Mechanisms of pathogenicity in the hypertrophic cardiomyopathy-associated TPM1 variant S215L.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder often caused by mutations to sarcomeric genes. Many different HCM-associated TPM1 mutations have been identified but they vary in their degrees of severity, prevalence, and rate of disease progression. The pathogenicity of many TPM1 variants detected in the clinical population remains unknown. Our objective was to employ a computational modeling pipeline to assess pathogenicity of one such variant of unknown significance, TPM1 S215L, and validate predictions using experimental methods. Molecular dynamic simulations of tropomyosin on actin suggest that the S215L significantly destabilizes the blocked regulatory state while increasing flexibility of the tropomyosin chain. These changes were quantitatively represented in a Markov model of thin-filament activation to infer the impacts of S215L on myofilament function. Simulations of in vitro motility and isometric twitch force predicted that the mutation would increase Ca2+ sensitivity and twitch force while slowing twitch relaxation. In vitro motility experiments with thin filaments containing TPM1 S215L revealed higher Ca2+ sensitivity compared with wild type. Three-dimensional genetically engineered heart tissues expressing TPM1 S215L exhibited hypercontractility, upregulation of hypertrophic gene markers, and diastolic dysfunction. These data form a mechanistic description of TPM1 S215L pathogenicity that starts with disruption of the mechanical and regulatory properties of tropomyosin, leading thereafter to hypercontractility and finally induction of a hypertrophic phenotype. These simulations and experiments support the classification of S215L as a pathogenic mutation and support the hypothesis that an inability to adequately inhibit actomyosin interactions is the mechanism whereby thin-filament mutations cause HCM.

Physiological calcium combined with electrical pacing accelerates maturation of human engineered heart tissue.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have wide potential application in basic research, drug discovery, and regenerative medicine, but functional maturation remains challenging. Here, we present a method whereby maturation of hiPSC-CMs can be accelerated by simultaneous application of physiological Ca2+ and frequency-ramped electrical pacing in culture. This combination produces positive force-frequency behavior, physiological twitch kinetics, robust ß-adrenergic response, improved Ca2+ handling, and cardiac troponin I expression within 25 days. This study provides insights into the role of Ca2+ in hiPSC-CM maturation and offers a scalable platform for translational and clinical research.

Sustained low-dose dexamethasone delivery via a PLGA microsphere-embedded agarose implant for enhanced osteochondral repair.

Articular cartilage defects are a common source of joint pain and dysfunction. We hypothesized that sustained low-dose dexamethasone (DEX) delivery via an acellular osteochondral implant would have a dual pro-anabolic and anti-catabolic effect, both supporting the functional integrity of adjacent graft and host tissue while also attenuating inflammation caused by iatrogenic injury. An acellular agarose hydrogel carrier with embedded DEX-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres (DLMS) was developed to provide sustained release for at least 99 days. The DLMS implant was first evaluated in an in vitro pro-inflammatory model of cartilage degradation. The implant was chondroprotective, as indicated by maintenance of Young's modulus (EY) (p = 0.92) and GAG content (p = 1.0) in the presence of interleukin-1ß insult. In a subsequent preliminary in vivo experiment, an osteochondral autograft transfer was performed using a pre-clinical canine model. DLMS implants were press-fit into the autograft donor site and compared to intra-articular DEX injection (INJ) or no DEX (CTL). Functional scores for DLMS animals returned to baseline (p = 0.39), whereas CTL and INJ remained significantly worse at 6 months (p < 0.05). DLMS knees were significantly more likely to have improved OARSI scores for proteoglycan, chondrocyte, and collagen pathology (p < 0.05). However, no significant improvements in synovial fluid cytokine content were observed. In conclusion, utilizing a targeted DLMS implant, we observed in vitro chondroprotection in the presence of IL-1-induced degradation and improved in vivo functional outcomes. These improved outcomes were correlated with superior histological scores but not necessarily a dampened inflammatory response, suggesting a primarily pro-anabolic effect. STATEMENT OF SIGNIFICANCE: Articular cartilage defects are a common source of joint pain and dysfunction. Effective treatment of these injuries may prevent the progression of osteoarthritis and reduce the need for total joint replacement. Dexamethasone, a potent glucocorticoid with concomitant anti-catabolic and pro-anabolic effects on cartilage, may serve as an adjuvant for a variety of repair strategies. Utilizing a dexamethasone-loaded osteochondral implant with controlled release characteristics, we demonstrated in vitro chondroprotection in the presence of IL-1-induced degradation and improved in vivo functional outcomes following osteochondral repair. These improved outcomes were correlated with superior histological cartilage scores and minimal-to-no comorbidity, which is a risk with high dose dexamethasone injections. Using this model of cartilage restoration, we have for the first time shown the application of targeted, low-dose dexamethasone for improved healing in a preclinical model of focal defect repair.

A Functional Tissue-Engineered Synovium Model to Study Osteoarthritis Progression and Treatment.

IMPACT STATEMENT: The synovium envelops the diarthrodial joint and plays a key regulatory role in defining the composition of the synovial fluid through filtration and biosynthesis of critical boundary lubricants. Synovium changes often precede cartilage damage in osteoarthritis. We describe a novel in vitro tissue engineered model, validated against native synovium explants, to investigate the structure-function of synovium through quantitative solute transport measures. Synovium was evaluated in the presence of a proinflammatory cytokine, interleukin-1, or the clinically relevant corticosteroid, dexamethasone. We anticipate that a better understanding of synovium transport would support efforts to develop more effective strategies aimed at restoring joint health.