Helen K. Graham

Characterization of an Extensive Transverse Tubular Network in Sheep Atrial Myocytes and its Depletion in Heart Failure

Background—In ventricular myocytes, the majority of structures that couple excitation to the systolic rise of Ca2+ are located at the transverse tubular (t-tubule) membrane. In the failing ventricle, disorganization of t-tubules disrupts excitation contraction coupling. The t-tubule membrane is virtually absent in the atria of small mammals resulting in spatiotemporally distinct profiles of intracellular Ca2+ release on stimulation in atrial and ventricular cells. The aims of this study were to determine (i) whether atrial myocytes from a large mammal (sheep) possess t-tubules, (ii) whether these are functionally important, and (iii) whether they are disrupted in heart failure. Methods and Results—Sheep left atrial myocytes were stained with di-4-ANEPPS. Nearly all control cells had an extensive t-tubule network resulting in each voxel in the cell being nearer to a membrane (sarcolemma or t-tubule) than would otherwise be the case. T-tubules decrease the distance of 50% of voxels from a membrane from 3.35±0.15 to 0.88±0.04 &mgr;m. During depolarization, intracellular Ca2+ rises simultaneously at the cell periphery and center. In heart failure induced by rapid ventricular pacing, there was an almost complete loss of atrial t-tubules. The distance of 50% of voxels from a membrane increased to 2.04±0.08 &mgr;m, and there was a loss of early Ca2+ release from the cell center. Conclusion—Sheep atrial myocytes possess a substantial t-tubule network that synchronizes the systolic Ca2+ transient. In heart failure, this network is markedly disrupted. This may play an important role in changes of atrial function in heart failure.

Extracellular matrix profiles in the progression to heart failure. European Young Physiologists Symposium Keynote Lecture-Bratislava 2007.

The myocardial extracellular matrix (ECM), which preserves the geometry and integrity of the myocardium, is a dynamic structure whose component proteins are maintained by a finely controlled homeostatic balance between deposition and degradation. One of the key targets in cardiology is the elucidation of the molecular mechanisms which mediate pathological remodelling of this matrix causing the transition from compensatory hypertrophy to congestive decompensated heart failure. In response to injury or increased workload, cardiac remodelling including myocyte hypertrophy, develops as the heart attempts to compensate for increased wall stresses. Persistence of these stresses over extended time periods leads to disruption of ECM homeostasis resulting in irreversible maladaptive cardiac remodelling, ventricular dilatation and finally heart failure. ECM remodelling is regulated by the matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). Clinical studies and experimental models of cardiac disease states have reported alterations in the balance between the MMPs and TIMPs in the failing heart and crucially at intermediate time points in the progression to failure. This article reviews the recent clinical, genetic and experimental approaches employed to compare ECM, MMP and TIMP profiles in healthy, compensated and failing hearts and identifies common themes in the perturbation of ECM homeostasis in the transition to heart failure.

Tissue section AFM: In situ ultrastructural imaging of native biomolecules.

Conventional approaches for ultrastructural high-resolution imaging of biological specimens induce profound changes in bio-molecular structures. By combining tissue cryo-sectioning with non-destructive atomic force microscopy (AFM) imaging we have developed a methodology that may be applied by the non-specialist to both preserve and visualize bio-molecular structures (in particular extracellular matrix assemblies) in situ. This tissue section AFM technique is capable of: i) resolving nm–µm scale features of intra- and extracellular structures in tissue cryo-sections; ii) imaging the same tissue region before and after experimental interventions; iii) combining ultrastructural imaging with complimentary microscopical and micromechanical methods. Here, we employ this technique to: i) visualize the macro-molecular structures of unstained and unfixed fibrillar collagens (in skin, cartilage and intervertebral disc), elastic fibres (in aorta and lung), desmosomes (in nasal epithelium) and mitochondria (in heart); ii) quantify the ultrastructural effects of sequential collagenase digestion on a single elastic fibre; iii) correlate optical (auto fluorescent) with ultrastructural (AFM) images of aortic elastic lamellae.

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.

Nanoindentation of histological specimens: Mapping the elastic properties of soft tissues

Although alterations in the gross mechanical properties of dynamic and compliant tissues have a major impact on human health and morbidity, there are no well-established techniques to characterize the micromechanical properties of tissues such as blood vessels and lungs. We have used nanoindentation to spatially map the micromechanical properties of 5-mum-thick sections of ferret aorta and vena cava and to relate these mechanical properties to the histological distribution of fluorescent elastic fibers. To decouple the effect of the glass substrate on our analysis of the nanoindentation data, we have used the extended Oliver and Pharr method. The elastic modulus of the aorta decreased progressively from 35 MPa in the adventitial (outermost) layer to 8 MPa at the intimal (innermost) layer. In contrast, the vena cava was relatively stiff, with an elastic modulus >30 MPa in both the extracellular matrix-rich adventitial and intimal regions of the vessel. The central, highly cellularized, medial layer of the vena cava, however, had an invariant elastic modulus of ~20 MPa. In extracellular matrix-rich regions of the tissue, the elastic modulus, as determined by nanoindentation, was inversely correlated with elastic fiber density. Thus, we show it is possible to distinguish and spatially resolve differences in the micromechanical properties of large arteries and veins, which are related to the tissue microstructure.

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.

Localised micro-mechanical stiffening in the ageing aorta

Highlights ► Age-related tissue stiffening has a profound effect on human morbidity and mortality. ► Scanning acoustic microscopy can quantify the localised stiffness of discrete tissue components in situ. ► Age-related aortic stiffening is both localised to collagen fibril-rich regions and associated with collagen fibrosis. ► Age-related structural remodelling events can be directly linked with in situ mechanical changes using scanning acoustic microscopy.

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.

Comparison of in vivo and in silico methods used for prediction of tissue:plasma partition coefficients in rat

Objectives  To use methods from the literature to predict rat tissue:plasma partition coefficients (Kps) and volume of distribution values. Determine which model provides the most accurate predictions to increase confidence in the use of predicted pharmacokinetic parameters in physiologically based pharmacokinetic modelling.

Localized micro- and nano-scale remodelling in the diabetic aorta.

Graphical abstract