Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that is characterized by severe and progressive muscle weakness and muscle wasting including respiratory muscles (Duan et al., 2021). This disease is caused by a mutation in the largest known human gene which encodes the protein, dystrophin. Dystrophin is a cytoskeletal protein connecting the inner cytoskeleton to the extracellular matrix and serves as a molecular shock absorber to maintain sarcolemmal stability and protect the muscles from injury (Le et al., 2018). Dystrophin deficiency leads to impairment of skeletal muscle sarcolemmal membrane integrity, including the diaphragm, rendering the membrane susceptible to mechanical damage during muscle contraction, leading to immune cell infiltration, necrosis, and fibrosis (Bez Batti Angulski et al., 2023). These histopathological abnormalities of the respiratory muscle have been shown to lead to reduced lung and chest wall compliance, decreased ventilation, and alveolar hypoventilation in many boys with DMD (Smith et al., 1989; Barbe et al., 1994). Patients with DMD have reduced ventilatory capacity beginning at a young age and will decline with age (Burns et al., 2019; Khirani et al., 2014). Respiratory insufficiency is ubiquitous in patients at older age and respiratory failure is one of the leading causes of morbidity and mortality in muscular dystrophy, with a mean life expectancy between 14.4 and 27.0 years unless artificially supported by mechanical ventilation (Pennati et al., 2021; Landfeldt et al., 2020).

A primary strategy to treat DMD would be to reverse the instability of muscle membranes by increasing dystrophin levels. However, a major obstacle to that approach is that the dystrophin gene is too large to be packaged into current gene therapy vectors. While Elevidys micro-dystrophin gene therapy has recently been approved by FDA, these highly truncated forms of dystrophin do not offer full protection (Elangkovan and Dickson, 2021). Moreover, exon-skipping therapies including Amondys 45, Exondys 51, Viltepso, and Vyondys 53 are only effective for some dystrophin mutations (Eser and Topaloglu, 2022). Therefore, it is critical to identify new molecules that are important for ameliorating dystrophic phenotypes and restoring diaphragm function in DMD.

Lipin1 is a phosphatidic acid (PA) phosphatase (PAP) that catalyzes the conversion of PA into diacylglycerol (DAG), which is a critical step in the synthesis of glycerophospholipids, the major structural lipid component of cellular membranes (Csaki and Reue, 2010). Lipin1 accounts for most of the PAP activity in skeletal muscles (Reue and Dwyer, 2009). We recently discovered that lipin1 is critical for maintaining membrane integrity, and plays complementary roles in myofiber stability and muscle function in dystrophic skeletal muscles (Jama et al., 2023). Lipin1 deficiency in skeletal muscle led to membrane damage indicated by increased Evans blue dye (EBD) leakage (Ramani et al., 2020; Schweitzer et al., 2019). Increased membrane permeability was associated with elevated cell death markers and inflammation. This is consistent with our cell culture study, lipin1 deficiency in differentiated primary myoblasts leads to increased membrane permeability and upregulated necroptotic markers (Jama et al., 2023). In contrast, increasing lipin1 expression levels suppressed the necroptotic markers (Jama et al., 2023).

In this study, we aimed to explore the role of lipin1 in dystrophic pathogenesis and respiratory dysfunction through loss-of-function and gain-of-function studies in the mdx mouse diaphragm. We assessed the effects of knocking out lipin1 in dystrophic diaphragm histology and function using dystrophin:lipin1-DKO mice, and the effects of restoring lipin1 in dystrophic diaphragm using a unique muscle-specific transgenic mouse model, mdx:lipin1 ^Tg/0 , in which lipin1 is selectively restored in the muscles of mdx mice. We found that DKO mice had increased dystrophic disease severity, whereas increasing lipin1 expression levels led to decreased muscle fiber degeneration, suppressed inflammation, reduced fibrosis, strengthened membrane integrity, and improved respiratory function.

Respiratory failure is a leading cause of premature death in DMD patients. Although respiratory malfunction in DMD patients is slightly alleviated by noninvasive mechanical ventilation, identifying therapeutic targets and improving respiratory function would greatly impact DMD patients’ quality of life. In this study, we explored the roles of lipin1 in the dystrophic diaphragm and respiratory function using loss-of-function and gain-of-function approaches.

We identified that lipin1 protein expression levels were reduced by 60% in the mdx diaphragm. This is consistent with our previous study, which found that lipin1 decreased by 60% in the mdx gastrocnemius muscle compared to WT controls (Jama et al., 2023). Our recent studies revealed that lipin1 plays a complementary role in myofiber stability and muscle function in dystrophic muscles (Jama et al., 2023; Ramani et al., 2020; Jama et al., 2024). Lipin1 deficiency alone leads to compromised plasma membrane integrity, elevated necroptotic markers, and inflammation (Jama et al., 2023; Ramani et al., 2020). It is possible that lipin1 expression levels were already very low, so, further knockout of the remaining 40% of lipin1 in the dystrophic diaphragm did not lead to a significant worsening phenotype in this study. However, we did find that NF-kB, a master transcriptional factor of inflammation, was highly activated in the mdx and DKO diaphragms as indicated by enhanced phosphorylation of NF-kB at serine 468 and serine 536.

Dystrophin deficiency leads to an increase in muscle damage. The imbalance between muscle damage and repair is associated with apoptotic and necrotic cell death contributing to muscle fiber loss. Previous studies suggested that apoptosis characterizes the onset of the pathology of dystrophin-deficient muscle and precedes necrosis (Tidball et al., 1995). Apoptosis has been identified by nuclei with DMA fragmentation in the muscles of DMD patients and mdx mice (Tidball et al., 1995; Matsuda et al., 1995; Sandri and Carraro, 1999). Necroptosis, programmed necrosis, is distinguished from other modes of cell death in that it is highly proinflammatory given that cell membrane integrity is lost, triggering the activation of the immune system and inflammation. Therefore, necroptosis has been shown to be a major contributor to muscle degeneration in dystrophic muscles contributing to the loss of muscle fibers (Oeckinghaus et al., 2011). In our study, highly elevated Bax, Bak, and cleaved caspase 3 may indicate elevated apoptotic death in the mdx diaphragm. We observed that RIPK1, RIPK3, and MLKL are elevated in the diaphragm muscle of mdx mice, and RIPK1 and RIPK3 are further upregulated in the DKO diaphragm. Elevated necroptosis is not the only mechanism leading to muscle degeneration. Sarcolemmal disruptions, abnormal calcium (Ca²⁺) homeostasis (Culligan et al., 2002; Doran et al., 2006; Matsumura et al., 2009), increased calpain activities (Spencer and Mellgren, 2002), and NF-κB³⁸ have been shown to contribute to muscle degeneration. The effects of loss of lipin1 in Ca²⁺ homeostasis and calpain activities remain to be investigated. Nevertheless, as a result of increased muscle degeneration, loss of lipin1 in the dystrophic diaphragm may lead to deterioration of respiratory function, indicated by a decrease in respiratory rate, which represents the most sensitive indicator of increasing respiratory difficulty.

Given the fact that loss of lipin1 plays an important role in dystrophic pathology, increasing lipin1 expression levels may have resulted in an improvement in muscle membrane integrity and respiratory function in mdx mice. Indeed, we found that the diaphragm of mdx:lipin1 ^Tg/0 mice had fewer EBD-positive muscle fibers compared to mdx controls, suggesting that compromised membrane integrity in the dystrophic diaphragm can be ameliorated by lipin1 restoration. This is consistent with our recent studies that lipin1 deficiency leads to increased sarcolemmal permeability, increased creatine kinase (CK) levels, and elevated necroptotic markers in animal models and cell culture systems (Jama et al., 2023; Jama et al., 2024). On the contrary, restoration of lipin1 in dystrophic muscle resulted in reduced sarcolemmal permeability, reduced serum CK levels, and downregulated necroptotic markers. Furthermore, restoration of lipin1 rescued the protein expression levels of α-sarcoglycan, dystroglycan, and nNOS, and promoted sarcolemmal stability (Jama et al., 2024).

Inflammation and cell death are effective hallmarks of membrane instability and dystrophin deficiency in DMD. NF-κB transcriptional activity is progressively elevated in DMD, and has been shown to contribute to disease onset and progression (Messina et al., 2011). We observed that the phosphorylation of NF-kB at serine 468 and serine 536 was suppressed in the diaphragm of mdx:lipin1 ^Tg/0 mice. In addition, CD86⁺ and CD206+ macrophages were highly elevated in mdx and DKO diaphragms compared to B10 controls suggesting that lipin1 restoration in dystrophic diaphragm significantly reduces myofiber inflammation. The protein expression levels of RIPK1, RIPK3, and MLKL were significantly suppressed in the diaphragm of mdx:lipin1 ^Tg/0 mice, suggesting that lipin1 restoration in dystrophic diaphragm reduced myofiber necrosis. Either inhibition of cell death or inflammation has been shown to improve dystrophic phenotype. Corticosteroids have been the first-line anti-inflammatory drugs approved by the FDA for the treatment of DMD. Inhibition of NF-κB in cardiomyocytes improved calcium handling and rescued cardiac function (Peterson et al., 2018). Genetic or pharmacologic inhibition of classical NF-κB DNA-binding subunit, p65, or its upstream activator, IκB kinase β (IKKβ), have been found to reduce inflammation and improve muscle regeneration in mdx mice (Acharyya et al., 2007). Genetic depletion of RIPK3 in dystrophic mdx mice reduced muscle degeneration and improved motor function (Morgan et al., 2018).

The ensuing loss of myofibers, largely mediated by a necrotic cell death process, is associated with the progressive replacement of the myofibers by fibrosis. Overall, our data suggest that the mdx diaphragm showed enhanced fibrosis indicated by Sirius red staining and activation of SMAD2. Activation of SMAD signaling via the canonical TGFβ pathway has been shown to promote collagen deposition and fibrosis (Coulis et al., 2023). Elevated diaphragm fibrosis results in muscle stiffness, prevents the diaphragm from achieving the excursion lengths required for respiration, and is the major contributor to the disease progression (Sahani et al., 1985). Increasing lipin1 expression in dystrophic muscle leads to substantially suppressed SMAD activation and reduced fibrosis development in mdx:lipin1 ^Tg/0 mice.

Patients with DMD experience progressive respiratory muscle weakness by 10–12 years of age (Spencer and Mellgren, 2002; Kumar and Boriek, 2003). As DMD progresses to later stages, patients often present a significant increase in respiratory rate, which is known as rapid shallow breathing (Lo Mauro and Aliverti, 2016). It has been suggested that rapid shallow breathing is adopted as a strategy to reduce the perception of labored breathing (Lo Mauro and Aliverti, 2016). We found that the respiratory rates, tidal volume, and minute volume were reduced in mdx and mdx:lipin1-DKO mice at 6 months of age measured by plethysmography. This is consistent with previous studies that mdx mice at 6 months old had significantly impaired respiratory function, indicated by reduced respiratory frequency, tidal volume, and minute volume (Huang et al., 2011). The impaired respiratory function is likely due to increased diaphragm sarcolemmal damage, inflammation, elevated cell death, and increased fibrosis which confer mechanical defects in the mdx diaphragm. We also found that restoration of lipin1 in the dystrophic diaphragm significantly improved the respiratory rate, tidal volume, and minute volume, suggesting that increasing lipin1 levels successfully improved respiratory function in mdx mice.

It should be noted that we observed only 4% of EBD⁺ myofibers in the skeletal muscle of mdx mice. The ratio was calculated by EBD⁺ myofiber areas versus the total muscle fiber area. The total muscle fiber areas were calculated by measuring the areas of the diaphragm ruling out the tissue gaps between borders of myofibers (Brown et al., 2023; Dubuisson et al., 2022). However, the amount of muscle damage does not seem to match the degree of impaired respiratory function and elevated cell death, inflammation, and fibrosis markers. These results suggest that the EBD staining may not represent muscle damage, but muscle sarcolemmal leakage. Although dystrophin-deficient muscle cells are vulnerable to membrane damage, not all muscle damage leads to sarcolemmal leakage when subjected to increased mechanical stress. Therefore, EBD staining needs to be combined with other techniques, such as plasma creatine kinase levels (Jama et al., 2023; Kobayashi et al., 2012), and the identification of intracellular fibronectin in muscle cells (Friden and Lieber, 1998) to provide a more comprehensive and integrated view of muscle damage. Indeed, in our recent study (Jama et al., 2024), we observed that mdx mice had elevated CK levels and increasing lipin1 expression levels in mdx:lipin1 ^Tg/0 mice reduced CK levels suggesting a critical role for lipin1 in maintaining myofiber stability and integrity. Another reason that leads to low EBD⁺ myofibers in 6-month-old mdx mice may be due to age. Previous studies found that EBD is significantly more abundant in the diaphragm of 4-week-old mdx than in the diaphragm of 24-week-old mdx mice (Pelosi et al., 2015). It is possible that younger mdx mice may exhibit more damaged muscle fibers. The variations of sarcolemmal leakage in mdx mice affected by age will need to be investigated in the future.

In conclusion, we found that lipin1 expression level is markedly reduced in the mdx diaphragm. Loss of lipin1 is associated with increased severity of pathology in the dystrophic diaphragm and contributes to respiratory dysfunction. In contrast, lipin1 overexpression significantly improves the primary defect and downstream pathology in the dystrophic diaphragm, resulting in improved membrane integrity, prevented inflammation, decreased muscle fiber degeneration, reduced fibrosis, and most importantly, improved respiratory function. These findings suggest that lipin1 is an important therapeutic target in the management and treatment of respiratory insufficiency and failure in DMD. In future studies, more functional assays will be introduced to highlight the protective effects of lipin1 on respiratory muscles. We will conduct ex vivo contractile assessments to measure the isometric force production of a thin diaphragm muscle strip. We will conduct exercise testing (e.g., running wheel; treadmill) on the mdx:lipin1 ^Tg/0 , mdx: mdx:lipin1-DKO, and mdx mice. This type of stress will provide interesting discrimination from a whole-body functional perspective if lipin1 is overexpressed versus reduced in the mdx diaphragm. We will also assess whether lipin1 overexpression can ameliorate functional impairments and cellular markers of damage in a more severe mouse model (e.g., D2.mdx or mdx-utrn dKO mice). To explore the potential of lipin1 overexpression for treating respiratory failure, we will use an AAV-based strategy as a therapeutic tool to efficiently deliver lipin1 to the diaphragm. We will investigate the effectiveness, possible adverse effects, and long-term safety of different expression levels of lipin1 as a potential therapy for respiratory dysfunction in DMD. Another future direction is to investigate if older aged mice also present rapid shallow breathing with the progression of the disease; and if lipin1 restoration could help to mitigate this response. Moreover, continued investigation is required to better understand the mechanisms behind these findings, and to determine the role of lipin1 in maintaining muscle membrane stability.