Jessica L. Caldwell

Dependence of Cardiac Transverse Tubules on the BAR Domain Protein Amphiphysin II (BIN-1)

Rationale: Transverse tubules (t-tubules) regulate cardiac excitation–contraction coupling and exhibit interchamber and interspecies differences in expression. In cardiac disease, t-tubule loss occurs and affects the systolic calcium transient. However, the mechanisms controlling t-tubule maintenance and whether these factors differ between species, cardiac chambers, and in a disease setting remain unclear. Objective: To determine the role of the Bin/Amphiphysin/Rvs domain protein amphiphysin II (AmpII) in regulating t-tubule maintenance and the systolic calcium transient. Methods and Results: T-tubule density was assessed by di-4-ANEPPS, FM4-64 or WGA staining using confocal microscopy. In rat, ferret, and sheep hearts t-tubule density and AmpII protein levels were lower in the atrium than in the ventricle. Heart failure (HF) was induced in sheep using right ventricular tachypacing and ferrets by ascending aortic coarctation. In both HF models, AmpII protein and t-tubule density were decreased in the ventricles. In the sheep, atrial t-tubules were also lost in HF and AmpII levels decreased. Conversely, junctophilin 2 levels did not show interchamber differences in the rat and ferret nor did they change in HF in the sheep or ferret. In addition, in rat atrial and sheep HF atrial cells where t-tubules were absent, junctophilin 2 had sarcomeric intracellular distribution. Small interfering RNA–induced knockdown of AmpII protein reduced t-tubule density, calcium transient amplitude, and the synchrony of the systolic calcium transient. Conclusions: AmpII is intricately involved in t-tubule maintenance. Reducing AmpII protein decreases t-tubule density, reduces the amplitude, and increases the heterogeneity of the systolic calcium transient.

Impaired β-adrenergic responsiveness accentuates dysfunctional excitation-contraction coupling in an ovine model of tachypacing-induced heart failure.

Non‐technical summary  Heart failure is where the heart is unable to pump sufficient blood in order to meet the requirements of the body. Symptoms of heart failure often first present during exercise. During exercise the blood levels of a hormone, noradrenaline, increase and activate receptors on the muscle cells of the heart known as β‐receptors causing the heart to contract more forcefully. We show that in heart failure the response to β‐receptor stimulation is reduced and this appears to be due to a failure of the β‐receptor to signal correctly to downstream targets inside the cell. However, by‐passing the β‐receptor and directly activating one of the downstream targets, an enzyme known as adenylyl cyclase, inside the cell restores the function of the muscle cells in failing hearts. These observations provide a number of potential targets for therapies to improve the function of the heart in patients with heart failure.

Calcium and Excitation-Contraction Coupling in the Heart

Cardiac contractility is regulated by changes in intracellular Ca concentration ([Ca2+]i). Normal function requires that [Ca2+]i be sufficiently high in systole and low in diastole. Much of the Ca needed for contraction comes from the sarcoplasmic reticulum and is released by the process of calcium-induced calcium release. The factors that regulate and fine-tune the initiation and termination of release are reviewed. The precise control of intracellular Ca cycling depends on the relationships between the various channels and pumps that are involved. We consider 2 aspects: (1) structural coupling: the transporters are organized within the dyad, linking the transverse tubule and sarcoplasmic reticulum and ensuring close proximity of Ca entry to sites of release. (2) Functional coupling: where the fluxes across all membranes must be balanced such that, in the steady state, Ca influx equals Ca efflux on every beat. The remainder of the review considers specific aspects of Ca signaling, including the role of Ca buffers, mitochondria, Ca leak, and regulation of diastolic [Ca2+]i.

Perturbed atrial calcium handling in an ovine model of heart failure: Potential roles for reductions in the L-type calcium current.

Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during β-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca2 + and the response to β-adrenergic (β-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca2 + concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca2 + transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca2 + removal (kSR, by 32%), L-type Ca2 + current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca2 + content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca2 + current (ICa-L) in control cells reproduced both the decrease in Ca2 + transient amplitude and increase of SR Ca2 + content observed in voltage-clamped HF cells. During β-AR stimulation Ca2 + transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca2 + content was highest in HF cells during β-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca2 + transient amplitude and increased SR Ca2 + content observed in voltage-clamped cells.

Sarcoplasmic Reticulum Ca-ATPase and Heart Failure 20 Years Later

This article reflects on the impact of a classic paper identifying the effects of loss of sarcoplasmic reticulum Ca-ATPase activity in heart failure. Understanding the effects of heart failure on contraction and Ca signaling in the heart has long been a priority. By the late 1980s, many studies on animal models had shown that heart failure resulted in a slowing of the decay of the systolic Ca transient1 because of a decrease in the expression of the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA).2 Work using human tissue found similar changes.3,4 In 1994, Hasenfuss et al5 published a now classic article in Circulation Research entitled “Relation between myocardial function and expression of sarcoplasmic reticulum Ca-ATPase in failing and non-failing human myocardium.” Previous work had shown that SERCA expression and activity were decreased in heart failure. Other work had shown that the increase of force seen on increasing the frequency of stimulation (the positive force–frequency curve) disappeared in heart failure. Hasenfuss et al5 showed that the degree of change in the force–frequency relationship correlated with the loss of SERCA. Hearts with low levels of SERCA developed maximum force at lower frequencies than those with higher levels. In addition, and suggested as causative of the reduced force–frequency responses, the failing hearts had reduced SERCA-mediated Ca uptake. The work in this article has influenced 2 areas of research: changes of SR function in heart failure and restoration of SERCA as a therapeutic strategy. We consider these in turn. Twenty years later, the role of changes of SERCA expression in heart failure is well-established. The majority of studies of heart failure in either humans or experimental animals show that SERCA activity is decreased in heart failure. This decrease of SERCA has 2 immediate effects on Ca signaling. First, it …

Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes

Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart. Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur. We report alterations to the atrial systolic Ca2+ transient that have implications for the function of the atrial in the elderly. We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca2+ in the old atria. The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops.

Phosphodiesterase-5 inhibitors and the heart: compound cardioprotection?

Novel cardioprotective agents are needed in both heart failure (HF) and myocardial infarction. Increasing evidence from cellular studies and animal models indicate protective effects of phosphodiesterase-5 (PDE5) inhibitors, drugs usually reserved as treatments of erectile dysfunction and pulmonary arterial hypertension. PDE5 inhibitors have been shown to improve contractile function in systolic HF, regress left ventricular hypertrophy, reduce myocardial infarct size and suppress ischaemia-induced ventricular arrhythmias. Underpinning these actions are complex but increasingly understood cellular mechanisms involving the cyclic GMP activation of protein kinase-G in both cardiac myocytes and the vasculature. In clinical trials, PDE5 inhibitors improve symptoms and ventricular function in systolic HF, and accumulating epidemiological data indicate a reduction in cardiovascular events and mortality in PDE5 inhibitor users at high cardiovascular risk. Here, we focus on the translation of underpinning basic science to clinical studies and report that PDE5 inhibitors act through a number of cardioprotective mechanisms, including a direct myocardial action independent of the vasculature. We conclude that future clinical trials should be designed with these mechanisms in mind to identify patient subsets that derive greatest treatment benefit from these novel cardioprotective agents.

PDE5 inhibition improves symptom-free survival and restores transverse tubule loss and catecholamine responsiveness in heart failure

Heart failure is characterized by poor survival, a loss of catecholamine reserve and cellular structural remodeling in the form of disorganization and loss of the transverse tubule network. Indeed, survival rates for heart failure are worse than many common cancers and have not improved over time. Tadalafil is a clinically relevant drug that blocks phosphodiesterase 5 with high specificity and is used to treat erectile dysfunction. Using a sheep model of advanced heart failure, we show that tadalafil treatment results in a marked improvement in symptom-free survival, reverses transverse tubule loss and restore cardiac myocyte systolic calcium transient amplitude and the hearts chronotropic and contractile response to catecholamines. These effects are independent of changes in myocardial cGMP content and are associated with upregulation of both monomeric and dimerized forms of protein kinase G and of the cGMP hydrolyzing phosphodiesterase 2 and 3. We propose that the molecular switch for the loss of transverse tubules in heart failure and their restoration following tadalafil treatment involves the BAR domain protein Amphiphysin II (BIN1) and the restoration of catecholamine sensitivity is through reductions in G-protein receptor kinase 2, protein phosphatase 1 and protein phosphatase 2A abundance following phosphodiesterase 5 inhibition.

Calcium in the pathophysiology of atrial fibrillation and heart failure

Atrial fibrillation (AF) is commonly associated with heart failure. A bidirectional relationship exists between the two—AF exacerbates heart failure causing a significant increase in heart failure symptoms, admissions to hospital and cardiovascular death, while pathological remodeling of the atria as a result of heart failure increases the risk of AF. A comprehensive understanding of the pathophysiology of AF is essential if we are to break this vicious circle. In this review, the latest evidence will be presented showing a fundamental role for calcium in both the induction and maintenance of AF. After outlining atrial electrophysiology and calcium handling, the role of calcium-dependent afterdepolarizations and atrial repolarization alternans in triggering AF will be considered. The atrial response to rapid stimulation will be discussed, including the short-term protection from calcium overload in the form of calcium signaling silencing and the eventual progression to diastolic calcium leak causing afterdepolarizations and the development of an electrical substrate that perpetuates AF. The role of calcium in the bidirectional relationship between heart failure and AF will then be covered. The effects of heart failure on atrial calcium handling that promote AF will be reviewed, including effects on both atrial myocytes and the pulmonary veins, before the aspects of AF which exacerbate heart failure are discussed. Finally, the limitations of human and animal studies will be explored allowing contextualization of what are sometimes discordant results.

171 Amphiphysin ii (bin1) driven transverse tubule formation in cardiac muscle

Transverse (t)-tubules are vital for maintaining normal contractility of the heart through the tight regulation of excitation coupling. In cardiac disease, such as heart failure, t-tubule loss is closely associated with decreased synchrony of calcium release from the sarcoplasmic reticulum, resulting in impaired contractility. Thus, determining the mechanisms that control t-tubule formation is essential for understanding cardiac disease. Evidence suggests that the protein Amphiphysin II (AmpII) controls t-tubule formation in cardiac muscle and thus, may play a vital role in calcium regulation. Several studies, including our own, have shown that gene silencing of AmpII causes t-tubule loss in both skeletal and cardiac muscle. Furthermore, in non-muscle cells that usually lack t-tubules, expression of some variants of AmpII led to tubule formation. We therefore aimed to extend these observations and determine if AmpII is sufficient to drive t-tubule formation in the heart. Neonate rat ventricular myocytes (NRVMs) were isolated from 2 day old rats and maintained in culture. Vectors encoding isoforms 5, 8 and 9 of the AmpII gene (Bin1) with a C-terminal mKate2 fluorescent protein tag were transiently expressed in NRVMs using FuGENE 6 lipofection. A vector containing the mKate2 fluorescent tag only was used as negative control. After 48 hours, over-expression of Bin1 was confirmed at both the mRNA and protein level. Tubule formation was assessed using the membrane dye FM-464 and confocal microscopy. Of cells successfully transfected with Bin1, 95% had developed tubule structures. Conversely, tubules were absent in cells only expressing the fluorescent tag (p<0.001). Furthermore, Bin1 isoform 8 expression led to formation of more tubule structures when compared to isoform 5 and 9 (p<0.05). To determine if Bin1 driven tubules are functional, transfected cells were loaded with the Ca2+ indicator Fluo-8 AM and field stimulated. When compared with untransfected myocytes, expression of Bin1 isoforms 5, 8 and 9 increased the amplitude of the systolic calcium transient (p<0.05). Furthermore, transfection with Bin1 isoforms 5 and 9 led to faster rise and decay of the systolic calcium transient (p<0.05). Transfection with the control vector only had no effect on the calcium handling when compared with untransfected cells. Over-expression of Bin1 isoforms 5, 8 and 9 led to the formation of tubular structures in NRVMs. Whilst Bin1 isoforms 8 appears to play more of a role in tubule formation in NRVMs, these data suggest that other Bin1 isoforms (5 and 9) may enhance calcium kinetics. These data therefore suggest that Bin1 plays a vital role in tubule formation and development in cardiac myocytes. Given the importance of t-tubules to normal excitation contraction coupling and their perturbation in heart failure we therefore suggest that Bin1 might be a novel therapeutic target.