Current Grant
09/25 – 08/26
Objective: Develop cellular models of LGMD2B muscle to enable studies of dysferlin’s function and preclinical testing of drug candidates.
We have previously developed “myobundles,” 3D cultures of myogenic progenitor cells derived from human induced pluripotent stem cells (hiPSCs) that contract like native muscle in response to electrical stimulation. Using this platform, we have successfully generated functional myobundles from three control dysferlin-positive and three dysferlin-negative (LGMD2B) hiPSC lines provided by Jain foundation. We demonstrated that compared to control myobundles, LGMD2B myobundles exhibit decreased contractile strength, abnormal calcium handling, impaired membrane repair, and altered response to lipid supplementation. Our recent studies have focused on determining why LGMD2B muscle cholesterol metabolism is altered, how this affects muscle strength and membrane repair, and whether treatments that restore normal cholesterol handling can improve LGMD2B muscle strength. Additionally, we are expanding the myobundle platform to study how non-muscle cells such as muscle interstitial cells and macrophages contribute to ectopic fat formation and inflammation in LGMD2B. Lastly, we have created the first in vitro human heart tissue model of LGMD2B (“cardiobundles”) to understand why heart muscle, but not skeletal muscle, is protected in LGMD2B. We expect that this work will establish personalized human 3D cell culture platforms for testing underlying pathogenic mechanisms of LGMD2B and discovering candidate therapeutics for patients with dysferlin deficiency.
Project Results
Previous Grant Period
09/24 – 08/25
In this funding period, we continued to compare human myobundles, cardiobundles, and macrophages (iMPs) made from 3 dysferlin-deficient and 3 healthy hiPSC lines for their tissue function, drug response, and biochemical differences. Our previous studies have suggested that effective cholesterol modulation can improve LGMD2B muscle function. In the last year, we have found that supplementing myobundles with (“bad”) LDL cholesterol worsens LGMD2B muscle weakness and disrupted mitochondrial function and structure. However, LDL treatment prevented the beneficial effects of statins, mirroring findings in BLAJ mice, and highlighting the need for alternative cholesterol lowering therapeutics. To guide future therapeutic development, lipidomic profiling of the mevalonate/HMGCR pathway further defined cholesterol-related abnormalities and identified potential pathway branchpoints for therapeutic targeting in the upcoming funding period. In contrast to myobundles, LGMD2B cardiobundles did not exhibit significant deficits in contractile force generation, calcium-handling, or membrane repair. RNA-sequencing of both cardiac and skeletal muscle in BLAJ mice and 3D bundles revealed transcriptomic differences that may confer cardioprotection. Lastly, we continued investigating dysferlin’s role in macrophages, performing proteomic profiling to characterize altered cytokine secretion and decreased endocytosis in LGMD2B iMPs. These findings suggest that an altered macrophage secretome and endocytosis of muscle debris may drive inflammation and LGMD2B disease progression. In the next funding period, we will continue to: 1) define dysferlin’s role in intramuscular cholesterol homeostasis, 2) investigate new cholesterol-lowering therapies in BLAJ mice, 3) determine whether cardioprotective pathways can be leveraged to improve LGMD2B myobundle function, 4) explore the effects of LGMD2B macrophages on myobundle function, and 5) test whether restoring altered cytokine secretion or endocytic capacity of macrophages can improve LGMD2B myobundle function. We anticipate these studies will help identify novel disease mechanisms and pharmacological interventions in dysferlinopathy.
Previous Grant Period
09/23 – 08/24
In this funding period, we continued to compare human myobundles, cardiobundles, and macrophages (iMPs) made from 3 dysferlin-deficient and 3 healthy hiPSC lines for their contractile function, metabolism, drug response, and transcriptomic differences. Specifically, our comparative gene expression profiling of myobundles, mouse, and human LGMD2B muscle revealed transcriptomic signatures characteristic of increased cholesterol accumulation. Pharmacologically decreasing this accumulation increased force generation and membrane repair of LGMD2B myobundles. To further explore the causes of increased cholesterol accumulation, we characterized cholesterol homeostasis and functional responses to pharmacological inhibitors (i.e. statins) and degraders of HMGCR, the rate-limiting enzyme involved in cholesterol biosynthesis. Reduced HMGCR activity specifically increased LGMD2B myobundle function and membrane repair, corroborating the finding that effective cholesterol modulation can improve LGMD2B muscle function.
During this funding period we also continued our exploration of roles of dysferlin in cardiac tissue function, which is largely unaffected in patients. In contrast to myobundles, we found that dysferlin deficiency in cardiobundles does not result in significant deficits in their contractile force generation, calcium-handling, or membrane repair. Our future studies will seek to determine fundamental differences in cardiac and skeletal muscle structure and physiology that result in cardioprotection despite loss of dysferlin. During this funding period, we also continued exploring the dysferlin roles in iMP function and validated that dysferlin-deficient iMPs show impaired endocytosis and phagocytosis of damaged myotubes. This suggests that LGMD2B MPs are less capable of clearing damaged muscle which may exacerbate LGMD2B pathology in patients.
Previous Grant Period
09/22 – 08/23
In this funding period, we continued to compare human myobundles made from 3 dysferlin-deficient and 3 healthy hiPSC lines for their drug response, metabolic function, and transcriptomic differences. Previously, we have found that LGMD2B myobundles display decreased contractile force generation, impaired membrane repair capacity, lipid droplet accumulation, and altered mitochondrial structure and function. As cholesterol esters accumulate within LGMD2B myobundles, we explored the functional consequences of inducing cholesterol accumulation in healthy myobundles by inhibiting lysosomal cholesterol export with the small molecule U18666A. Increased cholesterol accumulation in healthy myobundles resulted in no changes in force generation but significantly impaired membrane repair capacity and increased lipid droplet accumulation. To explore the role of cholesterol trafficking, we acutely decreased or increased plasma membrane (PM) cholesterol with methyl beta cyclodextrin (MβCD) or MβCD-cholesterol, respectively. Decreasing PM cholesterol levels impaired membrane repair capacity in healthy but had no effect in LGMD2B myobundles. In contrast, increasing PM cholesterol levels increased membrane repair capacity in LGMD2B myobundles to healthy levels. Together, this suggests that dysferlin deficiency impairs PM cholesterol trafficking which subsequently impairs membrane repair, without impacting force generation. During this funding period we also explored the role of dysferlin in macrophage function by generating hiPSC-derived macrophages (iMPs) from 3 healthy and 3 LGMD2B lines. Our findings suggest that dysferlin deficiency in IMPs alters secretion of cytokines related to muscle regeneration, immune cell chemoattraction, and chemotaxis, as well as impairs endocytosis and phagocytosis which are required for clearance of damaged muscle, all of which may contribute to LGMD2B progression. In the next funding period, we will continue to explore the roles of dysferlin in regulating intramuscular cholesterol homeostasis, macrophage function and metabolism, and function and stress response of engineered cardiac muscle (“cardiobundles”). We anticipate these studies will help identify novel disease mechanisms and pharmacological interventions in dysferlinopathy.
Previous Grant Period
09/21 – 08/22
In this funding period, we continued to compare human myobundles made from 3 dysferlin-deficient and 3 healthy hiPSC lines for their drug response, metabolic function, and transcriptomic differences. Previously, we found that LGMD2B myobundles display decreased contractile force generation, impaired membrane repair capacity, lipid droplet accumulation, and altered mitochondrial structure and function. Inhibiting calcium leakage from the sarcoplasmic reticulum with dantrolene or facilitating membrane repair with vamorolone restored contractile function, membrane repair capacity, and mitochondrial function, and significantly reduced lipid droplet accumulation in LGMD2B myobundles. Additionally, we found that inducing calcium leakage in healthy myobundles resulted in several disease signatures such as decreased force generation, lipid droplet accumulation, and mitochondrial dysfunction. Interestingly, membrane repair capacity was not compromised, suggesting that altered calcium homeostasis alone does not drive the LGMD2B phenotype. As our metabolomic studies confirmed that cholesterol esters accumulate within LGMD2B myobundles, we explored the effects of decreasing intracellular cholesterol biosynthesis with statins and reducing cholesterol esterification/storage with small molecule inhibitors. These cholesterol-lowering drugs improved LGMD2B contractile force and membrane repair capacity. Lastly, for the first time, we optimized cell culture conditions and injury protocols to induce intramuscular adipose tissue (IMAT) formation in tissue-engineered skeletal muscle. Our ongoing work in myobundles will further determine the role of dysferlin in regulating intramuscular cholesterol homeostasis and reveal mechanisms of IMAT formation and immune cell infiltration in LGMD2B. We anticipate that these studies will identify novel pharmacological interventions aimed at alleviating the pathology of dysferlin deficiency.
Previous Grant Period
09/20 – 08/21
In this funding period, we continued to compare human myobundles made from 3 dysferlin-deficient and 3 healthy hiPSC lines for their drug response, metabolic function, and transcriptomic differences. We found that the decrease in contractile force generation, consistently observed in dysferlin-deficient vs. healthy myobundles, is caused by impaired calcium homeostasis and excessive accumulation of intracellular calcium in muscle fibers. Inhibiting calcium leak from sarcoplasmic reticulum with dantrolene or increasing membrane repair with vamorolone restored contractile function, calcium transient amplitude, and recovery from osmotic shock injury. Interestingly, increased accumulation of lipid droplets in dysferlin-deficient myobundles in response to fatty acid supplementation was also decreased by application of dantrolene and vamorolone, suggesting a link between calcium handling and lipid metabolism. We have also performed analysis of mitochondrial function, metabolomic profiling, and RNA sequencing to better understand the mechanisms underlying insufficiencies in calcium and lipid handling in dysferlin-deficient myobundles. These datasets suggest a disease signature characterized by an intricate relationship between dysferlin deficiency, alterations in cholesterol metabolism, and mitochondrial dysfunction. Our ongoing work will further validate these findings and explore pharmacological interventions aimed at alleviating the dysferlin-deficient pathology.
