The effects of tumor necrosis factor-alpha on systolic and diastolic function in rat ventricular myocytes

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) is associated with myocardial dysfunction observed in sepsis and septic shock. There are two fundamental components to this dysfunction. (1) systolic dysfunction; and (2) diastolic dysfunction. The aim of these experiments was to determine if any aspect of whole-heart dysfunction could be explained by alterations to global intracellular calcium ([Ca(2+)]i), contractility, and [Ca(2+)]i handling, by TNF-α, at the level of the individual rat myocyte. We took an integrative approach to simultaneously measure [Ca(2+)]i, contractility and sarcolemmal Ca fluxes using the Ca indicator fluo-3, video edge detection, and the perforated patch technique, respectively. All experiments were performed at 37°C. The effects of 50 ng/mL TNF-α were immediate and sustained. The amplitude of systolic [Ca(2+)]i was reduced by 31% and systolic shortening by 19%. Diastolic [Ca(2+)]i, myocyte length and relaxation rate were not affected, nor were the activity of the [Ca(2+)]i removal mechanisms. The reduction in systolic [Ca(2+)]i was associated with a 14% reduction in sarcoplasmic reticulum (SR) content and a 11% decrease in peak L-type Ca current (IC a-L). Ca influx was decreased by 7% associated with a more rapid IC a-L inactivation. These data show that at the level of the myocyte, TNF-α reduces SR Ca which underlies a reduction in systolic [Ca(2+)]i and thence shortening. Although these findings correlate well with aspects of systolic myocardial dysfunction seen in sepsis, in this model, acutely, TNF-α does not appear to provide a cellular mechanism for sepsis-related diastolic myocardial dysfunction

Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response.

The Frank-Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16-a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank-Starling response

Disulfide-activated protein kinase G I alpha regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response

British Heart Foundation, European Research Council (ERC Advanced award), Medical Research Council and the Department of Health via the NIHR cBRC award to Guy’s & St Thomas’ NHS Foundation Trust. Part supported by the NIHR University College London Hospitals Biomedical Research Centre

Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes

Spatio-temporal dynamics of intracellular calcium, [Ca2+]i, regulate the contractile function of cardiac muscle cells. Measuring [Ca2+]i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca2+]i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca2+ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils.We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca2+]i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca2+]i transient and of the simulated fluorescence intensity signal, F/F0, reached values similar to that found in the literature ([Ca2+]i 1 μM; F/F0 5.5). However, our model predicted the variation in [Ca2+]i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F0 signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca2+]i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structureinduced heterogeneities in [Ca2+]i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca2+]i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes

The effects of 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on calcium handling in rat ventricular myocytes

2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) is a reversible inhibitor of SERCA, potentially making it a useful tool to study the effects of SERCA inhibition. It has been used in a variety of cell types including ventricular myocytes (1). However, it is currently unknown if TBQ also has effects on other components of ventricular intracellular Ca handling. The aim of these experiments was therefore to characterise the effects of TBQ on Ca handling in the rat ventricular myocyte and assess its suitability as specific inhibitor of SERCA.Male Wistar rats were killed in accordance with the The Home Office Animal (Scientific Procedures) Act 1986 for enzymatic isolation of ventricular myocytes which were subsequently loaded with the Ca indicator Fluo-3AM to measure global intracellular Ca. Myocytes were voltage clamped via perforated patch and paced at 0.5 Hz to measure sarcolemmal Ca fluxes.TBQ produced a concentration dependent decrease of the rate of decay of systolic Ca, consistent with a decrease in SERCA activity. This was fully reversible at all concentrations tested. For most experiments we used 10 µM TBQ as this produced a large effect without completely abolishing the systolic Ca transient. 10 µM TBQ produced a 48 ± 5 and 54 ± 6 % decrease in the amplitude and the rate of decay of the systolic Ca transient respectively. However, at 10 µM, TBQ also produced a 23 ± 7 % decrease of the peak Ca current without altering the voltage dependence. SR Ca content was also reduced by 62 ± 4 %. Interestingly, at higher concentrations (100 µM), TBQ also activated an outward current that demonstrated a current-voltage relationship consistent with a potassium current. This outward current was sensitive to Glibenclamide. Thapsigargin (another inhibitor of SERCA) did not produce this outward current, suggesting its activation is TBQ dependent, rather than as a result of SERCA inhibition.These data show that in rat ventricular myocytes, TBQ can be used to reversibly inhibit SERCA. However, at concentrations that decrease SERCA activity, TBQ also decreases the Ca current, and, at higher concentrations, activates an outward current which appears to be an ATP dependent potassium current. We conclude that TBQ cannot be used as a specific inhibitor of SERCA in rat ventricular myocytes

Co-creation and empowerment: pathways to better student engagement

Progression and employability are at the forefront of current conversations in higher education, with much effort invested in developing complex interventions to improve graduate outcomes. Pedagogy research indicates that factors such as a sense of belonging, impostor syndrome and a meaningful connection to campus life are contributing factors to student success. Furthermore, student outcomes correlate closely with socio-economic status and demographic data. Across all disciplines, academic teams work tirelessly to “close the gap” created by social inequalities, but it remains to be determined where efforts are best placed. For instance, many universities offer access programmes to low-participating neighbourhoods, while other institutions place great effort on the BTEC and BAME attainment gaps. Here we share guidance on effective student engagement based on our anecdotal experience and propose co-creation with and empowerment of all students as a desirable alternative pathway to success

Direct measurements of SR free Ca reveal the mechanism underlying the transient effects of RyR potentiation under physiological conditions

AIMS: Most of the calcium that activates contraction is released from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). It is controversial whether activators of the RyR produce a maintained increase in the amplitude of the systolic Ca transient. We therefore aimed to examine the effects of activation of the RyR in large animals under conditions designed to be as physiological as possible while simultaneously measuring SR and cytoplasmic Ca. METHODS AND RESULTS: Experiments were performed on ventricular myocytes from canine and ovine hearts. Cytoplasmic Ca was measured with fluo-3 and SR Ca with mag-fura-2. Application of caffeine resulted in a brief increase in the amplitude of the systolic Ca transient accompanied by an increase of action potential duration. These effects disappeared with a rate constant of ∼3 s(-1). Similar effects were seen in cells taken from sheep in which heart failure had been induced by rapid pacing. The decrease of Ca transient amplitude was accompanied by a decrease of SR Ca content. During this phase, the maximum (end-diastolic) SR Ca content fell while the minimum systolic increased. CONCLUSIONS: This study shows that, under conditions designed to be as physiological as possible, potentiation of RyR opening has no maintained effect on the systolic Ca transient. This result makes it unlikely that potentiation of the RyR has a maintained role in positive inotropy

Enhancing employability through building graduate capital: a multi-faceted approach

The University of Salford is home to a diverse community of learners, a high proportion of whom are from underprivileged socioeconomic backgrounds and/or underrepresented communities and many are commuters. A significant proportion of the cohort gain university entry through level three qualifications other than A-levels. It is widely documented that students from such backgrounds often achieve lower graduate outcomes and struggle to secure graduate jobs.1 We present here a series of interventions aimed at enhancing graduate capital through building confidence and competence. Availing this timely acquisition of information and opportunities has promoted a value-added student experience against the backdrop of a strong sense of belonging and ownership. Many of the projects described here have been co-designed with students as partners and involve collaboration with wider teams within the university. Using the graduate capital model as a framework, we designed interventions to build social, cultural, identity and psychological capital (Tomlinson, 2017). Collectively, these forms of capital facilitate information awareness, development of a personal, reflective, proactive employability narrative and the development of confidence and professionalism. Furthermore, we encouraged students to take ownership of their own career management, an important step towards enhancing students’ employability (Bridgstock, 2009). Given our student demographics, offering such support is fundamentally important to student success, as capitalising on the university experience, social situations and networks can often be a challenge (Tholen et al, 2013)

The effect of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBQ) on intracellular Ca(2+) handling in rat ventricular myocytes

2,5-Di-(tert-butyl)-1,4-benzohydroquinone (TBQ) is a reversible inhibitor of SERCA, potentially making it a useful tool to study the effects of SERCA inhibition in cardiac cells. However, it is unknown if TBQ also has effects on other components of ventricular Ca handling. The aim of these experiments was to characterise the effects of TBQ on Ca handling in rat ventricular myocytes and assess its suitability as a specific inhibitor of SERCA. This was achieved by voltage clamp via perforated patch and [Ca(2+)]i measurement using Fluo-3 AM. TBQ produced a fully reversible, concentration dependent decrease in the rate of systolic Ca decay. 10μM TBQ decreased the amplitude of the systolic Ca transient by 48±5% and the rate of decay by 54±6%. SR Ca content was also reduced by 62±4%. However, 10μM TBQ also decreased the peak L-type Ca current by 23±7%. At higher concentrations (100μM), TBQ also activated an outward current with a current-voltage relationship consistent with a potassium current. This outward current was abolished by Glibenclamide (100μM). These data show that TBQ can be used to reversibly inhibit SERCA. However, at concentrations that decrease SERCA activity, TBQ also decreases the L-type Ca current and (at higher concentrations) activates an outward current which appears to be an ATP dependent potassium current. We conclude that TBQ cannot be used as a specific inhibitor of SERCA in rat ventricular myocytes