Editing CaMKIIδ As A Therapy For Diabetic Cardiomyopathy

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Diabetes mellitus is a chronic disease affecting approximately 20% of the population over 60 years of age. Patients with diabetes have an increased risk of cardiovascular diseases, including arrhythmias and heart failure. Unfortunately, patients’ compliance with recommended treatment strategies is often low, resulting in hyperglycaemia and an increased risk for diabetic cardiomyopathy. Upon high glucose levels, the stress-responsive kinase CaMKIIδ, an indicator and inducer of cardiac disease, is overactivated by glycosylation at amino acid serine 280. The present project aims to develop a CRISPR-Cas9 gene editing strategy to ablate the glycosylation site of CaMKIIδ as a potential new therapeutic approach for diabetic cardiomyopathy. We will design and optimize a CRISPR-Cas9 gene editing strategy to precisely modify the CaMKIIδ gene. Our approach will compare adenine base editing, cytosine base editing, and prime editing in HEK293 cells. Based on the results, the most efficient editing strategies will be applied to human induced pluripotent stem cells (iPSCs). Homozygous iPSC-lines with edited CaMKIIδ will be differentiated into cardiomyocytes, then subjected to 10 days of high glucose treatment as an in vitro model for diabetes, and then assessed for cardioprotection. We will investigate several mechanisms that have been previously linked to CaMKII-dependent pathological signalling. Specifically, we will measure stimulated calcium transients (epifluorescence microscopy), diastolic sarcoplasmic reticulum calcium leak (confocal laser scanning microscopy), and late sodium current (patch clamp technique). These mechanisms are expected to be pathologically altered upon high glucose treatment, but not when the glycosylation site of CaMKIIδ is ablated as in the CaMKIIδ-edited cardiomyocytes. Depending on the editing efficiency and the degree of cardioprotection, we will further pursue the most promising editing strategy in vivo, using a humanized CaMKIIδ knockin mouse model that I have recently generated. Diabetes will be induced by streptozotocin treatment. The optimized gene editing construct with a cardiac troponin T promoter to ensure cardiomyocyte-specific editing will be packaged into an adeno-associated virus serotype-9 and administered to mice. Mice will be challenged by being subjected to transverse aortic constriction at a later timepoint. All mice will be analysed by echocardiography to measure cardiac function and structure at several timepoints during the experiment. In vivo arrhythmias will be assessed using programmed electrical stimulation. Wildtype mice with diabetes are expected to show impaired cardiac contractility and an increased risk of arrhythmias. I hypothesize that CRISPR-Cas9 gene editing to render CaMKIIδ insensitive to glycosylation in vivo will protect from these deleterious alterations, which may lead to a prospective therapy for diabetic cardiomyopathy.