Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure.

Gwathmey, Judith K.; Copelas, L; MacKinnon, Roderick; Schoen, F J; Feldman, Marc D.; Grossman, William; Morgan, James P.

doi:10.1161/01.res.61.1.70

Cited by 798 publications

(380 citation statements)

References 15 publications

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“…It is referred to as (I27) 5 throughout this study. (I27) 5 was expressed and purified as described in Brockwell et al (34). (0.1 mol) was mixed with an excess of sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) cross-linker (5 mol) in buffer containing 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2.…”

Section: Methodsmentioning

confidence: 99%

“…Final product was stored at Ϫ20°C. The same procedure was followed to conjugate SERCA2a peptide (YLE-PAILE) to (I27) 5 concatamer. Protein concentration was determined by a BCA assay (35).…”

Section: Methodsmentioning

confidence: 99%

“…34). Purified phosphoepitope peptide was conjugated to the (I27) 5 concatamer via the only cysteine residue in the protein (C47 in I27 domain 3, presented schematically in Fig. 3A), and the stoichiometry of covalent attachment of the peptide was evaluated by mass spectrometry.…”

Section: Serca Ps-38 Recognition Of a Calibration Standard-hav-mentioning

confidence: 99%

“…Fig. 3C shows that antibody SERCA PS-38 recognized the concatamer product decorated with the relevant phosphopeptide (calibration-38), but did not recognize the same concatamer (I27) 5 decorated with an irrelevant peptide (calibration-␣CLEP) even when 60 pmol of concatamer was presented. The calibration standard migrates as a single molecular species of ϳ60 kDa in SDS-PAGE.…”

Section: Serca Ps-38 Recognition Of a Calibration Standard-hav-mentioning

confidence: 99%

“…B, calibration-38 composition described by electrospray mass spectroscopy. The product (calibration-38) is separated from substrate (I27) 5 components and a contaminant. Components marked with * were included in the calculation of product yield (5.4%).…”

Section: Fig 3 Specific Recognition Of a Serine 38 Calibration Stanmentioning

confidence: 99%

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Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

Rodríguez¹,

Jackson²,

Colyer

2004

Journal of Biological Chemistry

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The phosphorylation of the cardiac muscle isoform of the sarcoplasmic reticulum (SR) Ca 2؉ -ATPase (SERCA2a) on serine 38 has been described as a regulatory event capable of very significant enhancement of enzyme activity (Hawkins, C., Xu, A., and Narayanan, N. (1994) J. Biol. Chem. 269, 31198 -31206). Independent confirmation of these observations has not been forthcoming. This study has utilized a polyclonal antibody specific for the phosphorylated serine 38 epitope on the Ca 2؉ -ATPase to evaluate the phosphorylation of SERCA2a in isolated sarcoplasmic reticulum vesicles and isolated rat ventricular myocytes. A quantitative Western blot approach failed to detect serine 38-phosphorylated Ca 2؉ -ATPase in either kinase-treated sarcoplasmic reticulum vesicles or suitably stimulated cardiac myocytes. Calibration standards confirmed that the detection sensitivity of assays was adequate to detect Ser-38 phosphorylation if it occurred on at least 1% of Ca 2؉ -ATPase molecules in SR vesicle experiments or on at least 0.1% of Ca 2؉ -ATPase molecules in cardiac myocytes. The failure to detect a phosphorylated form of the Ca 2؉ -ATPase in either preparation (isolated myocyte, purified sarcoplasmic reticulum vesicles) suggests that Ser-38 phosphorylation of the Ca 2؉ -ATPase is not a significant regulatory feature of cardiac Ca 2؉ homeostasis.Regulation of Ca 2ϩ sequestration by cardiac sarcoplasmic reticulum has been identified as a key control point in cardiac muscle contraction (1, 2). Stimulation of the rate of Ca 2ϩ uptake occurs during exercise, or following ␤-adrenergic stimulation (3) and is associated with an enhanced force of contraction and an increased rate of relaxation. This accounts for much of the positive inotropic and positive lusitropic effects of these interventions. In contrast, Ca 2ϩ sequestration by cardiac SR 1 is abnormally slow in the muscle of individuals with heart failure (4 -6). In animal studies, normalization of the rate of Ca 2ϩ sequestration has been shown to prevent progression of heart failure (7) and thus the molecular mechanism of control of Ca 2ϩ transport into the sarcoplasmic reticulum has become a focus for research into purposeful therapies to combat human heart failure (7,8).Ca 2ϩ transport into cardiac SR is an enzymatic process performed by the (Ca 2ϩ -Mg 2ϩ )-ATPase (9) (or SERCA2a, Ref. 10), a member of the P-type ATPase family (11). The transport process involves the movement of two Ca 2ϩ from the cytoplasm into the lumen of the SR following the hydrolysis of a single molecule of ATP (12), although significantly lower coupling efficiencies have been measured empirically (13). The reaction cycle is relatively slow and thus a large number of SERCA2 molecules are expressed in cardiac muscle (up to 45% of total SR protein content, 14) to achieve the rates of Ca 2ϩ sequestration required by the kinetics of contraction and relaxation.Regulation of Ca 2ϩ transport into the SR occurs on an acute time scale (seconds to minutes) through the transient modification of the proteins inv...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Serca Ps-38 Recognition Of a Calibration Standard-hav-mentioning

confidence: 99%

Section: Serca Ps-38 Recognition Of a Calibration Standard-hav-mentioning

confidence: 99%

Section: Fig 3 Specific Recognition Of a Serine 38 Calibration Stanmentioning

confidence: 99%

See 3 more Smart Citations

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

Rodríguez¹,

Jackson²,

Colyer

2004

Journal of Biological Chemistry

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Right ventricular diastolic performance: Compliance characteristics with focus on pulmonary hypertension, right ventricular hypertrophy, and calcium channel blockade

Ferlinz

1998

Cathet. Cardiovasc. Diagn.

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Gene Therapy in Heart Failure

Hayward¹,

Patel²,

Welch³

et al. 2017

Encyclopedia of Life Sciences

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Heart failure is a chronic condition leading to debilitating symptoms and reduced life expectancy. Several pharmacological therapies which improve symptoms and mortality have been developed over the past 30 years. These therapies are, however, limited in terms of both efficacy and side‐effect profile. Gene therapy represents a fundamental change in the way treatments are developed with the potential to directly target and correct individual molecular abnormalities. These therapies have the potential to offer long‐term beneficial effects from a single treatment and, with direct targeting, less off‐target effects. As with any new therapy, a number of challenges must be overcome before gene therapy can be considered a realistic option to patients with heart failure. Key Concepts Gene therapy could allow direct targeting of molecular abnormalities in patients with heart failure. Preclinical data suggests that in animal models of heart failure, SERCA2a gene therapy significantly improves haemodynamic parameters, reduces arrhythmia risk and corrects molecular abnormalities. Initial clinical data in a small number of patients suggested that SERCA2a gene therapy was beneficial to patients with advanced heart failure. However, a recent study in a larger number of patients was disappointingly neutral. A number of molecular abnormalities exist in the failing myocytes, each of which could represent a potential target to treat heart failure. Gene therapy requires a large financial commitment from pharmaceutical companies and cost is a major limitation to the development of gene therapy products.

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Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure.

Cited by 798 publications

References 15 publications

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

Right ventricular diastolic performance: Compliance characteristics with focus on pulmonary hypertension, right ventricular hypertrophy, and calcium channel blockade

Gene Therapy in Heart Failure

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