Fig 2: Inhibition of calpain activity attenuated the adverse effects of pacing. Flow cytometry analysis of cardiac troponin T (cTnT) and MLC2v (a) demonstrated that the pharmaceutical inhibition of calpain activation significantly increased the cTnT+ and MLC2v+ cells ratio compared with that in the paced VCMs (b). Patch-clamp studies revealed that ICa, L density was significantly increased after the calpeptin treatment (c). In the calpeptin (5 µM)-treated group, the protein levels of cTnT (d) and L-type calcium channel (g) were increased by western blot analysis. The expression of apoptosis proteins (caspase-3, e; Bax/Bcl-2, f) that are involved in ER stress decreased markedly upon the calpeptin treatment compared with 100% Pace group. * p < 0.05, ** p < 0.005, *** p < 0.001 according to a one-way ANOVA test

Fig 3: Long-term pacing disrupted the contractile structure of cardiomyocytes. a Compared with age-matched controls, the paced VCMs demonstrated decreased expression of cTnT, while cells from both the pacing and control groups showed robustly expressed Cx43 (scale bar 50 µm). TEM investigation illustrated significantly increased intracellular empty vacuoles and decreased myofibril density in paced VCMs (EV empty vacuoles, MF myofibrils, MI mitochondria, scale bar 500 nm, Myofibril panel, b); pacing also significantly increased the swelling mitochondria percentage, c; endoplasmic reticula (ER) with narrow cisterns were observed in the controls. By contrast, the paced VCMs demonstrated significantly increased aberrant dilated electron-dense ERs, which were swollen into circular shapes (*endoplasmic reticulum, scale bar 500 nm, ER panel, b?); qRT-PCR investigation revealed that the expression of TNNT2 was significantly lower in the pacing groups compared with the age-matched controls. However, the expression of Cx43 demonstrated a similar level among 40% Pace, 100% Pace and control VCMs (d). ** p < 0.005, *** p < 0.001 according to a one-way ANOVA test

Fig 4: Effects of chronic in vivo DAAO activation on ex vivo cardiac responses and signaling. a Representative traces of tension generated by paced papillary muscles isolated from control animals or animals expressing DAAO and treated with oral D-alanine for 4 weeks, with and without isoproterenol stimulation (100 nM). b, c Isometric contractile force and d, e relaxation times for papillary muscles at baseline and after treatment with the beta-adrenergic agonist isoproterenol (100 nM). Isoproterenol contractile force is represented as the fold change from the baseline developed tension in the absence of agonist. *p < 0.05 and **p < 0.01 by t-test. f Immunoblots of cardiac lysates from Ctrl and DAAO-infected animals and probed for phospho-phospholamban (PLN) at phospho-serine 16 and phospho-threonine 17, and for total cardiac troponin T (cTnT). g Densitometry of immunoblots shown in f. **p < 0.01 by ANOVA. Immunoblotting of the same lysates for YFP, a component of HyPer, did reveal some heterogeneity in efficiency of expression from animal to animal, but did not correlate with signaling or physiologic responses (Supplementary Figures 1A and 1B). h Relative changes in expression of alpha MHC (Myh6) and beta MHC (Myh7) in hearts from DAAO (red squares) vs. Ctrl (blue circles) infected animals. i Relative changes in expression of atrial natriuretic peptide (Nppa) in hearts of control (Ctrl) and DAAO-expressing animals treated with oral D-alanine for 4 weeks. ***p < 0.001 by t-test. j Relative expression of B-type natriuretic peptide (Nppb) in hearts from Ctrl and DAAO animals. *p < 0.05 by t-test. Direct measurement of ANP and BNP protein levels by ELISA in cardiac lysates from control (Ctrl) and DAAO-expressing animals. *p < 0.05 by t-test. Data are represented as mean ± standard error