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Highlights • GsMTx4-D is a mechanosensitive ion channel inhibitor. • GsMTx4-D has a one-week half-life. • Six-week treatment of D2.mdx dystrophic mice: ○ reduced muscle loss. ○ decrease susceptibility to contraction induced injury. Abstract Duchenne muscular dystrophy is a life-limiting muscle disease that has no current effective therapy. Despite mounting evidence that dysregulation of mechanosensitive ion channels is a significant contributor to dystrophy pathogenesis, effective pharmacologic strategies targeting these channels are lacking. GsMTx4, and its enantiomer GsMTx4-D, are peptide inhibitors of mechanosensitive channels with identical activity. In previous studies, acute in vitro application of GsMTx4 to dystrophic murine muscle effectively reduced the excess MSC dependent calcium influx linked to contraction-induced muscle damage. Here we sought to determine if in vivo treatment with GsMTx4-D proffered benefit in the D2.mdx mouse. GsMTx4-D showed a 1-week half-life when administered by subcutaneous injection over four weeks. Informed by these results, D2. mdx mice were then treated by a subcutaneous injection regimen of GsMTx4-D for six weeks followed by determination of muscle mass, muscle susceptibility to eccentric contraction injury and multiple histological indicators of disease progression. The mice showed a reduction in the loss of muscle mass and a decrease in susceptibility to contraction induced injury. These protective effects were realized without reduction in fibrosis, supporting a model where GsMTx4-D acts directly on muscle cells. We propose GsMTx4-D represents a promising new therapy to slow disease progression and may complement other therapies such as anti-inflammatory agents and gene-replacement strategies. [ABSTRACT FROM AUTHOR]

GsMTx4 is a selective inhibitor of cationic mechanosensitive ion channels (MSCs) and has helped establish the role of MSCs in cardiac physiology. Inhomogeneous local mechanical stresses due to hypercontracture and swelling during ischemic reperfusion injury (IRI) likely induce elevated MSC activity that can contribute to cation imbalance. The aim of this study was to determine if the D enantiomer of GsMTx4 can act as a cardioprotectant in a mouse IRI model. Ischemia and reperfusion involved ligating a coronary artery followed by release of the ligature. GsMTx4-D was tested by either acute intravenous injection during the ischemic event or by two day pretreatment by intraperitoneal injection, both methods achieving similar results. Based on pharmacokinetic studies, GsMTx4-D dosage was set to achieve expected plasma concentrations between 50 and 5000 nM and heart tissue concentrations between 1 and 200 nM by intravenous injection. Relative to vehicle injected animals, GsMTx4-D reduced infarct area by ~ 40% for acute and pretreated animals for both 20 and 45 min ischemic challenges. Many indicators of cardiac output were indistinguishable from sham-treated control hearts after GsMTx4-D treatment showing improvement at both 4 and 48 h post ischemia, and premature ventricular beats immediately following reperfusion were also significantly reduced. To determine if GsMTx4-D cardioprotection could act directly at the level of cardiomyocytes, we tested its effects in vitro on indicators of IRI damage like cation influx and activation of inflammatory kinases in isolated myocytes cultured under hypoxic conditions. Hypoxia challenged cardiomyocytes treated with 10 μM GsMTx4-D showed improved contractility and near normal contraction-related Ca 2 + influx. GsMTx4-D inhibited indicators of ischemic damage such as the apoptotic signaling system JNK/c-Jun, but also inhibited the energy response signaling system Akt kinase. We conclude that GsMTx4-D is a potent cardioprotectant in vivo that may act directly on cardiomyocytes and potentially be useful in multidrug strategies to treat IRI. [ABSTRACT FROM AUTHOR]