System and method for apical four-chamber view echocardiography

Abstract

Systems and methods to obtain high-quality apical 4-chamber-long-axis (A4CLAX) views of the mouse heart. The pathological progression of heart function following transverse aortic constriction (TAC) surgery is monitored using both a long-axis biplane (LAX-BP) and the conventional PSAX-M mode assays. A clear visualization of both mitral and tricuspid valve leaflets in A4CLAX view images enabled detecting dysfunctions such as valve regurgitation and desynchronized opening due lower chamber contractile desynchronies. Additionally, A4CLAX echocardiography facilitated the characterization of right ventricle (RV) dysfunction in mice, including RV dilation, higher diastolic filling pressures, and early characterization of decreased RV free wall longitudinal strain caused by pulmonary hypertension secondary to TAC procedure. Furthermore, A4CLAX imaging expands upon our capabilities to study left atrial and appendage function. The A4CLAX methodology allows significantly more clinically comparable echocardiographic analyses in murine heart, including a LAX-BP assay that provides CMR-comparable accuracy to evaluate the LV volumes.

Description

SYSTEM AND METHOD FOR APICAL FOUR-CHAMBER VIEWECHOCARDIOGRAPHYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 466,851, filed May 16, 2023, entitled “SYSTEM AND METHOD FOR APICAL FOUR-CHAMBER VIEW ECHOCARDIOGRAPHY” and U.S. Provisional Patent Application No. 63 / 513,695, filed July 14, 2023, entitled “SYSTEM AND METHOD FOR APICAL FOUR-CHAMBER VIEW ECHOCARDIOGRAPHY,” the disclosures of which are expressly incorporated herein by reference in their respective entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCHOR DEVELOPMENT

[0002] This invention was made with government support under grant / contract nos. HL139006 and OD031965 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Small animal models, particularly numerous transgenic mice, are extensively utilized in cardiovascular research. Accurately assessing cardiac performance in these models is needed for precise phenotyping of genetic variants and evaluating therapeutic interventions. For over two decades, the evaluation of systolic and diastolic functions in experimental cardiovascular disease models has predominantly relied on echocardiography. For example, cardiac magnetic resonance (CMR) imaging has been recently used in small animals, but its high cost and time requirements often lead research to opt for simpler measures of the left ventricle (LV) morphological and functional parameters using parasternal short-axis (PSAX) M-mode assays.

[0004] The PSAX M-mode assay assumes a spherical shape for the LV, a simplification that may compromise accuracy, especially in diseased hearts. Scientists have made strides in enhancing the precision of murine echocardiography, including the adoption of single-plane analysis from parasternal long-axis B-mode (PLAX) assays, PLAX speckle tracking, consecutiveshort-axis views, or a modified bi-plane Simpson method incorporating PLAX with multiple short-axis views. Clinically, the biplane Simpson’s method remains the most commonly used method for echocardiographic volume assessment. Considering the heart’s complex geometry, the LV length is measured from the apical point to the mitral valve ring bisector. Volume measurements require tracing the interface between the LV cavity and compacted myocardium. Consequently, the biplane Simpson’s calculation necessitates clear two-dimensional echocardiographic images from apical long-axis 2- and 4-chamber views.

[0005] Although the PLAX 2-chamber view is frequently used in murine echocardiography, acquiring clear on-axis apical 4-chamber view remains challenging, despite significant efforts in murine echocardiography. Further, transthoracic echocardiography remains the primary non- invasive method for efficient and comprehensive cardiac function assessment in clinical settings. However, challenges in acquiring accurate apical on-axis 4-chamber views have traditionally limited most mouse research to left ventricle (LV) evaluations using parasternal short-axis M (PSAX-M) mode assays.SUMMARY

[0006] Disclosed herein are systems and methods to obtain high-quality apical 4-chamber- long-axis (A4CLAX) views of the heart of a subject, such as a mouse. The present disclosure explores the extensive capabilities of comprehensive echocardiography assessments in subjects enabled by the A4CLAX imaging approach. The pathological progression of heart function was monitored following transverse aortic constriction (TAC) surgery using both a newly developed long-axis biplane (LAX-BP) and the PSAX-M mode assays. Findings showed that the LAX-BP sensitively detected a significant LV pathological progression mainly occurring within the first week post-TAC, which was not captured by PSAX-M-mode due to its lack of longitudinal assessment. Bland-Altman analyses confirmed that LAX-BP echocardiography offers accuracy comparable to CMR in assessing LV volume during systolic and diastolic phases in mouse hearts. Importantly, the clear visualization of both mitral and tricuspid valve leaflets in A4CLAX view images enabled clinically comparable Pulse Wave, Tissue, and Color Doppler, sensitively detecting dysfunctions such as valve regurgitation, restrictive filling, and desynchronized valve opening. Additionally, A4CLAX echocardiography facilitated the characterization of left atrial (LA) and right ventricle (RV) dysfunction in mice, including their size, shape, and fractional areachange (FAC). In conclusion, the innovative A4CLAX methodology allows clinically comparable echocardiographic analyses in murine heart, including a LAX-BP assay that provides CMR-comparable accuracy to evaluate the LV volumes. Beyond the LV, the A4CLAX echocardiography enables assessments of LA, RV, and valves to sensitively detect subtle changes in cardiac performance during disease progression in mouse models.

[0007] Thus, in accordance with an aspect the disclosure, A method for capturing apical 4- chamber-long-axis (A4CLAX) views of a heart of a subject is disclosed. The method includes positioning the subject on a heated echocardiography platform; placing a support under an upper abdominal area of the subject; tilting the heated echocardiography platform at a predetermined angle; and capturing the A4CLAX views using a transducer via subcostal imaging.

[0008] In accordance with other aspects of the disclosure, a method for echocardiography analysis is disclosed. The method includes capturing apical 4-chamber-long-axis (A4CLAX) views of a heart of a subject; obtaining parasternal long-axis B-mode (PLAX) images of the heart; creating a long axis biplane (LAX-BP) assay; and assessing parameters of the heart. In some implementations, the parameters include at least one of left ventricle (LV), left atrial (LA), and right ventricle (RV) parameters..

[0009] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The foregoing summary, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the embodiments, there is shown in the drawings example constructions of the embodiments; however, the embodiments are not limited to the specific methods and instrumentalities disclosed. In the drawings:

[0011] FIGS. 1A-1B illustrate key landmarks and representative images of echocardiography views in accordance with aspects of the present disclosure;

[0012] FIGS. 2A-2D illustrate A4CLAX image acquisition approach and that an A4CLAX- derived LAX-BP approach in accordance with aspects of the present disclosure shows superior accuracy in LV functional assessments;

[0013] FIGS. 3A-3D illustrate improved Doppler assays reveal LV restrictive fdling patterns and mitral regurgitation in transverse aortic constriction (TAC) subjects in accordance with aspects of the present disclosure;

[0014] FIGS. 4A-4E illustrate that A4CLAX imaging shows deteriorating trend of RV function secondary to LV pressure overload in subjects in accordance with aspects of the present disclosure;

[0015] FIGS. 5A-5B illustrate that A4CLAX imaging enables assessments of RV strain and regional contractile function, as well as lower chamber movement asynchrony in subjects in accordance with aspects of the present disclosure;

[0016] FIGS. 6A-6C illustrate that A4CLAX imaging allows for atria and atrial appendage assessments in subjects in accordance with aspects of the present disclosure; and

[0017] FIGS. 7A-7C illustrate aspects of TAC surgery in subjects in accordance with aspects of the present disclosure.DETAILED DESCRIPTION

[0018] Disclosed herein is an imaging system and methodology that allows for consistent collection of apical 4-chamber images of hearts of subjects, such as mice, thus enabling the biplane assessment of cardiac function by integrating long-axis and apical 4-chamber views. As will be evident to those of skill in the art, the disclosed methods may be applicable to other animal subjects in addition to mice. The method combines an imaging approach that consistently captures high-quality apical 4-chamber-long-axis (A4CLAX) views of the mouse heart with PLAX (2-chamber) images to create a modified Simpson’s method, termed the long axis biplane (LAX-BP) assay. The LAX-BP method of the present disclosure yields CMR-comparable assessments of LV volumetric parameters, including end diastolic (EDV) and end systolic (ESV) volumes, in both wildtype, TAC, and miRl -mutated transgenic mouse hearts.

[0019] As will be described herein, the LAX-BP methodology is applied to a common heart disease model - transverse aortic constriction (TAC) - to determine if it can sensitively detect subtle changes in the LV and monitor the progression of heart diseases in mice. Additionally, thepresent disclosure demonstrates how the A4CLAX imaging technique facilitates more holistic and clinically comparable echocardiography analyses in mouse hearts as compared to conventional methods, including assessments of the right ventricle, atria, and hemodynamics of the atrioventricular valves.

[0020] For example, the novel A4CLAX approach significantly improves the comprehensive capabilities of echocardiography for assessing cardiac function and detecting subtle changes in pathological progression in mouse hearts. Bland-Altman analysis revealed that LAX-BP echocardiography achieves CMR-comparable accuracy in assessing LV volume in diseased mouse hearts. Furthermore, A4CLAX echocardiography extends beyond LV assessments to sensitively detect subtle changes in the atrial, right ventricle, and valves, thereby revealing the pathological progression in mouse models of cardiovascular diseases. This A4CLAX technique effectively facilitates clinic comprehensive echocardiography assessments, enabling detailed evaluation of the morphology and functional performance of LV, RV, atria, and valves in mice. Other advantages will be evident in view of the disclosure herein.

[0021] INTRODUCTION

[0022] A4CLAX Echocardiography Provides Precise LV Assessments in Mice

[0023] In clinic, transthoracic echocardiography remains the primary non-invasive approach for measuring cardiac function and is routinely used to assess the morphology and hemodynamics of the LV and RV, as well as the function of the atria and valves. However, in cardiovascular research using mouse models, the PSAX-M mode has been extensively used to estimate the hemodynamic functions of LV, based on an assumption of LV sphere-morphology. This simplification inaccurately describes the longitudinal shape of the heart and raises concerns about the precision of PSAX-M mode in assessing cardiac performance of mouse heart in various disease models. Although single-plane PLAX-view analysis and PLAX speckle tracking assays have been adapted to improve the accuracy of mouse echocardiography, these monoplane assays still assess only one dimension of the heart. Acquiring and measuring consecutive short-axis views along with incorporated PLAX view may be more precise; however, similar to CMR, these echocardiography approaches are time-consuming and impractical with the increased number of animals typically used in basic research that requires data collection from relatively large numbers of mice within a limited time.

[0024] Significant effort has been invested with limited progress in acquiring an on-axis 4- chamber view of the mouse heart, which is required for the most practical and clinically recommended biplane Simpson’s method for echocardiographic LV volume assessment. The novel A4CLAX imaging approach of the present disclosure overcomes this challenge and others. The LAX-BP approach was developed as a modified Simpson’s assay from A4CLAX and PLAX images of mouse hearts. During the development of this A4CLAX dependent LAX-BP technique, LAX-BP and the conventional PSAX-M mode assays were compared with the gold standard CMR assay in a cohort of adult normal mice. Findings indicated that LAX- BP method could accurately evaluate LV volumes, including EDV and ESV, whereas M-mode assessments tended to overestimate these values, leading to inflated calculations of other functional parameters (e.g., SV and CO). Bland-Altman plot assays have demonstrated that LAX-BP and CMR assays exhibited comparable accuracy in assessing LV volume in both normal and diseased (e g., TAC) mouse hearts. Indeed, LAX-BP assays revealed rapid pathological LV remodeling in TAC mice that primarily occurred in the first week post-surgery. This was evidenced by a significant and swift decline in LV function, increased LV wall thickness, enlarged LA size, and reduced LA FAC. In contrast, PSAX-Mode assessments failed to capture this dynamic progression, only detecting LV volumetric functional parameter decline over a gradual and continuous process, although the rapid LV wall thickening was detected in PSAX-M assay. Thus, LAX-BP provides accurate LV functional evaluation and can sensitively detect subtle changes of heart systolic pumping function, including EF, SV, and CO parameters, during the pathological progression of TAC hearts.

[0025] The A4CLAX echocardiography enables clinically comparable Doppler assessments in mice

[0026] Adapted from clinical recommendation for Doppler assays, tissue Doppler from the PSAX view has been applied to measure endocardial and epicardial systolic velocities in mice. Pulse wave Doppler has also been conducted using non-long axis apical 4-chamber view images to evaluate the diastolic function of mouse hearts, where the measured flow movement has a certain degree angle from the real blood flow in the heart. This concern has been resolved by A4CLAX echocardiography, allowing perfect alignment of pulse wave Doppler assays with the direction of mitral and tricuspid blood flow and tissue Doppler assays with the longitudinal movement of the heart. Consistent with the LAX-BP assessments of the LV, pulse wave dopplerin A4CLAX images also revealed a rapid pathological progression in LV of TAC mice, evidenced by a significant increase in the E / A ratio and E wave deceleration, and decreases in the E wave deceleration time within the first week, which are consistent with the pathological remodeling of LV wall thickness and significant diastolic dysfunction with restrictive filling patterns, which are consistent with clinical observations.

[0027] Color Doppler using PLAX B-mode image has historically been applied to study the hemodynamics in the LV, RV, and the aortic flow in mice. The PLAX view color Doppler provides a good angle to assess the flow through the LV outflow tract and is very crucial for evaluating hypertrophic cardiomyopathy and aortic dilation or aneurysm. Importantly, the A4CLAX echocardiography provides an in plane horizontal cross-section of all four chambers, thus the A4CLAX view color Doppler assays can not only study the blood filling and emptying patterns of both the left and right sides of mouse hearts, but remarkably can better evaluate the hemodynamic function and patterns of mitral and tricuspid valves and quantify the severity of regurgitation if present. Indeed, as described below, A4CLAX echocardiography detected higher presence of turbulent diastolic inflow in the LV of TAC mice and mice with genetical mutations while also detecting mitral valve regurgitation in diseased TAC hearts.

[0028] In the clinic, pulse wave doppler of left atrial appendage has been employed as an indicator of diastolic function, to assess the risk of thrombus formation, and to predict maintenance of sinus rhythm in atrial fibrillation patient. While the atria and atrial appendage of the mouse heart was clearly captured by A4CLAX echocardiography, its feasibility to conduct atrial and atrial appendage assessments was explored and revealed that the size of left atrium and appendage was dramatically enlarged due to the TAC-induced pressure increase. Pulse wave doppler of the left atrial appendage showed sinus rhythm cardiac cycles in WT mice with sinus peaks aligning perfectly with ECG tracings (waveforms were inverted in Vevo LAB Software due to doppler gate directionality), indicating great potential of the A4CLAX technique application in atrial fibrillation studies using mouse models.

[0029] The A4CLAX echocardiography enable assessments of other chambers and the synchronization of chamber movements in mice

[0030] RV examinations are recommended for all comprehensive clinical studies. In addition to RV FAC, and TAPSE, RVFWLS assay was found to be more sensitive in detecting RV dysfunction in clinical settings. However, mouse RV assessments were only studied usingPSAX- M mode, PLAX-B mode, and color Doppler in the past, those clinical RV assessments were not available for mouse hearts due to the technique challenge of acquiring apical 4 chamber view imaging. The novel A4CLAX echocardiography allowed us to estimate the RV area during diastole and systole phases to calculate RV FAC and overlay A4CLAX recordings with M-Mode to acquire TAPSE in mouse hearts. Although pathological trends were observed in 4-week TAC mice, neither RV FAC nor TAPSE showed significant RV dysfunction in 4-week TAC mice. Importantly, RVFWLS assay did reveal that RV function was significantly declined within the first week post- TAC. This pathological progression aligns with the potential increase in pulmonary hypertension as a secondary consequence of the TAC procedure. In particular, the severe pressure overload induced by the 28G TAC procedure in this study would result in rapid LV dilatation.

[0031] Before the availability of a 4-chamber view of the mouse heart, murine LA function has been measured using speckle-tracking echocardiography from a modified PLAX window and non-long axis apical 4-chamber views. However, clinical LA echocardiography assessments are conducted from the A4CLAX view, which offers complete visualization of the LA and is necessary for a thorough evaluation of LA size, shape, and function. Indeed, the A4CLAX echocardiography revealed dramatic changes in LA morphology (e.g., size and shape) and a significant decline in LA FAC of TAC mice, mainly occurring in the first week post-surgery, which is consistent with the pathological progression of LV remodeling.

[0032] While all chambers of the mouse heart are clearly visualized, importantly, it is for the first time that the synchronization of all chamber movement can be studied in mice by echocardiography. For example, the A4CLAX echocardiography successfully captured delayed RV contraction compared to LV contraction. As a result, instead of the decline in RV pressure during diastole, the asynchronous contraction from the RV decreased the pressure gradient across the ventricle and atrium, causing the lack of tricuspid valve opening during the early filling phase. The tricuspid valve was asynchronously opened fully only after the ECG P-wave of atrial contraction. These pathological phenotypes would not be able to be identified without the A4CLAX technique.

[0033] Overall, the innovative A4CLAX technique of the present disclosure significantly facilitates the implementation of significantly more comprehensive clinical echocardiography assessments in basic cardiovascular research using mouse models. These clinically matchedassessments will significantly improve the translational applications of discoveries from numerous basic research studies utilizing mouse models.

[0034] METHODS

[0035] Conventional echocardiography

[0036] Echocardiography was performed by using a Vevo 3100 system (FUJIFILM VisualSonics) with a MX550D transducer. Traditional echocardiography of mouse heart was conducted as described previously. Briefly, mice (3-6 months) were anesthetized with 2.0% isoflurane in Oxygen. Anesthesia was maintained by administration of oxygen and -1.25% isoflurane on a heated pad during the whole imaging procedure. Animal heart rate (HR) was monitored throughout the imaging procedure; goal HR was kept at 450 bpm and above to maintain physiological relevance. Parasternal short-axis images at the level of the mid-papillary muscles, inclusive of M-mode imaging, were obtained to determine the left ventricular diastolic diameter (LVDD), as well as anterior (LVAW) and posterior wall thickness (LVPW) in both systole (s) and diastole (d). Fractional shortening (FS), ejection fraction (EF), stroke volume (SV), and cardiac output (CO) of the LV were calculated from the analyses of PSAX M-mode measurements. Parasternal long-axis images were taken with the aortic outflow tract in plane to maintain consistency of PLAX image acquisition and were subsequently used as a guidance view for finding the optimal orientation of PSAX view images, as well as to be used for the novel LAX-BP approach outlined below.

[0037] Novel A4CLAX view imaging approach and LAX-BP assessments for the LV

[0038] With reference to FIGS.. 1A-1B and 2A-2D, the A4CLAX view imaging approach and LAX-BP assessments will now be introduced. FIGS. 1A-1B illustrate key landmarks and representative images of echocardiography views. In FIG. 1A, an A4CLAX view diagram shows landmarks of full longitudinal 4 chamber of the heart (1), mitral valve and annulus (2), and RV tricuspid annulus and valve (3). The PLAX view was taken with clear apical form to LV (1) as well as aortic outflow tract (4). The PSAX view was taken perpendicular to PLAX view needs visualization of LV and RV as well as papillary muscles at the mid papillary level for consistent image acquisition. FIG. IB illustrates representative images taken of these three views showing all clear landmarks for consistent imaging. PSAX view also shows m-mode recording plane placement at mid-papillary level. FIG. 2A-2D illustrates that an A4CLAX-derived LAX-BP approach has superior accuracy in LV functional assessments. In particular, FIG. 2A is anexample diagram of A4CLAX image acquisition technique to be used for LAX-BP assessments of LV volumetric function, as well as study of other heart chambers and valves.

[0039] For A4CLAX view imaging, anesthetized mice were placed on a heated echocardiography platform (see, 210, FIG. 2 A). Under the mouse’s upper abdominal area, a 1 cm diameter soft support was positioned to elevate the back, to allow for better subcostal probe access and orienting the long axis of the heart towards the left subcostal region of the thoracic cavity. The platform was then tilted to a 50-degree angle, utilizing gravity to amend heart configurations and situation in the chest cavity due to the elevated chest, thus bringing it closer to the diaphragm and subsequently the transducer probe allowing for improved image clarity (220). Recording was conducted using an MX550D transducer to capture the A4CLAX view via subcostal imaging (230). Precise adjustment of the transducer is desirable to ensure it aligns directly with the heart’s axis. While this subcostal recording approach should generally obtain images of all four chambers, several key landmarks must be met to guarantee accurate A4CLAX view acquisition, as shown in FIGS. 1A-1B. In particular., a full longitudinal length of both the LV and RV must be visible, including the apex; the mitral annulus and mitral valves must be included within the plane; and the motion of the RV tricuspid annulus should be in view, however it’s important to note that tissue echo shadowing can sometimes obscure the tricuspid valve, posing visualization challenges.

[0040] To assess the volumetric parameters of the LV, the Vevo LAB software (FUJIFILM VisualSonics) were utilized to quantify the monoplane volumes from LV tracings in both the PLAX and A4CLAX views, which were subsequently used to develop a modified Simpson’s long- axis biplane (LAX-BP) assays. In brief, using the Vevo LAB software, the LV was traced from the mitral annulus to the apex along the LV wall in A4CLAX view, and from the aortic valve to the apex in the PLAX viewing plane. Visualization of wall movements aids in precise placement of the LV trace. The average LV volumes at the end of systole (ESV) and diastole (EDV) were calculated from each monoplane, resulting in two sets of values: 1) PLAX-ESV and PLAX- EDV, 2) A4CLAX- ESV and A4CLAX-EDV. Consequently, the following relationships were derived for LAX-BP-ESV and LAX-BP-EDV values.LAX - BP ESV = / (pLAX - ESV) ■ (A4CLAX - ESV)LAX - BP EDV = / (pLAX - EDV) ■ (A4CLAX - EDV)The LAX-BP-ESV and LAX-BP-EDV values were then utilized to estimate other volumetric parameters, such as EF, SV, and CO.

[0041] CMR imaging validation study

[0042] An MRI validation study was performed in 3 TAC mice (4 weeks post-op) on a 11.7-T (500 MHz) MR microimaging system comprising a vertical magnet of bore size 30 mm (Bruker Biospin, Ettlingen, Germany). Quadrature-driven birdcage coils with inner diameters of 25 mm were used to transmit / receive the NMR signals. Briefly, mice were anesthetized in an anesthetic chamber using 1.5% isofhirane in 95% oxygen and 5% carbon dioxide mixture. Mice were imaged using a 30-mmlH-imaging probe after scouting for long- and short-axis orientation of the heart, using a k-space segmented cardiac-triggered and ECG gated FLASH sequence. Cine MR images with 6 to 9 contiguous short-axis slices (slice thickness 1 mm) were acquired, comprising between 20 and 31 frames per cardiac cycle (echo time of 1.43ms, repetition time of 4.6ms, 2 averages per slice, matrix size of 256x256, and a field of view of 2.56cm, giving an inplane resolution of 100x100 pm). Finally, a single, long-axis, cine dataset was acquired. Total time for an MRI study was 60-80 minutes.

[0043] Statistical analysis

[0044] Data are expressed as mean±S.E.M, with “n” indicating the number of distinct biological samples. Statistical significance of differences in means was estimated using one-way ANOVA followed by one-tailed Student’s t tests conducted in GraphPad Prism version 9.4.1 for Windows (GraphPad Software). A p<0.05 indicates a statistical significance.

[0045] RESULTS

[0046] LAX-BP echocardiography effectively monitors the LV pathological progression in TAC mouse hearts

[0047] To determine if LAX-BP could sensitively detect subtle changes during the pathological progression in diseased hearts, both the LAX-BP and pSAX-M Mode methods were applied to monitor LV function in TAC mice at comparable heart rates as shown in FIG.2B, which illustrates that LAX-BP indicates more rapid decline of LV function 1-week post- surgery consistent with the rapid hypertrophic remodeling occurring in TAC hearts. Details of the TAC surgery are described below with reference to FIGS. 7A-7C. In FIG. 2B, the PSAX-M Mode shows decline over time in a more linear a steady pace, not accurately showing the true pathological progression of 28G TAC hearts. One female mouse was not recovered from TACsurgery; in addition, one male and one female mouse died in week 2 or 3 post-surgery and were excluded from subsequent analyses, resulting in cardiac performance evaluation of 5 male and 4 female mice post-TAC.

[0048] A significant increase in LV anterior (LVAW) and posterior (LVPD) wall thickness along the time was observed, particularly within the first week post-surgery (see, FIG. 2B). Specifically, the LVAW at diastolic phase was increased from 0.789±0.040mm to 0.969±0.064mm (weekl, p=0.0054 vs. pre-surgery) and 1.005±0.067mm (week 4, p=0.0053 vs. pre-surgery, p=0.223 vs. week 1), the LVPD at diastolic phase changed from 0.612±0.043 to 0.763±0.050mm (week 1, p=0.0052 vs. pre-surgery) and 0.812±0.059mm (week 4, p=0.0016 vs. pre-surgery, p=0.159 vs. week 1)). These results indicate that TAC-induced hypertrophic remodeling rapidly occurred within the first week.

[0049] With continued reference to FIG. 2B, consistent with the rapid progression of pathological remodeling, a significant increase of the LV diameter at diastolic (LVD-D) and systolic phases also rapidly occurred from week 1 to week 4. Both LAX-BP and PSAX-M mode assessments showed significant changes of LV function from volumetric parameters such as EF, SV, and CO from week 1 to week 4 post-TAC, while the assessments of PSAX-M mode were notably higher than that of LAX-BP assay. The PSAX-M mode assays showed that TAC hearts experienced a relatively slow progressing remodeling with even functional decline in LV EF, SV, and CO over time. However, LAX-BP assays revealed a rapid decline in EF (30.22±2.48% at week 1 vs. 51.33±2.82% pre- surgery, p<0.0001) within the first week post-surgery, followed by a further decline by week 4 (23.30±3.72%, p=0.020 vs. week 1). This is more obviously evidenced in the LV SV that decreased from 20.85±0.84 pl pre-surgery to 17.22±0.48 pl at week 1 (p=0.0005) and to 16.74±1.69 pl at week 4 (p= 0.030 vs. pre-surgery, p=0.400 vs. weekl), and the CO that decreased from 10.13±0.45 ml / min pre-surgery to 8.23±0.26 ml / min at week 1 (p=0.0043) and to 7.69±0.89 ml / min at week 4 (p= 0.021 vs. pre-surgery, p=0.297 vs. weekl). This quick and severe LV response in the first week, followed by a slow functional deterioration progression, is consistent with the hypertrophy remodeling progression. The LV ESV and EDV were estimated significantly higher by PSAX-M mode than LAX-BP assays, although both approaches could detect the increase of LV volume post-TAC. However, PSAX-M mode detected 13.36 pl increase in ESV and 7.31 pl increase in EDV within the first week post-TAC, while LAX-BP measurement revealed 22.00 pl ESV increase and 18.37 EDV increase. Thisdiscrepancy in detecting LV volumetric changes between two approaches could be the major factor determining the accuracy of echocardiography approaches.

[0050] To evaluate the accuracy of LAX-BP and PSAX-M mode approaches, 3 TAC mice underwent assessment with both LAX-BP and PSAX-M mode echocardiography methods as well as CMR. The Bland-Altman plot assay was employed to evaluate the overall agreement between the two echocardiographic approaches and CMR. The results revealed a high degree of agreement between LAX-BP and CMR assessments for LV volumes. In contrast, the PSAX-M mode approach tended to significantly overestimate LV volumes in TAC mouse hearts, especially the ESV (90.90±4.41 pl vs. 63.67±6.33 pl measured by CMR, p=0.038), whereas LAX-BP echocardiography provided comparable assessments to CMR as shown in FIG. 2C. FIG. 2C and FIG. 2D illustrate CMR validation of LAX-BP and PS AX M- Mode of ESV and EDV parameter showing LAX-BP has a much higher agreement with CMR when estimating diastolic and systolic volumes of diseased hearts, while PSAX-M mode overestimates both LV EDV and ESV.

[0051] This overestimated EDV and ESV consequently resulted in inaccurately elevated SV and CO in the PSAX-M mode (see, FIG. 2B). In summary, the CMR Bland-Altman analysis highlighted that LAX-BP echocardiography provides accuracy comparable to CMR in assessing LV volumetric functional parameters in TAC mouse hearts.

[0052] A4CLAX echocardiography enables clinically comparable assessments of atrioventricular hemodynamics and mitral valvular regurgitation

[0053] FIGS. 3A-3D generally illustrate improved doppler assays revealing LV restrictive filling patterns and mitral regurgitation in TAC Mice. FIG. 3 A illustrates a diagram of Pulsed Wave and Tissue Doppler gate placement on A4CLAX views as well as representative presurgery and post-surgery waveform recordings showing progression towards restrictive filling and LV stiffening. FIG. 3B illustrates Pulsed Wave Doppler assays show high LV filling patterns indicative of high atrial pressure and restrictive filling in 1-week TAC mice. FIG. 3C illustrates Tissue doppler results show LV stiffening from hypertrophic remodeling. FIG. 3D illustrates a diagram of normal cardiac cycle blood flow, and pathological mitral regurgitation above representative image of pre-op mice and recordings from a TAC mouse with mitral regurgitation. Increased Mitral Valve Area at 1 week post-surgery calculated from Pressure Half Time indicates mitral annular dilatation in TAC mice consistent with mitral regurgitation observations.

[0054] Doppler Imaging, including pulse wave, tissue, and color Doppler, is recommended for visualizing blood flow patterns and assessing hemodynamics and valvular function in clinic practice. The A4CLAX view displays the mitral valve and lateral annulus of mouse hearts. Consequently, pulse wave Doppler was conducted from the apical 4 chamber view in a clinical format, perfectly aligning the gate with the direction of mitral valve blood flow (see, FIG. 3A). The gate angle correction was set to parallel with the long axis of the LV. Additionally, blood flow directionality can be clearly observed from Color Doppler to make minor adjustments to this angle alignment of the pulsed wave doppler gate for most consistent results.

[0055] Pulse wave Doppler revealed a significant increase in mitral valve ejection time from 41.28±0.44 ms to 53.26±2.07 ms (n=4, p=0.0009) week 4 post-TAC, indicating that increased resistance from the constricted aorta prolongs blood ejection from the LV. The early diastolic mitral valve inflow velocity (E wave) was significantly increased from 641.34±77.85 mm / s to 816.91±37.34 mm / s (p=0.026), with a corresponding decline trend in the late inflow velocity (A wave) post-TAC (see, FIG. 3B), resulting in a significant rise in the E / A wave ratio from the first week post-TAC (1.81±0.18 vs. 1.19±0.12 pre-surgery, p=0.009). The E wave deceleration (E- Dec) was dramatically increased together with significant decrease of the E wave deceleration time within the first week. These results demonstrated that TAC-induced reduction in LV compliance and high filling pressures was rapidly occurring in the first week post-surgery, resulting in restrictive filling during the diastole. This rapid progression to restrictive filling and diastolic failure is consistent with LAX-BP assessments of declined LV function (see, FIGS. 3B and 2B).

[0056] Tissue Doppler on the LV lateral mitral annulus in the A4CLAX view (see, FIGS. 3A and 3C) showed a significant decrease in the peak velocity of late diastolic myocardial tissue motion (A’, from 29.70±2.44 mm / s to 18.08±3.91 mm / s, n=4, p=0.025 vs. pre-surgery), while the early diastolic myocardial tissue motion peak velocity (E’) had no significant changes. The E / E’ ratio notably increased from 26.03±2.92 to 32.63±1.53 (p=0.0054) during the four-week progression of TAC mouse hearts, indicating increased filling pressure and diastolic dysfunction in the hypertrophic LV.

[0057] Additionally, trans-mitral color Doppler in the A4CLAX view clearly visualize blood flowing into the LV during diastole and out to the aorta during systole of pre-TAC mouse hearts. However, TAC mice displayed a higher degree of turbulent diastolic inflow potentially due tohigher early filling velocities (See, FIGS. 3B and 3D). Starting from week 1 post-TAC, color Doppler detected backflow into the left atrium during LV systole, indicating mitral valve regurgitation. Additionally, pressure half time (PHT) analyzed from pulsed wave doppler was utilized to calculate mitral valve area (MV Area) which was significantly increased in mice 1 week post-TAC (46.00±5.47 mm2vs. 25.46±3.69 mm2pre-surgery, p=0.0104) indicating annular dilatation, a factor potentially causing MV regurgitation observed from color doppler (See, FIG. 3D).

[0058] In summary, the A4CLAX echocardiography revealed the rapid progression of LV remodeling in mouse TAC hearts. These findings also demonstrate that the novel A4CLAX echocardiography technique provides clinically comparable Doppler assays for precise evaluation of atrioventricular hemodynamics and mitral valve performance in mouse hearts.

[0059] A4CLAX echocardiography enables clinical evaluations of RV dysfunction secondary to LV pressure overload

[0060] FIGS. 4A-4E generally illustrate that A4CLAX imaging shows deteriorating trend of RV function secondary to LV pressure overload. FIG. 4A illustrates that RV systolic area shows increasing trend over time indicative of RV pressure increase. FIG. 4B illustrates that RV diastolic area also has increasing trend over time. FIG. 4C illustrates that RV Fractional Area Change declines at four weeks post-surgery, indicative of poor RV contractility. FIG. 4D illustrates that Tricuspid Annular Plane Systolic Excursion shows a non-significant increase in TAC mice, most likely caused by dilating trend of RV lumen. FIG. 4E illustrates a diagram showing RV pressure and volume increase resulting from TAC induced LV pressure overload.

[0061] In addition to LV assessments, it was investigated how the A4CLAX view can be used to assess right heart functions in mice. Although the current Vevo LAB software lacks a recommended protocol for RV assessments, the tracing tool typically applied in LV assays was utilized to trace the RV from the tricuspid lateral and medial annulus to the apex of the RV during both diastolic and systolic phases. The fractional area change (FAC) was calculated using the relationship:100 x ((Area Diastole - Area Systole) / Area Diastole).

[0062] A non-significant increase in RV volume was noted during diastole and systole phases in TAC mice at week 4 post-TAC (see, FIGS. 4A and 4B), while RV-FAC exhibited a declining trend (See, FIG. 4C).

[0063] To perform a tricuspid annular plane systolic excursion (TAPSE) assay, primarily used to assess overall RV longitudinal function, an anatomical M-Mode recording section was created using A4CLAX view images. In the Vevo LAB software, the M-Mode recording line was positioned over the RV, crossing the lateral RV annulus towards the apical aspect, and aligned parallel to the annulus movement (normally towards apex). The TAPSE assay was conducted by measuring the absolute annular distance traveled from diastole to systole. A non- significant increase in TAPSE among TAC mice was observed, rising from 0.785±0.078 mm to 0.964±0.055 mm within 4 weeks post-surgery (n=5, p=0.051) (See, FIG. 4D). This trend may be attributed to the enlargement of the RV lumen.

[0064] For a more comprehensive and precise study of RV function and remodeling, speckle tracking-based strain analysis was employed in the A4CLAX view images and studied the RV free wall longitudinal strain (RVFWLS). The RVFWLS assay, which necessitates exceptionally clear imaging of the entire RV free wall, has been shown to be more sensitive than RV-FAC and TAPSE in detecting RV dysfunction in clinical settings.15Indeed, the RVFWLS assay was conducted successfully in three TAC mice. Findings revealed a decrease in RVFWLS from 14.09±2.82% to 6.25±1.40% (week 1, p=0.034) and further to 2.19±0.69% (week 4, p=0.007), indicating rapid RV remodeling and dysfunction in TAC mice from the first week post-TAC (See, FIG. 5A). The speckle tracking assays also revealed regional desynchrony of the RV free wall in TAC mice.

[0065] A4CLAX echocardiography detects asynchronized chamber movements in mice

[0066] FIGS. 5A-5B illustrate that A4CLAX imaging enables assessments of chamber movement asynchrony in mice. FIG. 5 A is a Diagram of RV speckle tracking trace placement, and isolated RV free wall tracking results indicating decline of RV contractile function prior to dysfunction observed from RV FAC and TAPSE. Regional synchronicity assessment of the RV free wall indicates asynchronous free wall contraction and relaxation. FIG. 5B shows diagrams of pre-surgery, and two post-surgery lower cardiac chamber desynchrony phenotypes with representative traces indicative of septal and RV free wall contractile remodeling. The phenotype with Septal wall defect maintains normal diastolic opening patterns of atrioventricular valves due to normal LV free wall and apical function.

[0067] Using speckle tracking-based strain analysis, contractile synchronicity was studied throughout the septal and RV lateral walls (See, FIG. 5A). In the case of overall contractilefunction, septal wall motion is mostly attributed to LV function, while majority of RV contraction is from the RV free wall and longitudinal annular motion. In non-diseased hearts, both the RV free wall and septal wall have velocity vectors pointing in the same direction during cardiac phases, indicating LV and RV synchronized movements. However, two distinct phenotypes of RV and LV contractile remodeling was noted. In the first phenotype, LV septal wall movement during diastole (noted by MV opening) occurred towards the LV lumen, straying from the normal pre-operative septal wall relaxation pattern in which speckle vectors on the septum move away from the LV lumen indicating a radial relaxation (See, FIG. 5B). This inward septal motion occurred alongside normal RV free wall relaxation pattern, indicating LV desynchronized movements.

[0068] A second distinct ventricular remodeling pattern showed that the RV free wall in a TAC mouse 4 weeks post-surgery was contracting during normal LV diastolic phase (See, FIG. 5B). Due to RV free wall contraction being the largest component to RV function, this asynchronous contraction during diastolic phase caused a delayed opening of the tricuspid valve. Upon close inspection, by combining the A4CLAX recording with the ECG trace, it was observed that the tricuspid valve opened only after the ECG P-Wave. This indicates a lack of passive RV filling and highlights the necessity of right atrial kick to facilitate full valve opening.

[0069] The phenotype with RV free wall asynchronous contraction results in delayed opening of the tricuspid valve until right atrial contraction after P-Wave.

[0070] Improved left atrium and atrial appendage assessments reflect the pathological progression in TAC mouse hearts

[0071] FIGS. 6A-6B illustrate that A4CLAX imaging allows for atria and atrial appendage assessments in mice. FIG. 6A illustrates results from longitudinal left atrial tracings show significantly increased chamber size and decreased left atrial FAC 1 week post-surgery indicating rapid pressure increases in the LA. FIG. 6B illustrates LA monitoring in 1 mouse from pre-op, 1 week, and 4 weeks post-surgery showing severe dilation of the LA and atrial appendage over time.

[0072] A4CLAX echocardiography can capture clear images of the left atrium (LA), including the left atrial appendage (LAA), enabling us to study the LA pathological progression in TAC- mice (See, FIG. 6B). The LA of TAC mice was significantly enlarged from the first week postsurgery. Indeed, the LA FAC decreased from 30.99±1.61% to 20.67±1.30% by week 1 (n=6,p=0.0034) and further to 16.27±5.68% by week4 (p=0.0080 vs. pre-surgery, p=0.257 vs. week 1), suggesting that TAC surgery rapidly increased LA pressure.

[0073] In the late stage of the study, the capabilities of this new technique were explored and confirmed that A4CLAX echocardiography allows for pulse wave Doppler assessment of the LA appendage (LAA) (See, FIG. 6C). The LAA Doppler revealed sinus rhythm blood flow in the LAA of WT mice and successfully captured the sinus waveform peaks. Notably, waveform peaks aligned with the ECG tracings, indicating correct placement of pulsed doppler gate in the LAA. Doppler waveforms for LAA Doppler were inverted in Vevo LAB software to orient with clinical layout of normal LAA waveform since doppler gate recording directionality are inverted between transthoracic and transesophageal echo which is normally utilized for LAA doppler in clinical settings. These LAA assays, along with LV and RV echocardiography assessments, contribute to a comprehensive understanding of cardiac function and help elucidate the pathological progression in TAC mouse hearts.

[0074] Transverse Aortic Constriction (TAC) Surgery and Subject Mice

[0075] Adult C57BL / 6N mice were purchased from the Charles River at 10 weeks and housed in a temperature-controlled environment under an alternating 12-hour light / dark cycle at the Ohio State University. All animal handling procedures adhered strictly to the guidelines approved by the Institutional Animal Care and Use Committee under the protocol 2019A00000085. Both female and male mice with matched gender and ages were used in the studies.

[0076] TAC was performed to induce LV pressure overload conditions in 6 male and 6 female adult mice (10 weeks old). In brief, mice were anesthetized (isoflurane, 2.5%), intubated, and placed on a respirator (120 breaths per minute - 1; 0.1 ml tidal volume). A midline sternotomy was performed to expose the aorta, and a 7.0 Prolene suture 710 was placed around the aorta 720 distal to the brachiocephalic artery. The suture was tightened using a blunted 28-gauge needle next to the aorta as a guide for the degree of aortic constraint, the needle was removed, and the chest was closed. The success of TAC surgery was confirmed by observing restricted blood flow through the aortic arch using color doppler echocardiography imaging (See, FIGS. 7A-7C).

[0077] Alternatively, is noted that pulmonary artery catheter (PAC) surgery may be performed to induce RV pressure overload conditions in mice.

[0078] The construction and arrangement of the systems and methods as shown in the various implementations are illustrative only. Although only a few implementations have been described in detail in this disclosure, many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes, and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.). For example, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied. Accordingly, all such modifications are intended to be included within the scope of the present disclosure. The order or sequence of any process or method steps may be varied or re-sequenced according to alternative implementations. Other substitutions, modifications, changes, and omissions may be made in the design, operating conditions, and arrangement of the implementations without departing from the scope of the present disclosure.

[0079] The present disclosure contemplates methods, systems, and program products on any machine-readable media for accomplishing various operations. The implementations of the present disclosure may be implemented using existing computer processors, or by a special purpose computer processor for an appropriate system, incorporated for this or another purpose, or by a hardwired system. Implementations within the scope of the present disclosure include program products including machine-readable media for carrying or having machine-executable instructions or data structures stored thereon. Such machine-readable media can be any available media that can be accessed by a general purpose or special purpose computer or other machine with a processor. By way of example, such machine-readable media can comprise RAM, ROM, EPROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to carry or store desired program code in the form of machine-executable instructions or data structures, and which can be accessed by a general purpose or special purpose computer or other machine with a processor.

[0080] When information is transferred or provided over a network or another communications connection (either hardwired, wireless, or a combination of hardwired or wireless) to a machine, the machine properly views the connection as a machine-readable medium. Thus, any such connection is properly termed a machine-readable medium. Combinations of the above are also included within the scope of machine-readable media. Machine-executable instructions include, for example, instructions and data which cause a general-purpose computer, special purposecomputer, or special purpose processing machines to perform a certain function or group of functions.

[0081] Although the figures show a specific order of method steps, the order of the steps may differ from what is depicted. Also, two or more steps may be performed concurrently or with partial concurrence. Such variation will depend on the software and hardware systems chosen and on designer choice. All such variations are within the scope of the disclosure. Likewise, software implementations could be accomplished with standard programming techniques with rule-based logic and other logic to accomplish the various connection steps, processing steps, comparison steps and decision steps.

[0082] It is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular implementations only and is not intended to be limiting.

[0083] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another implementation includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another implementation. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0084] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0085] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal implementation. “Such as” is not used in a restrictive sense, but for explanatory purposes.

[0086] Although the subject matter has been described in language specific to structural features and / or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts described above are disclosed as example forms of implementing the claims.

Claims

AMENDED CLAIMS received by the International Bureau on 28 January 2025 (28.01.2025)What is claimed is:

1. A method for capturing apical 4-chamber-long-axis (A4CLAX) views of a heart of a subject: positioning the subject on a heated echocardiography platform; placing a support under an upper abdominal area of the subject; tilting the heated echocardiography platform at a predetermined angle; and capturing the A4CLAX views using a transducer via subcostal imaging.

2. The method of claim 1, wherein the heated echocardiography platform is tilted to a 50- degree angle.

3. The method of claim 1, further comprising aligning the transducer with an axis of the heart.

4. The method of claim 1, further comprising acquiring views of all four chambers of the heart.

5. The method of claim 4, further comprising acquiring a full longitudinal length of both a left ventricle (LV) and right ventricle (RV), an apex, mitral annulus and mitral valves, tricuspid annulus and tricuspid valve, and both right and left atria. .

6. The method of claim 1, further comprising: adjusting the support to elevate a back of the subject for better subcostal probe access; allowing for gravity to orient a long axis of the heart towards a left subcostal region of a thoracic cavity of the subject.

7. The method of claim 1, wherein the subject is a mouse.

8. A method for echocardiography analysis, comprising: capturing apical 4-chamber-long-axis (A4CLAX) views of a heart of a subject; obtaining parasternal long-axis B-mode (PLAX) images of the heart;26AMENDED SHEET (ARTICLE 19)creating a long axis biplane (LAX-BP) assay; and assessing left ventricle (LV) volumetric parameters.

9. The method of claim 8, further comprising: determining average LV volumes at an end of systole (ESV) and diastole (EDV) from each monoplane volume as two sets of values, PLAX-ESV and PLAX-EDV, and A4CLA -ESV and A4CLA -EDV; and deriving relationships for LA -BP- ESV and LAX-BP-EDV values as follows:

10. The method of claim 9, further comprising: tracing the LV from a mitral annulus to an apex along an LV wall in A4CLAX view; and tracing from an aortic valve to the apex in a PLAX viewing plane.

11. The method of claim 9, further comprising deriving EF, SV and CO volumetric parameters from the relationships.

12. The method of claim 8, assessing the LV volumetric parameters further comprising: quantifying monoplane volumes from LV tracings in both the PLAX and A4CLAX views.

13. The method of claim 12, wherein the LAX-BP is a modified Simpson’s method derived from the LV tracings.

14. The method of claim 8, wherein the LV volumetric parameters include at least one of end diastolic (EDV) and end systolic (ESV) volumes, in both wildtype, and disease model mice including but not limited to surgical and transgenic mouse models.

15. The method of claim 8, further comprising repeating the method to detect changes during a pathological progression in diseased hearts through longitudinal study.

16. A method of cardiac functional analysis that utilizes apical 4-chamber long axis (A4CLAX) views for analysis of cardiac function including at least one of:27AMENDED SHEET (ARTICLE 19)Left Ventricular analysis,Right Ventricular analysis,Left Atrial analysis,Right Atrial analysis,Mitral Valve analysis, andTricuspid Valve analysis.

17. The method of claim 16, further comprising assessing right heart function of the subject.

18. The method of claim 17, further comprising: tracing a right ventricle (RV) from a tricuspid lateral and medial annulus to an apex of the RV during both diastolic and systolic phases; and calculating a fractional area change (FAC).

19. The method of claim 17, wherein the FAC is determined using a relationship:100 x ((Area Diastole - Area Systole) / Area Diastole).

20. The method of claim 16, further comprising: determining LA pathological progression in mice using images of a left atrium (LA), including a left atrial appendage (LAA) captured from A4CLAX views.

21. The method of claim 16, wherein the cardiac analysis includes parameters of at least one of left ventricle (LV), left atrial (LA), right atrial (RA), right ventricle (RV), and valves of the heart.

22. The method of claim 16, further comprising repeating the method to detect changes during a pathological progression in diseased hearts23. The method of claim 16, further comprising detecting dysfunctions comprising at least one of valve regurgitation, restrictive filling, and de synchronized valve opening.

24. The method of claim 16, further comprising:28AMENDED SHEET (ARTICLE 19)using speckle tracking-based strain analysis to determine regional contractile synchronicity throughout the heart including septal wall, RV lateral wall, LV walls, and atrial walls.

25. The method of claim 16, wherein the analyses include functional parameters analyzed from A4CLAX views taken in wild type, and disease model mice.

26. The method of claim 16, wherein the subject is one of a mouse or a small animal subject.29AMENDED SHEET (ARTICLE 19)

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