Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.582642
Title: Structural and functional polymorphisms of troponin in failing heart
Author: Messer, Andrew Easton
Awarding Body: University of London
Current Institution: Imperial College London
Date of Award: 2007
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Abstract:
Previous work has indicated that in failing heart muscle the defect in contractility is primarily due to altered troponin function. Troponin has been isolated from non-failing (donor) and end-stage failing human heart muscle. Using in vitro motility assays we have found an increase in Ca2+ -sensitivity (EC50 decreased 2.6-fold) and a 13% reduction in cross-bridge turnover rate for troponin from failing heart muscle. In vitro motility analysis of thin filaments containing a replaced unphosphorylated troponin I resulted in a failing phenotype of a reduced sliding speed and an increased calcium sensitivity. When replaced with a PKA phosphorylated troponin I, motility parameters reverted to a pattern indistinguishable from non-failing troponin. Measurement of total phosphorylation using the Pro-Q Diamond phosphoprotein gel stain showed troponin T phosphorylation was 3.05 ± 0.20 mol Pi / mol protein in non- failing and 3.11 ± 0.47 in failing heart troponin. Troponin I phosphorylation level was 2.25 ± 0.31 mol Pi / mol protein in non-failing and 0.38 ± 0.16 in failing. Measurement of phosphorylation at troponin I serine 23/24 was determined using a phospho-specific antibody and yielded 1.14 ± 0.09 mol Pi / mol in non-failing and 0.19 ± 0.04 in failing. The difference between Pro-Q and serine 23/24 measurements is ascribed to phosphorylation at PKC sites on troponin. It was 1.11 ± 0.23 mol Pi / mol protein in non- failing heart and 0.18 ± 0.16 in failing. Thus the level of phosphorylation of troponin I in failing heart is substantially reduced at both PKA-specific and PKC-specific sites. It has been reported that PKC activity is increased in failing hearts and therefore, it is possible that phosphatase activity may be increased. Using recombinant human cardiac troponin I phosphorylated with PKA and [y32p]_ATP as a substrate we found substantial troponin I-specific phosphatase activity in washed myofibrils from human heart. We observed that the activity in myofibrils from failing heart was 19.3 ± 7.4% higher than from non-failing heart and that the dephosphorylating ability is predominantly type PP2a as it was 95% inhibited by 2nM okadaic acid. In conclusion, we showed that decreased troponin I phosphorylation in failing heart was correlated with higher Ca2+ -sensitivity and lower cross-bridge turnover rate and that troponin I is dephosphorylated at PKA specific sites in end-stage heart failure and could account for the contractile defect seen. We also studied HCM heart muscle obtained by surgical myectomy and several animal models of heart failure. It appears that this relationship between troponin function and phosphorylation does not apply in several of these cases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.582642  DOI: Not available
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