Time Course of Irreversible Electroporation Lesion Development Through Short- and Long-Term Follow-Up in Pulsed-Field Ablation-Treated Hearts.

BACKGROUND: Pulsed-field ablation (PFA) is a tissue-selective, nonthermal cardiac ablation modality. A novel PFA ablation system consisted of a multichannel irreversible electroporation generator system and a multielectrode circular irreversible electroporation catheter has been developed for catheter ablation. To understand the progression and immediate impacts of PFA, this study evaluated the subchronic (7±3 day) and chronic (30±3 day) safety and performance of the novel PFA system when simulating pulmonary vein and superior vena cava isolation in a porcine beating heart model. METHODS: Ten swine models were divided into subchronic (n=6) and chronic cohorts (n=4). Lesions were performed within the right and left atrium to conduct right pulmonary veins and superior vena cava isolations, in addition to creating stacked lesions in the left atrium roof and right atrium posterior wall. RESULTS: Acute pulmonary vein and superior vena cava isolation were achieved in 10 out of 10 swine and demonstrated 100% lesion durability in both cohorts, including sustained elimination of electrical activity at the left atrium roof and right atrium posterior wall. Histology demonstrated that all the cardiac sites ablated showed discrete zones of loss of myocardial fibers or smooth muscle cells with preservation of the tissue architecture with resultant fibrocellular replacement, neovascularization, and neocollagen deposition. Mineralization findings were present in association with residual necrotic muscle fibers. Only in 7 days group, areas of mineralization were frequently associated with inflammation. There were no treatment-related changes in other tissues, including complete sparing of the phrenic nerve. CONCLUSIONS: Pulsed-field ablation for pulmonary vein and superior vena cava isolation with the novel PFA system was feasible, safe with myocardial-specific ablative effect. Durable lesions were observed at the target areas. with inflammation phenomena mainly documented at 7 days.

In vivo porcine characterization of atrial lesion safety and efficacy utilizing a circular pulsed-field ablation catheter including assessment of collateral damage to adjacent tissue in supratherapeutic ablation applications.

INTRODUCTION: Pulsed-field ablation (PFA), an ablative method that causes cell death by irreversible electroporation, has potential safety advantages over radiofrequency ablation and cryoablation. Pulmonary vein (PV) isolation was performed in a porcine model to characterize safety and performance of a novel, fully-integrated biphasic PFA system comprising a multi-channel generator, variable loop circular catheter, and integrated PFA mapping software module. METHODS: Eight healthy porcine subjects were included. To evaluate safety, multiple ablations were performed, including sites not generally targeted for therapeutic ablation, such as the right inferior PV lumen, right superior PV ostium, and adjacent to the esophagus and phrenic nerve. To evaluate the efficacy, animals were recovered, followed for 30(±3) days, then re-mapped. Gross pathological and histopathological examinations assessed procedural injuries, chronic thrombosis, tissue ablation, penetration depth, healing, and inflammatory response. RESULTS: All eight animals survived follow-up. PV narrowing was not observed acutely nor at follow-up, even when ablation was performed deep to the PV ostium. No injury was seen grossly or histologically in adjacent structures. All PVs were durably isolated, confirmed by bidirectional block at re-map procedure. Histological examination showed complete, transmural necrosis around the circumference of the ablated section of right PVs. CONCLUSION: This preclinical evaluation of a fully-integrated PFA system demonstrated effective and durable ablation of cardiac tissue and PV isolation without collateral damage to adjacent structures, even when ablation was performed in more extreme settings than those used therapeutically. Histological staining confirmed complete transmural cell necrosis around the circumference of the PV ostium at 30 days.

Real-world safety of catheter ablation for atrial fibrillation with contact force or cryoballoon ablation.

PURPOSE: Real-world data can help medical administrators, physicians, and payers make evidence-based decisions regarding treatment choices. The objective of this study was to compare real-world safety outcomes with the latest catheter technologies used for the treatment of atrial fibrillation (AF). METHODS: The Vizient Health Systems database, a large US hospital database, was used to compare acute complications in AF ablation with the contact force sensing THERMOCOOL SMARTTOUCH® Catheter or the THERMOCOOL SMARTTOUCH® SF Catheter (ST) versus the second-generation Arctic Front Advance™ Cryoablation Catheter (CB2) between September 2015 and June 2017. The primary outcome was a composite safety endpoint of acute ablation-related complications defined via ICD-10 diagnosis and procedure codes, including tamponade and other pericardial events, respiratory complications, stroke, cerebral or pre-cerebral occlusion/stenosis without infarction, vascular access complications, hemorrhage, phrenic nerve injury, myocardial infarction, and pulmonary embolism. RESULTS: In total, 1473 ablations met all inclusion criteria (407 ST, 1066 CB2). Ablations for paroxysmal AF (PAF) had a lower complication rate than ablations for persistent AF (PsAF) (6.1% vs. 7.3%), as did ablations with ST compared with CB2 within each AF type (PAF 6.0% vs. 6.1%, PsAF 6.3% vs. 7.8%). Neither ablation catheter nor AF type was statistically significant after controlling for site volume, patient age, and comorbid conditions (ST vs. CB2: OR 0.86, p = 0.5544; PsAF vs. PAF: OR 1.08, p = 0.7376). CONCLUSION: Acute ablation-related complication rates were low and were not significantly associated with catheter technology. Increased risk of complication was attributable to low-volume sites and baseline patient characteristics.

Comparing rates of atrioesophageal fistula with contact force-sensing and non-contact force-sensing catheters: analysis of post-market safety surveillance data.

PURPOSE: There is limited data on the specific incidence of serious adverse events, such as atrioesophageal fistula (AEF), associated with either contact force (CF) or non-CF ablation catheters. Since the actual number of procedures performed with each type of catheter is unknown, making direct comparisons is difficult. The purpose of this study was to assess the incidence of AEF associated with the use of CF and non-CF catheters. Additionally, we aimed to understand the workflow present in confirmed AEF cases voluntarily provided by physicians. METHODS: The number of AEFs for 2014-2017 associated with each type of catheter was extracted from an ablation device manufacturer's complaint database. Proprietary device sales data, a proxy for the total number of procedures, were used as the denominator to calculate the incidence rates. Additional survey and workflow data were systematically reviewed. RESULTS: Both CF and non-CF ablation catheters have comparably low incidence of AEF (0.006 ± 0.003% and 0.005 ± 0.003%, respectively, p = 0.69). CF catheters are the catheter of choice for left atrium (LA) procedures which pose the greatest risk for AEF injury. Retrospective analysis of seven AEF cases demonstrated that high power and force and long RF duration were delivered on the posterior wall of the left atrium in all cases. CONCLUSIONS: CF and non-CF ablation catheters were found to have similar AEF incidence, despite CF catheters being the catheter of choice for LA procedures. More investigation is needed to understand the range of parameters which may create risk for AEF.

Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1.

BACKGROUND: The proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin (Ub) chain appendage. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn10, Rpn13, or Rpt5, or are shuttled to the proteasome by Rad23, Dsk2, or Ddi1. The latter proteins share an Ub association domain (UBA) for binding poly-Ub chains and an Ub-like-domain (UBL) for binding to the proteasome. It has been proposed that shuttling receptors dock on the proteasome via Rpn1, but the precise nature of the docking site remains poorly defined. RESULTS: To shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in Rpn1 that disrupt its binding to UBA-UBL proteins. Here we demonstrate that delivery of Ub conjugates and docking of Ddi1 (and to a lesser extent Dsk2) to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved D517 residue of Rpn1. Moreover, degradation of the Ddi1-dependent proteasome substrate, Ufo1, is blocked in rpn1-D517A yeast cells. By contrast, Rad23 recruitment to the proteasome is not affected by rpn1-D517A. CONCLUSIONS: These studies provide insight into the mechanism by which the UBA-UBL protein Ddi1 is recruited to the proteasome to enable Ub-dependent degradation of its ligands. Our studies suggest that different UBA-UBL proteins are recruited to the proteasome by distinct mechanisms.

A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals.

The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-D-glucose to GDP and D-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-D-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-D-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-D-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-D-glucose in the C10F3.4 mutant worms, suggesting that the GDP-D-glucose phosphorylase may function to remove GDP-D-glucose formed by GDP-D-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological D-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals.

Combined chemical and genetic approach to inhibit proteolysis by the proteasome.

Regulated protein destruction by the proteasome is crucial for the maintenance of normal cellular homeostasis. Much of our understanding of proteasome function stems from the use of drugs that inhibit its activity. Curiously, despite the importance of proteasomal proteolysis, previous studies have found that proliferation of the yeast Saccharomyces cerevisiae is relatively resistant to the effects of proteasome inhibitors such as MG132, even in the presence of mutations that increase inhibitor levels in cells. We reasoned that part of the resistance of S. cerevisiae to proteasome inhibitors stems from the fact that most proteasome inhibitors preferentially target the chymotryptic activity of the proteasome, and that the caspase-like and tryptic sites within the 20S core could compensate for proteasome function under these conditions. To test this hypothesis, we generated a strain of yeast in which the gene encoding the drug efflux pump Pdr5 is deleted, and the tryptic and caspase-like proteasome activities are inactivated by mutation. We find that this strain has dramatically increased sensitivity to the proteasome inhibitor MG132. Under these conditions, treatment of yeast with MG132 blocks progression through the cell cycle, increases the accumulation of polyubiquitylated proteins and decreases the ability to induce transcription of certain genes. These results highlight the contribution of the caspase-like and tryptic activities of the proteasome to its function, and provide a strategy to potently block proteasomal proteolysis in yeast that has practical applications.

Defective responses to oxidative stress in protein l-isoaspartyl repair-deficient Caenorhabditis elegans.

We have shown that Caenorhabditis elegans lacking the PCM-1 protein repair l-isoaspartyl methyltransferase are more sensitive to oxidative stress than wild-type nematodes. Exposure to the redox-cycling quinone juglone upon exit from dauer diapause results in defective egg-laying (Egl phenotype) in the pcm-1 mutants only. Treatment with paraquat, a redox-cycling dipyridyl, causes a more severe developmental delay at the second larval stage in pcm-1 mutants than in wild-type nematodes. Finally, exposure to homocysteine and homocysteine thiolactone, molecules that can induce oxidative stress via distinct mechanisms, results in a more pronounced delay in development at the first larval stage in pcm-1 mutants than in wild-type animals. Homocysteine treatment also induced the Egl phenotype in mutant but not wild-type nematodes. All of the effects of these agents were reversed upon addition of vitamin C, indicating that the developmental delay and egg-laying defects result from oxidative stress. Furthermore, we have demonstrated that a mutation in the gene encoding the insulin-like receptor DAF-2 suppresses the Egl phenotype in pcm-1 mutants treated with juglone. Our results support a role of PCM-1 in the cellular responses mediated by the DAF-2 insulin-like signaling pathway in C. elegans for optimal protection against oxidative stress.

The protein L-isoaspartyl-O-methyltransferase functions in the Caenorhabditis elegans stress response.

The efficient use of nutrients is important in development and aging. In this study, we asked if the protein repair methyltransferase has a related or additional role in energy metabolism and stress response in the nematode Caenorhabditis elegans. Worms lacking the pcm-1 gene encoding this enzyme exhibit reduced longevity as SDS-isolated dauer larvae and as arrested L1 larvae under starvation stress, while overexpression leads to increased adult longevity. These findings led us to question whether pcm-1 deficient C. elegans may have inappropriate metabolic responses to stress. We assayed dauer and dauer-like larvae for starvation survival and observed a two-fold reduction of median survival time for pcm-1 mutants compared to N2 wild-type worms. Under these conditions, pcm-1 deficient dauer larvae had reduced fat stores, suggesting that PCM-1 may have a role in the initiation of the correct metabolic responses to stress starvation. We show expression of the pcm-1 gene in neurons, body wall and reproductive tissues. Upon heat shock and dauer formation-inducing conditions, we observe additional pcm-1 expression in body wall muscle nuclei and actomyosin filaments and in hypodermal cells. These results suggest that this enzyme may be important in stress response pathways, including proper decision making for energy storage.

Protein-repair and hormone-signaling pathways specify dauer and adult longevity and dauer development in Caenorhabditis elegans.

Protein damage that accumulates during aging can be mitigated by a repair methyltransferase, the l-isoaspartyl-O-methyltransferase. In Caenorhabditis elegans, the pcm-1 gene encodes this enzyme. In response to pheromone, we show that pcm-1 mutants form fewer dauer larvae with reduced survival due to loss of the methyltransferase activity. Mutations in daf-2, an insulin/insulin-like growth factor-1-like receptor, and daf-7, a transforming growth factor-beta-like ligand, modulate pcm-1 dauer defects. Additionally, daf-2 and daf-7 mutant dauer larvae live significantly longer than wild type. Although dauer larvae are resistant to many environmental stressors, a proportionately larger decrease in dauer larvae life spans occurred at 25 degrees C compared to 20 degrees C than in adult life span. At 25 degrees C, mutation of the daf-7 or pcm-1 genes does not change adult life span, whereas mutation of the daf-2 gene and overexpression of PCM-1 increases adult life span. Thus, there are both overlapping and distinct mechanisms that specify dauer and adult longevity.

Autophagy and insulin/TOR signaling in Caenorhabditis elegans pcm-1 protein repair mutants.

Biological responses due to nutrient deprivation in the nematode Caenorhabditis elegans, including L1 diapause and autophagy during dauer formation, can be mediated through the linked DAF-2/insulin/IGF receptor and target-of-rapamycin (TOR) kinase pathways. Here we discuss how altered insulin/TOR signaling may underlie the previously reported phenotypes of worms with a null mutation in the pcm-1 gene that results in reduced autophagy during dauer formation and decreased L1 arrest survival. PCM-1 encodes a protein repair methyltransferase and mutants of the encoding pcm-1 gene are incapable of converting spontaneously damaged l-isoaspartyl residues in cellular proteins to normal forms by this pathway. We speculate that PCM-1 may function either directly or indirectly as an inhibitor of insulin/TOR signaling, perhaps in a role to balance autophagy with alternative protein degradation pathways that may be more specific for recognizing age-damaged proteins.

Arabidopsis VTC2 encodes a GDP-L-galactose phosphorylase, the last unknown enzyme in the Smirnoff-Wheeler pathway to ascorbic acid in plants.

The first committed step in the biosynthesis of L-ascorbate from D-glucose in plants requires conversion of GDP-L-galactose to L-galactose 1-phosphate by a previously unidentified enzyme. Here we show that the protein encoded by VTC2, a gene mutated in vitamin C-deficient Arabidopsis thaliana strains, is a member of the GalT/Apa1 branch of the histidine triad protein superfamily that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in a reaction that consumes inorganic phosphate and produces GDP. In characterizing recombinant VTC2 from A. thaliana as a specific GDP-L-galactose/GDP-D-glucose phosphorylase, we conclude that enzymes catalyzing each of the ten steps of the Smirnoff-Wheeler pathway from glucose to ascorbate have been identified. Finally, we identify VTC2 homologs in plants, invertebrates, and vertebrates, suggesting that a similar reaction is used widely in nature.

The L-isoaspartyl-O-methyltransferase in Caenorhabditis elegans larval longevity and autophagy.

The protein L-isoaspartyl-O-methyltransferase, coded by the pcm-1 gene in Caenorhabditis elegans, participates in the repair of age-damaged proteins. We tested the ability of pcm-1-deficient nematodes to survive starvation stress as developmentally-arrested L1 larvae. We found that pcm-1 mutant L1 larvae do not survive as well as wild-type L1 larvae when incubated in M9 medium without nutrients. We then tested whether the starved L1 larvae could continue development when allowed access to food in a recovery assay. A loss of recovery ability with age was observed for all larvae, with little or no difference between the pcm-1 mutant and wild-type N2 larvae. Interestingly, when L1 larvae were starved in cholesterol-containing S medium or M9 medium supplemented with cholesterol, the survival rates of both mutant and wild-type animals nearly doubles, with pcm-1 larvae again faring more poorly than N2 larvae. Furthermore, L1 larvae cultured in these cholesterol-containing media show an increase in Sudan Black staining over animals cultured in M9 medium. The longevity defects of pcm-1 mutants previously seen in dauer larvae and here in L1 larvae suggest a defect in the ability of pcm-1 mutants to recycle and reuse old cellular components in pathways such as autophagy. Using an autophagosomal marker, we found evidence suggesting that the pcm-1 mutation may inhibit autophagy during dauer formation, suggesting that the absence of protein repair may also interfere with protein degradation pathways.