PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility. METHODS: Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies. RESULTS: In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (P=2.0×10-2; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (P=1.4×10-6; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9×10-2 and 6.7×10-3, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (P=5.0×10-6; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (P=1.8×10-2 and 4.1×10-2, respectively; n=6, 6, and 5). CONCLUSIONS: Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Visualization of electrographic flow fields of increasing complexity and detection of simulated sources during spontaneously persistent AF in an animal model.

Background: Mapping algorithms have thus far been unable to localize triggers that serve as drivers of AF, but electrographic flow (EGF) mapping provides an innovative method of estimating and visualizing in vivo, near real-time cardiac wavefront propagation. Materials and Methods: One-minute unipolar EGMs were recorded in the right atrium (RA) from a 64-electrode basket catheter to generate EGF maps during atrial rhythms of increasing complexity. They were obtained from 3 normal, animals in sinus rhythm (SR) and from 6 animals in which persistent AF which was induced by rapid atrial pacing. Concurrent EGF maps and high-resolution bipolar EGMs at the location of all EGF-identified sources were acquired. Pacing was subsequently conducted to create focal drivers of AF, and the accuracy of source detection at the pacing site was assessed during subthreshold, threshold and high-output pacing in the ipsilateral or contralateral atria (n = 78). Results: EGF recordings showed strong coherent flow emanating from the sinus node in SR that changed direction during pacing and were blocked by ablation lesions. Additional passive rotational phenomena and lower activity sources were visualized in atrial flutter (AFL) and AF. During the AF recordings, source activity was not found to be correlated to dominant frequency or f wave amplitude observed in concurrently recorded EGMs. While pacing in AF, subthreshold pacing did not affect map properties but pacing at or above threshold created active sources that could be accurately localized without any spurious detection in 95% of cases of ipsilateral mapping when the basket covered the pacing source. Discussion: EGF mapping can be used to visualize flow patterns and accurately identify sources of AF in an animal model. Source activity was not correlated to spectral properties of f-waves in concurrently obtained EGMs. The locations of sources could be pinpointed with high precision, suggesting that they may serve as prime targets for focal ablations.

Myosin Light Chain Dephosphorylation by PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

Background: Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods: Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results: In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls ( P =2.0×10 -2 , n=12,12 in each group) with > 40% reduction in MLC2a phosphorylation ( P =1.4×10 -6 , n=12,12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF ( P =2.9×10 -2 and 6.7×10 -3 respectively, n=8,8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls ( P =5.0×10 -6 , n=12,8,12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls ( P =1.8×10 -2 and 4.1×10 -2 respectively, n= 6,6,5). Conclusions: AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Determination of single cryoablation outcome within 30 to 60 seconds of freezing based on ice impedance.

BACKGROUND: A direct indicator of effective pulmonary vein isolation (PVI) based on early ice formation is presently lacking. OBJECTIVE: The initial impedance rise within 30 to 60 seconds (sec) of single cryoablation relating to ice on the distal surface of the cryoballoon could; predict effective PVI with early termination, the need for prolonging the cryoablation, or failure to achieve effective ablation. METHODS: Impedance measurements were taken between two ring electrodes, at the anterior balloon surface and at the shaft behind the balloon. Ice covering the anterior ring leads to impedance rise. Single cryoablation (eight animals, 37 veins) was applied for 90 to 180 sec. Cryoapplication was terminated if the impedance reached ≥500 Ω. Impedance levels at ≤60 sec of cryoablation were divided into three groups based on the characteristics of the impedance rise. PVI was confirmed acutely and at 45 ± 9 days recovery by electrophysiology mapping and histopathology. RESULTS: At 60 sec of freezing, an impedance rise of 34.1 ± 15.2 Ω (13-50 Ω) and slope of the impedance rise (measured during 15-30 sec of cryoapplication) less than 1 Ω/sec resulted in failed PVI. An impedance rise of 104.4 ± 31.5 Ω (76-159 Ω) and slope of 2 Ω/sec resulted in 100% PVIs. An impedance rise of 130.9 ± 137.8 Ω (40-590 Ω) and slope of 10 Ω/sec resulted in 100% PVIs with early termination at 90 sec. CONCLUSION: The efficacy of single cryoablation can be defined within 30 to 60 sec based on ice impedance. Three unique impedance profiles described in this investigation are associated with the uniformity and thickness of the ice buildup on the anterior surface of the balloon. One cryoablation with an adequate impedance rise is needed for successful outcomes.

Characteristics of Ice Impedance Recorded From a Ring Electrode Placed at the Anterior Surface of the Cryoballoon: Novel Approach to Define Ice Formation and Pulmonary Vein Isolation.

BACKGROUND: The success of cryoablation of the pulmonary vein isolation (PVI) is dependent on transmural and circumferential ice formation. We hypothesize that rising impedance recorded from a ring electrode placed 2 mm from the cryoballoon signifies ice formation covering the balloon surface and indicates ice expansion. The impedance level enables titration of the cryoapplication time to avoid extracardiac damage while ensuring PVI. METHODS AND RESULTS: In 12 canines, a total of 57 pulmonary veins were targeted for isolation. Two cryoapplications were delivered per vein with a minimum of 90 and maximum of 180-second duration. Cryoapplication was terminated on reaching a 500 Ω change from baseline. Animals recovered 38±6 days post-procedure, and veins were assessed electrically for isolation. Heart tissue was histologically analyzed. Extracardiac structures were examined for damage. PVI was achieved in 100% of the veins if the impedance reached 500 Ω in <90 seconds with freeze time of 90 seconds. When 500 Ω was reached >90 to 180 seconds (142.60±29.3 seconds), 90% PVI was achieved. When the final impedance was between 200 and 500 Ω with 180 seconds of freeze time, PVI was achieved in 86.8%. For impedance of <200 Ω, PVI was achieved in 14%. No extracardiac damage was recorded. CONCLUSIONS: Impedance rise of 500 Ω at <90 seconds with freeze time of 90 seconds resulted in 100% PVI. Impedance measurements from the nose of the balloon is a direct measure of ice formation on the balloon. It provides real-time feedback on the quality of the ablation and defines the cryoapplication termination time based on ice formation, limiting ice expansion to extracardiac tissues.