Voltage Sensitive Protein 2.3: A Novel Tool to Study Sarcolemmal Structure and Electrical Activity in Mouse Hearts

Optical imaging of cardiac electrical activity is of considerable interest, especially in cardiac pathophysiology and pharmacology studies. Voltage sensitive fluorescent dyes are widely used in this context, but have a limited applicability in long-term studies. We hypothesized that novel genetically encoded voltage sensitive fluorescent proteins (VSFPs) can be stably expressed in mouse hearts to (1) specifically label sarcolemmal membranes and (2) monitor membrane voltage transients. Methods: cDNA encoding for the VSFP2.3 voltage probe (see W. Akemann et al., 2010 Nat Methods) was placed under the control of the cardiomyocyte-specific alpha myosin heavy chain (αMHC) promoter. Transgenic mice were generated by pronuclear injection. Myocardial structure and performance were assessed by echocardiography (in 16-week old mice). Myocyte- and sarcolemma-restricted transgene activity was studied in isolated cardiomyocytes using confocal laser scanning microscopy and FRET-imaging. Results: We established 4 independent αMHC-VSFP2.3 mouse lines (TG#97, #107, #108, #123). Heart morphology and function did not differ in transgenic and wildtype mice with an average heart-to-body weight ratio of 4.3±0.5 (n=26) and 4.2±0.2 (n=8), respectively. Fluorescent imaging of intact hearts showed homogeneous pattern of CFP and YFP expression throughout the ventricles. Fluorescence intensity varied between the established lines (#123>#97>#108>#107). On a single cell basis, prominent sarcolemmal targeting was observed. Interestingly, the T-tubular system was clearly labeled with a predicted periodicity of 1.88±0.02 and 1.82±0.02 µm (from 7/11 cells) in wildtype and transgenic mice, respectively. Voltage transients could be readily detected using optical imaging at the level of intact hearts and isolated myocytes. Conclusion: We have established the first mouse model with cardiac-restricted VSFP-expression. αMHC-VSFP2.3 mice demonstrated unimpaired myocardial structure and function. We anticipate that myocyte-specific VSFP-based voltage imaging will facilitate studies of cardiomyocyte and whole-heart functionality under minimally invasive conditions.