Heterotopic abdominal heart transplantation in rats for functional studies of ventricular unloading.

INTRODUCTION: Chronic changes in mechanical load regulate long-term cardiac function. Chronic overload of the ventricle results in myocardial failure. Clinical use of ventricular assist devices shows that chronic reduction in load has a number of different consequences on the myocardium, including beneficial reverse remodeling as well as undesired remodeling (e.g., myocardial atrophy and fibrosis, both of which could have negative functional implications). The complex response to mechanical unloading necessitates reproducible animal models of mechanical unloading for use in the laboratory. This article aims to describe the operative technique of two animal models of mechanical unloading in detail, to enable the reproducible use of these animal models. METHODS: In 1964, Abbott et al first described the heterotopic abdominal heart transplantation technique as a means to study the biology of transplanted cardiac grafts. This involves an aorto-aortic anastomosis and a pulmonary artery to inferior vena cava anastomosis. In this model, the left ventricle is virtually completely volume unloaded, receiving only thebesian venous return, and substantially but not entirely pressure unloaded. In this report we describe two refined techniques for mechanical unloading of healthy or failing hearts based on experience with over 500 operations. RESULTS: We describe an operative technique, including cardioprotective strategies, that provides a model of mechanical unloading with no immunological rejection and allows measurements of parameters of myocardial structure and function for many months. We describe a refined technique that achieves a lesser degree of left ventricular volume unloading, involving transplantation of both heart and lungs via a single aorto-aortic anastomosis. CONCLUSIONS: This article is the first to describe these two techniques in sufficient detail to enable novices to attempt and understand these operations and the differences between them. The technique we describe provides an effective and reproducible model of complete and partial mechanical unloading.

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 Ca(2+)-induced Ca(2+) 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. Ca(2+) release synchronicity was reduced at 8 weeks moderate unloading only. Ca(2+) 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 Ca(2+) transient, increased Ca(2+) 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.

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-ß 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-ß type 1 receptors, we determined that TGF-ß 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-ß, is crucial in this interaction.