If you like it, put a ring on it!

This editorial refers to ‘A novel method for high precision aortic constriction that allows for generation of specific cardiac phenotypes in mice’ by A.O. Melleby et al., pp. 1680–1690. Approximately 23 million people suffer from heart failure (HF) worldwide, and incidence is further increasing the coming decades.1 Human HF is a complex clinical syndrome, caused by a variety of underlying diseases. In long-standing systemic arterial hypertension or left ventricular (LV) outflow tract obstruction, an increase in the ratio of LV wall thickness to chamber radius increases the afterload against which the myocardium needs to contract during systole.2 Still regarded as an adaptive response, concentric hypertrophy inevitably leads to reduced LV chamber expansiveness and an increase in the elastic rigidity of the myocardium. Despite LV chamber dilation, preload reserve eventually becomes exhausted and basal contractility turns out to be mismatched to the level of afterload, provoking a minor degree, and speed of shortening of the LV chamber.3 Associated elevation of both the LV end-diastolic pressure and left atrial mean pressure increase pulmonary congestion. On the other hand, reduced systolic fractional shortening is associated with an insufficient cardiac output in general contributing directly to the clinical syndrome of congestive HF in patients with a pressure-overload condition.3 Research focusing on adverse remodelling in non-ischaemic HF often makes use of increasing the afterload of the heart to induce pressure overload. Usually the preferred model used for pressure-overload HF in rodents is transverse aortic constriction (TAC). First described by Rockman et al.4 in 1991, it was initially designed to study the molecular particulars driving cardiac hypertrophy. Besides hypertrophy, the model is characterized by cardiac fibrosis, a limited amount of inflammation and eventually leads to cardiac dilation and HF. Since the introduction of murine echocardiography, numerous studies using the TAC model in genetically engineered mice were reported with cardiac dimensions and function as outcome parameters. Whilst the TAC model was supposed to be a singular system allowing the studying of pressure overload in the rodent heart, the clinical phenotype showed to be highly variable between the different laboratories. Whereas in some groups, TAC surgery resulted in compensated hypertrophy, others showed a clinical outcome resembling more or less acute HF.5 These alterations in severity of adverse cardiac remodelling are partly due to differences in genetic background, gender, follow-up time, and severity of constriction. As these differences could account for distinct pathophysiological responses at the cellular level such as cardiomyocyte hypertrophy, fibrosis, and low-grade inflammation, the differential phenotypic outcome could also be explained by a much more simple reason. One of the most important technical aspects of the TAC model is the strength of the suture and the degree of stenosis, influenced by both ligation material as well as the technical ability of the micro-surgeon. Using either silk or the less tensile prolene threads together with inter-surgeon variability could dramatically affect the degree of stenosis. There are ample studies where the same genetically engineered mouse line subjected to TAC result in diametrically opposed results in different laboratories. Indeed, recent studies showed that the pathophysiological outcome of the pressure-overloaded heart directly correlates with the degree of constriction.6 For example, initially the drug sildenafil was described to be highly protective having an antihypertrophic effects in mice subjected to pressure overload.7 However, other laboratories could not confirm these effects for sildenafil. More in depth studies showed that the effect of sildenafil depends on the level of pressure-overload.8 To which extend these ‘technical’ variables affected the degree of cardiac remodelling in other studies are not fully understood and obviously material for re-interpretation of published data using the TAC procedure. A novel method for high precision aortic constriction in mice developed by Melleby et al.9 adds a valuable strategy to tackle this issue. Here, the authors tested different types of O-rings that represented three fixed inner diameters to constrict the ascending aorta of two commonly used mice lines, the C57BL/6J and FVB line. Functional measurement using echocardiography, MRT, invasive LV pressure–volume measurements combined with histological and molecular analysis were performed to characterize in depth the impact of the three different aortic constrictions on the haemodynamic, structural, and molecular remodelling of the heart. The authors used a minimally invasive surgery to apply the O-rings, which is a more elegant and less strenuous strategy compared to more invasive approaches that comprise partly cutting the sternum to access the aortic branch. Moreover, the ORAB technique allows standardization of the constriction and avoidance of total occlusion and circumvents excessive constrictions that are the source of the majority of early deaths reported after TAC and this beneficial feature is evidenced by a reported 99% survival of mice with an ORAB intervention. A minor note is the relatively high incidence of pre-operative deaths due to complications during intubation and thoracotomy (i.e. atria and lung injury), which can be lessened through proper practice. In addition to the technical aspect of the ORAB, the inert nitrile rubber material of the O-rings did not cause any adverse effects upon insertion and did not show any band internalization in the aortic lumen. This is particularly important since suture-based TAC is essentially dependent on reproducible constriction of a suture around a sizing needle. Although in theory constriction of a suture around a sizing needle seems to be easily performed, in reality the technique is quite complicated since the pressure used to constrict the suture is not always consistent and together with the type of suture used can result in heterogeneity in the diameter of the constriction after removing the needle. Indeed, and as also reported in the manuscript, the suture-based TAC procedure around a blunt needle resulted in various degrees of cardiac remodelling since the inner stenosis diameters showed a large range despite using exactly the same needle.10 Additionally, Furihata et al. demonstrated how tight the margins are of the diameters of the constriction to produce large effect on the outcome. In that study, three kinds of thin metal rods with a 0.400 mm, a 0.385 mm, and a 0.375 mm OD were tested and found that the use of 0.385 mm OD gauge was suitable for TAC surgery from the point of survival and caused the transition from LV hypertrophy in early phase to LV dysfunction and HF in later phases. Interestingly, the use of thicker rod (0.400 mm OD) led to LV hypertrophy and showed higher survival rate than 0.385 mm OD gauge, but did not lead to HF. Moreover, the use of the thinner rod (0.375 mm OD) induced HF, but showed low survival rate.6 Functional magnetic resonance imaging analysis showed that LV mass increased 1 week post-ORAB in the 0.61 mm and 0.66 mm groups, whereas all three ORAB groups showed increased LV mass from 8 to 20 weeks post-surgery. Interestingly, the ORAB 0.61 mm presented a rapid increase in LV wall thickness already after 1 week but gradually changed from concentric hypertrophic remodelling to congestive dilated HF in the course of 20 weeks. The ORAB 0.66 mm constriction resulted in a model of concentric hypertrophy with progressive LV hypertrophic growth, without pulmonary congestion over the 20 weeks of the study. Despite increased LV stiffness in this period, the mice maintained a stable cardiac contractility, rendering the use of this ring suitable to serve as a model for HF with mid-range reduced ejection fraction (HFmrEF), or even HF with preserved ejection fraction (HFpEF). Finally, the ORAB 0.71 mm constriction induced hypertrophic LV remodelling in mice without severely compromising systolic function. A considerable proportion of HF patients could be classified as suffering from HFpEF, a syndrome where standard HF therapies have yet to show to impact morbidity and mortality.11,12 Although animal models are invaluable to understand molecular mechanisms that distinguish HFrEF, HFmrEF, or HFpEF, a major limitation of pre-clinical models with aortic constriction to represent aspects of HFmrEF or HFpEF is that the majority of patients with HFpEF continue to have HF symptoms even in the absence of normal afterload, while only half of HFpEF patients have LV hypertrophy and frequently show no indication of LV dilatation.13 Taking this into account, the value of the current ORAB model as pre-clinical model for HFmrEF or HFpEF should be addressed with caution. Since its inception, the murine TAC model undoubtingly has been invaluable to simulate human cardiac disease and to clarify fundamental signalling processes involved in the cardiac hypertrophic response and HF development. However, apart from genetic backgrounds or gender differences of animals, a major confounding variability relies in the aortic diameter after ligation due to needle size, tightness, and/or composition of the ligature. The landmark publication by Melleby et al. introduces an alternative and standardized method to evoke afterload increases in rodents, thereby bypassing many of the technical issues introduced by conventional TAC surgery. Although this method still remains to be validated by other groups, the straightforward and simplicity of this surgical technique can and should be easily implemented in the community. Furthermore, the composition and the scalability of the O-rings make them perfectly amendable for use in other surgical models including pulmonary aorta banding. This study holds the potential to become an important reference paper as well as introducing a new ‘golden standard’ in rodent TAC studies by refining the pressure through O-rings. Conflict of interest: none declared. L.D.W. acknowledges support from the Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation, Dutch Federation of University Medical Centers, ZonMW, and the Royal Netherlands Academy of Sciences. L.D.W. was further supported by grant 311549 from the European Research Council (ERC) and a VICI award 918-156-47 from NWO. The opinions expressed in this article are not necessarily those of the Editors of Cardiovascular Research or of the European Society of Cardiology.