Micro-electrocardiograms to study post-ventricular amputation of zebrafish heart.

The zebrafish (Danio rerio) is an emerging model for cardiovascular research. The zebrafish heart regenerates after 20% ventricular amputation. However, assessment of the physiological responses during heart regeneration has been hampered by the small size of the heart and the necessity of conducting experiments in an aqueous environment. We developed a methodology to monitor a real-time surface electrocardiogram (ECG) by the use of micro-electrodes, signal amplification, and a low pass-filter at a sampling rate of 1 kHz. Wavelet transform was used to further remove ambient noises. Rather than paralyzing the fish, we performed mild sedation by placing the fish in a water bath mixed with MS-222 (tricane methanesulfonate). We recorded distinct P waves for atrial contraction, QRS complexes for ventricular depolarization, and QT intervals for ventricular repolarization prior to, and 2 and 4 days post-amputation (dpa). Sedation reduced the mean fish heart rate from 149 +/- 18 to 90 +/- 17 beats/min. The PR and QRS intervals remained unchanged in response to ventricular apical amputation (n = 6, p > 0.05). Corrected QT intervals (QTc) were shortened 4 dpa (n = 6, p < 0.05). In a parallel study, histology revealed that apical thrombi were replaced with fibrin clots and collagen fibers. Atrial arrhythmia was noted in response to prolonged sedation. Unlike the human counterpart, ventricular tachycardia or fibrillation was not observed in response to ventricular amputation 2 and 4 dpa. Taken together, we demonstrated a minimally invasive methodology to monitor zebrafish heart function, electrical activities, and regeneration in real-time.

Mechanical response of a calcified plaque model to fluid shear force.

Vascular calcification is associated with atherosclerosis, but whether it mechanically affects plaque stability remains controversial. To assess the effect of mineralization on plaque vulnerability to mechanical shear stress, we applied fluid shear to cultures of calcifying vascular cells (CVC), a subpopulation of smooth muscle cells that spontaneously mineralize. CVC cultures containing nodules were treated for 10 days with vehicle control or beta-glycerophosphate (BGP) to accelerate mineralization. Cultures were placed in a parallel-plate flow system and were subjected to increasing fluid shear stress (4.9 dyn/cm(2)/min up to 400 dyn/cm(2)). The number of nodules remaining attached was recorded every 10 min. Results showed that control cultures and BGP-treated cultures, which contained significantly greater calcium mineral than control cultures, had similar detachment thresholds (50-100 dyn/cm(2)), with linear portions of their stress/detachment curves from 100 to 275 dyn/cm(2). Based on repeated measure analysis of variance, BGP-treated nodules were no more likely to detach at a given shear than controls, although they showed a trend toward greater stability. Thus, calcification does not appear to increase plaque vulnerability to fluid shear stress, although it may contribute to a slight stabilization. This model may represent the first in vitro model of mechanical rupture of atherosclerotic plaque.