Development of Novel Therapeutics for Timothy Syndrome Using Human Induced Pluripotent Stem Cells

Cardiac disease is the leading cause of death in the United States, despite the continuing efforts contributed to scientific research and disease management in the past few decades. However many advances have been made in cardiovascular research in recent decades and one of the advances is the development of human induced pluripotent stem cell(iPSC)-based disease models. The human iPSC-based disease models are derived from the somatic cells of patients with cardiac diseases and capture the genotypes of the original patients, which make them more ideal for mimicking human diseases compared with conventional rodent models. So far, the iPSC-based disease models have been used to model several types of cardiac diseases, one of which is the focus of this work-Timothy syndrome. Timothy syndrome is caused by the missense mutations in the CACNA1C gene encoding the voltage-gated calcium channel CaV1.2, which plays an essential role in cardiac function. The disease is a multisystem disorder that is featured by long QT syndrome and syndactyly. Timothy syndrome patients are treated clinically with beta-adrenergic blockers, calcium channel blockers, and sodium channel blockers. However, these regimens are insufficient to prevent lethal arrhythmias in Timothy syndrome patients, especially infants with Timothy syndrome. Therefore, new therapeutics to prevent the lethal arrhythmias in Timothy syndrome patients are needed until the age when an implantable defibrillator is available. The iPSC-based model of Timothy syndrome was first reported in 2011. The previous report showed that the Timothy syndrome iPSC-derived cardiomyocytes demonstrated several cellular phenotypes including abnormal contractions, abnormal electrophysiological properties and abnormal calcium handling, which were consistent with the clinical features of the patients that the iPSCs were derived from. In addition, the authors demonstrated that Roscovitine, a cyclin-dependent kinase (CDK) inhibitor, could rescue the cellular phenotypes in Timothy syndrome cardiomyocytes. However, the mechanisms underlying the beneficial effects of Roscovitine on Timothy syndrome cardiomyocytes were not fully elucidated. This work will employ the iPSC-based model of Timothy syndrome to investigate the mechanisms underlying the beneficial effects of Roscovitine on Timothy syndrome cardiomyocytes and search for additional therapeutic compounds and targets for Timothy syndrome. In chapter 1 of this work, we presented new methods to generate iPSCs from human skin fibroblasts or hair keratinocytes, and to differentiate iPSCs into cardiomyocytes in a monolayer format. The major advantage of the two new methods is that they are technically simple and generally applicable for samples from healthy control donors and patients with cardiac diseases. The new methods enabled us to generate a sufficient amount of Timothy syndrome cardiomyocytes from iPSCs derived from the skin fibroblasts of Timothy syndrome patients, which became the foundation for the subsequent mechanistic study. Chapter 2 presents the identification of CDK5 as a new therapeutic target for Timothy syndrome. As introduced above, the previous report demonstrated that Roscovitine, a CDK inhibitor, could rescue the cellular phenotypes in Timothy syndrome cardiomyocytes. However, the mechanisms underlying the beneficial effects of Roscovitine on Timothy syndrome cardiomyocytes were not fully elucidated. To identify additional therapeutic compounds for Timothy syndrome and investigate the mechanisms underlying the therapeutic effects of Roscovitine on Timothy syndrome cardiomyocytes, we conducted a phenotypic screen using Timothy syndrome cardiomyocytes to screen through twenty Roscovitine analogs and four CDK inhibitors with different specificities for different CDKs. Four positive compounds were identified from the screen. When we summarized the CDK targets of the four positive compounds and the lead compound Roscovitine, it was found that four out of the five positive compounds shared a common CDK target, which is CDK5, indicating that CDK5 could be involved in the pathogenesis of Timothy syndrome as a therapeutic target. We next validated CDK5 as a new therapeutic target for Timothy syndrome using two independent approaches. The two approaches are expressing a dominant negative mutant of CDK5 and expressing short hairpin RNAs targeting CDK5 in Timothy syndrome cardiomyocytes using lentiviruses. Both approaches led to CDK5 inhibition in Timothy syndrome cardiomyocytes and we examined the changes in the cellular phenotypes in Timothy syndrome cardiomyocytes with CDK5 inhibition. The results indicated that CDK5 inhibition alleviated all the previously-reported phenotypes in Timothy syndrome cardiomyocytes. To investigate the mechanisms underlying the beneficial effects of CDK5 inhibition on Timothy syndrome cardiomyocytes, we examined the expression of CDK5 activator p35 and the activity of CDK5 in Timothy syndrome cardiomyocytes. We found that Timothy syndrome cardiomyocytes showed a higher expression of CDK5 activator p35 and a higher activity of CDK5 compared with control cardiomyocytes. When we over-expressed CDK5 in control cardiomyocytes, we found that CDK5 over-expression caused a change in the function of CaV1.2 channels in control cardiomyocytes that resembled the phenotype in Timothy syndrome cardiomyocytes. In summary of the results, we propose that in Timothy syndrome cardiomyocytes, the increased expression of CDK5 activator p35 causes CDK5 hyper-activation, which enhances the abnormal function of the mutant CaV1.2 channels, leading to more severe phenotypes. Thus, CDK5 inhibition alleviates the phenotypes in Timothy syndrome cardiomyocytes. The results in this chapter reveal that CDK5 is a new therapeutic target for Timothy syndrome and CDK5-specific inhibitors can potentially be developed into new therapeutics for Timothy syndrome. However, we found that the currently-available chemical inhibitors for CDK5 are not highly-selective and have several significant side effects that make them not ideal candidates to be developed into new therapeutics for cardiac diseases. Therefore new therapeutic compounds and targets are still needed for Timothy syndrome. Chapter 3 presents the identification of the sigma-1 receptor as a new therapeutic target for Timothy syndrome. Due to the side effects associated with the currently-available chemical inhibitors for CDK5, we made an effort to search for an additional therapeutic target and therapeutic compounds for Timothy syndrome. We reasoned that instead of directly inhibiting CDK5, we could potentially alleviate the phenotypes in Timothy syndrome cardiomyocytes by affecting the CDK5 activator p35 and this idea led us to the sigma-1 receptor. After we looked into the sigma-1 receptor, we found that in addition to being reported to modulate p35 protein level, the sigma-1 receptor had also been reported to modulate calcium homeostasis, which is another favorable effect for Timothy syndrome cardiomyocytes. Therefore we hypothesized that the activation of the sigma-1 receptor could be beneficial for Timothy syndrome cardiomyocytes, which feature an increased expression of p35 and a dysregulation of calcium homeostasis. To test this hypothesis, we examined the effects of two sigma-1 receptor agonists, one of which is a FDA-approved drug, on the phenotypes in Timothy syndrome cardiomyocytes. The results demonstrated that the treatment of the two sigma-1 receptor agonists alleviated the previously-reported phenotypes in Timothy syndrome cardiomyocytes. We also examined the effects of the two sigma-1 receptor agonists on the functions of control cardiomyocytes and found that the treatment of the two sigma-1 receptor agonists did not have significant side effects on the regular contractions and normal calcium transients in control cardiomyocytes. Overall, the results reveal that the sigma-1 receptor is a new therapeutic target for Timothy syndrome. The results also demonstrate that the two sigma-1 receptor agonists that we tested are promising lead compounds that can developed into novel therapeutics for Timothy syndrome in the future. Since one of the sigma-1 receptor agonists that we tested is a FDA-approved drug, this drug could potentially be used directly in Timothy syndrome patients for treating the cardiac arrhythmias in the near future. In summary, this work is a proof of concept that the iPSC-based models of cardiac diseases can be used to generate novel insights into disease pathogenesis, and to identify new therapeutic targets and compounds for cardiac diseases, and in particular for Timothy syndrome. The therapeutic targets and compounds that we have identified in this work would be helpful for the development of novel therapeutics for treating the lethal arrhythmias in Timothy syndrome patients in the future

Targeting lymphatic function in cardiovascular-kidney-metabolic syndrome: preclinical methods to analyze lymphatic function and therapeutic opportunities

The lymphatic vascular system spans nearly every organ in the body and serves as an important network that maintains fluid, metabolite, and immune cell homeostasis. Recently, there has been a growing interest in the role of lymphatic biology in chronic disorders outside the realm of lymphatic abnormalities, lymphedema, or oncology, such as cardiovascular-kidney-metabolic syndrome (CKM). We propose that enhancing lymphatic function pharmacologically may be a novel and effective way to improve quality of life in patients with CKM syndrome by engaging multiple pathologies at once throughout the body. Several promising therapeutic targets that enhance lymphatic function have already been reported and may have clinical benefit. However, much remains unclear of the discreet ways the lymphatic vasculature interacts with CKM pathogenesis, and translation of these therapeutic targets to clinical development is challenging. Thus, the field must improve characterization of lymphatic function in preclinical mouse models of CKM syndrome to better understand molecular mechanisms of disease and uncover effective therapies

Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype

Author ManuscriptThe application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to β-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR, and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); NSF grant 16478 (G.V.-N.); the University of Minho MD/PhD program (D.T.); a Japan Society for the Promotion of Science fellowship (K.M.); and the Columbia University Stem Cell Initiative (L.S., M.Y.

Advanced maturation of human cardiac tissue grown from pluripotent stem cells

Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1-6. However, the predictive power of these models is presently limited by the immature state of the cells1, 2, 5, 6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force-frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.The authors acknowledge funding support from the National Institutes of Health of the USA (NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); Columbia University MD/PhD program (S.P.M., T.C.); University of Minho MD/PhD program (D.T.); Japan Society for the Promotion of Science fellowship (K.M.); and Columbia University Stem Cell Initiative (D.S., L.S., M.Y.). We thank S. Duncan and B. Conklin for providing human iPSCs, M.B. Bouchard for assistance with image and video analysis, and L. Cohen-Gould for transmission electron microscopy services.info:eu-repo/semantics/publishedVersio

Inhibition of CDK5 Alleviates the Cardiac Phenotypes in Timothy Syndrome

L-type calcium channel CaV1.2 plays an essential role in cardiac function. The gain-of-function mutations in CaV1.2 have been reported to be associated with Timothy syndrome, a disease characterized by QT prolongation and syndactyly. Previously we demonstrated that roscovitine, a cyclin-dependent kinase (CDK) inhibitor, could rescue the phenotypes in induced pluripotent stem cell-derived cardiomyocytes from Timothy syndrome patients. However, exactly how roscovitine rescued the phenotypes remained unclear. Here we report a mechanism potentially underlying the therapeutic effects of roscovitine on Timothy syndrome cardiomyocytes. Our results using roscovitine analogs and CDK inhibitors and constructs demonstrated that roscovitine exhibits its therapeutic effects in part by inhibiting CDK5. The outcomes of this study allowed us to identify a molecular mechanism whereby CaV1.2 channels are regulated by CDK5. This study provides insights into the regulation of cardiac calcium channels and the development of future therapeutics for Timothy syndrome patients

SPAG7 deletion causes intrauterine growth restriction, resulting in adulthood obesity and metabolic dysfunction

From a forward mutagenetic screen to discover mutations associated with obesity, we identified mutations in the Spag7 gene linked to metabolic dysfunction in mice. Here, we show that SPAG7 KO mice are born smaller and develop obesity and glucose intolerance in adulthood. This obesity does not stem from hyperphagia, but a decrease in energy expenditure. The KO animals also display reduced exercise tolerance and muscle function due to impaired mitochondrial function. Furthermore, SPAG7-deficiency in developing embryos leads to intrauterine growth restriction, brought on by placental insufficiency, likely due to abnormal development of the placental junctional zone. This insufficiency leads to loss of SPAG7-deficient fetuses in utero and reduced birth weights of those that survive. We hypothesize that a ‘thrifty phenotype’ is ingrained in SPAG7 KO animals during development that leads to adult obesity. Collectively, these results indicate that SPAG7 is essential for embryonic development and energy homeostasis later in life

Muscle LIM Protein Force-Sensing Mediates Sarcomeric Biomechanical Signaling in Human Familial Hypertrophic Cardiomyopathy

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