Mechanical load induced by glass microspheres releases angiogenic factors from neonatal rat ventricular myocytes cultures and causes arrhythmias.

In the present study, we tested the hypothesis that similar to other mechanical loads, notably cyclic stretch (simulating pre-load), glass microspheres simulating afterload will stimulate the secretion of angiogenic factors. Hence, we employed glass microspheres (average diameter 15.7 microm, average mass 5.2 ng) as a new method for imposing mechanical load on neonatal rat ventricular myocytes (NRVM) in culture. The collagen-coated microspheres were spread over the cultures at an estimated density of 3000 microspheres/mm2, they adhered strongly to the myocytes, and acted as small weights carried by the cells during their contraction. NRVM were exposed to either glass microspheres or to cyclic stretch, and several key angiogenic factors were measured by RT-PCR. The major findings were: (1) In contrast to other mechanical loads, such as cyclic stretch, microspheres (at 24 hrs) did not cause hypertrophy. (2) Further, in contrast to cyclic stretch, glass microspheres did not affect Cx43 expression, or the conduction velocity measured by means of the Micro-Electrode-Array system. (3) At 24 hrs, glass microspheres caused arrhythmias, probably resulting from early afterdepolarizations. (4) Glass microspheres caused the release of angiogenic factors as indicated by an increase in mRNA levels of vascular endothelial growth factor (80%), angiopoietin-2 (60%), transforming growth factor-beta (40%) and basic fibroblast growth factor (15%); these effects were comparable to those of cyclic stretch. (5) As compared with control cultures, conditioned media from cultures exposed to microspheres increased endothelial cell migration by 15% (P<0.05) and endothelial cell tube formation by 120% (P<0.05), both common assays for angiogenesis. In conclusion, based on these findings we propose that loading cardiomyocytes with glass microspheres may serve as a new in vitro model for investigating the role of mechanical forces in angiogenesis and arrhythmias.

Electrophysiologic perturbations and arrhythmogenic activity caused by activation of the Fas receptor in murine ventricular myocytes: role of the inositol trisphosphate pathway.

INTRODUCTION: Experimental evidence suggests a major role for Fas receptor activation in a wide range of myocardial pathologies. Because clinical situations, which are likely to be associated with Fas activation, are accompanied by a variety of ventricular arrhythmias, the major goal of this study was to investigate the ionic mechanisms responsible for these phenomena. METHODS AND RESULTS: To delineate the origin of Fas-mediated electrophysiologic perturbations, the transient outward K+ current I(to) and the L-type Ca2+ current I(Ca,L) were studied in murine ventricular myocytes treated with the Fas-activating monoclonal antibody Jo2. Jo2 decreased I(to) (4.36 +/- 1.2 pA/pF vs 17.48 +/- 2.36 pA/pF in control, V(M) = +50 mV; P < 0.001) and increased I(Ca,L) (-13.17 +/- 1.38 pA/pF vs -3.94 +/- 0.78 pA/pF in control, V(M) = 0 mV; P < 0.001). Pretreatment of ventricular myocytes with ryanodine or thapsigargin prevented the electrophysiologic effects of Jo2, suggesting that [Ca2+]i elevation is important for Fas-mediated action. In agreement with our previous studies demonstrating dependence of Fas-based myocyte dysfunction on an intact inositol trisphosphate (1,4,5-IP3) pathway, the effects of Jo2 on I(to) and I(Ca,L) were prevented by the phospholipase C (generates 1,4,5-IP3) blocker U73122, and by xestospongin C (tested with I(to)), a specific blocker of IP3-operated sarcoplasmic reticulum Ca2+ release channels. Furthermore, intracellular perfusion with 1,4,5-IP3, but not with 1,3,4-IP3, caused electrophysiologic effects resembling those of Jo2. CONCLUSION: Decreased I(to) and increased I(Ca,L) underlie Fas-induced action potential alterations and arrhythmias in murine ventricular myocytes, effects that appear to be mediated by 1,4,5-IP3-induced intracellular calcium release.

Cardiac dysfunction in murine autoimmune myocarditis.

We have investigated the pathophysiological basis of cardiac dysfunction in autoimmune myocarditis and in the resulting dilated cardiomyopathy. To this end we utilized the myosin-induced autoimmune myocarditis model in BALB/c mice. Myocarditis has been found to be associated with massive ventricular lymphocyte infiltration and a 50% reduction in tail artery blood flow, reflecting the depressed cardiac function in myocarditis. Action potential characteristics of control and diseased isolated ventricular myocytes were (mean+/-SEM): resting potential: -68.1+/-1. 1,-68.3+/-0.7 mV; action potential amplitude: 96.5+/-10.4, 92.3+/-4. 4 mV; action potential duration at 80% repolarization (APD80) 38+/-5, 116+/-24* ms; * P<0.05. We utilized the whole cell voltage clamp technique to explore ion currents involved in APD prolongation and arrhythmogenic activity, and found that in diseased myocytes the transient outward current (Ito) was markedly attenuated. At a membrane potential of +40 mV, in control and in diseased myocytes, I(to) current density was 14.7+/-1.5 and 6.5+/-1.4 pA/pF, respectively, P<0.005. In contrast, the L-type Ca2+current (ICa,L) remained unchanged. To further explore the basis for cardiac impairment, we simultaneously measured [Ca2+]i transient and contraction in isolated normal and diseased myocytes. The major findings indicated that both the relaxation kinetics of [Ca2+]i transients and myocyte contraction were significantly faster in the diseased myocytes. In conclusion, substantial, potentially reversible, electrophysiological and mechanical perturbations in ventricular myocytes from mice with myosin-induced autoimmune myocarditis appear to contribute to disease-related cardiac dysfunction.

Fas (CD95/Apo-1)-mediated damage to ventricular myocytes induced by cytotoxic T lymphocytes from perforin-deficient mice: a major role for inositol 1,4,5-trisphosphate.

Cytotoxic T lymphocytes (CTLs) that infiltrate the heart are important immune effectors implicated in heart transplant rejection, myocarditis, and other cardiomyopathies. To investigate the mechanism(s) underlying CTL damage to the myocardium through activation of the Fas receptor (Fas/CD95/Apo-1) by the Fas ligand, we explored the interaction between peritoneal exudate CTLs (PELs), derived from perforin gene-knockout (P-/-) mice, and murine ventricular myocytes. Fas expression on isolated ventricular myocytes was demonstrated immunohistochemically. Action potentials, [Ca2+]i transients, and contractions of myocytes conjugated to P-/- PELs or treated with the apoptosis-inducing anti-Fas monoclonal antibody Jo2 were recorded. Action potential characteristics of nonconjugated myocytes and myocytes conjugated with P-/- PELs were, respectively, as follows: Vm, -73.2+/-1.5 and -53.6+/-6.4 mV (mean+/-SEM); action potential amplitude, 117.9+/-3.9 and 74.3+/-21.2 mV; and action potential duration at 80% repolarization, 17+/-6 and 42+/-13 milliseconds (all P<.05). P-/- PELs also induced early and delayed afterdepolarizations as well as arrhythmogenic activity. Diastolic [Ca2+]i increased during the cytocidal interaction with P-/- PELs, from a fluorescence ratio of 0.82+/-0.05 (n=7) to 1.98+/-0.09 (n=13) (P<.05). All of the effects caused by P-/- PELs were reproduced by incubating the myocytes with Jo2. Heparin (50 microg/mL), an antagonist of inositol trisphosphate (IP3)-operated sarcoplasmic reticulum Ca2+ channels, or U-73122 (2 micromol/L), a phospholipase C inhibitor, but not the inactive agonist U-73343, prevented Fas-mediated myocyte dysfunction. Additionally, intracellular application (through the patch pipette) of the active IP3 analogue, inositol 1,4,5-trisphosphate, but not the inactive analogue, inositol 1,3,4-trisphosphate, caused electrophysiological changes resembling those resulting from P-/- PELs and Jo2, suggesting that CTL-induced Fas-based myocyte dysfunction is mediated by IP3. We conclude that a Fas-based perforin-independent mechanism of CTL action can account for the immunopathology seen in the allotransplanted heart, myocarditis, and dilated cardiomyopathy.