Transcription profiling of HCN-channel isotypes throughout mouse cardiac development.

Hyperpolarization-activated ion channels, encoded by four mammalian genes (HCN1-4), contribute in an important way to the cardiac pacemaker current I(f). Here, we describe the transcription profiles of the four HCN genes, the NRSF, KCNE2 and Kir2.1 genes from embryonic stage E9.5 dpc to postnatal day 120 in the mouse. Embryonic atrium and ventricle revealed abundant HCN4 transcription but other HCN transcripts were almost absent. Towards birth, HCN4 was downregulated in the atrium and almost vanished from the ventricle. After birth, however, HCN isotype transcription changed remarkably, showing increased levels of HCN1, HCN2 and HCN4 in the atrium and of HCN2 and HCN4 in the ventricle. HCN3 showed highest transcription at early embryonic stages and was hardly detectable thereafter. At postnatal day 10, HCN4 was highest in the sinoatrial node, being twofold higher than HCN1 and fivefold higher than HCN2. In the atrium, HCN4 was similar to HCN1 and sevenfold higher than HCN2. In the ventricle, in contrast, HCN2 was sixfold higher than HCN4, while HCN1 was absent. Subsequently all HCN isotype transcripts declined to lower adult levels, while ratios of HCN isotypes remained stable. In conclusion, substantial changes of HCN isotype transcription throughout cardiac development suggest that a regulated pattern of HCN isotypes is required to establish and ensure a stable heart rhythm. Furthermore, constantly low HCN transcription in adult myocardium may be required to prevent atrial and ventricular arrhythmogenesis.

Highly sensitive cardiac troponin T values remain constant after brief exercise- or pharmacologic-induced reversible myocardial ischemia.

BACKGROUND: Using a new precommercial high-sensitivity cardiac troponin T (hsTnT) assay, we evaluated whether hsTnT increases after reversible myocardial ischemia. METHODS: In 195 patients undergoing nuclear stress testing (ST) using single-photon emission computed tomography (SPECT) for suspected ischemic heart disease, we measured hsTnT before and 18 min, 4 h, and 24 h after the stress test. Thirty patients were excluded before ST because of cardiac troponin T (cTnT) >30 ng/L (0.03 mug/L) as measured by the fourth-generation commercial test. Another 65 patients were excluded because of a combination of fixed and reversible perfusion defects (PDs) after SPECT. RESULTS: We studied 18 patients with reversible PDs, 41 patients with fixed PDs, and 41 patients without any PDs. Of these 100 patients, 61 received dynamic ST and 39 pharmacological ST. Median baseline hsTnT concentrations (25th, 75th percentile) were comparable in patients with reversible, fixed, and no PDs [5.57 (2.47, 12.60), 8.01 (4.55, 12.44), and 6.90 (4.63, 10.59) ng/L, respectively]. After ST, median hsTnT concentrations did not change in the reversible, fixed, or no PD groups from baseline to 18 min [-0.41 (-0.81, 0.01), 0.01 (-0.75, 0.79), and 0.36 (-0.42, 1.01) ng/L] or from baseline to 4 h [-0.56 (-1.82, 0.74), 0.24 (-0.60, 1.45), and 0.23 (-0.99, 1.15) ng/L]. Median baseline hsTnT concentrations tended to be higher in patients undergoing pharmacological vs dynamic ST; however, there were no significant increases in hsTnT concentrations after either type of ST. CONCLUSIONS: Elevation of cTnT is rather a consequence of irreversible myocyte death than reversible myocardial ischemia after exercise or pharmacologic myocardial ischemia.

Treatment of iatrogenic femoral pseudoaneurysm by ultrasound-guided compression therapy and thrombin injection.

Development of an arterial pseudoaneurysm is a common complication following cardiac catheterization. We analyzed data from 6,300 patients who received left heart catheterization at our institution. One day after the procedure, approximately 10% of the patients were examined with duplex sonography. In 204 patients (3.0%), a pseudoaneurysm of the femoral artery was diagnosed. All patients underwent compression therapy. Thereby, 159 of the pseudoaneurysms could be treated successfully. The remaining 45 pseudoaneurysms had a maximal diameter of more than 1.5 cm. Forty-two patients underwent ultrasound and biopsy-line-guided thrombin injection without complications. This strategy resulted in a successful occlusion in 41 cases. Pseudoaneurysms smaller than 2 cm can be treated with compression therapy. Larger pseudoaneurysms can be occluded by thrombin injection using ultrasound guidance. Patients with a pseudoaneurysm with a wide "neck" should be treated surgically, because the risk of an arterial occlusion following thrombin injection cannot be excluded.

Atrial-radiofrequency catheter ablation mediated targeting of mesenchymal stromal cells.

Sinus node dysfunction and high-degree heart block are the major causes for electronic pacemaker implantation. Recently, genetically modified mesenchymal stromal cells (MSCs, also known as "mesenchymal stem cells") were demonstrated to generate pacemaker function in vivo. However, experimental approaches typically use open thoracotomy for direct cell injection into the myocardium. Future clinical implementation, however, essentially requires development of more gentle methods to precisely and efficiently apply specified stem cells at specific cardiac locations. In a "proof of concept" study, we performed selective power-controlled radiofrequency catheter ablation (RFCA) with eight ablation pulses (30 W, 60 seconds each) to induce heat-mediated lesions at the auricles of the cardiac right atrium of four healthy foxhounds. The next day, allogeneic MSCs (4.3 x 10(5) cells per kilogram of body weight) labeled with superparamagnetic iron oxide particles (SPIOs) were infused intravenously. Hearts were explanted 8 days later. High numbers of SPIO-labeled cells were identified in areas surrounding the RFCA-induced lesions by Prussian blue staining. Antibody staining revealed SPIO-labeled cells being positive for the typical MSC surface antigen CD44. In contrast, low levels of calprotectin, an antigen found on monocytes and macrophages, indicated negligible infiltration of monocytes in MSC-positive areas. Thus, RFCA allows targeting of MSCs to the cardiac right atrium, adjacent to the sinoatrial node, providing an opportunity to rescue or generate pacemaker function without open thoracotomy and direct injection of MSCs. This method presents a new strategy for cardiac stem cell application leading to an efficient guidance of MSCs into the myocardium. Disclosure of potential conflicts of interest is found at the end of this article.

Skipping of Exon 1 in the KCNQ1 gene causes Jervell and Lange-Nielsen syndrome.

The Jervell and Lange-Nielsen syndrome (JLNS) is a rare autosomal recessive form of the long QT syndrome linked with a profound hearing loss caused by mutations affecting both alleles of either the KCNQ1 or the KCNE1 gene. We carried out a mutant screening of the KCNQ1 and KCNE1 genes in a clinical diagnosed German family with JLNS. Family members were examined by single strand conformation polymorphism analysis and PCR and amplified products were characterized by DNA sequence analysis. We identified a splice donor mutation of exon 1 in the KCNQ1 gene (G477+1A). Analysis of lymphocyte RNA by RT-PCR revealed that two symptomatic patients, homozygous for the mutant allele, exclusively produce KCNQ1 transcripts lacking exon 1 leading to a frameshift that introduced a premature termination codon at exon 4. Mutant subunits, functionally characterized in Xenpous oocytes, were unable to form homomeric channels but strongly reduced IKs (slowly activating delayed rectifier potassium current) in vitro (mutant isoforms 1 and 2 by 62 and 86%, respectively), a fact supposed to lead to severely affected heterozygous individuals. However, individuals heterozygous for the mutant allele exhibit an asymptomatic cardiac phenotype. Thus, the observed dominant-negative effect of mutant subunits in vitro is absent in vivo leaving heterozygous individuals unaffected. These data suggest mechanisms that prevent production of truncated KCNQ1 channel subunits in cardiomyocytes of individuals heterozygous for the mutant allele.

Real-time myocardial perfusion imaging for pharmacologic stress testing: added value to single photon emission computed tomography.

BACKGROUND: Little is known about the incremental value of real-time myocardial contrast echocardiography (MCE) as an adjunct to pharmacologic stress testing. This study was performed to evaluate the diagnostic value of MCE to detect abnormal myocardial perfusion by technetium Tc 99m sestamibi-single photon emission computed tomography (SPECT) and anatomically significant coronary artery disease (CAD) by angiography. METHODS: Myocardial contrast echocardiography was performed at rest and during vasodilator stress in consecutive patients (N = 120) undergoing SPECT imaging for known or suspected CAD. Myocardial opacification, wall motion, and tracer uptake were visually analyzed in 12 myocardial segments by 2 pairs of blinded observers. Concordance between the 2 methods was assessed using the kappa statistic. RESULTS: Of 1356 segments, 1025 (76%) were interpretable by MCE, wall motion, and SPECT. Sensitivity of wall motion was 75%, specificity 83%, and accuracy 81% for detecting abnormal myocardial perfusion by SPECT (kappa = 0.53). Myocardial contrast echocardiography and wall motion together yielded significantly higher sensitivity (85% vs 74%, P < .05), specificity of 83%, and accuracy of 85% (kappa = 0.64) for the detection of abnormal myocardial perfusion. In 89 patients who underwent coronary angiography, MCE and wall motion together yielded higher sensitivity (83% vs 64%, P < .05) and accuracy (77% vs 68%, P < .05) but similar specificity (72%) compared with SPECT for the detection of high-grade, stenotic (> or = 75%) coronary lesions. CONCLUSION: Assessment of myocardial perfusion adds value to conventional stress echocardiography by increasing its sensitivity for the detection of functionally abnormal myocardial perfusion. Myocardial contrast echocardiography and wall motion together provide higher sensitivity and accuracy for detection of CAD compared with SPECT.

Dominant-negative I(Ks) suppression by KCNQ1-deltaF339 potassium channels linked to Romano-Ward syndrome.

OBJECTIVE: Hereditary long QT syndrome (LQTS) is a genetically heterogeneous disease characterized by prolonged QT intervals and an increased risk for ventricular arrhythmias and sudden cardiac death. Mutations in the voltage-gated potassium channel subunit KCNQ1 induce the most common form of LQTS. KCNQ1 is associated with two different entities of LQTS, the autosomal-dominant Romano-Ward syndrome (RWS), and the autosomal-recessive Jervell and Lange-Nielsen syndrome (JLNS) characterized by bilateral deafness in addition to cardiac arrhythmias. In this study, we investigate and discuss dominant-negative I(Ks) current reduction by a KCNQ1 deletion mutation identified in a RWS family. METHODS: Single-strand conformation polymorphism analysis and direct sequencing were used to screen LQTS genes for mutations. Mutant KCNQ1 channels were heterologously expressed in Xenopus oocytes, and potassium currents were recorded using the two-microelectrode voltage clamp technique. RESULTS: A heterozygous deletion of three nucleotides (CTT) identified in the KCNQ1 gene caused the loss of a single phenylalanine residue at position 339 (KCNQ1-deltaF339). Electrophysiological measurements in the presence and absence of the regulatory beta-subunit KCNE1 revealed that mutant and wild type forms of an N-terminal truncated KCNQ1 subunit (isoform 2) caused much stronger dominant-negative current reduction than the mutant form of the full-length KCNQ1 subunit (isoform 1). CONCLUSION: This study highlights the functional relevance of the truncated KCNQ1 splice variant (isoform 2) in establishment and mode of inheritance in long QT syndrome. In the RWS family presented here, the autosomal-dominant trait is caused by multiple dominant-negative effects provoked by heteromultimeric channels formed by wild type and mutant KCNQ1-isoforms in combination with KCNE1.

Comparison of real-time myocardial contrast echocardiography for the assessment of myocardial viability with fluorodeoxyglucose-18 positron emission tomography and dobutamine stress echocardiography.

Little is known about the diagnostic value of real-time myocardial contrast echocardiography (MCE) for the assessment of myocardial viability. We compared the diagnostic accuracy of MCE with that of low-dose dobutamine stress echocardiography (DSE) and of combined technetium-99 sestamibi single-photon emission computed tomography and fluorodeoxyglucose-18 positron emission tomography and investigated whether quantitative assessment of myocardial blood flow could increase its diagnostic value. Cardiac imaging was performed with these 3 methods in 41 patients who had ischemic heart disease (ejection fraction < 40%) and were being considered for revascularization. Follow-up echocardiograms were obtained after 3 to 6 months in revascularized patients, and increased regional function served as a standard reference for assessment of myocardial viability. A control group of 25 patients who had no coronary artery disease underwent MCE to assess normal values of myocardial perfusion parameters. Recovery of myocardial function was predicted by MCE with a sensitivity of 86% and a specificity of 43%. Nuclear imaging was comparably sensitive (90%) and specific (44%), whereas low-dose DSE was similarly sensitive (83%) but more specific (76%). Normalization of myocardial signal intensity to that of the control group significantly increased the specificity of MCE from 43% to 64% and the accuracy from 73% to 81%. A combination of quantitative MCE and DSE provided the best diagnostic characteristics, with a sensitivity of 96%, a specificity of 63%, and an accuracy of 83%. Thus, MCE is useful for assessing myocardial viability. Normalization of contrast intensity to that of a reference control group is a valid approach for detection of myocardial viability and expands on information obtained from visual MCE and DSE.

Biventricular hypertrophy in dogs with chronic AV block: effects of cyclosporin A on morphology and electrophysiology.

Chronic atrioventricular (AV) block (CAVB) and biventricular hypertrophy in dogs increase susceptibility to drug-induced polymorphic ventricular tachycardia (PVT). In various rodent models, cyclosporin A (CsA) prevented hypertrophy. A similar effect in the CAVB model would allow us to determine whether hypertrophy represents an epiphenomenon, the cause of electrophysiological changes, and/or the anatomic substrate for PVTs. Upon AV node ablation, 6 dogs were studied acutely (AAVB), 25 dogs were kept for 6 (6W) and 12 wk (12W), receiving no treatment [CTL-CAVB-6W (n=6) and CTL-CAVB-12W (n=7)] or a daily oral dose of 10-20 mg/kg CsA directly (n=6, CsA-CAVB-6W) or 6 wk after radio-frequency ablation (n=6, CsA-CAVB-12W). For the final study, dogs were anesthetized, and 60 needles were inserted into both ventricles and connected to a multiplexer mapping system. Local effective refractory periods (ERPs) were determined at 56 +/- 22 randomly selected sites (extrastimulus technique, basic cycle length=800 ms). Arrhythmias within 30 min after application of almokalant (0.34 micromol/kg iv) were registered. The hearts were then excised to obtain the heart weight-body weight index (HBWI). Compared with AAVB, CTL-CAVB-6W and CTL-CAVB-12W showed increased HBWI and ERP associated with PVT inducibility in none of six AAVB dogs, four of six CTL-CAVB-6W dogs, and one of seven CTL-CAVB-12W dogs. Compared with CTL-CAVB-6W and CTL-CAVB-12W, CsA-CAVB-6W and CsA-CAVB-12W partially prevented hypertrophy or led to a regression of hypertrophy without reducing ERP prolongation. Despite ERP prolongation, PVTs were no longer inducible with CsA treatment. Thus prolongation of refractoriness seems to provide the trigger, but hypertrophy provides the essential substrate for the induction of PVTs in this model.

Real-time myocardial contrast echocardiography for pharmacologic stress testing: is quantitative estimation of myocardial blood flow reserve necessary?

BACKGROUND: Little is known about the diagnostic accuracy of quantitative real-time myocardial contrast echocardiography (MCE) as an adjunct to stress testing. This study was performed to evaluate the agreement between MCE and technetium 99m-sestamibi single photon emission computed tomography (SPECT) for detection of perfusion defects and to investigate whether quantitative assessment of myocardial perfusion can increase the diagnostic value of MCE. METHODS: MCE was performed at rest and during peak adenosine stress in 50 unselected patients undergoing SPECT imaging. Concordance between the 2 methods was assessed using kappa statistics. MCE images were analyzed quantitatively, measuring peak intensity (A) and maximal rise of signal intensity (beta). Myocardial blood flow reserve was estimated by calculating the ratios of A(adenosine)/A(baseline) (A reserve), beta(adenosine)/beta(baseline) (beta reserve), and A x beta(adenosine)/A x beta(baseline) (A x beta reserve). RESULTS: Visual analysis of MCE agreed well with SPECT (kappa = 0.67) with sensitivity of 64%, specificity of 97%, and overall accuracy of 87%. Quantitative analysis showed that peak signal intensity A significantly increased under adenosine stress in SPECT-normal segments (2.6 +/- 1.9 vs 3.0 +/- 1.6 dB, P <.0001), tendencially decreased in reversible (3.0 +/- 2.0 vs 2.4 +/- 1.2 dB, P =.07) and remained unchanged in fixed (0.9 +/- 0.9 vs 0.8 +/- 0.9 dB) defects. beta Increased markedly under adenosine in normal segments (0.4 +/- 0.4 vs 1.4 +/- 1.3, P <.0001) but not in segments with reversible or fixed defects. Receiver operating characteristic showed that beta reserve and A x beta reserve, but not A reserve, are sensitive parameters for detecting perfusion defects with areas under the curve of 0.84, 0.85, and 0.61, respectively. Cut-off values of 1.9 and 2.3, respectively, for beta and A x beta reserve yielded sensitivity rates of 79% and 80%, specificity rates of 75% and 78%, and overall accuracy rates of 76% and 79%, respectively. CONCLUSION: Quantitative estimation of myocardial blood flow reserve by MCE parameters corresponds to the evaluation of myocardial perfusion by nuclear imaging and can increase the sensitivity but not the overall accuracy of contrast echocardiography.

Fourier phase and amplitude analysis for automated objective evaluation of myocardial contrast echocardiograms.

AIMS: Objective methods for evaluating myocardial contrast echocardiography (MCE) are not yet widely available. We applied a Fourier analysis to myocardial contrast echocardiograms to identify myocardial perfusion defects. METHODS: Harmonic power-Doppler contrast echocardiograms were performed in 21 patients undergoing Tl-201-SPECT imaging and in 13 controls. Images were transformed using Fourier analysis to obtain phase of the first harmonic sinusoidal curve displayed as color coded sequence of myocardial intensity changes. Means and standard deviations of regional phase angles were measured. The method was validated in an in vitro model. A contrast filled latex balloon was imaged at different gain settings mimicking defined time-intensity curves. An intraoperative porcine infarction model served to prove feasibility of Fourier transformation to analyze real-time pulse inversion contrast echocardiography. RESULTS: In patients, phase imaging and intensity analysis showed focal areas with marked phase shifts (106 +/- 90 degrees) and heterogeneous distribution of phase angles (SD 66 +/- 17 degrees), correctly identifying 13/14 perfusion defects. The in vitro validation yielded increasing phase angles with increasing beta-values. This method was successfully applied to real-time MCE, identifying all infarction areas during occlusion of the left anterior descending artery. CONCLUSION: Phase analysis can be used to display dynamics of myocardial opacification.