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Dystrophin-deficient cardiomyocytes are abnormally vulnerable to mechanical stress-induced contractile failure and injury¹

GAWIYOU DANIALOU^*, ALAIN S. COMTOIS, ROY DUDLEY^*, GEORGE KARPATI, GENEVIÈVE VINCENT, CHRISTINE DES ROSIERS and BASIL J. PETROF^*,2

^* Respiratory Division, McGill University Health Centre, and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada H3A 1A1;
Laboratoire de Physiologie Respiratoire, Centre de Recherche du CHUM, Université de Montréal, Québec, Canada H2L 4M1;
Neuromuscular Research Group, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4; and
Laboratoire du Métabolisme Intermédiaire, Départements de Biochimie et Nutrition, Université de Montréal, Québec, Canada H2L 4M1

²Correspondence: Royal Victoria Hospital, Room L4.11, 687 Pine Ave. West, Montreal, Quebec, Canada H3A 1A1. E-mail: basil.petrof{at}muhc.mcgill.ca

PRINCIPAL AIMS

The role of the cytoskeletal protein dystrophin in providing protection against mechanical stress-induced cardiomyocyte damage and dysfunction has been investigated using an ex vivo perfused working heart preparation, together with in vivo approaches, to experimentally manipulate the level of mechanical stress imposed on hearts of normal dystrophin-expressing and dystrophin-deficient, X-linked muscular dystrophic (mdx) mice. We have addressed the hypothesis that cardiac muscle cells lacking dystrophin, the absence of which is associated with dilated cardiomyopathy in humans, have a reduced threshold for the development of sarcolemmal injury and attendant contractile dysfunction under conditions of increased biomechanical stress.

PRINCIPAL FINDINGS

A. Experiments performed in ex vivo perfused working hearts:
1. Dystrophin-deficient hearts working against a normal afterload level develop severe left ventricular dysfunction
Standard hemodynamic conditions (preload=12.5 mmHg, afterload=50 mmHg) previously shown to be appropriate for the normal function of working mouse hearts perfused ex vivo were used to perfuse working hearts obtained from 8- to 10-wk-old dystrophin-expressing (C57BL10) and dystrophin-deficient (mdx) mice. The mdx hearts were incapable of sustaining left ventricular function at the same level as normal hearts under these standard physiological conditions. Reducing the afterload from 50 to 40 mmHg stabilized or improved left ventricular function in mdx hearts, whereas cardiac function worsened at the lower afterload in the C57 group.

2. Dystrophin-deficient hearts working against a normal afterload suffer excessive cardiomyocyte injury in a workload-related manner
To evaluate whether hearts lacking dystrophin are more susceptible to workload-induced injury, LDH release into the coronary circulation was correlated with the level of mechanical work performed by hearts perfused ex vivo. At the 50 mmHg afterload level in the mdx group, there was a significant correlation between the work rate and the rate of coronary LDH release (P=0.03). No significant correlation between cardiac work rate and LDH release was found for C57 hearts.

B. Experiments performed in vivo:
1. Dystrophin-deficient cardiomyocytes exhibit an abnormally increased susceptibility to sarcolemmal damage upon exposure to acute mechanical stress
To evaluate the in vivo situation, sarcolemmal injury was sought (ascertained by the ability of a vital dye, Evans blue, to penetrate into individual cardiomyocytes) after an acute increase in cardiac mechanical stress induced by either isoproterenol administration or 10 s clamping of the aorta (Fig. 1 ). Although there was no significant sarcolemmal injury produced by isoproterenol infusion in C57 mice, the mdx mice suffered a ninefold increase in the proportion of cardiomyocytes exhibiting a loss of sarcolemmal integrity. In addition, essentially identical findings were obtained after brief clamping of the aorta, with only the mdx group showing a significant degree of Evans blue dye penetration into cardiomyocytes.

View larger version (51K): [in this window] [in a new window]   Figure 1. Quantitation of in vivo cardiomyocyte sarcolemmal injury induced by an acute increase in cardiac mechanical stress. Values are means ± SE. A) Note that a significant increase in cardiomyocyte sarcolemmal injury after acute exposure to isoproterenol or 10 s aortic clamping was observed in the mdx group only. B, C) Representative photomicrographs demonstrating increased Evans blue dye uptake (red fluorescence within the cytoplasm) by mdx cardiomyocytes after acute exposure to isoproterenol or aortic clamping. *P < 0.05, comparison of isoproterenol or aortic clamping effects (-vs.+) within the same mouse strain. P < 0.05, comparison of the two mouse strains (C57 vs. mdx) under the same isoproterenol or aortic clamping conditions.

3. Dystrophin-deficient mice demonstrate excessive mortality and cardiac necrosis upon exposure to a chronic increase in cardiac mechanical stress
To further ascertain the response of dystrophin-deficient hearts to biomechanical stress in vivo, C57 and mdx hearts were subjected to chronically increased mechanical stress via partial constriction of the aorta. There was a significant difference in the ability of C57 and mdx animals to survive this intervention (P<0.05). In the C57 group, the majority of mice (5/6) remained alive and clinically well up to the time of euthanasia 7 days postaortic constriction. In contrast, all mdx mice (6/6) died within the 7 day period, and this occurred primarily within the first 24 h. In mdx animals that survived beyond 24 h, histological sections revealed extensive areas of cardiac necrosis by hematoxylin-eosin staining; no such lesions were found in C57 hearts.

CONCLUSIONS
This study addressed the hypothesis that cardiomyocytes lacking dystrophin have a reduced threshold for the development of sarcolemmal injury and attendant contractile dysfunction upon exposure to increased levels of mechanical stress. For this purpose, we used an animal model of dystrophin deficiency, the mdx mutant mouse. In humans, the cardiomyopathies associated with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, and X-linked dilated cardiomyopathy are all caused by defects in the dystrophin gene. Dystrophin is a 427 kDa protein that is attached to the F-actin cytoskeletal network through its amino-terminal region, whereas domains near the carboxyl terminus of the molecule mediate binding to a group of subsarcolemmal and transmembrane proteins referred to collectively as the dystrophin-associated protein complex (DPC). The dystrophin–DPC interaction confers a link between cytoskeletal F-actin inside the muscle cell and the extracellular matrix. Although the precise function of this linkage remains unclear, there is strong evidence that it must be maintained entirely intact in skeletal muscles in order to structurally stabilize and thus safeguard the physical integrity of the sarcolemma. In cardiac muscle, however, such a role for dystrophin has not been established, and several alternative functions for dystrophin and/or its associated complex have been proposed including roles in calcium channel function, cell signaling, and the regulation of coronary blood flow.

The results of the present study provide the first direct evidence that at least one critical function of cardiac dystrophin is to protect the myocardium from mechanical stress and workload-induced damage. Thus, using both ex vivo and in vivo approaches, we find that cardiomyocytes lacking dystrophin are abnormally vulnerable to mechanical stress-induced injury, thereby resulting in a loss of sarcolemmal integrity and attendant contractile dysfunction. In agreement with other investigators, we also find that the histological appearance of mdx hearts is largely normal in the absence of experimental manipulations that impose an abnormally high level of mechanical stress on the heart. Despite this benign histological appearance, mdx hearts demonstrated major deficits of left ventricular contractility during both systole and diastole when exposed to a normal afterload level ex vivo. One possible explanation for the apparent discrepancy between histological and physiological findings is that mdx cardiomyocytes may suffer microdisruptions of the sarcolemma that are quickly resealed and hence nonlethal for the cell. In this regard, it has been reported that transient plasma membrane disruption is a relatively common event even in normal tissues subjected to high mechanical stress on a regular basis. Under these conditions, it appears that cell membrane resealing generally occurs within a matter of seconds via targeted fusion of exocytic vesicles with the damaged membrane segment. Although not necessarily lethal to the cell, transient sarcolemmal disruptions of this nature may nonetheless permit excessive entry of extracellular calcium into the intracellular milieu. With respect to the abnormalities of mdx cardiac contractility found in the present study, such increases in intracellular calcium could potentially activate proteases involved in the degradation of contractile proteins or prolong thin filament activation, thereby leading to impaired ventricular pressure generation and relaxation, respectively. In addition, more severe calcium overload likely contributes to cardiomyocyte death when membrane repair mechanisms become overwhelmed by the magnitude of sarcolemmal disruptions, which is likely to have been the case in mdx hearts subjected to chronic aortic constriction.

Insights from this study into pathogenetic mechanisms underlying the development of cardiac disease in the setting of dystrophin deficiency could extend beyond patients with dystrophinopathies. Dilated cardiomyopathy has been found to be associated with mutations in desmin, metavinculin, and cardiac actin. A common feature of these different proteins as well as dystrophin is that each participates in the mechanical coupling of force transmission between the sarcomeric contractile apparatus and cytoskeletal elements of the muscle cell. However, exactly how this predisposes to the development of a dilated cardiomyopathy is unclear. Our results suggest the possibility of a general paradigm in which aberrant mechanical coupling between the force-generating elements of the cardiomyocyte and its noncontractile cytoskeletal scaffolding components creates an increased vulnerability to mechanical stress-induced sarcolemmal injury, thereby leading to eventual dilatation and failure of the heart (see Fig. 2 ). The approaches used in the current study could provide a useful experimental framework for further exploration of this hypothesis in murine models of other diseases involving the cardiomyocyte cytoskeletal network.

View larger version (31K): [in this window] [in a new window]   Figure 2. Schematic diagram illustrating the proposed role of dystrophin in mechanical coupling between the contractile apparatus and cytoskeleton of cardiac muscle cells. A) Normally, mechanical stresses imposed on the sarcolemma by muscle contraction are partially redistributed to the actin-based cytoskeleton via dystrophin (in red) and its associated complex (in green). B) In the absence of dystrophin, the sarcolemma bears a greater brunt of these mechanical stresses, leading to recurrent episodes of sarcolemmal injury and eventual dilated cardiomyopathy.

Finally, our findings may have important clinical implications for patients with dystrophin gene defects. Based on our findings, excessive increases in cardiac workload in the dystrophinopathies would be predicted to induce cardiomyocyte injury and thus accelerate progression of the cardiomyopathic disease process. Therefore, whereas it has been proposed that ß-adrenergic agonists may improve skeletal muscle strength in dystrophin deficiency, the results of the present study suggest that such a strategy could have significant deleterious effects on the heart in DMD patients. Conversely, the use of pharmacologic interventions that reduce cardiac work (e.g., cardioselective ß-adrenergic antagonists, afterload-reducing agents) could be beneficial not only in treating established cardiomyopathy, as is the current practice, but also in preventing the onset of cardiomyopathy in these patients if instituted sufficiently early in life.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0030fje ; to cite this article, use FASEB J. (May 29, 2001) 10.1096/fj.01-0030fje

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