暂无摘要。

Phospholamban (PLN) is a 52 amino acid regulin that allosterically modulates the activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) in the heart muscle. In its unphosphorylated form, PLN binds SERCA within its transmembrane (TM) domains, approximately 20 Å away from the Ca2+ binding site, reducing SERCA\'s apparent Ca2+ affinity (pKCa) and decreasing cardiac contractility. During the enzymatic cycle, the inhibitory TM domain of PLN remains anchored to SERCA, whereas its cytoplasmic region transiently binds the ATPase\'s headpiece. Phosphorylation of PLN at Ser16 by protein kinase A increases the affinity of its cytoplasmic domain to SERCA, weakening the TM interactions with the ATPase, reversing its inhibitory function, and augmenting muscle contractility. How the structural changes caused by pathological mutations in the PLN cytoplasmic region are transmitted to its inhibitory TM domain is still unclear. Using solid-state NMR spectroscopy and activity assays, we analyzed the structural and functional effects of a series of mutations and their phosphorylated forms located in the PLN cytoplasmic region and linked to dilated cardiomyopathy. We found that these missense mutations affect the overall topology and dynamics of PLN and ultimately modulate its inhibitory potency. Also, the changes in the TM tilt angle and cytoplasmic dynamics of PLN caused by these mutations correlate well with the extent of SERCA inhibition. Our study unveils new molecular determinants for designing variants of PLN that outcompete endogenous PLN to regulate SERCA in a tunable manner.

Risk of cardiovascular disease mortality rises in women after menopause. While increased cardiovascular risk is largely attributed to postmenopausal declines in estrogens, the molecular changes in the heart that contribute to risk are poorly understood. Disruptions in intracellular calcium handling develop in ovariectomized mice and have been implicated in cardiac dysfunction. Using a mouse model of menopause in which ovarian failure occurs over 120 days, we sought to determine if perimenopause impacted calcium removal mechanisms in the heart and identify the molecular mechanisms. Mice were injected with 4-vinylcyclohexene diepoxide (VCD) to induce ovarian failure over 120 days, mimicking perimenopause. Hearts were removed at 60 and 120 days after VCD injections, representing the middle and end of perimenopause. SERCA2a function was significantly diminished at the end of perimenopause. Neither SERCA2a nor phospholamban expression changed at either time point, but phospholamban phosphorylation at S16 and T17 was dynamically altered. Intrinsic SERCA inhibitors sarcolipin and myoregulin increased >4-fold at day 60, as did the native activator DWORF. At the end of perimenopause, sarcolipin and myoregulin returned to baseline levels while DWORF was significantly reduced below controls. Sodium-calcium exchanger expression was significantly increased at the end of perimenopause. These results show that the foundation for increased cardiovascular disease mortality develops in the heart during perimenopause and that regulators of calcium handling exhibit significant fluctuations over time. Understanding the temporal development of cardiovascular risk associated with menopause and the underlying mechanisms is critical to developing interventions that mitigate the rise in cardiovascular mortality that arises after menopause.

In cardiac myocytes, the type 2a sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) plays a key role in intracellular Ca regulation. Due to its critical role in heart function, SERCA2a activity is tightly regulated by different mechanisms, including micropeptides. While phospholamban (PLB) is a well-known SERCA2a inhibitor, dwarf open reading frame (DWORF) is a recently identified SERCA2a activator. Since PLB phosphorylation is the most recognized mechanism of SERCA2a activation during adrenergic stress, we studied whether PLB phosphorylation also affects SERCA2a regulation by DWORF. By using confocal Ca imaging in a HEK293 expressing cell system, we analyzed the effect of the co-expression of PLB and DWORF using a bicistronic construct on SERCA2a-mediated Ca uptake. Under these conditions of matched expression of PLB and DWORF, we found that SERCA2a inhibition by non-phosphorylated PLB prevails over DWORF activating effect. However, when PLB is phosphorylated at PKA and CaMKII sites, not only PLB\'s inhibitory effect was relieved, but SERCA2a was effectively activated by DWORF. Förster resonance energy transfer (FRET) analysis between SERCA2a and DWORF showed that DWORF has a higher relative affinity for SERCA2a when PLB is phosphorylated. Thus, SERCA2a regulation by DWORF responds to the PLB phosphorylation status, suggesting that DWORF might contribute to SERCA2a activation during conditions of adrenergic stress.

This study aimed to determine the prevalence and clinical features of Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) caused by pathogenic mutations in the Phospholamban (PLN) gene. The study included 170 patients who had a confirmed diagnosis of ARVC and underwent PLN genetic screening using next-generation sequencing. The findings of this study provide valuable insights into the association between PLN mutations and ARVC, which can aid in the development of more effective diagnostic and treatment strategies for ARVC patients. Out of the patients evaluated, six had a rare pathogenic mutation in PLN with the same p.R14del variant. Family screening revealed that heterozygous carriers of p.R14del exhibited a definite ARVC phenotype. In clinical studies, individuals with the p.R14del mutation experienced a similar rate of malignant arrhythmia events as those with classic desmosome mutations. After adjusting for covariates, individuals with PLN mutations had a two point one seven times greater likelihood of experiencing transplant-related risks compared to those who did not possess PLN mutations (95% CI 1.08-6.82, p = 0.035). The accumulation of left ventricular fat and fibers is a pathological marker for ARVC patients with p.R14del mutations. In a cohort of 170 Chinese ARVC patients, three point five percent of probands had the PLN pathogenic variant (p.R14del) and all were female. Our data shows that PLN-related ARVC patients are at high risk for ventricular arrhythmias and heart failure, which requires clinical differentiation from classic ARVC. Furthermore, carrying the p.R14del mutation can be an independent prognostic risk factor in ARVC patients.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s43657-023-00126-w.

OBJECTIVE: Recently, a genetic variant-specific prediction model for phospholamban (PLN) p.(Arg14del)-positive individuals was developed to predict individual major ventricular arrhythmia (VA) risk to support decision-making for primary prevention implantable cardioverter defibrillator (ICD) implantation. This model predicts major VA risk from baseline data, but iterative evaluation of major VA risk may be warranted considering that the risk factors for major VA are progressive. Our aim is to evaluate the diagnostic performance of the PLN p.(Arg14del) risk model at 3-year follow-up.
RESULTS: We performed a landmark analysis 3 years after presentation and selected only patients with no prior major VA. Data were collected of 268 PLN p.(Arg14del)-positive subjects, aged 43.5 ± 16.3 years, 38.9% male. After the 3 years landmark, subjects had a mean follow-up of 4.0 years (± 3.5 years) and 28 (10%) subjects experienced major VA with an annual event rate of 2.6% [95% confidence interval (CI) 1.6-3.6], defined as sustained VA, appropriate ICD intervention, or (aborted) sudden cardiac death. The PLN p.(Arg14del) risk score yielded good discrimination in the 3 years landmark cohort with a C-statistic of 0.83 (95% CI 0.79-0.87) and calibration slope of 0.97.
CONCLUSIONS: The PLN p.(Arg14del) risk model has sustained good model performance up to 3 years follow-up in PLN p.(Arg14del)-positive subjects with no history of major VA. It may therefore be used to support decision-making for primary prevention ICD implantation not merely at presentation but also up to at least 3 years of follow-up.

The gastrointestinal system is a hollow organ affected by fibrostenotic diseases that cause volumetric compromise of the lumen via smooth muscle hypertrophy and fibrosis. Many of the driving mechanisms remain unclear. Yes-associated protein-1 (YAP) is a critical mechanosensory transcriptional regulator that mediates cell hypertrophy in response to elevated extracellular rigidity. In the type 2 inflammatory disorder, eosinophilic esophagitis (EoE), phospholamban (PLN) can induce smooth muscle cell hypertrophy. We used EoE as a disease model for understanding a mechanistic pathway in which PLN and YAP interact in response to rigid extracellular substrate to induce smooth muscle cell hypertrophy. PLN-induced YAP nuclear sequestration in a feed-forward loop caused increased cell size in response to a rigid substrate. This mechanism of rigidity sensing may have previously unappreciated clinical implications for PLN-expressing hollow systems such as the esophagus and heart.

BACKGROUND: Phospholamban (PLN) p.(Arg14del) variant carriers are at risk for development of malignant ventricular arrhythmia (MVA). Accurate risk stratification allows timely implantation of intracardiac defibrillators and is currently performed with a multimodality prediction model.
OBJECTIVE: This study aimed to investigate whether an explainable deep learning-based approach allows risk prediction with only electrocardiogram (ECG) data.
METHODS: A total of 679 PLN p.(Arg14del) carriers without MVA at baseline were identified. A deep learning-based variational auto-encoder, trained on 1.1 million ECGs, was used to convert the 12-lead baseline ECG into its FactorECG, a compressed version of the ECG that summarizes it into 32 explainable factors. Prediction models were developed by Cox regression.
RESULTS: The deep learning-based ECG-only approach was able to predict MVA with a C statistic of 0.79 (95% CI, 0.76-0.83), comparable to the current prediction model (C statistic, 0.83 [95% CI, 0.79-0.88]; P = .054) and outperforming a model based on conventional ECG parameters (low-voltage ECG and negative T waves; C statistic, 0.65 [95% CI, 0.58-0.73]; P < .001). Clinical simulations showed that a 2-step approach, with ECG-only screening followed by a full workup, resulted in 60% less additional diagnostics while outperforming the multimodal prediction model in all patients. A visualization tool was created to provide interactive visualizations (https://pln.ecgx.ai).
CONCLUSIONS: Our deep learning-based algorithm based on ECG data only accurately predicts the occurrence of MVA in PLN p.(Arg14del) carriers, enabling more efficient stratification of patients who need additional diagnostic testing and follow-up.

Hypertrophic cardiomyopathy (HCM) is a heterogeneous genetic disorder, most often caused by sarcomeric gene mutations, with a small proportion due to variants in non-sarcomeric loci. Phospholamban (PLN) is a phosphoprotein associated with the cardiac sarcoplasmic reticulum, a major determinant of cardiac contractility and relaxation. We conducted a retrospective study to determine the prevalence, phenotypical spectrum and clinical course of patients carrying the PLN p.Leu39* variant. A cohort including 11 PLN patients was identified among all patients with HCM (9/189, 4.8%) and DCM (2/62, 3.2%) who underwent genetic testing from two tertiary centers and five more were detected through cascade screening. Complete phenotyping was performed. PLN p.Leu39* variant-driven cardiomyopathy presented mostly as hypertrophic, with frequent progression to end-stage dilated HCM. We proceeded to compare these results to a similar analysis of a control cohort consisting of age-matched individuals that inherited pathogenic or likely pathogenic variants in common sarcomeric genes (MYBPC3/MYH7). Overall, the clinical characteristics and examination findings of patients carrying PLN p.Leu39* were not different from patients with cardiomyopathy related to sarcomeric mutations except for the presence of pathological Q waves and the incidence of non-sustained ventricular arrhythmias, which were higher in PLN patients than in those with MYBPC3/MYH7-related diseases.

As the genetic landscape of cardiomyopathies continues to expand, the identification of missense variants in disease-associated genes frequently leads to a classification of variant of uncertain significance (VUS). For the proper reclassification of such variants, functional characterization is an important contributor to the proper assessment of pathogenic potential. Several missense variants in the calcium transport regulatory protein phospholamban have been associated with dilated cardiomyopathy. However, >40 missense variants in this transmembrane peptide are currently known and most remain classified as VUS with little clinical information. Similarly, missense variants in cardiac myosin binding protein have been associated with hypertrophic cardiomyopathy. However, hundreds of variants are known and many have low penetrance and are often found in control populations. Herein, we focused on novel missense variants in phospholamban, an Ala15-Thr variant found in a 4-year-old female and a Pro21-Thr variant found in a 60-year-old female, both with a family history and clinical diagnosis of dilated cardiomyopathy. The patients also harbored a Val896-Met variant in cardiac myosin binding protein. The phospholamban variants caused defects in the function, phosphorylation, and dephosphorylation of this calcium transport regulatory peptide, and we classified these variants as potentially pathogenic. The variant in cardiac myosin binding protein alters the structure of the protein. While this variant has been classified as benign, it has the potential to be a low-risk susceptibility variant because of the structural change in cardiac myosin binding protein. Our studies provide new biochemical evidence for missense variants previously classified as benign or VUS.