Matriglycan maintains t-tubule structural integrity in cardiac muscle

Significance Cardiac muscle has a unique membrane organelle called transverse tubule (t-tubule) system which is an invagination of the surface membrane. Preserving the structural integrity of the t-tubule system is necessary for cardiac muscle contraction and, thus, overall heart function. Here, we report that O-glycosylation of dystroglycan (DG) and the formation of matriglycan are essential for maintaining the structural integrity of the cardiac t-tubule system. Importantly, our findings unveil the physiological significance of O-glycosylated DG within cardiac muscle t-tubules, thereby providing evidence of an extracellular matrix-interacting, transmembrane protein that plays a vital role in preserving the structural integrity of t-tubule membranes. Collectively, our findings provide essential advances in elucidating the contribution of cardiac t-tubule disruption in heart disease.


Fig. S1 .
Fig. S1.The presence of basement membrane within the t-tubule lumen is specific to cardiac muscle fibers.A-B Fluorescent labeling of myofiber lipid membranes with FM464-FX and basement membranes with WGA-AlexaFluor488 on cardiac ventricular myofibers A, and extensor digitorum longs (EDL) skeletal muscle fibers B, from control mice.120x magnification; Scale bar = 20 μm.Illustrations depict the differences between t-tubules in cardiac muscle and skeletal muscle.The lumen of cardiac muscle t-tubules contains extracellular basement membrane and generally has a greater width compared to that of skeletal muscle t-tubules.

Fig. S6 .
Fig. S6.Catecholamine-induced stress promotes progressive cardiomyopathy in Pomt1 cKO hearts.A-B Control and Pomt1 cKO mice were injected with a single bolus of ISO (10 mg/kg body weight).A, Kaplan-Meier survival curve of controls and Pomt1 cKO mice.Experiments were performed with mice of both sexes.Controls, n = 11; Pomt1 cKO, n = 17.B, Histological analysis of cardiac tissues 28 days after injection.Cardiac sections were stained with Picrosirius red and Fast Green.Scale = 100 μm.Quantification of ventricular fibrosis of tissue stained with Picrosirius red.Experiments were performed with mice of both sexes.Controls, n = 5; Pomt1 cKO, n = 6.Statistical analyses were performed with unpaired t-test with Holm-Sidak post-hoc.Data expressed

Fig. S7 .
Fig. S7.Daily, low dose β-adrenergic challenge leads to death and disrupts the ventricular morphology in Pomt1 cKO mice.A, Schematic of experimental design.Control and Pomt1 cKO mice received repeated i.p. injections of ISO (2.5 mg/kg body weight).B, Kaplan-Meier survival curve of control and Pomt1 cKO mice treated as in A. (Controls, n = 8; Pomt1 cKO, n = 10).C, Heart weight normalized to body weight (left panel) and heart weight normalized to tibial length (right panel) of mice in B. **, p = 0.0037, as determined by unpaired two-tailed t-test.D, Immunofluorescence on ventricular cross-sections from mice in B. WGA-488 (green) was used as a marker of the extracellular matrix and dystrophin (purple) was used as a myocyte membrane marker.Scale bar = 1 mm.Graph displays quantification of ventricular lesions.Experiments were

Fig. S8 .
Fig. S8.Presence of matriglycan in human cardiac muscle t-tubule membranes.Immunofluorescence on frozen human heart cryosections to detect the presence of matriglycan and extracellular matrix laminin in cardiac muscle fibers.Top row shows matriglycan and laminin immunofluorescence in longitudinally oriented cardiac muscle fibers.Scale bar = 5 μm.Middle and bottom rows show matriglycan and laminin immunofluorescence in transverse oriented cardiac muscle fibers.Scale bars = 10 μm.Results were collected from two human heart samples.

Fig. S11 .
Fig. S11.Lipophilic dye, FM464-FX, infiltrates myocytes that suffered catastrophic sarcolemma damage.A, Low magnification (10x) examination identified regions of fibers that displayed myocyte uptake of FM464-FX, while many of the cardiac fibers were spared of catastrophic membrane damage in stressed Pomt1 cKO hearts.Scale bar = 300 μm.B, Variations in myocyte damage are shown.Left panel displays cardiac fibers that did not accumulate intramyocyte FM464-FX but were susceptible to t-tubule disruption.Right panel shows cardiac fibers on the border of an ischemic region.Myocytes that were infiltrated with FM464-FX dye are intensely red, whereas fibers that avoided catastrophic membrane damage did not show myocyte infiltration of the dye.

Fig. S14 .
Fig. S14.Cardiac muscle matriglycan is essential to prevent stress-induced t-tubule damage.A, Labeling of control and Large1 cKO whole left ventricles stained with FM 464-FX under baseline conditions.Scale bar = 20 µm.B, Percentage of myofibers that showed either normal or disrupted patterns of t-tubules.C, Line scan analysis to determine the peak signal intensity of FM 464-FX labeled t-tubules (TT).D, Number of t-tubules observed within a 16 µm regions.Image analysis was performed on hearts of mice from both sexes.Unpaired t-test with Holm-Sidak post-hoc were performed.Data expressed as mean ± standard deviation.E, Labeling of control and Large1 cKO whole left ventricle stained with FM 464-FX 24-h after ISO challenge.F-H, Quantification as performed in B-C.Image analysis was performed on hearts from both sexes.Unpaired t-test with Holm-Sidak post-hoc were performed.Data expressed as mean ± standard deviation.**** p = 0.0001.