Depolarization of Cellular Resting Membrane Potential Promotes Neonatal Cardiomyocyte Proliferation In Vitro
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Cardiomyocytes (CMs) undergo a rapid transition from hyperplastic to hypertrophic growth soon after birth, which is a major challenge to the development of engineered cardiac tissue for pediatric patients. Resting membrane potential (V mem) has been shown to play an important role in cell differentiation and proliferation during development. We hypothesized that depolarization of neonatal CMs would stimulate or maintain CM proliferation in vitro. To test our hypothesis, we isolated postnatal day 3 neonatal rat CMs and subjected them to sustained depolarization via the addition of potassium gluconate or Ouabain to the culture medium. Cell density and CM percentage measurements demonstrated an increase in mitotic CMs along with a ~twofold increase in CM numbers with depolarization. In addition, depolarization led to an increase in cells in G2 and S phase, indicating increased proliferation, as measured by flow cytometry. Surprisingly depolarization of V mem with either treatment led to inhibition of proliferation in cardiac fibroblasts. This effect is abrogated when the study was carried out on postnatal day 7 neonatal CMs, which are less proliferative, indicating that the likely mechanism of depolarization is the maintenance of the proliferating CM population. In summary, our findings suggest that depolarization maintains postnatal CM proliferation and may be a novel approach to encourage growth of engineered tissue and cardiac regeneration in pediatric patients.
KeywordsCell cycle Potassium gluconate Ouabain Cardiac fibroblasts Akt pathway
We gratefully acknowledge Professor Qiaobing Xu and Yuji Takeda for the use of the flow cytometer and assistance with protocols and data analysis techniques. We also acknowledge the NIH-NHLBI for funding this work via an NRSA individual postdoctoral fellowship (F32 HL112538) to CW and awards R00HL093358 and R21HL115570 to LDB. M. L. gratefully acknowledges support of the G. Harold and Leila Y. Mathers Charitable Foundation, DARPA (W911NF-09-1-0125), and the W. M. Keck Foundation.
Conflict of interest
The authors (J.L., C.W., M.L. and L.B.) have no conflicts of interest to report.
No human studies were carried out by the authors for this article. All procedures involving animals were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at Tufts University.
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