Introduction: Phospholamban (PLB) is an integral sarcoplasmic reticulum (SR) membrane protein, which directly regulates cardiac Ca 2+ handling and contractility by reversibly inhibiting SR Ca 2+ ATPase (SERCA). Our previous studies have suggested that the naturally occurring human heart failure mutation of PLB, L39X disrupts membrane localization. Hypothesis: We hypothesize that the membrane localization of PLB is a prerequisite for PLB oligomerization and interaction with SERCA. The truncation mutations in C-terminus of PLB will disrupt membrane localization, PLB oligomerization, and SERCA regulation. Results and Methods: To identify the minimum length of PLB required for membrane localization and function, we generated a series of C-terminal transmembrane truncation mutants of PLB (tagged N-terminally with Cer or YFP) including L51X, M50X, V49X, I48X, I38X, I33X, and the heart-failure mutant L39X. Confocal microscopy revealed that progressive truncation of the C-terminal residues of PLB resulted in escalating increase in mislocalization of PLB to the cytoplasm and nucleus. In addition, we observed an increased solubilization of PLB as indicated by loss of YFP fluorescence after selective permeabilization of the plasma membrane by saponin. As expected, there was no change in localization of Cer-SERCA upon saponin permeabilization. Next, western blot analysis exhibited a decrease in molecular weight corresponding to the relative sizes of truncation mutants compared to full length PLB, indicating that protein degradation is not the cause of membrane mislocalization. Fluorescence resonance energy transfer analysis revealed that truncating the C-terminal residues of PLB results in a progressive decrease in apparent affinity of PLB oligomerization and interaction with SERCA. Finally, molecular dynamics simulations exhibited that the heart failure mutant L39X was unstable compared to full length PLB pentamer and started protruding out of the bilayer until complete solubilization. Conclusions: Truncating only two C-terminal residues of PLB resulted in significant mislocalization, while deleting five or more residues profoundly disrupted membrane localization, PLB oligomerization and SERCA regulation.
Phospholamban
SERCA
Endoplasmic-reticulum-associated protein degradation
Rationale: A naturally-occurring, missense Arg9-to-Cys (R9C) mutation of phospholamban (PLB) triggers cardiomyopathy and premature death in humans. However, the fundamental molecular mechanism underlying the cardiotoxic role of R9C-PLB in sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) regulation and cardiomyocyte Ca 2+ handling is not clear. Objective: The goal of this study was to investigate the acute physiological consequences of R9C-PLB mutation on cardiomyocyte Ca 2+ kinetics and contractility and identify the molecular mechanism underlying R9C pathology. Methods and Results: We measured the physiological consequences of R9C-PLB mutation on Ca 2+ transients and sarcomere shortening in adult cardiomyocytes at increasing pacing frequencies. In contrast to studies of chronic R9C-PLB expression in transgenic mice, we found that acute expression of R9C-PLB exerts a positively inotropic and lusitropic effect in cardiomyocytes. Importantly, R9C-PLB exhibited blunted sensitivity to frequency potentiation and β-adrenergic stimulation, two major physiological mechanisms for the regulation of cardiac performance. To identify the molecular mechanism of R9C pathology, we fused fluorescent protein tags to PLB and SERCA, and compared the effect of R9C and pentamer-destabilizing mutation (SSS) on PLB oligomerization and PLB-SERCA interaction. Fluorescence resonance energy transfer (FRET) measurements in live cells revealed that R9C exhibited an increased affinity of PLB oligomerization, and a decreased binding affinity to SERCA due to an oxidative modification which mimics phosphorylation. Real-time FRET analysis in cardiomyocytes revealed that R9C-PLB exhibits enhanced sensitivity to oxidative stress, which is a prevailing condition in heart failure. Conclusions: We conclude that the primary mechanism of R9C pathology is a phosphomimetic effect of PLB cysteine oxidation, manifested as increased oligomerization and a change in the structure of the PLB-SERCA regulatory complex.
To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val(49)-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-Å decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.
Rationale: A naturally occurring missense Leu-39stop (L39X) mutation in phospholamban (PLB) results in truncation of the C-terminal transmembrane domain, leading to cardiomyopathy and premature death in humans. Objective: The goal of this study was to determine the structural and regulatory role of the C-terminal residues of PLB in the membranes of living cells. Methods and Results: We fused fluorescent protein tags to PLB and cardiac Ca 2+ ATPase (SERCA) to investigate the role of PLB C-terminal residues for membrane localization, PLB oligomerization and SERCA regulation. Alanine substitution of C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB. Notably, substitution mutation V49A had profound effects on pentamer structure and regulatory complex conformation, increasing and decreasing probe separation distance, respectively. Progressive deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. Selective permeabilization of the plasma membrane by saponin resulted in diffusion of fluorescently labeled PLB out of the cells, consistent with solubilization of truncated proteins. Molecular dynamics simulations recapitulated decreased bilayer anchoring for truncated PLB. C-terminal truncations resulted in progressive loss of PLB-PLB FRET, due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the SERCA-PLB binding affinity, and loss of inhibitory potency as quantified by Ca 2+ -dependent ATPase activity. However, despite decreased SERCA-PLB binding, intermolecular FRET was paradoxically increased as a result of a 14.5 Å decrease in the distance between donor and acceptor fluorophores. Conclusions: We conclude that PLB C-terminal residues are critical for membrane anchoring and quaternary structure determination of PLB pentamer and PLB-SERCA regulatory complex. The loss of membrane registration restraint by C-terminal residues (especially V49) causes displacement of PLB to an alternative position on SERCA. The data are compatible with a model in which PLB binds to the canonical inhibitory binding site and an additional novel site.
Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.
Abstract Background Heart failure is a leading cause of death worldwide and is associated with the rising prevalence of obesity, hypertension and diabetes. O- GlcNAcylation, a post-translational modification of intracellular proteins, serves as a potent transducer of cellular stress. Failing myocardium is marked by increased O -GlcNAcylation, but it is unknown if excessive O- GlcNAcylation contributes to cardiomyopathy and heart failure. The total levels of O- GlcNAcylation are determined by nutrient and metabolic flux, in addition to the net activity of two enzymes, O- GlcNAc transferase (OGT) and O -GlcNAcase (OGA). Methods We developed two new transgenic mouse models with myocardial overexpression of OGT and OGA to control O-GlcNAclyation independent of pathological stress. Results We found that OGT transgenic hearts showed increased O- GlcNAcylation, and developed severe dilated cardiomyopathy, ventricular arrhythmias and premature death. In contrast, OGA transgenic hearts had O- GlcNAcylation and cardiac function similar to wild type littermate controls. However, OGA trangenic hearts were resistant to pathological stress induced by pressure overload and had attenuated myocardial O- GlcNAcylation levels, decreased pathological hypertrophy and improved systolic function. Interbreeding OGT with OGA transgenic mice rescued cardiomyopathy and premature death despite persistant elevation of myocardial OGT. Transcriptomic and functional studies revealed disrupted mitochondrial energetics with impairment of complex I activity in hearts from OGT transgenic mice. Complex I activity was rescued by OGA transgenic interbreeding, suggesting an important role for mitochondrial complex I in O -GlcNAc mediated cardiac pathology. Conclusions Our data provide evidence that excessive O- GlcNAcylation causes cardiomyopathy, at least in part, due to defective energetics. Enhanced OGA activity is well tolerated and attenuation of O- GlcNAcylation is an effective therapy against pressure overload induced heart failure. Attenuation of excessive O- GlcNAcylation may represent a novel therapeutic approach for cardiomyopathy. Clinical Perspective What is new? Cardiomyopathy from diverse causes is marked by increased O -GlcNAcylation. Here we provide new genetic mouse models to control myocardial O -GlcNAcylation independent of pathological stress. Genetically increased myocardial O- GlcNAcylation causes progressive dilated cardiomyopathy and premature death, while genetic reduction of myocardial O -GlcNAcylation is protective against pathological hypertrophy caused by transaortic banding. Excessive myocardial O -GlcNAcylation decreases activity and expression of mitochondrial complex I. What are the clinical implications? Increased myocardial O- GlcNAcylation has been shown to be associated with a diverse range of clinical heart failure including aortic stenosis, hypertension, ischemia and diabetes. Using novel genetic mouse models we have provided new proof of concept data that excessive O- GlcNAcylation is sufficient to cause cardiomyopathy. We have shown myocardial over-expression of O -GlcNAcase, an enzyme that reverses O -GlcNAcylation, is well tolerated at baseline, and improves myocardial responses to pathological stress. Our findings suggest reversing excessive myocardial O- GlcNAcylation could benefit diverse etiologies of heart failure.
