3.1. Myocardial infarction model
A total of 19, 8 month old rabbits (9 TG and 10 NTG) with significant LVFW infarcts (% infarct 32 ± 3% TG versus 31 ± 4% NTG, p
= 0.95) survived for study. An additional 8 rabbits (4 TG and 4 NTG) underwent sham operations. All animals had an echocardiogram performed immediately preceding surgery, then three and six weeks post-surgery. The NTG rabbits were significantly heavier than the TG group (4.7 ± .6 kg NTG versus 3.7 ± .5 kg TG, p
= .002, likely due to a preponderance of females in the NTG group), so echocardiographic variables affected by body mass, including LV dimensions, wall thickness and mass were indexed to body weight. As previous studies have shown no gender-related differences in echocardiography parameters ([12
] and unpublished data) and the number of rabbits relatively small, data were not analyzed for potential gender-related effects. The pre-surgery data from the TG and NTG shams were combined as, aside from aortic ejection time (ET) and VCFc, the differences between the two sham groups failed to reach statistical significance. We found subtle baseline differences between genotypes, with the TG rabbits having higher IVS, LVFW and LVEDD compared to NTG (). The ET was consistently shorter in TG animals, likely a manifestation of the faster crossbridge cycling rate inherent to the α-MHC [20
]. Accordingly, VCFc, a preload independent measure of contractility with ET in the equation's denominator, was also increased.
Echocardiographic assessment of cardiac structure and function following coronary artery ligation.
Based on our previous results with the TIC model, we hypothesized that significant differences between TG and NTG would likely present early (i.e., 6 weeks following coronary artery ligation). However, we found no significant difference in LV morphometry, and functional parameters were not strikingly different aside from a significantly shorter ET and higher VCFc in the infarcted TG rabbits (). Additionally, we performed Millar catheterization and tissue harvest in all shams and a subset of infarcted animals (3 TG and 2 NTG) at 6 weeks post-surgery and found no significant differences between TG and NTG (data not shown).
We posited that perhaps 6 weeks was too early in the compensatory process for MHC alteration to exert an affect on ventricular structure and function, and thus elected to follow the remaining animals (8 TG and 6 NTG) with serial echocardiograms until 9 months post-surgery. Even 9 months after coronary artery ligation, the effects of genotype on cardiac function remained subtle. Serial echocardiography over the follow-up period demonstrated persistent differences in ET and VCFc ( and ) and the TG rabbits had mildly elevated LV dimensions 9 months post-infarct (). Cardiac catheterization 9 months after coronary ligation showed a slight elevation in peak LV systolic pressure at baseline in TG rabbits (75 ± 11 mmHg TG versus 63 ± 8 mmHg NTG, p = 0.02). However, the NTG rabbits were able to achieve identical peak LV pressure at DOB10, and there were no significant differences between genotypes at the higher infusion rates of DOB20 and DOB30. Interestingly, we noted mildly elevated LV end diastolic pressure (LVEDP) in TG rabbits compared to NTG at all points in the Millar protocol, with LVEDP of 12 ± 3 mmHg in the TG rabbits versus 9 ± 2 mmHg in the NTG animals (p = 0.02). There was no significant difference in ±dP/dt between TG and NTG rabbits, with both groups showing comparable measurements at baseline and in response to DOB infusion ().
Fig 1 Left ventricular functional parameters following myocardial infarction. (A) Assessment of post-infarction left ventricular function by serial echocardiography, including shortening fraction (top), aortic valve ejection time (middle) and VCFc (bottom). (more ...)
As was the case with the serial echocardiograms, in the final echo studies the only difference between TG and NTG MI rabbits was in the VCFc, which was slightly increased at baseline in the TG rabbits (1.70 ± .27 circ/s TG versus 1.42 ± .15 circ/s NTG, p = 0.03). The disparity between genotypes was most apparent at DOB10, when the VCFc in the TG rabbits increased to 2.25 ± .18 circ/s TG versus 1.64 ± .21 circ/s NTG (p = 0.0001). In TG rabbits, escalating DOB above 10 μg/kg/min did not result in any further increase in VCFc. In the NTG group, the VCFc increased slightly between DOB0 and DOB10, with the greatest increase occurring between DOB10 and DOB20 (from 1.64 ± .21 circ/sec to 2.01 ± .20 circ/sec). Since there was no significant difference between genotypes in heart rate or SF at any DOB dose (data not shown), the observed VCFc differences between TG and NTG can be accounted for by the shorter ET in the TG rabbits.
Seven to ten days after cardiac catheterization, rabbits underwent cMRI as a terminal procedure (). The advantages of cMRI include the evaluation of those portions of the LV not assessed by echocardiography (i.e., the infarcted LV lying below the mid-cavity papillary muscles), quantification of ventricular volumes and cardiac output and assessment of regional wall motion abnormalities. While the LV end-diastolic volume, LV end-systolic volume and ejection fraction were not significantly different, the infarcted TG rabbits had a significantly higher cardiac index compared to infarcted NTG (26 ± 8 mL/min/kg TG versus 16 ± 5 mL/kg/min NTG, p = 0.02).
Cardiac MRI measurements 9 months after coronary artery ligation
Using semi-quantitative PCR, we compared the relative LVFW expression of α-MHC, β-MHC, BNP, SERCA and PLN among 4 groups: NTG and TG sham (6 weeks post-surgery) and NTG and TG MI (9 months post-surgery), normalizing to GAPDH and expressed as X-fold NTG sham 6 weeks post-surgery (). Comparable results were obtained with samples derived from MI rabbits 6 weeks post-surgery (data not shown). The only significant change noted was in α-MHC expression, which was increased both at baseline and after MI in the TG rabbits (sham and MI TG 4.3-fold and 5.7-fold compared to 6 week post-surgery NTG sham rabbits, p ≤ 0.002 for both). The increase in α-MHC expression was confirmed using SDS-PAGE (data not shown), with α-MHC comprising 42 ± 8% of total MHC in the LVFW of TG sham rabbits, increasing to 63 ± 5% in TG MI rabbits (p = 0.03). In contrast, α-MHC composition of LVFW samples from NTG sham rabbits was 4 ± 3% and 4 ± 1% in NTG MI rabbits (p = NS).
Fig. 2 Molecular and histological consequences of coronary artery ligation. (A) Semi-quantitative real-time PCR analysis comparing relative expression of the MHC isoforms and molecular markers of hypertrophy 9 months after coronary ligation (normalized to GAPDH (more ...)
Light microscopy was performed on samples of viable LVFW 9 months post-surgery to assess for changes in ventricular architecture that might be associated with genotype. We found no striking differences between TG and NTG MI hearts using H&E and trichrome staining ().
3.2. Aortic banding model
The different outcomes noted between myocardial dysfunction models (i.e., a conclusive benefit from persistent α-MHC expression in TIC versus a subtle, if any benefit in MI) suggested that the effects of α-MHC replacement on ventricular function and remodeling might be dependent upon the mode of cardiac stress. Accordingly, we created LV pressure overload in 10 day old rabbits by placing an initially non-obstructive suture around the descending aorta. We chose 10 day old rabbits for surgery as their size at that age enhanced surgical survival and yet was early enough to take advantage of the rapid increase in size that rabbits experience in the first 6 weeks of life. Compared to transverse aortic constriction commonly performed in mature mice, in neonatal aortic banding the suture becomes increasingly more obstructive with growth, resulting in escalating LV pressure overload with time. This model allows for compensatory ventricular remodeling stimulated by progressive obstruction, comparable to that experienced by humans with conditions such as coarctation of the aorta or aortic stenosis.
Surgeries were performed on day-of-life (DOL) 10 with tissue taken at the time of operation for subsequent genotyping. Only three rabbits required supplemental (intraperitoneal) fluids in the first 24 hours post-surgery, with complete recovery by 72 hours. Our cohort consisted of 61 banded animals (16 TG and 45 NTG, designated TG-B and NTG-B, respectively) with an additional 17 rabbits randomly selected for sham operations (8 TG and 9NTG, TG-S and NTG-S, respectively). Since we ultimately found no detectable anatomic or functional differences between TG-S and NTG-S at the ages studied in this model, TG-S and NTG-S echocardiography and catheterization data were combined for comparison to banded rabbits. The 3 groups had similar weights on DOL 10 () and comparable weight gains over the course of the study (data not shown). Serial echocardiography demonstrated a highly reproducible phenotype with LV hypertrophy and fairly well preserved systolic function persisting in both TG-B and NTG-B animals up to three weeks post-surgery (). Thereafter, LV dimensions increased as systolic performance worsened. While the banded rabbits were clearly compromised compared to sham operated controls, there were no significant differences between TG-B and NTG-B in echocardiographic parameters, including LV systolic and diastolic dimensions, SF and septal and LVFW thickness. As we observed previously [12
], at most time points the TG-B rabbits had a significantly shorter LV ET (p
≤ 0.02) compared to NTG-B (data not shown), but this did not translate into a consistently higher VCFc in the TG-B cohort.
Aortic banding invasive hemodynamics
Fig. 3 Left ventricular parameters and survival following aortic banding at 10 days of age. (A) Serial echocardiographic assessment of left ventricular size (top) and systolic performance (shortening fraction and VCFc, middle and bottom panels, respectively). (more ...)
Rabbits were removed from the cohort for terminal invasive hemodynamic assessment and tissue harvest once the SF dropped to ≤ 20% or VCFc ≤ 1.0 circ/sec. Sham operated animals were randomly selected for cardiac catheterization each week until the last of the banded animals went into failure, at which time all remaining control rabbits underwent cardiac catheterization and tissue harvest. Due to unexpected death in some banded animals during the course of the study, we collected complete invasive hemodynamic data on 38 NTG-B, 10 TG-B and 12 sham controls. Time to failure was not different between genotypes (), nor was the peak pressure gradient between ascending and abdominal aorta (). Banded rabbits of both genotypes had significantly higher LVSP (p < 0.05 NTG-B versus sham and p < 0.001 TG-B versus sham) and higher LVEDP (p < 0.001 NTG-B versus sham and p < 0.05 TG-B versus sham). Between the banded groups, the TG-B LVSP was significantly higher (120 ± 19 mmHg TG-B versus 103 ± 119 mmHg NTG-B, p < 0.05), but there was no genotype-dependent difference in LVEDP. Both NTG-B and TG-B showed significantly depressed dP/dtmax compared to shams (p < 0.001 NTG-B versus sham and p < 0.01 for TG-B versus sham), but the difference between NTG-B and TG-B rabbits was not significant. Interestingly there was a significant difference in dP/dtmin with NTG-B showing impairment in this measure of diastolic performance compared to both TG-B and sham (-2394 ± 1000 mmHg/sec for NTG-B, -3229± 747 mmHg/sec for TG-B and 3171 ± 717 mmHg/sec for sham, p < 0.05 NTG-B versus both sham and TG-B, p = NS for TG-B versus sham).
At the time of tissue harvest, pericardial and pleural effusions, left atrial enlargement and ascites were common observations in both TG-B and NTG-B rabbits. However, we found no striking differences by light microscopy in the histological appearance of TG-B and NTG-B hearts with qualitatively equivalent myocyte size and interstitial fibrosis ().
Fig. 4 Effects of aortic banding. (A) Representative histology of failing 6 week old TG (i, ii) and NTG (iii, iv) banded rabbits with H&E (i, iii) and trichrome (ii, iv) staining. All images 400× magnification. (B) Comparison of hypertrophy marker (more ...)
Molecular markers of hypertrophy were assessed by semi-quantitative real time PCR. To determine relative expression of α- and β-MHC at the time of surgery, left ventricular RNA was isolated from 10 day old TG and NTG rabbits and used in real-time experiments. At this age, the TG rabbits demonstrated 3.9-fold overexpression of α-MHC compared to NTG, with both genotypes showing equivalent expression of β-MHC (). Transcript levels of ANF, SERCA2a and PLN were not significantly different between genotypes, indicating that at the time of operation no differences existed in the molecular phenotype aside from the expected upregulation of α-MHC in TG rabbits.
Real-time PCR analyses were likewise performed on RNA isolated from left ventricular tissue harvested at the time of terminal study (). The levels of α-MHC, β-MHC, SERCA2a, PLN and brain natiuretic peptide (BNP) were normalized to GAPDH expression. Comparisons were made among TG-B, NTG-B, TG-S and NTG-S using one-way ANOVA and the Tukey multiple comparisons post test. Values are expressed as X-fold relative to NTG-S samples. The only difference between the two sham genotypes was in α-MHC expression, with 3.4-fold overexpression in TG-S compared to NTG-S (P ≤ 0.05).
As expected, the greatest α-MHC levels were found in TG-B rabbits, with 4.8-fold overexpression compared to NTG-S (P ≤ 0.001), while in TG-S α-MHC was upregulated 3.4-fold. NTG-B rabbits had essentially non-detectable levels of α-MHC at 0.04-fold compared to NTG-S (P ≤ 0.01 versus NTG-S and ≤ 0.001 versus TG-B). We found no difference among the groups in β-MHC or PLN expression. SERCA2a was significantly downregulated in both TG-B and NTG-B rabbits compared to NTG-S (P ≤ 0.01), but there was no genotype-dependent difference between the banded or sham groups. Not unexpectedly given the severity of cardiac dysfunction, BNP expression was markedly increased in both TG-B and NTG-B animals compared to NTG-S, but again no genotype dependent differences presented.
One explanation for the lack of a dramatic phenotypic difference between pressure-overloaded TG and NTG rabbits is that transgenic overexpression of α-MHC at the RNA level may not directly translate into protein accumulation in young rabbits during developmental stage modulation of endogenous MHC isoforms. While we have consistently found excellent correlation between MHC transcript and protein levels in older rabbits, the MHC isoform composition of the ventricles varies significantly in the first few months of post-natal life as the fetal gene program is down-regulated [21
]. To quantify accumulation of α-MHC we used an isoform-specific antibody for Western blot analysis of LV protein extracts from 6 week old NTG-B, TG-B, NTG-S and TG-S rabbits (). NTG-S LA samples were used as a representation of 100% α-MHC. Unexpectedly, we found relatively high levels of α-MHC accumulation in 6 week old NTG-S animals (35 ± 1%), exceeding the typical 5 – 10% α-MHC in mature rabbits and in contrast to the relatively low α-MHC message level determined by real-time PCR. The 6 week old TG-S rabbits (with 3.4-fold α-MHC RNA) message showed 45 ± 9% LV α-MHC accumulation compared to 62 ± 6% in TG-B rabbits (4.9-fold α-MHC RNA). NTG-B rabbits had significantly less but generally still detectable α-MHC (8 ± 6%), even though the α-MHC message level as determined by real-time PCR was barely detectable. Taken together, the lack of perfect congruence between message and protein levels in 6 week old NTG groups is likely due the persistence of α-MHC protein synthesized earlier in life and not yet replaced by β-MHC.