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Background: Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. Methods: Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell–derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations. Results: Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain ( MYH7 ) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin–nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein–W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. Conclusions: Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

Tissue engineered vascular grafts possess several advantages over synthetic or autologous grafts, including increased availability and reduced rates of infection and thrombosis. Engineered grafts constructed from human induced pluripotent stem cell derivatives further offer enhanced reproducibility in graft production. One notable obstacle to clinical application of these grafts is the lack of elastin in the vessel wall, which would serve to endow compliance in addition to mechanical strength. This study establishes the ability of the polyphenol compound epigallocatechin gallate, a principal component of green tea, to facilitate the extracellular formation of elastin fibers in vascular smooth muscle cells derived from human induced pluripotent stem cells. Further, this study describes the creation of a doxycycline-inducible elastin expression system to uncouple elastin production from vascular smooth muscle cell proliferative capacity to permit fiber formation in conditions conducive to robust tissue engineering.

Tissue engineered vascular grafts (TEVGs) represent a promising therapeutic option for emergency vascular intervention. Although the application of small-diameter TEVGs using patient-specific primary endothelial cells (ECs) to prevent thrombosis and occlusion prior to implantation could be hindered by the long time course required for in vitro endothelialization, human induced pluripotent stem cells (hiPSCs) provide a robust source to derive immunocompatible ECs (hiPSC-ECs) for immediate TEVG endothelialization. To achieve clinical application, hiPSC-ECs should be derived under culture conditions without the use of animal-derived reagents (xenogeneic-free conditions), to avoid unwanted host immune responses from xenogeneic reagents. However, a completely xenogeneic-free method of hiPSC-EC generation has not previously been established. Herein, we substituted animal-derived reagents used in a standard method of xenogeneic hiPSC-EC differentiation with functional counterparts of human origin. As a result, we generated xenogeneic-free hiPSC-ECs (XF-hiPSC-ECs) with similar marker expression and function to those of human primary ECs. Furthermore, XF-hiPSC-ECs functionally responded to shear stress with typical cell alignment and gene expression. Finally, we successfully endothelialized decellularized human vessels with XF-hiPSC-ECs in a dynamic bioreactor system. In conclusion, we developed xenogeneic-free conditions for generating functional hiPSC-ECs suitable for vascular tissue engineering, which will further move TEVG therapy toward clinical application.

Development of mechanically advanced tissue-engineered vascular grafts (TEVGs) from human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (hiPSC-VSMCs) offers an innovative approach to replace or bypass diseased blood vessels. To move current hiPSC-TEVGs toward clinical application, it is essential to obtain hiPSC-VSMC-derived tissues under xenogeneic-free conditions, meaning without the use of any animal-derived reagents. Many approaches in VSMC differentiation of hiPSCs have been reported, although a xenogeneic-free method for generating hiPSC-VSMCs suitable for vascular tissue engineering has yet to be established. Based on our previously established standard method of xenogeneic VSMC differentiation, we have replaced all animal-derived reagents with functional counterparts of human origin and successfully derived functional xenogeneic-free hiPSC-VSMCs (XF-hiPSC-VSMCs). Next, our group developed tissue rings via cellular self-assembly from XF-hiPSC-VSMCs, which exhibited comparable mechanical strength to those developed from xenogeneic hiPSC-VSMCs. Moreover, by seeding XF-hiPSC-VSMCs onto biodegradable polyglycolic acid (PGA) scaffolds, we generated engineered vascular tissues presenting effective collagen deposition which were suitable for implantation into an immunodeficient mice model. In conclusion, our xenogeneic-free conditions for generating hiPSC-VSMCs produce cells with the comparable capacity for vascular tissue engineering as standard xenogeneic protocols, thereby moving the hiPSC-TEVG technology one step closer to safe and efficacious clinical translation.

Pharmacologic inhibition of acetyl-CoA carboxylase (ACC) enzymes, ACC1 and ACC2, offers an attractive therapeutic strategy for nonalcoholic fatty liver disease (NAFLD) through simultaneous inhibition of fatty acid synthesis and stimulation of fatty acid oxidation. However, the effects of ACC inhibition on hepatic mitochondrial oxidation, anaplerosis, and ketogenesis in vivo are unknown. Here, we evaluated the effect of a liver-directed allosteric inhibitor of ACC1 and ACC2 (Compound 1) on these parameters, as well as glucose and lipid metabolism, in control and diet-induced rodent models of NAFLD. Oral administration of Compound 1 preferentially inhibited ACC enzymatic activity in the liver, reduced hepatic malonyl-CoA levels, and enhanced hepatic ketogenesis by 50%. Furthermore, administration for 6 days to high-fructose-fed rats resulted in a 20% reduction in hepatic de novo lipogenesis. Importantly, long-term treatment (21 days) significantly reduced high-fat sucrose diet-induced hepatic steatosis, protein kinase C epsilon activation, and hepatic insulin resistance. ACCi treatment was associated with a significant increase in plasma triglycerides (approximately 30% to 130%, depending on the length of fasting). ACCi-mediated hypertriglyceridemia could be attributed to approximately a 15% increase in hepatic very low-density lipoprotein production and approximately a 20% reduction in triglyceride clearance by lipoprotein lipase (P ≤ 0.05). At the molecular level, these changes were associated with increases in liver X receptor/sterol response element-binding protein-1 and decreases in peroxisome proliferator-activated receptor-α target activation and could be reversed with fenofibrate co-treatment in a high-fat diet mouse model. Conclusion: Collectively, these studies warrant further investigation into the therapeutic utility of liver-directed ACC inhibition for the treatment of NAFLD and hepatic insulin resistance.

Development of autologous tissue-engineered vascular constructs using vascular smooth muscle cells (VSMCs) derived from human induced pluripotent stem cells (iPSCs) holds great potential in treating patients with vascular disease. However, preclinical, large animal iPSC-based cellular and tissue models are required to evaluate safety and efficacy prior to clinical application. Herein, swine iPSC (siPSC) lines were established by introducing doxycycline-inducible reprogramming factors into fetal fibroblasts from a line of inbred Massachusetts General Hospital miniature swine that accept tissue and organ transplants without immunosuppression within the line. Highly enriched, functional VSMCs were derived from siPSCs based on addition of ascorbic acid and inactivation of reprogramming factor via doxycycline withdrawal. Moreover, siPSC-VSMCs seeded onto biodegradable polyglycolic acid (PGA) scaffolds readily formed vascular tissues, which were implanted subcutaneously into immunodeficient mice and showed further maturation revealed by expression of the mature VSMC marker, smooth muscle myosin heavy chain. Finally, using a robust cellular self-assembly approach, we developed 3D scaffold-free tissue rings from siPSC-VSMCs that showed comparable mechanical properties and contractile function to those developed from swine primary VSMCs. These engineered vascular constructs, prepared from doxycycline-inducible inbred siPSCs, offer new opportunities for preclinical investigation of autologous human iPSC-based vascular tissues for patient treatment.

Summary Vascular smooth muscle cells (VSMCs) can be derived in large numbers from human induced pluripotent stem cells (hiPSCs) for producing tissue-engineered vascular grafts (TEVGs). However, hiPSC-derived TEVGs are hampered by low mechanical strength and significant radial dilation after implantation. Here, we report generation of hiPSC-derived TEVGs with mechanical strength comparable to native vessels used in arterial bypass grafts by utilizing biodegradable scaffolds, incremental pulsatile stretching, and optimal culture conditions. Following implantation into a rat aortic model, hiPSC-derived TEVGs show excellent patency without luminal dilation and effectively maintain mechanical and contractile function. This study provides a foundation for future production of non-immunogenic, cellularized hiPSC-derived TEVGs composed of allogenic vascular cells, potentially serving needs to a considerable number of patients whose dysfunctional vascular cells preclude TEVG generation via other methods.