Myh6 Knockout Syrian Golden Hamster Dilated Cardiomyopathy Research Via CRISPR/Cas9 System

Objects and methodsIn the world,there are many people suffering from inherited cardiomyopathies,which have a severe clinical outcome,what is worse,given the familial hereditary it will influence the general population potentially and badly.Hypertrophic cardiomyopathy(HCM)and dilated cardiomyopathy(DCM)as the top 2 common inherited cardiomyopathies are still big challenge for the world.HCM it is recognized that affects the general population about 1/500 to 1/200,and the clinical manifestations can be different over persons,but they can be concluded as palpation,short of breath,thoracalgia.Some patients with severe manifestations can accompany with left ventricular outflow tract obstruction(LVOT),heart failure even sudden cardiac death(SCD).As for DCM,its morbidity is about 1/2500,with very a bad five-year survive rate less than 50%.Meanwhile DCM patients are easier to be found accompanied by heart failure,cardiac arrhythmia and even strock events.Myosin heavy chain is the most abundant sarcomere protein in the heart,and the main mutation type found till now is missense mutation,meanwhile that is supposed to be responsible for either HCM or DCM because of dominant negative effect.However,other mutations in myosin heavy chain such as loss of function(LOF)(including Truncation Variation as well as Nonsense Variation and so on)and Non-Frameshift Deletion Mutation are still obscure now.LOF mutations in this gene are usually thought not the strong evidence responsible for the cardiovascular diseases in the American Society of Medical Genetics(ACMG)guideline.But given that more than 95%of the mutations in this gene are missense mutations,other mutations count only less than 5%.What is more,there are few clinical data about other mutation types,even more the incomplete penetrance of cardiovascular diseases,so it is hard to confirm the pathogenicity of other mutations in myosin heavy chain.Myosin heavy chain and Titin(TTN)are the most important components of myocytes sarcomere structure.LOF including Truncation Variation in TTN will result in serious DCM but it is unknown for Myosin heavy chain Truncation Variation and Deletion Variation.Of note,there are no animal models for us to further explore.In order to clarify the pathogenicity of myosin heavy chain Truncation Variation and Deletion Variation,meanwhile,Syrian golden hamster is closer to human in cardiovascular disease than rats and mice,we used CRISPR/Cas9 technology to establish Myh6 knockout Syrian golden hamster models including Truncation Variation and Deletion Variation.We performed cardiac B-ultrasounds on wild-type(WT)and knockout hamsters to analyze their systolic function and determine the thickness of the wall as well as the septum.Subsequently,Truncation Variation hamsters and WT hamsters were sacrificed,and the heart-to-body weight ratio of hamsters was compared.HE and Masson staining were also performed,and heart failure and fibrosis index were tested by qRT-PCR.At the same time,WT and Myh6 Truncation Variation hamsters were intraperitoneally injected with the cardiotoxic chemotherapeutic drug doxorubicin(DOX),and the cardiotoxic effects of hamsters in each group were compared.ResultsUsing CRISPR/Cas9 gene editing system,for the first time,the Syrian golden hamster Myh6 gene Truncation and Deletion models were obtained.Notable,this is also the first Myh6 gene Truncation and Deletion models established in the world.We have proved that the Myh6 knockout hamsters manifested dilated cardiomyopathy symptoms,including reduced ejection fraction,dilated heart cavity,thinner wall thickness,and myocardial fibrosis.We have demonstrated that myocardial cell sarcomere structure and mitochondria size were also destroyed significantly in Myh6 Truncation ones,such as shorter sarcomere length and smaller mitochondria size.At the same time,we used the cardiotoxic chemical compound doxorubicin to treat wild-type and Myh6 Truncation Syrian hamsters and found that Myh6 Truncation hamsters are more sensitive to the cardiotoxic effects of doxorubicin,for example we observed doxorubicin accelerated the symptom of DCM,and resulted in sarcomere structure destroy and mitochondrial dysfunction in Myh6 Truncation hamsters.ConclusionsMyosin heavy chain Truncation Variation and Deletion Variation will result in DCM,meanwhile more sensitive to the cardiotoxic effects of doxorubicin.Furthermore,we demonstrated that Myosin heavy chain Truncation Variation and Deletion Variation Syrian golden hamsters are superb animal models to study DCM.