Recurrent atrial arrhythmia in a randomised controlled trial comparing contact force-guided and contact force-blinded ablation for typical atrial flutter.

BACKGROUND: Contact force (CF)-guided catheter ablation (CA) is a novel technology developed to improve efficacy and reduce complications. In a randomised controlled trial (RCT), we previously documented that after 3 months, rate of persistent conduction block was similar with and without using CF while performing CA for typical atrial flutter (AFL). Clinical effect of CF on recurrent arrhythmia is unknown. Our objective is to study recurrent atrial arrhythmia during 12-month follow-up in a RCT investigating whether CF-guided CA for typical AFL is superior to CF-blinded CA. METHODS: Patients were randomised 1:1 to CA guided by CF (intervention group) or blinded to CF (control group). After 12 months, patients attended clinical check-up preceded by a 5-day ambulatory Holter monitor recording. Primary outcome was any recurrent atrial arrhythmia ≥ 30 s within 12 months and documented in 12-lead ECG or Holter monitor recording. RESULTS: We included 156 patients, four patients withdrew consent and two died during follow-up. Thus, 150 patients were included in final analysis. Recurrent arrhythmia was detected in 36 of 77 (47%) patients in the intervention group, and 32 of 73 patients (44%) in the control group (p = 0.51). Atrial fibrillation was detected in 23 (30%) and 29 (40%) patients in the intervention and control groups respectively. AFL was detected in 11 (14%) and 5 (7%) patients in the intervention and control groups respectively. CONCLUSIONS: Contact force-guided ablation for typical atrial flutter does not reduce recurrent atrial arrhythmia after 12-month follow-up as compared with ablation blinded for contact force.

Impact of Administration Time and Kv7 Subchannels on the Cardioprotective Efficacy of Kv7 Channel Inhibition.

PURPOSE: The mechanism of cardioprotection by Kv7.1-5 (KCNQ1-5) channels inhibition by XE991 is unclear. We examined the impact of administration time on the cardioprotective efficacy of XE991, the involvement of key pro-survival kinases, and the importance of the Kv7 subchannels. METHODS: Isolated perfused rat hearts were divided into five groups: 1) vehicle, 2) pre-, 3) post- or 4) pre- and post-ischemic administration of XE991 or 5) chromanol 293B (Kv7.1 inhibitor) followed by infarct size quantification. HL-1 cells undergoing simulated ischemia/reperfusion were exposed to either a) vehicle, b) pre-, c) per-, d) post-ischemic administration of XE991 or pre-, per- and post-ischemic administration of e) XE991, f) Chromanol 293B or g) HMR1556 (Kv7.1 inhibitor). HL-1 cell injury was evaluated by propidium iodide/Hoechst staining. Pro-survival kinase activation of Akt, Erk and STAT3 in XE991-mediated HL-1 cell protection was evaluated using phosphokinase inhibitors. Kv7 subtype expression was examined by RT-PCR and qPCR. RESULTS: XE991, but not Chromanol 293B, reduced infarct size and improved hemodynamic recovery in all isolated heart groups. XE991 protected HL-1 cells when administered during simulated ischemia. Minor activation of the survival kinases was observed in cells exposed to XE991 but pharmacological inhibition of kinase activation did not reduce XE991-mediated protection. Kv7 subchannels 1-5 were all present in rat hearts but predominately Kv7.1 and Kv7.4 were present in HL-1 cells and selective Kv7.1 did not reduce ischemia/reperfusion injury. CONCLUSION: The cardioprotective efficacy of XE991 seems to depend on its presence during ischemia and early reperfusion and do not rely on RISK (p-Akt and p-Erk) and SAFE (p-STAT3) pathway activation. The protective effect of XE991 seems mainly mediated through the Kv7.4 subchannel.

A randomized trial of contact force in atrial flutter ablation.

AIMS: Contact force (CF) sensing has emerged as a tool to guide and improve outcomes for catheter ablation (CA) for cardiac arrhythmias. The clinical benefit on patient outcomes remains unknown. To study whether CF-guided CA for typical atrial flutter (AFL) is superior to CA not guided by CF. METHODS AND RESULTS: In a double-blinded controlled superiority trial, we randomized patients 1:1 to receive CA for typical AFL guided by CF (intervention group) or blinded to CF (control group). In the intervention group, a specific value of the lesion size index (LSI), estimating ablation lesions size was targeted for each ablation lesion. Patients underwent electrophysiological study (EPS) after 3 months to assess occurrence of the primary endpoint of re-conduction across the cavo-tricuspid isthmus (CTI). We included 156 patients with typical AFL, median age was 68 [interquartile range (IQR) 61-74] years and 120 (77%) patients were male. At index procedure median LSI was higher in the intervention group [6.4 (IQR 5.1-7) vs. 5.6 (IQR 4.5-6.9), P < 0.0001]. After 3 months, 126 patients (58 in intervention group) underwent EPS for primary endpoint assessment. Thirty (24%) patients had CTI re-conduction, distributed with 15 patients in each treatment group (P = 0.62). We observed no difference between treatment groups with regard to fluoroscopy, ablation, or procedure times, nor peri-procedural complications. CONCLUSION: Contact force-guided ablation does not reduce re-conduction across the CTI after 3 months, nor does CF-guided ablation shorten fluoroscopy, ablation, or total procedure times.

Recurrent atrial flutter ablation and incidence of atrial fibrillation ablation after first-time ablation for typical atrial flutter: A nation-wide Danish cohort study.

BACKGROUND: Cavo tricuspid isthmus ablation (CTIA) is considered an effective first-line treatment for typical atrial flutter (AFL). However, many patients develop atrial fibrillation (AF) after successful CTIA. Knowledge about recurrent arrhythmia after CTIA mainly comes from small cohort studies with limited follow-up. OBJECTIVE: To describe incidences of re-ablation for AFL and ablation for AF after first-time CTIA in a nation-wide cohort. METHODS: In the Danish National Ablation Registry we identified patients undergoing first-time CTIA during 2010-2016. Subsequent CTIA and AF-ablation procedures were identified until March 1st, 2018. We collected information on patient comorbidities in the Danish National Patient Registry. RESULTS: We identified 2409 patients undergoing first-time CTIA. Median age was 66 (IQR 58-72) years, 1952 (81%) were men, and 78 (3%) patients had a history of previous ablation for AF. Acute procedural success was achieved in 2288 (95%) patients. During mean follow-up of 4.0 ±â€¯1.7 years, 242 (10%) patients underwent CTI re-ablation and 326 (13.5%) underwent ablation for AF. Baseline characteristics associated with CTI re-ablation included prolonged procedural time, unsuccessful index CTIA, age <75 years and CHA2DS2-VASc score <2. Hypertension, history of AF-ablation, age <65 years use of a contact force sensing catheter and CHA2DS2-VASc score <2 were associated with later ablation for AF. CONCLUSION: In a nation-wide cohort undergoing first-time CTIA for AFL, 10% of patients underwent CTI re-ablation and 13.5% ablation for AF during mean follow-up of 4.0 ±â€¯1.7 years. Probability of a second procedure was higher in younger patients with less comorbidities.

Outcome after catheter ablation for left atrial flutter.

Objectives. Left atrial flutter has been reported in up to 10% of patients following pulmonary vein isolation or cardiac surgery. Left atrial flutter is typically highly symptomatic, responds poorly to medical antiarrhythmic treatment, and is often treated by catheter ablation. We aimed to investigate midterm freedom from recurrent arrhythmia after catheter ablation for left atrial flutter. Design. In the National Danish Ablation Registry, we identified consecutive patients, who had undergone catheter ablation for left atrial flutter between 1 January 2014 and 1 April 2017 at our centre. Results. A total of 53 patients (median age 68 years (IQR 60-71) 37 (70%) male) were included. Forty-two patients had prior left atrial catheter ablation procedures (79%), one patient prior ablation for classic atrial flutter (2%), four patients had prior surgery for congenital heart disease (8%), and six patients (11%) had no previous cardiac intervention. Acute procedural success, defined as non-inducibility of any atrial arrhythmia, was achieved in 45 of 53 patients (85%). During midterm follow-up (mean 20 ± 12 months), 26 patients experienced an episode of recurrent atrial arrhythmia. Median EHRA-score was 3 (range 2-4) before catheter ablation and reduced to median 1 (range 1-3) evaluated at follow-up visits after three and twelve months (both p < .001, Wilcoxon rank test). Conclusion. Left atrial flutter is preceded by catheter ablation or cardiac surgery in 89% of patients. Acute procedural success is achieved in majority of patients and ablation reduces symptoms effectively. During midterm follow-up, almost half the patients experience recurrent atrial arrhythmia.

Multicenter Study of Ischemic Ventricular Tachycardia Ablation With Decrement-Evoked Potential (DEEP) Mapping With Extra Stimulus.

OBJECTIVES: The authors conducted a multicenter study of decrement-evoked potential (DEEP)-based functional ventricular tachycardia (VT) substrate modification to evaluate if such a mechanistic and physiological strategy is feasible and efficient in clinical practice and provides reduction in the VT burden. BACKGROUND: Only a fraction of the myocardium targeted in current VT substrate modification procedures is involved in the initiation and perpetuation of VT. The physiological basis of the DEEP strategy for identification of areas of initiation and maintenance of VT was recently established. METHODS: We included 20 consecutive patients with ischemic cardiomyopathy. During substrate mapping, fractionated and late potentials (LPs) were tagged, and an extra stimulus was performed to determine which LPs displayed decrement (DEEPs). All patients underwent DEEP-focused ablation: elimination of DEEP + further radiofrequency (RF) if VT was still inducible. Patients were followed during 6 months. RESULTS: Patients were predominantly male (95%), and their mean age was 64.6 ± 17.1 years. Mean left ventricular ejection fraction was 33.4 ± 11.4%. Mean ablation time was 30.6 ± 20.4 min. Specificity of DEEPs to detect the isthmus of VT was better than that of LPs (0.97 [95% confidence interval [CI]: 0.95 to 0.98] vs. 0.82 [95% CI: 0.73 to 0.89]), without significant differences in terms of sensitivity (0.61 [95% CI: 0.52 to 0.69] vs. 0.60 [95% CI: 0.44 to 0.74], respectively). Fifteen of 20 (75%) patients were free of any VT after DEEP-RF at 6 months of follow-up and there was a strong reduction in VT burden compared to 6 months pre-ablation. CONCLUSIONS: In a multicenter prospective study, DEEP substrate mapping identified the functional substrate critical to the VT circuit with high specificity. DEEP-guided VT ablation, by its physiological nature, may enable greater access to focused ablation therapy for patients requiring VT treatment.

Recurrence after pulmonary vein isolation is associated with low contact force.

OBJECTIVES: Recurrent arrhythmia after pulmonary vein isolation (PVI) by radiofrequency (RF) ablation in patients with atrial fibrillation (AFIB) remains a significant challenge. Using contact force (CF) sensing ablation catheters, we aimed to identify procedure related parameters associated with recurrence after de-novo PVI in patients with AFIB. METHODS: Consecutive patients undergoing a de-novo PVI procedure (n = 120, 63% paroxysmal and 37% persistent AFIB) employing a force-sensing ablation catheter were included. A clinical control including electrocardiogram and 120 hour of Holter-recording at 12-months was performed in all patients. Recurrence was defined as any documented AFIB or atrial flutter more than 30 seconds on Holter-recording after an initial blanking period of three months. RESULTS: Recurrence occurred in 44 patients (37%). Mean CF was lower in patients with recurrent arrhythmia (22.2 ± 9.5 vs. 28.8 ± 9.3 g, p < .001). In multi-variable analyses lower mean CF (OR 0.9 (95% CI 0.8-1.0), p = .03), and higher percentage of ablation time with a CF <10 grams (OR 1.1 (95% CI 1.0-1.1), p = .004) were both associated with recurrence in two distinct models. Dragging during ablation compared with point-by-point ablation technique was associated with recurrence in both models (OR 19.2 (95% CI 2.9-130.0), p = .002, and OR 21.7 (95% CI 2.7-176.2), p = .004). CONCLUSIONS: Low CF and dragging during ablation as compared with point-by-point ablation technique were associated with recurrent arrhythmia in patients with AFIB undergoing de-novo PVI by RF ablation.

Untargeted metabolomics reveals a mild impact of remote ischemic conditioning on the plasma metabolome and α-hydroxybutyrate as a possible cardioprotective factor and biomarker of tissue ischemia.

INTRODUCTION: Remote ischemic conditioning (RIC) is a maneuver by which short non-lethal ischemic events are applied on distant organs or limbs to reduce ischemia and reperfusion injuries caused by e.g. myocardial infarct. Although intensively investigated, the specific mechanism of this protective phenomenon remains incompletely understood and in particular, knowledge on the role of small metabolites is scarce. OBJECTIVES: In this study, we aimed to study perturbations in the plasma metabolome following RIC and gain insight into metabolic changes by the intervention as well as to identify potential novel cardio-protective metabolites. METHODS: Blood plasma samples from ten healthy males were collected prior to and after RIC and tested for bioactivity in a HL-1 based cellular model of ischemia-reperfusion damage. Following this, the plasma was analyzed using untargeted LC-qTOF-MS and regulated metabolites were identified using univariate and multivariate statistical analysis. Results were finally verified in a second plasma study from the same group of volunteers and by testing a metabolite ester in the HL-1 cell model. RESULTS: The analysis revealed a moderate impact on the plasma metabolome following RIC. One metabolite, α-hydroxybutyrate (AHB) however, stood out as highly significantly upregulated after RIC. AHB might be a novel and more sensitive plasma-biomarker of transient tissue ischemia than lactate. Importantly, it was also found that a cell permeable AHB precursor protects cardiomyocytes from ischemia-reperfusion damage. CONCLUSION: Untargeted metabolomics analysis of plasma following RIC has led to insight into metabolism during RIC and revealed a possible novel metabolite of relevance to ischemic-reperfusion damage.

Effect of paroxetine on left ventricular remodeling in an in vivo rat model of myocardial infarction.

Left ventricular (LV) remodeling following a myocardial infarction (MI) involves formation of reactive oxygen species (ROS). Paroxetine, a selective serotonin reuptake inhibitor, has an antioxidant effect in the vascular wall. We investigated whether paroxetine reduces myocardial ROS formation and LV remodeling following a MI. In a total of 32 Wistar rats, MI was induced by a 30-min ligation of the left anterior descending artery followed by 7- or 28-day reperfusion. During the 28 days of reperfusion, LV remodeling was evaluated by magnetic resonance imaging (MRI) and echocardiography (n = 20). After 28 days of reperfusion, the susceptibility to ventricular tachycardia was evaluated prior to sacrifice and histological assessment of myocyte cross-sectional area, fibrosis, and presence of myofibroblasts. Myocardial ROS formation was measured with dihydroethidium after 7 days of reperfusion in separate groups (n = 12). Diastolic LV volume, evaluated by MRI (417 ± 60 vs. 511 ± 64 µL, p < 0.05), and echocardiography (515 ± 80 vs. 596 ± 83 µL, p < 0.05) as well as diastolic LV internal diameter evaluated with echocardiography (7.2 ± 0.6 vs. 8.1 ± 0.7 mm, p < 0.05) were lower in the paroxetine group than in controls. Furthermore, myocyte cross-sectional area was reduced in the paroxetine group compared with controls (277 ± 26 vs. 354 ± 23 mm3, p < 0.05) and ROS formation was reduced in the remote myocardium (0.415 ± 0.19 normalized to controls, p < 0.05). However, no differences in the presence of fibrosis or myofibroblasts were observed. Finally, paroxetine reduced the susceptibility to ventricular tachycardia (induced in 2/11 vs. 6/8 rats, p < 0.05). Paroxetine treatment following MI decreases LV remodeling and susceptibility to arrhythmias, probably by reducing ROS formation.

Inotropic Effects of Prostacyclins on the Right Ventricle Are Abolished in Isolated Rat Hearts With Right-Ventricular Hypertrophy and Failure.

BACKGROUND: Prostacyclin mimetics are vasodilatory agents used in the treatment of pulmonary arterial hypertension. The direct effects of prostanoids on right-ventricular (RV) function are unknown. We aimed to investigate the direct effects of prostacyclin mimetics on RV function in hearts with and without RV hypertrophy and failure. METHODS: Wistar rats were subjected to pulmonary trunk banding to induce compensated RV hypertrophy (n = 32) or manifest RV failure (n = 32). Rats without banding served as healthy controls (n = 30). The hearts were excised and perfused in a Langendorff system and subjected to iloprost, treprostinil, epoprostenol, or MRE-269 in increasing concentrations. The effect on RV function was evaluated using a balloon-tipped catheter inserted into the right ventricle. RESULTS: In control hearts, iloprost, treprostinil, and MRE-269 improved RV function. The effect was, however, absent in hearts with RV hypertrophy and failure. Treprostinil and MRE-269 even impaired RV function in hearts with manifest RV failure. CONCLUSIONS: Iloprost, treprostinil, and MRE-269 improved RV function in the healthy rat heart. RV hypertrophy abolished the positive inotropic effect, and in the failing right ventricle, MRE-269 and treprostinil impaired RV function. This may be related to changes in prostanoid receptor expression and reduced coronary flow reserve in the hypertrophic and failing right ventricle.

[Radiofrequency ablation therapy of intractable ventricular tachycardia present with a left ventricular assist device]. / Radiofrekvensablation af ventrikulær takykardi hos en patient med kunstig hjertepumpe.

Ventricular tachycardia (VT) occurs in up to 59% of patients with left ventricular assist devices (LVAD). In some of these patients, the VT cannot be managed medically or by implantable cardioverter-defibrillator. In this case, a 66-year-old male was successfully treated with radiofrequency ablation of intractable VT that developed months after implantation of an LVAD (Heartware). The LVAD provided haemodynamical support during mapping and did not interfere with the ablation.

Change of cardiac function, but not form, in postprandial pythons.

Pythons are renowned for a rapid and pronounced postprandial growth of the heart that coincides with a several-fold elevation of cardiac output that lasts for several days. Here we investigate whether ventricular morphology is affected by digestive state in two species of pythons (Python regius and Python molurus) and we determine the cardiac right-to-left shunt during the postprandial period in P. regius. Both species experienced several-fold increases in metabolism and mass of the digestive organs by 24 and 48 h after ingestion of meals equivalent to 25% of body mass. Surprisingly there were no changes in ventricular mass or dimensions as we used a meal size and husbandry conditions similar to studies finding rapid and significant growth. Based on these data and literature we therefore suggest that postprandial cardiac growth should be regarded as a facultative rather than obligatory component of the renowned postprandial response. The cardiac right-to-left shunt, calculated on the basis of oxygen concentrations in the left and right atria and the dorsal aorta, was negligible in fasting P. regius, but increased to 10-15% during digestion. Such shunt levels are very low compared to other reptiles and does not support a recent proposal that shunts may facilitate digestion in reptiles.

Decreased expression of natriuretic peptides associated with lipid accumulation in cardiac ventricle of obese mice.

Plasma B-type natriuretic peptide (BNP) and proBNP are established markers of cardiac dysfunction. Even though obesity increases the risk of cardiovascular disease, obese individuals have reduced plasma concentrations of natriuretic peptides. The underlying mechanism is not established. We used cultured cardiomyocytes and three different mouse models to examine the impact of obesity and cardiac lipid accumulation on cardiac natriuretic peptide expression. The cardiac ventricular expression of atrial natriuretic peptide (ANP) and BNP mRNA and ANP peptide was decreased 36-72% in obese ob/ob, db/db, and fat-fed C57BL/6 mice as compared with their respective controls. The db/db and ob/ob mice displayed impaired cardiac function, whereas the fat-fed mice had almost normal cardiac function. Moreover, the ventricular expression of hypertrophic genes (α- and ß-myosin heavy chain and α-actin) and natriuretic peptide receptor genes were not consistently altered by obesity across the three mouse models. In contrast, cardiac ventricular triglycerides were similarly increased by 60-115% in all three obese mouse models and incubation with oleic acid caused triglyceride accumulation and an approximately 35% (P < 0.005) depression of ANP mRNA expression in cultured HL-1 atrial myocytes. The data suggest that obesity and altered cardiac lipid metabolism are associated with reduced production of ANP and BNP in the cardiac ventricles in the setting of normal as well as impaired cardiac function.

Inhibition of the malate-aspartate shuttle by pre-ischaemic aminooxyacetate loading of the heart induces cardioprotection.

AIMS: Preserved mitochondrial function is essential for protection against ischaemia-reperfusion (IR) injury. The malate-aspartate (MA) shuttle constitutes the principal pathway for transport of reducing cytosolic equivalents for mitochondrial oxidation. We hypothesized that a transient shut-down of the MA-shuttle by aminooxyacetate (AOA) during ischaemia and early reperfusion modulates IR injury by mechanisms comparable to ischaemic preconditioning (IPC). METHODS AND RESULTS: Isolated perfused rat hearts exposed to 40 min global no-flow ischaemia were studied in: (i) control, (ii) pre-ischaemic AOA (0.1 mM), (iii) IPC, and (iv) AOA+IPC hearts. IR injury was evaluated by infarct size and haemodynamic recovery. Tracer-estimated glucose oxidation and metabolic changes in glycogen, lactate, pyruvate, tricarboxylic acid (TCA) cycle intermediates, and ATP degradation products were measured. The effects of AOA on complex I respiration and reactive oxygen species (ROS) production were examined in isolated rabbit mitochondria. Treatment with AOA, IPC, or AOA+IPC induced significant infarct reduction; 28 ± 6, 30 ± 3, and 18 ± 1%, respectively, vs. 52 ± 5% of left ventricular (LV) mass for control (P < 0.01 for all). LV-developed pressure improved to 60 ± 3, 63 ± 5 and 53 ± 4 vs. 31 ± 5 mmHg (P < 0.01 for all) after 2 h reperfusion. Pre-ischaemic AOA administration inhibited glycolysis and increased glucose oxidation during post-ischaemic reperfusion similar to IPC, and suppressed complex I respiration and ROS production in the non-ischaemic heart. Changes in lactate, pyruvate, TCA intermediates, and ATP end products suggested an AOA inhibition of the MA-shuttle during late ischaemia and early reperfusion. CONCLUSION: Inhibition of the MA-shuttle during ischaemia and early reperfusion is proposed as a mechanism to reduce IR injury.

Effects of phosphodiesterase-5 inhibition by sildenafil in the pressure overloaded right heart.

BACKGROUND: Sustained pressure overload of the right ventricle (RV) causes RV hypertrophy and failure. Cyclic-GMP has previously been shown to modulate left ventricular hypertrophy. AIM: To evaluate the effects of sildenafil, a phosphodiesterase-5 (PDE5) inhibitor elevating c-GMP, on myocardial hypertrophy and function in rats with RV hypertrophy. METHODS: Rats were pulmonary trunk banded (PTB) and randomized to receive sildenafil (SIL) or vehicle (VEC) for three (n=14) and nine weeks (n=18). In addition, rats with established RV hypertrophy were randomized to SIL or VEC (n=17) three weeks after PTB. Right ventricular function was evaluated by echocardiography and RV hypertrophy by histology and RV weight. RESULTS: Sildenafil failed to inhibit the development of RV hypertrophy when given for both 3 and 9 weeks. On the contrary, sildenafil increased RV hypertrophy after 3 weeks (RV/bodyweight: SIL 0.099+/-0.016 vs. VEC 0.081+/-0.011; p<0.05) and total heart weight after 9 weeks (SIL 1.05+/-0.10 vs. VEC 0.93+/-0.08 g; p<0.05). Sildenafil also failed to reverse established RV hypertrophy, but significantly improved RV myocardial function as measured by Tricuspid Annular Plane Systolic Excursion (TAPSE: SIL 1.85+/-0.027 vs. VEC 1.39+/-0.037 mm; p<0.05). CONCLUSION: PDE5 inhibition by sildenafil failed to prevent or reverse RV hypertrophy in rats operated by pulmonary trunk banding. It actually increased RV hypertrophy and improved RV contractile function when given to rats with established RV hypertrophy.

Left ventricular volume measurement in mice by conductance catheter: evaluation and optimization of calibration.

The conductance catheter (CC) allows thorough evaluation of cardiac function because it simultaneously provides measurements of pressure and volume. Calibration of the volume signal remains challenging. With different calibration techniques, in vivo left ventricular volumes (V(CC)) were measured in mice (n = 52) with a Millar CC (SPR-839) and compared with MRI-derived volumes (V(MRI)). Significant correlations between V(CC) and V(MRI) [end-diastolic volume (EDV): R(2) = 0.85, P < 0.01; end-systolic volume (ESV): R(2) = 0.88, P < 0.01] were found when injection of hypertonic saline in the pulmonary artery was used to calibrate for parallel conductance and volume conversion was done by individual cylinder calibration. However, a significant underestimation was observed [EDV = -17.3 microl (-22.7 to -11.9 microl); ESV = -8.8 microl (-12.5 to -5.1 microl)]. Intravenous injection of the hypertonic saline bolus was inferior to injection into the pulmonary artery as a calibration method. Calibration with an independent measurement of stroke volume decreased the agreement with V(MRI). Correction for an increase in blood conductivity during the in vivo experiments improved estimation of EDV. The dual-frequency method for estimation of parallel conductance failed to produce V(CC) that correlated with V(MRI). We conclude that selection of the calibration procedure for the CC has significant implications for the accuracy and precision of volume estimation and pressure-volume loop-derived variables like myocardial contractility. Although V(CC) may be underestimated compared with MRI, optimized calibration techniques enable reliable volume estimation with the CC in mice.