This protocol describes the preparation of highly viable adult ventricular myocardial slices from the hearts of small and large mammals, including rodents, pigs, dogs and humans. Adult ventricular ...myocardial slices are 100- to 400-μm-thick slices of living myocardium that retain the native multicellularity, architecture and physiology of the heart. This protocol provides a list of the equipment and reagents required alongside a detailed description of the methodology for heart explantation, tissue preparation, slicing with a vibratome and handling of myocardial slices. Supplementary videos are included to visually demonstrate these steps. A number of critical steps are addressed that must be followed in order to prepare highly viable myocardial slices. These include identification of myocardial fiber direction and fiber alignment within the tissue block, careful temperature control, use of an excitation-contraction uncoupler, optimal vibratome settings and correct handling of myocardial slices. Many aspects of cardiac structure and function can be studied using myocardial slices in vitro. Typical results obtained with hearts from a small mammal (rat) and a large mammal (human) with heart failure are shown, demonstrating myocardial slice viability, maximum contractility, Ca
handling and structure. This protocol can be completed in ∼4 h.
Functional endothelial cells (EC) are a critical interface between blood vessels and the thrombogenic flowing blood. Disruption of this layer can lead to early thrombosis, inflammation, vessel ...restenosis, and, following coronary (CABG) or peripheral (PABG) artery bypass graft surgery, vein graft failure. Blood-derived ECs have shown potential for vascular tissue engineering applications. Here, we show the development and preliminary testing of a method for deriving porcine endothelial-like cells from blood obtained under clinical conditions for use in translational research. The derived cells show cobblestone morphology and expression of EC markers, similar to those seen in isolated porcine aortic ECs (PAEC), and when exposed to increasing shear stress, they remain viable and show mRNA expression of EC markers similar to PAEC. In addition, we confirm the feasibility of seeding endothelial-like cells onto a decellularised human vein scaffold with approximately 90% lumen coverage at lower passages, and show that increasing cell passage results in reduced endothelial coverage.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) represent an in vitro model of cardiac function. Isolated iPSC-CMs, however, exhibit electrophysiological heterogeneity which ...hinders their utility in the study of certain cardiac currents. In the healthy adult heart, the current mediated by small conductance, calcium-activated potassium (SK) channels (I
) is atrial-selective. Functional expression of I
within atrial-like iPSC-CMs has not been explored thoroughly. The present study therefore aimed to investigate atrial-like iPSC-CMs as a model system for the study of I
. iPSCs were differentiated using retinoic acid (RA) to produce iPSC-CMs which exhibited an atrial-like phenotype (RA-iPSC-CMs). Only 18% of isolated RA-iPSC-CMs responded to SK channel inhibition by UCL1684 and isolated iPSC-CMs exhibited substantial cell-to-cell electrophysiological heterogeneity. This variability was significantly reduced by patch clamp of RA-iPSC-CMs in situ as a monolayer (iPSC-ML). A novel method of electrical stimulation was developed to facilitate recording from iPSC-MLs via In situ Monolayer Patch clamp of Acutely Stimulated iPSC-CMs (IMPASC). Using IMPASC, > 95% of iPSC-MLs could be paced at a 1 Hz. In contrast to isolated RA-iPSC-CMs, 100% of RA-iPSC-MLs responded to UCL1684, with APD
being prolonged by 16.0 ± 2.0 ms (p < 0.0001; n = 12). These data demonstrate that in conjunction with IMPASC, RA-iPSC-MLs represent an improved model for the study of I
. IMPASC may be of wider value in the study of other ion channels that are inconsistently expressed in isolated iPSC-CMs and in pharmacological studies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Objective Off-pump coronary artery bypass grafting reduces postoperative morbidity and uses fewer resources than conventional surgical intervention with cardiopulmonary bypass. However, only 15% to ...20% of coronary artery bypass grafting operations use off-pump coronary artery bypass. One reason for not using off-pump coronary artery bypass might be the surgeon's concern about the long-term patency of grafts performed with this technique. Therefore our objective was to compare long-term outcomes in patients randomized to off-pump coronary artery bypass or coronary artery bypass grafting with cardiopulmonary bypass. Methods Participants in 2 randomized trials comparing off-pump coronary artery bypass and coronary artery bypass grafting with cardiopulmonary bypass were followed up for 6 to 8 years after surgical intervention to assess graft patency, major adverse cardiac-related events, and health-related quality of life. Patency was assessed by using multidetector computed tomographic coronary angiographic analysis with a 16-slice scanner. Two blinded observers classified proximal, body, and distal segments of each graft as occluded or not. Major adverse cardiac-related events and health-related quality of life were obtained from questionnaires given to participants and family practitioners. Results Patency was studied in 199 and health-related quality of life was studied in 299 of 349 survivors. There was no evidence of attrition bias. The likelihood of graft occlusion was no different between off-pump coronary artery bypass (10.6%) and coronary artery bypass grafting with cardiopulmonary bypass (11.0%) groups (odds ratio, 1.00; 95% confidence interval, 0.55–1.81; P > .99). Graft occlusion was more likely at the distal than the proximal anastomosis (odds ratio, 1.11; 95% confidence interval, 1.02–1.20). There were also no differences between the off-pump coronary artery bypass and coronary artery bypass grafting with cardiopulmonary bypass groups in the hazard of death (hazard ratio, 1.24; 95% confidence interval, 0.72–2.15) or major adverse cardiac-related events or death (hazard ratio, 0.84; 95% confidence interval, 0.58–1.24), or mean health-related quality of life across a range of domains and instruments. Conclusions Long-term health outcomes with off-pump coronary artery bypass are similar to those with coronary artery bypass grafting with cardiopulmonary bypass when both operations are performed by experienced surgeons.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Typically, subgroup analyses in clinical trials are conducted by comparing the intervention effect in each subgroup by means of an interaction test. However, trials are rarely, if ever, adequately ...powered for interaction tests, so clinically important interactions may go undetected. We discuss the application of Bayesian methods by using expert opinions alongside the trial data. We applied this methodology to the VeRDiCT trial investigating the effect of preoperative volume replacement therapy (VRT) versus no VRT (usual care) in diabetic patients undergoing cardiac surgery. Two subgroup effects were of clinical interest, a) preoperative renal failure and b) preoperative type of antidiabetic medication.
Clinical experts were identified within the VeRDiCT trial centre in the UK. A questionnaire was designed to elicit opinions on the impact of VRT on the primary outcome of time from surgery until medically fit for hospital discharge, in the different subgroups. Prior beliefs of the subgroup effect of VRT were elicited face-to-face using two unconditional and one conditional questions per subgroup analysis. The robustness of results to the 'community of priors' was assessed. The community of priors was built using the expert priors for the mean average treatment effect, the interaction effect or both in a Bayesian Cox proportional hazards model implemented in the STAN software in R.
Expert opinions were obtained from 7 clinicians (6 cardiac surgeons and 1 cardiac anaesthetist). Participating experts believed VRT could reduce the length of recovery compared to usual care and the greatest benefit was expected in the subgroups with the more severe comorbidity. The Bayesian posterior estimates were more precise compared to the frequentist maximum likelihood estimate and were shifted toward the overall mean treatment effect.
In the VeRDiCT trial, the Bayesian analysis did not provide evidence of a difference in treatment effect across subgroups. However, this approach increased the precision of the estimated subgroup effects and produced more stable treatment effect point estimates than the frequentist approach. Trial methodologists are encouraged to prospectively consider Bayesian subgroup analyses when low-powered interaction tests are planned.
ISRCTN, ISRCTN02159606 . Registered 29th October 2008.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pain triggers a homeostatic alarm reaction to injury. It remains unknown, however, whether nociceptive signaling activated by ischemia is relevant for progenitor cells (PC) release from bone marrow. ...To this end, we investigated the role of the neuropeptide substance P (SP) and cognate neurokinin 1 (NK1) nociceptor in PC activation and angiogenesis during ischemia in mice and in human subjects.
The mouse bone marrow contains sensory fibers and PC that express SP. Moreover, SP-induced migration provides enrichment for PC that express NK1 and promote reparative angiogenesis after transplantation in a mouse model of limb ischemia. Acute myocardial infarction and limb ischemia increase SP levels in peripheral blood, decrease SP levels in bone marrow, and stimulate the mobilization of NK1-expressing PC, with these effects being abrogated by systemic administration of the opioid receptor agonist morphine. Moreover, bone marrow reconstitution with NK1-knockout cells results in depressed PC mobilization, delayed blood flow recovery, and reduced neovascularization after ischemia. We next asked whether SP is instrumental to PC mobilization and homing in patients with ischemia. Human PC express NK1, and SP-induced migration provides enrichment for proangiogenic PC. Patients with acute myocardial infarction show high circulating levels of SP and NK1-positive cells that coexpress PC antigens, such as CD34, KDR, and CXCR4. Moreover, NK1-expressing PC are abundant in infarcted hearts but not in hearts that developed an infarct after transplantation.
Our data highlight the role of SP in reparative neovascularization. Nociceptive signaling may represent a novel target of regenerative medicine.
Sulfide-releasing compounds reduce reperfusion injury by decreasing mitochondria-derived reactive oxygen species production. We previously characterised ammonium tetrathiomolybdate (ATTM), a ...clinically used copper chelator, as a sulfide donor in rodents. Here we assessed translation to large mammals prior to clinical testing. In healthy pigs an intravenous ATTM dose escalation revealed a reproducible pharmacokinetic/pharmacodynamic (PK/PD) relationship with minimal adverse clinical or biochemical events. In a myocardial infarction (1-h occlusion of the left anterior descending coronary artery)-reperfusion model, intravenous ATTM or saline was commenced just prior to reperfusion. ATTM protected the heart (24-h histological examination) in a drug-exposure-dependent manner (r2 = 0.58, p < 0.05). Blood troponin T levels were significantly (p < 0.05) lower in ATTM-treated animals while myocardial glutathione peroxidase activity, an antioxidant selenoprotein, was elevated (p < 0.05). Overall, our study represents a significant advance in the development of sulfides as therapeutics and underlines the potential of ATTM as a novel adjunct therapy for reperfusion injury. Mechanistically, our study suggests that modulating selenoprotein activity could represent an additional mode of action of sulfide-releasing drugs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Volume indices and left ventricular ejection fraction (LVEF) are routinely used to assess cardiac function. Ventricular strain values may provide additional diagnostic information, but their ...reproducibility is unclear. This study therefore compares the repeatability and reproducibility of volumes, volume fraction, and regional ventricular strains, derived from cardiovascular magnetic resonance (CMR) imaging, across three software packages and between readers.
Seven readers analysed 16 short-axis CMR stacks of a porcine heart. Endocardial contours were manually drawn using OsiriX and Simpleware ScanIP and repeated in both softwares. The images were also contoured automatically in Circle CVI42. Endocardial global, apical, mid-ventricular, and basal circumferential strains, as well as end-diastolic and end-systolic volume and LVEF were compared.
Bland-Altman analysis found systematic biases in contour length between software packages. Compared to OsiriX, contour lengths were shorter in both ScanIP (-1.9 cm) and CVI42 (-0.6 cm), causing statistically significant differences in end-diastolic and end-systolic volumes, and apical circumferential strain (all p<0.006). No differences were found for mid-ventricular, basal or global strains, or left ventricular ejection fraction (all p<0.007). All CVI42 results lay within the ranges of the OsiriX results. Intra-software differences were found to be lower than inter-software differences.
OsiriX and CVI42 gave consistent results for all strain and volume metrics, with no statistical differences found between OsiriX and ScanIP for mid-ventricular, global or basal strains, or left ventricular ejection fraction. However, volumes were influenced by the choice of contouring software, suggesting care should be taken when comparing volumes across different software.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Myocardial healing following myocardial infarction (MI) toward either functional tissue repair or excessive scarring/heart failure, may depend on a complex interplay between nervous and immune system ...responses, myocardial ischemia/reperfusion injury factors, as well as genetic and epidemiological factors. Hence, enhancing cardiac repair post MI may require a more patient-specific approach targeting this complex interplay and not just the heart, bearing in mind that the dysregulation or modulation of just one of these systems or some of their mechanisms may determine the outcome either toward functional repair or toward heart failure.
In this review we have elected to focus on existing preclinical and clinical in-vivo studies aimed at testing novel therapeutic approaches targeting the nervous and immune systems to trigger myocardial healing toward functional tissue repair. To this end, we have only selected clinical and preclinical in-vivo studies reporting on novel treatments targeting neuro-immune systems to ultimately treat MI. Next, we have grouped and reported treatments under each neuro-immune system. Finally, for each treatment we have assessed and reported the results of each clinical/preclinical study and then discussed their results collectively. This structured approach has been followed for each treatment discussed. To keep this review focused, we have deliberately omitted to cover other important and related research areas such as myocardial ischemia/reperfusion injury, cell and gene therapies as well as any ex-vivo and in-vitro studies.
The review indicates that some of the treatments targeting the neuro-immune/inflammatory systems appear to induce beneficial effects remotely on the healing heart post MI, warranting further validation. These remote effects on the heart also indicates the presence of an overarching synergic response occurring across the nervous and immune systems in response to acute MI, which appear to influence cardiac tissue repair in different ways depending on age and timing of treatment delivery following MI. The cumulative evidence arising from this review allows also to make informed considerations on safe as opposed to detrimental treatments, and within the safe treatments to ascertain those associated with conflicting or supporting preclinical data, and those warranting further validation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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