A mutation in the human phospholamban gene, deleting arginine 14, results in lethal, hereditary cardiomyopathy

Abstract

The sarcoplasmic reticulum Ca(2+)-cycling proteins are key regulators of cardiac contractility, and alterations in sarcoplasmic reticulum Ca(2+)-cycling properties have been shown to be causal of familial cardiomyopathies. Through genetic screening of dilated cardiomyopathy patients, we identified a previously uncharacterized deletion of arginine 14 (PLN-R14Del) in the coding region of the phospholamban (PLN) gene in a large family with hereditary heart failure. No homozygous individuals were identified. By middle age, heterozygous individuals developed left ventricular dilation, contractile dysfunction, and episodic ventricular arrhythmias, with overt heart failure in some cases. Transgenic mice overexpressing the mutant PLN-R14Del recapitulated human cardiomyopathy exhibiting similar histopathologic abnormalities and premature death. Coexpression of the normal and mutant-PLN in HEK-293 cells resulted in sarcoplasmic reticulum Ca(2+)-ATPase superinhibition. The dominant effect of the PLN-R14Del mutation could not be fully removed, even upon phosphorylation by protein kinase A. Thus, by chronic suppression of sarcoplasmic reticulum Ca(2+)-ATPase activity, the nonreversible superinhibitory function of mutant PLN-R14Del may lead to inherited dilated cardiomyopathy and premature death in both humans and mice.

Fig. 1.

Analysis of inheritance of the PLN-R14Del mutation and the cardiomyopathic phenotype and cardiac histology of affected individuals. (A) Pedigree for the presence or absence of AGA (R14) in the PLN gene. Squares represent males, and circles represent females. Slash denotes deceased. (B) Histological analysis of cardiac biopsies from probands V-2 and V-6 heterozygous for the R14 deletion mutation, stained with Masson's-Trichrome, illustrating the massive interstitial fibrosis and myocardial disarrangement (arrow). All images are at equal magnification. (Scale bar, 50 μm.)

Fig. 2.

Expression and localization of WT-PLN and PLN-R14Del mutant in HEK-293 cells. (A) Immunoblot analyses of WT-PLN and PLN-R14Del in transfected cell lysates. (Left) Immunoblots of boiled microsomes from cells transfected with WT-PLN and PLN-R14Del and detected by using anti-PLN antibody, 1D11. (Right) Immunoblots of boiled microsomes isolated from cells transfected with NF-PLN or NF-PLNR14Del and detected with anti-flag antibody, M2. (B) Immunoblots of unboiled microsomes of NF-PLN-R14Del (homozygote) and NF-PLN-R14Del plus WT-PLN (heterozygote) and probed with anti-flag antibody. (B Lower) Blots were striped and reprobed with anti-SERCA1 antibody. (C) Immunofluorescence of NF-PLN and NF-PLN-R14Del (heterozygote) transfected cells analyzed by confocal microscopy 48 h after transfection. In both cases, immunofluorescence is exclusively in the ER. (Scale bar, 30 μm.)

Fig. 3.

Effect of PLN and mutant PLN on the Ca²⁺ affinity of SERCA1a in HEK-293 cells. Cells were cotransfected with wild-type, homozygous, or heterozygous mutant PLN cDNA and SERCA1 cDNA, and the rates of Ca²⁺ uptake were measured. V_max, maximum velocity of Ca²⁺ uptake. (A) SERCA1 Ca²⁺ affinity is highly inhibited when cells are cotransfected with heterozygous mutant PLN-R14Del cDNA. PKA phosphorylation partially relieves the inhibitory effect of NF-PLN-R14Del plus WT-PLN (heterozygote) mutant, leaving it in the superinhibitory range. Phosphorylation of NF-PLN and NF-PLN-R14Del mutant (pPLN, phosphorylated PLN) was detected in boiled (B) and unboiled (C) microsomes in the presence and absence of PKA.

Fig. 4.

Mortality and cardiac morphology of a mouse model expressing the PLN-R14Del mutant PLN. (A) Mice carrying the PLN-R14Del mutant exhibited mortality at a young age. (B) A comparison of the hearts of strain and age-matched WT-PLN and TgPLN-R14Del overexpression mice at 6 weeks of age showed enlargement for TgPLN-R14Del only. (C) A comparison of the histopathology of left ventricular heart tissue from 6-week-old, strain-matched WT-PLN and TgPLN-R14Del mice by using Masson's trichrome for collagen showed massive interstitial fibrosis in the PLN-R14Del mouse only (arrow). All images are at equal magnification. (Scale bar, 50 μm.)

Fig. 5.

Effect of wild-type and mutant PLN-R14Del on the Ca²⁺ affinity of SERCA2a. Cardiac homogenates from WT-PLN and TgPLN-R14Del (heterozygote) were incubated in the presence or absence of ATP and PKA catalytic subunit, and then the initial rates of SR Ca²⁺ transport were measured. V_max, maximum velocity of Ca²⁺ uptake.