Browsing by Subject "Cardiomyopathy"

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    Modeling and rescue of Duchenne muscular dystrophy cardiomyopathy using human induced pluripotent-derived cardiomyocytes
    (2021-01) Kamdar, Forum
    Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and affects 1:5000 boys born in the United States. DMD is a result of mutations in the dystrophin (DMD) gene that leads to the absence of the full length cytoskeletal protein dystrophin, which is expressed in skeletal muscle, brain, and heart. The absence of dystrophin leads to weakness of not only the skeletal muscle but also the heart. With advances in treatment for DMD, patients are living longer but a cardiomyopathic phenotype has been uncovered. DMD associated cardiomyopathy is nearly ubiquitous and is the leading cause of death with adults with DMD. There have been limited studies and therapies for dystrophic heart failure thus far, and there is a critical need to identify the pathophysiology and develop effective therapeutic regimens. In this thesis, I hypothesized that DMD cardiomyopathy could be modeled using DMD patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-derived CMs) to identify physiological changes and future drug therapies. To explore and define therapies for DMD cardiomyopathy, we used DMD patient-specific and dystrophin null isogenic hiPSC-derived CMs to examine the physiological response to adrenergic agonists and -blocker treatment. We further examined these agents in vivo using wildtype and the mdx mouse model. At baseline and following adrenergic stimulation, DMD hiPSC-derived CMs had a significant increase in arrhythmic calcium traces compared to isogenic controls. Further, these arrhythmias were significantly decreased with propranolol treatment. Using telemetric monitoring, we observed that mdx mice, which lack dystrophin, and were stimulated with isoproterenol had an arrhythmic death and the lethal arrhythmias were rescued, in part, by propranolol pretreatment. Using single cell and bulk RNA-seq, we compared DMD and control hiPSC-derived CMs, mdx mice and control mice (in the presence or absence of propranolol and isoproterenol) and defined pathways that were perturbed under baseline conditions and pathways that were normalized following propranolol treatment in the mdx model. We also undertook transcriptome analysis of human DMD left ventricle samples and found that DMD hiPSC-derived CMs have similar dysregulated pathways as the human DMD heart. We further determined that relatively few DMD patients see a cardiovascular specialist or receive β-blocker therapy. The results of these experiments highlight important mechanisms and therapeutic interventions from human to animal and back to human in the dystrophic heart. Importantly, these results may serve as a platform to elucidate further mechanisms of DMD cardiomyopathy and serve as a platform for testing novel therapies. Our results also provide a rationale for an adequately powered clinical study that examines the impact of β-blocker therapy in patients with dystrophinopathies.
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    Tuning the Equilibrium: A Biophysical Approach to Controlling Cardiac Contractility through SERCA and Phospholamban
    (2017-05) Soller, Kailey
    Heart disease is the leading cause of death throughout the world and one of the major hallmarks is dysfunctional muscle contractility. Contractility is a highly regulated and complex process which involves multiple proteins. This network can be easily disrupted by mutation or changes in protein expression level and thus, proteins involved in this process are key drug targets. Muscle contractility is controlled by the calcium concentration in the cytosol. In cardiomyocytes, the sarco(endo)plasmic reticulum Ca2+ -ATPase, SERCA, and its regulatory protein, phospholamban (PLN) are responsible for ~70% of Ca2+ reuptake into the SR. While unphosphorylated, PLN inhibits SERCA by lowering its apparent Ca2+ affinity. Upon phosphorylation by PKA at Ser16, PLN inhibition is relieved. Mutations or disruptions in this complex have found in many forms of heart disease; thus, understanding the molecular interactions between SERCA and PLN, along with possible regulatory molecules, is essential. Understanding the molecular mechanisms that occur on a beat-to-beat basis will be essential for developing therapeutics to treat cardiomyopathies. In this dissertation work, I studied the structural, biochemical and biophysical properties of SERCA and PLN. We found that single-stranded DNA (ssDNA), RNA, and DNA analogs bind the cytoplasmic domain of PLN with low nanomolar dissociation constants, relieving inhibition of SERCA. The relief of inhibition is length-dependent, while affinity is constant for oligonucleotides longer than 10 bases. Solution and solid-state NMR experiments have provided residue specific information that ssDNA targets the cytoplasmic domain of PLN and does not affect SERCA in the absence of PLN. In-cell FRET and NMR experiments determined that addition of ssDNA does not dissociate PLN from SERCA. Additionally, I started moving this work into a sustainable, in vivo system using cardiomyocytes derived from induced pluripotent stem cells (iPSCs) to investigate the functional effects of these molecules with PLN in cell. The establishment of this cell line in our laboratory will allow for future characterization of not only XNAs with PLN, but also experiments with any cell type obtainable through differentiation of iPSCs. Finally, early work with the R14del hereditary mutant of PLN helped to determine the structural changes this mutant imposes on PLN as well as when in complex with SERCA. We found that the R14del mutant is loss-of-function and also if phosphorylated, will not relieve inhibition of SERCA. We believe that the knowledge gained here on the SERCA-PLN complex contributes to the overall understanding of how calcium handling can be modulated to change protein function and demonstrates a novel avenue of oligonucleotide action in the body. miRNAs may have evolved to also directly interact with non-transcription related proteins to modulate their function. These results and our future experiments will provide a promising avenue for development of novel therapeutic regulators of the SERCA-PLN complex to help treat heart disease, as well as provide some of the needed mechanistic insight on how hereditary mutants like R14del cause aberrant regulation in the heart.