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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 flavoprotein oxidation in vitro. A, Representative traces of flavoprotein autofluorescence 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. Carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP; 10 μmol/L) was added to increase flavoprotein signal, confirming the signal was mitochondrial in origin. B, Mean±SEM of changes flavoprotein autofluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, 10 μmol/L of nisoldipine (Nisol), and 10 μmol/L of FCCP as indicated. C, Representative traces of flavoprotein autofluorescence 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 derived against the AID of the L‐type calcium channel as indicated. Vertical arrows indicate when drugs were added. FCCP (10 μmol/L) was added to increase flavoprotein signal, confirming the signal was mitochondrial in origin. D, Mean±SEM of changes in flavoprotein autofluorescence 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. E, Exposure of guinea pig myocytes to BayK(–) causes a significant increase in formation of formazan from tetrozolium salt that can be attenuated by nisoldipine and by AID‐TAT peptide.19 AID indicates alpha‐interacting domain; TAT, transactivator of transcription.
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fig07: Effect of AID‐TAT peptide on flavoprotein oxidation in vitro. A, Representative traces of flavoprotein autofluorescence 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. Carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP; 10 μmol/L) was added to increase flavoprotein signal, confirming the signal was mitochondrial in origin. B, Mean±SEM of changes flavoprotein autofluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, 10 μmol/L of nisoldipine (Nisol), and 10 μmol/L of FCCP as indicated. C, Representative traces of flavoprotein autofluorescence 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 derived against the AID of the L‐type calcium channel as indicated. Vertical arrows indicate when drugs were added. FCCP (10 μmol/L) was added to increase flavoprotein signal, confirming the signal was mitochondrial in origin. D, Mean±SEM of changes in flavoprotein autofluorescence 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. E, Exposure of guinea pig myocytes to BayK(–) causes a significant increase in formation of formazan from tetrozolium salt that can be attenuated by nisoldipine and by AID‐TAT peptide.19 AID indicates alpha‐interacting domain; TAT, transactivator of transcription.

Mentions: We assessed effects of the peptide on Ψm assessed as changes in JC‐1 fluorescence. Consistent with previous work,10,19,30 application of H2O2 increased JC‐1 signal and the response could be attenuated with nisoldipine (Figure 6A and 6B). Collapse of the signal with application of NaCN demonstrated that the signal was mitochondrial in origin. Application of 10 μmol/L of AID‐TAT peptide significantly decreased the signal induced by H2O2 (Figure 6C and 6D). Addition of H2O2 to myocytes also increased flavoprotein oxidation measured as flavoprotein autofluorescence (Figure 7A and 7B). An increase in flavoprotein autofluorescence is indicative of an increase in flavoprotein oxidation in myocytes. It is also a measure of oxygen consumption in myocytes. The response is dependent on Ψm, and addition of the cyanide derivative, carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP), significantly increased flavoprotein signal (Figure 7A through 7C).25 Application of 10 μmol/L of AID‐TAT significantly decreased flavoprotein fluorescence, indicating that the peptide decreased flavoprotein oxidation induced by H2O2 (Figure 7C and 7D). Consistent with this result, we have previously demonstrated that application of 1 μmol/L of AID‐TAT peptide decreased the formation of formazan from tetrozolium salt in guinea pig ventricular myocytes induced by activation of the channel with the dihydropyridine agonist, BayK(–) (Figure 7E).19 This is a measure of oxygen consumption, suggesting that the peptide can decrease metabolic activity 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 flavoprotein oxidation in vitro. A, Representative traces of flavoprotein autofluorescence 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. Carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP; 10 μmol/L) was added to increase flavoprotein signal, confirming the signal was mitochondrial in origin. B, Mean±SEM of changes flavoprotein autofluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, 10 μmol/L of nisoldipine (Nisol), and 10 μmol/L of FCCP as indicated. C, Representative traces of flavoprotein autofluorescence 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 derived against the AID of the L‐type calcium channel as indicated. Vertical arrows indicate when drugs were added. FCCP (10 μmol/L) was added to increase flavoprotein signal, confirming the signal was mitochondrial in origin. D, Mean±SEM of changes in flavoprotein autofluorescence 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. E, Exposure of guinea pig myocytes to BayK(–) causes a significant increase in formation of formazan from tetrozolium salt that can be attenuated by nisoldipine and by AID‐TAT peptide.19 AID indicates alpha‐interacting domain; TAT, transactivator of transcription.
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fig07: Effect of AID‐TAT peptide on flavoprotein oxidation in vitro. A, Representative traces of flavoprotein autofluorescence 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. Carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP; 10 μmol/L) was added to increase flavoprotein signal, confirming the signal was mitochondrial in origin. B, Mean±SEM of changes flavoprotein autofluorescence for all myocytes exposed to 40 μmol/L of H2O2, 10 U/mL of catalase, 10 μmol/L of nisoldipine (Nisol), and 10 μmol/L of FCCP as indicated. C, Representative traces of flavoprotein autofluorescence 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 derived against the AID of the L‐type calcium channel as indicated. Vertical arrows indicate when drugs were added. FCCP (10 μmol/L) was added to increase flavoprotein signal, confirming the signal was mitochondrial in origin. D, Mean±SEM of changes in flavoprotein autofluorescence 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. E, Exposure of guinea pig myocytes to BayK(–) causes a significant increase in formation of formazan from tetrozolium salt that can be attenuated by nisoldipine and by AID‐TAT peptide.19 AID indicates alpha‐interacting domain; TAT, transactivator of transcription.
Mentions: We assessed effects of the peptide on Ψm assessed as changes in JC‐1 fluorescence. Consistent with previous work,10,19,30 application of H2O2 increased JC‐1 signal and the response could be attenuated with nisoldipine (Figure 6A and 6B). Collapse of the signal with application of NaCN demonstrated that the signal was mitochondrial in origin. Application of 10 μmol/L of AID‐TAT peptide significantly decreased the signal induced by H2O2 (Figure 6C and 6D). Addition of H2O2 to myocytes also increased flavoprotein oxidation measured as flavoprotein autofluorescence (Figure 7A and 7B). An increase in flavoprotein autofluorescence is indicative of an increase in flavoprotein oxidation in myocytes. It is also a measure of oxygen consumption in myocytes. The response is dependent on Ψm, and addition of the cyanide derivative, carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP), significantly increased flavoprotein signal (Figure 7A through 7C).25 Application of 10 μmol/L of AID‐TAT significantly decreased flavoprotein fluorescence, indicating that the peptide decreased flavoprotein oxidation induced by H2O2 (Figure 7C and 7D). Consistent with this result, we have previously demonstrated that application of 1 μmol/L of AID‐TAT peptide decreased the formation of formazan from tetrozolium salt in guinea pig ventricular myocytes induced by activation of the channel with the dihydropyridine agonist, BayK(–) (Figure 7E).19 This is a measure of oxygen consumption, suggesting that the peptide can decrease metabolic activity 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