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(Circulation. 1999;100:974-980.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Effects of Mutant and Antisense RNA of Phospholamban on SR Ca²⁺-ATPase Activity and Cardiac Myocyte Contractility

Huaping He, PhD; Markus Meyer, MD; Jody L. Martin, PhD; Patrick M. McDonough, PhD; Peter Ho, BS; Xiaojing Lou, MD; Wilbur Y. W. Lew, MD; Randa Hilal-Dandan, PhD; Wolfgang H. Dillmann, MD

From the Department of Medicine, University of California San Diego, La Jolla (H.H., M.M., J.L.M., X.L., R.H.-D., W.H.D.); the Department of Biology, San Diego State University (P.M.M., P.H.); and the Department of Medicine, Veteran's Affairs Medical Center–San Diego (W.Y.W.L.), Calif. Dr He is now at the Scripps Research Institute, La Jolla, Calif.

Correspondence to Wolfgang H. Dillmann, MD, Department of Medicine, 0618, University of California San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0618. E-mail wdillmann{at}ucsd.edu

Background—The delayed cardiac relaxation in failing hearts has been attributed to a reduced activity of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2). Phospholamban (PLB) inhibits SERCA2 activity and is therefore a potential target to improve the cardiac performance in heart failure.

Methods and Results—Mutants of PLB (Adv/mPLB) or antisense RNA of PLB (Adv/asPLB) was expressed in cardiac myocytes by recombinant adenovirus, and their effects on SERCA2 activity and myocyte contractility were studied. One mPLB, K3E/R14E, pentamerized with endogenous PLB in neonatal myocytes and resulted in a 45% increase in the affinity of SERCA2 for Ca²⁺ and 27% faster diastolic Ca²⁺ decline as determined by SR ⁴⁵Ca uptake assays and by indo 1–facilitated Ca²⁺ transient measurement, respectively. Edge-detection analysis of adult myocyte contractility showed a 74% increase in fractional shortening, accompanied by 115% increase in velocity of relengthening and 25% decrease in time to half-maximal relengthening. In parallel, infection of neonatal cardiac myocytes by Adv/asPLB decreased the endogenous PLB level by 54%, which was associated with a 35% increase in Ca²⁺ affinity of SERCA2 and 21% faster diastolic Ca²⁺ decline. However, in adult cardiac myocytes, Adv/asPLB failed to significantly alter the endogenous PLB level, the SERCA2 activity, or most of the contractile parameters.

Conclusions—K3E/R14E is a dominant negative mutant of PLB that disrupts the structural integrity and function of the endogenous PLB and consequently enhances SERCA2 activity and myocyte contractility. In neonatal myocytes, the decrease in steady-state abundance of PLB by asPLB also leads to increased SERCA2 activity.

Key Words: phospholamban • enzymes • sarcoplasmic reticulum • RNA • viruses

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