Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers

Tropomodulins, cytoplasmic γ-actin, and small ankyrin 1.5 mechanically stabilize the sarcoplasmic reticulum and maintain myofibril alignment in skeletal muscle fibers

Anchoring skeletal muscle development and disease: the role of ankyrin repeat domain containing proteins in muscle physiology

The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies

Prevention of Protease-Induced Degradation of Desmoplakin via Small Molecule Binding

Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, the subcellular structure that links the intermediate filament network of one cell to its neighbor. A mutation “hot-spot” within the NH2-terminal of the DSP protein (residues 299–515) is associated with arrhythmogenic cardiomyopathy. In a subset of DSP variants, disease is linked to calpain hypersensitivity. Previous studies show that calpain hypersensitivity can be corrected in vitro through the addition of a bulky residue neighboring the cleavage site, suggesting that physically blocking calpain accessibility is a viable strategy to restore DSP levels. Here, we aim to find drug-like molecules that also block calpain-dependent degradation of DSP. To do this, we screened ~2500 small molecules to identify compounds that specifically rescue DSP protein levels in the presence of proteases. We find that several molecules, including sodium dodecyl sulfate, palmitoylethanolamide, GW0742, salirasib, eprosarten mesylate, and GSK1838705A prevent wildtype and disease-variant-carrying DSP protein degradation in the presence of both trypsin and calpain without altering protease function. Computational screenings did not predict which molecules would protect DSP, likely due to a lack of specific DSP–drug interactions. Molecular dynamic simulations of DSP–drug complexes suggest that some long hydrophobic molecules can bind in a shallow hydrophobic groove that runs alongside the protease cleavage site. Identification of these compounds lays the groundwork for pharmacological treatment for individuals harboring these hypersensitive DSP variants

Creating a ‘Molecular Band-Aid’; Blocking an Exposed Protease Target Site in Desmoplakin

Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, a subcellular complex that links the cytoskeleton of one cell to its neighbor. A mutation ‘hot-spot’ within the NH2-terminal third of the DSP protein (specifically, residues 299–515) is associated with both cardiomyopathies and skin defects. In select DSP variants, disease is linked specifically to the uncovering of a previously-occluded calpain target site (residues 447–451). Here, we partially stabilize these calpain-sensitive DSP clinical variants through the addition of a secondary single point mutation—tyrosine for leucine at amino acid position 518 (L518Y). Molecular dynamic (MD) simulations and enzymatic assays reveal that this stabilizing mutation partially blocks access to the calpain target site, resulting in restored DSP protein levels. This ‘molecular band-aid’ provides a novel way to maintain DSP protein levels, which may lead to new strategies for treating this subset of DSP-related disorders

Humanized Dsp ACM Mouse Model Displays Stress-Induced Cardiac Electrical and Structural Phenotypes

Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder characterized by fibro-fatty infiltration with an increased propensity for ventricular arrhythmias and sudden death. Genetic variants in desmosomal genes are associated with ACM. Incomplete penetrance is a common feature in ACM families, complicating the understanding of how external stressors contribute towards disease development. To analyze the dual role of genetics and external stressors on ACM progression, we developed one of the first mouse models of ACM that recapitulates a human variant by introducing the murine equivalent of the human R451G variant into endogenous desmoplakin (DspR451G/+). Mice homozygous for this variant displayed embryonic lethality. While DspR451G/+ mice were viable with reduced expression of DSP, no presentable arrhythmogenic or structural phenotypes were identified at baseline. However, increased afterload resulted in reduced cardiac performance, increased chamber dilation, and accelerated progression to heart failure. In addition, following catecholaminergic challenge, DspR451G/+ mice displayed frequent and prolonged arrhythmic events. Finally, aberrant localization of connexin-43 was noted in the DspR451G/+ mice at baseline, becoming more apparent following cardiac stress via pressure overload. In summary, cardiovascular stress is a key trigger for unmasking both electrical and structural phenotypes in one of the first humanized ACM mouse models

Altered Expression of Zonula occludens-1 Affects Cardiac Na⁺ Channels and Increases Susceptibility to Ventricular Arrhythmias

Zonula occludens-1 (ZO-1) is an intracellular scaffolding protein that orchestrates the anchoring of membrane proteins to the cytoskeleton in epithelial and specialized tissue including the heart. There is clear evidence to support the central role of intracellular auxiliary proteins in arrhythmogenesis and previous studies have found altered ZO-1 expression associated with atrioventricular conduction abnormalities. Here, using human cardiac tissues, we identified all three isoforms of ZO-1, canonical (Transcript Variant 1, TV1), CRA_e (Transcript Variant 4, TV4), and an additionally expressed (Transcript Variant 3, TV3) in non-failing myocardium. To investigate the role of ZO-1 on ventricular arrhythmogenesis, we generated a haploinsufficient ZO-1 mouse model (ZO-1+/−). ZO-1+/− mice exhibited dysregulated connexin-43 protein expression and localization at the intercalated disc. While ZO-1+/− mice did not display abnormal cardiac function at baseline, adrenergic challenge resulted in rhythm abnormalities, including premature ventricular contractions and bigeminy. At baseline, ventricular myocytes from the ZO-1+/− mice displayed prolonged action potential duration and spontaneous depolarizations, with ZO-1+/− cells displaying frequent unsolicited (non-paced) diastolic depolarizations leading to spontaneous activity with multiple early afterdepolarizations (EADs). Mechanistically, ZO-1 deficient myocytes displayed a reduction in sodium current density (INa) and an increased sensitivity to isoproterenol stimulation. Further, ZO-1 deficient myocytes displayed remodeling in ICa current, likely a compensatory change. Taken together, our data suggest that ZO-1 deficiency results in myocardial substrate susceptible to triggered arrhythmias