James E. Cartledge

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.

Reduced Na+ and higher K+ channel expression and function contribute to right ventricular origin of arrhythmias in Scn5a+/− mice

Brugada syndrome (BrS) is associated with ventricular tachycardia originating particularly in the right ventricle (RV). We explore electrophysiological features predisposing to such arrhythmic tendency and their possible RV localization in a heterozygotic Scn5a+/− murine model. Nav1.5 mRNA and protein expression were lower in Scn5a+/− than wild-type (WT), with a further reduction in the RV compared with the left ventricle (LV). RVs showed higher expression levels of Kv4.2, Kv4.3 and KChIP2 in both Scn5a+/− and WT. Action potential upstroke velocity and maximum Na+ current (INa) density were correspondingly decreased in Scn5a+/−, with a further reduction in the RV. The voltage dependence of inactivation was shifted to more negative values in Scn5a+/−. These findings are predictive of a localized depolarization abnormality leading to slowed conduction. Persistent Na+ current (IpNa) density was decreased in a similar pattern to INa. RV transient outward current (Ito) density was greater than LV in both WT and Scn5a+/−, and had larger time constants of inactivation. These findings were also consistent with the observation that AP durations were smallest in the RV of Scn5a+/−, fulfilling predictions of an increased heterogeneity of repolarization as an additional possible electrophysiological mechanism for arrhythmogenesis in BrS.

Cardiomyocyte Ca2+ handling and structure is regulated by degree and duration of mechanical load variation

Cardiac transverse (t)‐tubules are altered during disease and may be regulated by stretch‐sensitive molecules. The relationship between variations in the degree and duration of load and t‐tubule structure remains unknown, as well as its implications for local Ca2+‐induced Ca2+ release (CICR). Rat hearts were studied after 4 or 8 weeks of moderate mechanical unloading [using heterotopic abdominal heart–lung transplantation (HAHLT)] and 6 or 10 weeks of pressure overloading using thoracic aortic constriction. CICR, cell and t‐tubule structure were assessed using confocal‐microscopy, patch‐clamping and scanning ion conductance microscopy. Moderate unloading was compared with severe unloading [using heart‐only transplantation (HAHT)]. Mechanical unloading reduced cardiomyocyte volume in a time‐dependent manner. Ca2+ release synchronicity was reduced at 8 weeks moderate unloading only. Ca2+ sparks increased in frequency and duration at 8 weeks of moderate unloading, which also induced t‐tubule disorganization. Overloading increased cardiomyocyte volume and disrupted t‐tubule morphology at 10 weeks but not 6 weeks. Moderate mechanical unloading for 4 weeks had milder effects compared with severe mechanical unloading (37% reduction in cell volume at 4 weeks compared to 56% reduction after severe mechanical unloading) and did not cause depression and delay of the Ca2+ transient, increased Ca2+ spark frequency or impaired t‐tubule and cell surface structure. These data suggest that variations in chronic mechanical load influence local CICR and t‐tubule structure in a time‐ and degree‐dependent manner, and that physiological states of increased and reduced cell size, without pathological changes are possible.

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.

Exercise, load and remodelling: do we know what we think we know?

The mammalian heart is exquisitely stretch sensitive and is ordinarily capable of profound changes from the subcellular to systems levels in order to handle changes in cardiac workload encountered during pregnancy, exercise training and initially also when challenged by disease processes such as aortic stenosis and hypertension (Ellison et al. 2012). Classically, increases in cardiac load may be physiological (e.g. exercise) or pathological (e.g. hypertension), the former resulting in reversible physiological hypertrophy without diminished cardiac function, and the latter in maladaptive, irreversible changes. The last two to three decades have seen multiple studies which challenge this dogma. In a recent issue of The Journal of Physiology da Costa Rebelo et al. describe their study of the biochemical and physiological consequences of high-intensity but voluntary exercise in spontaneously hypertensive rats (SHRs) (da Costa Rebelo et al. 2012). They find that exercise in SHRs induces no change in systolic blood pressure, increases fibrosis and impairs cardiac function (e.g. left ventricular (LV) developed pressure). Exercise is widely thought to be beneficial in the setting of heart disease in general and hypertension in particular (Wexler et al. 2012). On the hypothesis that the exercise-induced maladaptive changes are angiotensin II dependent, the authors tested whether captopril was able to ameliorate these effects. The addition of captopril partially prevented the increased fibrosis and the impaired LV function; moreover, the combined use of captopril and exercise training resulted in reverse remodelling at the cellular level (sarcoendoplamic reticulum Ca2+ ATPase/Na+ Ca2+ exchanger (SERCA/NCX) ratio). Sedentary SHRs developed further hypertension. Captopril alone was the only intervention to significantly reduce the final systolic blood pressure and reduce LV hypertrophy (indeed training induces LV hypertrophy, unless captopril is used). These results raise a number of interesting issues.