Micron-scale voltage and [Ca(2+)]i imaging in the intact heart

Studies in isolated cardiomyocytes have provided tremendous information at the cellular and molecular level concerning regulation of transmembrane voltage (Vm) and intracellular calcium ([Ca(2+)]i). The ability to use the information gleaned to gain insight into the function of ion channels and Ca(2+) handling proteins in a more complex system, e.g., the intact heart, has remained a challenge. We have developed laser scanning fluorescence microscopy-based approaches to monitor, at the sub-cellular to multi-cellular level in the immobilized, Langendorff-perfused mouse heart, dynamic changes in [Ca(2+)]i and Vm. This article will review the use of single- or dual-photon laser scanning microscopy [Ca(2+)]i imaging in conjunction with transgenic reporter technology to (a) interrogate the extent to which transplanted, donor-derived myocytes or cardiac stem cell-derived de novo myocytes are capable of forming a functional syncytium with the pre-existing myocardium, using entrainment of [Ca(2+)]i transients by the electrical activity of the recipient heart as a surrogate for electrical coupling, and (b) characterize the Ca(2+) handling phenotypes of cellular implants. Further, we will review the ability of laser scanning fluorescence microscopy in conjunction with a fast-response voltage-sensitive to resolve, on a subcellular level in Langendorff-perfused mouse hearts, Vm dynamics that typically occur during the course of a cardiac action potential. Specifically, the utility of this technique to measure microscopic-scale voltage gradients in the normal and diseased heart is discussed

Efficacy of Gene Therapy Is Dependent on Disease Progression in Dystrophic Mice with Mutations in the FKRP Gene

Loss-of-function mutations in the Fukutin-related protein ( ) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) and other forms of congenital muscular dystrophy-dystroglycanopathy that are associated with glycosylation defects in the α-dystroglycan (α-DG) protein. Systemic administration of a single dose of recombinant adeno-associated virus serotype 9 (AAV9) vector expressing human to a mouse model of LGMD2I at various stages of disease progression was evaluated. The results demonstrate rescue of functional glycosylation of α-DG and muscle function, along with improvements in muscle structure at all disease stages versus age-matched untreated cohorts. Nevertheless, mice treated in the latter stages of disease progression revealed a decrease in beneficial effects of the treatment. The results provide a proof of concept for future clinical trials in patients with -related muscular dystrophy and demonstrate that AAV-mediated gene therapy can potentially benefit patients at all stages of disease progression, but earlier intervention would be highly preferred