Computed tomography (CT) assessment of the membranous septal anatomy prior to transcatheter aortic valve replacement (TAVR) with the balloon-expandable SAPIEN 3 valve.

OBJECTIVES: The lower limit of the membranous septum (MS) is considered an anatomic landmark for the emergence of the Bundle of His into the left ventricle. Computed tomography (CT) assessment of MS anatomy may provide useful information about the risk of conduction abnormalities following transcatheter aortic valve replacement (TAVR). METHODS AND RESULTS: The study included 102 consecutive patients undergoing TAVR with the Edwards Sapien 3 (S3) valve. Using pre-TAVR CT and post-procedure angiography we evaluated for the presence of calcium in the left ventricular outflow tract (LVOT), calcium depth (CD), implantation depth (ID) and MS length. The MS length minus the prosthesis ID was calculated (Delta MSID). Outcomes included new left bundle branch block (LBBB) or permanent pacemaker (PPM) within 30days. Seventeen patients (17%) received a PPM and 28 (27%) developed new LBBB following TAVR. Mean (±SD) MS length and delta MSID were 7.5mm (2) and 0.9mm (4.5), respectively. Twenty-one patients (20%) had calcium in the device landing zone and the mean (SD) CD was 6.8mm (±4). Calcium in the device landing zone (37% versus 16%, p=0.02) and implantation depth (6mm (4-8) versus 4mm (4-5), p=0.02) predicted new conduction abnormalities after TAVR. CONCLUSIONS: The presence of calcium in the device landing zone is associated with increased risk of conduction abnormalities after TAVR with S3. In such cases, a more aortic deployment of the prosthesis may be warranted.

Periodontal Bacterial DNA and Their Link to Human Cardiac Tissue: Findings of a Pilot Study.

BACKGROUND: The aim of this pilot study was to detect correlations of microbiological DNA, inflammatory proteins, and infection parameters in patients with periodontal disease (PD) and valvular heart disease (VHD). METHODS: A perioperative comprehensive dental examination for the investigation of periodontal status, including sampling of specific subgingival bacteria, was performed in 10 patients with indication for surgery of aortic valve stenosis with or without concomitant myocardial revascularization. Standard protocol biopsies were taken from right atrium (A), left septal myocardium (M), and aortic valve (V). Eleven periodontal pathogens DNA in oral and cardiac tissue samples (A/M/V) were analyzed using polymerase chain reaction. For cardiac tissue samples, Western blot analysis of LPS-binding protein (LBP), immunohistochemical (IHC) detection of LBP-big42, LPS-binding protein receptor (CD14), and macrophages (CD68), as well as inflammation scoring measurement were performed. RESULTS: Periodontitis was present in all patients with severe intensity in 7, moderate in 2 and mild in one patient. Same bacterial DNA was detected in A, M, and V in different distribution, and detection was more often in atrium than in myocardium or valve tissue. Morphological investigation revealed increased extracellular inflammatory cell migration. In IHC markers of LBP, CD68 and CD14 showed positive findings for all patients in atrium and myocardium. CONCLUSION: Our results demonstrate the presence of oral bacterial DNA in human cardiac tissue, as well as inflammatory markers potentially indicating connection of PD and VHD. Further investigation is necessary to confirm these preliminary data.

Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor.

G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.

A functional genetic study identifies HAND1 mutations in septation defects of the human heart.

Heart and neural crest derivatives expressed 1 (HAND1) is a basic helix-loop-helix (bHLH) transcription factor essential for mammalian heart development. Absence of Hand1 in mice results in embryonal lethality, as well as in a wide spectrum of cardiac abnormalities including failed cardiac looping, defective chamber septation and impaired ventricular development. Therefore, Hand1 is a strong candidate for the many cardiac malformations observed in human congenital heart disease (CHD). Recently, we identified a loss-of-function frameshift mutation (p.A126fs) in the bHLH domain of HAND1 frequent in hypoplastic hearts. This finding prompted us to continue our search for HAND1 gene mutations in a different cohort of malformed hearts affected primarily by septation defects. Indeed, in tissue samples of septal defects, we detected 32 sequence alterations leading to amino acid change, of which 12 are in the bHLH domain of HAND1. Interestingly, 10 sequence alterations, such as p.L28H and p.L138P, had been identified earlier in hypoplastic hearts, but the frequent p.A126fs mutation was absent except in one aborted case with ventricular septal defect and outflow tract abnormalities. Functional studies in yeast and mammalian cells enabled translation of sequence alterations to HAND1 transcriptional activity, which was reduced or abolished by certain mutations, notably p.L138P. Our results suggest that HAND1 may also be affected in septation defects of the human hearts, and thus has a broader role in human heart development and CHD.

Creatine depletion and altered fatty acid metabolism in diseased human hearts: clinical investigation using 1H magnetic resonance spectroscopy and 123I BMIPP myocardial scintigraphy.

BACKGROUND: In the heart, the creatine kinase system plays an important role in energy reserves, and myocardial energy production essentially depends upon fatty acid metabolism. PURPOSE: To examine myocardial creatine (CR) concentration and altered cardiac fatty acid metabolism in various forms of heart disease. MATERIAL AND METHODS: Myocardial CR concentration of the septum was measured by gated 1H magnetic resonance spectroscopy (MRS), applying a point-resolved spectroscopy (PRESS) sequence in 34 patients with heart disease. Of these patients, 14 underwent 123I BMIPP (radioactive fatty acid analogue) myocardial scintigraphy to evaluate myocardial fatty acid metabolism. Cardiac 123I BMIPP uptake was calculated as the heart-to-mediastinum count ratio. RESULTS: Myocardial CR concentration correlated positively with the left ventricular ejection fraction (LVEF) by echocardiography (R = 0.61, P<0.001, n = 34), suggesting that the degree of reduced CR is related to the severity of contractile dysfunction. Cardiac 123I BMIPP uptake also correlated positively with LVEF (initial image, R = 0.60, P<0.05; delayed image, R = 0.63, P<0.05; n = 14). There was a significant correlation between myocardial CR and cardiac 123I BMIPP uptake (initial image, R = 0.77, P<0.01; delayed image, R = 0.82, P<0.001; n = 14). CONCLUSION: Our study suggests an association between CR depletion and impaired fatty acid metabolism in various forms of heart diseases.

Normal distribution of melanocytes in the mouse heart.

We report the consistent distribution of a population of pigmented trp-1-positive cells in several important septal and valvular structures of the normal mouse (C57BL/6) heart. The pigmented cell population was first apparent by E16.5 p.c. in the right atrial wall and extended into the atrium along the interatrial septum. By E17.5, these cells were found along the apical membranous interventricular septum near or below the surface of the endocardium. The most striking distribution of dark pigmented cells was found in the tricuspid and mitral valvular leaflets and chordae tendineae. The normal distribution of pigmented cells in the valvuloseptal apparatus of C57BL/6 adult heart suggests that a premelanocytic lineage may participate in the earlier morphogenesis of the valve leaflets and chordae tendineae. The origin of the premelanocyte lineage is currently unknown. The most likely candidate populations include the neural crest and the epicardially derived cells. The only cell type in the heart previously shown to form melanocytes is the neural crest. The presence of neural crest cells, but not melanocytes, in some of the regions we describe has been reported by others. However, previous reports have not shown a contribution of melanocytes or neural crest derivatives to the atrioventricular valve leaflets or chordae tendineae in mouse hearts. If these cells are of neural crest origin, it would suggest a possibly greater contribution and persistence of neural crest cells to the valvuloseptal apparatus than has been previously understood.

Severe aortic stenosis and myocardial function: diagnostic and prognostic usefulness of ultrasonic integrated backscatter analysis.

BACKGROUND: The aim of this study was to assess the myocardial reflectivity pattern in severe aortic valve stenosis through the use of integrated backscatter (IBS) analysis. Patients with aortic stenosis (AS) were carefully selected in the Department of Cardiology. METHODS AND RESULTS: Thirty-five subjects (AS: valve orifice < or =1 cm2; 12 female; mean age, 71.8+/-6.2 years) and 25 healthy subjects were studied. All subjects of the study had conventional 2D-Doppler echocardiography and IBS. Backscatter signal was sampled at the septum and posterior wall levels. Patients with AS were divided into 2 groups: 16 patients with initial signs of congestive heart failure and a depressed left ventricular systolic function (DSF) (ejection fraction [EF] range, 35% to 50%) and 19 asymptomatic patients with normal left ventricular systolic function (NSF) (EF >50%). Myocardial echo intensity (pericardium related) was significantly higher at the septum and posterior wall levels in DSF than in NSF and in control subjects. IBS variation, as an expression of variation of the signal, appeared to be significantly lower in AS with DSF than in NSF and in control subjects, at both the septum and posterior wall levels. Patients with DSF underwent aortic valve replacement, and, during surgical intervention, a septal myocardial biopsy was made for evaluation of myocardium/fibrosis ratio. Abnormally increased echo intensity was detected in left ventricular pressure overload by severe aortic stenosis and correlated with increase of myocardial collagen content (operating biopsy). CONCLUSIONS: One year after aortic valve replacement, we observed a significant reduction of left ventricular mass, and, only if pericardial indexed IBS value (reduction of interstitial fibrosis) decreased, it was possible to observe an improvement of EF and of IBS variation.

Prichard's structures of the fossa ovalis are not histogenetically related to cardiac myxoma.

AIMS: Cardiac myxomas are neoplasms of unknown histogenesis. They are thought to arise from hypothetical subendothelial vasoformative reserve cells or from primitive cells which reside in the fossa ovalis and surrounding endocardium. In 1951 Prichard described a kind of microscopic endocardial structure with a predilection for the interatrial septum, which were suggested to be related to cardiac myxomas. To confirm the existence of Prichard's structures and to clarify their role in the genesis of cardiac myxomas, we examined histologically the fossa ovalis and we performed an immunohistochemical study of the endocardial abnormalities that were found. METHODS AND RESULTS: A prospective histological study of 100 interatrial septa and an immunohistochemical study of three out of the 12 endocardial abnormalities that were detected, as well as of four conventional cardiac myxomas were accomplished. Antibodies were used to vimentin, CD31, CD34, alpha-smooth muscle actin, S100 protein, thrombomodulin, calretinin and c-kit (CD117), a tyrosine kinase growth factor receptor for stem cell factor usually expressed by embryonic/fetal endothelium. Structures similar to the ones described by Prichard were found in 12% of septa, most of them in the left side of the fossa ovalis. The hearts with these structures were from patients 10 years older than the ones without them (72 +/- 10 versus 62 +/- 16 years, P=0.006). Immunohistochemically the cells comprising Prichard's structures were positive for vimentin, CD31, CD34 and thrombomodulin, and negative for alpha-smooth muscle actin, S100 protein, calretinin and c-kit. Therefore these cells seem to be mature endothelial cells, but not primitive multipotential mesenchymal cells. Furthermore, these cells were not found in the atrial tissue from the bases of any of the conventional cardiac myxomas. CONCLUSIONS: Our study suggests that there is no apparent relation between Prichard's structures and cardiac myxomas, and that Prichard's minute endocardial deformities are age-related phenomena.

Regional contributions of Kv1.4, Kv4.2, and Kv4.3 to transient outward K+ current in rat ventricle.

The aim of the present study was to assess differences in transient outward potassium current (Ito) between the right ventricular free wall and the interventricular septum of the adult rat ventricle and to evaluate the relative contributions of Kv4.2, Kv4.3, and Kv1.4 to Ito in these regions. The results show that Ito is composed of both rapidly and slowly recovering components in the right wall and septum. The fast component had a significantly higher density in the right free wall than in the septum, whereas the slow component did not differ between the two sites. Kv4.2 mRNA and protein levels were also highest in the right wall and correlated with Ito density, whereas Kv4.3 was expressed uniformly in these regions. The kinetics of the rapidly recovering component of Ito in myocytes was similar to that recorded in tsa-201 cells expressing Kv4.2 and Kv4.3 channels. Kv1.4 mRNA and protein expression correlated well with the density of the slowly recovering Ito, whereas the recovery kinetics of the slow component were identical to Kv1.4 expressed in tsa-201 cells. In conclusion, expression of Kv1.4, Kv4.2, and Kv4.3 differs between regions in rat hearts. Regionally specific differences in the genetic composition of Ito can account for the region-specific properties of this current.

Fibulin-1, vitronectin, and fibronectin expression during avian cardiac valve and septa development.

BACKGROUND: Extracellular matrix (ECM) proteins have been implicated as mediators of events important to valvuloseptal development (reviewed by Little and Rongish, Experentia, 51:873-882, 1995). The aim of this study was to identify connective tissue ECM proteins present at sites of valvuloseptal morphogenesis, and to determine how their patterns of expression change during the developmental process. METHODS: Immunofluorescence microscopy was used to examine the distribution of fibulin-1, vitronectin, and fibronectin in the embryonic chicken heart over a broad developmental time frame (Hamburger and Hamilton stages 14 to 44), emphasizing stages that illustrate endocardial cushion formation, growth, fusion, and development into valvuloseptal components. RESULTS AND CONCLUSIONS: Fibulin-1 immunolabeling was concentrated in endocardial cushions, notably at boundaries with the myocardium, during stages when the cushions are differentiating into valvular and septal components. Fibulin-1 was detected in the endocardial cushions prior to their seeding with cushion cells, but became undetectable by early midgestation. Vitronectin expression was similar to fibulin-1, but less restricted in its distribution. Vitronectin was observed before endocardial cushion cell migration commenced and persisted until the formation of prevalvular structures (early midgestation) in the atrioventricular cushions. Vitronectin remained detectable in the semilunar valves until late midgestation. Fibronectin was present in the endocardial cushion region and in portions of the endocardium and myocardium throughout the stages presented. Our data suggests that the ECM of the endocardial cushions undergoes remodelling in a regionally and temporally specific manner which corresponds with morphogenetic changes during valvuloseptal development.

Analysis of beta-adrenergic receptor mRNA levels in human ventricular biopsy specimens by quantitative polymerase chain reactions: progressive reduction of beta 1-adrenergic receptor mRNA in heart failure.

OBJECTIVES: This study investigated the relation between the severity of heart failure and the extent of the reduction of beta 1-adrenergic receptor messenger ribonucleic acid (mRNA) levels in biopsy specimens from the ventricular septum obtained during cardiac catheterization of patients with various degrees of heart failure. BACKGROUND: Heart failure is accompanied by desensitization of the beta-adrenergic receptor system, which is in part due to downregulation of beta 1-adrenergic receptors. Downregulation of beta 1-adrenergic receptors has been suggested to be caused by reductions in mRNA levels. METHODS: Because biopsy specimens were small and receptor mRNAs not abundant, mRNA levels were determined by quantitative reverse transcription/polymerase chain reactions. This method was validated by measuring synthetic ribonucleic acid (RNA) standards and samples from explanted hearts by solution hybridization assays. Both methods yielded similar results, but the polymerase chain reaction method was approximately 1,000-fold more sensitive. Sources of variations in the polymerase chain reaction were quantitated and found to be best controlled for by determination of the glyceraldehyde phosphate dehydrogenase mRNA as an endogenous control. RESULTS: Beta 1-adrenergic receptor mRNA levels in the biopsy specimens were decreased by 7% in mild (New York Heart Association functional class II), 26% in moderate (functional class III) and > 50% in severe heart failure (functional class IV). There was a good correlation between hemodynamic indicators of heart failure and beta 1-adrenergic receptor mRNA levels. In contrast, beta 2-adrenergic receptor mRNA levels were apparently unaffected by heart failure. CONCLUSIONS: Reduced beta 1-adrenergic receptor mRNA levels occur early in heart failure and can be detected in septal biopsy specimens during right heart catheterization. The reduction in beta 1-adrenergic receptor expression may contribute to further loss of cardiac function.

Autoradiographic localization and quantitation of beta 1- and beta 2-adrenoceptors in the human atrioventricular conducting system: a comparison of patients with idiopathic dilated cardiomyopathy and ischemic heart disease.

The density and distribution of beta 1- and beta 2-adrenoceptors in the atrioventricular conducting system and interatrial and interventricular septa from human hearts with idiopathic dilated cardiomyopathy and ischemic heart disease was determined by quantitative autoradiography using (-)[125I]cyanopindolol and the selective beta 1-adrenoceptor antagonist CGP 20712A and the selective beta 2-adrenoceptor antagonist ICI 118,551. Both beta 1- and beta 2-adrenoceptors were present in the atrioventricular node, bundle of His, interatrial and interventricular septa. No differences in the density or proportions of beta 1- and beta 2-adrenoceptors in the atrioventricular node, bundle of His, interatrial septum and interventricular septum were observed between hearts with idiopathic dilated cardiomyopathy or ischemic heart disease (P > 0.05) so further analysis did not distinguish between the two aetiologies. The density of beta 1-adrenoceptors was lower in the bundle of His (5.0 +/- 1.7 fmol/mg protein) than in the atrioventricular node (22.2 +/- 5.7 fmol/mg protein, P < 0.05), the interatrial septum (29.6 +/- 4.5 fmol/mg protein, P < 0.001) and interventricular septum (24.9 +/- 5.2 fmol/mg protein, P < 0.005, n = 8 for all values). The atrioventricular node, interatrial and interventricular septa had similar densities of beta 1-adrenoceptors (P = 0.60, ANOVA). The distribution of beta 2-adrenoceptors in the atrioventricular node (21.5 +/- 4.1 fmol/mg protein), bundle of His (12.9 +/- 2.6 fmol/mg protein) and atrial (16.7 +/- 2.3 fmol/mg protein) and septal myocardium (13.8 +/- 2.5 fmol/mg protein, n = 8 for all values) was uniform (P = 0.18, ANOVA). The percentage of beta 1- and beta 2-adrenoceptors in the atrioventricular node, bundle of His, interatrial and interventricular septa was uneven (P < 0.001, ANOVA). There was a higher proportion of beta 2-adrenoceptors in the bundle of His (72 +/- 6%) than in the atrioventricular node (51 +/- 3%, P < 0.01), interatrial septum (36 +/- 1%, P < 0.001) and interventricular septum (36 +/- 1%, P < 0.001).

Conventional and confocal fluorescence microscopy of collagen fibers in the heart.

The arrangement of collagen fibers has previously been studied with picrosirius red (PSR) staining and brightfield microscopy. We discovered that PSR staining can also be visualized by fluorescence microscopy. PSR-stained collagen was strongly fluorescent using excitation and barrier filters for rhodamine, and distracting background cytoplasmic fluorescence was drastically reduced with phosphomolybdic acid (PMA) treatment before PSR staining. The PMA-PSR fluorescence method was more sensitive than the brightfield PSR or PMA-PSR method, and permitted confocal microscopic study. We applied the method to the study of collagen fiber three-dimensional arrangement in perimysial and endomysial septa of the heart, showing the three-dimensional course of the fibers in stereo views generated by confocal microscopy. The PMA-PSR fluorescence method should be generally useful for accurately determining collagen fiber three-dimensional arrangement, a necessary prelude to mechanical modeling of collagen-reinforced tissues.

Increased nerve growth factor levels in spontaneously hypertensive rats.

OBJECTIVE: Increased sympathetic innervation has been reported in spontaneously hypertensive rats (SHR); however, the precise mechanisms involved are not yet clear. Nerve growth factor (NGF), a neurotrophic peptide in peripheral sympathetic neurons, is believed to contribute to this phenomenon. METHODS: We measured the content of NGF in SHR and control Wistar-Kyoto (WKY) rats during development. Mesenteric artery, spleen, heart and sciatic nerve were isolated and homogenized. NGF content in the supernatant fractions was measured using a highly sensitive and specific two-site enzyme immunoassay. RESULTS: At 3 weeks of age, SHR had a greater NGF content in the spleen, the sciatic nerve and the mesenteric artery than WKY rats. However, these differences disappeared completely at 12 weeks of age. Cardiac NGF content was slightly lower in 3-week-old SHR and, conversely, higher in 12-week-old SHR than in age-matched WKY rats. CONCLUSIONS: These findings suggest that, except for the heart, the SHR tissues observed overproduce NGF at a young age, leading to enhancement of peripheral sympathetic nervous system activity and the production of vasoconstrictive catecholamines.

The idiopathic dilated cardiomyopathy in man. A biochemical and molecular study on myosin.

We studied subunit composition and Ca(++)-activated ATPase activity of myosin isolated from atria and ventricles of hearts explanted from patients suffering from idiopathic dilated cardiomyopathy. At variance with previously published data, we have been unable to detect in the ventricular subendocardial layers a significant amount of myosin atrial-like light chain 1 (ALC1), which has been reported to be related to some hemodynamic features of the hypertrophied and failing heart. Such a subunit was not visible in the septum and in the subepicardial layers either. On the contrary, in both atria a ventricular-like light chain 2 (VLC2) was found. The nature of this additional light chain was confirmed on the basis of two-dimensional electrophoresis and immunoblotting techniques with polyclonal antibodies reacting with VLC2. In these patients we also observed a depressed Ca(++)-activated ATPase activity, both in atrial and ventricular myosin. The explanation for this finding in ventricles still remains obscure since neither myosin light chains, nor myosin heavy chains showed any difference between patients with dilated cardiomyopathy and controls. On the contrary, in atria we clearly identified changes consistent with the expression of myosin heavy chains of ventricular type and VLC2, which can account for the depressed Ca(++)-activated ATPase activity.