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The molecular phenotype of human cardiac myosin associated with hypertrophic obstructive cardiomyopathy.

Jacques AM, Briceno N, Messer AE, Gallon CE, Jalilzadeh S, Garcia E, Kikonda-Kanda G, Goddard J, Harding SE, Watkins H, Esteban MT, Tsang VT, McKenna WJ, Marston SB - Cardiovasc. Res. (2008)

Bottom Line: Failing heart myosin showed 18% reduced motility.One myectomy myosin sample produced a consistently higher sliding speed than donor heart myosin and was identified with a disease-causing heavy chain mutation (V606M).Contractility in myectomy samples resembles the hypocontractile phenotype found in end-stage failing heart muscle irrespective of the primary stimulus, and this phenotype is not a direct effect of the hypertrophy-inducing mutation.

View Article: PubMed Central - PubMed

Affiliation: Cardiac Medicine, National Heart and Lung Institute, Imperial College London, Guy Scadding Building, Dovehouse Street, London, UK.

ABSTRACT

Aim: The aim of the study was to compare the functional and structural properties of the motor protein, myosin, and isolated myocyte contractility in heart muscle excised from hypertrophic cardiomyopathy patients by surgical myectomy with explanted failing heart and non-failing donor heart muscle.

Methods: Myosin was isolated and studied using an in vitro motility assay. The distribution of myosin light chain-1 isoforms was measured by two-dimensional electrophoresis. Myosin light chain-2 phosphorylation was measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using Pro-Q Diamond phosphoprotein stain.

Results: The fraction of actin filaments moving when powered by myectomy myosin was 21% less than with donor myosin (P = 0.006), whereas the sliding speed was not different (0.310 +/- 0.034 for myectomy myosin vs. 0.305 +/- 0.019 microm/s for donor myosin in six paired experiments). Failing heart myosin showed 18% reduced motility. One myectomy myosin sample produced a consistently higher sliding speed than donor heart myosin and was identified with a disease-causing heavy chain mutation (V606M). In myectomy myosin, the level of atrial light chain-1 relative to ventricular light chain-1 was 20 +/- 5% compared with 11 +/- 5% in donor heart myosin and the level of myosin light chain-2 phosphorylation was decreased by 30-45%. Isolated cardiomyocytes showed reduced contraction amplitude (1.61 +/- 0.25 vs. 3.58 +/- 0.40%) and reduced relaxation rates compared with donor myocytes (TT(50%) = 0.32 +/- 0.09 vs. 0.17 +/- 0.02 s).

Conclusion: Contractility in myectomy samples resembles the hypocontractile phenotype found in end-stage failing heart muscle irrespective of the primary stimulus, and this phenotype is not a direct effect of the hypertrophy-inducing mutation. The presence of a myosin heavy chain mutation causing hypertrophic cardiomyopathy can be predicted from a simple functional assay.

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Related in: MedlinePlus

Isolation of myosin from human heart muscle. SDS–PAGE of (A) high salt extract of muscle myofibrils, (B) supernatant containing myosin and troponin–tropomyosin after ultracentrifugation, and (C) pure myosin sedimented at low salt concentration.
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CVN094F2: Isolation of myosin from human heart muscle. SDS–PAGE of (A) high salt extract of muscle myofibrils, (B) supernatant containing myosin and troponin–tropomyosin after ultracentrifugation, and (C) pure myosin sedimented at low salt concentration.

Mentions: Fifty milligrams of human cardiac tissue were crushed using a liquid-nitrogen-cooled percussion mortar and homogenized in 0.25 mL ice-cold high salt extraction buffer (0.3 mol/L KCl, 0.15 mol/L K2HPO4, 10 mmol/L Na4P2O7, 1 mmol/L MgCl2, 1 mmol/L MgATP, 10 mmol/L DTT, 3 µg/mL each of protease inhibitors, chymostatin, pepstatin A, leupeptin, and E64, pH 6.8). The homogenate was extracted at 4°C for 1 h on a rotating mixer and then centrifuged for 20 min at 337 000 g. The supernatant was collected and diluted ×20 with ice-cold 5 mmol/L DTT and allowed to precipitate on ice for 1 h. It was then spun for 20 min at 26 000 g at 4°C to collect the precipitated myosin.8 The pellet obtained was dissolved in 250 µL of resuspension buffer (0.3 mol/L KCl, 25 mmol/L imidazole, pH 7.4, 1 mmol/L EGTA, 4 mmol/L MgCl2, 10 mmol/L DTT, 1.4 µL each of 2.5 mg/mL chymostatin, pepstatin, leupeptin, and E64). The concentration of myosin was determined using the Bradford assay and the purity was assessed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE; Figure 2). Using this method, 1 mg of myosin can be obtained from 50 mg of tissue. Skeletal muscle actin and human cardiac muscle tropomyosin and troponin were prepared as previously described.17 The preparation of myofibrils from human heart samples is described in the Supplementary material.


The molecular phenotype of human cardiac myosin associated with hypertrophic obstructive cardiomyopathy.

Jacques AM, Briceno N, Messer AE, Gallon CE, Jalilzadeh S, Garcia E, Kikonda-Kanda G, Goddard J, Harding SE, Watkins H, Esteban MT, Tsang VT, McKenna WJ, Marston SB - Cardiovasc. Res. (2008)

Isolation of myosin from human heart muscle. SDS–PAGE of (A) high salt extract of muscle myofibrils, (B) supernatant containing myosin and troponin–tropomyosin after ultracentrifugation, and (C) pure myosin sedimented at low salt concentration.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2492731&req=5

CVN094F2: Isolation of myosin from human heart muscle. SDS–PAGE of (A) high salt extract of muscle myofibrils, (B) supernatant containing myosin and troponin–tropomyosin after ultracentrifugation, and (C) pure myosin sedimented at low salt concentration.
Mentions: Fifty milligrams of human cardiac tissue were crushed using a liquid-nitrogen-cooled percussion mortar and homogenized in 0.25 mL ice-cold high salt extraction buffer (0.3 mol/L KCl, 0.15 mol/L K2HPO4, 10 mmol/L Na4P2O7, 1 mmol/L MgCl2, 1 mmol/L MgATP, 10 mmol/L DTT, 3 µg/mL each of protease inhibitors, chymostatin, pepstatin A, leupeptin, and E64, pH 6.8). The homogenate was extracted at 4°C for 1 h on a rotating mixer and then centrifuged for 20 min at 337 000 g. The supernatant was collected and diluted ×20 with ice-cold 5 mmol/L DTT and allowed to precipitate on ice for 1 h. It was then spun for 20 min at 26 000 g at 4°C to collect the precipitated myosin.8 The pellet obtained was dissolved in 250 µL of resuspension buffer (0.3 mol/L KCl, 25 mmol/L imidazole, pH 7.4, 1 mmol/L EGTA, 4 mmol/L MgCl2, 10 mmol/L DTT, 1.4 µL each of 2.5 mg/mL chymostatin, pepstatin, leupeptin, and E64). The concentration of myosin was determined using the Bradford assay and the purity was assessed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE; Figure 2). Using this method, 1 mg of myosin can be obtained from 50 mg of tissue. Skeletal muscle actin and human cardiac muscle tropomyosin and troponin were prepared as previously described.17 The preparation of myofibrils from human heart samples is described in the Supplementary material.

Bottom Line: Failing heart myosin showed 18% reduced motility.One myectomy myosin sample produced a consistently higher sliding speed than donor heart myosin and was identified with a disease-causing heavy chain mutation (V606M).Contractility in myectomy samples resembles the hypocontractile phenotype found in end-stage failing heart muscle irrespective of the primary stimulus, and this phenotype is not a direct effect of the hypertrophy-inducing mutation.

View Article: PubMed Central - PubMed

Affiliation: Cardiac Medicine, National Heart and Lung Institute, Imperial College London, Guy Scadding Building, Dovehouse Street, London, UK.

ABSTRACT

Aim: The aim of the study was to compare the functional and structural properties of the motor protein, myosin, and isolated myocyte contractility in heart muscle excised from hypertrophic cardiomyopathy patients by surgical myectomy with explanted failing heart and non-failing donor heart muscle.

Methods: Myosin was isolated and studied using an in vitro motility assay. The distribution of myosin light chain-1 isoforms was measured by two-dimensional electrophoresis. Myosin light chain-2 phosphorylation was measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using Pro-Q Diamond phosphoprotein stain.

Results: The fraction of actin filaments moving when powered by myectomy myosin was 21% less than with donor myosin (P = 0.006), whereas the sliding speed was not different (0.310 +/- 0.034 for myectomy myosin vs. 0.305 +/- 0.019 microm/s for donor myosin in six paired experiments). Failing heart myosin showed 18% reduced motility. One myectomy myosin sample produced a consistently higher sliding speed than donor heart myosin and was identified with a disease-causing heavy chain mutation (V606M). In myectomy myosin, the level of atrial light chain-1 relative to ventricular light chain-1 was 20 +/- 5% compared with 11 +/- 5% in donor heart myosin and the level of myosin light chain-2 phosphorylation was decreased by 30-45%. Isolated cardiomyocytes showed reduced contraction amplitude (1.61 +/- 0.25 vs. 3.58 +/- 0.40%) and reduced relaxation rates compared with donor myocytes (TT(50%) = 0.32 +/- 0.09 vs. 0.17 +/- 0.02 s).

Conclusion: Contractility in myectomy samples resembles the hypocontractile phenotype found in end-stage failing heart muscle irrespective of the primary stimulus, and this phenotype is not a direct effect of the hypertrophy-inducing mutation. The presence of a myosin heavy chain mutation causing hypertrophic cardiomyopathy can be predicted from a simple functional assay.

Show MeSH
Related in: MedlinePlus