded in a myocyte before and after 5 minutes of exposure to 40 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase and a myocyte before and after 5 minutes of exposure to 0 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase. Vertical arrows indicate when drugs were added. B, Mean±SEM of changes in ratiometric Fura‐2 fluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, and 10 μmol/L of nisoldipine (Nisol) as indicated. C, Representative traces of ratiometric Fura‐2 fluorescence recorded in myocytes before and after 5 minutes of exposure to 40 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase in the presence of either 1 or 10 μmol/L of AID(S)‐TAT or AID‐TAT peptide as indicated. Vertical arrows indicate when drugs were added. D, Mean±SEM of changes in ratiometric Fura‐2 fluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, 1 or 10 μmol/L of AID(S)‐TAT or AID‐TAT peptide, and 10 μmol/L of nisoldipine (Nisol) as indicated. Statistical significance was determined using the Kruskal‐Wallis test followed by Dunn's multiple comparison test. Inset above: mean±SEM of changes in ratiometric Fura‐2 fluorescence for myocytes before and 30 and 60 minutes after exposure to 10 μmol/L of AID‐TAT peptide as indicated. AID indicates alpha‐interacting domain; TAT, transactivator of transcription.
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Related In: Results  -  Collection

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fig03: Effect of AID‐TAT peptide on calcium influx in vitro. A, Representative traces of ratiometric Fura‐2 fluorescence recorded in a myocyte before and after 5 minutes of exposure to 40 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase and a myocyte before and after 5 minutes of exposure to 0 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase. Vertical arrows indicate when drugs were added. B, Mean±SEM of changes in ratiometric Fura‐2 fluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, and 10 μmol/L of nisoldipine (Nisol) as indicated. C, Representative traces of ratiometric Fura‐2 fluorescence recorded in myocytes before and after 5 minutes of exposure to 40 μmol/L of H2O2 followed by 5 minutes of exposure to 10 U/mL of catalase in the presence of either 1 or 10 μmol/L of AID(S)‐TAT or AID‐TAT peptide as indicated. Vertical arrows indicate when drugs were added. D, Mean±SEM of changes in ratiometric Fura‐2 fluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, 1 or 10 μmol/L of AID(S)‐TAT or AID‐TAT peptide, and 10 μmol/L of nisoldipine (Nisol) as indicated. Statistical significance was determined using the Kruskal‐Wallis test followed by Dunn's multiple comparison test. Inset above: mean±SEM of changes in ratiometric Fura‐2 fluorescence for myocytes before and 30 and 60 minutes after exposure to 10 μmol/L of AID‐TAT peptide as indicated. AID indicates alpha‐interacting domain; TAT, transactivator of transcription.
Mentions: Because AID‐TAT peptide slows the inactivation rate of ICa‐L current,19–20 we examined the effect of the peptide on changes in [Ca2+]i after activation of the channel. We and others have shown previously that application of H2O2 can directly increase ICa‐L current.4,10–11 As little as 5 minutes of exposure to 30 to 50 μmol/L of H2O2 can cause further release of superoxide from the mitochondria without inducing apoptosis or necrosis and is sufficient to increase protein synthesis, consistent with the development of myocyte hypertrophy.10,12 ROS‐induced ROS‐release is proposed to occur during reperfusion and can lead to mitochondrial permeability transition pore opening and arrhythmia.27–29 We mimicked the OS associated with reperfusion by applying 40 μmol/L of H2O2 to ventricular myocytes for 5 minutes, followed by 10 U/mL of CAT to degrade the H2O2. Consistent with previous studies,10 addition of H2O2 resulted in a small, but significant, increase in [Ca2+]i assessed as changes in Fura‐2 that could be attenuated with addition of the ICa‐L antagonist, nisoldipine (Figure 3A and 3B). Application of 1 μmol/L of AID‐TAT peptide had no effect on the increase in Fura‐2 signal induced by 40 μmol/L of H2O2. However, the addition of 10 μmol/L of AID‐TAT peptide attenuated the increase in Fura‐2 signal induced by H2O2 by ≈75% (Figure 3C and 3D). Application of AID‐TAT peptide did not alter basal Fura‐2 signal, indicating that the peptide did not prevent calcium influx through the alpha subunit of the channel (Figure 3D, inset right). These data suggest that 10 μmol/L, but not 1 μmol/L, of AID‐TAT peptide can alter ICa‐L‐activated calcium influx in myocytes.

Bottom Line: Activation of the channel also alters mitochondrial function.In search of the mechanism for the effect, we found that intracellular calcium ([Ca(2+)]i, Fura-2), superoxide production (dihydroethidium fluorescence), mitochondrial membrane potential (Ψm, JC-1 fluorescence), reduced nicotinamide adenine dinucleotide production, and flavoprotein oxidation (autofluorescence) are decreased after application of AID-TAT peptide.Application of AID-TAT peptide significantly decreased infarct size and supported contractility up to 12 weeks postcoronary artery occlusion as a result of a decrease in metabolic demand during reperfusion.

View Article: PubMed Central - PubMed

Affiliation: School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, WA, Australia (H.M.V., L.C.H.).

Show MeSH
Related in: MedlinePlus