Priyanthi Dias

Mechanical unloading reverses transverse tubule remodelling and normalizes local Ca2+-induced Ca2+release in a rodent model of heart failure

Ca2+‐induced Ca2+ release (CICR) is critical for contraction in cardiomyocytes. The transverse (t)‐tubule system guarantees the proximity of the triggers for Ca2+ release [L‐type Ca2+ channel, dihydropyridine receptors (DHPRs)] and the sarcoplasmic reticulum Ca2+ release channels [ryanodine receptors (RyRs)]. Transverse tubule disruption occurs early in heart failure (HF). Clinical studies of left ventricular assist devices in HF indicate that mechanical unloading induces reverse remodelling. We hypothesize that unloading of failing hearts normalizes t‐tubule structure and improves CICR.

Functional crosstalk between cardiac fibroblasts and adult cardiomyocytes by soluble mediators

AIMS Crosstalk between cardiomyocytes and fibroblasts in physiological conditions and during disease remains poorly defined. Previous studies have shown that fibroblasts and myocytes interact via paracrine communication, but several experimental confounding factors, including the use of immature myocytes and the induction of alpha-smooth muscle actin (α-SMA) expression in fibroblasts by prolonged culture, have hindered our understanding of this phenomenon. We hypothesize that fibroblasts and myofibroblasts differentially affect cardiomyocytes viability, volume, and Ca(2+) handling via soluble mediators. More specifically here: (i) we compare the effects of freshly isolated fibroblasts and cultured fibroblasts from normal rat hearts on adult cardiomyocytes; (ii) we compare the effects of (freshly isolated) normal fibroblasts and myofibroblasts from pressure-overloaded hearts; and (iii) we study the contribution of TGF-β and the importance of the crosstalk between the two cell types. METHODS AND RESULTS We used co-culture methods and conditioned medium to investigate paracrine interaction between fibroblasts and cardiomyocytes. All fibroblast types reduce cardiomyocyte viability and increase cardiomyocyte volume but α-SMA-negative fibroblasts increase cardiomyocyte Ca(2+) transient amplitude, whereas cultured fibroblasts and myofibroblasts from pressure-overloaded hearts decrease Ca(2+) transient amplitude. In turn, cardiomyocytes release soluble mediators that affect fibroblast proliferation. Using SB431542 to block TGF-β type 1 receptors, we determined that TGF-β directly causes cardiomyocyte hypertrophy and participates in bi-directional regulatory signalling between fibroblasts and cardiomyocytes. CONCLUSIONS Fibroblasts have different roles during physiology and disease in regulating myocardial function via soluble mediators. A crosstalk between fibroblasts and cardiomyocytes, controlled by TGF-β, is crucial in this interaction.

Characterisation of Connexin Expression and Electrophysiological Properties in Stable Clones of the HL-1 Myocyte Cell Line

Abstract The HL-1 atrial line contains cells blocked at various developmental stages. To obtain homogeneous sub-clones and correlate changes in gene expression with functional alterations, individual clones were obtained and characterised for parameters involved in conduction and excitation-contraction coupling. Northern blots for mRNAs coding for connexins 40, 43 and 45 and calcium handling proteins (sodium/calcium exchanger, L- and T-type calcium channels, ryanodine receptor 2 and sarco-endoplasmic reticulum calcium ATPase 2) were performed. Connexin expression was further characterised by western blots and immunofluorescence. Inward currents were characterised by voltage clamp and conduction velocities measured using microelectrode arrays. The HL-1 clones had similar sodium and calcium inward currents with the exception of clone 2 which had a significantly smaller calcium current density. All the clones displayed homogenous propagation of electrical activity across the monolayer correlating with the levels of connexin expression. Conduction velocities were also more sensitive to inhibition of junctional coupling by carbenoxolone (∼80%) compared to inhibition of the sodium current by lidocaine (∼20%). Electrical coupling by gap junctions was the major determinant of conduction velocities in HL-1 cell lines. In summary we have isolated homogenous and stable HL-1 clones that display characteristics distinct from the heterogeneous properties of the original cell line.

Influence of ivabradine on reverse remodelling during mechanical unloading

AIMS Ivabradine (Iva) has shown beneficial structural and functional effects in clinical and experimental heart failure (HF), but its action in combination with mechanical unloading (MU), such as during treatment with left ventricular assist devices (LVAD), is unknown. The aim of this study was to investigate the effects of Iva during MU, in a rodent model of HF. METHODS AND RESULTS We studied the chronic effects (4 weeks) of Iva (10 mg/kg/day) alone and in combination with MU [induced by heterotopic abdominal heart transplantation (HATx)] on whole-heart and cellular structure, function, and excitation-contraction (E-C) coupling in a rodent (rat) model of HF, 12 weeks post-left coronary artery (LCA) ligation. Effects of Iva were compared with those of β-blockade using metoprolol [(Met), 250 mg/kg/day]. Iva, but not Met, reversed myocardial fibrosis, alone and in combination with MU. MU-induced restoration of deranged E-C coupling was enhanced by Iva to a greater extent than Met: both Iva and Met enhanced the recovery of the Ca(2+) transient amplitude and the sarcoplasmic reticulum (SR) Ca(2+) content, but Iva alone maintained MU-induced normalization of L-type Ca(2+) current and t-tubule abnormalities. Met prevented MU-induced reduction in the myocardial size (myocardial atrophy); Iva had no effect on this parameter. CONCLUSION Iva shows beneficial structural and E-C coupling effects during MU: Iva reverses myocardial fibrosis and enhances the restoration of deranged E-C coupling, displaying more beneficial effects than that of Met. These results suggest that Iva may prove effective in enhancing functional recovery in heart failure patients receiving LVAD therapy.

Hyperpolarization‐Activated Cyclic Nucleotide‐Gated Channels and Ventricular Arrhythmias in Heart Failure: A Novel Target for Therapy?

Arrhythmias are a common and often fatal complication of heart failure accounting for 50% to 70% of cardiac deaths due to tachyarrhythmic mechanisms.[1][1] In the failing heart, disturbances in cardiac rhythm are the likely result of cardiac remodeling consisting of structural and/or

Mechanical unloading reverses transverse tubule remodelling and normalises local calcium-induced calcium release in a rodent model of heart failure

Abstract Calcium-induced calcium release (CICR) is crucial for contraction in cardiomyocytes. The transverse (t)-tubule system guarantees the proximity of the triggers for calcium release (L-type calcium channel, dihydropyridine receptors) and the sarcoplasmic reticulum calcium-release channels (ryanodine receptors). Transverse tubule disruption occurs early in heart failure. Clinical studies of left ventricular assist devices in heart failure indicate that mechanical unloading induces reverse remodelling. We hypothesise that unloading of failing hearts normalises t-tubule structure and improves CICR. Heart failure was induced in Lewis rats by left coronary artery ligation for 12 weeks; sham-operated animals were used as controls. Failing hearts were mechanically unloaded for 4 weeks by heterotopic abdominal heart transplantation (HF-UN). Heart failure reduced the t-tubule density as measured by di-8-ANEPPS staining in isolated left ventricular myocytes, and this was reversed by unloading. The deterioration in the regularity of the t-tubule system in heart failure was also reversed in HF-UN. Scanning ion conductance microscopy showed the reappearance of normal surface striations in HF-UN. Electron microscopy revealed recovery of normal t-tubule microarchitecture in HF-UN. L-type calcium current density, measured with whole-cell patch clamping, was reduced in heart failure but unaffected by unloading. The variance of the time-to-peak of the calcium transient, an index of CICR dyssynchrony, was increased in heart failure and normalised by unloading. The increased calcium spark frequency observed in heart failure was reduced in HF-UN. These results could be explained by the recoupling of orphaned ryanodine receptors in heart failure, as indicated by immunofluorescence. Our data show that mechanical unloading of the failing heart reverses the pathological remodelling of the t-tubule system and improves CICR. Funding British Heart Foundation.

9 Ivabradine Alters Fibroblast Number and Transforming Growth Factor beta 1 Expression in Heart Failure

Cardiac fibrosis is associated with disruptions of the myocardial architecture through changes in volume, composition and organisation of the extracellular matrix. Ivabradine (IVA), a selective inhibitor of the pacemaker current, has shown to have beneficial effects on pathological remodelling. As fibroblasts are the predominant mediators of fibrosis we investigated whether IVA has direct effects on fibroblasts. Heart failure (HF) was induced by permanent coronary artery ligation in rats; sham-operated animals (S) were used as controls. After 12 weeks, HF animals were treated either with IVA or saline (HF-S) for a further 4 weeks. Discoidin domain-containing receptor-2 (DDR-2) and α-smooth muscle actin (α-SMA) were measured as markers of fibroblasts and activated fibroblasts respectively. IVA reduced the DDR-2 (in arbitrary units (a.u.), S: 0.33 ± 0.03, n = 4; HF-S: 0.77 ± 0.06, n = 5; HF-IVA: 0.57 ± 0.02, n = 5; p < 0.05) and α-SMA (S: 0.50 ± 0.13, n = 5; HF-S: 1.00 ± 0.13, n = 6; HF-IVA: 0.44 ± 0.08, n = 4; p < 0.05) expression observed in HF to sham levels. Chronic effects (4 weeks) of IVA (30 nM and 1 uM) were assessed on left ventricular fibroblasts isolated from a patient with dilated cardiomyopathy investigating α-SMA expression. IVA decreased α-SMA expression in fibroblasts (control: 535 ± 41; IVA 30 nM: 363 ± 20; IVA 1 uM: 181 ± 36; n = 4; p < 0.05). Expression of TGF-b1 (S: 0.26 ± 0.03, n = 4; HF-S: 0.49 ± 0.02, n = 5; HF-IVA: 0.34 ± 0.01, n = 4; p < 0.05) and SMAD-2 (S: 0.23 ± 0.01, n = 4; HF-S: 0.40 ± 0.04, n = 5; HF-IVA: 0.25 ± 0.03, n = 5; p < 0.05) was up-regulated during HF but IVA normalised this to sham levels. Our results on reduced TGF-b1 and α-SMA expression highlight a beneficial role of IVA in the reparative process of the failing heart.