Elspeth B. Elliott

Excessive Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase Expression Causes Increased Sarcoplasmic Reticulum Ca2+ Uptake but Decreases Myocyte Shortening

Background—Increasing sarcoplasmic/endoplasmic reticulum (SR) Ca2+-ATPase (SERCA) uptake activity is a promising therapeutic approach for heart failure. We investigated the effects of different levels of SERCA1a expression on contractility and Ca2+ cycling. We tested whether increased SERCA1a expression levels enhance myocyte contractility in a gene-dose–dependent manner. Methods and Results—Rabbit isolated cardiomyocytes were transfected at different multiplicities of infection (MOIs) with adenoviruses encoding SERCA1a (or &bgr;-galactosidase as control). Myocyte relaxation half-time was decreased by 10% (P=0.052) at SERCA1a MOI 10 and by 28% at MOI 50 (P<0.05). Myocyte fractional shortening was increased by 12% at MOI 10 (P<0.05) but surprisingly decreased at MOI 50 (−22%, P<0.05) versus control. SR Ca2+ uptake (in permeabilized myocytes) demonstrated a gene-dose–dependent decrease in Km by 29% and 46% and an increase in Vmax by 37% and 72% at MOI 10 and MOI 50, respectively (all P<0.05 versus control). Ca2+ transient amplitude was increased in Ad-SERCA1a–infected myocytes at MOI 10 (by 121%, P<0.05), but at MOI 50, the Ca2+ transient amplitude was not significantly changed. Caffeine-induced Ca2+ transients indicated significantly increased SR Ca2+ content in Ad-SERCA1a–infected cells, by 72% at MOI 10 and by 87% at MOI 50. Mathematical simulations demonstrate that the functional increase in SR Ca2+-ATPase uptake activity at MOI 50 (and increased cytosolic Ca2+ buffering) is sufficient to curtail the Ca2+ transient amplitude and explain the reduced contraction. Conclusions—Moderate SERCA1a gene transfer and expression improve contractility and Ca2+ cycling. However, higher SERCA1a expression levels can impair myocyte shortening because of higher SERCA activity and Ca2+ buffering.

Two candidates at the heart of dysfunction: the ryanodine receptor and calcium/calmodulin protein kinase II as potential targets for therapeutic intervention-An in vivo perspective

At the start of a new decade (2011), heart failure and sudden cardiac death are still leading causes of mortality worldwide. There is a very obvious need for improved treatment strategies. Research over the past decade has focused on understanding and realising the therapeutic potential of molecular mechanisms that underlie the pathophysiology of cardiac dysfunction. There is now recognition that cell- and gene-based therapies could prove beneficial if aimed at the appropriate molecular targets. Two cardiac proteins that have received considerable attention over the last decade, have been identified as possible therapeutic targets. The cardiac sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor) and calcium/calmodulin dependent kinase II (CaMKIIδ) can act independently and in partnership, to regulate cardiac Ca(2+) handling. CaMKIIδ, by the very nature of its core function as a kinase, also modulates cardiac function globally, promoting effects on gene transcription and modulating inflammatory and proliferative responses, all events that are associated with both the functional and dysfunctional heart. In vivo approaches using genetic and pharmacologic strategies have revealed the prominent role of both proteins in cardiac dysfunction. More excitingly, they have also shown the potential for cardioprotection that modulation at the level of each protein can have. Translating these effects to the human heart is in its infancy. Whether intervention at these targets could result in clinical application is unknown at present, however current in vivo research has proved invaluable in revealing the potential that targeting of RyR and CaMKIIδ could have in limiting cardiac dysfunction.

Na+/Ca2+ Exchanger Expression and Function in a Rabbit Model of Myocardial Infarction

Introduction: In general, sarcolemmal Na+/Ca2+ exchanger (NCX) protein and activity is increased in hearts with ventricular dysfunction. However, in a subset of studies, reduced activity of NCX has been reported. Left ventricular dysfunction (LVD) was induced in the rabbit eight weeks after an apical myocardial infarction.

Trypanosoma brucei cathepsin-L increases arrhythmogenic sarcoplasmic reticulum-mediated calcium release in rat cardiomyocytes

Aims African trypanosomiasis, caused by Trypanosoma brucei species, leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, the cardiac pathogenesis remains unknown. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter cardiac function independent of a systemic inflammatory/immune response. Methods and results Using confocal imaging, T. brucei and T. brucei culture media (supernatant) caused an increased frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves) in isolated adult rat ventricular cardiomyocytes. Studies utilising inhibitors, recombinant protein and RNAi all demonstrated that this altered SR function was due to T. brucei cathepsin-L (TbCatL). Separate experiments revealed that TbCatL induced a 10–15% increase of SERCA activity but reduced SR Ca2+ content, suggesting a concomitant increased SR-mediated Ca2+ leak. This conclusion was supported by data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide, highlighting a role for CaMKII in the TbCatL action on SR function. Isolated Langendorff perfused whole heart experiments confirmed that supernatant caused an increased number of arrhythmic events. Conclusion These data demonstrate for the first time that African trypanosomes alter cardiac function independent of a systemic immune response, via a mechanism involving extracellular cathepsin-L-mediated changes in SR function.

Isolated Rabbit Working Heart Function During Progressive Inhibition of Myocardial SERCA Activity

Rationale: The extent to which sarcoplasmic reticulum Ca2+ATPase (SERCA) activity alone determines left ventricular (LV) pump function is unknown. Objective: To correlate SERCA activity with hemodynamic function of rabbit LV during thapsigargin perfusion. Methods and Results: Isolated rabbit hearts were perfused in working heart configuration, and LV pump function was assessed using a pressure-volume catheter. Rapid and complete (>95%) inhibition of SERCA was associated with a moderate decrease in cardiac function (to 70%–85% of control). Further decrease in cardiac function to 50%–75% of control occurred over the next ≈30 minutes despite no detectable further inhibition of SERCA activity. Analysis of the 20 seconds prior to pump failure revealed a rapid decrease in end diastolic volume. Intermediate levels of SERCA function (≈50% of control) had only minor hemodynamic effects. Parallel experiments in field-stimulated isolated ventricular cardiomyocytes monitored intracellular Ca2+ and cell shortening. On perfusion with thapsigargin, Ca2+ transient amplitude and cell shortening fell to ≈70% of control followed by increased diastolic Ca2+ concentration and diastolic cell shortening to achieve a new steady state. Conclusions: The relationship between SERCA activity and LV function in the rabbit is highly nonlinear. In the short term, only moderate effects on LV pump function were observed despite almost complete (>95%) reduction in SERCA activity. The terminal decline of function was associated with sudden sustained increase in diastolic tone comparable to the sustained contraction observed in isolated cardiomyocytes. Secondary increases of intracellular Ca2+ and Na+ following complete SERCA inhibition eventually limit contractile function and precipitate LV pump failure.

The effect of K201 on isolated working rabbit heart mechanical function during pharmacologically induced Ca2+ overload

Reduced cardiac contractility has been associated with disrupted myocardial Ca2+ signalling. The 1,4 benzothiazepine K201 (JTV‐519) acts on several Ca2+ handling proteins and improves cardiac contractility in vivo in a variety of animal models of myocardial dysfunction. However, it is unclear whether this improvement depends on the systemic effects of K201 or if K201 reverses the effects of Ca2+ dysregulation, regardless of the cause.

Runx1 Deficiency Protects Against Adverse Cardiac Remodeling After Myocardial Infarction

Background: Myocardial infarction (MI) is a leading cause of heart failure and death worldwide. Preservation of contractile function and protection against adverse changes in ventricular architecture (cardiac remodeling) are key factors to limiting progression of this condition to heart failure. Consequently, new therapeutic targets are urgently required to achieve this aim. Expression of the Runx1 transcription factor is increased in adult cardiomyocytes after MI; however, the functional role of Runx1 in the heart is unknown. Methods: To address this question, we have generated a novel tamoxifen-inducible cardiomyocyte-specific Runx1-deficient mouse. Mice were subjected to MI by means of coronary artery ligation. Cardiac remodeling and contractile function were assessed extensively at the whole-heart, cardiomyocyte, and molecular levels. Results: Runx1-deficient mice were protected against adverse cardiac remodeling after MI, maintaining ventricular wall thickness and contractile function. Furthermore, these mice lacked eccentric hypertrophy, and their cardiomyocytes exhibited markedly improved calcium handling. At the mechanistic level, these effects were achieved through increased phosphorylation of phospholamban by protein kinase A and relief of sarco/endoplasmic reticulum Ca2+-ATPase inhibition. Enhanced sarco/endoplasmic reticulum Ca2+-ATPase activity in Runx1-deficient mice increased sarcoplasmic reticulum calcium content and sarcoplasmic reticulum–mediated calcium release, preserving cardiomyocyte contraction after MI. Conclusions: Our data identified Runx1 as a novel therapeutic target with translational potential to counteract the effects of adverse cardiac remodeling, thereby improving survival and quality of life among patients with MI.

African Trypanosomes Increase Calcium Wave Frequency in Isolated Adult Rat Cardiomyocytes via Secretion of Cathepsin L

African trypanosomes are blood-borne extracellular parasites which have recently been linked to cardiac dysfunction in ∼70% of sleeping sickness patients. Although this may result from an indirect effect of the parasite (e.g. myocarditis), a direct effect of the parasite on the heart has not been investigated. Adult rat cardiomyocytes were incubated with trypanosome growth media containing Trypanosoma brucei Lister427 (30min). A population assay assessed the percentage of cells demonstrating Ca2+ waves within a 1min period. Incubation with live trypanosomes led to a significant increase in the percentage of cells demonstrating Ca2+ waves (54.8±2.8% vs. 79.2±5.1%; media vs. live trypanosomes, P 0.05), whereas CA074 (specific inhibitor of cathepsin-B) had no effect on Ca2+ wave frequency. These data suggest trypanosomes interact with cardiomyocytes leading to increased Ca2+ wave production via cathepsin-L. This may contribute to the cardiac abnormalities observed in patients with trypanosomiasis.