Reviewer 2's Review

“The additional experiments (listed below) are meant to be suggestions for future follow-up studies and are not required as essential control experiments.

The manuscript by Asai et al, investigates the role of functional ischemia in a mouse model of Duchenne Muscular Dystrophy (´mdx mice`). The authors conclude that functional ischemia plays a primary role in the disease process and is part of a two-hit mechanism that ultimately leads to muscle fiber damage. This conclusion is based on in vivo imaging experiments which provide direct evidence that (i) mdx mice fail to increase RBC flux after tetanic stimulation, (ii) failure to upregulate blood flow is not due to defective blood vessel but rather to deficient release/production of endogenous vasodilatory signals like NO and H202 and (iii) exogenous application of vasodilatory drugs like SNAP, 8-CPT cGMP and clenbuterol can prevent muscle fiber damage induced by tetanic stimulation in mdx mice. Follow-up experiments using pharmacological induction of ischemia in wild-type mice suggest that functional ischemia alone however is not sufficient to cause contraction-induced myofiber damage. This is in line with the two-hit mechanisms of muscle fiber damage suggested by the authors. As a last experiment the authors then present evidence that therapy with the vasodilatory drug (tadalafil) can ameliorate the course of muscular dystrophy in mdx mice.

Overall the study appears to be well performed, the experiments are carefully controlled and the manuscript is suitably written and illustrated. While some conclusions of this work have been proposed in the past, this is the first study that provides direct insight into the mechanisms that underlie muscle fiber damage in muscular dystrophy in vivo.”

N.B. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.