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. 2015 Jul 1;107(1):164-74.
doi: 10.1093/cvr/cvv127. Epub 2015 Apr 7.

A novel human R25C-phospholamban mutation is associated with super-inhibition of calcium cycling and ventricular arrhythmia

Guan-Sheng Liu  1 , Ana Morales  2 , Elizabeth Vafiadaki  3 , Chi Keung Lam  1 , Wen-Feng Cai  4 , Kobra Haghighi  1 , George Adly  1 , Ray E Hershberger  5 , Evangelia G Kranias  6
Affiliations

A novel human R25C-phospholamban mutation is associated with super-inhibition of calcium cycling and ventricular arrhythmia

Guan-Sheng Liu et al. Cardiovasc Res. .

Abstract

Aims: Depressed sarcoplasmic reticulum (SR) Ca(2+) cycling, a universal characteristic of human and experimental heart failure, may be associated with genetic alterations in key Ca(2+)-handling proteins. In this study, we identified a novel PLN mutation (R25C) in dilated cardiomyopathy (DCM) and investigated its functional significance in cardiomyocyte Ca(2+)-handling and contractility.

Methods and results: Exome sequencing identified a C73T substitution in the coding region of PLN in a family with DCM. The four heterozygous family members had implantable cardiac defibrillators, and three developed prominent ventricular arrhythmias. Overexpression of R25C-PLN in adult rat cardiomyocytes significantly suppressed the Ca(2+) affinity of SR Ca(2+)-ATPase (SERCA2a), resulting in decreased SR Ca(2+) content, Ca(2+) transients, and impaired contractile function, compared with WT-PLN. These inhibitory effects were associated with enhanced interaction of R25C-PLN with SERCA2, which was prevented by PKA phosphorylation. Accordingly, isoproterenol stimulation relieved the depressive effects of R25C-PLN in cardiomyocytes. However, R25C-PLN also elicited increases in the frequency of Ca(2+) sparks and waves as well as stress-induced aftercontractions. This was accompanied by increased Ca(2+)/calmodulin-dependent protein kinase II activity and hyper-phosphorylation of RyR2 at serine 2814.

Conclusion: The findings demonstrate that human R25C-PLN is associated with super-inhibition of SERCA2a and Ca(2+) transport as well as increased SR Ca(2+) leak, promoting arrhythmogenesis under stress conditions. This is the first mechanistic evidence that increased PLN inhibition may impact both SR Ca(2+) uptake and Ca(2+) release activities and suggests that the human R25C-PLN may be a prognostic factor for increased ventricular arrhythmia risk in DCM carriers.

Keywords: Calcium cycling; Dilated cardiomyopathy; Mutation.

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Figures

Figure 1
Figure 1
Familial DCM Pedigree with a PLN mutation. Squares represent males and circles represent females. Slash denotes deceased. Darkened symbols indicate idiopathic DCM with implantable cardiac defibrillator (ICD) and grey symbols represent a significant cardiovascular abnormality. Open symbols represent negative cardiovascular history. The presence of the PLN mutation is denoted with (+) and the presence of the LMNA mutation with asterisk.
Figure 2
Figure 2
Quantitative immunoblots from infected cardiomyocytes and Ca2+ uptake assays. (A) Representative blots of PLN and SERCA. PLNp, pentameric PLN; PLNm, monomeric PLN; (B) PLN protein levels in GFP, WT and R25C cardiomyocytes expressed as relative ratio of PLN/SERCA2a (n = 4 hearts); (C) Effects of wild-type and mutant R25C-PLN on the apparent Ca2+ affinity of SERCA2a. After 24-h infection with adenoviruses, cardiomyocytes were homogenized and the initial rates of oxalate-supported SR Ca2+ uptake were measured. Data are expressed as per cent of maximal uptake rates in each group (Vmax: 99 ± 4 in GFP, 101 ± 3 in WT, and 96 ± 5 in R25C, nmol/mg/min). Inset: The average EC50 values for the three groups (n = 6 hearts). *P < 0.05, vs. GFP; †P < 0.05, vs. WT. Values are mean ± SE.
Figure 3
Figure 3
Contractile parameters in Ad.GFP, Ad.WT-PLN, and Ad.R25C-PLN infected cardiomyocytes. (A) Representative cell-shortening traces of Ad.GFP, Ad.WT-PLN, and Ad.R25C-PLN cardiomyocytes (cell-length trace represents the percentage of resting cell length); (B) Fractional shortening (FS%); (C) Maximum rates of contraction (+dL/dt); (D) Maximum rates of relengthening (−dL/dt) (20–25 cells were measured per experiment or each heart; n = 4 hearts for GFP, WT-PLN, and R25C-PLN groups). *P < 0.05, vs. GFP; †P < 0.05, vs. WT. Values are mean ± SE.
Figure 4
Figure 4
Ca2+ kinetics in Ad.GFP, Ad. WT-PLN, and Ad.R25C-PLN cardiomyocytes. Infected myocytes were incubated with Fura-2/AM for half an hour and Ca2+ transients were measured. (A) Representative tracings of Ca2+ transients; (B) Ca2+ transient amplitude in infected cardiomyocytes; (C) Time to 50% decay (T50) of Ca2+ signal; (D) Intracellular diastolic Ca2+; (E) Representative tracings of caffeine (10 mM)-induced Ca2+ transient; (F) Caffeine-induced Ca2+ transient amplitude; (G) Time to 50% decay (T50) of caffeine-induced Ca2+ transient peak (20–25 cells were measured per experiment or each heart; n = 4 hearts for GFP, WT-PLN, and R25C-PLN groups). *P < 0.05, vs. GFP; †P < 0.05, vs. WT. Values are mean ± SE.
Figure 5
Figure 5
(A) The R25C-PLN mutant co-localizes with SERCA2 in transfected HEK 293 cells, similar to WT-PLN. Nuclei are stained with DAPI. Scale bar, 5 μm; (B and C) R25C-PLN mutant exhibits enhanced association to SERCA2. Immunoprecipitation assays in HEK 293 cells that co-express GFP-PLN and SERCA2 were performed using GFP antibody. Quantification of SERCA2 levels revealed a significant increase in the SERCA2/R25C-PLN protein complex compared with GFP-WT-PLN (n = 4) values are means ± SE; *P < 0.05, compared with WT-PLN); (D and E) enhanced binding of R25C-PLN with SERCA2a was abolished upon PKA phosphorylation. Immunoprecipitations were performed in lysates from HEK 293 transfected cells that had been previously phosphorylated with PKA. Western blot analysis (D) determined similar levels of SERCA2 in both WT-PLN and R25C-PLN samples and quantitative analysis (E) showed no difference in SERCA2 binding between WT-PLN and R25C-PLN (n = 4).
Figure 6
Figure 6
Ca2+ sparks, waves, diastolic SR Ca2+ leak, and stress-induced aftercontractions (Acs) in GFP, WT-PLN, and R25C-PLN cardiomyocytes. (A) Representative line-scan and three-dimensional (3D) images of Ca2+ sparks acquired in infected cardiomyocytes; (B) Cumulative data on Ca2+ spark frequency; (C) Representative line-scan and 3D images of Ca2+ waves acquired in R25C-PLN cardiomyocytes; (D) Percentage of cells showing Ca2+ waves (10–15 cells were measured per experiment or each heart; n = 6 hearts for GFP, WT-PLN, and R25C-PLN groups); (E) Representative traces of SR Ca2+ leak were obtained from the three groups. Ca2+ leak was determined as the tertacaine sensitive drop in diastolic −2 ratio; (F) Comparison of average diastolic SR Ca2+ leak; (G) Quantification of leak/SR load relationships in GFP, WT, and R25C myocytes (ratio of twitch Ca2+ transient/caffeine-induced Ca2+ transient (10–12 cells were measured per experiment or each heart; n = 4 hearts for GFP, WT-PLN and R25C-PLN groups); *P < 0.05, vs. WT and GFP. Values are mean ± SE. (H) Representative traces of Acs; (I) Percentage of the infected cardiomyocytes that developed Acs (10–12 cells were measured per experiment or each heart; n = 6 hearts for GFP, WT-PLN, and R25C-PLN groups). *P < 0.05, vs. GFP and WT. Values are mean ± SE.
Figure 7
Figure 7
Phosphorylation of RyR2 and CaMKII activity. (A) Representative blots of phosphorylation and total levels of RyR2; (B and C) Percentage of phosphorylated Ser2808 (pSer2808) and Ser2814 (pSer2814) RyR2 in infected cardiomyocytes (n = 6 hearts); (D) CaMKII activity in GFP, WT, and R25C cardiomyocytes (n = 7 hearts); (E) Representative blots of phosphorylation and total levels of CaMKII; (F) Percentage of phosphorylated Thr286 (pT286) CaMKII in infected cardiomyocytes (n = 4 hearts); (G and H) Ca2+ spark frequency and percentage of Ca2+ waves recorded in GFP, WT, and R25C cardiomyocytes in the absence or presence of CaMKII inhibitor KN93 (1 µmol/L), with KN92 (1 µmol/L) used as a control (15–20 cells were measured per experiment or each heart; n = 4 hearts for GFP, WT-PLN, and R25C-PLN groups). *P < 0.05, vs. WT-Basal, †P < 0.05, vs. R25C-Basal. Values are mean ± SE.
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