Y of pressure overload between the two studies, judging from the hypertrophy data. Increasing the pressure overload intensity in our model may result in a significant decrease in mtDNA copy number in TAC. In the present study, we used a 26-gauge needle to induce pressure overload, which produced stable hypertrophy but rather mild effect on heart failure. On the other hand, using a 27- or 28-gauge needle as in previous study [13] Title Loaded From File resulted in higher surgical mortality but produces greater pressure overload in our preliminary experiments. We found that increasing mtDNA copy by Twinkle overexpression did not affect mitochondrial enzyme activity, which is consistent with a previous report [16]. Furthermore, TAC also did not affect mitochondrial enzyme activity (Figure 1B and C). These results suggest that mitochondrial electron transport complex activity is not directly related to the cardioprotective effect of Twinkle overexpression. The mechanism by which increased Twinkle expression prevents heart failure under pressure overload condition remains unknown. In this study we showed that Twinkle overexpression prevented Title Loaded From File cardiac fibrosis in vivo and in vitro (to be discussed in detail below). We therefore speculate that Twinkle overexpression somehow inhibits cardiac profibrogenic signals. We need to conduct further investigation about the mechanism.Figure 5. Effects of the upregulation or donwregulation of Twinkle on TGF-b1 mRNA expression. TGF-b1 expression in cardiac fibroblasts stimulated with AngII (1 mM) for 24 hours, quantified by realtime PCR relative to housekeeping gene (18S gene). Cells were preinfected with AxCAhTwinkle (Twinkle), AxCAsi-rTwinkle (siTwinkle) or AxCALacZ (LacZ). Values are mean 6 SEM. Data are presented as ratio to LacZ-vehicle. **; P,0.01 vs LacZ-vehicle, {; P,0.05 vs LacZAngII. doi:10.1371/journal.pone.0067642.gA major limitation of the present study is that we cannot elucidate the mechanism of how increased mtDNA reduces fibrosis in the pressure overload model. We should conduct further investigations to reveal the molecular mechanisms of how Twinkle overexpression or increased mtDNA decreases fibrosis or fibrosisrelated signaling.Clinical ImplicationWe speculate that increased mtDNA copy number by Twinkle overexpression is responsible for the cardioprotective effects. Previous studies have proposed various strategies such as resveratrol intake [14], exercise training [27], and caloric restriction [28] to increase mtDNA copy number systematically. We have also reported that exogenously administered recombinant mitochondrial transcription factor A protein increases mtDNA copy number in cardiac myocytes [15]. Increasing mtDNA copy number in clinical situation using these methods would be beneficial for the prevention of heart failure caused by pressure overload. Further investigations, especially in human studies, are anticipated.Cardiac Hypertrophy, Function, and FibrosisTwinkle overexpression ameliorated TAC-induced decreases in LV fractional shortening and ejection fraction, as well as increase in LV end-diastolic pressure (Table 1 and Figure 2). These changes were significant although the 23977191 magnitudes were small. As mentioned earlier, the relatively mild pressure overload produced in our model may partially explain the small amelioration of cardiac dysfunction by Twinkle overexpression. Nevertheless, the significant improvements in cardiac function indicate the benefit of Twinkle overexpression in preventing hear.Y of pressure overload between the two studies, judging from the hypertrophy data. Increasing the pressure overload intensity in our model may result in a significant decrease in mtDNA copy number in TAC. In the present study, we used a 26-gauge needle to induce pressure overload, which produced stable hypertrophy but rather mild effect on heart failure. On the other hand, using a 27- or 28-gauge needle as in previous study [13] resulted in higher surgical mortality but produces greater pressure overload in our preliminary experiments. We found that increasing mtDNA copy by Twinkle overexpression did not affect mitochondrial enzyme activity, which is consistent with a previous report [16]. Furthermore, TAC also did not affect mitochondrial enzyme activity (Figure 1B and C). These results suggest that mitochondrial electron transport complex activity is not directly related to the cardioprotective effect of Twinkle overexpression. The mechanism by which increased Twinkle expression prevents heart failure under pressure overload condition remains unknown. In this study we showed that Twinkle overexpression prevented cardiac fibrosis in vivo and in vitro (to be discussed in detail below). We therefore speculate that Twinkle overexpression somehow inhibits cardiac profibrogenic signals. We need to conduct further investigation about the mechanism.Figure 5. Effects of the upregulation or donwregulation of Twinkle on TGF-b1 mRNA expression. TGF-b1 expression in cardiac fibroblasts stimulated with AngII (1 mM) for 24 hours, quantified by realtime PCR relative to housekeeping gene (18S gene). Cells were preinfected with AxCAhTwinkle (Twinkle), AxCAsi-rTwinkle (siTwinkle) or AxCALacZ (LacZ). Values are mean 6 SEM. Data are presented as ratio to LacZ-vehicle. **; P,0.01 vs LacZ-vehicle, {; P,0.05 vs LacZAngII. doi:10.1371/journal.pone.0067642.gA major limitation of the present study is that we cannot elucidate the mechanism of how increased mtDNA reduces fibrosis in the pressure overload model. We should conduct further investigations to reveal the molecular mechanisms of how Twinkle overexpression or increased mtDNA decreases fibrosis or fibrosisrelated signaling.Clinical ImplicationWe speculate that increased mtDNA copy number by Twinkle overexpression is responsible for the cardioprotective effects. Previous studies have proposed various strategies such as resveratrol intake [14], exercise training [27], and caloric restriction [28] to increase mtDNA copy number systematically. We have also reported that exogenously administered recombinant mitochondrial transcription factor A protein increases mtDNA copy number in cardiac myocytes [15]. Increasing mtDNA copy number in clinical situation using these methods would be beneficial for the prevention of heart failure caused by pressure overload. Further investigations, especially in human studies, are anticipated.Cardiac Hypertrophy, Function, and FibrosisTwinkle overexpression ameliorated TAC-induced decreases in LV fractional shortening and ejection fraction, as well as increase in LV end-diastolic pressure (Table 1 and Figure 2). These changes were significant although the 23977191 magnitudes were small. As mentioned earlier, the relatively mild pressure overload produced in our model may partially explain the small amelioration of cardiac dysfunction by Twinkle overexpression. Nevertheless, the significant improvements in cardiac function indicate the benefit of Twinkle overexpression in preventing hear.
