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Introduction: Studies on isolated cardiomyocytes and ventricular wedges have shown contradicting data on the presence of transmural repolarization gradients in the left ventricle (LV). Here, we investigate electrophysiological heterogeneities of the canine LV free wall, using myocardial slices. Methods: Vibratome- cut myocardial slices (300 μm thick) were prepared from transmural LV biopsies (10x10x9 mm; n=4 hearts), tangentially to the wall surface. Multiphoton microscopy was used to visualise muscle fiber orientation. Multi-electrode arrays (60 microelectrodes, total area 4.9x4.9 mm) were used to map electrophysiological parameters. Preparations were paced at cycle lengths (CL) ranging from 1 to 4s. Values are means ± S.E.M. Results: Slices had longitudinal muscle fiber orientation, independent from transmural location. Activation-recovery intervals (ARI; Fig) correlated with transmural site at all pacing CL (p<0.001; ANOVA), with the longest values found in a 1.2 mm layer of the deep midmyocardium (4.8 to 6 mm from the epicardial surface; total LV thickness 9 mm). Longer pacing CL heterogeneously prolonged ARI (Fig) and increased transmural repolarization gradient from 63±7 to 106±3 ms (1s versus 4s CL; p<0.01 t-test). In plane ARI dispersion ranged from 9±1 to 18±3 ms, and was independent from transmural location and CL (p>0.05; ANOVA), suggesting the presence of electrophysiologically homogeneous and well coupled cells in each slice. Conclusion: The deep midmyocardium contains a well-defined homogeneous region of prolonged ARI, which may contribute to repolarisation gradients in the canine LV. [ABSTRACT FROM AUTHOR]

In the failing heart, the extracellular matrix (ECM) undergoes structural remodelling with changes in the accumulation and organisation of ECM proteins. Ivabradine (IVA) an inhibitor of the pacemaker (If) current has been shown to have beneficial effects in the structure and function of the failing heart including the reversal of myocardial fibrosis. However, whether IVA alters the expression of specific ECM proteins during the progression of heart failure (HF) is unknown. In addition, changes in the fibroblast number and phenotype before and after IVA treatment have not been studied. HF was induced by permanent coronary artery ligation in rats anaesthetized with Isoflurane. Sham-operated animals (S) were used as controls. After 12 weeks, HF (HF-12) animals (ejection fraction<40%) were treated either with oral IVA (HF-IVA) (10mg/kg/day) or saline (HF-S) for a further 4 weeks. Values are presented as means±S.E.M. and compared by an ANOVA. Using immunofluorescence and confocal microscopy, sections were studied for specific ECM proteins and the percentage area fraction was quantified on 8-10 fields per section (3-4 hearts per group) using ImageJ software. IVA significantly reduced the levels of collagen I (S: 0.76±0.07%, n=69; HF-12: 1.12±0.08%, n=57; HFS: 1.59±0.12%, n=70; HF-IVA: 0.68±0.06%, n=52; p<0.05), collagen III (S: 0.88±0.06%, n=78; HF-12: 1.26±0.09%, n=90; HFS: 1.91±0.13%, n=74; HF-IVA: 0.75±0.65%, n=60; p<0.05) and elastin (S: 0.62±0.06%, n=52; HF-12: 1.16±0.06%, n=85; HF-S: 1.93±0.22%, n=68; HF-IVA: 0.52±0.09%, n=29; p<0.05) compared with HF-12. Vimentin, a marker for cardiac fibroblasts was measured using western blotting. IVA reduced the vimentin expression observed in HF-S to sham levels (in arbitrary units, S: 0.33±0.04, n=5; HF-S: 0.59±0.05, n=6; HF-IVA: 0.37±0.02, n=5; p<0.05). Since vimentin is also expressed in endothelial cells, von Willebrand factor (vWf) was measured. No change in vWf was observed suggesting that endothelial cell number was not affected. a-smooth muscle actin, a marker used to identify myofibroblasts but also present in smooth muscle cells, was also measured. IVA normalised the levels of a-smooth muscle actin to sham levels (S: 0.50±0.13, n=5; HFS: 1.00±0.13, n=6; HF-IVA: 0.44±0.08, n=4; p<0.05). Expression of the hyperpolarisation-activated cyclic nucleotide-gated channel isoform 4 which encodes the If channels was up-regulated during HF but IVA down-regulated this overexpression (S: 0.31±0.05, n=5; HF-S: 0.57±0.05, n=6; HF-IVA: 0.38±0.05, n=5; p<0.05). Our results demonstrate that IVA reverses the accumulation of several components of the ECM and reduce fibroblast number and phenotype which may explain the changes observed in the ECM. Thus IVA appears to play a beneficial role in the structural integrity of the ECM which can be important in the reparative process of the failing heart. [ABSTRACT FROM AUTHOR]

An abstract of the article "Mapping regional repolarisation gradients in the failing human ventricle using cardiac slices" by P. Camelliti, S. Al Ayoubi and C. Terracciano is presented.

An abstract of the article "Manipulation of SR Ca2+-release in heart failure through mechanical intervention" by M. Ibrahim and C. Terracciano is presented.

The interaction of cardiac fibroblasts (FBs) and myocytes (CMs) is gaining considerable interest due to growing evidence that fibroblasts can affect myocyte structure and function. This interaction can occur through direct cell contact or paracrine communication. Very limited data exists regarding paracrine communication, particularly between adult cells. We examined the effect of fibroblasts from normal and overloaded hearts on adult myocytes. TGF-ß is a potential paracrine mediator and was investigated. Adult Lewis rat CMs were co-cultured with FBs from pressure overloaded (10 week thoracic aortic constriction (TAC)) (T+CM) or sham operated hearts (S+CM) at 1:2 CM/FB ratio in a co-culture system that allows paracrine communication but prevents direct cell contact. CMs co-cultured with CMs (CM+CM) served as control. After 24 hours co-culture, CM viability was measured as the % rod shaped cells. CM volume was measured using di-8-ANEPPS membrane staining. Ca2+ transients were recorded using the Ca2+ sensitive dye Fluo-4. 10 µM SB431542 (SB) was used to block TGF- ß type 1 receptors. Data is shown as mean±SEM (n number) and analysed by 1 way ANOVA with Tukeys post hoc analysis. Co-culture with sham or TAC FBs reduced the viability of co-cultured CMs but this was blocked by SB (in %: CM+CM 70±1.1 (5); CM+CM+SB 72±1.7 (5); S+CM 61±2.3 (6); S+CM+SB 74±2.4 (6); T+CM 52±2.2 (6); T+CM+SB 66±2.8 (6). p<0.05: S+CM vs CM+CM, T+CM vs CM+CM, S+CM+SB vs S+CM, T+CM+SB vs T+CM). Both sham and TAC FBs induced CM hypertrophy which was also prevented by SB (in x103µm3: CM+CM 48±1.2 (60); CM+CM+SB 49±1.7 (43); S+CM 53±1.3 (48); S+CM+SB 48±1.4 (47); T+CM 56±1.7 (44); T+CM+SB 50±1.8 (42). p<0.05: S+CM vs CM+CM, T+CM vs CM+CM, S+CM+SB vs S+CM, T+CM+SB vs T+CM). Ca2+ transient amplitude was increased after co-culture with sham FBs but reduced after co-culture with TAC FBs. These effects were also prevented by SB (in F/F0: CM+CM 3.4±0.1 (42); CM+CM+SB 3.2±0.1 (33); S+CM 4.6±0.2 (50); S+CM+SB 3.3±0.1 (60); T+CM 2.8±0.1 (36); T+CM+SB 3.3±0.2 (45). p<0.05: S+CM vs CM+CM, T+CM vs CM+CM, S+CM+SB vs S+CM, T+CM+SB vs T+CM). Time to 90% decay of the Ca2+ transient was reduced after co-culture with sham or TAC FBs but unaffected by SB (in ms: CM+CM 359±8.5 (42); CM+CM+SB 390±17.2 (33); S+CM 322±6.2 (50); S+CM+SB 339±7.0 (60); T+CM 295±13.5 (36); T+CM+SB 317±9.3 (45). p<0.05: S+CM vs CM+CM, T+CM vs CM+CM). Time to peak and time to 50% decay were unaffected. These results show that FBs affect viability and Ca2+ cycling of CMs by paracrine TGF-ß signalling. FBs from overloaded hearts alter Ca2+ cycling of CMs differently from normally loaded FBs, suggesting that this paracrine interaction is sensitive to chronic variations in load and may play a role in the pathophysiology of cardiac disease. [ABSTRACT FROM AUTHOR]

Chronic variations in load are clinically relevant as mechanical overload can result in heart failure and unloading with left ventricular assist devices is used in the management of patients with heart failure. Previous studies have investigated the effect of chronic load variation on the electrophysiological properties of the left ventricle using isolated cardiomyocytes and whole hearts. Here, we study the load-dependent remodeling using myocardial slices, an intact multicellular preparation that allows the investigation of functional and structural regional heterogeneities. Lewis rats were anaesthetized with isoflurane and pressure overload was induced by transverse aortic constriction (TAC) for 10 and 20 weeks. Sham-operated animals (S) served as control. Mechanical unloading (MU) was obtained by heterotopic abdominal heart-lung transplantation for 8 weeks. The recipient's native heart acted as control. Vibratome-cut myocardial slices (350µm thick) were prepared from the left ventricle, tangentially to the epicardial surface. Slices were electrically stimulated and studied using a multi-electrode array (MEA) system. Extracellular field potentials, recorded from 60 MEA microelectrodes, were analysed to measure field potential duration (FPD)-an index of action potential duration. Conduction velocity (CV) was also measured. Values are mean ± S.E.M., compared by t-test. The measurement of heart weight/body weight ratio confirmed the presence of hypertrophy in the TAC group (10 weeks: 4.9±0.2, n=5; S: 3.8±0.1, n=5; p<0.01, 20 weeks: 4.1±0.2; n=4; S: 3.1±0.1 n=5; p<0.05) and atrophy in the unloading group (MU: 1.4±0.1, n=3; control: 3.5±0.1, n=3; p<0.05). Myocardial slices prepared from the TAC group showed significantly faster CV compared with the SHAM group at 10 weeks (TAC: 49±4 cm/s, n=25 slices; S: 35±4 cm/s, n=17 slices; p<0.05) but no significant change at 20 weeks (TAC: 67±10 cm/s, n=17 slices; S: 56±6 cm/s, n=22 slices; p>0.05). FPD did not change at 10 weeks TAC (TAC: 124±3 ms, n=16 slices; S: 122±2 ms; n=13 slices; p>0.05), but was significantly prolonged at 20 weeks (TAC: 122±2 ms, n=18 slices; S: 114±3 ms, n=20 slices; p<0.05). Slices obtained from the MU group showed significantly prolonged FPD (MU: 116±2 ms, n=11 slices; control: 108±2 ms, n=13 slices; p<0.05), but unchanged CV (MU: 30±3 cm/s, n=10 slices; control: 32±5cm/s, n=13 slices; p>0.05). Our data show that chronic mechanical overloading temporarily increases CV with delayed repolarisation. The latter is also observed during unloading without effects on CV. Delayed repolarisation may represent a compensatory response to variations in mechanical load and underlie possible arrhythmogenic effects. Future studies will employ myocardial slices to determine the structural alterations responsible for the observed electrophysiological remodeling. [ABSTRACT FROM AUTHOR]

Popeye domain containing (Popdc) genes encode a novel class of cAMP-binding proteins. Functional analysis of Popdc1 and-2 genes in mice indicate a potential role in the chronotropic response to β-adrenergic signalling, with single null mutants displaying a stress-induced bradycardia.1 The overlapping phenotype in both mutants is age-dependent, suggesting the presence of genetic redundancy. Here we report our preliminary analysis of Popdc1;Popdc2 double knockout (P12dKO) mice and the functional interactions of Popdc1 and -2 at the protein level. Popdc1;Popdc2 double heterozygous (P12dHET), P12dKO, and wild type (WT) age-matched control male mice were subjected to telemetric ECG analysis at rest and during physical stress (6min swim, 15min recovery). In accordance with our single null mutant analysis, no significant difference was observed in the heart rates of 3 month old mice at rest (WT 514±34bpm, n=7; P12dHET 547±36bpm, n=5, two-tailed t-test p=0.14; P12dKO 552±36bpm, n=, two-tailed t-test p=0.20). However, in contrast to the single null mutants where the stress-induced bradycardia was not observed until 5.5 months of age, the 3 month old P12dKO mice had a blunted response to physical stress (WT39% increase in heart rate, n=7; P12dHET32%, n=5, two tailed t-test p=0.35; P12dKO 18%, n=, two-tailed t-test p=0.01). A blunted increase in heart rate in response to physical stress is consistently seen in P12dKO older mice (>9 months), although not exacerbated compared to P1KO mice (WT 719±14bpm, n=5; P1KO 593±39bpm, n=5; P12dKO 665±24bpm, n=6, WT vs P12dKO two-tailed t-test p<0.01), which suggests that during early postnatal development the two Popdc genes can functionally compensate each other and act in the same functional context since the loss of either gene is causing a pacemaker deficiency and phenotypic severity does not increase if both Popdc genes are missing. Immunohistochemistry revealed that both Popdc1 and -2 proteins are expressed throughout the heart, with enhanced expression in the cardiac conduction system. At the single cell level both proteins are located at the plasma membrane, intercalated disks and in T-tubules. To identify new interaction partners for Popdc proteins, we performed co-immunoprecipitation followed by mass spectrometry. By this screen Ankyrin-G and -B and the Na+/Ca2+ exchanger NCX1 were identified and subsequently confirmed by Western Blot as interacting proteins. Furthermore, we were able to demonstrate an interaction of Popdc1 and, suggesting that members of the Popdc family are interacting with each other and thereby acting in the same functional context. This may explain the overlapping phenotypes of Popdc1 and -2 mutants and the earlier manifestation of the bradycardia in P12dKO mice. Further analysis at the protein, cell, and organismic level, will help to define the role of Popdc proteins in cardiac stress adaptation. Froese Aet al (2012) Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice. J Clin Invest. 12, 1119-30. [ABSTRACT FROM AUTHOR]