P2Y12 Inhibitor Switching in Response to Routine Notification of CYP2C19 Clopidogrel Metabolizer Status Following Acute Coronary Syndromes.

Importance: Physician behavior in response to knowledge of a patient's CYP2C19 clopidogrel metabolizer status is unknown. Objective: To investigate the association of mandatory reporting of CYP2C19 pharmacogenomic testing, provided to investigators with no direct recommendations on how to use these results, with changes in P2Y12 inhibitor use, particularly clopidogrel, in the Randomized Trial to Compare the Safety of Rivaroxaban vs Aspirin in Addition to Either Clopidogrel or Ticagrelor in Acute Coronary Syndrome (GEMINI-ACS-1) clinical trial. Design, Setting, and Participants: The GEMINI-ACS-1 trial compared rivaroxaban, 2.5 mg twice daily, with aspirin, 100 mg daily, plus open-label clopidogrel or ticagrelor (provided), in patients with recent acute coronary syndromes (ACS). The trial included 371 clinical centers in 21 countries and 3037 patients with ACS. Data were analyzed between May 2017 and February 2019. Interventions: Investigators were required to prestipulate their planned response to CYP2C19 metabolizer status. In response to a regulatory mandate, results for all patients were reported to investigators approximately 1 week after randomization. Main Outcomes and Measures: Reasons for switching P2Y12 inhibitors and occurrence of bleeding and ischemic events were collected. Results: Of 3037 patients enrolled (mean [SD] age, 62.8 [9.0] years; 2275 men [74.9%], and 2824 white race/ethnicity [93.0%]), investigators initially treated 1704 (56.1%) with ticagrelor and 1333 (43.9%) with clopidogrel. Investigators prestipulated that they would use CYP2C19 metabolizer status to change P2Y12 inhibitor in 48.5% of genotyped clopidogrel-treated patients (n = 642 of 1324) and 5.5% of genotyped ticagrelor-treated patients (n = 93 of 1692). P2Y12 inhibitor switching for any reason occurred in 197 patients and was more common in patients treated with ticagrelor (146 of 1704 [8.6%]) compared with clopidogrel (51 of 1333 [3.8%]). Of patients initially treated with ticagrelor, only 1 (0.1% overall; 0.7% of all who switched) was switched based on CYP2C19 status. Of patients initially treated with clopidogrel, 23 (1.7% overall,;45.1% of all who switched) were switched owing to metabolizer status. Of 48 patients (3.6%) with reduced metabolizer status treated initially with clopidogrel, 15 (31.3%) were switched based on metabolizer status, including 48.1% (13 of 27) in which switching was prestipulated. Conclusions and Relevance: Physicians were evenly split on how to respond to knowledge of CYP2C19 metabolizer status in clopidogrel-treated patients. Mandatory provision of this information rarely prompted P2Y12 inhibitor switching overall, including a minority of patients with reduced metabolizer status. These findings highlight the clinical equipoise among physicians regarding use of this information and the reluctance to use information from routine genotyping in the absence of definitive clinical trial data demonstrating the efficacy of this approach. Clinical Trial Registration: ClinicalTrials.gov identifier: NCT02293395.

HCN212-channel biological pacemakers manifesting ventricular tachyarrhythmias are responsive to treatment with I(f) blockade.

BACKGROUND: A potential concern about biological pacemakers is their possible malfunction, which might create ventricular tachycardias (VTs). OBJECTIVE: The purpose of this study was to test our hypothesis that should VTs complicate implantation of HCN-channel-based biological pacemakers, they would be suppressed by inhibitors of the pacemaker current, I(f). METHODS: We created a chimeric channel (HCN212) containing the N- and C-termini of mouse HCN2 and the transmembrane region of mouse HCN1 and implanted it in HEK293 cells. Forty-eight hours later, in whole-cell patch clamp recordings, mean steady state block induced by 3 microM ivabradine (IVB) showed HCN1 = HCN212 > HCN2 currents. The HCN212 adenoviral construct was then implanted into the canine left bundle branch in 11 dogs. Complete AV block was created via radiofrequency ablation, and a ventricular demand electronic pacemaker was implanted (VVI 45 bpm). Electrocardiogram, 24-hour Holter monitoring, and pacemaker log record check were performed for 11 days. RESULTS: All dogs developed rapid VT (>120 bpm, maximum rate = 285 +/- 37 bpm) at 0.9 +/- 0.3 days after implantation that persisted through 5 +/- 1 days. IVB, 1 mg/kg over 5 minutes, was administered during rapid VT, and three dogs received a second dose 24 hours later. While VT terminated with IBV in all instances within 3.4 +/- 0.6 minutes, no effect of IVB on sinus rate was noted. CONCLUSION: We conclude that (1) I(f)-associated tachyarrhythmias-if they occur with HCN-based biological pacemakers-can be controlled with I(f)-inhibiting drugs such as IVB; (2) in vitro, IVB appears to have a greater steady state inhibiting effect on HCN1 and HCN212 isoforms than on HCN4; and (3) VT originating from the HCN212 injection site is suppressed more readily than sinus rhythm. This suggests a selectivity of IVB at the concentration attained for ectopic over HCN4-based pacemaker function. This might confer a therapeutic benefit.

Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart.

BACKGROUND: Biological pacemaking has been performed with viral vectors, human embryonic stem cells, and adult human mesenchymal stem cells (hMSCs) as delivery systems. Only with human embryonic stem cells are data available regarding stability for >2 to 3 weeks, and here, immunosuppression has been used to facilitate survival of xenografts. The purpose of the present study was to determine whether hMSCs provide stable impulse initiation over 6 weeks without the use of immunosuppression, the "dose" of hMSCs that ensures function over this period, and the catecholamine responsiveness of hMSC-packaged pacemakers. METHODS AND RESULTS: A full-length mHCN2 cDNA subcloned in a pIRES2-EGFP vector was electroporated into hMSCs. Transfection efficiency was estimated by GFP expression. I(HCN2) was measured with patch clamp, and cells were administered into the left ventricular anterior wall of adult dogs in complete heart block and with backup electronic pacemakers. Studies encompassed 6 weeks. I(HCN2) for all cells was 32.1+/-1.3 pA/pF (mean+/-SE) at -150 mV. Pacemaker function in intact dogs required 10 to 12 days to fully stabilize and persisted consistently through day 42 in dogs receiving > or =700,000 hMSCs (approximately 40% of which carried current). Rhythms were catecholamine responsive. Tissues from animals killed at 42 days manifested neither apoptosis nor humoral or cellular rejection. CONCLUSIONS: hMSCs provide a means for administering catecholamine-responsive biological pacemakers that function stably for 6 weeks and manifest no cellular or humoral rejection at that time. Cell doses >700,000 are sufficient for pacemaking when administered to left ventricular myocardium.

I(Kr) contributes to the altered ventricular repolarization that determines long-term cardiac memory.

OBJECTIVE: Cardiac memory (CM) is characterized by an altered T-wave morphology, which reflects altered repolarization gradients. We hypothesized that the delayed rectifier currents, I(Kr) and I(Ks), might contribute to these repolarization changes. METHODS: We studied conscious, chronically instrumented dogs paced from the postero-lateral left ventricular (LV) wall at rates 5-10% faster than sinus rate for 3 weeks. ECGs during sinus rhythm were recorded on days 0, 7, 14 and 21 of pacing. Within 3 weeks, CM achieved steady state, hearts were excised, and epicardial and endocardial tissues and myocytes were studied. RESULTS: In unpaced controls, action potential duration to 50% and 90% repolarization (APD) in epicardium was shorter than in endocardium (P < 0.05); in CM epicardial APD increased at CL > or = 500 ms, while endocardial APD was either unchanged or decreased such that the transmural gradient seen in controls diminished (P < 0.05). A transmural I(Kr) gradient occurred in controls (epicardium>endocardium, P < 0.05) and was reversed in CM. No I(Ks) transmural gradient was found in controls, while in CM endocardial I(Ks) was greater than epicardial at greater than +50 mV. Canine ERG (cERG) mRNA and protein in epicardium > endocardium in controls (P < 0.05), and this difference was lost in CM. Expression levels of KCNQ1 and KCNE1 protein were similar in all groups. CONCLUSIONS: A transcriptionally induced change in epicardial I(Kr) contributes to the altered ventricular repolarization that characterizes CM.

The cAMP response element binding protein modulates expression of the transient outward current: implications for cardiac memory.

OBJECTIVE: Long-term cardiac memory (LTCM), expressed as a specific pattern of T-wave change on ECG, is associated with 1) reduced transient outward potassium current (I(to)), 2) reduced mRNA for the pore-forming protein of I(to), Kv4.3, 3) reduced cAMP response element binding protein (CREB), and 4) diminished binding to its docking site on the DNA, the cAMP response element (CRE). We hypothesized a causal link between the decrease of the transcription factor CREB and down-regulation of I(to) and one of its channel subunits, KChIP2, in LTCM. METHODS: After three weeks of left ventricular pacing to induce LTCM (8 paced, 7 sham control dogs), epicardial KChIP2 mRNA and protein levels were assessed by real-time PCR and Western blotting. Mimicking the CREB down-regulation in LTCM, CREB was knocked down in situ in other dogs using adenoviral anti-sense. Effects on the action potential notch, reflecting I(to), were investigated in situ using monophasic action potential (MAP) recordings and at the cellular level by the whole-cell patch clamp technique. CREB binding in the KChIP2 promoter region was ascertained by electrophoretic mobility-shift assays. RESULTS: In LTCM, epicardial KChIP2 mRNA and protein were reduced by 62% and 76%, respectively, compared to shams (p < 0.05). CREB binding by the canine KChIP2 promoter region was demonstrated. CREB knockdown led to disappearance of the phase1 notch in MAP and ablation of I(to). CONCLUSIONS: These results strengthen the hypothesis that down-regulation of CREB-mediated transcription underlies the attenuation of epicardial I(to) in LTCM. They also emphasize that ventricular pacing exerts effects at a subcellular level contributing to memory and conceivably to other forms of cardiac remodeling.

Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates.

BACKGROUND: We hypothesized that administration of the HCN2 gene to the left bundle-branch (LBB) system of intact dogs would provide pacemaker function in the physiological range of heart rates. METHODS AND RESULTS: An adenoviral construct incorporating HCN2 and green fluorescent protein (GFP) as a marker was injected via catheter under fluoroscopic control into the posterior division of the LBB. Controls were injected with an adenoviral construct of GFP alone or saline. Animals were monitored electrocardiographically for up to 7 days after surgery, at which time they were anesthetized and subjected to vagal stimulation to permit emergence of escape pacemakers. Hearts were then removed and injection sites visually identified and removed for microelectrode study of action potentials, patch clamp studies of pacemaker current, and/or immunohistochemical studies of HCN2. For 48 hours postoperatively, 7 of 7 animals subjected to 24-hour ECG monitoring showed multiple ventricular premature depolarizations and/or ventricular tachycardia attributable to injection-induced injury. Thereafter, sinus rhythm prevailed. During vagal stimulation, HCN2-injected dogs showed rhythms originating from the left ventricle, the rate of which was significantly more rapid than in the controls. Excised posterior divisions of the LBB from HCN2-injected animals manifested automatic rates significantly greater than the controls. Isolated tissues showed immunohistochemical and biophysical evidence of overexpressed HCN2. CONCLUSIONS: A gene-therapy approach for induction of biological pacemaker activity within the LBB system provides ventricular escape rhythms that have physiologically acceptable rates. Long-term stability and feasibility of the approach remain to be tested.

Expression and function of a biological pacemaker in canine heart.

BACKGROUND: We hypothesized that localized overexpression of the hyperpolarization-activated, cyclic nucleotide-gated (HCN2) pacemaker current isoform in canine left atrium (LA) would constitute a novel biological pacemaker. METHODS AND RESULTS: Adenoviral constructs of mouse HCN2 and green fluorescent protein (GFP) or GFP alone were injected into LA, terminal studies performed 3 to 4 days later, hearts removed, and myocytes examined for native and expressed pacemaker current (I(f)). Spontaneous LA rhythms occurred after vagal stimulation-induced sinus arrest in 4 of 4 HCN2+GFP dogs and 0 of 3 GFP dogs (P<0.05). Native I(f) in nonexpressed atrial myocytes was 7+/-4 pA at -130 mV (n=5), whereas HCN2+GFP LA had expressed pacemaker current (I(HCN2)) of 3823+/-713 pA at -125 mV (n=10) and 768+/-365 pA at -85 mV. CONCLUSIONS: HCN2 overexpression provides an I(f)-based pacemaker sufficient to drive the heart when injected into a localized region of atrium, offering a promising gene therapy for pacemaker disease.