# 4D ultrasound reveals regional left atrial strain remodelling in a murine model of reversible pressure overload

John Salvas, Thomas Moore-Morris, Craig Goergen, Pierre Sicard

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John Salvas, Thomas Moore-Morris, Craig Goergen, Pierre Sicard. 4D ultrasound reveals regional left atrial strain remodelling in a murine model of reversible pressure overload. The Journal of Physiology, In press, Online ahead of print. 10.1113/JP289361 hal-05355898

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# 4D ultrasound reveals regional left atrial strain remodelling in a murine model of reversible pressure overload

John P. Salvas<fnref n="1" />,<fnref n="2" />, Thomas Moore-Morris<fnref n="3" />, Craig J. Goergen<fnref n="1" /> ID and Pierre Sicard<fnref n="4" />

Handling Editors: Bjorn Knollmann & Leonardo Sacconi

The peer review history is available in the Supporting Information section of this article (https://doi.org/10.1113/JP289361#support-information-section).

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**Abstract figure legend** Traditional planar imaging approaches of the left atrium do not capture complex geometry. Four-dimensional imaging allows the acquisition of 3D data + time to visualize left atrial geometry and kinematics.

J. P. Salvas received B.S. and M.S. degrees in biomedical engineering from Purdue University, West Lafayette, IN, USA. He has been a Researcher with the Cardiovascular Imaging Research Laboratory, Weldon School of Biomedical Engineering, Purdue University, for a number of years. After his B.S. degree, he interned with the Institut National de la Santé et de la Recherche Médicale (INSERM), Montpellier, France. Later he returned to Purdue to finish his M.S. degree with research interests in left atrial mechanics, cardiovascular imaging and heart failure. In the future, he plans to complete his medical degree.

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<footnote n="1">¹Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, USA</footnote>

<footnote n="2">²Indiana University School of Medicine, Indianapolis, Indiana, USA</footnote>

<footnote n="3">³IGF, Univ Montpellier, CNRS, INSERM, Montpellier, France</footnote>

<footnote n="4">⁴PhyMedExp, Univ Montpellier, Inserm, CNRS, Montpellier, France</footnote>
We designed an advanced analysis technique to investigate left atrial biomechanics in a model of pressure overload and release. The data are first reoriented in a standardized fashion. Next, the atrial endocardium is manually segmented over the cardiac cycle to create a 4D mesh. Functional metrics including volume and strain are extracted from the mesh. We found left atrial volume was increased and global longitudinal strain was decreased after induced pressure overload. The analysis technique also allows regional strain quantification which exhibited heterogeneous remodelling in pressure overload and release.

**Abstract** As a marker of atrial mechanics, left atrial (LA) strain offers unique insight into the functional impact of heart failure treatments. However, given its complex geometry and contra-ctile pattern, the evolution of global and regional LA biomechanics during pressure overload and subsequent release remains unclear. We performed four-dimensional (4D) ultrasound in mice (n = 8) to evaluate global and regional LA kinematics at baseline, 3 weeks after transverse aortic constriction (TAC), and 1 and 4 weeks after aortic debanding (deTAC). LA maximum volume increased from 11.8 ± 0.8 µL at baseline to 30.4 ± 9.4 µL after TAC (P = 0.0024), returning to baseline after deTAC. LA ejection fraction declined from 68.0 ± 5.8 to 29.1 ± 9.2% after TAC (P < 0.0001), partially recovering after deTAC. Global longitudinal strain dropped from 24.8 ± 3.6% at baseline to 10.2 ± 3.7% after TAC (P < 0.0001), improving to 18.9 ± 3.5% by 4 weeks after deTAC (P = 0.0073 vs baseline). Despite partial functional recovery, fibrosis remained elevated after pressure release. Notably, regional strain recovery was heterogeneous, with anterior strain measurements showing stronger correlations with delayed ventricular functional improvement than global LA strain, highlighting the superior predictive value of regional analysis. This approach reveals incomplete and region-specific atrial recovery following pressure overload reversal, offering new perspectives on reverse remodelling and potential prognostic value in heart failure management.

(Received 30 May 2025; accepted after revision 23 September 2025; first published online 24 October 2025)

**Corresponding authors** P. Sicard: INSERM, CNRS, Université de Montpellier, PHYMEDEXP CHU Arnaud De Villeneuve Bât Crastes de Paulet, 371 Avenue du Doyen Gaston Giraud, 34295 Montpellier cedex, France. Email: pierre.sicard@inserm.fr

C. J. Goergen: Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA. Email: cgoergen@purdue.edu

**Key points**

* Advanced 4D ultrasound enables precise assessment of regional left atrial strain dynamics throughout cardiac remodelling.

* Left atrial strain reveals region-specific remodelling patterns during both pressure overload and its reversal.

* Functional changes in the atrium, particularly in the anterior wall, serve as early predictors of subsequent ventricular remodelling.

**Introduction**

Despite having a thinner and weaker wall compared to the left ventricle (LV), the left atrium (LA) plays a crucial role in maintaining optimal cardiac performance (Barbier et al., 1999). In conditions such as heart failure with pressure overload, the LA is one of the first chambers to undergo acute structural and functional changes (Habibi et al., 2014; Lim et al., 2024; Salvas et al., 2025). These changes involve an increase in LA volume, hyper-trophy of cardiomyocytes and, ultimately, fibrosis, all of which contribute to a decline in LA function (de Jong

et al., 2013; Hanif et al., 2017; Inciardi et al., 2022). Recent advances in specific cardiac pharmacological therapies have demonstrated the potential to induce reverse remodelling, improving both the structure and function of the left ventricle and atrium. This process of reverse remodelling has only recently been studied and is becoming an important measure for assessing the effectiveness of treatments, allowing for differentiation between responsive and non-responsive patients (Moon et al., 2022). LA strain, in particular, has emerged as a valuable indicator of treatment response and prognosis (Barki et al., 2024; Jia et al., 2022; Moon et al., 2022).
However, a deeper understanding of LA plasticity during the reverse remodelling process is essential for optimizing patient outcomes and guiding therapeutic strategies. Given the complex geometry and contraction patterns of the LA (Colazzo et al., 2015), limited clinical and pre-clinical information is available on how LA strain changes in response to pressure overload and subsequent reversal. Four-dimensional (4D) strain imaging is an advanced technology that can be used to capture intricate details of cardiac deformation (Damen et al., 2017). 4D ultrasound images can be spatiotemporally compiled and analysed to create volumetric segmentations that provide insights into global and regional mechanics of the cardiovascular system (Damen et al., 2021). To further investigate gaps in LA mechanics, we used state-of-the-art high-resolution echocardiography and developed an advanced strain analysis algorithm to investigate the 4D biomechanics of the LA in a murine model of reversible cardiac pressure overload.

# Materials and methods

## Ethical approval

Experiments were conducted following European Directive (2010/63/EU) and French laws for laboratory animal use. The local institutional ethics committee (CCEA no. 36) approved this study (no. 2 023 013 113 485 359). We followed the ARRIVE guidelines 2.0 for animal reporting (Percie du Sert et al., 2020) and the following experiments comply with the policies and regulations of *The Journal of Physiology* (O'Halloran, 2024). Briefly, we used 8-week-old C57BL/6JRj male mice (n = 8). The animal habitat was held at a constant 21 ± 1°C with 55 ± 1% humidity. Animals had access to food and water *ad libitum*. High-resolution ultrasound was performed at baseline, 3 weeks after transverse aortic constriction (post-TAC), 1 week after aortic de-banding (deTAC) and 4 weeks after deTAC (Fig. A1). A histological cohort underwent a sham (n = 4) or TAC (n = 4) procedure prior to tissue collection at 3 weeks. Four of the 4 week deTAC animals were also used for histology (Fig. A1). All animals were killed via cervical dislocation under deep anaesthesia (2.5% isoflurane mixed with room air).

## Transverse aortic constriction and aortic debanding

As previously described, a left thoracotomy was performed in the third intercostal space to induce a TAC (Eichhorn et al., 2018; Ghajar-Rahimi et al., 2025; Moore-Morris et al., 2014). Briefly, we anaesthetized the mice using a mixture of 3.0% isoflurane and room air. Once adequate anaesthesia was confirmed via tail

pinch reflex, we intubated the mice and placed them on a ventilator (MiniVent Type 845, Hugo Sachs Elektronik, Germany) with a mixture of 2.0% isoflurane and room air. Respiratory rate was maintained at 140 breaths/min with a tidal volume of 225 µL. We then injected a mixture of lidocaine 5 mg/kg and 0.5% bupivacaine 3.5 mg/kg (50/50–100 µL) subcutaneously at the incision site. In addition, an intraperitoneal injection (IP) of buprenorphine was administered 30 min prior to surgery. We made a 0.5–1.0 cm transverse incision in the third left intercostal space. We dissected the pectoralis muscle with an electrosurgical unit, followed by a blunt dissection technique to visualize the aortic arch. We used a homemade J-hook wire to isolate the aortic arch and placed a 27-gauge needle next to the arch. We tied a 6.0 PVDF mooring hitch suture around the needle and aorta distal to the innominate artery but proximal to the left common carotid artery. After successful tightening of the suture, we removed the 27-gauge needle. We then closed the thoracic cavity and skin with two 5.0 resorbable simple sutures (OptimeR, Peters Surgical, Plymouth, MA, USA). After stopping the isoflurane and extubating the mice, the animal's spontaneous respiration returned. Mice were placed on a heating pad to recover and monitored continuously throughout the perioperative period. Following the recovery period, we injected mice once daily with buprenorphine (0.1 mg/kg, i.P.) for three consecutive days following surgery.

Three weeks after initial TAC, as previously described (Ghajar-Rahimi et al., 2025), we performed the aortic de-banding (deTAC) procedure. Briefly, we anaesthetized the mice using a mixture of 2.5% isoflurane and room air without intubation. After confirmation of adequate anaesthesia with a tail pinch reflex, we injected local anaesthetic (lidocaine 5 mg/kg and 0.5% bupivacaine 3.5 mg/kg, 50/50–100 µL) and made a small incision to expose the previously enclosed suture. Forceps were used to gently pull on one end of the suture to release the aortic constriction. We closed the skin with a 5.0 resorbable suture and allowed the mice to recover as described above.

## Ultrasound imaging

We used a Vevo 3100 high-frequency ultrasound system (FUJIFILM VisualSonics) with a 40 MHz centre frequency MX550D linear array probe with axial resolution of 40 µm. Animals were anaesthetized with a mixture of 2.5% isoflurane and room air, without intubation, as previously described (Salvas et al., 2025). Ventral thoracic hair was removed using a depilatory cream, and the mice were positioned supine and secured onto the imaging stage. We then performed 2D echocardiography following American Physiological Society guidelines for assessment of murine LV cardiac function (Lindsey et al., 2018).
The core temperature was maintained at 36–37.5°C, and the heart rate was monitored and maintained at >450 beats/min throughout the 15 min procedure. We used the parasternal long-axis view to collect B-mode images of the left ventricle at a frame rate of 250 frames/s. Offline images were used to calculate 2D LV strain and LV mass using Vevo Strain Software 1.0 (FUJIFILM VisualSonics).

To assess LA volume and function, we used 4D echocardiography at (1) baseline, (2) 3 weeks post-TAC, (3) 1 week post-deTAC and (4) 4 weeks post-deTAC. We used a linear step motor with a step size of 0.13 mm, frame rate of 300 frames/s, and gating from cardiac and respiratory signals using ECG-gated kilohertz visualization. We scanned from the apex of the left ventricle up to the aortic arch to ensure the entire LA was imaged (Fig. 1A). Ultrasound data were then spatiotemporally compiled into an average 4D (3D + time) dataset using MATLAB 2023a (Fig. 1B).

## 4D strain analysis

A previously described in-house graphical user interface (GUI) for left ventricular 4D strain analysis was adapted for left atrial analysis (Damen et al., 2021; Leyba et al., 2023). Each data set was set to baseline at the onset of atrial diastole, indicated by the QRS complex. We oriented the data along a standard longitudinal axis through the mitral valve. We then estimated the motion of the mitral valve and defined superior of the LA along the longitudinal axis (Fig. 1C). We interpolated short-axis slices from the stacked data corresponding to locations from the mitral valve to the superior LA. Depending on LA geometry, up to 10 short-axis slices were used for manual segmentation. For each short axis slice, six equally spaced points were placed. Each point in each slice was tracked over the entire cardiac cycle and splines were used to interpolate LA myocardium position between defined points (Fig. 1D).

Once all points were tracked, a 4D volumetric mesh, including time, was created in order to calculate global function measurements (max and min volumes, ejection fraction, strain and strain rate) as well as extract regional function metrics (Fig. 1E). We calculated left atrial ejection fraction (EF) as:

$$EF = \frac{\text{max volume} - \text{min volume}}{\text{max volume}} \times 100\%$$

We then extracted and mapped circumferential, longitudinal and surface area strain as defined below for improved visualization of regional differences (Fig. 1F and G). Peak LA strain values are measured from the highest peak, which may represent peak reservoir strain, and strain rates represent the slope of the curve of the highest strain peak, although no formal distinction between atrial phases was made.

## Regional strain calculation

Longitudinal strain ($E_{ll}$) was estimated using the Green–LaGrange strain tensor at each point around the circumference of the atrium:

$$E_{ll}(\theta, t) = \frac{1}{2} \left[ \left( \frac{L(\theta, t)}{L_M(\theta)} \right)^2 - 1 \right] \times 100\%$$

where L is the length from the superior LA down to the mitral valve along the endocardial boundary at a rotation $\theta$ over time t in a representative cardiac cycle. $L_M$ is the length at the maximum atrial volume (i.e. $t = 0$). Regional longitudinal peak strain and strain rate estimates were extracted from strain curves from specific $\theta$ values corresponding to the anterior, anterolateral, posterolateral, posterior, posterior septal and anterior septal walls of the atrium. Global longitudinal strain and strain rate were calculated by taking the average over all $\theta$.

To obtain estimates for circumferential strain ($E_{cc}$), we calculated the circumferential component of the Green–LaGrange strain tensor from the 4D mesh at each short axis slice (Damen et al., 2021; Morrison et al., 2009):

$$E_{cc}(z, t) = \frac{1}{2} \left[ \left( \frac{C(z, t)}{C_M(z)} \right)^2 - 1 \right] \times 100\%$$

where C is the relative circumference along the short-axis slice perpendicular to the longitudinal z axis over time t in a representative cardiac cycle. $C_M$ is the circumference at the maximum atrial volume. Regional circumferential strain estimates in the superior, mid-LA and mitral annulus regions were calculated by generating strain curves at specific defined slices (Damen et al., 2021). Peak circumferential strain and strain rate were extracted from each curve. Global circumferential strain and strain rate were calculated by taking the average over all slices.

Surface area strain ($E_a$) was estimated by using the engineering strain approximation in the Lagrangian reference frame:

$$E_a(z, \theta, t) = \left[ \frac{A(z, \theta, t) - A_M(z, \theta)}{A_M(z, \theta)} \right] \times 100\%$$

where A is the surface area on the endocardial volume between sequential slices on the longitudinal z-axis at specific rotational $\theta$ location over time t in a representative cardiac cycle. $A_M(z, \theta)$ is surface area at specific z slices and $\theta$ rotations at the maximum atrial volume. Briefly, the 3D mesh generated from manual contouring was sampled at 60 time points over the cardiac cycle, with 60 rotations around the longitudinal axis, and 60 short-axis slices from the superior LA to the mitral annulus for a total of 3600 nodes at each time point. Surface area of the LA endocardium was calculated from the area between nodes.
<figure><img src="image_5.png" /><figcaption>Figure 1. Left atrial imaging and strain analysis steps</figcaption></figure>

A, four-dimensional (4D) imaging acquisition process. B, specialized atrial short-axis image stack of a representative cardiac cycle compiled into 3D + time data. C, schematic of data reorientation for standardized analysis. The left ventricle is outlined with the red continuous line while the left atrium is outlined with the yellow continuous line. The long-axis view of the atrium and mitral valve are visualized with yellow dotted lines. D, manual segmentation of the LA. Blue dots represent the atrial endocardium which are tracked over the cardiac cycle. The yellow line represents the long-axis view seen in C. E, after manual segmentation of all points, a 4D volume is extracted. F, representative global longitudinal (GLS), circumferential (GCS) and surface area strain (GSAS) curves in conjunction with the left atrial and ventricular cycles. G, 4D strain extraction with representative surface area and longitudinal strain maps (circumferential strain is not shown).
Surface area strain was then calculated at each time point based on the change in surface area at each time point relative to the initial surface area. Regional surface area strain and strain rates were calculated by generating an average metric in each defined region. Global surface area strain and strain rate were calculated by taking the average over all *z* and *θ*.

## Immunofluorescence

Sham (n = 4), 3 week TAC (n = 4) and 4 week deTAC (n = 4) hearts were fixed overnight in 4% PFA, subjected to a sucrose gradient (5, 10 and 20% sucrose in PBS) and included in optimal cutting temperature compound. Sections 12 µm thick were washed (PBS), permeabilized (PBS, 0.1% Triton), blocked in blocking solution or BS (PBS, 0.1% Triton, 2% BSA) and incubated with primary antibody (Goat anti-Collagen type I, Southern Biotech 1310-01, Homewood, AL, USA) overnight. Sections were then washed (PBS) and incubated for 2 h with secondary antibody in BS (Anti-Goat, Alexa 647, ThermoFisher A32849, Waltham, MA, USA) and DAPI. Sections were then washed (PBS) and mounted (Fluoromount, Sigma, St Louis, MO, USA). Images were acquired with a Zeiss Axioscan 7 and were processed with Zen and ImageJ software (Schneider et al., 2012). Collagen Type I+ area was measured globally throughout the LA as a percentage of collagen I-positive area over total tissue area. We then measured the epicardial collagen thickness in both the superior and inferior left atrial appendage (LAA) in —two to three consecutive sections per individual heart.

## Statistical analysis

All statistical analysis was performed in prism 10.4.1 (GraphPad Software, La Jolla, CA, USA), and the data are presented as mean ± SD. We used a one-way repeated measures ANOVA with Tukey *post hoc* multiple comparisons test to compare baseline, 3 week TAC, 1 week deTAC and 4 week deTAC global parameters, checking for normality with a Shapiro-Wilk test. A Friedman test with Dunn's multiple comparisons test was used for non-normally distributed data. For regional strain analyses, we normalized the data for each metric to its baseline value. We then used a two-way repeated measures ANOVA with Tukey *post hoc* multiple comparisons test to compare regional strain and strain rates over time. We used a Pearson correlation to describe relationships between 3 week TAC and 4 week post-deTAC variables. Lastly, an ordinary one-way ANOVA or two-way ANOVA was used to compare histological results. We set the threshold for statistical significance at *P* < 0.05.

## Results

### Longitudinal 4D ultrasound assessment of LA geometry and remodelling

We used high-resolution 4D ultrasound images (Fig. 2A) to evaluate LA geometry and global function before and after (post-) TAC and after (post-) deTAC (Fig. 2B). LA max and min volumes were increased 3 weeks post-TAC (max: 30.5 ± 9.4 µL; min: 22.2 ± 9.0 µL) compared to baseline (max: 11.8 ± 0.8 µL, *P* = 0.002; min: 3.8 ± 0.9 µL, *P* = 0.002). While LA max volume recovered to baseline levels after deTAC [4 weeks (4w)-deTAC: 16.3 ± 4.4 µL], LA min volume was significantly greater than baseline after deTAC (4w-deTAC: 7.7 ± 2.9 µL; Fig. 2C and D). Baseline LA EF calculated from volumetric data was 68.0 ± 5.8% and significantly decreased to 29.1 ± 9.2% after TAC (*P* < 0.001). This quickly recovered to 49.2 ± 8.5% 1 week and 53.8 ± 5.4% 4 weeks post-deTAC but remained significantly lower compared to baseline (*P* = 0.025; Fig. 2E).

We also assessed LV function using a parasternal long axis view to obtain measurements of LV longitudinal strain. LV longitudinal strain was decreased 3 weeks after TAC but recovered to baseline levels 4 weeks post-deTAC (Fig. A2).

### Global LA function partially recovers after deTAC

Next, we used advanced 4D image analysis techniques to evaluate global LA strain trajectories (Fig. 3A–C). LA peak global longitudinal strain was 24.8 ± 3.6% at baseline and decreased to 10.2 ± 3.7% 3 weeks post TAC (*P* < 0.001). LA longitudinal strain increased to 18.0 ± 3.5% 1 week post-deTAC (*P* = 0.001) and remained constant at 18.9 ± 3.5% 4 weeks post-deTAC (*P* = 0.863), which was lower than baseline (*P* = 0.007; Fig. 3D). LA global longitudinal strain rate was 1.51 ± 0.20 at baseline and decreased to 0.65 ± 0.28 3 weeks post-TAC (*P* < 0.001). LA longitudinal strain rate increased to 1.09 ± 0.20 at 1 week (*P* = 0.016) and 1.23 ± 0.27 at 4 weeks post-deTAC which was similar to baseline (*P* = 0.075; Fig. 3E). Additionally, circumferential and surface area strain and strain rates showed similar reductions 3 weeks post-TAC followed by a partial recovery by 4 weeks deTAC (Appendix and Fig. A3). Longitudinal, circumferential and surface area strain curves and strain maps for each mouse can be found in Figs A4–A9.

We then sought to determine if global LA maladaptation was associated with LV dysfunction (LV functional and morphometric trajectories are detailed in the Supplementary Material). LA ejection fraction (*r* = 0.768, *P* < 0.001), global longitudinal strain (*r* = 0.698, *P* < 0.001) and global longitudinal strain rate (*r* = 0.705, *P* < 0.001) all correlated strongly with LV
longitudinal strain at all time points (Fig. 3F–H). Further, circumferential and surface area strain and strain rates all also strongly correlated with LV longitudinal strain (Fig. A3).

## 4D LA strain mapping uncovers heterogeneous regional recovery

Four-dimensional strain mapping enables the visualization of regional differences during cardiac contraction and the quantification of heterogeneous remodelling (Videos S1–S3). We tested the hypothesis that LA reverse remodelling is regionally heterogeneous.

To assess this, we quantified regional differences by extracting peak strain and strain rate values from the six defined regions around the circumference of the LA (Fig. 4A–C). At baseline, longitudinal peak strain and strain rate appeared similar across regions (Fig. A10). However, at 3 weeks post-TAC, after normalizing to baseline values, regional differences became apparent particularly between the anterior and posterior areas (Fig. 4D and E). Specifically, the anterior and anterior septal regions exhibited a smaller reduction in peak longitudinal strain compared to the posterior and posterior lateral regions (Fig. 4D). During the first week after deTAC, recovery patterns were non-specific across regions. However, at 4 weeks post-deTAC, the anterior

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Global atrial volume and function</figcaption></figure>

A, representative short-axis images of the left atrium at baseline, 3 weeks post-TAC and 4 weeks post-deTAC. Blue dots represent endocardial points used for tracking over the cardiac cycle. Scale bar is 1 mm. B, representative left atrial 3D volumes at baseline, 3 weeks TAC and 4 weeks deTAC. Scale bar is 1 mm. C–E, left atrial (LA) max volume (C), min volume (D) and ejection fraction (E). Sample size *n* = 8 for each time point. Results expressed as mean ± SD. Statistical comparisons were conducted with one-way repeated measures ANOVA with Tukey's *post hoc* multiple comparisons tests. A + indicates significant difference from all other time points. Asterisks indicate \*P < 0.05, \*\*P < 0.01 and \*\*\*P < 0.001, respectively.
regions demonstrated significantly greater recovery compared to the posterior regions (Fig. 4E). A similar trend was observed in the normalized longitudinal strain rate where the posterior regions were significantly lower at 3 weeks TAC and did not recover as strongly as anterior regions after 4 weeks deTAC (Fig. 4E). Additionally, circumferential strain and surface area strain mapping revealed regional strain differences that can be quantified numerically (Appendix Fig. A11). Non-normalized data trends are provided in Fig. A10. Overall, reverse remodelling was associated with progressively increasing regional differences in the degree of strain recovery, both between and within anterior and posterior regions.

## 4D mapping of regional LA strain enhances prediction of ventricular remodelling compared to global LA strain

We then investigated whether global LA strain decreases could predict LV functional trajectories following pressure overload and release by examining the correlation between 3 weeks post-TAC global LA strains and 4 weeks deTAC LV kinematics. We found that relative reductions in global longitudinal (r = 0.720, P = 0.044), circumferential (r = 0.611, P = 0.107) and surface area (r = 0.673, P = 0.067) strain rates at 3 weeks TAC associated with LV longitudinal strain 4 weeks post-deTAC (Fig. 5A–C).

<figure><img src="image_7.png" /><figcaption>Figure 3. LA global longitudinal strain after TAC and deTAC</figcaption></figure>

A, representative long-axis images of the left atrium at baseline, 3 weeks post-TAC and 4 weeks post-deTAC. Blue dots represent endocardial points used for tracking over the cardiac cycle. Scale bar is 1 mm. B, representation of longitudinal strain calculation. C, representative global longitudinal strain curves at each time point. D, global longitudinal strain evolution. E, global longitudinal strain rate evolution. Sample size *n* = 8 for each time point. Results are expressed as mean ± SD. Statistical comparisons were conducted with one-way ANOVA with Tukey's *post hoc* multiple comparisons tests. A + indicates a significant difference from all other time points. *F-H*, Pearson correlation of left ventricular longitudinal strain with LA ejection fraction (*F*), global longitudinal strain (*G*) and global longitudinal strain rate (*H*). Sample sizes *n* = 32, which includes measurements from all time points. Correlation coefficients and corresponding *P*-values are displayed.
Next, we investigated whether LA regional kinematics provided further insights into LV function trajectories given the heterogeneity of LA kinematics in pressure overload and after release. We found that anterior septal longitudinal strain rate (r = 0.760, P = 0.028), superior LA circumferential strain rate (r = 0.930, P < 0.001) and anterior septal surface area strain rate (r = 0.722, P = 0.043) showed stronger associations with 4 weeks deTAC LV function compared to global strain rates (Fig. 5D–F). Lastly, we assessed whether LA parameters could predict LV structural remodelling by correlating parameters from 3 weeks TAC to LV mass at 4 weeks deTAC. We found that 3 weeks TAC LA max volume (r = 0.724, P = 0.043) was predictive of long-term LV hypertrophy while 3 weeks TAC LV mass (r = 0.452, P = 0.261) was not (Fig. A2).

## Global fibrotic expansion and regional heterogeneity in LA structural remodelling

We then assessed the LA myocardium histologically in sham (n = 4), 3 weeks TAC (n = 4) and 4 weeks deTAC (n = 4) animals (Fig. 6A–C). At baseline there was 5.2 ± 0.6% area type I+ collagen. There was increased atrial fibrosis with 16.4 ± 5.0% area type I+ collagen 3 weeks post-TAC (16.4 ± 5.0%, P = 0.012). Interestingly, the global level of fibrosis was consistently elevated in the 4 weeks deTAC group (16.0 ± 5.4%, P = 0.991; Fig. 6D). We measured fibrosis thickness in two different segments of the LAA epicardium in sham animals, after 3 weeks of TAC and after 4 weeks of de-TAC. In sham animals there was no difference in regional epicardial thickness. However, the superior region epicardium trended towards greater thickness compared to the inferior epicardium in

<figure><img src="image_8.png" /><figcaption>**Figure 4.** Regional longitudinal strain trajectory during reverse remodelling</figcaption></figure>

A, longitudinal strain map interpretation key. The vertical axis represents how the regions of the LA are unwrapped along the axis. The horizontal axis represents the time of one cardiac cycle. B, mean longitudinal strain maps at baseline, 3 weeks post-TAC, 1 week post-deTAC and 4 weeks post-deTAC. C, difference between time points in mean longitudinal strain maps. Blue indicates decreased strain compared to the prior time point, and red indicates increased strain compared to the previous time point. D and E, regional longitudinal strain (D) and longitudinal strain rate (E) normalized to baseline levels. Statistical comparisons were conducted with two-way ANOVA with Tukey's *post hoc* multiple comparisons tests. Asterisks indicate \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001 and \*\*\*\*P < 0.0001, respectively.
3w TAC (P = 0.143) and 4w deTAC animals (P = 0.055; Fig. 6E–G), but these differences were not statistically significant.

Discussion

Our study demonstrates that advanced atrial strain analysis not only captures detailed spatial and temporal variations in LA kinematics but also reveals critical insights into reverse remodelling in a murine model of pressure overload – a breakthrough that could help to reshape how heart failure is evaluated and managed. Specifically, our results demonstrate that mice subjected to TAC followed by debanding exhibit signs of LA maladaptation (decreased strain and LA fibrosis), followed by only partial recovery. This reverse remodelling pattern is effectively captured through a novel 4D ultrasound and strain mapping technique, providing a detailed view of regional LA strain dynamics that are not possible with traditional 2D imaging methods.

Since the development of real-time volumetric
ultrasound imaging (Fraser et al., 2022; von Ramm
& Smith, 1990), tremendous efforts have been made
to integrate 3D/4D ultrasound with strain analysis to

provide novel insights into the trajectory of LV function
during heart failure, both in patients and more recently
in rodent models (Cebull et al., 2019; Soepriatna et al.,
2017). Unlike traditional 2D techniques that rely on
planar views and interpolation, 3D ultrasound has
been used to more accurately assess volume and LA
strain patterns in atrial fibrillation patients (Mochizuki
et al., 2013; Schaaf et al., 2017) and detect early signs of
cardiac remodelling (Toprak et al., 2016). In addition, 4D
imaging, which offers gated volumetric data, represents a
significant advancement in cardiac imaging technology,
enhancing the precision and resolution of cardiac strain
measurements by providing comprehensive spatial
and temporal data. This has enabled a more accurate
characterization of the temporal evolution of LV hyper-
trophy (Damen et al., 2021), heart failure with pre-
served ejection fraction (Leyba et al., 2023; Nagueh
et al., 2017) and reverse LV remodelling (Cimino
et al., 2019; Ghajar-Rahimi et al., 2025). Here, we
specifically developed and implemented an advanced
4D LA image analysis technique. Our preclinical study
further demonstrates the feasibility and usefulness of the
high temporal resolution of 4D ultrasound in capturing
specific dynamic changes in atrial strain throughout heart
failure and the reverse remodelling process. Building on

<figure><img src="image_9.png" /><figcaption>Figure 5. Regional left atrial strain rate predicts left ventricular function</figcaption></figure>

A–C, Pearson correlation of normalized 3 weeks post-TAC left atrial global longitudinal strain rate (A), global circumferential strain rate (B) and global surface area strain rate (C) with 4 weeks post-deTAC global left ventricular (LV) longitudinal strain. D–F, Pearson correlation of normalized 3 weeks post-TAC left atrial anterior septal longitudinal strain rate (D), superior circumferential strain rate (E) and anterior septal surface area strain rate (F) with 4 weeks post-deTAC LV longitudinal strain. Sample sizes *n* = 8. Correlation coefficients and corresponding *P*-values are displayed.
<figure><img src="image_10.png" /><figcaption>Figure 6. Left atrial fibrosis in TAC and deTAC animals</figcaption></figure>

A, representative histological cross-sections of the left atrium and left atrial appendage (LAA) in sham, 3 weeks post-TAC and 4 weeks post-deTAC animals. Scale bar is 1 mm. B and C, insets showing higher magnification histological cross-section of the superior (B) and inferior LAA (C) in sham, 3 weeks post-TAC and 4 weeks post-deTAC animals. Scale bar is 50 µm. Immunofluorescence staining shows collagen type I+ (red) and DAPI/nuclei (blue). D, global left atrial fibrosis quantification in sham, 3 weeks post-TAC and 4 weeks post-deTAC animals. Sample size *n* = 4 for each group. Statistical comparison was conducted with one-way ANOVA with Tukey's *post hoc* multiple comparisons tests. E–G, superior and inferior LAA epicardial thickness in sham (E), 3 weeks TAC (F) and 4 weeks deTAC animals (G). Statistical comparisons were conducted with a paired *t* test.
these technical advancements, our findings offer valuable insights into the pathophysiological processes underlying atrial remodelling and recovery.

Pressure overload after TAC induces structural, molecular and functional alterations in the atrium, reflecting the progression of heart failure (Salvas et al., 2025). Although the transported LA volume remains unchanged, the concomitant decrease in LA strain and ejection fraction suggests a reduction in atrial mechanical efficiency (Melenovsky et al., 2015). This decrease in efficiency may be due to a compensatory adaptation in response to altered LV diastolic properties versus intrinsic atrial failure in pressure overload. Without invasive atrial contractility measurements, it is difficult to distinguish whether decreases in function relate to compensation or intrinsic failure. Nonetheless, our advanced 4D ultrasound image analysis techniques allow for non-invasive global and regional function assessments which may hold clinical utility in heart failure (Barki et al., 2024; Jia et al., 2022). Upon pressure relief in the deTAC phase, reverse remodelling occurs (Thomas & Abhayaratna, 2017). Despite clinical evidence suggesting that LA size and function provide valuable markers of reduced risk for adverse cardiovascular events during heart disease treatment (Thomas & Abhayaratna, 2017), little is known about the functional and molecular trajectories of atrial reverse remodelling (Abe et al., 2025) and whether it occurs heterogeneously across the atrium.

The TAC-deTAC model offers a robust framework to study the remodelling dynamics and the interplay between the LV and LA geometry, mimicking the reverse remodelling process in patients (Aidara et al., 2024; Perera-Gonzalez et al., 2021; Weinheimer et al., 2018). Here, our findings confirm that global LA strain correlates with LV dysfunction (Salvas et al., 2025) and that LA dilatation in pressure overload predicts the degree of residual LV hypertrophy after release of pressure overload (Hatani et al., 2016). In addition, 4D strain mapping enables the identification of specific atrial areas with differential recovery, highlighting regions of effective reverse remodelling versus other regions that may remain impaired (Mehringer & McVeigh, 2025). We observed that global atrial function did not return to baseline and exhibits heterogeneous regional remodelling. Specifically, anterior regional LA strain showed improved recovery compared to the posterior region, which could offer clinical utility. Moreover, we found regional LA strain rates, again the anterior LA (anterior septal and superior), were more effective than global LA strain rates in predicting ventricular functional reverse remodelling, further highlighting potential utility of regional LA analyses. This is particularly relevant given the close but complex interplay between myocardial dysfunction and structural abnormalities, as recent studies have shown an inverse correlation between the

level of LA strain and LA fibrosis content during the development of heart failure (Lisi et al., 2022). Based on histological data, we found that pressure overload induces atrial fibrotic remodelling with a heterogeneous pattern. Clinical studies have demonstrated regional variations in atrial strain and function due to atrial fibrillation (Kuraoka et al., 2021). Peters et al. (2017) further suggested a correlation between reduced regional atrial strain and possible focal atrial fibrosis, as assessed by late gadolinium enhancement magnetic resonance imaging in atrial fibrillation patients (Peters et al., 2017). In addition, recent findings in a porcine ischaemic heart failure model highlight that the heterogeneous distribution of fibrosis across LA regions appears to determine atrial fibrillation susceptibility in ischaemic heart failure rather than the overall level of fibrosis (Zhang et al., 2024). While fibrosis evolution during reverse remodelling is not well understood, functional recovery seems to occur more quickly than the regression of myocardial fibrosis (Sato et al., 2017; Treibel et al., 2018; Villari et al., 1995), as fibrosis evolution is complex and progresses slowly, often with incomplete resolution. Along these lines, we did not observe a significant decrease in LA fibrosis despite early LA functional recovery after release of pressure overload. Together, these findings highlight the complex and heterogeneous nature of atrial remodelling, emphasizing that regional variations in fibrosis and strain may critically influence functional recovery and arrhythmogenic susceptibility in heart failure. In line with this heterogeneity, atrial remodelling during disease development and recovery follows a non-uniform trajectory in both timing and structure across the myocardium.

These temporal and spatial observations of LA kinematics are essential for understanding the mechanisms behind atrial functional recovery and the evolution of fibrosis, while also potentially helping guide the development of targeted therapeutic strategies. However, the application of 4D strain imaging to map these changes in clinical settings remains an emerging field (Mehringer & McVeigh, 2025), requiring further investigation to validate preclinical studies and translate findings into treatment of human disease.

There are several limitations to our study. We considered the LA and LAA as a single chamber with contractile properties due to the relatively large size of the LAA and its synergistic contribution to overall LA function in mice (Colazzo et al., 2015). In humans, the LAA may have a unique contraction profile (Pollick & Taylor, 1991), and thus distinct global and regional LA and LAA analyses may be warranted. We did not consider the various phases of LA contraction due to difficulty in distinguishing between the reservoir and contra-ctile phases. Further, we used a Green-LaGrange strain definition for calculation of LA strain which assumes a
cylindrical reference geometry. While Green–LaGrange
strain can be used to quantify both small and large
deformations, the complex geometry of the LA, especially
at the minimum volume, makes it difficult to pre-
cisely estimate strain. Finally, although LA function
has been shown to correlate with fibrosis levels, our
findings emphasize the complexity of this relationship
(Kuppahally et al., 2010; Salvas et al., 2025), as we did not
directly investigate this association. To better understand

this correlation, future work will be needed to merge
4D strain data with 3D LA fibrosis levels to determine
correlative and causative effects.

In conclusion, our study highlights the potential of advanced 4D strain analyses as a powerful tool for studying the heterogeneous patterns of atrial remodelling. This methodology not only enhances our understanding of the pathophysiological of heart failure, but it also paves the way for additional targeted therapeutic strategies.

**Appendix**

Figures A1–A11

<figure><img src="image_11.png" /><figcaption>Figure A1. Study design</figcaption></figure>

A, main cohort study design with *n* = 8 animals followed over time. *B*, histological cohort study design with *n* = 8 additional animals for sham and 3 weeks TAC analyses and *n* = 4 animals from the main cohort used for 4 weeks deTAC analyses. Created in BioRender. Goergen, C. (2025) https://BioRender.com/i02j746
<figure><img src="image_12.png" /><figcaption>**Figure A2. Left ventricular (LV) longitudinal strain (A) and mass (B)**</figcaption></figure>

Statistical comparisons conducted with one-way ANOVA with Tukey's *post hoc* multiple comparisons tests. A + indicates significant difference from all other time points. Asterisks indicate \*P < 0.05, \*\*P < 0.01 and \*\*\*P < 0.001, respectively. C and D, Pearson correlation between 4 week deTAC LV longitudinal strain and 3 weeks post-TAC LA max volume (C) and LV mass (D). Sample sizes *n* = 8. Correlation coefficients and corresponding *P*-values are displayed.
<figure><img src="image_13.png" /><figcaption>Figure A3. Global circumferential and surface area strain evolution after TAC and deTAC</figcaption></figure>

A, representative global circumferential strain curves at each time point. B, global circumferential strain evolution. C, global circumferential strain rate evolution. Sample size *n* = 8 for each time point. Results expressed as mean ± SD. D, representation of circumferential strain calculation. E and F, Pearson correlation of left ventricular longitudinal strain with LA global circumferential strain (E) and global circumferential strain rate (F). Sample sizes *n* = 32, which include measurements from all time points. G, representative global surface area strain curves at each time point. H, global surface area strain evolution. I, global surface area strain rate evolution. Sample size *n* = 8 for each time point. Results are expressed as mean ± SD. J, representation of surface area strain calculation. K and L, Pearson correlation of left ventricular longitudinal strain with LA global surface area strain (K) and global surface strain rate (L). Sample sizes *n* = 32, which include measurements from all time points. Statistical comparisons were conducted with one-way ANOVA with Tukey's *post hoc* multiple comparisons tests. A + indicates significant difference from all other time points. Asterisks indicate \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001 and \*\*\*\*P < 0.0001. Pearson correlation coefficients and corresponding *P*-values are displayed.
Individual Global and Regional Longitudinal Strain Curves

<figure><img src="image_14.png" /><figcaption>Figure A4. Individual global and regional longitudinal strain curves from all time points</figcaption></figure>
Individual Longitudinal Strain Maps

<figure><img src="image_15.png" /><figcaption>Figure A5. Individual longitudinal strain maps from all time points</figcaption></figure>
Individual Global and Regional Circumferential Strain Curves

<figure><img src="image_16.png" /><figcaption>Figure A6. Individual global and regional circumferential strain curves from all time points</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>Figure A7. Individual circumferential strain maps from all time points</figcaption></figure>
Individual Global and Regional Surface Area Strain Curves

<figure><img src="image_18.png" /><figcaption>Figure A8. Individual global and regional surface area strain curves from all time points</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>Figure A9. Individual peak surface area strain maps from all time points</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>Figure A10. Non-normalized data trends of regional longitudinal, circumferential and surface area strain and strain rate</figcaption></figure>

A–F, left atrial regional longitudinal strain (A), longitudinal strain rate (B), circumferential strain (C), circumferential strain rate (D), surface area strain (E) and surface area strain rate over time (F). Statistical comparisons were conducted with two-way ANOVA with Tukey's *post hoc* multiple comparisons tests. A + indicates significant difference from all other time points. Asterisks indicate \*P < 0.05, \*\*P < 0.01 and \*\*\*P < 0.001.
<figure><img src="image_21.png" /><figcaption>Figure A11. Regional circumferential and surface area strain</figcaption></figure>

A, circumferential strain map interpretation key. The vertical axis represents regions of the LA from the defined superior LA down to the mitral valve. The horizontal axis represents the time of one cardiac cycle. B, mean circumferential strain maps at baseline, 3 weeks post-TAC, 1 week post-deTAC and 4 weeks post-deTAC. C, difference between time points in mean circumferential strain maps. Blue indicates decreased strain compared to the prior time point, and red indicates increased strain compared to the previous time point. D and E, regional peak circumferential strain (D) and circumferential strain rate normalized to baseline levels (E). F, surface area peak strain map interpretation key. G, representative peak surface area strain maps at baseline, 3 weeks post-TAC, 1 week post-deTAC and 4 weeks post-deTAC. H and I, regional peak surface area strain (H) and surface area strain rate (I) normalized to baseline levels. Statistical comparisons were conducted with two-way ANOVA with Tukey's post hoc multiple comparisons tests. \*P < 0.05.
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## Additional information

### Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

### Competing interests

All authors have read the journal's policy on disclosure of potential conflicts of interest. C. J. Goergen serves on the Scientific Advisory Board for FUJIFILM VisualSonics Inc. None of the other authors have conflicts of interest, financial or otherwise, to disclose. Disclaimers: FUJIFILM VisualSonics Inc. had no role in the study's design, execution, interpretation or writing.

### Author contributions

J.P.S. performed formal analysis, investigation, and visualization, and contributed to writing – original draft, review and editing.

T.M-M. contributed to conceptualization, investigation, methodology, and supervision, and participated in writing – original draft, review and editing. C.J.G. contributed to conceptualization, methodology and supervision, and participated in writing – review and editing. P.S. contributed to conceptualization, investigation, methodology and supervision, and participated in writing – original draft, review and editing. All authors have approved the final version of the manuscript and agreed to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

### Funding

Indiana University School of Medicine (J.P.S.), Fulbright United States Scholar Program (C.J.G.), Thomas Jefferson Fund from the French-American Cultural Exchange Foundation (C.J.G., P.S.), Biocampus (P.S.), I-Site Muse (P.S.).

### Acknowledgements

We thank PhyMedExp staff for animal housing. We acknowledge the technical support provided by Salomé Bélec from Imagerie du Petit Animal de Montpellier (IPAM) for accessing high-resolution ultrasound [LRQA Iso9001; France Life Imaging (grant ANR-11-INBS-0006); IBISA; Leducq Foundation (RETP), I-Site Muse].

### Keywords

atrium, heart failure, imaging, strain, 4D ultrasound

### Supporting information

Additional supporting information can be found online in the Supporting Information section at the end of the HTML view of the article. Supporting information files available:

#### Peer Review History

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