The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K-ATPase

Kobra Haghighi, Tracy Pritchard, Julie B C Bossuyt, Jason R. Waggoner, Qunying Yuan, Guo Chang Fan, Hanna Osinska, Ahmad Anjak, Jack Rubinstein, Jeffrey Robbins, Donald M Bers, Evangelia G. Kranias

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO, and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman. Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.

Original languageEnglish (US)
Pages (from-to)773-782
Number of pages10
JournalJournal of Molecular and Cellular Cardiology
Volume52
Issue number3
DOIs
StatePublished - Mar 2012

Fingerprint

Cell Membrane
Sarcoplasmic Reticulum
Cardiac Myocytes
phospholamban
sodium-translocating ATPase
Dilated Cardiomyopathy
Basic Amino Acids
Sarcolemma
Fluorescence Resonance Energy Transfer
Oxalates
Consensus Sequence
Cardiomegaly
Isoproterenol
Confocal Microscopy
Nucleic Acids
Arginine
Heart Failure
Phosphorylation

Keywords

  • Calcium cycling
  • Heart failure
  • Mutation
  • Phospholamban
  • SERCA

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

Cite this

The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K-ATPase. / Haghighi, Kobra; Pritchard, Tracy; Bossuyt, Julie B C; Waggoner, Jason R.; Yuan, Qunying; Fan, Guo Chang; Osinska, Hanna; Anjak, Ahmad; Rubinstein, Jack; Robbins, Jeffrey; Bers, Donald M; Kranias, Evangelia G.

In: Journal of Molecular and Cellular Cardiology, Vol. 52, No. 3, 03.2012, p. 773-782.

Research output: Contribution to journalArticle

Haghighi, K, Pritchard, T, Bossuyt, JBC, Waggoner, JR, Yuan, Q, Fan, GC, Osinska, H, Anjak, A, Rubinstein, J, Robbins, J, Bers, DM & Kranias, EG 2012, 'The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K-ATPase', Journal of Molecular and Cellular Cardiology, vol. 52, no. 3, pp. 773-782. https://doi.org/10.1016/j.yjmcc.2011.11.012
Haghighi, Kobra ; Pritchard, Tracy ; Bossuyt, Julie B C ; Waggoner, Jason R. ; Yuan, Qunying ; Fan, Guo Chang ; Osinska, Hanna ; Anjak, Ahmad ; Rubinstein, Jack ; Robbins, Jeffrey ; Bers, Donald M ; Kranias, Evangelia G. / The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K-ATPase. In: Journal of Molecular and Cellular Cardiology. 2012 ; Vol. 52, No. 3. pp. 773-782.
@article{30ced9d592494e6aa4ea0c91b0e86323,
title = "The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K-ATPase",
abstract = "Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO, and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman. Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.",
keywords = "Calcium cycling, Heart failure, Mutation, Phospholamban, SERCA",
author = "Kobra Haghighi and Tracy Pritchard and Bossuyt, {Julie B C} and Waggoner, {Jason R.} and Qunying Yuan and Fan, {Guo Chang} and Hanna Osinska and Ahmad Anjak and Jack Rubinstein and Jeffrey Robbins and Bers, {Donald M} and Kranias, {Evangelia G.}",
year = "2012",
month = "3",
doi = "10.1016/j.yjmcc.2011.11.012",
language = "English (US)",
volume = "52",
pages = "773--782",
journal = "Journal of Molecular and Cellular Cardiology",
issn = "0022-2828",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K-ATPase

AU - Haghighi, Kobra

AU - Pritchard, Tracy

AU - Bossuyt, Julie B C

AU - Waggoner, Jason R.

AU - Yuan, Qunying

AU - Fan, Guo Chang

AU - Osinska, Hanna

AU - Anjak, Ahmad

AU - Rubinstein, Jack

AU - Robbins, Jeffrey

AU - Bers, Donald M

AU - Kranias, Evangelia G.

PY - 2012/3

Y1 - 2012/3

N2 - Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO, and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman. Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.

AB - Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO, and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman. Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.

KW - Calcium cycling

KW - Heart failure

KW - Mutation

KW - Phospholamban

KW - SERCA

UR - http://www.scopus.com/inward/record.url?scp=84857637211&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84857637211&partnerID=8YFLogxK

U2 - 10.1016/j.yjmcc.2011.11.012

DO - 10.1016/j.yjmcc.2011.11.012

M3 - Article

VL - 52

SP - 773

EP - 782

JO - Journal of Molecular and Cellular Cardiology

JF - Journal of Molecular and Cellular Cardiology

SN - 0022-2828

IS - 3

ER -