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

Abstract

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...

I K1 and I f in ventricular myocytes isolated from control and hypertrophied rat hearts

“…The resulting cell suspensions were filtered through a 250-μm nylon mesh and centrifuged for 4 min at 20×g. Finally, the cell pellets were divided into two fractions; one was suspended in a storage solution containing 0.1 mM of CaCl 2 and 2 mg/ml of BSA (Sigma) and stored at room temperature until use for electrophysiological experiments (within 5 h of the isolation), and another fraction was counted using a Neubauer chamber and resuspended in concentrated Laemmli (5×) sample buffer [19]. Cell suspensions were immediately stored at −40°C until Western blot analysis.…”

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

“…Some reports attributed enhanced SERCA V max to CaMKII dependent SERCA phosphorylation [43,44]. However, such a direct CaMKIIdependent SERCA phosphorylation has been strongly challenged by others [45][46][47].…”

Integrative omics analysis of the mechanisms underlying left ventricular diastolic dysfunction in cynomolgus monkeys with spontaneous type 2 diabetes mellitus

“…They indicated that decreased SUMOylation and increased acetylation of SERCA2a were observed in human and animal heart failure models and correlated with the reduced activity of SERCA2a. Moreover, SERCA2a phosphorylation at Ser38, which is part of the CaMKII consensus site, has been identified [59,60]; however, Ser38-phosphorylated SERCA2a has not been detected in the sarcoplasmic reticulum vesicles, stimulated cardiac myocytes or in in vivo animal models [61,62]. Ours is the first study, however, to report of a new phosphosite, SERCA2a Ser626 in DDD monkeys, of which we found the phosphorylation to be downregulated despite no changes in phospholamban expression, and its potential correlation with cardiac contraction and relaxation.…”