5. Ferlins in Human Diseases: Dysferlinopathies and Their Pathomechanisms
Dysferlinopathies are diseases caused by mutations in
DYSF, affecting mainly skeletal muscles
[80][180]. There are two common dysferlinopathy phenotypes—limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM)—along with several more rare conditions: distal myopathy with anterior tibialis onset (distal anterior compartment myopathy), congenital muscular dystrophy and isolated hyperCKemia, an elevated concentration of serum creatine kinase (CK)
[80][180]. Onset and progression of the disease as well as distribution of muscle weakness and wasting may vary significantly between individuals affected by dysferlinopathies. Several different clinical phenotypes can occur even within families carrying the same pathogenic variants of
DYSF [5][81][82][83][5,181,182,183]. These observations emphasize the importance of investigating potential modifier genes
[5][81][5,181].
Dysferlinopathies are characterized by late onset and slow progression. In carriers of pathogenic gene variants disease usually manifests in the second or third decade of life. The first symptoms are lower limb weakness accompanied by an increase in serum CK levels. The patients with the most severe phenotype of LGMD2B can become confined to wheelchair after two or three decades of disease progression, while most MM patients preserve ambulation
[84][184]. Histological signs of the diseases are degeneration and regeneration of skeletal muscle
[85][185] and in the more severe cases, fibrotic and adipogenic replacement of myofibers. On the protein level, dysferlinopathies are diagnosed by a complete loss or severe reduction of dysferlin in muscle biopsies or peripheral blood monocytes
[86][186].
Mouse models lacking dysferlin develop muscular dystrophy, however, with an apparently less severe phenotype than humans and do not lose ambulation with age
[87][88][187,188]. In dysferlin-deficient mice, the earliest symptoms are centrally nucleated fibers and marked differences in the myofiber diameter as well as 4- to 6-fold elevated CK levels at four weeks of age
[89][189].
Latent cardiac dysfunction has been reported in dysferlinophathies, but patients do not primarily suffer from cardiomyopathies
[90][91][190,191]. In a retrospective analysis, cardiac and respiratory functions were studied in dysferlinopathy patients
[91][191]. Thus, a cardiovascular magnetic resonance analysis of LGMD2B patients showed indications of mild structural and functional cardiomyopathy
[92][192]. One fifth of the patients developed respiratory problems and 9% required non-invasive ventilation. Accordingly, heart function in dysferlinopathy mouse models is either not or mildly affected
[87][90][93][94][95][187,190,193,194,195]. However, also membrane repair in cardiomyocytes is dependent on dysferlin
[96][196] and in a model of ischemia/reperfusion injury, dysferlin was shown to be cardioprotective
[96][97][196,197]. Furthermore, physical stress exercise, or β-adrenergic activation provoke various symptoms of cardiac dysfunction in dysferlinopathy mice
[90][94][96][97][98][99][190,194,196,197,198,199]. Mice with dysferlin inactivation show increased susceptibility to coxsackie virus infection and virus-induced myocardial damage
[100][200], suggesting that pathways of viral infection and muscle repair may overlap.
It is generally assumed that disease causing mutations are more or less uniformly distributed along the dysferlin-coding sequence
[101][177] (). Two pathogenic missense mutations in human dysferlin FerA do destabilize the domain in differential scanning calorimetry (DSC) experiments
[18][35] and a similar prediction was made for the three most frequent out of 15 missense mutations in the inner DysF domain
[102][55]. These and other mutations may result in the poor dysferlin folding and degradation of the protein via different pathways. For example, missense mutation L1341P in C2E domain causes dysferlin aggregation in the ER and degradation by the additional autophagy/lysosome ER-associated degradation system
[103][201]. Dysferlin lacking C2C domain or carrying patient mutation L344P within FerI domain demonstrate accelerated endocytosis, protein lability and endosomal proteolysis
[104][47].
At present it is not clear how exactly loss-of-function mutations of
DYSF and a decrease of the corresponding protein expression level lead to the development of dysferlinopathies. The following mechanisms may contribute to the development of the disease: (a) a defect in sarcolemma repair; (b) changes in Ca
2+-homeostasis; (c) impaired muscle growth and regeneration and (d) inflammatory processes. Below, we discussed these factors in turn. To which degree, however, these mechanisms contribute to the development of the disease is still not known. The situation becomes even more complex when considering the existence of the various clinical manifestations of dysferlinopathies.
5.1. Defective Repair of Myofiber Sarcolemma and, Possibly, T-Tubules
The pathomechanism could be the following: dysferlin deficiency decreases the efficiency of sarcolemma and, probably, T-tubule system repair
[48][19][105][77,83,202]. This increases (i) influx of Ca
2+ into injured muscle fibers, (ii) leakage of the muscle fiber contents such as muscle enzymes, e.g., CK, and (iii) the probability of death of damaged myofibers
[5][19][5,83]. The latter promotes cycles of muscle degeneration and regeneration. In parallel, inefficient sarcolemma repair changes the properties of the regenerative niche by enhanced accumulation of dysferlin partner protein annexin A2 in the myofiber matrix
[24][106][86,203]. The formation of a regenerative niche requires the absence of annexin A2 in the myofiber matrix, and if it is progressively accumulating, fibro/adipogenic precursors are escaping apoptotic signal. This leads to their differentiation into adipocytes and the substitution of muscle fibers in dysferlinopathy
[106][203].
5.2. Changes in Muscle Fibers Ca2+ Homeostasis
Overloading of the cells with Ca
2+ or abnormal intracellular distribution of these ions can lead to autophagic, necrotic or apoptotic cell death
[107][204]. In muscles of dysferlinopathy patients, altered Ca
2+ homeostasis and Ca
2+-mediated cytotoxicity can result from (i) impaired sarcolemma and T-tubule system repair contributing to the leakage of extracellular Ca
2+ into the sarcoplasm through a lesion and DHPRs as well as sarcoplasmic reticulum-stored Ca
2+ through RyRs, (ii) abnormalities in the biogenesis of the T-tubule system and triads as well as a decrease in their plasticity in response to stress and (iii) enhanced X-ROS (NADPH oxidase 2-dependent reactive oxygen species, ROS) signaling activating mechano-sensitive Ca
2+ channels in the T-tubule system, coupling mechanical stress to changes in the intracellular Ca
2+ concentration
[108][205].
Under normal physiological conditions, ROS production is linked to both signaling and metabolism (as a side product of the latter)
[109][206]. In the mouse model of dysferlinopathy (A/J strain), X-ROS signaling is amplified and contributes to the development of myopathy in aged animals (> 6 month). Thus, in the stretched dysferlin-deficient muscle fibers of A/J mice, intracellular ROS and Ca
2+ concentrations increased in comparison to wild-type controls
[108][205], implying that X-ROS signaling could be enhanced as a result of dysferlin dysfunction.
Altered Ca
2+ homeostasis in dysferlinopathy can lead to myofibers death and cycles of regeneration via (i) activation of endonucleases, phospholipases and proteases like calpains leading to unwanted cleavage of cellular components; (ii) triggering multiple signaling cascades affecting gene expression or cell survival
[107][204].
5.3. Impaired Muscle Growth and Regeneration
The potential effect of dysferlin deficiency on muscle growth and regeneration was discussed above in
Section 6.3. Additionally, dysferlin functions in these processes could be linked to T-tubule development in regenerating muscle, abnormalities in Ca
2+-homeostasis and inflammatory processes (). Formation of an irregular T-tubule network upon myofiber regeneration may disturb Ca
2+-homeostasis. The impaired Ca
2+-compartmentalization and signaling may lead to myofiber death, promote cycles of muscle regeneration and reduce secretion of cytokines by surviving myoblasts or myofibers
[67][107][110][122,204,207]. The defects in secretion of chemotactic molecules leads to a decrease in the number of recruited neutrophils, delayed removal of necrotic fibers, prolonged inflammatory responses, incomplete regeneration cycles and development of muscular dystrophy
[67][122].
Figure 4. The network of dysferlin functions. Malfunctioning of one or several aspects contributes to the pathology of dysferlinopathies (red arrows). (1) Impaired sarcolemma repair leads to changes in Ca2+ homeostasis and in turn could be affected by intracellular Ca2+ compartmentalization and signaling. (2) The T-tubule system is necessary to maintain Ca2+ homeostasis and in turn could be affected by abnormalities in Ca2+ signaling. (3) Deficits in sarcolemma and T-tubule system repair may cause death of damaged myofibers and promote cycles of the muscle regeneration. Leakage of the muscle fibers contents may change properties of the regenerative niche. (4) Changes in Ca2+ compartmentalization and signaling in myofibers can result in dysregulation of, e.g., cytokines secretion and prolonged inflammatory responses. (5) Dysregulation of Ca2+ homeostasis may lead to myofibers death, which promotes cycles of muscle regeneration. (6) Sarcolemma repair may depend on the function of T-tubule system as a membrane reservoir and affect T-tubule system function via changes in Ca2+ homeostasis. (7) T-tubule system function may be affected by abnormalities in its structure arising during dysferlin-deficient muscle regeneration. (8) Malfunctioning of sarcolemma repair enhances leakage of damage-associated molecules, e.g., annexin A2, promoting inflammation. (9) Prolonged inflammation may result in incomplete cycles of regeneration and pro-inflammatory signaling may inhibit myogenesis.
5.4. Inflammatory Processes
Dysferlinopathies are often accompanied by muscle inflammation and dysferlinopathy patients can be misdiagnosed as having polymyositis
[111][112][113][139,208,209]. The role of dysferlin in the inflammatory response was reviewed by several authors
[114][115][138,210]. Inflammation could originate from:
-
Leakage of damage-associated molecules such as annexin A2 from dysferlin-deficient myofibers
[116][211] through sarcolemma lesions
[115][210],
-
Intrinsic pro-inflammatory signaling of dysferlin-deficient muscle fibers
[62][117][118,212],
-
Deregulation of cytokine secretion
[67][122], and/or,
-
Activation of dysferlin-deficient monocytes or macrophages
[111][139].
However, there is also evidence that inflammation in dysferlinopathies originates autonomously within the skeletal muscle and not due to dysferlin function in other cell types. For example, it was shown by means of bone marrow transplantation that inflammation in SJL/J mice does not depend on the genotype of the leukocytes
[118][213]. Along the same lines, transgenic mice generated from the A/J mouse model of dysferlinopathy, expressing dysferlin under a skeletal muscle-specific promoter are indistinguishable from dysferlin-sufficient mice
[119][70]. Lastly, it was shown that macrophage infiltration is a consequence of myofiber damage and not vice versa
[120][214].
In summary, reduced efficiency of sarcolemma and, likely, T-tubule system repair can lead to changes in Ca
2+ homeostasis, myofiber necrosis, inflammation and cycles of regeneration followed by fibro-adipogenic substitution of the muscles, resulting in their weakness ()
[121][215]. However, pathomechanisms leading to the development of dysferlinopathies are, likely, not restricted to the defects in sarcolemma repair, since rescue of PM repair malfunctioning by myoferlin overexpression does not improve muscle histology
[122][216]. This means that dysferlin functions other than sarcolemma repair are also indispensable for skeletal muscle health.