Mutations in the gene encoding for dystrophin leads to structural and functional deterioration of cardiomyocytes and is a hallmark of cardiomyopathy in Duchenne muscular dystrophy (DMD) patients. Administration of recombinant adeno-associated viral vectors delivering microdystrophin or ribonucleotide reductase (RNR), under muscle-specific regulatory control, rescues both baseline and high workload-challenged hearts in an aged, DMD mouse model. However, only RNR treatments improved both systolic and diastolic function under those conditions. Cardiac-specific recombinant adeno-associated viral treatment of RNR holds therapeutic promise for improvement of cardiomyopathy in DMD patients.
CK8, miniaturized murine creatine kinase regulatory cassette; CMV, cytomegalovirus; DMD, Duchenne muscular dystrophy; RNR, ribonucleotide reductase; cTnT, cardiac troponin T; cardiomyopathy; dADP, deoxy-adenosine diphosphate; dATP, deoxy-adenosine triphosphate; diastolic dysfunction; dystrophin; mdx, mouse muscular dystrophy model; rAAV, recombinant adeno-associated viral vector; recombinant adeno-associated virus vectors; ribonucleotide reductase; μDys, microdystrophin.
© 2019 The Authors.
Overview of Experimental Design for the Treatment of Aged (22-24 Month)
mdx Mice 4cv (A) Graphic representation of rAAV6 vectors used in the present study. The ribonucleotide reductase vector contains the human cDNA for the RRM1 and RRM2 subunits whose expression is driven by the cardiac specific (cTnT455) regulatory cassette (RC). The human microdystrophin (ΔR2-R15/ΔR18-R22/ΔCT) vector has expression driven by the CK8 muscle-specific RC. The control vector used in the present study carries the firefly luciferase transgene whose CMV early promoter/enhancer RC has been deleted. (B) Outline of animal enrollment, vector administration, and experimental protocols implemented following a treatment period of 5 months. cDNA = complementary DNA; CMV = cytomegalovirus; ITR = inverted terminal repeat; dATP = deoxy-adenosine triphosphate; p2A = porcine teschovirus-1 2A; pA = poly-adenylation; qPCR = quantitative polymerase chain reaction; rAAV6 = recombinant adeno-associated viral vector 6; RC = regulatory cassette; WT = wild-type.
Pressure-Volume Relationships in Isolated Perfused Hearts From Vector-Treated
mdx Mice Hearts were isolated from 4cv mdx mice treated with control vector ( 4cv mdx , n = 6), ribonucleotide reductase ( 4cv mdx + RNR, n = 6), or microdystrophin ( 4cv mdx 4cv + μDys, n = 5). Age-matched, nondiseased, nontreated wild-type (WT) mice were used as control subjects (n = 8). The pressure-volume relationship (i.e., Frank-Starling mechanism) was evaluated by gradually increasing the volume of the left ventricular (LV) balloon. Hearts were paced at 450 beats/min throughout the protocol. (A) Left ventricular systolic pressure (LVSP). (B) LV end-diastolic pressure (LVEDP). (C) LV developed pressure (LVDevP, the difference between systolic and diastolic pressures). (D) Positive (pos) rate of pressure change was calculated by the first derivative of the ascending LV pressure wave (+dP/dt), and is used as an index of ventricular contractility. (E) Negative (neg) rate of pressure change calculated by the first derivative of the descending LV pressure wave (–dP/dt), and is used as an index of ventricular relaxation. * p < 0.05 mdx versus WT; # p < 0.05 4cv mdx + RNR versus 4cv mdx ; † p < 0.05 4cv mdx + μDys versus 4cv mdx . 4cv
The Response of Vector-Treated
mdx Mice to High Workload Challenge in Langendorff Isolated Heart Preparations Hearts were isolated from 4cv mdx mice treated with control vector ( 4cv mdx , n = 6), ribonucleotide reductase ( 4cv mdx + RNR, n = 6), or microdystrophin ( 4cv mdx + μDys, n = 5). Age-matched, nondiseased, nontreated wild-type (WT) mice were used as control subjects (n = 4). All hearts were perfused with a glucose-pyruvate buffer containing high calcium (4.0 mmol/l) to simulate a high workload challenge for 20 min. Hearts were paced at 450 beats per minute throughout the protocol. 4cv (A) LVDevP (the difference between systolic and diastolic pressures). (B) Rate pressure product (RPP, the product of LVDevP and HR). (C) Positive rate of pressure change calculated by the first derivative of the ascending LV pressure wave (+dP/dt), is used as an index of ventricular contractility. (D) Negative rate of pressure change calculated by the first derivative of the descending LV pressure wave (–dP/dt), is used as an index of ventricular relaxation. * p < 0.05 mdx versus WT; # p < 0.05 4cv mdx + RNR versus 4cv mdx ; † p < 0.05 4cv mdx + μDys versus 4cv mdx . Abbreviations as in Figures 1 and 2. 4cv
Cardiac Transduction Following Intravenous Delivery of Ribonucleotide Reductase or Microdystrophin At 5 months after vector administration, cryosections were prepared and immunostained with antisera against dystrophin or ribonucleotide reductase. A considerable level of protein is detected for each ribonucleotide reductase subunit-1 (human specific) as indicated by immunofluorescent staining
(red) localized primarily within the cytoplasm of cardiomyocytes with occasional perinuclear accumulation (upper panel). On the lower panel, the robust level of expression for dystrophin in WT and in aged mdx mice treated with AAV6-CK8-μDys (laminin staining, 4cv inset image). Abbreviations as in Figures 1 and 2.
Heart Histologic Staining of
mdx Mice Suggests No Morphologic Alterations From RNR Therapy Representative full-view photomicrographs of Masson trichrome staining of the hearts from mdx 4cv 4cv mice displaying control vector (ΔCMV), and rAAV6-treated with either RNR or μDys from mdx4cv mice. Similarly, a 20× enlarged view of the corresponding images (*) is shown. Abbreviations as in Figures 1 and 2.
Protein Expression Levels for μDys, RNR, and Resultant Vector Genomes Due to rAAV6-Mediated Gene Transfer
(A) RNR and μDys protein expression detection as revealed by immunoblotting of cardiac whole tissue lysates using either RRM1, RRM2, or antidystrophin antibody. (B) HPLC-MS/MS intracellular [dNTP] quantification from methanol extracted cardiac tissue. (C) qPCR analysis of vector genomes from cardiac tissue revealed similar vector genomes being represented for all treated cohorts. dNTP = deoxynucleotide triphosphate; HPLC-MS/MS = high-performance liquid chromatography–tandem mass spectrometry; qPCR = quantitative polymerase chain reaction; other abbreviations as in Figures 1 and 2.
All figures (7)
Microdystrophin gene therapy of cardiomyopathy restores dystrophin-glycoprotein complex and improves sarcolemma integrity in the mdx mouse heart.
Circulation. 2003 Sep 30;108(13):1626-32. doi: 10.1161/01.CIR.0000089371.11664.27. Epub 2003 Sep 2.
12952841 Free PMC article.
Adeno-associated virus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice.
Version 2. Hum Gene Ther. 2008 Aug;19(8):851-6. doi: 10.1089/hum.2008.058.
Hum Gene Ther. 2008.
18666839 Free PMC article.
The AAV-mediated and RNA-guided CRISPR/Cas9 system for gene therapy of DMD and BMD.
Brain Dev. 2017 Aug;39(7):547-556. doi: 10.1016/j.braindev.2017.03.024. Epub 2017 Apr 5.
Brain Dev. 2017.
Emery A.E., Muntoni F. 3rd ed. Oxford University Press; Oxford, United Kingdom: 2003. Duchenne Muscular Dystrophy; p. 270.
Ervasti J.M. Structure and function of the dystrophin-glycoprotein complex. In: Winder S.J., editor. Molecular Mechanisms of Muscular Dystrophies. Landes Biosciences; Georgetown, Texas: 2006. pp. 1–13.
Cox G.A., Phelps S.F., Chapman V.M., Chamberlain J.S. New mdx mutation disrupts expression of muscle and nonmuscle isoforms of dystrophin. Nature Genet. 1993;4:87–93.
Brooks S.V., Faulkner J.A. Contractile properties of skeletal muscles from young, adult and aged mice. J Physiol (London) 1988;404:71–82.
Campbell K.P. Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage. Cell. 1995;80:675–679.