Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human
Kobra Haghighi, … , Dimitrios T. Kremastinos, Evangelia G. Kranias
Kobra Haghighi, … , Dimitrios T. Kremastinos, Evangelia G. Kranias
Published March 15, 2003
Citation Information: J Clin Invest. 2003;111(6):869-876. https://doi.org/10.1172/JCI17892.
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Article Cardiology

Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human

Abstract

In human disease and experimental animal models, depressed Ca2+ handling in failing cardiomyocytes is widely attributed to impaired sarcoplasmic reticulum (SR) function. In mice, disruption of the PLN gene encoding phospholamban (PLN) or expression of dominant-negative PLN mutants enhances SR and cardiac function, but effects of PLN mutations in humans are unknown. Here, a T116G point mutation, substituting a termination codon for Leu-39 (L39stop), was identified in two families with hereditary heart failure. The heterozygous individuals exhibited hypertrophy without diminished contractile performance. Strikingly, both individuals homozygous for L39stop developed dilated cardiomyopathy and heart failure, requiring cardiac transplantation at ages 16 and 27. An over 50% reduction in PLN mRNA and no detectable PLN protein were noted in one explanted heart. The expression of recombinant PLN-L39stop in human embryonic kidney (HEK) 293 cells and adult rat cardiomyocytes showed no PLN inhibition of SR Ca2+-ATPase and the virtual absence of stable PLN expression; where PLN was expressed, it was misrouted to the cytosol or plasma membrane. These findings describe a naturally-occurring loss-of-function human PLN mutation (PLN null). In contrast to reported benefits of PLN ablation in mouse heart failure, humans lacking PLN develop lethal dilated cardiomyopathy.

Authors

Kobra Haghighi, Fotis Kolokathis, Luke Pater, Roy A. Lynch, Michio Asahi, Anthony O. Gramolini, Guo-Chang Fan, Dimitris Tsiapras, Harvey S. Hahn, Stamatis Adamopoulos, Stephen B. Liggett, Gerald W. Dorn II, David H. MacLennan, Dimitrios T. Kremastinos, Evangelia G. Kranias

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Figure 5

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Expression and localization of PLN-WT and PLN-L39stop mutant in HEK-293 ...
Expression and localization of PLN-WT and PLN-L39stop mutant in HEK-293 cells. (a) Immunoblot analyses of endoplasmic reticulum microsomes (left panel) and insoluble fractions (right panel) obtained at 24 and 48 hours after transfections. Note the absence of PLN-L39stop in the endoplasmic reticulum fraction, although detectable protein levels are found in the insoluble fraction. PLNp, PLN pentamer; PLNm, PLN monomer. (b) Quantitation of the number of fluorescent cells on PLN-WT and PLN-L39stop transfected coverslips. Cultures were transfected with equal amounts of plasmid DNA, and 48 hours later the number of fluorescent cells was counted on the entire coverslip. (c) Immunofluorescence of PLN-WT and PLN-L39stop transfected cells analyzed by confocal microscopy 48 hours after transfection. In PLN-WT transfected cells, immunofluorescence is exclusively in the endoplasmic reticulum, whereas a clearly distinct and plasma membrane–associated staining pattern is observed in the PLN-L39stop transfected cells. Scale bar, 30 μm. (d) Fluorescence intensity was quantitated using available Leica software. For these assays, a straight profile line was drawn across the center of the cell and fluorescence amplitude was plotted. Arrows mark the edge of the cell and asterisks mark the ER. In PLN-WT transfected cells (upper panel), immunofluorescence is found within the interior of the cell, whereas in PLN-L39stop transfected cells (lower panel), staining is found enriched at the outer edge of the cell.