Heart on a plate: histological and functional assessment of isolated adult zebrafish hearts maintained in culture.
The zebrafish is increasingly used for cardiovascular genetic and functional studies. We present a novel protocol to maintain and monitor whole isolated beating adult zebrafish hearts in culture for long-term experiments. Excised whole adult zebrafish hearts were transferred directly into culture dishes containing optimized L-15 Leibovitz growth medium and maintained for 5 days. Hearts were assessed daily using video-edge analysis of ventricle function using low power microscopy images. High-throughput histology techniques were used to assess changes in myocardial architecture and cell viability. Mean spontaneous Heart rate (HR, min(-1)) declined significantly between day 0 and day 1 in culture (96.7 ± 19.5 to 45.2 ± 8.2 min-1, mean ± SD, p = 0.001), and thereafter declined more slowly to 27.6 ± 7.2 min(-1) on day 5. Ventricle wall motion amplitude (WMA) did not change until day 4 in culture (day 0, 46.7 ± 13.0 µm vs day 4, 16.9 ± 1.9 µm, p = 0.08). Contraction velocity (CV) declined between day 0 and day 3 (35.6 ± 14.8 vs 15.2 ± 5.3 µms(-1), respectively, p = 0.012) while relaxation velocity (RV) declined quite rapidly (day 0, 72.5 ± 11.9 vs day 1, 29.5 ± 5.8 µms(-1), p = 0.03). HR and WMA responded consistently to isoproterenol from day 0 to day 5 in culture while CV and RV showed less consistent responses to beta-agonist. Cellular architecture and cross-striation pattern of cardiomyocytes remained unchanged up to day 3 in culture and thereafter showed significant deterioration with loss of striation pattern, pyknotic nuclei and cell swelling. Apoptotic markers within the myocardium became increasingly frequent by day 3 in culture. Whole adult zebrafish hearts can be maintained in culture-medium for up to 3 days. However, after day-3 there is significant deterioration in ventricle function and heart rate accompanied by significant histological changes consistent with cell death and loss of cardiomyocyte cell integrity. Further studies are needed to assess whether this preparation can be optimised for longer term survival.
UoE/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
Sebastian
S
Pieperhoff
UoE/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
Kathryn S
KS
Wilson
UoE/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
James
J
Baily
UoE/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
Kim
K
de Mora
UoE/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
Sana
S
Maqsood
UoE/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
Sharron
S
Vass
School of Physics and Astronomy, University of Glasgow, Glasgow, Scotland, United Kingdom.
Jonathan
J
Taylor
Royal Dick School of Veterinary Studies, Division of Veterinary Clinical Sciences, The University of Edinburgh, Hospital for Small Animals, Easter Bush Veterinary Centre, Roslin, Midlothian, Scotland, United Kingdom.
Jorge
J
Del-Pozo
Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, The Broad Institute of MIT and Harvard, Boston, Massachusetts, United States of America.
Calum A
CA
MacRae
UoE/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
John J
JJ
Mullins
UoE/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
Martin A
MA
Denvir
D000818:Animals; D002316:Cardiotonic Agents / Q000494:pharmacology; D006321:Heart / Q000033:anatomy & histology / Q000187:drug effects / Q000502:physiology*; D006339:Heart Rate / Q000187:drug effects / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism; D009924:Organ Culture Techniques / Q000379:methods*; D015027:Zebrafish / Q000502:physiology*
D002316:Cardiotonic Agents; D007545:Isoproterenol