Heterogeneous abnormalities of in-vivo left ventricular calcium influx and function in mouse models of muscular dystrophy cardiomyopathy
© Greally et al.; licensee BioMed Central Ltd. 2013
Received: 15 June 2012
Accepted: 14 December 2012
Published: 16 January 2013
Manganese-enhanced cardiovascular magnetic resonance (MECMR) can non-invasively assess myocardial calcium influx, and calcium levels are known to be elevated in muscular dystrophy cardiomyopathy based on cellular studies.
Left ventricular functional studies and MECMR were performed in mdx mice (model of Duchenne Muscular Dystrophy, 24 and 40 weeks) and Sgcd−/− mice (Limb Girdle Muscular Dystrophy 2 F, 16 and 32 weeks), compared to wild type controls (C57Bl/10, WT).
Both models had left ventricular hypertrophy at the later age compared to WT, though the mdx mice had reduced stroke volumes and the Sgcd−/− mice increased heart rate and cardiac index. Especially at the younger ages, MECMR was significantly elevated in both models (both P<0.05 versus WT). The L-type calcium channel inhibitor diltiazem (5 mg/kg i.p.) significantly reduced MECMR in the mdx mice (P<0.01), though only with a higher dose (10 mg/kg i.p.) in the Sgcd−/− mice (P<0.05). As the Sgcd−/− mice had increased heart rates, to determine the role of heart rate in MECMR we studied the hyperpolarization-activated cyclic nucleotide-gated channel inhibitor ZD 7288 which selectively reduces heart rate. This reduced heart rate and MECMR in all mouse groups. However, when looking at the time course of reduction of MECMR in the Sgcd−/− mice at up to 5 minutes of the manganese infusion when heart rates were matched to the WT mice, MECMR was still significantly elevated in the Sgcd−/− mice (P<0.01) indicating that heart rate alone could not account for all the increased MECMR.
Despite both mouse models exhibiting increased in-vivo calcium influx at an early stage in the development of the cardiomyopathy before left ventricular hypertrophy, there are distinct phenotypical differences between the 2 models in terms of heart rates, hemodynamics and responses to calcium channel inhibitors.
KeywordsMuscular dystrophy Cardiomyopathy Magnetic resonance imaging Calcium
Muscular dystrophies are frequently associated with cardiomyopathies. In Duchenne muscular dystrophy (DMD), which is caused by the absence of dystrophin, either clinical or subclinical dilated cardiomyopathy is invariably present [1, 2]. Recessive mutations in one of the genes for α-, β-, γ- or δ-sarcoglycan cause a heterogeneous group of autosomal recessive limb girdle muscular dystrophies (LGMD2C-F), and these also frequently have dilated cardiomyopathy, especially those patients with LGMD2F caused by mutations in the δ-sarcoglycan gene .
A hypothesis to explain the underlying pathophysiology of cardiomyopathies associated with muscular dystrophy is that recurrent membrane injury leads to an increased influx of calcium  which then causes downstream effects such as activation of calcium-dependent hypertrophic pathways , reactive oxygen species  and cell death through necrosis with mitochondrial defects . Membrane injury may not be the only mechanism whereby calcium enters cardiomyocytes. Cohn et al.  have shown that the L-type calcium channel antagonist verapamil restores left ventricular function in Sgcd−/− mice, though has no effects in mdx mice. Whereas this was explained at that time on the basis of effects on coronary vasculature, subsequent studies have suggested that this was a direct cardiomyocyte effect [7, 9]. That there are differences between the Sgcd−/− and mdx mice has also recently been highlighted by a study showing marked hemodynamic differences in response to β-blockers .
Manganese-enhanced cardiovascular magnetic resonance (MECMR) uses 2 properties of the manganese ion to allow in-vivo assessment of calcium influx . Manganese enters cardiomyocytes through calcium channels, and is also a T1 contrast agent with CMR. Thus, increased calcium influx with dobutamine increases contrast-enhancement, and decreased influx with the calcium channel blocker diltiazem reduces contrast. We used MECMR to test the hypothesis that in-vivo calcium influx in muscular dystrophy cardiomyopathy mice is increased at an early stage in the development of the cardiomyopathy and so is an important factor in disease progression. To test this hypothesis we studied in-vivo left ventricular function, and MECMR in Sgcd−/− and mdx mice, determining the roles of ageing, L-type calcium channel inhibition and heart rate in the altered manganese contrast enhancement pattern of these models.
Animals: Two separate age groups of male mice were used in this study. C57BL/10ScSnOlaHsd (wild type, WT, Harlan Laboratories, Indianapolis, USA) and mdx mice (C57BL/10ScSn-mdx/J, Jackson, Maine USA) were scanned at ages 24 and 40 weeks, and Sgcd−/− mice were scanned at 16 and 32 weeks (on C57Bl/6 background, a kind gift from Dr Kevin Campbell, University of Iowa). These ages were chosen based on previous data reflecting the earlier development of the cardiomyopathy in the Sgcd−/− compared to the mdx mice [12, 13]. Numbers of mice in the various experiments are detailed in the table and figure legends. The investigation conforms with Directive 2010/63/EU of the European Parliament and was performed under the terms of the Animals (Scientific Procedures) Act 1986, authorized by the Home Secretary, Home Office UK. The Sgcd−/− mice were compared with the 8 week older C57/BL10 mice as controls, as preliminary data showed no differences in any measure of left ventricular function or manganese uptake between 16 and 24 week old C57/BL10 mice (data not shown).
Mice were anaesthetized using 5% isofluorane and anaesthesia maintained at 1.5% in oxygen with a flow rate of 0.5 L/min. The tail-vein was cannulated and mice placed on a MR compatible sled with surface ECG electrodes on the chest wall, respiration pillow and cutaneous temperature probe (Dazai Research Instruments, MICe, Toronto, Canada). This was connected to MR compatible monitoring equipment (SA Instruments Inc., Stony Brook, NY 11790). Mice on the sled were then placed on a bed holding the nose cone for anaesthesia delivery and then all of this slid into a 39 mm diameter quadrature birdcage volume coil (Rapid Biomedical GmbH). Images were acquired on a 7 Tesla horizontal bore microimaging system equipped with a 12-cm microimaging gradient insert (maximum gradient 40 gauss/cm) (Varian Inc., Palo Alto, CA, USA). Following power calibration and global shimming a series of four pilot transverse images were acquired over the heart. Single slice coronal and sagittal images were then obtained in order to view the apex and mitral valve planes. These images were used to plan for the true short axis plane . To measure left ventricular function, contiguous short axis slices were acquired to cover the entire left ventricle using a spoiled gradient-echo cine sequence (TR=5 ms, TE=1.42 ms, flip angle 15°, FOV 30×30 mm, data matrix 128×128, 1 mm slice thickness, max. of 10 slices depending on heart size, 4 averages). Images were ECG triggered to the R wave with a cine delay of 15 ms and typically 30 phases were acquired distributed through the cardiac cycle (depending on HR). Images were zero-filled to a matrix size of 256×256. Scans were converted to matfiles (using a matlab script kindly provided by Dr Johannes Riegler, University College London, UK) and analysed using the freely available analysis software Segment v1.8 (http://segment.heiberg.se)  to measure left ventricular mass and the LV functional parameters – end-systolic and end-diastolic volumes, stroke volume, ejection fraction and cardiac output.
Manganese enhanced CMR (MECMR)
Pharmaceutical modification of manganese contrast enhancement
The L-type calcium channel antagonist diltiazem (5–10 mg/kg body weight i.p., 10 mg/kg being maximal tolerated dose, Sigma Chemical Co., St Louis, MO) was given separately to subgroups of the younger mice. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel inhibitor ZD 7288 (0.3 mg/kg body weight i.p., Sigma Chemical Co., St Louis, MO) was also given to separate groups to study the effects of selective heart rate reduction on manganese contrast enhancement [16, 17]. For the ZD 7288 experiments we also included a 2.5 minute scan as we were particularly interested in the early kinetics of manganese uptake related to heart rate. These medications were given prior to placing the animal in the scanner which was approximately 30–40 minutes before the manganese infusion.
To test effects of time and age on manganese-contrast enhancement, repeated measures of analysis of variance was used combining data from young and old groups. Within each age group, analysis of variance was used to test differences amongst groups comparing an average value of steady state manganese contrast enhancement, and MR functional data. Post-hoc testing was performed with the Scheffe′ test. Single comparisons were made with Student’s t-tests. Data are expressed as mean ± standard error of the mean.
MR haemodynamics: Left ventricular hypertrophy in older Sgcd−/− and mdx mice, increased heart rates and cardiac index in Sgcd−/− mice, and reduced stroke volume in mdx mice
Haemodynamics in young and older mice
Body weight, g
Heart rate, bpm
LV mass index, (×10 -3 )
EDV index, μl/g
ESV index, μl/g
Stroke volume index, μl/g
Ejection fraction, %
Cardiac index, ml/min/g
MECMR: Slower kinetics of manganese uptake in all older mice, and increased contrast enhancement in younger Sgcd−/− and mdx mice
Pharmaceutical modification of calcium channels: Resistance to L-type calcium channel blockade in young Sgcd−/− mice
The role of heart rate in manganese contrast enhancement
In muscular dystrophy cardiomyopathy at an early stage in the development of the cardiomyopathy before significant left ventricular hypertrophy in both the mdx and Sgcd−/− mouse models, in-vivo calcium influx is increased (as determined by manganese contrast enhancement). However, there are clear differences in both left ventricular function and myocardial calcium influx between the 2 models. The mdx mouse has left ventricular systolic dysfunction with a reduction in stroke volume, whereas the Sgcd−/− mice have increased heart rates, cardiac index and so a hyperdynamic circulation. Ejection fractions are lower in the mdx compared to the Sgcd−/− mice. These are also differences in the responses to L-type calcium channel blockade in that there is a relative resistance in the Sgcd−/− mice though mdx mice exhibit a similar response to wild type mice. As heart rate is increased in the Sgcd−/− mice, this is potentially a cause of the increased contrast enhancement, and selective heart rate reduction reduces contrast enhancement in all groups. Nevertheless, it is clear in the Sgcd−/− mice that when heart rates are reduced to wild type levels, there is still increased manganese contrast enhancement indicating a primary cellular defect in calcium influx. This increase in early contrast enhancement after heart rate reduction is not seen with the mdx mice.
L-type calcium channels in muscular dystrophy cardiomyopathy
We were interested in L-type calcium channel blockers for 2 reasons – 1) they can reduce manganese contrast enhancement , and 2) have differential effects in the mdx and Sgcd−/− mice . It is known that intracellular calcium is increased in muscular dystrophy cardiomyopathy [7, 18]. Cohn et al.  have shown that the L-type calcium channel blocker verapamil had significant beneficial effects in the Sgcd−/− mice on troponin I levels and cardiac histology, though not in the mdx mice. To reconcile the findings in this study and our findings it is necessary to examine doses used. The doses used in Cohn et al. compared to the present one are different when corresponding human doses calculated by weight are examined, though a consistent theme is that high doses are required. The doses of diltiazem in the current study of 5–10 mg/kg would equal 350–700 mg in a 70 kg human, the upper limit of a normal human dose being 240 mg. The dose of verapamil used by Cohn et al. was 3.5 mg/day, which would be approximately 35 fold higher than an appropriate human dose. One possible consistent explanation is that only higher doses of L-type calcium channel antagonists are effective in the Sgcd−/− mice, as we only show responses to higher doses of diltiazem. There are strain differences in the backgrounds of the wild type and mdx (both C57Bl/10) versus the Sgcd−/− mice (C57Bl/6). Though we could find no differences between Bl/10 and Bl/6 mice in terms of manganese uptake, we cannot rule out some strain effect in the response to L-type calcium channel blockade.
Role of heart rate in manganese contrast enhancement
When comparing models with different heart rates, it may be difficult to determine whether a difference in manganese contrast enhancement is due to the increased heart rate resulting in more action potentials over the period of the manganese infusion or whether there is an underlying cellular defect with an altered calcium influx per action potential . To determine whether the increased heart rate in the Sgcd−/− mice accounts for the increased manganese contrast enhancement we have used the HCN channel inhibitor ZD 7288 which inhibits the I f channel producing a selective reduction in heart rate [16, 17]. These studies clearly show that at matched heart rates, the Sgcd−/− mice have increased manganese contrast enhancement compared to wild type, indicating a primary cellular defect in calcium influx. These experiments also show clearly the important role that heart rate plays in manganese contrast enhancement, so that for instance, in our experiments with diltiazem it is reasonable to assume that some of the reduction in contrast enhancement is due to a reduction in heart rate. Of note, a small amount of calcium may enter cardiac myocytes through the I f channel (0.5% of the current from the HCN2 channel) , though this small amount is unlikely to have significantly effected our results. Furthermore, as the mdx mice do not have significantly different heart rates compared to wild type mice the increase in manganese contrast enhancement will be due to a primary cellular defect in calcium influx. Surface temperatures of the mice were below physiological levels during the scans, and so heart rates were also low. Surface temperatures are approximately 1°C lower than core body temperature . Higher temperatures would have likely resulted in higher heart rates and this may alter manganese uptake rates.
Age-related changes in myocardial calcium influx
Isenberg et al.  have shown that in aged mice calcium transients peak late and decay slowly. Salameh et al.  have shown that the L-type calcium (ICa.L) current is abnormal in aging rabbit hearts with the ICa.L density normalised to cell volume significantly reduced, maximum conductance also significantly decreased and steady state inactivation shifted to more positive potentials in aged hearts. However, expression of the α1c subunit of the L-type channel, the SERCA2a-ATPase, and the Na+/Ca2+ exchanger did not differ significantly between the two age groups. Direct comparison of cellular calcium handling and MECMR would be very informative in studying age-related changes in calcium influx. Our young and old mice were separate groups and in future, serial MECMR studies within the same mouse will also be important.
Left ventricular function in mouse models of muscular dystrophy cardiomyopathy
We have previously shown that there are differences in function between the mdx and Sgcd−/− mouse models using the conductance catheter , and these results are very consistent despite the very different techniques, though adding information about left ventricular mass and calcium influx. Townsend et al.  have compared these mouse models and also found distinct differences (at 6 – 8 months of age). Isolated myocytes from the mdx mice show reduced compliance and are susceptible to terminal contracture, whereas the Sgcd−/− mice had normal compliance. There was increased fibrosis in the Sgcd−/− hearts, though not in the mdx. We have also shown that mdx mice exhibit improved hemodynamics with 8 weeks of β-blocker treatment though there is significant deterioration in the Sgcd−/− mice with reduced heart rate, cardiac output and impaired active relaxation . Despite this information a specific molecular pathway by which these differences occur is still unclear.
Conclusions and clinical relevance
We have shown at an early stage in the development of muscular dystrophy cardiomyopathy that in-vivo calcium influx is increased. Increased calcium influx and left ventricular hypertrophy are seen in both models, though other important haemodynamic and pharmacological differences are apparent, so that all these factors need to be considered together when assessing mechanisms of the cardiomyopathy and response to therapies. A limitation of these in-vivo techniques is that it is difficult to say definitively if there are specific abnormalities in channels such as the L-type calcium channel in these models. Thus, the future utility of these non-invasive in-vivo techniques will likely be in serially assessing responses to novel preclinical treatments.
Supported by grants from Heart Research UK (RG2585), British Heart Foundation FS/11/89/29162, and the Newcastle upon Tyne Hospitals ‘Special Trustees’ We are grateful to Dr Tom Hu, U.S. Department of Health & Human Services and Medical College of Georgia for helpful suggestions.
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