Cardiac Sarcoplasmic Reticulum Function in Heart Failure

Heart failure is characterised by abnormalities of myocardial systolic and diastolic function. While there are changes in the structure of the heart which could account, in part, for these abnormalities, there is strong evidence to suggest that the regulation of calcium within the failing cardiac myocyte is abnormal. A diminished ability to release and sequester calcium could give rise to such contractile abnormalities and so the sarcoplasmic reticulum (SR), as the main site for calcium uptake and release, is the major focus of study in this thesis. Two rabbit models of heart failure were studied: adriamycin-induced cardiomyopathy and coronary artery ligation. The adriamycin-treated animals were studied 2 weeks after stopping 8 weeks of chronic intravenous administration. The coronary ligation animals were studied in two groups : 8 weeks after ligation and 15 weeks after ligation. The effects of down- regulation of adrenoreceptors on SR function was studied in a rabbit model following 7 days continuous administration of isoprenaline by mini-osmotic pump. Lastly, human ventricular trabeculae were studied from patients undergoing cardiac transplantation and are presented as a separate chapter. The severity of heart failure was assessed in each rabbit by echocardiography and by invasive measurement of cardiac output and left ventricular pressures. The preparation chosen to study the SR was the saponin-treated cardiac ventricular trabecula. This preparation has a number of advantages in that it leaves the SR anatomically undisrupted, the SR can load and leak calcium in a physiological way, the permeabilised sarcolemma allows free access to drugs and chemicals and the myofilaments may be assessed both in association with the SR and independently following treatment with the detergent Triton-X100. Furthermore, in our hands, this technique is very reliable and reproducible which is extremely important since very few animals which started the protocol, for any of the models, failed to produce results from the saponin-treated fibres. This has important implications for animal suffering. Sarcoplasmic reticulum function was assessed in the rabbit model of adriamycin-induced cardiomyopathy. The method for inducing cardiomyopathy is described in chapter 2 (methods 1) and the results are described in chapter 3. Unfortunately few animals with significant left ventricular dysfunction were produced but those with ejection fractions of less than 45% demonstrated enhanced SR Calcium loading compared with saline-treated controls and with adriamycin treated animals with higher ejection fractions. The ability of the myofilaments to produce force was unaffected by chronic treatment with adriamycin and the calcium responsiveness of the myofilaments was also unaffected by adriamycin treatment. 8 weeks after coronary ligation, rabbits with significant left ventricular dysfunction demonstrated enhanced SR calcium uptake compared with controls. A group of ligation animals with less severe left ventricular dysfunction, assessed by a scoring system, also demonstrated enhanced SR calcium loading but the difference between this group and sham-operated controls was less marked. Additionally , this enhanced SR calcium loading was correlated with a number of indices of heart failure including ejection fraction, left ventricular end-diastolic pressure and resting cardiac output. Enhanced SR calcium loading was associated with an increased predisposition to calcium overload, revealed as spontaneous tension oscillations. These oscillations were observed more frequently in the ligated animals and at lower loading calcium concentrations than in the control animals. This pattern of SR loading was observed in both left and right ventricular preparations although the greatest increase in SR loading was observed in the left ventricle. The ability of the myofilaments to produce force was not altered in the left ventricle but there was a fall in the maximum force produced by trabeculae from the right ventricle in the ligated group. The calcium responsiveness of the myofilaments was not different between groups. Chapter 4 examines SR calcium loading 15 weeks after coronary ligation. In these animals there was no significant difference in SR calcium loading ability between ligation and control trabeculae. The ligated animals demonstrated significant left ventricular dysfunction but this was not more severe than that observed in 8 week animals. Myofilament force production was unaltered as was calcium responsiveness of the myofilaments. Chapter 6 examines possible changes in the SR calcium release mechanism. There was no difference in the sensitivity of the mechanism of caffeine- induced calcium release in ligation animals compared with controls. Trabeculae from ligation animals appeared more sensitive to the calcium channel blocking agent, ryanodine, when applied during sustained oscillations. In a separate set of experiments SR calcium leak was assessed. (Abstract shortened by ProQuest.)

Cardiomyocyte proliferation in zebrafish and mammals: lessons for human disease

Cardiomyocytes proliferate profusely during early development and for a brief period after birth in mammals. Within a month after birth, this proliferative capability is dramatically reduced in mammals unlike lower vertebrates where it persists into adult life. The zebrafish, for example, retains the ability to regenerate the apex of the heart following resection by a mechanism predominantly driven by cardiomyocyte proliferation. Differences in proliferative capacity of cardiomyocytes in adulthood between mammals and lower vertebrates are closely liked to ontogenetic or phylogenetic factors. Elucidation of these factors has the potential to provide enormous benefits if they lead to the development of therapeutic strategies that facilitate cardiomyocyte proliferation. In this review, we highlight the differences between Mammalian and Zebrafish cardiomyocytes, which could explain at least in part the different proliferative capacities in these two species. We discuss the advantages of the zebrafish as a model of cardiomyocyte proliferation, particularly at the embryonic stage. We also identify a number of key molecular pathways with potential to reveal key steps in switching cardiomyocytes from a quiescent to a proliferative phenotype. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00018-016-2404-x) contains supplementary material, which is available to authorized users

Systolic and diastolic ventricular function in zebrafish embryos: Influence of norepenephrine, MS-222 and temperature

<p>Abstract</p> <p>Background</p> <p>Zebrafish are increasingly used to study the influences of gene mutation and manipulation on cardiac development, structure and function. In this study, a video edge detection system was used to characterise, continuously, cardiac ventricle function in 2–5 days old zebrafish embryos embedded in 0.6% agar and examined under light microscopy at room temperature (22°C). Using video edge detection software (IonOptix Inc), the motion of a small region of the cardiac ventricle wall was converted to a continuous chart trace allowing analysis of wall motion amplitude (WMA) and myocardial wall velocity during systole (MWVs) and diastole (MWVd).</p> <p>Results</p> <p>Cardiac wall motion characteristics changed progressively from day 2 to 5 (WMA, 2-days, 17.6 ± 4.4 μm vs 5-days, 24.6 ± 4.7 μm, p < 0.01). MWVd was more rapid than MWVs at all developmental time points. Embryonic hearts were also assessed after increasing concentrations of norepenephrine (NE) and the anaesthetic agent MS222 (tricaine) were added to the bathing water. In response to NE, WMA increased significantly more in 4 day embryos compared with 2 day embryos (change in WMA,13.6 ± 8.2 μm vs 4.0 ± 8.8 μm, p = 0.01, respectively) while the decrease in WMA in response to MS222 was similar in both 2 and 4-day embryos. Heart rate, MWVs and MWVd were significantly higher at 28°C compared with 22°C. No differences in cardiac function were observed between AB and Golden strains.</p> <p>Conclusion</p> <p>Video edge detection appears sufficiently sensitive to detect subtle changes in diastolic and systolic cardiac function during development and changes resulting from pharmacological and environmental interventions. Such measurements could be valuable in assessment of altered cardiac function after genetic manipulation.</p

Imaging the Developing Heart: Synchronized Timelapse Microscopy During Developmental Changes

How do you use imaging to analyse the development of the heart, which not only changes shape but also undergoes constant, high-speed, quasi-periodic changes? We have integrated ideas from prospective and retrospective optical gating to capture long-term, phase-locked developmental time-lapse videos. In this paper we demonstrate the success of this approach over a key developmental time period: heart looping, where large changes in heart shape prevent previous prospective gating approaches from capturing phase-locked videos. We use the comparison with other approaches to in vivo heart imaging to highlight the importance of collecting the most appropriate data for the biological question.Comment: Carl J. Nelson and Charlotte Buckley and John J. Mullins and Martin A. Denvir and Jonathan Taylor, "Imaging the Developing Heart: Synchronized Timelapse Microscopy During Developmental Changes", Proc. SPIE (10499), 10499-41 (2018). Copyright 2018 Society of Photo Optical Instrumentation Engineers (SPIE

FLI1+ cells transcriptional analysis reveals LMO2-PRDM16 axis in angiogenesis

A network of molecular factors drives the development, differentiation, and maintenance of endothelial cells. Friend leukemia integration 1 transcription factor (FLI1) is a bona fide marker of endothelial cells during early development. In zebrafish Tg(fli1:EGFP)(y1), we identified two endothelial cell populations, high-fli1(+) and low-fli1(+), by the intensity of green fluorescent protein signal. By comparing RNA-sequencing analysis of non-fli1 expressing cells (fli1(−)) with these two (fli1(+)) cell populations, we identified several up-regulated genes, not previously recognized as important, during endothelial development. Compared with fli1(−) and low-fli1(+) cells, high-fli1(+) cells showed up-regulated expression of the zinc finger transcription factor PRDI-BF1 and RIZ homology domain containing 16 (prdm16). Prdm16 knockdown (KD) by morpholino in the zebrafish larva was associated with impaired angiogenesis and increased number of low-fli1(+) cells at the expense of high-fli1(+) cells. In addition, PRDM16 KD in endothelial cells derived from human-induced pluripotent stem cells impaired their differentiation and migration in vitro. Moreover, zebrafish mutants (mut) with loss of function for the oncogene LIM domain only 2 (lmo2) also showed reduced prdm16 gene expression combined with impaired angiogenesis. Prdm16 expression was reduced further in endothelial (CD31(+)) cells compared with CD31(−) cells isolated from lmo2-mutants (lmo2-mut) embryos. Chromatin immunoprecipitation–PCR demonstrated that Lmo2 binds to the promoter and directly regulates the transcription of prdm16. This work unveils a mechanism by which prdm16 expression is activated in endothelial cells by Lmo2 and highlights a possible therapeutic pathway by which to modulate endothelial cell growth and repair

Palliative care needs in patients hospitalized with heart failure (PCHF) study: rationale and design

Abstract Aims The primary aim of this study is to provide data to inform the design of a randomized controlled clinical trial (RCT) of a palliative care (PC) intervention in heart failure (HF). We will identify an appropriate study population with a high prevalence of PC needs defined using quantifiable measures. We will also identify which components a specific and targeted PC intervention in HF should include and attempt to define the most relevant trial outcomes. Methods An unselected, prospective, near-consecutive, cohort of patients admitted to hospital with acute decompensated HF will be enrolled over a 2-year period. All potential participants will be screened using B-type natriuretic peptide and echocardiography, and all those enrolled will be extensively characterized in terms of their HF status, comorbidity, and PC needs. Quantitative assessment of PC needs will include evaluation of general and disease-specific quality of life, mood, symptom burden, caregiver burden, and end of life care. Inpatient assessments will be performed and after discharge outpatient assessments will be carried out every 4 months for up to 2.5 years. Participants will be followed up for a minimum of 1 year for hospital admissions, and place and cause of death. Methods for identifying patients with HF with PC needs will be evaluated, and estimates of healthcare utilisation performed. Conclusion By assessing the prevalence of these needs, describing how these needs change over time, and evaluating how best PC needs can be identified, we will provide the foundation for designing an RCT of a PC intervention in HF

Identifying Acute Coronary Syndrome Patients Approaching End-of-Life

Background: Acute coronary syndrome (ACS) is common in patients approaching the end-of-life (EoL), but these patients rarely receive palliative care. We compared the utility of a palliative care prognostic tool (Gold Standards Framework (GSF)) and the Global Registry of Acute Coronary Events (GRACE) score, to help identify patients approaching EoL. Methods and Findings: 172 unselected consecutive patients with confirmed ACS admitted over an eight-week period were assessed using prognostic tools and followed up for 12 months. GSF criteria identified 40 (23%) patients suitable for EoL care while GRACE identified 32 (19%) patients with $10 % risk of death within 6 months. Patients meeting GSF criteria were older (p = 0.006), had more comorbidities (1.660.7 vs. 1.260.9, p = 0.007), more frequent hospitalisations before (p = 0.001) and after (0.0001) their index admission, and were more likely to die during follow-up (GSF+ 20 % vs GSF- 7%, p = 0.03). GRACE score was predictive of 12-month mortality (C-statistic 0.75) and this was improved by the addition of previous hospital admissions and previous history of stroke (C-statistic 0.88). Conclusions: This study has highlighted a potentially large number of ACS patients eligible for EoL care. GSF or GRACE could be used in the hospital setting to help identify these patients. GSF identifies ACS patients with more comorbidity and at increased risk of hospital readmission

Isolated left ventricular non-compaction as an unusual cause of heart failure: a case report

<p>Abstract</p> <p>Introduction</p> <p>Isolated left ventricular non-compaction is a recently described form of cardiomyopathy that is associated with a significant risk of life-threatening arrhythmia and thromboembolic complications.</p> <p>Case presentation</p> <p>We report the presentation, diagnosis and management of isolated left ventricular non-compaction in a 54-year-old Caucasian woman presenting with progressive symptoms of heart failure.</p> <p>Conclusion</p> <p>Advances in diagnostic imaging have undoubtedly led to an increase in the detection of isolated left ventricular non-compaction. Diagnosing and differentiating this uncommon condition from other forms of cardiomyopathy are important as treatment and prognosis may differ significantly. Our current understanding of isolated left ventricular non-compaction, including diagnostic criteria, management and prognosis, is discussed.</p