Cardiac involvement in Beagle-based canine X-linked muscular dystrophy in Japan (CXMD_J): electrocardiographic, echocardiographic, and morphologic studies

Duchenne muscular dystrophy (DMD) is a common and lethal genetic disease characterized by progressive muscle wasting. It is an X-linked recessive disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin [1]. The absence of dystrophin is accompanied by a loss of dystrophin-glycoprotein complex at the sarcolemma and results in progressive degeneration of skeletal and cardiac muscle with fibrotic tissue replacement and fatty infiltration [2, 3]. The onset of the disease occurs between 2 and 5 years of age, and most patients die of respiratory or cardiac failure [4, 5]. Cardiac involvement, which occurs commonly in DMD patients, has increasingly become an important cause of death because recent clinical progress has reduced the risk of death due to respiratory failure [6, 7].

Like dystrophin-deficient skeletal muscle, dystrophin-deficient cardiac muscle is replaced by fibrotic or fatty tissue, especially in the left ventricular posterobasal wall region [8‐11]. Atrophic changes with loss of striation, vacuolation, fragmentation, or nuclear degeneration in the myocardium have also been reported [12]. Progressive involvement of the left ventricle leads to wall motion abnormality and results in dilated cardiomyopathy. In DMD patients, the electrocardiogram (ECG) may show tall R-waves in the right precordial leads, deep Q-waves in leads I, aVL, V5-6 or II, III, and aVF [8‐13], as well as an increased heart rate, shortened PQ (PR) interval, conduction abnormalities or arrhythmias such as sinus arrhythmia, atrial ectopic beats, and ventricular premature complexes in DMD patients [13‐16]. One of the electrocardiographic abnormalities, deep Q-waves, has been considered to be attributable to myocardial fibrosis [8, 9, 17]. Echocardiography indicates myocardial thickening, wall motion abnormalities, enlargement of the left ventricle, and left ventricular systolic and diastolic dysfunction. Hypokinesis of the posterobasal wall is consistent with the spreading fibrosis and significant decrease in the internal dimensions of the ventricles [14, 15]. There are, however, many unresolved issues in cardiac involvement, such as the reason why the posterobasilar segment of the left ventricle is consistently the first lesion, whether extensive fibrosis involves the conduction system, the pathogenesis of inappropriate tachycardia or electrocardiographic abnormalities, and whether abnormal smooth muscle regulation affects the cardiomyopathy [18]. One way to clarify these problems is to study suitable animal models.

To date, the X-linked muscular dystrophy (mdx) mouse and the Golden retriever-based muscular dystrophy dog (GRMD) have been used for elucidation of the pathogenesis and development of therapy for DMD. The phenotypes of GRMD are more similar to DMD than that of the mdx mouse [19‐21], and GRMD also shows similar electrocardiographic findings and progressive cardiomyopathy comparable to the cardiac involvement of DMD patients [20‐23]. In this respect, GRMD is a useful model to explore cardiac involvement, but GRMD is very difficult to maintain because of their severe phenotypes. Mild phenotypes can be expected in small sized dogs such as Beagle, indicated by the cross-bred study by Valentine et al. [20]. Moreover, medium-sized Beagle is easy to handle or raise than GRMD, therefore they have definite advantages in animal housing or welfare. Therefore, we established a Beagle-based dog colony named canine X-linked muscular dystrophy in Japan (CXMD_J) [24]. In CXMD_J, involvement of the temporalis and limb muscles is observed from 2 months of age, and macroglossia, dysphagia, drooling, and joint contracture are apparent from 4 months of age; the phenotypes of CXMD_J are thus almost comparable to GRMD [25]. In this study, we investigated the cardiac phenotypes in CXMD_J using electrocardiography, echocardiography, and pathological examinations. Abnormalities on echocardiogram and cardiac pathology were detected from 12 months of age; however, the distinct deep Q-waves in leads II, III, and aVF on ECG were consistently observed by 6–7 months of age in all CXMD_J dogs examined. The cardiac phenotypes of CXMD_J were identical to but milder than those of GRMD described in the literature. Thus, CXMD_J may also be a suitable animal model for elucidation of the above-mentioned problems.

In electrocardiographic findings, an increased HR and a shortened PQ interval have been reported in both DMD patients [13] and GRMD [22]. These findings were also observed in CXMD_J dogs. Increased sympathetic activity and decreased parasympathetic activity have been observed in DMD patients and are associated with disease progression [30]; therefore, autonomic dysfunction in dystrophin deficiency might affect these parameters. It has been reported that HR is negatively correlated with PQ interval in normal Beagle dogs and it may be ascribed to a parasympathetic input at the level of the AV node [31]. The negative correlation between HR and PQ intervals was also found in affected dogs, indicating the parasympathetic input was maintained well even in affected dogs at AV node level. The QRS duration was within normal limits in the CXMD_J dogs, which is compatible with most cases of DMD [13]. Another peculiar electrocardiographic finding in DMD is the deep and narrow Q-waves in I, aVL and V6 or in II, III and aVF [10, 13, 16, 32]. CXMD_J dogs also showed prominent Q-waves and increases in the Q/R ratio in leads II, III, and aVF, findings that are consistent with those in GRMD [23]. In all CXMD_J dogs examined, the distinct deep Q-waves were recognized by 6–7 months of age, which is earlier than the other abnormal electrocardiographic parameters, and the Q/R ratio in affected dogs remained high from 6 to 21 months of age. Actually, the prominent Q-wave and increase in Q/R ratio were also detected in some of the CXMD_J dogs at around 2 months of age (Fig. 1C), but it is difficult to evaluate the degree of the Q/R ratio increase before 3 months of age because the QRS vector is almost exclusively directed to the right and varies significantly in the weeks after birth [33]. A previous report described GRMD dogs ranging from 6 months to > 2 years as having deep Q-waves and increased Q/R ratios in leads II, III, and aVF [23]. The Q-waves, however, might have been seen earlier and regarded as normal variants or not have been considered important for the reasons mentioned above.

Hyperechoic lesions indicating myocardial fibrosis in the posterobasal left ventricular wall have been detected by echocardiography in GRMD dogs as well as DMD patients [22, 23]. Moise et al. reported that hyperechoic lesions were first detected in eight of eleven GRMD dogs (73%) by 6–7 months of age and that they correlated with histologically recognizable areas of mineralization and corresponded to the progression of fibrosis [23]. In our study, one of eight of CXMD_J dogs showed a hyperechoic lesion in the left ventricular posterior wall, but the rest had not by the age of 6–7 months (Table 3, Fig. 4). The hyperechoic lesion in the left ventricular posterior walls was detected in both III-302MA and III-303MA, but not early as 12 months of age (Table 3). The results of echocardiography indicated that the cardiac involvement in CXMD_J is milder than that in GRMD. Echocardiography did not reveal particular left ventricular dysfunction in any CXMD_J dog by 21 months of age, but a mild hypokinesis of the left ventricular wall was observed in III-302MA at 21 months of age (Fig. 3B). The dysfunction found in the dog, however, was mild and the dog had no cardiac symptom. Moise et al. reported that three of the six GRMD dogs > 2 years of age showed a decrease in fractional shortening, but did not mention at what age the abnormal cardiac findings appeared.

Previous studies of morphology in GRMD showed that myocardial involvement is initially found in the left posterobasal ventricular wall, similar to that of patients with DMD [21‐23]. Valentine et al. reported that GRMD dogs at 6.5 months of age had acute severe lesions with focal myocardial mineralization associated macrophages and giant cells in the left ventricular papillary muscle and left ventricular wall [22]. Moreover, GRMD dogs at 12 months of age or older demonstrated prominent myocardial fibrosis in more widespread lesions [22]. The myocardial fibrosis of the left ventricular wall in the older stage of CXMD_J dogs was consistent with that in DMD patients and GRMD dogs. The change was detected at 15 months of age or older in the CXMD_J (III-D08MA, III-302MA, and III-303MA), although III-D08MA showed a hyperechoic lesion at 5 months of age or older (Table 3). The cardiac involvement in CXMD_J, therefore, was milder and slowly progressed than that in GRMD, although a longer period evaluation of large numbers of CXMD_J will be needed to conclude the mild cardiac phenotypes of CXMD_J.

Why is the cardiac involvement in CXMD_J milder than that in GRMD? Valentine et al. reported that skeletal muscle involvement in small dystrophic dogs was milder than that in large ones [19]. Several reports on dystrophic features have hypothesized that the clinical severity may be associated with growth rate [34] or muscle fiber diameter [35]. Living in a cage rather than running free could also affect the cardiac phenotypes of CXMD_J because physical exercise promotes cardiac involvement in dystrophin-deficient mdx mice [36]. The difference in the genetic background between GRMD, golden retriever and CXMD_J, Beagle might also affect the disease progression.

The prominent deep Q-waves seen in both DMD and GRMD have been attributed to a reduction in or a loss of electromotive force caused by scarring of the posterobasal region of the left ventricle [8, 9, 17]. In our study, the deep Q-waves and increases in the Q/R ratio in CXMD_J preceded the lesions seen on echocardiogram and histopathology, as shown in Fig. 6. Considering this result, the origin of the distinctive Q-waves might not be associated with the myocardial lesion in the posterobasal left ventricular wall. It has recently been reported that expression of a transgene in mdx mice for neuronal nitric oxide synthase (nNOS), which occurs as a secondary loss in dystrophin deficiency, decreased cardiac inflammation and fibrosis resulting in amelioration of both cardiac function and electrocardiographic abnormalities, including deep Q-waves [37]. Perloff et al. suggested that the alteration of a particular ionic current by lack of specific membrane proteins associated with dystrophin might participate in electrocardiographic changes [17]. We will not therefore deny that minimal myocardial damage could be associated with the pathogenesis of deep Q-waves, but our results suggest that an investigation of the conduction and cardiovascular systems will also be needed to explore the pathophysiology of the deep Q-waves in dystrophin-deficient heart. In this regard, CXMD_J will be very useful to elucidate aspects of the dystrophin-deficient heart, but we may recognize that a longer period of time would be required to complete cardiac phenotypes in CXMD_J.

We demonstrated that the cardiac phenotypes of CXMD_J are comparable to but milder than those of GRMD. Furthermore, we found for the first time that the distinct deep Q-waves precede detection of the left ventricular posterobasal lesion by echocardiography or histopathology. CXMD_J may provide not only new insights into the mechanisms causing the abnormal Q-waves but also more information on the pathogenesis in the dystrophin-deficient heart.