We derived four stable TG lines, the goal being to create a collection of lines in which varying proportions of the normal β-MHC protein complement was replaced by the transgenically encoded α-MHC isoform. As is the case for the mouse,14,15
total myofilament myosin is controlled such that expression of the transgenically encoded transcript and α-MHC protein results in reduced steady state levels of β-MHC. The degree of replacement is dependent upon the level of TG expression and normal sarcomeric myosin levels are maintained. TG expression varied somewhat between the four lines, with α-MHC replacement ranging from 15%–43% (). We focused on lines 18 and 45, which showed, within the limits of individual variation, essentially identical levels of replacement: approximately 40% ± 3% of the endogenous β-MHC was replaced with α-MHC. At all stages of development the animals appeared to be healthy. Detailed histological analyses were performed to assess for cardiac pathology resulting from persistent expression of α-MHC. Age- and gender-matched TG and NTG myocardium were examined throughout the adult period without striking differences in hypertrophy, interstitial fibrosis and myocyte disarray being found (). We also performed RNA dot blots and quantitative real time PCR on samples isolated from line 18 TG and NTG littermates to look for activation of molecular markers of hypertrophy or failure. As expected, the TG rabbits showed significant increases in ventricular α-MHC (P
<0.001) and decreases in β-MHC (P
=0.01). Transcript levels for ANF, PLN and SERCA2a showed no statistically significant variations ().
Figure 1 Protein levels and basic characteristics of the α-MHC TG rabbits. A, TG protein replacement and separation of the MHC isoforms via PAGE. The LV NTG sample used in this gel has no detectable α-MHC. LV samples from the four TG lines show (more ...)
To confirm activity of the TG protein, actin-activated ATPase rates were determined with NTG atrial and LV samples as controls. TG LV samples, with a 40/60 mix of α-MHC and β-MHC showed maximal ATPase activity that was approximately 40% that of NTG atrium, which has essentially 100% α-MHC (). These data confirmed that transgenically encoded α-MHC is biochemically active and influences the overall actin-activated ATPase activity as predicted based upon the α-:β-MHC ratio.
We then assessed cardiac structure and function. Serial echocardiograms were performed at 4, 8–9 and 12–15 months on cohorts of TG and NTG littermates (). At 9 months, the TG animals had slightly smaller LV end-diastolic dimensions and very mild septal hypertrophy. These differences were not apparent in 12–15 month rabbits. At no time did we find differences between the TG and NTG rabbits with respect to ventricular systolic or diastolic function, and no structural heart defects were detected. We also performed cardiac catheterization to directly measure LV pressure. The cohort consisted of 5 TG and 6 NTG mixed gender, 12 month (lines 18 and 45) rabbits. There were no significant differences in systemic blood pressure, LV peak systolic and end diastolic pressures, or maximum and minimum dP/dt (). Taking the histological, non-invasive and invasive data together, we conclude that persistent TG expression of α-MHC at high levels beyond the juvenile period is benign in the unstressed animal.
Baseline Echocardiographic Data
Baseline Invasive Hemodynamics
As no pathology or alterations at the molecular and cellular levels could be detected, we reasoned that they were a suitable model to test the hypothesis that α-MHC in the ventricle is cardioprotective. Rapid ventricular pacing results in depressed myocardial function in many species with clinical and laboratory findings very similar to that seen in human DCM.16,17
We applied tachycardia induced cardiomyopathy (TIC) to the α-MHC TG rabbits and developed a protocol similar to that of Jeron et al ().18
The initial cohort consisted of 6 TG and 7 NTG 15 month line 45 rabbits of mixed gender (2 females and 4 males in the TG group and 2 females and 5 males in the NTG group). All but one animal survived the pacing regimen. One clinically asymptomatic NTG male rabbit died 5 minutes after the pacemaker rate was increased to 380 bpm. A necropsy showed ascites, pleural and pericardial effusions and a large, dilated heart. The remaining TG and NTG animals appeared outwardly normal throughout the protocol with normal cage behavior, grooming, appetite and respiratory effort.
To assess the effects of TIC at the molecular level, we compared RNA isolated from paced TG and NTG rabbits to unpaced NTG rabbits and found that both TG and NTG paced rabbits had significant increases in atrial ANF and BNP expression and increased ventricular BNP transcript (). The differential levels of ANF and BNP between the cardiac compartments are similar to those noted by investigators using TIC in rabbits.19
Paced TG and NTG rabbits had significant decreases in atrial SERCA and PLN RNA compared to non-paced NTG, but transcript levels were not significantly down-regulated in the ventricles.
Figure 3 Relative RNA levels in unpaced and paced atria and ventricles. RNAs were collected at the end of the protocol and blotted onto nitrocellulose. Transcript levels were determined via hybridization to transcript specific oligonucleotides and those data used (more ...)
We predicted a significant difference between paced TG and NTG in the relative levels of α- and β-MHC at both the RNA and protein levels. As expected, RNA dot blots showed significant increases in ventricular β-MHC message in the paced NTG rabbits and ventricular α-MHC RNA for the TG paced rabbits. At the protein level, this correlated with a MHC complement of 100% β-MHC in myofibril preparations from paced NTG animals and persistent α-MHC accumulation in paced TG samples (), with the ratio of α-:β-MHC in the TG animals shifting from 40:60 under basal conditions to 60:40 after pacing.
Figure 4 Post-pacing characteristics of TG and NTG rabbits. A, PAGE of ventricular myosin showing the α- and β-MHC isoforms after completion of the pacing protocol. The ratio of α-MHC:β-MHC increased slightly in the paced TG ventricles (more ...)
Light microscopy revealed that rapid ventricular pacing produced histological changes consistent with dilated cardiomyopathy in both TG and NTG rabbits (). We found that while both groups of paced animals exhibited considerable variation in cardiomyocyte size, compared to a non-paced NTG control, both had a significantly smaller mean cardiomyocyte cross sectional area (NTG-paced 90.0 ± 40.9 μm3, TG-paced 88.8 ± 40.9 μm3, NTG-control 108.6 ± 46.8 μm3, P < 0.0001 for both paced groups vs. control, ) and evidence of more extensive interstitial fibrosis.
At the whole organ level, there were no differences between TG and NTG discerned by the pre-operative echocardiogram (). At the protocol’s termination, the change in IVS thickness was significantly different between NTG and TG, with a decrease in septal thickness seen in NTG rabbits (ΔNTG −0.06 ± .04 cm vs. ΔTG 0.03 ± .05 cm, P=.02). At the final study, the change in shortening fraction in paced NTG rabbits was significantly different than that of TG rabbits (ΔNTG −19 ± 5% vs. ΔTG −14 ± 4%, P=.02). The pre- to post-pacing change in heart rate-corrected velocity of circumferential fiber shortening (VCFc), a pre-load independent measure of contractility, was not statistically different (ΔNTG −0.74 ± .3 circ/sec vs. ΔTG −0.52 ± .41, P = .36), which was likely due to greater variability in the TG cohort. shows representative M-mode tracings from the final echocardiograms.
Echocardiographic Indices Of Paced Cohort
To further investigate cardiac function, 10 rabbits (5 NTG and 5 TG of mixed gender) underwent the pacing protocol and Millar catheterization at the terminal study. Compared to NTG paced rabbits, TG paced animals had higher systemic blood pressure, higher peak left ventricular systolic pressure, a higher +dP/dtmax and lower −dP/dtmin(). There was no significant difference in heart rate or left ventricular end diastolic pressure. These invasive measures of ventricular function are consistent with the echocardiographic data
Invasive Hemodynamics After Pacing Protocol