Animal Model

The PAB surgery was performed as previously described and in detail in the Appendix . Three weeks after surgery, mice underwent echocardiographic investigation followed by invasive hemodynamic measurements. After completion of the study, heart tissue samples were collected for histologic and molecular analysis. From a subgroup of mice, hearts were separated into the right and left ventricles and used for RV/(LV + septum) ratio measurements. A detailed description of the experimental plan is provided in Supplemental Figure 1S .

Echocardiography

A Vevo 770 High Resolution Imaging System with a 30-MHz RMV-707B scan head (VisualSonics, Toronto, ON, Canada) was used for transthoracic echocardiographic measurements. Investigations were performed under mild anesthesia with isoflurane (0.8%–1.2%) as previously described and wherever possible in accordance with the guidelines for echocardiographic assessment of the right hearts. A detailed description is provided in the Appendix .

Hemodynamic Measurements

RV systolic pressure (RVSP), blood pressure, and LV systolic pressure were measured under isoflurane anesthesia (2%–4%) using a closed-chest technique, as described previously. Briefly, a 1.4-F pressure catheter (SPR-671; Millar Instruments, Houston, TX) was inserted into the right jugular vein and directed to the right ventricle to measure RVSP. Systemic measurements were obtained via catheterization of the carotid artery. The catheter was then directed into the left ventricle to obtain LV systolic pressure. Measurements were performed and recorded for ≥5 min. Analysis was performed only for the pressure recordings with stable hemodynamics. Care was taken to ensure that the measurements for hemodynamics and echocardiography were made at similar heart rates. Online tracings and analysis were made using LabChart 7 software (ADInstruments, Spechbach, Germany). We calculated +dP/dt, −dP/dt, end-diastolic pressure (EDP), pressure-time index, and τ from pressure tracings using software analysis module. We calculated (+dP/dt)/Pmax and (−dP/dt)/P as described previously. Hemodynamic measurements were performed by experienced personnel blinded to the experimental group.

Tissue Collection and Analysis

After invasive hemodynamic measurements, animals were immediately sacrificed, and tissue samples were collected for gene expression and histologic analysis. Briefly, the chest was opened and the heart and lungs were flushed with ice-cold phosphate-buffered saline. The lungs and heart were excised, the atria were removed, and the right ventricle was separated from the left ventricle and septum. Tissue pieces were weighed and then snap-frozen in liquid nitrogen and stored at −80°C until analysis. For immunohistochemical analysis, the hearts were removed, fixed in formalin, and embedded in paraffin. For determination of congestion, internal organs (lungs, kidneys, and livers) were removed and weighed immediately. Values are presented after normalization to tibia length. No difference in tibia length was observed. For wet-to-dry ratio measurements, pieces of organs were weighed and then kept at 40°C for 2 to 3 weeks and reweighed until stable values were obtained.

Morphologic Analysis

For assessment of fibrosis, formalin-fixed, paraffin-embedded heart tissue was cut in 2-μm-thick sections and stained for collagen content using Masson’s trichrome stain according to standard protocols (red, tissue; black, nuclei; blue, collagen). Slides were scanned (Scanscope T3; Aperio, Oxford, United Kingdom) and the level of fibrosis was quantified using semiautomated image analysis (Visiomorph DP software; Visiopharm, Horsholm, Denmark). The percentage fibrosis in the right ventricle was calculated by dividing the total area of collagen (fibrosis, blue staining) by the total analyzed tissue area multiplied by 100.

For quantification of cardiomyocyte cross-sectional area, formalin-fixed, paraffin-embedded heart tissue was cut in 2-μm-thick sections and stained for laminin (Abcam, Cambridge, United Kingdom). Staining was detected using AlexaFluor Dye 555 conjugated secondary antibody (Life Technologies, Amstetten, Austria). Nuclei were counterstained using 4′,6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich, St Louis, MO). Slides were scanned (Olympus BX61US; Olympus, Melville, NY) and obtained images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). In the inner layer of RV myocardium, 20 to 25 cells were identified as a nucleus containing areas of laminin staining with clearly identifiable borders.

Gene Expression Analysis

Total RNA was isolated from RV tissue samples using the peqGOLD Total RNA kit (PeqLab, Erlangen, Germany) and reverse-transcribed using the iScript kit (BioRad, Hercules, CA) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction was performed using the QuantiFast SYBR PCR kit (Qiagen, Hilden, Germany) on a LightCycler 480 (Roche, Mannheim, Germany). Expression data are normalized to the reference genes β ₂ -microglobulin and porphobilinogen deaminase using the following formula ΔCt = Ct reference gene − Ct gene of interest. Data are presented as ΔΔCt (ΔCt PAB sample − mean ΔCt of sham group). Higher ΔΔCt values correspond to higher relative gene expression. All samples were assayed in duplicate, and the average value was used for quantification. Primer sequences are given in Supplemental Table 2S .

Statistical Analysis

Echocardiographic measurements were performed by B.E., and data were saved for offline analysis. Analysis was done by two investigators (B.E. and S.C.). Primary variables were pressure gradient across the clip, RV free wall (RVFW) thickness, and LV eccentricity index. For calculation of variability, five mice were randomly selected from the PAB group. For the determination of intraobserver variability, B.E. analyzed data with reanalysis after 2 to 3 weeks. To determine interobserver variability, S.C. analyzed data from the same mice, and his results were compared with those of B.E. To ensure blinding, results of analysis were not saved on original recording files. Variability was defined as the absolute difference between two sets of measurements divided by the mean of the measurements and expressed in percentage form. Intraobserver variability of B.E. for pressure gradient across the clip was 1.5%, for RVFW thickness was 9.7%, and for LV eccentricity index was 3.7%. Interobserver variability for these parameters was 8.3%, 14.0%, and 4.1%, respectively. Results presented in this report were analyzed by B.E. Data are expressed as mean ± SD. Statistical analysis was performed using SPSS version 21 (SPSS, Inc, Chicago, IL). Mann-Whitney tests were used for intergroup comparisons. Correlation analysis was performed using Spearman’s nonparametric test. Correlation analysis included data from both experimental groups. P values < .05 were considered to indicate statistical significance.

Structural and Functional Changes in the Right Ventricle Due to PAB

Three weeks after operation, the PAB and sham-operated mice were investigated by echocardiography and subsequently by right-heart catheterization. Echocardiographic characterization revealed increased RVFW thickness, enlarged right ventricle, and reduced LV eccentricity index ( Table 1 and Figure 1 ). Tricuspid annular plane systolic excursion (TAPSE) and S′ were significantly reduced in the PAB group ( Table 1 ). Isovolumic contraction time was shortened and isovolumic relaxation time (IVRT) prolonged in the PAB group, resulting in similar Tei index values between the groups. The amplitude of the E wave was not significantly changed, and the amplitude of the A wave was elevated in PAB mice. The resulting E/A ratios were not different between the groups. Significant reductions of E′ in PAB mice were found by Doppler tissue imaging (DTI). There were no differences in the amplitude of A′ between the groups ( Table 1 ). Cardiac index was reduced in PAB mice ( Table 1 ). Right-heart catheterization revealed elevated RVSP, +dP/dt, and pressure-time index in PAB mice, confirming the increased RV afterload ( Table 2 ). Additionally, (+dP/dt)/Pmax, a parameter reflecting systolic function, was significantly decreased. RV EDP was elevated and τ prolonged in PAB mice, indicating diastolic dysfunction, although (−dP/dt)/P was unchanged. By gross morphologic measurements, a significant increase in the RV/(LV + septum) ratio was observed ( Figure 2 A). No signs of congestion were observed, as demonstrated by similar liver weights and lung and liver wet-to-dry ratios ( Figures 2B–2D ).

Pathologic changes in the heart induced by PAB. (A,B) Representative images of modified left parasternal long-axis view demonstrating RVFW thickness and RV internal diameter. (C,D) Representative images of parasternal short-axis view examination at the level of the papillary muscles. Note D-shaped LV deformation due to RV pressure overload. Measurements of LV internal diameters for eccentricity index calculation are shown. D1 , Diameter of the left ventricle measured from left parasternal short-axis view from the middle of the interventricular septum to the left ventricular free wall; D2 , diameter of the left ventricle measured from the same position perpendicular to D1, corresponding approximately to the points of attachment of the RVFW to the left ventricle.

Gross morphologic changes induced by PAB. (A) Ratio of right ventricular mass to left ventricular mass (RV/[LV+S]). (B) Lung wet-to-dry (W/D) ratio. (C) Liver weight values normalized to tibia length. (D) Liver W/D ratio. Blue dots represent sham animals, red dots represent PAB animals. Mean ± SD values are shown. * P < .01.

Pressure Overload–Induced RV Remodeling

We next investigated how the physiologic alterations in the right ventricle were reflected by gene expression changes of several molecular pathways representing diverse aspects of cardiac remodeling. Consistent with myocardial remodeling, decreased expression of α-Mhc (myosin heavy chain) and increased β-Mhc was detected ( Figures 3A and 3B ). Furthermore, increased messenger ribonucleic acid expression of atrial and brain natriuretic peptides was detected in the RV tissue ( Figures 3C and 3D ). Analysis of expression of Ca ²⁺ -handling proteins demonstrated downregulation of Serca2 and phospholamban but no changes in Ryr2 expression ( Figures 3E–3G ). Compared with sham mice, the messenger ribonucleic acid levels of collagen types 1a1 and 3a1 as well as connective tissue growth factor were significantly elevated in the right ventricles of PAB mice ( Figures 3H–3J ). Masson’s trichrome staining revealed significantly increased levels of RV fibrosis in PAB mice ( Figures 4A–4C ), which was restricted to the right ventricle only ( Supplemental Figure 2S ). Laminin staining revealed increased cross-sectional area of RV cardiomyocytes in PAB mice ( Figures 4D–4F ).

Gene expression changes in the RV tissue after PAB. Expression of (A) markers of ventricular wall stress (Anp and Bnp), (B) fetal gene program activation (αMhc and βMhc), (C) Ca ²⁺ -handling proteins (Ryr2, Serca2, and Pln), (D) collagens (Col1a1 and Col3a1) and profibrotic factor Ctgf was determined by real-time polymerase chain reaction in RV tissue samples. Results are presented as relative change in expression (ΔΔCt) compared with mean expression values in the sham group. Blue dots represent sham animals, red dots represent PAB animals. Mean ± SD values are shown. * P < .05; ** P < .01; *** P < .001.

Right ventricular remodeling in PAB mice. (A,B) Representative images of Masson’s trichrome staining visualizing areas of fibrosis ( blue , fibrosis; red , muscle tissue). Note stronger blue staining in the PAB group, indicating fibrotic changes. Scale bar represents 100 μm. (C) Quantification of RV fibrosis (sham n = 4, PAB n = 5) stained with Masson’s trichrome. (D,E) Representative images of laminin staining to determine cell size. Scale bar on fluorescence images represents 50 μm. (F) Mean cardiomyocyte cross-sectional area of sham and PAB hearts. Blue dots represent sham animals, red dots represent PAB animals. Mean ± SD values are shown. * P < .05; ** P < .01.

Correlation between Parameters of RV Function and Morphologic Changes

TAPSE and S′ have been suggested for quantification of ventricular contractile function and were strongly correlated with each other ( Supplemental Figure 3S ). Both TAPSE and S′ were negatively correlated with +dP/dt ( Supplemental Table 2S ), reflecting the afterload dependency of these parameters. TAPSE was also moderately positively correlated ( rs = 0.499, P = .011) with the contractility index, suggesting that it indicates systolic function and thus the adaptation to the increased afterload. However, TAPSE was even more strongly negatively correlated with EDP ( rs = −0.574, P = .003) and τ ( rs = −0.674, P < .001), suggesting that decreased TAPSE indicates diastolic dysfunction. Moreover, TAPSE was significantly negatively correlated with the expression level of Col1a1 and Col3a1 ( rs = −0.636 and rs = −0.729; Supplemental Table 2S ). In line with this observation, TAPSE was strongly negatively correlated with the percentage of fibrosis ( rs = −0.755, P = .007; Supplemental Table 2S and Supplemental Figure 3S ).

Among the tested echocardiographic parameters describing diastolic function, E′, E/E′ ratio, and IVRT were significantly correlated with invasively measured parameters of diastolic function EDP and τ ( Figures 5A–5C and 5E–5G ). E′ and the E/E′ ratio and IVRT were significantly correlated with the level of Col1a1 ( Supplemental Table 2S and Figures 5I and 5J ). Like TAPSE, E′ was also strongly negatively correlated with the percentage of fibrosis ( rs = −0.717, P = .03; Supplemental Table 2S ).

Correlation of noninvasively measured parameters with invasive parameters of diastolic function and the level of collagen expression. Correlation of (A–D) EDP, (E–H) time constant of diastolic pressure decay (τ), and (I–L) collagen 1a1 expression with IVRT, early peak of RV diastolic relaxation (E′), ratio of the early peak of diastolic tricuspid inflow to the peak of RV diastolic relaxation (E/E′ ratio), and RVFW thickness. Blue dots represent sham animals, red dots represent pulmonary arterial banding animals.

RVFW thickness estimated by echocardiography was very strongly correlated with RV mass normalized to LV + septum ( rs = 0.786, P = .021) or body weight ( rs = 0.952, P < .001). RVFW thickness was strongly positively correlated with RVSP ( rs = 0.630, P = .001) and moderately negatively with (+dP/dt)/Pmax ( rs = −0.526, P = .007), suggesting that increased RVFW thickness could indicate both RV afterload and systolic dysfunction ( Supplemental Table 2S ). In line with this observation, RVFW thickness was strongly correlated with TAPSE ( Supplemental Figure 3S ). RVFW thickness was also strongly correlated with EDP ( rs = 0.663, P < .001) and τ ( rs = 0.830, P < .01), suggesting that it indicates diastolic dysfunction. The correlation with Col1a1 was only moderate ( Supplemental Table 2S , Figure 5 ).

Technical Aspects of Tricuspid Flow Measurements and DTI

We observed high variability in the tricuspid inflow profile depending on respiratory cycle ( Supplemental Figure 4SA ). This resulted in significant changes in the E/A ratio depending on the respiratory phase ( Supplemental Figure 4SB ). In contrast, the DTI-derived parameters E′ and A′ showed less variation due to respiration ( Supplemental Figures 4SC and 4SD ). Both tricuspid flow and DTI demonstrated partial or complete fusion of early (E or E′) and late (A or A′) waves depending on heart rate ( Supplemental Figure 5S ). Amplitudes of A and A′ were measured at heart rates < 420 beats/min, at which they were clearly identifiable. Thus, these technical aspects interfered with measurements of deceleration time of the E wave and the E/A ratio, making them unreliable for the characterization of RV diastolic function in our model.

Structural and Functional Changes in the Right Ventricle Due to PAB

Three weeks after operation, the PAB and sham-operated mice were investigated by echocardiography and subsequently by right-heart catheterization. Echocardiographic characterization revealed increased RVFW thickness, enlarged right ventricle, and reduced LV eccentricity index ( Table 1 and Figure 1 ). Tricuspid annular plane systolic excursion (TAPSE) and S′ were significantly reduced in the PAB group ( Table 1 ). Isovolumic contraction time was shortened and isovolumic relaxation time (IVRT) prolonged in the PAB group, resulting in similar Tei index values between the groups. The amplitude of the E wave was not significantly changed, and the amplitude of the A wave was elevated in PAB mice. The resulting E/A ratios were not different between the groups. Significant reductions of E′ in PAB mice were found by Doppler tissue imaging (DTI). There were no differences in the amplitude of A′ between the groups ( Table 1 ). Cardiac index was reduced in PAB mice ( Table 1 ). Right-heart catheterization revealed elevated RVSP, +dP/dt, and pressure-time index in PAB mice, confirming the increased RV afterload ( Table 2 ). Additionally, (+dP/dt)/Pmax, a parameter reflecting systolic function, was significantly decreased. RV EDP was elevated and τ prolonged in PAB mice, indicating diastolic dysfunction, although (−dP/dt)/P was unchanged. By gross morphologic measurements, a significant increase in the RV/(LV + septum) ratio was observed ( Figure 2 A). No signs of congestion were observed, as demonstrated by similar liver weights and lung and liver wet-to-dry ratios ( Figures 2B–2D ).

Table 1

Echocardiographic characterization of mice 3 weeks after operation

	Sham ( n = 13)	PAB ( n = 12)	P
Body weight (g)	27.1 ± 1.3	26.8 ± 1.7	.661
Heart rate (beats/min)	409 ± 51	419 ± 27	.580
RVFW thickness (mm)	0.21 ± 0.06	0.43 ± 0.12	<.001
RVIDd (mm)	1.67 ± 0.41	2.58 ± 0.38	<.001
D1 (mm)	4.18 ± 0.17	3.25 ± 0.4	<.001
D2 (mm)	4.53 ± 0.18	4.35 ± 0.47	.201
Eccentricity index (D1/D2)	0.93 ± 0.04	0.70 ± 0.05	<.001
TAPSE (mm)	1.02 ± 0.1	0.60 ± 0.19	<.001
Gradient velocity (mm/sec)	668 ± 94	2,877 ± 372	<.001
E (mm/sec)	227 ± 92	316 ± 142	.097
A (mm/sec)	412 ± 51	504 ± 202	.007
E/A ratio	0.50 ± 0.11	0.57 ± 0.26	.471
ET (msec)	61.1 ± 3.6	71.2 ± 6.3	<.001
TCOT	81.2 ± 4.7	90.6 ± 9.0	.008
IVCT (msec)	11.3 ± 1.2	8.3 ± 0.9	<.001
IVRT (msec)	8.8 ± 3.0	16.4 ± 4.3	<.001
Tei index	0.33 ± 0.05	0.27 ± 0.11	.138
E′ (mm/sec)	22.2 ± 5.4	15.8 ± 4.2	.007
A′ (mm/sec)	29.3 ± 7.6	26.1 ± 9.0	.37
S′ (mm/sec)	27.5 ± 4.1	19.8 ± 4.1	<.001
E/E′ ratio	10.5 ± 3.4	20.2 ± 8.8	.003
CI (mL/min/g)	0.61 ± 0.11	0.48 ± 0.07	.003

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related

Related posts:

1. The Miniaturized Multiplane Micro-Transesophageal Echocardiographic Probe: A Comparative Evaluation of Its Accuracy and Image Quality
2. Mitral Annular Dynamics in Mitral Annular Calcification: A Three-Dimensional Imaging Study
3. The Effect of Thrombolytic Use and Mechanical Ventilation on Echocardiographic Parameters of Survival after Acute Pulmonary Embolism
4. Passing the Torch of Leadership—The Bridge of Continued Excellence

Stay updated, free articles. Join our Telegram channel

Join