Introduction: Background and Motivation

• Congenital heart defects (CHDs) are common and life-threatening; innovative solutions are needed for treatment.
• Neonatal cardiomyocytes (heart muscle cells) naturally have the ability to proliferate (increase in number) soon after birth, which is crucial for heart growth.
• This study investigates whether depolarization—making the cell membrane less negative—can stimulate or maintain cardiomyocyte proliferation in vitro (in a lab setting).

Key Concepts and Definitions

• Depolarization: A change in the cell’s resting membrane potential that makes it less negative. Think of it like “turning up the voltage” on a battery to energize the cell.
• Resting Membrane Potential (Vmem): The natural voltage difference across a cell’s membrane when it is not active.
• Cardiomyocytes (CMs): Specialized heart muscle cells that contract to pump blood.
• Cardiac Fibroblasts (CFs): Support cells in the heart that produce the framework (extracellular matrix) keeping cells together.
• Hyperplasia vs. Hypertrophy: Hyperplasia is an increase in the number of cells (like baking many small cupcakes), while hypertrophy is an increase in the size of cells (like making one giant cupcake).

Materials and Methods (Step-by-Step Recipe)

• Cell Isolation:
  • Neonatal rat hearts were harvested on postnatal day 3 (P3) and day 7 (P7).
  • The ventricular tissue was minced and digested with collagenase to release individual cells.
• Cell Culture:
  • A mixed population of cardiomyocytes and cardiac fibroblasts was seeded into culture dishes.
  • Cells were grown in a nutrient-rich medium called Myo Media.
• Treatment to Alter Membrane Potential:
  • Depolarizing agents used: potassium gluconate and ouabain.
  • These agents were added at various concentrations (optimal: 40 mM for potassium gluconate and 10 μM for ouabain) for 72 hours.
• Validation of Depolarization:
  • A voltage-sensitive dye, DiBAC4(3), was used to measure changes in membrane potential.
  • Increased dye fluorescence indicated that the cells’ membranes had become less negative.
• Assessment Techniques:
  • Immunocytochemistry: Staining with cardiac α-actin identified cardiomyocytes and PHH3 marked cells undergoing mitosis (cell division).
  • Cell Counting: ImageJ software was used to count the total cells and specifically the cardiomyocytes.
  • Flow Cytometry: Measured cell cycle phases to determine if more cells were entering division (e.g., G2 and S phase).
  • Western Blot: Analyzed protein levels to check activation of key growth pathways such as Akt and MAPK/ERK.

Results: What Happened

• Validation of Depolarization:
  • Both potassium gluconate and ouabain successfully depolarized the cells at their optimal concentrations.
  • Enhanced fluorescence confirmed that the resting membrane potential was effectively altered.
• Effects on Cardiomyocytes (CMs):
  • Depolarization significantly increased the number of cardiomyocytes.
  • Optimal doses resulted in roughly a twofold increase in CM numbers compared to untreated control cells.
  • Flow cytometry showed more cells in the G2 and S phases, which are stages of DNA synthesis and division, indicating increased proliferation.
• Effects on Cardiac Fibroblasts (CFs):
  • In contrast, depolarization inhibited the proliferation of cardiac fibroblasts.
  • This selective effect is beneficial as it promotes heart muscle growth without encouraging excess fibrous tissue formation.
• Age-Dependent Response:
  • P3 cells (from younger rats) showed a more robust proliferative response than P7 cells, which are naturally less capable of division.
• Signaling Pathways:
  • Western blot analysis revealed increased activation of the Akt and MAPK/ERK pathways, which are crucial for promoting cell growth and survival.

Discussion: What It Means

• Depolarization as a Stimulus:
  • The study indicates that altering the electrical state of cells can maintain or boost the proliferative capacity of cardiomyocytes.
  • This is similar to giving the cells a gentle electrical “nudge” to keep them in a youthful and active state.
• Therapeutic Implications:
  • This method could be applied to grow engineered cardiac tissue for pediatric patients with congenital heart defects.
  • By enhancing the growth of heart muscle cells while limiting the growth of fibroblasts, overall heart function might be improved.
• Selective Effects:
  • Inhibiting fibroblast proliferation is advantageous because too many fibroblasts can lead to scar tissue formation, which impairs heart function.

Conclusions: Key Takeaways

• Depolarization using potassium gluconate or ouabain increases neonatal cardiomyocyte proliferation in vitro.
• There is an optimal concentration for these agents to achieve maximal proliferative effects.
• This strategy maintains a population of proliferative heart cells, offering a potential therapeutic approach for cardiac regeneration in young patients.
• The activation of growth pathways (Akt and MAPK/ERK) provides a link between the bioelectric changes and the cell division process.

Step-by-Step Summary (Cooking Recipe Style)

• Step 1: Isolate neonatal rat heart cells and seed them into a nutrient-rich medium.
• Step 2: Add depolarizing agents (potassium gluconate or ouabain) at optimal concentrations.
• Step 3: Verify depolarization using a voltage-sensitive dye that increases in fluorescence when the cell membrane becomes less negative.
• Step 4: Stain the cells to identify cardiomyocytes and cells undergoing division.
• Step 5: Use imaging software and flow cytometry to count cells and determine the proliferation rate.
• Step 6: Perform Western blot analysis to check for the activation of key growth pathways (Akt and MAPK/ERK).
• Step 7: Compare results between younger (P3) and older (P7) cells to understand age-related differences in proliferation.

Overall Impact

• This study introduces a novel approach to stimulate heart cell growth by manipulating bioelectric signals.
• The findings open new avenues for tissue engineering and regenerative medicine, especially for treating congenital heart defects in pediatric patients.

引言：背景和动机

• 先天性心脏缺陷（CHDs）常见且危及生命，亟需创新治疗方法。
• 新生儿心肌细胞（心脏肌肉细胞）在出生后不久具备增殖能力，这对心脏生长至关重要。
• 本研究探讨通过去极化（使细胞膜电位变得不那么负）是否能刺激或维持心肌细胞在体外增殖。

关键概念和定义

• 去极化：细胞静息膜电位发生改变，使其不那么负。可比作“加电”让细胞更活跃。
• 静息膜电位 (Vmem)：细胞在静止状态下膜内外的电压差。
• 心肌细胞 (CMs)：负责收缩、泵血的心脏肌肉细胞。
• 心脏成纤维细胞 (CFs)：支撑心脏结构的细胞，产生维持细胞连接的基质。
• 增生与肥大：增生指细胞数量增加（如烤出许多小杯子蛋糕），而肥大指细胞体积变大（如做一个大杯子蛋糕）。

材料与方法（步骤详解）

• 细胞分离：
  • 从新生大鼠（出生后第3天和第7天）的心脏中取出心脏组织。
  • 将心室组织切碎，并用胶原酶消化以分离出单个细胞。
• 细胞培养：
  • 将混合的心肌细胞与心脏成纤维细胞接种于培养皿中。
  • 细胞在富含营养的培养基（Myo Media）中生长。
• 改变膜电位的处理：
  • 使用去极化剂：葡萄糖酸钾和毒草甙（ouabain）。
  • 在不同浓度下（最佳浓度：40 mM 葡萄糖酸钾和10 μM 毒草甙）处理72小时。
• 去极化验证：
  • 采用电压敏感染料 DiBAC4(3) 测量膜电位变化。
  • 染料荧光增强表明细胞膜电位成功去极化。
• 检测方法：
  • 免疫细胞化学：用心脏α-肌动蛋白标记心肌细胞，用PHH3标记正在分裂的细胞。
  • 细胞计数：利用ImageJ软件统计总细胞数及心肌细胞数。
  • 流式细胞术：检测细胞周期阶段，观察更多细胞进入分裂期（如G2和S期）。
  • 蛋白质印迹（Western Blot）：检测蛋白水平，确认关键生长通路（Akt和MAPK/ERK）的激活情况。

结果：实验发现

• 去极化验证：
  • 葡萄糖酸钾和毒草甙在各自最佳浓度下均成功去极化细胞膜。
  • 荧光增强证明细胞膜电位已被有效改变。
• 对心肌细胞的影响：
  • 去极化显著增加了心肌细胞的数量。
  • 最佳处理下，心肌细胞数量较对照组约增加了两倍。
  • 流式细胞术显示更多细胞进入DNA合成和分裂阶段（G2和S期），表明增殖增强。
• 对心脏成纤维细胞的影响：
  • 与心肌细胞不同，去极化抑制了心脏成纤维细胞的增殖。
  • 这种选择性作用有助于促进心肌生长，同时避免过多纤维组织形成。
• 年龄相关反应：
  • 出生后第3天（P3）的细胞比第7天（P7）的细胞显示出更强的增殖反应，因为P7细胞天生增殖能力较低。
• 信号通路：
  • Western Blot显示Akt及MAPK/ERK通路激活增强，这些通路对细胞生长和存活起关键作用。

讨论：意义与影响

• 去极化作为刺激因素：
  • 研究表明，改变细胞的电状态可以维持或增强心肌细胞的增殖能力。
  • 这类似于给细胞一个轻微的电刺激，使其保持年轻和活跃。
• 治疗意义：
  • 该方法有望用于为先天性心脏缺陷患儿培养工程化心脏组织。
  • 通过促进心肌细胞生长并抑制成纤维细胞过度增殖，可望改善整体心脏功能。
• 选择性效应：
  • 抑制成纤维细胞增殖有利，因为过多的成纤维细胞会导致疤痕组织形成，从而降低心脏功能。

结论：主要收获

• 利用葡萄糖酸钾或毒草甙去极化能在体外增加新生儿心肌细胞的增殖。
• 实验确定了最佳浓度，以达到最优增殖效果。
• 这种方法有助于维持心肌细胞的增殖状态，为儿童心脏再生提供了一种潜在的治疗策略。
• Akt和MAPK/ERK通路的激活将生物电变化与细胞分裂过程联系起来。

步骤总结（烹饪食谱式）

• 步骤1：分离新生大鼠心脏细胞，并将其接种于富含营养的培养基中。
• 步骤2：加入去极化剂（葡萄糖酸钾或毒草甙），使用最佳浓度进行处理。
• 步骤3：用电压敏感染料验证去极化效果，观察细胞膜是否变得不那么负。
• 步骤4：通过染色识别心肌细胞和正在分裂的细胞。
• 步骤5：利用图像软件和流式细胞仪统计细胞数量，分析增殖情况。
• 步骤6：检测蛋白水平，确认关键生长信号（如Akt和MAPK/ERK）的激活。
• 步骤7：比较P3与P7细胞的反应，了解年龄对增殖的影响。

整体影响

• 本研究为通过调控生物电信号刺激心肌细胞增殖提供了一种新思路。
• 这些发现为组织工程和再生医学开辟了新途径，尤其在治疗先天性心脏缺陷方面具有潜在应用价值。