Normal Myocardium Research Articles

Preparation of Cell-Seeded Heart Patch In Vitro; Co-Culture of Adipose-Derived Mesenchymal Stem Cell and Cardiomyocytes in Amnion Bilayer Patch.

Cardiovascular disease is the second killer across the globe, while coronary disease is the major cause. Cell therapy is one alternative to regenerate the infarcted heart wall. In this study, the cardiomyogenesis capacity of human adipose stem cells (hAdSC) and human cardiomyocytes (hCardio) cultured in a 3-D biological scaffold (decellularised amnion bilayer) for nine days in a static condition was investigated. The cardiomyogenesis capacity of hAdSC were identified using immunohistochemistry and RT-PCR. The population of the cells isolated from the heart tissue expressed cTnT-1 (13.38 ± 11.38%), cKit (7.85 ± 4.2%), ICAM (85.53 ± 8.69%), PECAM (61.63 ± 7.18%) and VCAM (35.9 ± 9.11%), while from the fat tissue expressed the mesenchymal phenotypes (CD73, CD90, CD105, but not CD45, CD34, CD11b, CD19 and HLA-DR). Two age groups of hAdSC donors were compared, the youngsters (30-40yo) and the elderly (60-70 yo). The co-culture showed that after 5-day incubation, the seeded graft in the hAdSC-30 group had a tube-like appearance while the hAdSC-60 group demonstrated a disorganised pattern, despite of the MSC expressions of the hAdSC-60 were significantly higher. Initial co-culture showed no difference of ATP counts among all groups, however the hAdSC-30 group had the highest ATP count after 9 days culture (p = 0.004). After normalising to the normal myocardium, only the hAdSC-60 group expressed cTnT and MHC, very low, seen during the initial cultivation, but then disappeared. Meanwhile, the hAdSC-30 group expressed α-actinin, MHC and cTnT in the Day-5. The PPAR also was higher in the Day-5 compared to the Day-9 (p <0.005). Cardiomyogenesis capacity of hAdSC co-cultured with hCardio in a 3-D scaffold taken from the 30-40yo donor showed better morphology and viability than the 60-70yo group, but maintained less than 5 days in this system.

B-PO02-133 HISTOLOGICAL CHARACTERIZATION OF REVERSIBLE AND IRREVERSIBLE VENTRICULAR LESION BOUNDARIES PRODUCED BY PULSED FIELD ABLATION

During pulsed field ablation (PFA), electric pulses induce reversible (R) or irreversible (IR) injury on cardiac myocytes. To perform histological examination of acute ventricular lesions produced by PFA, determining R and IR injury boundaries in a swine heart model. A 7F catheter with a 3.5mm ablation electrode (TactiCath, Abbott) was connected to a pulsed electric field (PEF) generator (CENTAURI, Galaxy Medical) and positioned in RV and LV under EnSite guidance in 2 closed chest pigs. Biphasic PEF current was delivered between the ablation electrode and a skin patch at 13 RV sites (28Amp, total pulse width of 1.4ms, 4 pulses) and 19 LV sites (35Amp, 1.6ms, 7 pulses). Two hours after ablation, triphenyl tetrazolium chloride (TTC) was administered. Pigs were sacrificed and hearts were excised and fixed in formalin. Hearts were sectioned and stained with hematoxylin and eosin (H&E) and Masson trichrome. Cytochrome c oxidase (COX) staining was also performed to examine mitochondrial activity to delineate R and IR lesion boundaries. Fig. Ablation lesions were well demarcated with TTC staining, showing a dark central zone surrounded by pale boundaries (PB). Histology showed destruction of myocyte architecture within the PB. A hyperstained (dark red) rim beyond the PB indicates R zone. COX staining showed no or low mitochondrial activity within the PB, consistent with IR ablated region. Enhanced activity of COX staining extended to unaffected normal myocardium, consistent with R zone. Acute ventricular lesions produced by PEF ablation show clear demarcation by TTC staining. COX staining suggests that IR lesions are surrounded by R zone.

Performance of a machine-learning algorithm for fully automatic LGE scar quantification in the large multi-national derivate registry

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): J. Schwitter receives research support by “ Bayer Schweiz AG “. C.N.C. received grant by Siemens. Gianluca Pontone received institutional fees by General Electric, Bracco, Heartflow, Medtronic, and Bayer. U.J.S received grand by Astellas, Bayer, General Electric. This work was supported by Italian Ministry of Health, Rome, Italy (RC 2017 R659/17-CCM698). This work was supported by Gyrotools, Zurich, Switzerland. Background Late Gadolinium enhancement (LGE) scar quantification is generally recognized as an accurate and reproducible technique, but it is observer-dependent and time consuming. Machine learning (ML) potentially offers to solve this problem. Purpose to develop and validate a ML-algorithm to allow for scar quantification thereby fully avoiding observer variability, and to apply this algorithm to the prospective international multicentre Derivate cohort. Method The Derivate Registry collected heart failure patients with LV ejection fraction &amp;lt;50% in 20 European and US centres. In the post-myocardial infarction patients (n = 689) quality of the LGE short-axis breath-hold images was determined (good, acceptable, sufficient, borderline, poor, excluded) and ground truth (GT) was produced (endo-epicardial contours, 2 remote reference regions, artefact elimination) to determine mass of non-infarcted myocardium and of dense (≥5SD above mean-remote) and non-dense scar (&amp;gt;2SD to &amp;lt;5SD above mean-remote). Data were divided into the learning (total n = 573; training: n = 289; testing: n = 284) and validation set (n = 116). A Ternaus-network (loss function = average of dice and binary-cross-entropy) produced 4 outputs (initial prediction, test time augmentation (TTA), threshold-based prediction (TB), and TTA + TB) representing normal myocardium, non-dense, and dense scar (Figure 1).Outputs were evaluated by dice metrics, Bland-Altman, and correlations. Results In the validation and test data sets, both not used for training, the dense scar GT was 20.8 ± 9.6% and 21.9 ± 13.3% of LV mass, respectively. The TTA-network yielded the best results with small biases vs GT (-2.2 ± 6.1%, p &amp;lt; 0.02; -1.7 ± 6.0%, p &amp;lt; 0.003, respectively) and 95%CI vs GT in the range of inter-human comparisons, i.e. TTA yielded SD of the differences vs GT in the validation and test data of 6.1 and 6.0 percentage points (%p), respectively (Fig 2), which was comparable to the 7.7%p for the inter-observer comparison (n = 40). For non-dense scar, TTA performance was similar with small biases (-1.9 ± 8.6%, p &amp;lt; 0.0005, -1.4 ± 8.2%, p &amp;lt; 0.0001, in the validation and test sets, respectively, GT 39.2 ± 13.8% and 42.1 ± 14.2%) and acceptable 95%CI with SD of the differences of 8.6 and 8.2%p for TTA vs GT, respectively, and 9.3%p for inter-observer. Conclusions In the large Derivate cohort from 20 centres, performance of the presented ML-algorithm to quantify dense and non-dense scar fully automatically is comparable to that of experienced humans with small bias and acceptable 95%-CI. Such a tool could facilitate scar quantification in clinical routine as it eliminates human observer variability and can handle large data sets.

Prolonged Right Ventricular Outflow Tract Endocardial Activation Duration and Presence of Deceleration Zones in Patients With Idiopathic Premature Ventricular Contractions. Association With Low Voltage Areas.

Background and AimsThe wavefront propagation velocity in the myocardium with fibrosis is characterized by the presence of deceleration zones and late activated zones, that are absent in the normal myocardium. Our aim was to study the right ventricular outflow tract (RVOT) endocardial activation duration in sinus rhythm, and assess the presence of deceleration zones, in patients with premature ventricular contractions (PVCs) and in controls.MethodsWe studied 29 patients with idiopathic PVCs from the outflow tract, subjected to catheter ablation that had an activation and voltage map of the RVOT in sinus rhythm. A control group of 15 patients without PVCs that underwent ablation of supraventricular arrhythmias was also studied. RVOT endocardial activation duration and number of 10 ms isochrones across the RVOT were assessed. Propagation speed was calculated at the zone with the higher number of isochrones per cm radius. Deceleration zones were defined as zones with >3 isochrones within 1 cm radius. Low voltage areas were defined as areas with local electrogram with amplitude <1.5 mV.ResultsThe two groups did not differ in relation to age, gender or number of points in the map. RVOT endocardial activation duration and number of 10 ms isochrones were higher in the PVC group; 56 (41–66) ms vs. 39 (35–41) ms, p = 0.001 and 5 (4–8) vs. 4 (4–5), p = 0.001. Presence of deceleration zones and low voltage areas were more frequent in the PVC group; 20 (69%) vs. 0 (0%), p < 0.0001 and 21 (72%) vs. 0 (0%), p < 0.0001. The wavefront propagation speed was significantly lower in patients with PVCs than in the control group, 0.35 (0.27–0.40) vs. 0.63 (0.56–0.66) m/s, p < 0.0001. Patients with low voltage areas had longer activation duration 60 (52–67) vs. 36 (32–40) ms, p < 0.0001, more deceleration zones, 20 (95%) vs. 0 (0%), p < 0.0001, and lower wavefront propagation speed, 0.30 (0.26–0.36) vs. 0.54 (0.36–0.66) m/s, p = 0.002, than patients without low voltage areas.ConclusionRight ventricular outflow tract endocardial activation duration was longer, propagation speed was lower and deceleration zones were more frequent in patients with PVCs than in controls and were associated with the presence of low voltage areas.

Diagnostic accuracy of cardiac magnetic resonance tissue tracking technology for differentiating between acute and chronic myocardial infarction.

This study aimed to explore the diagnostic accuracy of cardiac magnetic resonance tissue tracking (CMR-TT) technology in the quantitative evaluation of left myocardial infarction for differentiating between acute and chronic myocardial infarction. A total of 104 human subjects were enrolled in this prospective study. Among them, 64 healthy subjects and 40 patients with left ventricular myocardial infarction and 7 days and 6 months' follow-up CMR studies, including steady-state free precession (SSFP) sequence and late gadolinium enhancement MR imaging, were enrolled. The strain parameters of the infarcted myocardium, its corresponding remote segments, and global right ventricular strain were analyzed using tissue tracking technology, and CMR-TT 3D strain parameters in radial, circumferential, and longitudinal directions were obtained. Receiver operating characteristic (ROC) analysis was used to determine the diagnostic accuracy of the CMR-TT strain parameters for discriminating between acute and chronic myocardial infarction. Peak radial strain (RS) of infarcted myocardium increased from 12.99±7.28 to 18.57±6.66 at 6 months (P<0.001), whereas peak circumferential strain (CS) increased from -8.82±4.71 to -12.78±3.55 (P<0.001). CS yielded the best areas under the ROC curve (AUC) of 0.751 in showing differentiation between acute and chronic myocardial infarction of all the strain parameters obtained. The highest significant differences between acute myocardial infarction and normal myocardium, both in the left and right ventricles, were also found in the RS (P<0.001) and CS (P<0.001). RS and CS obtained by CMR-TT have high sensitivity and specificity in the differential diagnosis of acute versus chronic myocardial infarction, and their use is thus worth popularizing in clinical application.

Identification of Underlying Hub Genes Associated with Hypertrophic Cardiomyopathy by Integrated Bioinformatics Analysis.

BackgroundConsidered as one of the major reasons of sudden cardiac death, hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease. However, effective treatment for HCM is still lacking. Identification of hub gene may be a powerful tool for discovering potential therapeutic targets and candidate biomarkers.MethodsWe analysed three gene expression datasets for HCM from the Gene Expression Omnibus. Two of them were merged by “sva” package. The merged dataset was used for analysis while the other dataset was used for validation. Following this, a weighted gene coexpression network analysis (WGCNA) was performed, and the key module most related to HCM was identified. Based on the intramodular connectivity, we identified the potential hub genes. Then, a receiver operating characteristic curve analysis was performed to verify the diagnostic values of hub genes. Finally, we validated changes of hub genes, for genetic transcription and protein expression levels, in datasets of HCM patients and myocardium of transverse aortic constriction (TAC) mice.ResultsIn the merged dataset, a total of 455 differentially expressed genes (DEGs) were identified from normal and hypertrophic myocardium. In WGCNA, the blue module was identified as the key module and the genes in this module showed a high positive correlation with HCM. Functional enrichment analysis of DEGs and key module revealed that the extracellular matrix, fibrosis, and neurohormone pathways played important roles in HCM. FRZB, COL14A1, CRISPLD1, LUM, and sFRP4 were identified as hub genes in the key module. These genes showed a good predictive value for HCM and were significantly up-regulated in HCM patients and TAC mice. We also found protein expression of LUM and sFRP4 increased in myocardium of TAC mice.ConclusionThis study revealed that five hub genes are involved in the occurrence and development of HCM, and they are potentially to be used as therapeutic targets and biomarkers for HCM.

Abstract 705: SARS-CoV-2 infection of the human heart governs intracardiac innate immune response

Abstract Human coronavirus, hCoV-19 (SARS-CoV-2) is associated with pneumonia, severe systemic disease and, in fatal cases, a high prevalence of cardiac dysfunction. Cellular entry requires a cell-surface receptor, angiotensin-converting enzyme 2 (ACE2), and activation of the spike protein by host proteases, including TMPRSS2 and cathepsins. Cardiac toxicity is a serious cause of morbidity and mortality from SARS-CoV-2 with chronic sequelae reducing cardiac function in many survivors. We determined mRNA expression for genes related to SARS-Cov-2 infection in iPSC-derived cardiac myocytes treated with the widely used chemotherapeutic, Doxorubicin (DOX). DOX induced expression of TMPSS2, but not Ace2 or Furin, 4- to 5-fold. Further, DOX treatment enhanced expression of cathepsins A, B and F between 1.2-1.5 fold. Overall these changes promote an environment of enhanced SARS-CoV-2 susceptibility in the myocardium placing cancer patients on DOX therapy at increased risk of cardiac damage. To further characterize the effects of SARS-CoV-2 on the myocardium we examined the local spatial gene expression response in human post-mortem cardiac samples. A genome wide (1,864 genes) and matching proteome analysis on SARS-CoV-2 infected myocardium was conducted using Digital spatial profiling (DSP) (NanoString GeoMxTM). RNAscopeTM, a sensitive form of fluorescence in situ hybridization (FISH), identified SARS-CoV-2 spike RNA in cells enriched for ACE2 and TMPRSS2. Immunohistochemistry on serial sections identified the SARS-CoV-2 spike protein in both myocyte (desmin+) and monocyte (CD45+) populations suggesting multiple targets for viral infection in the myocardium. SCANscopeTM was used to determine the effects of SARS-CoV-2 on cardiac gene. Probes directed to the spike protein were used to identify regions of interest (ROI) for SARS-CoV-2-infection. Unsupervised principal component analysis (PCA) of the differentially expressed gene transcripts separated gene expression of SARS-CoV-2 ROI from control ROI areas in the same patient and was distinct from the PCA of normal myocardium control ROI. PCA of the SARS-CoV-2-associated gene expression of the ROI from male vs. female myocardium identified distinct gender-related gene expression patterns. Further, PCA based on SARS-Cov-2 spike RNA abundance correlated with induction of innate and acquired immunity signaling in the myocardial cells. Moreover, gene ontology (Reactome) analysis of SARS-CoV-2 infected regions of the myocardium displayed characteristic signatures indicating enhanced apoptosis and autophagy, chromatin remodeling and reduced DNA repair, and reduced oxidoreductase activity in regions of SARS-CoV-2 infection compared to uninfected myocardium. These data probe the underlying molecular mechanisms that enhance the risk of death from SARS-CoV-2 for cancer patients and the cardiac remodeling that results in death for many patients with COVID-19. Citation Format: Anthony W. Ashton, Liang Zhang, Yan Liang, Prajan Divakar, Carolos Cordon-Cardo, Richard G. Pestell. SARS-CoV-2 infection of the human heart governs intracardiac innate immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 705.

The impact of myocardial compressibility on organ-level simulations of the normal and infarcted heart

Myocardial infarction (MI) rapidly impairs cardiac contractile function and instigates maladaptive remodeling leading to heart failure. Patient-specific models are a maturing technology for developing and determining therapeutic modalities for MI that require accurate descriptions of myocardial mechanics. While substantial tissue volume reductions of 15–20% during systole have been reported, myocardium is commonly modeled as incompressible. We developed a myocardial model to simulate experimentally-observed systolic volume reductions in an ovine model of MI. Sheep-specific simulations of the cardiac cycle were performed using both incompressible and compressible tissue material models, and with synchronous or measurement-guided contraction. The compressible tissue model with measurement-guided contraction gave best agreement with experimentally measured reductions in tissue volume at peak systole, ventricular kinematics, and wall thickness changes. The incompressible model predicted myofiber peak contractile stresses approximately double the compressible model (182.8 kPa, 107.4 kPa respectively). Compensatory changes in remaining normal myocardium with MI present required less increase of contractile stress in the compressible model than the incompressible model (32.1%, 53.5%, respectively). The compressible model therefore provided more accurate representation of ventricular kinematics and potentially more realistic computed active contraction levels in the simulated infarcted heart. Our findings suggest that myocardial compressibility should be incorporated into future cardiac models for improved accuracy.

Feasibility of somatostatin receptor-targeted imaging for detection of myocardial inflammation: A pilot study

BackgroundGallium-68 Dotatate binds preferentially to somatostatin receptor (sstr) subtype-2 (sstr-2) on inflammatory cells. We aimed at investigating the potential clinical use of sstr-targeted imaging for the detection of myocardial inflammation. MethodsThirteen patients, with suspected cardiac sarcoidosis (CS) based on clinical history and myocardial uptake on recent fluorine-18 fluorodeoxyglucose (FDG) PET, were enrolled to undergo Dotatate PET after FDG-PET (median time 37 days [IQR 25-55]). Additionally, we investigated ex-vivo the immunohistochemistry expression of sstr-2 in 3 explanted sarcoid hearts. ResultsAll FDG scans showed cardiac uptake (focal/multifocal = 6, focal on diffuse/heterogeneous = 7), and 46% (n = 6) extra-cardiac uptake (mediastinal/hilar). In comparison, Dotatate scans showed definite abnormal cardiac uptake (focal/multifocal) in 4 patients, probably abnormal (heterogenous/patchy) in 3, and negative uptake in 6 cases. Similarly, 6 patients had increased mediastinal/hilar Dotatate uptake. Overall concordance of FDG and Dotatate uptake was 54% in the heart and 100% for thoracic nodal activity. Quantitatively, FDG maximum standardized uptake value was 5.0 times [3.8-7.1] higher in the heart, but only 2.25 times [1.7-3.0; P = .019] higher in thoracic nodes relative to Dotatate. Ex-vivo, sstr-2 immunostaining was weakly seen within well-formed granulomas in all 3 examined sarcoid heart specimens with no significant staining of background myocardium or normal myocardium. ConclusionOur preliminary data suggest that, compared to FDG imaging, somatostatin receptor-targeted imaging may be less sensitive for the detection of myocardial inflammation, but comparable for detecting extra-cardiac inflammation.

The Impact of Vendor-Specific Ultrasound Beam-Forming and Processing Techniques on the Visualization of In Vitro Experimental “Scar”: Implications for Myocardial Scar Imaging Using Two-Dimensional and Three-Dimensional Echocardiography

Myocardial scar appears brighter compared with normal myocardium on echocardiography because of differences in tissue characteristics. The aim of this study was to test how different ultrasound pulse characteristics affect the brightness contrast (i.e., contrast ratio [CR]) between tissues of different acoustic properties, as well as the accuracy of assessing tissue volume. An experimental invitro "scar" model was created using overheated and raw pieces of commercially available bovine muscle. Two-dimensional and three-dimensional ultrasound scanning of the model was performed using combinations of ultrasound pulse characteristics: ultrasound frequency, harmonics, pulse amplitude, steady pulse (SP) emission, power modulation (PM), and pulse inversion modalities. On both two-dimensional and three-dimensional imaging, the CR between the "scar" and its adjacent tissue was higher when PM was used. PM, as well as SP ultrasound imaging, provided good "scar" volume quantification. When tested on 10 "scars" of different size and shape, PM resulted in lower bias (-9.7 vs 54.2mm3) and narrower limits of agreement (-168.6 to 149.2mm3 vs -296.0 to 404.4mm3, P=.03). The interobserver variability for "scar" volume was better with PM (intraclass correlation coefficient=0.901 vs 0.815). Two-dimensional and three-dimensional echocardiography with PM and SP was performed on 15 individuals with myocardial scar secondary to infarction. The CR was higher on PM imaging. Using cardiac magnetic resonance as a reference, quantification of myocardial scar volume showed better agreement when PM was used (bias, -645mm3; limits of agreement, -3,158 to 1,868mm3) as opposed to SP (bias, -1,138mm3; limits of agreement, -5,510 to 3,233mm3). The PM modality increased the CR between tissues with different acoustic properties in an experimental invitro "scar" model while allowing accurate quantification of "scar" volume. By applying the invitro findings to humans, PM resulted in higher CR between scarred and healthy myocardium, providing better scar volume quantification than SP compared with cardiac magnetic resonance.

Assessment of wavefront propagation speed on the right ventricular outflow tract: deceleration zones associated with the presence of low voltage areas

Abstract Funding Acknowledgements Type of funding sources: None. Background and aims Activation wavefront is rapid and uniform in normal myocardium. Fibrosis is associated with deceleration zones (DZ) and late activated zones. The presence of low voltage areas (LVAs) in the right ventricular outflow tract (RVOT) of patients with premature ventricular contractions (PVCs) from this origin has been described previously. The aim of this study was to evaluate in sinus rhythm, the RVOT endocardial activation duration (EAD) and the presence of DZs, in patients with PVCs and in controls. Methods Consecutive patients with frequent (&amp;gt;10.000/24 h) idiopathic PVCs with inferior axis subjected to ablation that had an activation and voltage map of the RVOT performed in sinus rhythm. A control group of patients without PVCs that underwent ablation of supraventricular arrhythmias was also studied. Patients with structural heart disease, previous ablation or conduction disease were excluded. The RVOT EAD was measured as the time interval between the earliest and the latest activated region. Also evaluated the number of 10 ms isochrones throughout the RVOT and the maximal number of 10 ms isochrones within 1 cm, and a DZ was defined as a zone with &amp;gt; 3 isochrones within 1 cm. Low voltage areas (LVA) were defined as areas with local electrogram amplitude &amp;lt;1.5 mV. Results 42 patients, 29 in the PVC group and 13 control subjects. The site of origin of the PVCs was the RVOT in 23 patients and the LVOT in 6. The characteristics of the two groups are displayed in the Table. Patients with PVCS had longer RVOT EAD, total number of isochrones and presence of DZ was also significantly higher (See table). LVAs were more frequent in PVCs from the RVOT than from the LVOT (83% vs 33%, p = 0.033). Patients with LVA had longer EAD 60 (52-67) vs 36 (34-40) ms, p &amp;lt; 0.0001 (Figure A) and more DZ than patients without LVA 95% vs 0%, p &amp;lt; 0.0001 (Figure B and C). Conclusions The velocity of the wavefront propagation was slower and DZs were more frequently present in patients with PVCs and were associated with presence of LVAs. All sampleN= 42PVCsN = 29ControlsN = 13p-valueAge in years, median (Q1-Q3)56 (35-65)58 (38-66)53 (28-67)0.648Male gender, n (%)19 (45)14 (48)5 (39)0.401Nº points in the map, median (Q1-Q3)410 (338-589)467 (345-660)345 (333-465)0.056Activation duration in ms, median (Q1-Q3)41.8 (36-61)56 (41-66)39 (35-41)0.001Nº isochrones, median (Q1-Q3)4 (4-6)5 (4-6)4 (4-4)0.037Presence of DZs, n (%)20 (48)20 (69)0 (0)&amp;lt;0.0001Presence of LVAs, n(%)21 (50)21 (72)0 (0)&amp;lt;0.0001Abstract Figure.

Cardiomyopathic mutations in essential light chain reveal mechanisms regulating the super relaxed state of myosin.

In this study, we assessed the super relaxed (SRX) state of myosin and sarcomeric protein phosphorylation in two pathological models of cardiomyopathy and in a near-physiological model of cardiac hypertrophy. The cardiomyopathy models differ in disease progression and severity and express the hypertrophic (HCM-A57G) or restrictive (RCM-E143K) mutations in the human ventricular myosin essential light chain (ELC), which is encoded by the MYL3 gene. Their effects were compared with near-physiological heart remodeling, represented by the N-terminally truncated ELC (Δ43 ELC mice), and with nonmutated human ventricular WT-ELC mice. The HCM-A57G and RCM-E143K mutations had antagonistic effects on the ATP-dependent myosin energetic states, with HCM-A57G cross-bridges fostering the disordered relaxed (DRX) state and the RCM-E143K model favoring the energy-conserving SRX state. The HCM-A57G model promoted the switch from the SRX to DRX state and showed an ∼40% increase in myosin regulatory light chain (RLC) phosphorylation compared with the RLC of normal WT-ELC myocardium. On the contrary, the RCM-E143K-associated stabilization of the SRX state was accompanied by an approximately twofold lower level of myosin RLC phosphorylation compared with the RLC of WT-ELC. Upregulation of RLC phosphorylation was also observed in Δ43 versus WT-ELC hearts, and the Δ43 myosin favored the energy-saving SRX conformation. The two disease variants also differently affected the duration of force transients, with shorter (HCM-A57G) or longer (RCM-E143K) transients measured in electrically stimulated papillary muscles from these pathological models, while no changes were displayed by Δ43 fibers. We propose that the N terminus of ELC (N-ELC), which is missing in the hearts of Δ43 mice, works as an energetic switch promoting the SRX-to-DRX transition and contributing to the regulation of myosin RLC phosphorylation in full-length ELC mice by facilitating or sterically blocking RLC phosphorylation in HCM-A57G and RCM-E143K hearts, respectively.

Dynamic spatial dispersion of repolarization is present in regions critical for ischemic ventricular tachycardia ablation.

BackgroundThe presence of dynamic substrate changes may facilitate functional block and reentry in ventricular tachycardia (VT).ObjectiveWe aimed to study dynamic ventricular repolarization changes in critical regions of the VT circuit during sensed single extrastimulus pacing known as the Sense Protocol (SP).MethodsTwenty patients (aged 67 ± 9 years, 17 male) underwent VT ablation. A bipolar voltage map was obtained during sinus rhythm (SR) and right ventricular SP pacing at 20 ms above ventricular effective refractory period. Ventricular repolarization maps were constructed. Ventricular repolarization time (RT) was calculated from unipolar electrogram T waves, using the Wyatt method, as the dV/dtmax of the unipolar T wave. Entrainment or pace mapping confirmed critical sites for ablation.ResultsThe median global repolarization range (max-min RT per patient) was 166 ms (interquartile range [IQR] 143–181 ms) during SR mapping vs 208 ms (IQR 182–234) during SP mapping (P = .0003 vs intrinsic rhythm). Regions of late potentials (LP) had a longer RT during SP mapping compared to regions without LP (mean 394 ± 40 ms vs 342 ± 25 ms, P < .001). In paired regions of normal myocardium there was no significant spatial dispersion of repolarization (SDR)/10 mm2 during SP mapping vs SR mapping (SDR 11 ± 6 ms vs 10 ± 6 ms, P = .54). SDR/10 mm2 was greater in critical areas of the VT circuit during SP mapping 63 ± 29 ms vs SR mapping 16 ± 9 ms (P < .001).ConclusionVentricular repolarization is prolonged in regions of LP and increases dynamically, resulting in dynamic SDR in critical areas of the VT circuit. These dynamic substrate changes may be an important factor that facilitates VT circuits.

Interstitial Fibrosis in HFpEF Attenuates Diastolic Circumferential Strain and the Transcriptional Response to Left Ventricular Pressure Overload

Objective We previously demonstrated thattransient repetitive pressure overload (RPO) in swine leads to myocardial interstitial fibrosis and reduced diastolic left ventricular (LV) compliance. While this phenotype is similar to that found in some patients with heart failure with preserved ejection fraction (HFpEF), it occurs in the absence of anatomic LV hypertrophy, persistent hypertension, or comorbidities. The present study was designed to determine if reduced LV diastolic distensibility alters the acute transcriptional response to transient pressure overload. Methods A total of 21 swine were studied. Hemodynamic assessment, echocardiography, and blood sampling were performed before and after a 60 minute intravenous infusion of phenylephrine (PE; 300 μg/min) to elevate LV end-diastolic pressure (EDP) to ~30 mmHg. Myocardial RNA from control animals (n=7) was compared to tissue isolated 24 hours after a single episode of pressure overload (n=6) or after 14 days of RPO (n=8). We assessed LV diastolic strain and compliance via echocardiography and quantified interstitial fibrosis with picrosirius red staining. Genes previously demonstrated to be altered after pressure overload including natriuretic peptides (ANP and BNP), inflammatory mediators (CD68 and CCR2) and pro-fibrotic factors (LOXL2 and Fibronectin) were assessed with quantitative PCR and normalized to β2 microglobulin. Results Transient PE infusion increased LV EDP from 14 ± 1 to 30 ± 1 mmHg and systolic arterial pressure from 120 ± 6 to 205 ± 6 mmHg (both p<0.01). The hemodynamic response to PE was similar during a single episode of pressure overload and after 14 days of RPO. There was concentric LV remodeling (LV mass/EDV: 1.3 ± 0.1 vs. 1.1 ± 0.1 g/mL; p<0.05) without anatomic hypertrophy after RPO (LV mass/body mass: 2.4 ± 0.1 vs. 2.3 ± 0.1 g/kg; p=0.37), yet interstitial fibrosis increased from 6.6 ± 0.7% to 12.9 ± 1.8% (p<0.05) and LV diastolic compliance (∆EDV/∆EDP) decreased from 1.7 ± 0.2 to 0.6 ± 0.2 mL/mmHg (p<0.05). As a result, myocardial diastolic circumferential strain after RPO was markedly reduced in comparison to a single episode of pressure overload in the normal heart (1.5 ± 2.7% after RPO vs. 11.1 ± 3.0% in controls; p<0.05). The reduction in diastolic circumferential strain after RPO markedly attenuated the transcriptional response to acute pressure overload (Figure). In normal myocardium, transient pressure overload upregulated natriuretic peptides (ANP 26-fold and BNP 12-fold; both p<0.01), pro-inflammatory genes (CD68 2.3-fold and CCR2 4-fold; p=0.06), and pro-fibrotic genes (LOXL2 3.4-fold and fibronectin 3.9-fold; p<0.05). In contrast, the reduction in diastolic strain after RPO prevented any increase in gene expression despite a similar magnitude of transient pressure overload. Conclusions These results dissociate the effects of myocardial strain from systolic and end-diastolic pressure on the transcriptional response to LV pressure overload. The dependence of gene expression on diastolic strain rather than LV pressure may explain observations such as the frequent absence of natriuretic peptide elevation in patients with HFpEF.

Melatonin Treatment Does Not Modify Ectopic Activity During Ischemia and Reperfusion in Rats

Introduction Ventricular tachycardia and/or ventricular fibrillation (VT/VF), a life-threatening complication of acute myocardial ischemia are based on the interaction between a vulnerable arrhythmogenic substrate and an arrhythmic trigger. Melatonin was proposed as a potential antiarrhythmic medication and was shown to ameliorate the arrhythmogenic substrate. Also, melatonin provides a sympatholytic effect and thereby can attenuate ectopic activity, which provides the triggers for VT/VF. However, little is known about a melatonin effect on the arrhythmic triggering. In the present study, we aimed at evaluation of the melatonin effects on catecholamine synthesis in normal and ischemic myocardium and heart rhythm disturbances during an episode of ischemia and reperfusion. Methods Experiments were done in 30 control and 32 melatonin-treated (10 mg/kg, daily, for 7 days) male rats. The hearts from 10 control and 10 treated animals were taken for histochemical analysis of sympathetic fibers (glyoxylic acid-induced fluorescence). Continuous ECG recording was performed in 20 control and 22 treated anesthetized animals before and during an ischemia-reperfusion episode induced by reversible 5-min occlusion of the left anterior descending coronary artery. Tyrosine hydroxylase expression in normal and ischemic myocardial regions was assessed by Western blotting analysis in 12 control and 12 melatonin-treated animals. Results No differences in the state of sympathetic innervation (density and fluorescence intensity) were observed in histochemical analysis. However, Western blotting demonstrated that melatonin treatment suppressed tyrosine hydroxylase expression in normal (p<0.05 vs control) but not in ischemic regions of myocardium. Melatonin-treated animals had longer RR-intervals in the baseline state, but this difference progressively disappeared during the period of ischemia due to tachycardic response to ischemia in the treated group (Figure). No differences in the number of extrasystoles were found between the control and treated groups during ischemia and reperfusion periods (Figure). Conclusion Chronic melatonin treatment led to the peripheral sympatholytic effect in myocardium, which was associated with decreased heart rate in preischemic conditions. However, this effect attenuated during ischemia and no differences in heart rate or extrasystolic burden were found between the control and treated animals during ischemia and reperfusion.

Electroactive Polymeric Composites to Mimic the Electromechanical Properties of Myocardium in Cardiac Tissue Repair.

Due to the limited regenerative capabilities of cardiomyocytes, incidents of myocardial infarction can cause permanent damage to native myocardium through the formation of acellular, non-conductive scar tissue during wound repair. The generation of scar tissue in the myocardium compromises the biomechanical and electrical properties of the heart which can lead to further cardiac problems including heart failure. Currently, patients suffering from cardiac failure due to scarring undergo transplantation but limited donor availability and complications (i.e., rejection or infectious pathogens) exclude many individuals from successful transplant. Polymeric tissue engineering scaffolds provide an alternative approach to restore normal myocardium structure and function after damage by acting as a provisional matrix to support cell attachment, infiltration and stem cell delivery. However, issues associated with mechanical property mismatch and the limited electrical conductivity of these constructs when compared to native myocardium reduces their clinical applicability. Therefore, composite polymeric scaffolds with conductive reinforcement components (i.e., metal, carbon, or conductive polymers) provide tunable mechanical and electroactive properties to mimic the structure and function of natural myocardium in force transmission and electrical stimulation. This review summarizes recent advancements in the design, synthesis, and implementation of electroactive polymeric composites to better match the biomechanical and electrical properties of myocardial tissue.

Metabolic Scar Assessment with18F-FDG PET: Correlation to Ischemic Ventricular Tachycardia Substrate and Successful Ablation Sites.

The functional and molecular imaging characteristics of ischemic ventricular tachycardia (VT) substrate are incompletely understood. Our objective was to compare regional 18F-FDG PET tracer uptake with detailed electroanatomic maps (EAMs) in a more extensive series of postinfarction VT patients to define the metabolic properties of VT substrate and successful ablation sites. Methods: Three-dimensional (3D) metabolic left ventricular reconstructions were created from perfusion-normalized 18F-FDG PET images in consecutive patients undergoing VT ablation. PET defects were classified as severe (defined as <50% uptake) or moderate (defined as 50%-70% uptake), as referenced to the maximal 17-segment uptake. Color-coded PET scar reconstructions were coregistered with corresponding high-resolution 3D EAMs, which were classified as indicating dense scarring (defined as voltage < 0.5 mV), normal myocardium (defined as voltage > 1.5 mV), or border zones (defined as voltage of 0.5-1.5 mV). Results: All 56 patients had ischemic cardiomyopathy (ejection fraction, 29% ± 12%). Severe PET defects were larger than dense scarring, at 63.0 ± 48.4 cm2 versus 13.8 ± 33.1 cm2 (P < 0.001). Similarly, moderate/severe PET defects (≤70%) were larger than areas with abnormal voltage (≤1.5 mV) measuring 105.1 ± 67.2 cm2 versus 56.2 ± 62.6 cm2 (P < 0.001). Analysis of bipolar voltage (23,389 mapping points) showed decreased voltage among severe PET defects (n = 10,364; 0.5 ± 0.3 mV) and moderate PET defects (n = 5,243; 1.5 ± 0.9 mV, P < 0.01), with normal voltage among normal PET areas (>70% uptake) (n = 7,782, 3.2 ± 1.3 mV, P < 0.001). Eighty-eight percent of VT channel or exit sites (n = 44) were metabolically abnormal (severe PET defect, 78%; moderate PET defect, 10%), whereas 12% (n = 6) were in PET-normal areas. Metabolic channels (n = 26) existed in 45% (n = 25) of patients, with an average length and width of 17.6 ± 12.5 mm and 10.3 ± 4.2 mm, respectively. Metabolic channels were oriented predominantly in the apex or base (86%), harboring VT channel or exit sites in 31%. Metabolic rapid-transition areas (>50% change in 18F-FDG tracer uptake/15 mm) were detected in 59% of cases (n = 33), colocalizing to VT channels or exit sites (15%) or near these sites (85%, 12.8 ± 8.5 mm). Metabolism-voltage mismatches in which there was a severe PET defect but voltage indicating normal myocardium were seen in 21% of patients (n = 12), 41% of whom were harboring VT channel or exit sites. Conclusion: Abnormal 18F-FDG uptake categories could be detected using incremental 3D step-up reconstructions. They predicted decreasing bipolar voltages and VT channel or exit sites in about 90% of cases. Additionally, functional imaging allowed detection of novel molecular tissue characteristics within the ischemic VT substrate such as metabolic channels, rapid-transition areas, and metabolism-voltage mismatches demonstrating intrasubstrate heterogeneity and providing possible targets for imaging-guided ablation.

Layer-By-Layer Fabrication of Large and Thick Human Cardiac Muscle Patch Constructs With Superior Electrophysiological Properties.

Engineered cardiac tissues fabricated from human induced pluripotent stem cells (hiPSCs) show promise for ameliorating damage from myocardial infarction, while also restoring function to the damaged left ventricular (LV) myocardium. For these constructs to reach their clinical potential, they need to be of a clinically relevant volume and thickness, and capable of generating synchronous and forceful contraction to assist the pumping action of the recipient heart. Design prerequisites include a structure thickness sufficient to produce a beneficial contractile force, prevascularization to overcome diffusion limitations and sufficient structural development to allow for maximal cell communication. Previous attempts to meet these prerequisites have been hindered by lack of oxygen and nutrient transport due to diffusion limits (100–200 μm) resulting in necrosis. This study employs a layer-by-layer (LbL) fabrication method to produce cardiac tissue constructs that meet these design prerequisites and mimic normal myocardium in form and function. Thick (>2 mm) cardiac tissues created from hiPSC-derived cardiomyocytes, -endothelial cells (ECs) and -fibroblasts (FBs) were assessed, in vitro, over a 4-week period for viability (<6% necrotic cells), cell morphology and functionality. Functional performance assessment showed enhanced t-tubule network development, gap junction communication as well as previously unseen, physiologically relevant conduction velocities (CVs) (>30 cm/s). These results demonstrate that LbL fabrication can be utilized successfully to create prevascularized, functional cardiac tissue constructs from hiPSCs for potential therapeutic applications.

Direct Comparison of Bayesian and Fermi Deconvolution Approaches for Myocardial Blood Flow Quantification: In silico and Clinical Validations.

Cardiac magnetic resonance myocardial perfusion imaging can detect coronary artery disease and is an alternative to single-photon emission computed tomography or positron emission tomography. However, the complex, non-linear MR signal and the lack of robust quantification of myocardial blood flow have hindered its widespread clinical application thus far. Recently, a new Bayesian approach was developed for brain imaging and evaluation of perfusion indexes (Kudo et al., 2014). In addition to providing accurate perfusion measurements, this probabilistic approach appears more robust than previous approaches, particularly due to its insensitivity to bolus arrival delays. We assessed the performance of this approach against a well-known and commonly deployed model-independent method based on the Fermi function for cardiac magnetic resonance myocardial perfusion imaging. The methods were first evaluated for accuracy and precision using a digital phantom to test them against the ground truth; next, they were applied in a group of coronary artery disease patients. The Bayesian method can be considered an appropriate model-independent method with which to estimate myocardial blood flow and delays. The digital phantom comprised a set of synthetic time-concentration curve combinations generated with a 2-compartment exchange model and a realistic combination of perfusion indexes, arterial input dynamics, noise and delays collected from the clinical dataset. The myocardial blood flow values estimated with the two methods showed an excellent correlation coefficient (r2 > 0.9) under all noise and delay conditions. The Bayesian approach showed excellent robustness to bolus arrival delays, with a similar performance to Fermi modeling when delays were considered. Delays were better estimated with the Bayesian approach than with Fermi modeling. An in vivo analysis of coronary artery disease patients revealed that the Bayesian approach had an excellent ability to distinguish between abnormal and normal myocardium. The Bayesian approach was able to discriminate not only flows but also delays with increased sensitivity by offering a clearly enlarged range of distribution for the physiologic parameters.

Classifying Microscopic Acute and Old Myocardial Infarction Using Convolutional Neural Networks.

Convolutional neural network (CNN) has advanced in recent years and translated from research into medical practice, most notably in clinical radiology and histopathology. Research on CNNs in forensic/postmortem pathology is almost exclusive to postmortem computed tomography despite the wealth of research into CNNs in surgical/anatomical histopathology. This study was carried out to investigate whether CNNs are able to identify and age myocardial infarction (a common example of forensic/postmortem histopathology) from histology slides. As a proof of concept, this study compared 4 CNNs commonly used in surgical/anatomical histopathology to identify normal myocardium from myocardial infarction. A total of 150 images of the myocardium (50 images each for normal myocardium, acute myocardial infarction, and old myocardial infarction) were used to train and test each CNN. One of the CNNs used (InceptionResNet v2) was able to show a greater than 95% accuracy in classifying normal myocardium from acute and old myocardial infarction. The result of this study is promising and demonstrates that CNN technology has potential applications as a screening and computer-assisted diagnostics tool in forensic/postmortem histopathology.