Beneficial effect of voluntary physical exercise in Plakophilin2 transgenic mice

Fig 1. Echocardiographic evaluation of PKP2 mice during 16 weeks of physical activity. Exemplary echocardiographic original traces from mice recorded pre run/sed (A), after 8 weeks of running (B) and after 16 weeks of running (C). There was no difference between the genotypes at baseline in the functional characteristics ejection fraction (EF) and fractional shortening (FS) (D). Heterozygous PKP2 mice (he) showed a reduction in EF after 8 weeks of running, which was normalized after 16 weeks (E). Heterozygous animals also showed a reduction in FS after 8 weeks of running which normalized again at 16 weeks (F).

Fig 2. Electrophysiological in vivo study of mice after long-term training. (A) The electrophysiological study revealed that long-term training in mice did not cause an increased vulnerability in wt mice under basal conditions (18% in sedentary animals, n = 11 vs. 29% after 8 weeks, n = 7 and 25% after 16 weeks of running, n = 4). An additional ISO challenge did cause a slightly increased occurrence of arrhythmia in sedentary mice from 18% at basal conditions to 27% under ISO, which was increased to 57% under ISO after 8 weeks of training. This vulnerability was reduced after 16 weeks of training to 50%. The heterozygous animals showed increased occurrence of arrhythmia under basal conditions in sedentary mice of 50% (n = 10), which even increased after 8 weeks of training (80%; n = 5), but was reduced to only 17% after 16 weeks of training (n = 6). Interestingly the additional ISO challenge did not cause an increase of arrhythmia in heterozygous animals at rest or after 16 weeks of training. After 8 weeks of training the rate of arrhythmia observed dropped slightly. B Protein expression levels of β1-adrenergic receptor in cardiac tissue of wt mice was unaffected by voluntary running…

Fig 3. Cellular Ca cycling and contractions after long-term training. Fura-2 loaded cardiac myocytes were electrically stimulated and CaT measured. The CaT amplitude significantly increased after 16 weeks of training in both genotypes with p<0.0001 (A). While the CaT rise time was significantly faster in cells from wt mice after 16 weeks training, this trend was already significant in cells from the PKP2+/- animals after 8 weeks (B). However, the decay time to reach 50% of the maximal amplitude did change significantly in wt mice during 8 weeks of training (p = 0.04), while the PKP2+/- mice showed a significant (p = 0.001) acceleration after 8 weeks of running that was abolished again after 16 weeks (C). Diastolic Ca values did not change in PKP2+/- mice during the course of training after 8 and 16 weeks, but the wt had a distinct increase in diastolic Ca values after 16 weeks of training (p = 0.004) (D). Conversely, the SR Ca load measured by caffeine application, was unchanged in PKP2+/- mice and in wt animals (E). In parallel to the fluorescent measurement of cytosolic Ca, we recorded the contractility by measuring the sarcomere length with a bright field video camera system. The…

Fig 4. Histological analysis of cardiac tissue after long-term training. (A) Representative images for sedentary, 8 weeks of running and 16 weeks of running tissue from wt (upper row) and PKP2+/- mice (lower row) are shown for Trichrome-stained ventricular slices. No change of fibrous tissue could be detected in either genotype or in response to long-term training. (B) The oil stain in frozen sections from cardiac tissue with oil red did not reveal an increase in fatty replacements. (C) We labeled the gap junctions with a Cx43 specific antibody and analyzed the images by confocal microscopy. The connexins were predominantly located at the intercalated discs as seen in the representative images shown. There was no shift in subcellular distribution due to genotype or long-term training observed. The single data points indicate the number of animals analyzed.

Fig 5. Protein expression levels of ECC key players. We analyzed the protein expression levels by Western Blot to reveal the potential changes underlying the functional alterations we have found in cells from wt and PKP2+/- mice with and without training. First, we analyzed the RYR2 protein levels and found no changes neither between the genotypes nor due to training (A). In addition, the phosphorylation of the RyR by CaMKII at S2814 is not affected (B) but the PKA mediated phosphorylation at S2030 is higher in PKP2+/- animals but unaffected by training (C), indicating that the Ca release from the store is unaffected by physical activity. The protein level of the Na-Ca-exchanger NCX is also unchanged and thus most likely does not play a significant role in shifted Ca removal processes (D). In accordance with the SR-Ca load data, we found a slight shift towards higher expression of SERCA protein in wt animals after 16 weeks of training, albeit not significant (E). The overall PLB protein level as well as the PLB-phosphorylation was unchanged in wt animals (F-H), whereas the PLB protein level was significantly reduced in PKP2+/- animals after 16 weeks (p = 0.001) of training. In addition…