A new multi-institution study spearheaded by researchers at Florida State University and the University of California, Los Angeles suggests a tiny protein could play a major role in combating heart failure related to Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder among children.
In collaboration with scientists from across the nation, FSU researchers found that increased levels of the protein sarcospan improve cardiac function by reinforcing cardiac cell membranes, which become feeble in patients with DMD.
Their findings were published in the journal JCI Insight.
The condition, which typically afflicts young boys, is caused by a mutation that prevents the body from producing dystrophin, a protein crucial to the health of skeletal, respiratory and cardiac muscles.
Advances in treatment for certain types of DMD-related muscle degradation have helped to prolong patients’ lifespans.
However, as DMD patients age, their heart function declines dramatically.
“Patients typically live to 20 or 30 years of age,” said lead author Michelle Parvatiyar, an assistant professor in the Department of Nutrition, Food and Exercise Sciences in FSU’s College of Human Sciences.
“There have been important improvements in respiratory care, which used to be what a majority of patients would succumb to. Now, in their 20s and 30s, they’re often succumbing to cardiomyopathy.
The heart is functioning with a major component of the cell membrane missing. Over time, it wears out.”
The study was part of continued efforts by UCLA biologist Rachelle H. Crosbie, the study’s corresponding author, who previously identified sarcospan as a protein that could improve mechanical support in skeletal cell membranes lacking dystrophin.
Her finding buoyed DMD researchers and affirmed sarcospan’s potential as an effective tool in the fight against the condition.
“But nobody had really looked at how increasing the levels of this protein might affect the heart,” Parvatiyar said.
Using a unique mouse model with a dearth of dystrophin, Parvatiyar and her collaborators did just that.
In their study, the team found that while it’s is not a like-for-like replacement for dystrophin, an overexpression of sarcospan in cardiac cells seems to do the job of stabilizing cell membranes.
Even under stress, researchers found, sarcospan overexpression was able to improve the membrane defect in dystrophin-deficient cells.
“Sarcospan doesn’t quite do the job of dystrophin, but it acts as a glue to stabilize the membrane and hold protein complexes together when dystrophin is lacking,” said Parvatiyar, explaining a concept developed by Crosbie.
Cardiac measurements confirmed that sarcospan does protect the cell membrane even when the heart is placed under stress.
Study co-author and FSU College of Medicine Associate Professor Jose Pinto performed the measurements, along with FSU graduate student Karissa Dieseldorff Jones and University of Miami Miller School of Medicine research assistant Rosemeire Takeuchi Kanashiro.
In addition to serving as a kind of stabilizing glue, researchers said sarcospan could also act as a scaffold that supports other essential proteins at the cell membrane.
That function could allow sarcospan to carry mini versions of dystrophin – which, in its normal state, has a long and unwieldy genetic code – to the edges of cardiac cells, where they could buttress the fragile membranes.
“The idea is that you could administer the sarcospan and the dystrophin at the same time, and the sarcospan could facilitate mini dystrophin localizing to the cell membrane and help hold those complexes in place,” Parvatiyar said.
Sarcospan’s two possible functions could augment existing DMD treatments, Parvatiyar said, or they could give rise to novel therapies that fortify weakened cardiac cell membranes and improve the quality of life for people with DMD.
In her previous position at UCLA, Parvatiyar had frequent interactions with DMD patients and their families.
She said these interactions, and the unshakeable hope she’s witnessed in those suffering from DMD, continue to drive her and her colleagues in the search for new ways to combat this debilitating condition.
“Those were the first times in my life I’d ever had someone come up to me and thank me for my work,” she said. “Sometimes you can feel removed from it in the laboratory day after day. You see incremental progress. But to see people who are really yearning for help is motivating. Their positivity is incredibly inspiring.”
Duchenne muscular dystrophy (DMD) is a lethal childhood neuromuscular disorder that results in progressive muscle weakness, respiratory difficulties, and cardiovascular dysfunction that dramatically shortens life span. DMD is the most common lethal genetic disorder in children and affects 1 in approximately 5500 males (1, 2).
Mutations in the X-linked dystrophin gene DYS1 cause loss of the functional protein, along with loss of the entire dystrophin-glycoprotein complex (DGC) from the sarcolemma.
In DMD, dystrophin protein is degraded (3, 4) and/or not properly transported to the membrane (5).
In Becker muscular dystrophy, in-frame deletion mutations produce a less functional protein that localizes to the cell membrane. X-linked dilated cardiomyopathy manifests only in the heart and is caused by specific mutations located in the N-terminus of the dystrophin gene (6).
Individuals with DMD develop dilated cardiomyopathy by their second decade of life.
The typical disease course of DMD-associated cardiomyopathy initially manifests as ECG abnormalities, development of diastolic dysfunction, fibrosis detected by MRI, dilatation of cardiac cavities, and systolic dysfunction followed by end-stage heart failure (7).
Improvements in respiratory care have extended the life span of DMD patients, increasing the likelihood that patients will develop cardiomyopathy.
Management of patient cardiac symptoms largely includes angiotensin converting enzyme inhibitors, steroids and beta blockers that delay the onset of cardiac dysfunction, and development of adverse remodeling.
Many therapeutic platforms are being developed to combat DMD disease: exon-skipping strategies, gene editing, and development of adenoviral deliverable miniaturized utrophin and dystrophin constructs as well as membrane stabilizers.
In 2016, the FDA accelerated approval of the first DMD-specific drug eteplirsen (Exondys 51), an exon-skipping therapeutic that promotes the production of functional dystrophin (8).
However, eteplirsen only targets 13% of the DMD population, and current formulations and delivery strategies do not effectively target the heart.
The current study focuses on investigating the therapeutic potential of sarcospan (SSPN), which associates with dystrophin and contributes to normal cardiac function.
We sought to determine whether therapeutic approaches that benefit dystrophic skeletal muscle are also efficacious for the treatment of DMD cardiomyopathy (9, 10).
Findings from our DMD studies in mice have elucidated additional roles of structural proteins that also provide cell membrane support.
Dystrophin is a member of the DGC consisting of integral and peripheral membrane proteins (10, 11).
Dystrophin is located on the cytoplasmic face of the sarcolemma and connects intracellular actin with β-dystroglycan (β-DG) of the DGC complex (11, 12). β-DG is complexed with α-DG, which is heavily glycosylated in its mucin domain with O-linked glycans that bind to laminin in the extracellular matrix (13).
Also associated with the complex are 4 sarcoglycan proteins (α-SG, β-SG, δ-SG, γ-SG) that contain a single transmembrane domain. The protein SSPN (14) contains 4 transmembrane domains and tightly associates with the SGs (15, 16) and β-DG (17).
Other laminin-binding complexes are also situated at the cell membrane, including the α7/β1D integrin complex and the utrophin-glycoprotein complex (UGC), largely similar to the DGC with the exception that utrophin is substituted for dystrophin and shares 80% sequence homology with dystrophin (18).
The UGC is localized at neuromuscular junctions in skeletal muscle (19); however, it colocalizes with dystrophin at the cardiomyocyte sarcolemma (19).
Utrophin exhibits distinctive localization from dystrophin in cardiac muscle, as it is additionally expressed in Purkinje fibers and is localized to intercalated discs (20).
In dystrophic mdx mice (the genetic DMD mouse model), utrophin is upregulated in skeletal muscle and assumes a sarcolemmal distribution as a compensatory measure to protect the dystrophin-devoid sarcolemma from contraction-induced injury (21).
Humans exhibit more severe DMD disease, which is postulated in part to result from a diminished ability to upregulate utrophin to compensate for dystrophin deficiency.
During development, utrophin is expressed at high levels at the sarcolemma and is able to function in place of dystrophin (22, 23).
A number of efforts have been directed toward the development of drug-based utrophin therapies that can reactivate the fetal isoform utrophin-A in DMD patients based on amelioration of dystrophic symptoms in mdx mice engineered to overexpress utrophin (24–26).
The Phase II PhaseOut DMD trial (NCT02858362), testing the efficacy of the utrophin-upregulating compound ezutromid in ambulatory DMD boys, was discontinued after failing to show efficacy.
Utrophin deficiency has been shown to exacerbate cardiac dysfunction in mdx mice. Studies performed with double-knockout (dko) mice lacking both dystrophin and utrophin exhibit similar courses of dilated cardiomyopathy compared with mdx mice (27, 28).
However, the timeframe for developing cardiac function is much shorter, and by 20 weeks the dko mice died early (27, 28).
To assess the dependence of SSPN rescue on robust utrophin upregulation, we utilized mdx:utr-heterozygous (mdx:utr-het) mice, the most relevant mouse model available for DMD cardiac studies.
These mice offer a more severe manifestation of cardiac disease that develops over a much shorter time period and allow testing of whether SSPN addresses sarcolemma instability in a more DMD relevant context (29).
This experimental design allows us to examine SSPN overexpression as an alternative method for induction of utrophin expression in DMD hearts and as a strategy to ameliorate cardiac dysfunction.
Our studies exploring the therapeutic benefit of SSPN-based sarcolemma stabilization of DMD skeletal and cardiac muscle have revealed that SSPN overexpression is effective at improving numerous aspects of DMD pathology (5, 30, 31).
Even low levels of SSPN overexpression can enhance mdx cardiac membrane stability, upregulate utrophin expression, and improve cardiac function (30), in addition to addressing skeletal muscle pathology and contraction-induced injury to myofibers (5, 31–34).
Furthermore, we have demonstrated effective transduction of the heart using adeno-associated virus type 6–expressing SSPN (AAV6-SSPN).
AAV6-SSPN effectively delivers this small 25-kDa protein, therefore establishing, its suitability for gene therapy approaches.
The current study addresses the mechanistic basis of SSPN rescue of cardiac pathology in several relevant genotypic and phenotypic DMD mouse models and serves as a preclinical study, assessing the effectiveness of SSPN in upregulating utrophin and UGC-associated proteins in human DMD disease in order to preserve cardiac muscle integrity and contractility.
More information: Michelle S. Parvatiyar et al, Stabilization of the cardiac sarcolemma by sarcospan rescues DMD-associated cardiomyopathy, JCI Insight (2019). DOI: 10.1172/jci.insight.123855
Provided by Florida State University