Incremental benefit in correlation with histology of native T1 mapping, partition coefficient and extracellular volume fraction in patients with aortic stenosis

Background: We investigated the histological correlation of native T1 maps, partition coefficient and extracellular volume fraction (ECV) using an 11 heart beat (11 HB) MOLLI for identification of overall burden of fibrosis. Methods: Ten patients (8 male, age 73 ± 7 years; all in sinus rhythm, 2 with ventricular ectopy) with severe aortic stenosis (3 with coexisting coronary artery disease) scheduled for surgical aortic valve replacement underwent CMR on a 1.5T scanner (MAGNETOM Avanto, Siemens Healthcare, Erlangen). The 11HB MOLLI sequence (Siemens investigational prototype WIP 448B) was acquired before and 15 minutes post 0.1 mmol/kg gadolinium administration. Incorporating hematocrit results from the same day. This allowed native T1 maps, partition coefficient and ECV calculation. Images were obtained twice at end diastole at basal, and twice at mid left ventricular level. The average of all measurements was used to calculate ECV using the standard formula Partition Coefficient= [(1/T1myocardium post contrast-1/T1 myocardium native)]/[(1/T1 blood post contrast-1/T1 blood native)] with x(1-HCt) for ECV. Similar regions of interest were drawn in the septum at both levels for T1 values. Intraoperatively, trucut biopsies were taken from the left ventricular apical anterior/ lateral wall through the epicardium to allow histological characterization of the full myocardial wall, and fixed in warm buffered formalin. Histological analysis of formalin-fixed paraffin-embedded, transmural myocardial biopsies of the left ventricle was performed on hematoxylin/eosin and Picrosirius red-stained 3-micron-thick sections by a blinded experienced cardiac pathologist. Images were analysed using a purpose-built software (Nikon NIS elements BR) on a NIKON Eclipse light projection microscope to determine the extent of overall and reactive interstitial fibrosis, which was expressed as collagen volume fraction (%) per square millimetre. Results: Native T1 mapping, partition coefficient and ECV all correlated with histologically measured fibrosis. However, native T1 mapping showed the least accuracy (panel A, R2 = 0.42) and ECV showed the highest accuracy (panel B, R2 = 0.83). Partition coefficient was more accurate than native T1 mapping but only very marginally less so than ECV (panel C, R2 = 0.80). Conclusions: These results suggest that native T1 mapping is less accurate than partition coefficient and ECV for overall fibrosis. Therefore, post gadolinium images to enable calculation of partition coefficient and ECV should be routinely obtained to increase accuracy

Identification of myocardial diffuse fibrosis by 11 heartbeat MOLLI T1 mapping: averaging to improve precision and correlation with collagen volume fraction

Objectives: Our objectives involved identifying whether repeated averaging in basal and mid left ventricular myocardial levels improves precision and correlation with collagen volume fraction for 11 heartbeat MOLLI T1 mapping versus assessment at a single ventricular level. Materials and methods: For assessment of T1 mapping precision, a cohort of 15 healthy volunteers underwent two CMR scans on separate days using an 11 heartbeat MOLLI with a 5(3)3 beat scheme to measure native T1 and a 4(1)3(1)2 beat post-contrast scheme to measure post-contrast T1, allowing calculation of partition coefficient and ECV. To assess correlation of T1 mapping with collagen volume fraction, a separate cohort of ten aortic stenosis patients scheduled to undergo surgery underwent one CMR scan with this 11 heartbeat MOLLI scheme, followed by intraoperative tru-cut myocardial biopsy. Six models of myocardial diffuse fibrosis assessment were established with incremental inclusion of imaging by averaging of the basal and mid-myocardial left ventricular levels, and each model was assessed for precision and correlation with collagen volume fraction. Results: A model using 11 heart beat MOLLI imaging of two basal and two mid ventricular level averaged T1 maps provided improved precision (Intraclass correlation 0.93 vs 0.84) and correlation with histology (R2 = 0.83 vs 0.36) for diffuse fibrosis compared to a single mid-ventricular level alone. ECV was more precise and correlated better than native T1 mapping. Conclusion: T1 mapping sequences with repeated averaging could be considered for applications of 11 heartbeat MOLLI, especially when small changes in native T1/ECV might affect clinical management

Fine Mapping of the 1p36 Deletion Syndrome Identifies Mutation of PRDM16 as a Cause of Cardiomyopathy

Deletion 1p36 syndrome is recognized as the most common terminal deletion syndrome. Here, we describe the loss of a gene within the deletion that is responsible for the cardiomyopathy associated with monosomy 1p36, and we confirm its role in nonsyndromic left ventricular noncompaction cardiomyopathy (LVNC) and dilated cardiomyopathy (DCM). With our own data and publically available data from array comparative genomic hybridization (aCGH), we identified a minimal deletion for the cardiomyopathy associated with 1p36del syndrome that included only the terminal 14 exons of the transcription factor PRDM16 (PR domain containing 16), a gene that had previously been shown to direct brown fat determination and differentiation. Resequencing of PRDM16 in a cohort of 75 nonsyndromic individuals with LVNC detected three mutations, including one truncation mutant, one frameshift null mutation, and a single missense mutant. In addition, in a series of cardiac biopsies from 131 individuals with DCM, we found 5 individuals with 4 previously unreported nonsynonymous variants in the coding region of PRDM16. None of the PRDM16 mutations identified were observed in more than 6,400 controls. PRDM16 has not previously been associated with cardiac disease but is localized in the nuclei of cardiomyocytes throughout murine and human development and in the adult heart. Modeling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish resulted in both contractile dysfunction and partial uncoupling of cardiomyocytes and also revealed evidence of impaired cardiomyocyte proliferative capacity. In conclusion, mutation of PRDM16 causes the cardiomyopathy in 1p36 deletion syndrome as well as a proportion of nonsyndromic LVNC and DCM

An experimental ultrastructural study

Ziel der vorliegenden Arbeit war es, mittels qualitativer und quantitativer Untersuchungen zu ausgewählten Zeitpunkten der postnatalen Entwicklung den Zeitpunkt herauszuarbeiten, in welchem die Unterschiede in der Ultrastruktur der Kardiomyozyten zwischen den Gruppen der gesunden und kardiomyopathischen Hamster am stärksten repräsentiert sind, um zu diesem Zeitpunkt die Auswirkungen der Hypoxie auf die Kardiomyozyten in beiden Gruppen zu untersuchen. Als Versuchstiere dienten 35 gesunde männliche Hamster, unterteilt in 7 Kontrollgruppen im Alter von 3 bis 540 Tagen, sowie 45 gleichaltrige kardiomyopathische Hamster männlichen Geschlechts in den Kontrolltieren. Aus der Versuchsgruppe der kardiomyopathischen Hamster wurden 7 Tiere, und aus der Kontrollgruppe 5 gesunde Hamster einer 25-minütigen normobaren Hypoxie ausgesetzt. Die Tiere wurden unter Äthernarkose durch Genickschlag getötet. Nach Thorakotomie wurden Gewebeproben von der apikalen Region des Herzens jedes einzelnen Tieres entnommen und histologisch und elektronenmikroskopisch untersucht Für die Ermittlung der Messwerte der Kardiomyozytenstrukturen wurden sowohl das konventionelle Punktzählverfahren, als auch das Schnittpunktzählverfahren sowie das Auszählen von Strukturanschnitten genutzt Bis zum 1. Monat der postnatalen Entwicklung konnten auf lichtoptischer Ebene keine morphologischen Unterschiede zwischen den gesunden und den kardiomyopathischen Tieren festgestellt werden. Erst zum 45. Tag der postnatalen Entwicklung traten bei den kardiomyopathischen Hamstern qualitative ultrastrukturelle Veränderungen in Form von Zellödem und destruktiven Veränderungen der Mitochondrien und der Myofibrillen auf. Mit zunehmendem Alter traten geschädigte Z-Streifen und degenerierte Mitochondrien hervor. Form, Ausstattung und Lokalisation der Mitochondrien zeigten deutliche Abweichungen von der Norm, wie Kalkablagerungen, kleine, geschwollene und Riesenmitochondrien und solche mit konzentrischer Anordnung der Cristae und Ausbildung von Myelinfiguren. Das sarkoplasmatische Retikulum und die t-Tubuli waren nach dem ersten Monat der postnatalen Entwicklung oft stark dilatiert. In einigen Kardiomyozytenkernen ließen sich Chromatinverklumpungen nachweisen. Die Ergebnisse der im lichtoptischen Vergrößerungsbereich durchgeführten morphometrischen Untersuchungen zeigten, dass der Durchmesser der Kardiomyozyten bei den kardiomyopathischen Tieren mit zunehmendem Alter stärker als bei den Kontrolltieren anstieg. Dahingegen nahm die Kardiomyozyten/Interstitiumrelation bei den kardiomyopathischen Tieren ab. Die ultrastrukturellen morphometrischen Untersuchungen an 2, 6 und 27 Wochen alten kardiomyopatischen Hamstern ergaben, dass sich in der 6. und 27. Woche die Mitochondrienpopulation, verglichen mit der 2. Woche der postnatalen Entwicklung, aus einer geringeren Anzahl größerer, weiter auseinander liegender Mitochondrien zusammensetzte. Dies war als Folge der stärkeren Vergrößerung der Kardiomyozyten selbst anzusehen, welche quantitativ durch die Zunahme des Durchmessers der Kardiomyozyten ausgewiesen war. Sowohl die Anzahl der Schnittpunkte der Cristae pro Mitochondrion, als auch die Volumendichte der Cristae nahmen signifikant ab, welches auf eine Verschmälerung der intracristaeren Räume hinwies. Das relative Gesamtvolumen der geschädigten Bereiche der Mitochondrien zeigte sich in der 6. Woche nahezu halbiert, um sich in der Woche 27 wiederum mehr als verdoppelt darzustellen. Wie erwartet, nahm die Anzahl der geschädigten Mitochondrienareale pro Einheitsfläche Kardiomyozyt stetig zu. Gemessen an den zwei Wochen alten Tieren, wies die Mitochondrien/Myofibrillenrelation sowohl bei den 6 Wochen alten, als auch bei den 27 Wochen alten Tieren geringere Werte auf, welches als Ausdruck der Hypertrophie der Myofibrillen anzusehen ist. Der ultrastrukturelle quantitative Vergleich zwischen den 100 Tage alten gesunden und kardiomyopathischen Hamstern zeigte, dass die numerische Dichte der Mitochondrien bei den kardiomyopathischen Tieren tendenziell größer war als im Vergleich zu den gesunden Tieren. Die nahezu doppelt so große Volumendichte des Sarkoplasma bei der kardiomyopathischen Versuchsgruppe wirkte sich ausgleichend auf die Unterschiede in der numerischen und Volumendichte der Mitochondrien aus. Das mittlere Volumen der Mitochondrien war gleichwohl bei den kardiomyopathischen Tieren in Relation zu den gesunden Hamstern erhöht. Die Mitochondrienpopulation setzte sich bei den kardiomyopathischen Tieren aus einer vermehrten Anzahl größerer Mitochondrien zusammen. Daraus resultierend, waren die Oberflächen-/Volumenrelation der Mitochondrien gleichsam in beiden Gruppen auf ein und demselben Niveau anzutreffen. Die Oberflächen/Volumenrelation der Cristae war bei den kardiomyopathischen Tieren deutlich gesteigert. Dies wies auf eine Verschmälerung der Cristae hin. Diese Umstrukturierung der Cristae war bei den kardiomyopathischen Tieren möglicherweise als eine Reaktion auf die Hypertrophie der Mitochondrien, diesbezüglich auf die Verlängerung ihrer Zentrum- zu Zentrumdistanz durch die Vergrößerung des relativen Gesamtvolumens des Sarkoplasmas, anzusehen. Ein ausgeprägt größeres relatives Gesamtvolumen und eine eminent vermehrte Anzahl der degenerativen Mitochondrienareale waren in der Gruppe der kardiomyopathischen Tiere, gemessen an der Kontrollgruppe, feststellbar. Als Folge der Hypoxie war eine Steigerung des relativen Gesamtvolumens des Sarkoplasma und der t-Tubuli in beiden Gruppen der Hamstern belegbar. Die Mitochondrienpopulation setzte sich in beiden Gruppen aus einer geringeren Anzahl vergrößerter Mitochondrien zusammen, welche durch eine verkleinerte spezifische Oberflächendichte der Außenmembran und der Cristae charakterisiert waren. Hypoxie- bedingt, war in beiden das relative Gesamtvolumen der degenerierten Mitochondrienareale, verglichen mit den Versuchsgruppen, welche keine hypoxische Exposition erfahren haben, signifikant erhöht. Die Hypoxie exponierten gesunden Hamster, mit den Hypoxie exponierten kardiomyopathischen Hamstern in Relation setzend, war bei den an DCM Tieren ein größeres relatives Gesamtvolumen der Mitochondrien, ein nahezu verdoppeltes Mitochondrieneinzelvolumen und eine kleinere numerische Dichte sowie Steigerung war bei der spezifischen Oberflächendichte der Cristae dieser Zellorganelle erkennbar. Die Mitochondrienpopulation in der Gruppe der Hamster mit DCM und Hypoxieexposition bestand demzufolge aus einer geringeren Anzahl größerer Mitochondrien, welche durch eine kleinere spezifische Oberflächendichte und einen größeren Anteil an degenerierten Mitochondrienarealen charakterisiert waren und deren intracristäre Räume, gemessen an den gesunden Tieren, deutlich verschmälert imponierten. Die Volumendichte des Sarkoplasma war bei den Hamstern mit DCM nach Hypoxie deutlich größer und das relative Gesamtvolumen der Myofibrillen deutlich kleiner im Vergleich mit den gesunden Hamstern nach Hypoxie.The aim of the study was to determine by means of qualitative and quantitative examinations during post-natal development the age at which the differences in the ultrastructure of cardiomyocytes between Syrian hamsters (strain BIO 8262) as a model of human cardiomyopathy (DCM) and healthy hamsters at corresponding stages of development are most pronounced. We then compared the effects of normobaric hypoxia on the cardiomyocytes in the two groups at that age. At the light optical level, no morphological differences could be determined between healthy and cardiomyopathic animals up to the first month of postnatal development. Qualitative ultrastructural changes in the form of cell oedema and destructive changes in the mitochondria and myofibrils of cardiomyopathic hamsters only occurred after day 45. Damaged Z-lines and abnormalities (e.g. size variation, degeneration) within mitochondria occurred with increasing age. The results of morphometric investigations conducted in the light optical magnification range showed that the diameter of the cardiomyocytes of the healthy and cardiomyopathic animals increased during postnatal development (2, 6 and 27 weeks), but that this increase appeared to be more pronounced in the cardiomyopathic than in the control animals. By contrast, the cardiomyocyte to interstitium ratio diminished in the cardiomyopathic animals. Ultrastructural morphometric examinations of 2-, 6- and 27-week-old cardiomyopathic hamsters showed that the mitochondria population in the 6thand 27 th week consisted of a smaller number of larger mitochondria situated further apart when compared to animals in the 2nd week of postnatal development. This is considered to be the result of the more pronounced enlargement of the cardiomyocytes themselves, accounted for by the quantitative increase in the diameter of the cardiomyocytes. Both the number of intersections of cristae per mitochondrion, which represented the length and surface area of the cristae membrane, and the volume density of the cristae diminished significantly. This change suggests a reduction in intercristal spaces. The relative total volume of damaged regions of mitochondria was almost halved in the 6th week, only to more than double in week 27. As expected, the number of damaged mitochondria areas per unit area of cardiomyocyte increased steadily. Compared with 2-week-old animals, the mitochondria to myofibril ratios of both 6-week-old and 27-week-old animals were lower, interpreted as expression of hypertrophy of the myofibrils. The ultrastructural quantitative comparison of 100-day-old healthy versus cardiomyopathic hamsters showed that the numerical density of mitochondria (Nvm) of the cardiomyopathic animals tended to be higher than that of healthy animals. The fact that the volume density of sarcoplasm was almost twice as high in the cardiomyopathic test group has an equalising effect on the differences in the numerical and volume density of the mitochondria. However, the mean volume of mitochondria (Vm) in the cardiomyopathic animals was elevated in relation to that of the healthy hamsters. The mitochondria population in the cardiomyopathic animals showed an increased number of larger mitochondria. As a result, the surface area to volume ratio of the mitochondria in the two groups virtually equalised. The surface area to volume ratio of the cristae was markedly elevated in the cardiomyopathic animals. This points to a narrowing of the cristae and a diminished volume of the intercristal space. This restructuring of the cristae in the cardiomyopathic animals is seen as a possible reaction to the hypertrophy of the mitochondria, in this case to an elongation of their centre-to-centre distance as a result of the increase in the relative total volume of the sarcoplasm. A markedly enlarged relative total volume and a pronounced increase in the number of degenerative mitochondrial areas relative to the control group were evident. In the two groups, the mitochondria population comprised of a smaller number of enlarged mitochondria characterised by a diminished specific surface area density of the outer membrane and of the cristae. Following hypoxia, the relative total volume of the degenerative mitochondrial areas was significantly elevated in both experimental groups of healthy and cardiomyopathic animals compared with that of the healthy and cardiomyopathic animals that had not been subjected to hypoxia. Comparing healthy and cardiomyopathic hamsters that had been exposed to hypoxia, a larger relative total volume of mitochondria, an individual mitochondrial volume almost twice as large and a smaller numerical density of these cell organelles are demonstrable in animals suffering from DCM. An increase in specific surface area density of the cristae is observed in the DCM hamsters. Consequently, the mitochondrial population in the group of hamsters with DCM and exposure to hypoxia consists of a smaller number of larger mitochondria, which are characterised by a smaller specific surface area density and a larger proportion of degenerative mitochondrial areas whose intercristal spaces are manifestly narrower than those of the healthy animals

The Impact of Ventricular Assist Device Prior to Transplantation on Morphological Parameters in Cardiac Allografts /// Въздействие на вентрикуларните помощни устройства преди трансплантация върху морфологичните параметри при сърдечните алографи

Due to the shortage of donor organs, mechanical circulatory support systems (MCS) are now widely used as a treatment option to bridge the failing heart to transplantation. There are limited data, suggesting that prolonged use of ventricular assist device (VAD) therapy may result in cardiac allograft dysfunction, as MCS patients show a higher frequency of antibody-mediated rejection (AMR) episodes. We aimed to analyze the effects of MCS on cardiac AMR with regards to capillary C3d and C4d depositions. Regarding the functional parameters, both acute cellular rejection (ACR) and an increase of interstitial fibrosis (IF) often correlate with impaired ventricular function. The innate immune system, in particular macrophages, plays an important role in the resorptive process of ACR and is, on the other hand, known to promote IF. In this study we aimed to analyze the effect of ACR and specifically of the level of macrophages on the degree of IF in right ventricular endomyocardial biopsies (EMBs) of cardiac allografts. Methods: We evaluated all consecutive EMBs of cardiac allografts from 254 patients taken between 01/2011 and 12/2012.With regard to pre-transplant MCS treatment, patients were divided into two groups (MCS group n=82 patients, non-MCS group n=138 patients). The MCS patients were subdivided into groups regarding the device used (HeartMate II, HeartWare, Novacor, Incor, Excor). Patients were excluded from the analysis if they had an MCS system that was used in fewer than five of the study population patients. Tissue sections of formalin-fixed paraffin-embedded EMBs were evaluated histologically using conventional histology (hematoxylin-eosin stained slides) and immunohistochemical stains for a lymphocyte marker (CD3) and a pan- macrophage marker (CD68) for ACR, which has been classified according to the ISHLT. Statistical analysis was done using Fisher’s exact tests, bootstrapped Cochran-Mantel-Haenzsel test, GLM methods and tree analysis. In addition, immunohistochemical stains (complements C3d and C4d) were used to evaluate the EMBs regarding AMR. The amount of macrophages was assessed by eyeballing irrespective of the presence of ACR. The EMBs were also evaluated for level of IF on a collagen stain (Sirius red) and further quantified using NIKON software (NIS elements AR 4.10.02). Fisher’s exact test was performed to analyze the relationship between C4d and C3d in both patient groups. The Cochran-Mantel-Haenzsel test was applied to assess significance of the differences in interactions between groups. To evaluate the impact of bridge- to- transplant mechanical circulatory support on development on transplant vasculopathy in cardiac allografts, the intramyocardial terminal arterial network was assessed using conventional histology and immunohistochemistry (CD31, alpha actin). Results: In the study population as a whole there is a positive correlation between C3d and C4d (p=0.007), with 64% concordant cases (107 negative and 33 positive out of 220 in total). Analyzing the two groups separately a significant C3d/C4d correlation was found only for the MCS group (p=0.002, odds ratio=4.185, non-MCS group p=0.418, odds ratio=1.392). While there was a significant difference in C4d deposition between the overall MCS group (n=82) and the non-MCS-group (p=0.021, odds ratio=0.524), the Excor patients (39/82) did not show an increase in C4d depositions (p=0.838, odds ratio 1.11). Patients with the remaining MCS types (43/82) showed signs of antibody-mediated rejection in the form of C4d depositions in significantly more cases than the non-MCS group (p=0.003, odds ratio=2.97). The density of macrophages correlated with ACR (p=0.000) and with both pure IF (p= 0.017) and overall IF (p= 0.001). Interestingly, ACR appeared to have no direct impact on pure or overall IF (p=0.749, p=0.760) In addition, there was an adverse correlation between MCS-treatment and transplant vasculopathy of the myocardial terminal vascular network with a significant difference in presence of transplant vasculopathy in 62% of MCS-group compared 75% of patients without MCS support (p = 0.03, odds- ratio= 0.45). The Excor patients (39/82) did not show a decrease in transplant vasculopathy (p=0.31, odds ratio 0.66) on the contrary to the remaining MCS types (p=0.03, odds ratio=0.45). Conclusions: Only in patients with pre-transplant MCS does C3d correlate with capillary C4d depositions. This circumstance may beneficially complement existing diagnostic criteria for AMR. As well, the results suggest that not the MCS treatment as such, but the type of device used, may influence the outcome of cardiac allografts with regard to AMR, which may be due to allosensitation to the different device materials. Our results suggest that both the density of tissue macrophages and the macrophages of the inflammatory infiltrate in the resorptive process of ACR are important predictive factors for the remodeling in cardiac allografts. In addition, the Excor MCS system appears to have a protective effect on the development of transplant vasculopathy in cardiac allografts