SERCA2a-phospholamban interaction monitored by an interposed circularly permutated green fluorescent protein.

The interaction of phospholamban (PLB) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) is a key regulator of cardiac contractility and a therapeutic target in heart failure (HF). PLB-mediated increases in SERCA2a activity improve cardiac function and HF. Clinically, this mechanism can only be exploited by a general activation of the proteinkinase A (PKA), which is associated with side effects and adverse clinical outcomes. A selective interference of the PLB-SERCA2a interaction is desirable but will require novel tools that allow for an integrated assessment of this interaction under both physiological and pathophysiological conditions. A circularly permutated green fluorescent protein (cpGFP) was interposed between SERCA2a and PLB to result into a single SERCA2a-cpGFP-PLB recombinant protein (SGP). Expression, phosphorylation, fluorescence, and function of SGP were evaluated. Expression of SGP-cDNA results in a functional recombinant protein at the predicted molecular weight. The PLB domain of SGP retains its ability to polymerize and can be phosphorylated by PKA activation. This increases the fluorescent yield of SGP by between 10% and 165% depending on cell line and conditions. In conclusion, a single recombinant fusion protein that combines SERCA2a, a circularly permutated green fluorescent protein, and PLB can be expressed in cells and can be phosphorylated at the PLB domain that markedly increases the fluorescence yield. SGP is a novel cellular SERCA2a-PLB interaction monitor.NEW & NOTEWORTHY This study describes the design and characterization of a novel biosensor that can visualize the interaction of SERCA2a and phospholamban (PLB). The biosensor combines SERCA2a, a circularly permutated green fluorescent protein, and PLB into one recombinant protein (SGP). Proteinkinase A activation results in phosphorylation of the PLB domain and is associated with a marked increase in the fluorescence yield to allow for real-time monitoring of the SERCA2a and PLB interaction in cells.

Effects of a Higher Heart Rate on Quality of Life and Functional Capacity in Patients With Left Ventricular Diastolic Dysfunction.

There is no evidence-based treatment for heart failure with preserved ejection fraction. Although lower heart rates (HRs) provide an unequivocal benefit for patients with HF with reduced ejection fraction, higher HR might convey important hemodynamic and substrate-modifying benefits in patients with diastolic dysfunction. In a prospective study of 20 stable outpatients with diastolic dysfunction and pacemakers, we evaluated the effects of a 4-week increase in the lower pacemaker rate to 80 beats/min followed by reversal to the previous lower HR setting from weeks 4 to 6. We assessed quality of life (Minnesota Living with Heart Failure Questionnaire), 6-minute walk test and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels. Pacing at 80 beats/min significantly improved quality of life and the 6-minute walk test (p ≤0.05). There was a strong positive correlation between the pacing-induced changes in NT-proBNP and baseline QRS intervals (r2 = 0.31, p <0.01). Stratification by QRS duration revealed that pacing at 80 beats/min led to -21 ± 26% reduction in NT-proBNP in patients with QRS ≤150 ms, whereas QRS >150 ms was associated with a 26 ± 35% increase in NT-proBNP (p <0.01). Patients physiologically paced from the conduction system had a -46 ± 26% reduction in NT-proBNP at 80 beats/min as compared with 4 ± 26% and 13 ± 26% change with pacing from the right atrial appendage and right ventricular apical septum (pinteraction = 0.04). In conclusion, increasing the lower rate setting of pacemakers to 80 beats/min in patients with diastolic dysfunction improves quality of life, functional capacity, and NT-proBNP for those patients with a baseline QRS ≤150 ms. These findings suggest that higher HRs may provide meaningful benefits to patients with left ventricular diastolic dysfunction and heart failure with preserved ejection fraction.