The past decade has witnessed an explosion in the tools, resources, and publicly available data for human-based discovery implicating multiple new candidate pathways and potential therapeutic targets in cardiovascular disease. In addition to documenting associations between genetic sequence variants or circulating biomarkers and disease or clinically relevant phenotypes, techniques such as Mendelian randomization can—with some caveats—help determine whether a candidate is likely part of a causal pathway, contributing to disease pathogenesis, or simply associated with disease. The distinction is crucial as pathways mechanistically important in disease are potential therapeutic targets, whereas markers with no functional role are not.

There are ample examples of these principles, but one of the best documented is the strong, quantitative relationship between both positive and negative genetic determinants of circulating LDL (low-density lipoprotein) levels and the risk of clinically important atherosclerotic coronary artery disease.^1,2 In contrast, similar analyses of genetic determinants of CRP (C-reactive protein), a marker of inflammation that, like LDL, is powerfully predictive of coronary artery disease risk, suggest that CRP is not in the causal pathway.³ To be clear, as demonstrated in the CANTOS trial (Canakinumab Anti-Inflammatory Thrombosis Outcome),⁴ enormous evidence supports the role of inflammation in the pathogenesis of atherosclerotic vascular disease and consequent clinical events. However, CRP itself appears more a reporter of the inflammatory state than a mechanistically important part of this sequence. Advances in bioinformatic approaches combined with the ever-expanding scale of clinical data sets with detailed genetic, phenotypic, and outcome data available have led to a breathtaking acceleration of the pace of discovery with profound implications for our understanding of disease, as well as the development of therapeutic approaches.

Based on this remarkable progress, it may be tempting to argue that human evidence, particularly genetic data supporting a causal role, should be the essential foundation for identifying, evaluating, and advancing all therapeutic targets.⁵ In this view, the value of in vitro and in vivo biological models may appear diminished. Most basic scientists hope their discoveries will have implications for understanding human biology and disease and likely feel there is little or no value in discovering a cure that works only in animals. The road to failed therapies is littered with interventions that appeared promising in animal models. However, on deeper inspection, some of these encouraging preclinical results involved models with limited track record of predicting translational success or required interventions unrealistic clinically in timing or approach. For example, in the past, considerable effort was devoted to identifying treatments that reduced the development of restenosis in rodent vascular injury models, the vast majority of which failed to translate to large animal models or clinically. The combination of advances in human-based discovery and past disappointments based on animal models understandably leads some to advocate a focus driven by human genetics or genomics with little or no emphasis on nuanced investigation in animal or in vitro models.

In contrast, I would argue that both in vitro and in vivo models continue to play essential roles in our understanding of biology, disease pathogenesis, and pathophysiology and target validation for several reasons. First, genetic data sets for many phenotypes remain incomplete. Previous work has suggested that only a small minority of Food and Drug Administration–approved drug overlap is implicated by human genetic studies.⁶ While this number will undoubtedly continue to expand with the increasing scale and scope of available genetic studies, it is a fitting reminder that the absence of genetic data supporting a pathophysiological role should still not be taken as evidence that such a role is absent. Second, in many ways, the explosion of candidates fueled by genetics and genomics has only increased the need for mechanistic models to assess their functional roles, elucidate the responsible mechanisms, and optimize strategies for intervention. This nuanced understanding is often essential to the successful development of new therapies. Finally, genomic and epigenetic studies generally require relevant tissues, which may be readily available for some conditions, such as cancer, but are less so for many cardiovascular phenotypes, particularly early in the course of disease. Thus basic models of cardiovascular disease provide a crucial complement to human discovery efforts whose importance has only expanded with the explosion of putative candidates.

That said, it is important to choose the model most appropriate to the goals at hand and recognize the limitations inherent to that model. Often the goal is to mimic a clinical condition with the goal of elucidating mechanisms and validating pathways as therapeutic targets. Of course, no model perfectly recapitulates the nuanced biology of the human cardiovascular system or disease. Moreover, such studies are often performed in young, otherwise healthy animals in contrast to the generally older patients with multiple comorbidities who present clinically with cardiovascular disease. While large animal models often come closer to mimicking human biology, practical considerations including expense, access, and the challenges of genetically manipulating such models steer most investigators toward smaller, often genetically tractable species, particularly in the early stages of projects, reserving large animal studies for late preclinical development. As noted earlier, there are multiple humbling examples where success in small animals has not presaged clinical translation. However, there are also examples where they have. These include the seminal studies of Drs Marc and Janice Pfeffer along with Dr Eugene Braunwald documenting the benefits angiotensin-converting enzyme inhibition in mitigating adverse ventricular remodeling after myocardial infarction.⁷ These initial studies were performed in rats and ultimately laid a foundation for what became one of the pillars of modern therapy for heart failure with reduced ejection fraction. Recognizing the differences among models in their track record of predicting translational success as well as the importance of testing clinically relevant interventions can help place the results of experimental models in the appropriate context.

Less commonly, animal models are used not because they recapitulate human biology but precisely because of the interesting and potentially informative differences they exhibit. Examples include the remarkable regenerative capacity of the zebra fish, including the ability to regrow healthy heart tissue with minimal scarring after cardiac injury. Understanding the basis of this ability and how it was lost later in evolution could provide clues to how it might be at least partially restored. Similarly, the Burmese python’s ability to cyclically grow and shrink its organs—including the heart—after feeding in association with a remarkable increase in its metabolic oxygen consumption may provide insights that help us modulate more common forms of cardiac hypertrophy and regression. Thus we have included discussion of these models not because they resemble human biology but because they display unusual responses that may inform our understanding of more common phenotypes.

With these concepts in mind, we have invited experts to share their perspectives on a broad range of cardiovascular models. Our hope is that these reviews will serve as a resource for our community, providing an authoritative reference for cardiovascular investigators interested in learning more or potentially using these models. In addition, we have asked each of the authors to share insights based on their wealth of their experience as to the advantages and disadvantages of these models, as well as their translational relevance. We begin with 2 reviews related to sex differences in cardiovascular disease—an important and historically understudied area that was the subject of a prior Compendium in Circulation Research.⁸ The first review by Reue and Wiese⁹ discusses powerful tools available to illuminate the mechanisms responsible for sex differences in cardiovascular disease, including enabling investigators to parse the relative contributions of hormonal and chromosomal effects. The authors describe cardiovascular conditions with well-documented sex-based differences in prevalence or presentation and highlight some of the mechanistic insights garnered to date, acknowledging that much remains to be learned in this area. Cardiovascular disease is the most common cause of pregnancy-related death in women, and in the next review, Arany et al¹⁰ discuss models of pregnancy-related cardiovascular diseases including preeclampsia and peripartum cardiomyopathy. These models have provided some surprising new insights into the biology of these conditions and stimulated ongoing trials of new therapeutic approaches.

The next 5 reviews describe tools and systems used by many investigators across diverse model systems. The first by Thomas et al¹¹ describes the use of human induced pluripotent stem cells to generate cardiomyocytes and other cardiac cell types that can be studied in isolation in vitro or in structured multicellular organoid systems. This approach has the advantage of using human cells that can be derived from patients, and the authors thoughtfully discuss the potential advantages and limitations of current systems, as well as advise us on how best to select the right model for a particular application. Gonzales-Rosa¹² then discusses zebra fish cardiac models, describing both the unique advantages of this system and its limitations. As noted above, the regenerative capacity of the adult zebra fish heart is remarkable but is only one of several features that have stimulated interest in this model. Genome editing is briefly discussed in these reviews as it has enhanced the power of both induced pluripotent stem cell–derived cellular models and zebra fish studies. Liu and Olson¹³ then provide a more comprehensive review of genome editing to generate in vivo cardiovascular disease models, as well as the potential and challenges in developing this as a therapeutic strategy for cardiovascular disease. An important aspect of evaluating many animal models is the assessment of in vivo cardiac function. Sosnovik and Scherrer-Crosbie¹⁴ discuss advanced imaging approaches—including echocardiography, magnetic resonance, and positron emission tomography—to visualization of left ventricular function in small animal models.

The next 4 reviews illustrate the utility of animal models for investigating 4 different disease processes. Gisterå et al¹⁵ discussed models of atherosclerotic vascular disease. They provide a valuable perspective on legacy and current models across a broad range of species and discuss in detail murine models, including the advantages and disadvantages of actively used strains. Two reviews on heart failure follow. The first, by Pilz et al,¹⁶ discusses large and small animal models of heart failure with reduced ejection fraction. The second, by Roh et al,¹⁷ is focused on heart failure with preserved ejection fraction.¹⁷ Given our still-limited understanding of heart failure with preserved ejection fraction pathophysiology and the absence of any therapies to date that reduce mortality in this high-risk and growing population, the hope is that these models can help. Finally, Blackwell et al¹⁸ provide a detailed review of animal models of arrhythmia. The authors provide a useful review of electrophysiological principles followed by discussion of models relevant to genetic arrhythmia syndromes, genetic cardiomyopathies, and acquired arrhythmias.

The last 2 reviews in the series address conceptual issues relevant to multiple models. Given the relentless, energy-consuming work that the heart must perform, it is no surprise that cardiac metabolism has long been a topic of great interest. Bugger et al¹⁹ discuss the tools available to study both genetic and acquired models of dysregulated metabolism. Of course, some abnormalities may not be apparent in unperturbed animals at rest. Many of the reviews touch on surgical or other interventions that can be used to induce pathophysiologically relevant stress that elicits more subtle cardiac abnormalities. In the last review, Hastings et al²⁰ discuss exercise as a physiological stress that can also elicit phenotypes not apparent at rest. They also discuss exercise training protocols that can serve to elucidate both the impact of specific interventions on cardiovascular adaptations to training and as a platform for discovery of pathways mediating the benefits of exercise. While many of the reviews examine what goes wrong in models of cardiovascular disease, we believe there is also value in understanding the mechanisms that keep the heart healthy, using the exercised heart as a model, and determining whether these can be exploited to prevent or treat disease.

Of course, there are many useful models that could not be included due to space constraints, and even where models are discussed, often only an overview could be provided. We apologize to those whose work or favorite models we were unable to include. We also acknowledge that the insights shared, particularly regarding advantages and disadvantages of individual models, necessarily reflect the experience and perspectives of the authors, which may differ from those of the reader. Nevertheless, we hope these reviews will provide a useful starting place and conceptual framework, as well as a reference resource for investigators seeking to learn more about particular models and determine which might be best suited to their own needs.

This work was supported by the National Institutes of Health (R01AG061034 and R35HL155318).

Disclosures None.

The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.

For Sources of Funding and Disclosures, see page 1745.

在过去的十年中，以人类为基础的发现涉及多种新的候选途径和心血管疾病的潜在治疗靶点，工具、资源和公开可用数据呈爆炸式增长。除了记录基因序列变异或循环生物标志物与疾病或临床相关表型之间的关联外，孟德尔随机化等技术还可以帮助确定候选者是否可能是因果途径的一部分，促成疾病发病机制，或者仅仅是与疾病有关。这种区别是至关重要的，因为在疾病中具有重要机制的途径是潜在的治疗靶点，而没有功能性作用的标记则不是。

这些原则有很多例子，但最好的记录之一是循环 LDL（低密度脂蛋白）水平的阳性和阴性遗传决定因素与临床上重要的动脉粥样硬化性冠状动脉疾病的风险之间的强烈定量关系。^1,2相比之下，对 CRP（C 反应蛋白）的遗传决定因素的类似分析表明，CRP 不在因果途径中，CRP 是一种炎症标志物，与 LDL 一样，可有力预测冠状动脉疾病风险。³需要明确的是，正如 CANTOS 试验（卡纳金单抗抗炎血栓形成结果）^{4所证明的那样}大量证据支持炎症在动脉粥样硬化血管疾病和随后的临床事件的发病机制中的作用。然而，CRP 本身似乎更像是炎症状态的报告者，而不是该序列的重要机制部分。生物信息学方法的进步与不断扩大的临床数据集规模以及可用的详细遗传、表型和结果数据相结合，导致发现速度惊人地加快，对我们对疾病的理解以及发展产生深远影响的治疗方法。

基于这一显着进展，人们可能很想争辩说，人类证据，尤其是支持因果作用的遗传数据，应该是识别、评估和推进所有治疗目标的必要基础。^5个从这个角度来看，体外和体内生物模型的价值可能会降低。大多数基础科学家希望他们的发现能够对理解人类生物学和疾病产生影响，并且可能认为发现仅对动物有效的治疗方法几乎没有价值或没有价值。治疗失败的道路上到处都是在动物模型中看起来很有希望的干预措施。然而，在更深入的检查中，这些令人鼓舞的临床前结果中的一些涉及的模型在预测转化成功或所需干预在时间或方法上在临床上不切实际。例如，在过去，相当大的努力致力于确定减少啮齿动物血管损伤模型再狭窄发展的治疗方法，其中绝大多数未能转化为大型动物模型或临床。基于人类的发现的进步和过去基于动物模型的失望相结合，可以理解地导致一些人提倡由人类遗传学或基因组学驱动的焦点，很少或根本不强调动物或体外模型的细微调查。

相比之下，我认为体外和体内模型在我们理解生物学、疾病发病机制、病理生理学和目标验证方面继续发挥着重要作用，原因有几个。首先，许多表型的遗传数据集仍然不完整。先前的研究表明，只有一小部分食品和药物管理局批准的药物重叠与人类基因研究有关。^6个虽然这一数字无疑会随着可用基因研究规模和范围的扩大而继续扩大，但它提醒我们，缺乏支持病理生理作用的遗传数据仍不应被视为不存在这种作用的证据。其次，在许多方面，由遗传学和基因组学推动的候选人激增只会增加对机制模型的需求，以评估其功能作用、阐明负责任的机制并优化干预策略。这种细致入微的理解通常对于新疗法的成功开发至关重要。最后，基因组和表观遗传学研究通常需要相关组织，这些组织对于某些疾病（例如癌症）可能很容易获得，但对于许多心血管表型而言则不然，特别是在病程的早期。因此，心血管疾病的基本模型为人类发现工作提供了重要的补充，其重要性随着推定候选者的激增而扩大。

也就是说，重要的是选择最适合手头目标的模型并认识到该模型固有的局限性。通常，目标是模拟临床状况，目的是阐明机制和验证通路作为治疗目标。当然，没有任何模型可以完美地概括人类心血管系统或疾病的细微差别。此外，此类研究通常是在年轻的、其他方面健康的动物身上进行的，这与临床上表现为心血管疾病的患有多种合并症的老年患者形成鲜明对比。虽然大型动物模型通常更接近于模仿人类生物学，但包括费用、获取和基因操纵此类模型的挑战在内的实际考虑因素促使大多数研究人员转向较小的、通常在基因上易于处理的物种，特别是在项目的早期阶段，为后期的临床前开发保留大型动物研究。如前所述，有多个令人沮丧的例子表明，在小动物身上的成功并没有预示着临床转化。但是，也有他们拥有的例子。其中包括 Marc 博士和 Janice Pfeffer 博士以及 Eugene Braunwald 博士的开创性研究，记录了抑制血管紧张素转换酶在减轻心肌梗死后不良心室重构方面的益处。⁷这些初步研究是在老鼠身上进行的，最终为射血分数降低的心力衰竭现代疗法的支柱之一奠定了基础。认识到模型在预测转化成功方面的差异以及测试临床相关干预措施的重要性可以帮助将实验模型的结果置于适当的背景下。

不太常见的是，使用动物模型不是因为它们概括了人类生物学，而是因为它们表现出有趣和潜在的信息差异。例子包括斑马鱼非凡的再生能力，包括在心脏损伤后以最小的疤痕再生健康的心脏组织的能力。了解这种能力的基础以及它是如何在进化后期丢失的，可以为它如何至少部分恢复提供线索。同样，缅甸蟒蛇在进食后循环生长和收缩其器官（包括心脏）的能力与其代谢耗氧量的显着增加相关，这可能提供帮助我们调节更常见形式的心脏肥大和退化的见解。

考虑到这些概念，我们邀请了专家分享他们对广泛的心血管模型的看法。我们希望这些评论将成为我们社区的资源，为有兴趣了解更多或可能使用这些模型的心血管研究人员提供权威参考。此外，我们已要求每位作者根据他们对这些模型的优缺点及其转化相关性的丰富经验分享见解。我们从 2 篇与心血管疾病性别差异相关的评论开始——这是一个重要且历史上未被充分研究的领域，是先前《循环研究纲要》的主题。⁸ Reue 和 Wiese 的第一次评论⁹讨论了可用于阐明导致心血管疾病性别差异的机制的强大工具，包括使研究人员能够解析激素和染色体效应的相对贡献。作者描述了心血管疾病，在患病率或表现方面存在有据可查的基于性别的差异，并强调了迄今为止获得的一些机制见解，承认在这一领域仍有很多有待学习。心血管疾病是女性妊娠相关死亡的最常见原因，在下一篇综述中，Arany 等人¹⁰讨论妊娠相关心血管疾病模型，包括先兆子痫和围产期心肌病。这些模型为这些疾病的生物学提供了一些令人惊讶的新见解，并刺激了正在进行的新治疗方法试验。

接下来的 5 篇评论描述了许多研究人员在不同模型系统中使用的工具和系统。Thomas 等人¹¹的第一个描述了使用人类诱导多能干细胞生成心肌细胞和其他心肌细胞类型，这些细胞可以在体外或在结构化多细胞类器官系统中进行分离研究。这种方法的优点是可以使用来自患者的人体细胞，作者深思熟虑地讨论了当前系统的潜在优势和局限性，并建议我们如何最好地为特定应用选择正确的模型。冈萨雷斯-罗莎¹²然后讨论斑马鱼心脏模型，描述该系统的独特优势及其局限性。如上所述，成年斑马鱼心脏的再生能力非常出色，但这只是激发人们对该模型兴趣的几个特征之一。这些评论中简要讨论了基因组编辑，因为它增强了诱导多能干细胞衍生细胞模型和斑马鱼研究的能力。Liu 和 Olson ¹³随后更全面地回顾了基因组编辑以生成体内心血管疾病模型，以及将其开发为心血管疾病治疗策略的潜力和挑战。评估许多动物模型的一个重要方面是评估体内心脏功能。Sosnovik 和 Scherrer-Crosbie¹⁴讨论了先进的成像方法——包括超声心动图、磁共振和正电子发射断层扫描——以可视化小动物模型中的左心室功能。

接下来的 4 篇评论说明了动物模型在研究 4 种不同疾病过程中的效用。Gisterå 等人¹⁵讨论了动脉粥样硬化性血管疾病的模型。他们提供了关于广泛物种的遗留和当前模型的宝贵观点，并详细讨论了小鼠模型，包括积极使用的菌株的优缺点。以下是关于心力衰竭的两篇评论。第一个由 Pilz 等人¹⁶讨论了射血分数降低的心力衰竭的大型和小型动物模型。第二种方法由 Roh 等人¹⁷着重研究射血分数保留的心力衰竭。¹⁷鉴于我们对射血分数保留的心力衰竭病理生理学的了解仍然有限，而且迄今为止还没有任何疗法可以降低这一高风险和不断增长的人群的死亡率，希望这些模型能有所帮助。最后，Blackwell 等人¹⁸详细回顾了心律失常动物模型。作者对电生理学原理进行了有益的回顾，随后讨论了与遗传性心律失常综合征、遗传性心肌病和获得性心律失常相关的模型。

该系列的最后 2 篇评论解决了与多个模型相关的概念问题。鉴于心脏必须进行不间断的、耗能的工作，心脏代谢长期以来一直是人们非常感兴趣的话题也就不足为奇了。Bugger 等人¹⁹讨论了可用于研究失调代谢的遗传和后天模型的工具。当然，有些异常在静止的未受干扰的动物中可能并不明显。许多评论涉及手术或其他干预措施，这些干预措施可用于诱发病理生理相关的压力，从而引发更细微的心脏异常。在上一篇综述中，Hastings 等人²⁰将运动作为一种生理压力进行讨论，它也可以引发在休息时不明显的表型。他们还讨论了运动训练方案，这些方案可以用来阐明特定干预措施对心血管适应训练的影响，并作为发现调节运动益处的途径的平台。虽然许多评论检查心血管疾病模型出了什么问题，但我们认为了解保持心脏健康的机制、使用运动过的心脏作为模型并确定是否可以利用这些机制来预防或治疗也很有价值疾病。

当然，由于篇幅所限，许多有用的模型未能包括在内，即使在讨论模型的地方，通常也只能提供一个概述。对于那些我们无法收录其作品或最喜欢的模特的人，我们深表歉意。我们还承认，分享的见解，特别是关于个别模型的优缺点，必然反映作者的经验和观点，这可能与读者的不同。尽管如此，我们希望这些评论能够提供一个有用的起点和概念框架，并为寻求更多地了解特定模型并确定最适合他们自己需求的研究人员提供参考资源。

这项工作得到了美国国立卫生研究院 (R01AG061034 和 R35HL155318) 的支持。

披露无。

本文表达的观点不一定代表编辑或美国心脏协会的观点。

有关资金来源和披露信息，请参见第 1745 页。