The human heart beats over 2 billion times during an average lifetime, driven by rhythmic depolarizations initiated by cardiac pacemaker cells within the sinoatrial node (SAN; Figure 1A). These electrical impulses are distributed throughout the heart via the cardiac conduction system (CCS), which synchronizes the excitation-contraction coupling of atrial and ventricular myocytes.¹ Failure to generate or propagate the electrical impulses results in slow heart rates and insufficient support for circulation, requiring the implantation of electronic pacemakers.² Here, we will review current approaches to generate biological pacemakers and discuss recent advances in refining engineered biological pacemakers as an alternative therapy for CCS disorders.

Figure 1. Cardiac conduction system: physiology and disease. A, Representative anatomy of the cardiac conduction system. The cardiac electric impulse originates in the sinoatrial node (SAN) and travels across the atrioventricular node (AVN), the His bundle, left and right bundle branches, and Purkinje fibers (PFs). (Right inset) Action potential morphologies at different levels of the conduction system. B, The coupled-clock system generates spontaneous diastolic depolarizations by functional interplay between ion channels at the plasma membrane (membrane clock) and local diastolic Ca²⁺ release (calcium clock). C, SAN pacemaker cell action potential and ionic fluxes responsible for automaticity. D, Disease etiologies associated with conduction system disorders. AV indicates atrioventricular; HCN, hyperpolarization-activated cyclic nucleotide-gated; RyR, ryanodine receptor; SDD, slow diastolic depolarization; SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase; and SR, sarcoplasmic reticulum.

The cardiac pacemaker is the SAN, located at the junction of the superior vena cava and right atrium, comprising a few thousand cells highly specialized in generating spontaneous action potentials. These electrical impulses rapidly propagate to the right and left atria, slowing down when encountering the atrioventricular node. The atrioventricular node delay allows the atria to contract, priming the ventricles with blood for their contraction. Then, electrical impulses proceed along the His bundle (left and right bundle branches) and rapidly spread throughout the ventricles via a network of Purkinje fibers to trigger the contraction of both ventricles.¹ Although pacemaker cells populate the entire CCS, the rate of spontaneous depolarizations in SAN cells is faster compared with atrioventricular, His, and Purkinje fiber, making the SAN the primary cardiac pacemaker (Figure 1A, inset).

The automaticity of SAN pacemaker cells relies on the activity of dual oscillators to generate rhythmic action potentials (Figure 1B). The synchronized activity of these oscillators, referred to as the membrane clock and the calcium clock, generates the unique action potential profile of pacemaker cells (Figure 1C). The membrane clock operates within the plasma membrane and relies on the hyperpolarization-activated funny current (I_f), which supplies inward current throughout diastolic depolarization via hyperpolarization-activated cyclic nucleotide-gated channels (mainly, HCN1 and HCN4). The depolarization of membrane potential activates T-type (mainly, Ca_v3.1 and Ca_v3.2) and L-type (mainly, Ca_v1.2 and Ca_v1.3) calcium channel currents (I_Ca). Ionic influx generated by I_f and I_Ca synchronizes the rhythmic calcium release from the sarcoplasmic reticulum through ryanodine receptors, facilitating the diastolic depolarization via the NCX1 (electrogenic sodium-calcium exchanger; Figure 1B and 1C).³

Despite the presence of voltage-gated tetrodotoxin (TTX)-sensitive (I_Na,TTX) and TTX-insensitive sodium (I_Na) currents, these currents do not play a major role in SAN automaticity. However, they contribute to conducting electric signals out of the SAN into the surrounding atrial tissue and intranodal conduction. Repolarization of membrane potential occurs through rapid and slow delayed rectifier potassium currents (I_Kr and I_Ks, respectively). Other I_K, including I_K,ACh, I_K,Ado, and I_K,Ca, also contributes to SAN repolarization. The intracellular sarcoplasmic reticulum calcium uptake through the SERCA (sarcoplasmic/endoplasmic reticulum calcium ATPase) in conjunction with the repolarizing currents sets the maximum diastolic membrane potential, reinitiating a new heartbeat via I_f activation.³

The SAN is a heterogeneous structure in terms of cell morphology, ionic current densities, and autonomic regulation.⁴ Furthermore, SAN pacemaker cells are enmeshed within dense fibrotic strands, electrically insulating SAN cells from hyperpolarizing currents from adjacent atrial tissue.^4,5 This unique architecture of the SAN creates conduction pathways that facilitate the unidirectional conduction of electrical signals towards surrounding atrial tissue.⁵ While the features of SAN tissue are fairly conserved across different species, variations exist^4–6; therefore, structural and functional characteristics of species-dependent SAN should be considered when developing novel biological pacemakers.

Disturbances of the CCS are intricate and multifactorial leading to failure in impulse generation and propagation often creating clinical symptoms due to bradycardias or conduction blocks (Figure 1D). While electronic pacemakers remain the mainstay therapy for these conditions, limitations and complications from electronic devices exist, and they have been discussed elsewhere.¹ Hence, the development of biological pacemakers could offer a promising therapeutic alternative to electronic devices.

Over the past 2 decades, efforts in creating biological pacemakers have been centered on overexpressing or suppressing ionic currents (functional reengineering) and differentiation of pluripotent stem cells. More recently, converting normal working myocytes into SAN-like cells by somatic reprogramming has gained interest (Figure 2A). The first de novo biological pacemaker by gene therapy was described by Dr Eduardo Marbán’s group; as reported by Miake et al⁷ recombinant adenovirus-based expression of Kir2.1AAA (which suppresses I_K1) in ventricular myocytes elicited biological pacemaker activity in vivo. Likewise, the overexpression of HCN2 (which enhances I_f) has been pioneered by Dr Michael Rosen and coworkers to create biological pacemakers.⁸

Figure 2. Current and future of biological pacemakers. A, Chronological overview of biological pacemaker targets. B, Advantages and limitations of current biological pacemaker approaches. C, Next-generation of biological pacemakers. AAV indicates adeno-associated virus; iPSC, induced pluripotent stem cells; and LNP, lipid nanoparticle. Created with BioRender.com.

Unlike functional reengineering, which manipulates a single ionic current, somatic reprogramming has advanced the development of biological pacemakers by recreating genuine replicas of pacemaker cells. The overexpression of TBX18, a gene involved in the embryonic development of the SAN, has enabled the conversion of cardiomyocytes into functional SAN-like cells, resembling fundamental physiological and morphological features of native SAN pacemaker cells.^9,10 Initial studies demonstrated that TBX18-induced reprogramming created electrical automaticity in guinea pigs with atrioventricular block,⁹ while its clinical potential was verified in a large-porcine model of complete heart block.¹⁰ Recently, we have optimized the TBX18-induced reprogramming approach using chemically modified human TBX18 mRNA in conjunction with endogenous suppressive microRNAs. This method unleashes the biological pacemaker activity using a virus-free delivery system.¹¹

Human-induced pluripotent stem cells (hiPSCs) and embryonic stem cells have also been used to generate spontaneously beating cellular clusters, demonstrating pacemaker activity after transplantation into the ventricle.^12–14 While the success of cell-based therapy depends on the number of engrafted cells,¹³ whether the engrafted cells are electrically mature and stable remains to be determined. The applicability of cell-based therapy for biological pacemakers has been discussed elsewhere.^1,15Figure 2B illustrates the advantages and limitations of current biological pacemaker approaches.

Gene and cell therapies have made tremendous advances; however, new technologies must satisfy well-defined efficacy and safety requisites prior to translation to humans. Here, we will discuss how existing and emerging technologies could lead to improved biological pacemaker strategies, paving the way for the first biological pacemaker trial in humans. As for current biological pacemaker approaches, the implementation of new methodologies should create functional pacemakers capable of generating reliable cardiac pacemaker activity with appropriate and self-adjusted responsiveness to a variety of biological stimuli.

Nonviral strategies, including polymers, lipids, inorganic nanoparticles, and encapsulated RNAs hold immense potential. mRNA-based therapy with lipid nanoparticles gained popularity with COVID-19 mRNA vaccines, demonstrating efficient gene delivery and limited immune reactivity.¹⁶ Therefore, for future biological pacemaker applications, careful considerations about the selection of RNA sequence, chemical modifications, composition of lipid nanoparticle formulations, and specific cell targets are recommended. The duration of the biological pacemaker activity remains a major concern. While adenoviral vectors are suitable for temporary applications, extending the biological activity of reprogrammed cells with mRNA or adeno-associated virus may be a feasible alternative. Nevertheless, future studies are needed to establish how persistent the biological effect remains physiologically relevant to support the circulation, and to assess for potential long-term complications related to ectopic pacing. However, transient cell reprogramming affords the advantage of redosing as needed.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing tools emerged as a prospective therapy for monogenic disorders.¹⁷ The development of new CRISPR/Cas9 systems has enabled novel applications such as transcriptional regulation and RNA editing, opening the possibilities for new modalities of biological pacemakers. CRISPR-Cas9 gene editing components are generally packaged into adeno-associated viruses. Hence, nonviral systems such as lipid nanoparticles, polymeric nanoparticles, and exosomes could alternatively function as carriers for CRISPR systems.^16,18 Although definitive changes in the genome must be approached with great caution, adenine and cytosine base editors may also be considered for creating biological pacemakers.

The clinical applicability of the biological pacemaker depends on its delivery approach to patients. Minimally invasive delivery techniques have successfully mitigated many procedural risks associated with open-chest or transarterial approaches. However, limiting the diffusion of the vector, increasing the concentration at the focal injection site could potentiate the biological activity. Formulations of viral, nonviral, or cell-based systems combined with clinical-grade biomaterials with higher viscosity could aid in controlling the reprogramming of cells in a more restricted area.

Although cell therapy strategies have shown encouraging outcomes,^19,20 additional efforts are necessary to create hiPSCs with homogeneous maturity and consistent phenotypes. Single-cell RNA sequencing platforms have been instrumental in generating transcriptional roadmaps of human pacemaker cell differentiation. These transcriptional profiles may inspire the development of hiPSC-derived biological pacemakers based on developmental or lineage cues, resulting in more mature and stable SAN-like characteristics. From a manufacturing perspective, reducing the ex vivo production time and the possibility of an off-the-shelf universal cell line provides promise for affordable and scalable applications. Additionally, minimizing immune responses to hiPSCs through autologous or matched cells could enhance the biological pacemaker activity while reducing the risks associated with arrhythmogenicity and tumorigenicity.

Tissue engineering technologies have made significant advances though numerous challenges persist in developing tissues and organs suitable for clinical translation.^21,22 Engineered cardiac organoids, generated using hiPSC-derived embryoid bodies or somatic reprogrammed cardiac pacemaker spheroids, could create multicellular tissues that resemble the nodal architecture. The promise of 3-dimensional printing technologies lies in their ability to produce complex composite tissue constructs with precise placement of biocompatible cell-laden hydrogels in a layer-by-layer fashion.^21–23 However, the efficacy and safety of these approaches are yet to be fully tested. Practical and regulatory hurdles include the utilization of multiple bioactive materials responsive to biophysical, mechanical, and biochemical stimuli, engraftment efficacy, high manufacturing costs, and potential immunoreactivity. Addressing such challenges is crucial for the successful implementation of tissue engineering in creating functional and safe biological pacemakers.

Current biological pacemaker approaches offer significant promise as temporary pacing alternatives for patients with complications from permanent electronic devices requiring hardware extraction. The implementation of novel biological pacemaker modalities that create biological twins of native pacemaker cells signals a new era in cardiac pacing. Moreover, it remains to be determined whether the current and future biological pacemaker modalities can be applied in other regions of the CCS, for different disorders such as native SAN dysfunction. Nonetheless, efficacy and safety studies should be conducted to meet the standards of regulatory agencies. Thus, optimism prevails that ongoing research efforts will overcome challenges, making its clinical translation a reality in the near future.

This research was supported by the National Institutes of Health (grant R01 HL147570 to E. Cingolani), the American Heart Association (Career Development Award 940033 to T. Mesquita), the California Institute for Regenerative Medicine (grant EDUC4-12751 to R. Miguel-dos-Santos), and the Cedars-Sinai Board of Governors.

Disclosures None.

The American Heart Association celebrates its 100^th anniversary in 2024. This article is part of a series across the entire AHA Journal portfolio written by international thought leaders on the past, present, and future of cardiovascular and cerebrovascular research and care. To explore the full Centennial Collection, visit https://www.ahajournals.org/centennial.

*T. Mesquita and R. Miguel-dos-Santos contributed equally.

For Sources of Funding and Disclosures, see page 841.

人的心脏在平均一生中跳动超过 20 亿次，由窦房结内的心脏起搏细胞引发的节律性去极化驱动（SAN；图 1A）。这些电脉冲通过心脏传导系统（CCS）分布在整个心脏，同步心房和心室肌细胞的兴奋-收缩耦合。¹无法产生或传播电脉冲会导致心率缓慢且对循环的支持不足，需要植入电子起搏器。²在这里，我们将回顾当前生成生物起搏器的方法，并讨论改进工程生物起搏器作为 CCS 疾病替代疗法的最新进展。

图 1. 心脏传导系统：生理学和疾病。 A，心脏传导系统的代表性解剖结构。心脏电脉冲起源于窦房结 (SAN)，并穿过房室结 (AVN)、希氏束、左束支和右束支以及浦肯野纤维 (PF)。 （右插图）传导系统不同水平的动作电位形态。B，耦合时钟系统通过质膜离子通道（膜钟）和局部舒张期 Ca ²⁺释放（钙钟）之间的功能相互作用产生自发舒张期去极化。C，SAN 起搏器细胞动作电位和离子通量负责自动化。D，与传导系统障碍相关的疾病病因。 AV表示房室； HCN，超极化激活环核苷酸门控； RyR，兰尼碱受体； SDD-慢舒张除极； SERCA，肌浆/内质网钙 ATP 酶；和 SR，肌浆网。

心脏起搏器是 SAN，位于上腔静脉和右心房的交界处，由数千个高度专门产生自发动作电位的细胞组成。这些电脉冲快速传播到右心房和左心房，在遇到房室结时减慢。房室结延迟使心房收缩，为心室的收缩提供血液。然后，电脉冲沿着希氏束（左右束支）传播，并通过浦肯野纤维网络迅速传播到整个心室，引发两个心室的收缩。¹虽然起搏细胞遍布整个 CCS，但 SAN 细胞的自发去极化速率比房室纤维、希氏纤维和浦肯野纤维更快，这使得 SAN 成为主要的心脏起搏器（图 1A，插图）。

SAN 起搏器细胞的自动性依赖于双振荡器的活动来产生节律性动作电位（图 1B）。这些振荡器的同步活动（称为膜时钟和钙时钟）产生起搏细胞独特的动作电位曲线（图 1C）。膜时钟在质膜内运行，依赖于超极化激活的滑稽电流 (I _f )，该电流通过超极化激活的环核苷酸门控通道（主要是 HCN1 和 HCN4）在整个舒张期去极化过程中提供内向电流。膜电位的去极化激活T型（主要是Ca _v 3.1和Ca _v 3.2）和L型（主要是Ca _v 1.2和Ca _v 1.3）钙通道电流（I _Ca）。 I _f和 I _Ca产生的离子流入通过兰尼碱受体使肌浆网有节奏的钙释放同步，通过 NCX1（生电钠钙交换器；图 1B 和 1C）促进舒张期去极化。³

尽管存在电压门控河豚毒素 (TTX) 敏感 (I _Na,TTX ) 和 TTX 不敏感钠 (I _Na ) 电流，但这些电流在 SAN 自动化中并不起主要作用。然而，它们有助于将电信号从 SAN 传导到周围的心房组织和结内传导。膜电位的复极化通过快速和慢速延迟整流钾电流（分别为 I _Kr和 I _Ks）发生。其他_IK，包括 IK _,ACh、IK _,Ado和 IK _,Ca，也有助于 SAN 复极化。通过 SERCA（肌浆/内质网钙 ATP 酶）的细胞内肌浆网钙摄取与复极电流相结合，设定最大舒张膜电位，通过 I _f激活重新启动新的心跳。³

SAN在细胞形态、离子电流密度和自主调节方面是一种异质结构。⁴此外，SAN 起搏器细胞陷入致密的纤维束中，使 SAN 细胞与邻近心房组织的超极化电流电绝缘。^4,5 SAN 的这种独特架构创建了传导通路，促进电信号向周围心房组织的单向传导。⁵虽然 SAN 组织的特征在不同物种中相当保守，但存在差异^4-6；因此，在开发新型生物起搏器时应考虑物种依赖性SAN的结构和功能特征。

CCS 的干扰是复杂的、多因素的，导致脉冲生成和传播失败，通常会因心动过缓或传导阻滞而产生临床症状（图 1D）。虽然电子起搏器仍然是这些疾病的主要治疗方法，但电子设备的局限性和并发症仍然存在，并且已在其他地方进行了讨论。¹因此，生物起搏器的发展可以为电子设备提供一种有前景的治疗替代方案。

在过去的二十年里，创造生物起搏器的努力一直集中在过度表达或抑制离子电流（功能重组）和多能干细胞的分化上。最近，通过体细胞重编程将正常工作的肌细胞转化为 SAN 样细胞引起了人们的兴趣（图 2A）。 Eduardo Marbán 博士的团队描述了第一个通过基因疗法从头制造的生物起搏器；正如 Miake 等人报道的^7，心室肌细胞中基于重组腺病毒的 Kir2.1AAA（抑制 I _K1）表达引发了体内生物起搏器活性。同样， Michael Rosen 博士及其同事率先通过HCN2 的过度表达（增强 I _{f ）来创建生物起搏器。} ⁸

图 2. 生物起搏器的现状和未来。 A，生物起搏器目标的时间概述。B，当前生物起搏器方法的优点和局限性。C，下一代生物起搏器。 AAV表示腺相关病毒； iPSC，诱导多能干细胞；和LNP，脂质纳米颗粒。使用 BioRender.com 创建。

与操纵单一离子电流的功能重组不同，体细胞重编程通过重建起搏细胞的真实复制品，促进了生物起搏器的发展。TBX18 （一种参与 SAN 胚胎发育的基因）的过表达使心肌细胞能够转化为功能性 SAN 样细胞，类似于天然 SAN 起搏细胞的基本生理和形态特征。^9,10初步研究表明，TBX18诱导的重编程在患有房室传导阻滞的豚鼠中产生了电自动性，⁹而其临床潜力在完全心脏传导阻滞的大型猪模型中得到了验证。¹⁰最近，我们使用化学修饰的人TBX18 mRNA 与内源性抑制性 microRNA 结合优化了TBX18诱导的重编程方法。该方法使用无病毒的递送系统释放生物起搏器活性。¹¹

人类诱导的多能干细胞（hiPSC）和胚胎干细胞也被用来产生自发跳动的细胞簇，证明移植到心室后具有起搏器活性。^12–14虽然基于细胞的治疗的成功取决于移植细胞的数量，¹³移植细胞是否电成熟和稳定仍有待确定。基于细胞的疗法对生物起搏器的适用性已在其他地方讨论过。^1,15图 2B 说明了当前生物起搏器方法的优点和局限性。

基因和细胞疗法取得了巨大进步；然而，新技术在转化为人类之前必须满足明确的功效和安全要求。在这里，我们将讨论现有和新兴技术如何改进生物起搏器策略，为首次人体生物起搏器试验铺平道路。对于当前的生物起搏器方法，新方法的实施应该创造出功能性起搏器，能够产生可靠的心脏起搏器活动，并对各种生物刺激具有适当和自我调节的响应能力。

非病毒策略，包括聚合物、脂质、无机纳米粒子和封装的 RNA，具有巨大的潜力。基于 mRNA 的脂质纳米粒子疗法因 COVID-19 mRNA 疫苗而受到欢迎，证明了有效的基因传递和有限的免疫反应性。¹⁶因此，对于未来的生物起搏器应用，建议仔细考虑 RNA 序列的选择、化学修饰、脂质纳米颗粒配方的组成以及特定的细胞靶标。生物起搏器活动的持续时间仍然是一个主要问题。虽然腺病毒载体适合临时应用，但用 mRNA 或腺相关病毒扩展重编程细胞的生物活性可能是一种可行的替代方案。然而，未来的研究需要确定生物效应在生理上与支持循环的相关性有多持久，并评估与异位起搏相关的潜在长期并发症。然而，瞬时细胞重编程具有根据需要重新给药的优点。

成簇规则间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 ( Cas9 ) 基因编辑工具成为单基因疾病的前瞻性疗法。¹⁷新的 CRISPR/Cas9 系统的开发实现了转录调控和 RNA 编辑等新应用，为生物起搏器的新模式开辟了可能性。CRISPR-Cas9基因编辑组件通常被包装到腺相关病毒中。因此，脂质纳米颗粒、聚合物纳米颗粒和外泌体等非病毒系统也可以作为 CRISPR 系统的载体。^16,18尽管必须非常谨慎地对待基因组中的决定性变化，但腺嘌呤和胞嘧啶碱基编辑器也可以考虑用于创建生物起搏器。

生物起搏器的临床适用性取决于其向患者提供的方式。微创输送技术已成功降低了与开胸或经动脉途径相关的许多手术风险。然而，限制载体的扩散、增加局部注射部位的浓度可以增强生物活性。病毒、非病毒或基于细胞的系统的制剂与具有较高粘度的临床级生物材料相结合，可以帮助控制更受限区域内的细胞重编程。

尽管细胞治疗策略已显示出令人鼓舞的结果，^19,20还需要付出额外的努力来创建具有同质成熟度和一致表型的 hiPSC。单细胞 RNA 测序平台在生成人类起搏细胞分化的转录路线图方面发挥了重要作用。这些转录谱可能会激发基于发育或谱系线索的 hiPSC 衍生生物起搏器的开发，从而产生更成熟和稳定的 SAN 样特征。从制造角度来看，减少离体生产时间和现成通用细胞系的可能性为经济实惠且可扩展的应用提供了希望。此外，通过自体或匹配细胞最大限度地减少对 hiPSC 的免疫反应可以增强生物起搏器活性，同时降低与心律失常和致瘤性相关的风险。

尽管在开发适合临床转化的组织和器官方面仍然存在许多挑战，但组织工程技术已经取得了重大进展。^21,22使用 hiPSC 衍生的胚状体或体细胞重新编程的心脏起搏器球体生成的工程心脏类器官，可以创建类似于节点结构的多细胞组织。 3D打印技术的前景在于它们能够生产复杂的复合组织结构，并以逐层的方式精确放置生物相容性的充满细胞的水凝胶。^21-23然而，这些方法的有效性和安全性尚未得到充分测试。实际和监管障碍包括利用对生物物理、机械和生化刺激敏感的多种生物活性材料、植入功效、高制造成本和潜在的免疫反应性。解决这些挑战对于成功实施组织工程来创建功能性且安全的生物起搏器至关重要。

目前的生物起搏器方法为患有需要硬件提取的永久性电子设备并发症的患者提供了作为临时起搏替代方案的巨大前景。新型生物起搏器模式的实施创造了天然起搏器细胞的生物双胞胎，标志着心脏起搏的新时代。此外，目前和未来的生物起搏器模式是否可以应用于 CCS 的其他区域，以治疗不同的疾病，例如天然 SAN 功能障碍，仍有待确定。尽管如此，仍应进行功效和安全性研究，以满足监管机构的标准。因此，人们乐观地认为正在进行的研究工作将克服挑战，使其临床转化在不久的将来成为现实。

这项研究得到了美国国立卫生研究院（授予 E. Cingolani 的 R01 HL147570 拨款）、美国心脏协会（授予 T. Mesquita 职业发展奖 940033）、加州再生医学研究所（授予 R. Miguel EDUC4-12751）的支持-多斯-桑托斯）和雪松-西奈理事会。

披露无。

美国心脏协会将于 2024 年庆祝成立 100 周年。^本文是国际思想领袖撰写的整个 AHA 期刊系列文章的一部分，内容涉及心脑血管研究和护理的过去、现在和未来。要探索完整的百年纪念收藏，请访问 https://www.ahajournals.org/centennial。

*T。 Mesquita 和 R. Miguel-dos-Santos 的贡献相等。

有关资金来源和披露信息，请参阅第 841 页。