David J. Greensmith

Age-related divergent remodeling of the cardiac extracellular matrix in heart failure: collagen accumulation in the young and loss in the aged

The incidence of heart failure (HF) increases with age. This study sought to determine whether aging exacerbates structural and functional remodeling of the myocardium in HF. HF was induced in young (~18 months) and aged sheep (>8 years) by right ventricular tachypacing. In non-paced animals, aging was associated with increased left ventricular (LV) end diastolic internal dimensions (EDID, P<0.001), reduced fractional shortening (P<0.01) and an increase in myocardial collagen content (P<0.01). HF increased EDID and reduced fractional shortening in both young and aged animals, although these changes were more pronounced in the aged (P<0.05). Age-associated differences in cardiac extracellular matrix (ECM) remodeling occurred in HF with collagen accumulation in young HF (P<0.001) and depletion in aged HF (P<0.05). MMP-2 activity increased in the aged control and young HF groups (P<0.05). Reduced tissue inhibitor of metalloproteinase (TIMP) expression (TIMPs 3 and 4, P<0.05) was present only in the aged HF group. Secreted protein acidic and rich in cysteine (SPARC) was increased in aged hearts compared to young controls (P<0.05) while serum procollagen type I C-pro peptide (PICP) was increased in both young failing (P<0.05) and aged failing (P<0.01) animals. In conclusion, collagen content of the cardiac ECM changes in both aging and HF although; whether collagen accumulation or depletion occurs depends on age. Changes in TIMP expression in aged failing hearts alongside augmented collagen synthesis in HF provide a potential mechanism for the age-dependent ECM remodeling. Aging should therefore be considered an important factor when elucidating cardiac disease mechanisms.

Diastolic Spontaneous Calcium Release From the Sarcoplasmic Reticulum Increases Beat-to-Beat Variability of Repolarization in Canine Ventricular Myocytes After β-Adrenergic Stimulation

Rationale: Spontaneous Ca2+ release (SCR) from the sarcoplasmic reticulum can cause delayed afterdepolarizations and triggered activity, contributing to arrhythmogenesis during &bgr;-adrenergic stimulation. Excessive beat-to-beat variability of repolarization duration (BVR) is a proarrhythmic marker. Previous research has shown that BVR is increased during intense &bgr;-adrenergic stimulation, leading to SCR. Objective: We aimed to determine ionic mechanisms controlling BVR under these conditions. Methods and Results: Membrane potentials and cell shortening or Ca2+ transients were recorded from isolated canine left ventricular myocytes in the presence of isoproterenol. Action-potential (AP) durations after delayed afterdepolarizations were significantly prolonged. Addition of slowly activating delayed rectifier K+ current (IKs) blockade led to further AP prolongation after SCR, and this strongly correlated with exaggerated BVR. Suppressing SCR via inhibition of ryanodine receptors, Ca2+/calmodulin-dependent protein kinase II inhibition, or by using Mg2+ or flecainide eliminated delayed afterdepolarizations and decreased BVR independent of effects on AP duration. Computational analyses and voltage-clamp experiments measuring L-type Ca2+ current (ICaL) with and without previous SCR indicated that ICaL was increased during Ca2+-induced Ca2+ release after SCR, and this contributes to AP prolongation. Prolongation of QT, Tpeak-Tend intervals, and left ventricular monophasic AP duration of beats after aftercontractions occurred before torsades de pointes in an in vivo dog model of drug-induced long-QT1 syndrome. Conclusions: SCR contributes to increased BVR by interspersed prolongation of AP duration, which is exacerbated during IKs blockade. Attenuation of Ca2+-induced Ca2+ release by SCR underlies AP prolongation via increased ICaL. These data provide novel insights into arrhythmogenic mechanisms during &bgr;-adrenergic stimulation besides triggered activity and illustrate the importance of IKs function in preventing excessive BVR.

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.

Longstanding complex regional pain syndrome is associated with activating autoantibodies against alpha-1a adrenoceptors.

&NA; Serum immunoglobulin G from 18 patients with longstanding complex regional pain syndrome (CRPS) and 57 controls were examined for antiautonomic autoantibodies. CRPS preparations often contained &agr;‐1a adrenoceptor‐activating autoantibodies. &NA; Complex regional pain syndrome (CRPS) is a limb‐confined posttraumatic pain syndrome with sympathetic features. The cause is unknown, but the results of a randomized crossover trial on low‐dose intravenous immunoglobulins (IVIG) treatment point to a possible autoimmune mechanism. We tested purified serum immunoglobulin G (IgG) from patients with longstanding CRPS for evidence of antibodies interacting with autonomic receptors on adult primary cardiomyocytes, comparing with control IgG from healthy and diseased controls, and related the results to the clinical response to treatment with low‐dose IVIG. We simultaneously recorded both single‐cell contractions and intracellular calcium handling in an electrical field. Ten of 18 CRPS preparations and only 1/57 control preparations (P < 0.0001) increased the sensitivity of the myocytes to the electric field, and this effect was abrogated by preincubation with &agr;‐1a receptor blockers. By contrast, effects on baseline calcium were blocked by preincubation with atropine. Interestingly, serum‐IgG preparations from all 4 CRPS patients who had responded to low‐dose IVIG with meaningful pain relief were effective in these assays, although 4/8 of the nonresponders were also active. To see if there were antibodies to the &agr;‐1a receptor, CRPS‐IgG was applied to &agr;‐1a receptor‐transfected rat‐1 fibroblast cells. The CRPS serum IgG induced calcium flux, and fluorescence‐activated cell sorting showed that there was serum IgG binding to the cells. The results suggest that patients with longstanding CRPS have serum antibodies to &agr;‐1a receptors, and that measurement of these antibodies may be useful in the diagnosis and management of the patients.

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.

The effects of hydrogen peroxide on intracellular calcium handling and contractility in the rat ventricular myocyte.

Elevations in reactive oxygen species are implicated in many disease states and cause systolic and diastolic myocardial dysfunction. To understand the underlying cellular dysfunction, we characterised the effects of H₂O₂ on [Ca(2+)](i) handling and contractility in the rat ventricular myocyte. This was achieved using patch clamping, [Ca(2+)](i) measurement using Fluo-3, video edge detection and confocal microscopy. All experiments were performed at 37°C. 200 μM H₂O₂ resulted in a 44% decrease in the [Ca(2+)](i) transient amplitude, a 30% increase in diastolic [Ca(2+)](i) and an 18% decrease in the rate of systolic Ca(2+) removal. This was associated with a 61% reduction in systolic shortening, a contracture of 3 μm and a 42% increase in relaxation time respectively. The decrease in the [Ca(2+)](i) transient amplitude could be explained by a 27% decrease in SR Ca(2+) content. This, in turn results from a 22% decrease of SERCA activity. The decreased SR Ca(2+) content also provides a mechanism for a reduction in [Ca(2+)](i) spark frequency with no evidence for a Ca(2+) independent modification of ryanodine receptor open probability. We conclude that decreased SERCA activity is the major factor responsible for the changes of the systolic [Ca(2+)](i) transient.

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.

Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak

Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay. In the presence of β‐adrenergic stimulation, RyR‐mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase. Two forms of Ca leak have been studied, Ca‐sensitising (induced by caffeine) and non‐sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient. Only Ca‐sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine. Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient.

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.

Ca analysis

Here I present an Excel based program for the analysis of intracellular Ca transients recorded using fluorescent indicators. The program can perform all the necessary steps which convert recorded raw voltage changes into meaningful physiological information. The program performs two fundamental processes. (1) It can prepare the raw signal by several methods. (2) It can then be used to analyze the prepared data to provide information such as absolute intracellular Ca levels. Also, the rates of change of Ca can be measured using multiple, simultaneous regression analysis. I demonstrate that this program performs equally well as commercially available software, but has numerous advantages, namely creating a simplified, self-contained analysis workflow.