**6. Experimental Therapeutic Strategies to Improve Calcium Handling and Decrease Calcium Overload in DCM and DMD Cardiomyopathy**

Although the mechanisms of cardiac dysfunction in DMD are complex and multifactorial, impaired calcium handling and calcium overload are key contributors to both early and late stage pathogenesis, as previously described. Currently utilized clinical therapies for DMD cardiomyopathy do not directly target calcium handling defects. Therefore, development of novel therapeutic strategies should focus on targeting various aspects of these pathways. Upstream targets include repair of the damaged sarcolemma and restoration of hyperactive stretch-activated channel (SAC) activity and RyR2 function. These targets will mitigate calcium overload from both the extracellular space and intracellular stores. Downstream targets include normalization of Serca2a/PLN activity and calcium cycling. These targets will mitigate the contraction and relaxation deficits characteristic of the dilated phenotype in late stage DMD cardiomyopathy. A summary of studies that specifically target various aspects of calcium mishandling and overload in cardiac tissue of muscular dystrophy models is shown in Table 2.


**Table 2.** Summary of research investigating experimental therapeutic strategies for calcium mishandling and overload in muscular dystrophy cardiomyopathy.


**Table 2.** *Cont*.

KO: knockout; cTnI: cardiac troponin I; LV: left ventricle; RyR2: ryanodine receptor 2; AAV: adeno-associated virus; ECG: electrocardiogram; PLN: phospholamban; EBD: Evan's Blue Dye.

#### *6.1. Membrane Stabilization*

Membrane stabilization has been extensively studied as a therapeutic strategy for DMD cardiomyopathy in both small and large animal models [41,68] to mitigate sarcolemmal damage and calcium overload occurring from mechanical stress in the absence of dystrophin. Triblock copolymers, of which Poloxamer 188 (P188) has been most widely studied, consist of a hydrophobic polypropylene oxide (PPO) core, flanked by two hydrophilic polyethylene oxide (PEO) chains [63]. P188 is thought to insert into damaged membranes to provide stability until intrinsic repair mechanisms are able to restore integrity of the membrane [63]. P188 treatment of isolated dystrophic cardiac myocytes improved membrane compliance and decreased calcium influx and hypercontracture during passive physiological stretch [41]. *Ex vivo mdx* heart function was also improved after ischemia reperfusion injury with P188 treatment [159]. Finally, P188-treated *mdx* mice undergoing chronic isoproterenol stress had some improvements in *in vivo* cardiac function after two and four weeks [158], and a large animal model of DMD showed significant improvement in *in vivo* cardiac function after chronic P188 therapy [68]. The reader is referred to excellent recent reviews for more details on this topic [63,166].

Repairing damaged membranes using muscle-specific TRIM (tripartite motif) protein mitsugumin 53 (MG53) provides an additional avenue for targeting membrane instability in DMD. Recombinant MG53 administration to *mdx* mice improved skeletal muscle pathology and decreased damage after downhill treadmill running [167].
