Vity of proteasomes was 
significantly increased in atria and in the ventricles of one-month old TG mice. 9 / 15 Threonine 5 Modulates Sarcolipin Function Fig 5. TG 
mice hearts show decreased ISO response. Representative M-mode echocardiographic images of the left ventricle in NTG and TG mice at baseline and post ISO infusion. Dose response curves showing the changes in heart rate and MedChemExpress Endoxifen (E-isomer hydrochloride) ejection fraction after infusion of ISO at 0.02 and 0.04 g/Kg/min in the TG and NTG mice. NTG vs. TG; p<0.05, NTG vs. TG; p<0.005, #baseline vs. ISO; p<0.05. doi:10.1371/journal.pone.0115822.g005 To determine whether the increased ubiquitin-proteasome activity was due to the increased expression of UPS components, we measured the protein levels of 20S, 19S and 11S subunits. The quantitative Western blot analyses indicated that the protein levels of both 20S and 19S subunits were significantly increased in atria and in the ventricles of the TG mice. The protein levels of 11S and 11S subunits were unaltered in the TG hearts compared to that of NTG mice. Discussion The major goal of this study is to determine the functional importance of threonine 5 in modulating SLN function in the heart in vivo. To achieve this goal, T5A mutant SLN was created to abrogate SLN phosphorylation and expressed in the mouse heart by transgenesis. Data from the studies on this TG mouse model demonstrated that the cardiac specific expression of SLNT5A results in: 1) a replacement of the endogenous SLN without changes in the total SLN content in atria, 2) cardiac structural remodeling and bi-atrial enlargement, 3) a reduction in the expression of SR Ca2+ handling proteins and Ca2+ uptake both in atria and in the ventricles, 10 / 15 Threonine 5 Modulates Sarcolipin Function LVSP-LV systolic XMU-MP-1 supplier pressure; LVEDP-LV end diastolic pressure; SBP- Systolic blood pressure; DBP-Diastolic blood PubMed ID:http://jpet.aspetjournals.org/content/119/3/343 pressure; # MBP-Mean blood pressure; HR- Heart rate; bpm- beats per minute. p = 0.06 p<0.05 p<0.005 n=4 doi:10.1371/journal.pone.0115822.t002 4) a slower AP propagation and impaired atrial function, 5) a decreased ISO response and diastolic dysfunction, and 6) an activation of UPS in the heart. Previously, we and others have demonstrated that overexpression of NF-wildtype SLN in mouse heart decreased the cardiac SERCA pump affinity for Ca2+ and the contractile function. Although one of these mouse models develops mild ventricular hypertrophy, both TG mouse models did not show any atrial pathology. On the other hand, the cardiac specific expression of SLNT5A results in severe atrial structural remodeling including collagen accumulation and cell necrosis, followed by atrial enlargement. These changes are progressive upon aging. Structural remodeling is also apparent in the TG ventricles but to a lesser extent in comparison with that of atria. In the TG atria, SLNT5A replaces the endogenous protein without altering the total SLN content. We also made similar observations in the atria of TG mice expressing NF-SLN , in which the NF-SLN replaces the endogenous SLN to maintain the total SLN content. Thus, the atrial remodeling observed in the SLNT5A TG mouse model could be due to the functional consequences of mutant SLN rather than non-specific effect of transgene expression. At this juncture, it is also of interest to note that the cardiac specific overexpression of PLN impaired the atrial contractility but did not cause any atrial pathology. To determine the effect of SLNT5A on SERCA activity and Ca2+ transient.Vity of proteasomes was significantly increased in atria and in the ventricles of one-month old TG mice. 9 / 15 Threonine 5 Modulates Sarcolipin Function Fig 5. TG mice hearts show decreased ISO response. Representative M-mode echocardiographic images of the left ventricle in NTG and TG mice at baseline and post ISO infusion. Dose response curves showing the changes in heart rate and ejection fraction after infusion of ISO at 0.02 and 0.04 g/Kg/min in the TG and NTG mice. NTG vs. TG; p<0.05, NTG vs. TG; p<0.005, #baseline vs. ISO; p<0.05. doi:10.1371/journal.pone.0115822.g005 To determine whether the increased ubiquitin-proteasome activity was due to the increased expression of UPS components, we measured the protein levels of 20S, 19S and 11S subunits. The quantitative Western blot analyses indicated that the protein levels of both 20S and 19S subunits were significantly increased in atria and in the ventricles of the TG mice. The protein levels of 11S and 11S subunits were unaltered in the TG hearts compared to that of NTG mice. Discussion The major goal of this study is to determine the functional importance of threonine 5 in modulating SLN function in the heart in vivo. To achieve this goal, T5A mutant SLN was created to abrogate SLN phosphorylation and expressed in the mouse heart by transgenesis. Data from the studies on this TG mouse model demonstrated that the cardiac specific expression of SLNT5A results in: 1) a replacement of the endogenous SLN without changes in the total SLN content in atria, 2) cardiac structural remodeling and bi-atrial enlargement, 3) a reduction in the expression of SR Ca2+ handling proteins and Ca2+ uptake both in atria and in the ventricles, 10 / 15 Threonine 5 Modulates Sarcolipin Function LVSP-LV systolic pressure; LVEDP-LV end diastolic pressure; SBP- Systolic blood pressure; DBP-Diastolic blood PubMed ID:http://jpet.aspetjournals.org/content/119/3/343 pressure; # MBP-Mean blood pressure; HR- Heart rate; bpm- beats per minute. p = 0.06 p<0.05 p<0.005 n=4 doi:10.1371/journal.pone.0115822.t002 4) a slower AP propagation and impaired atrial function, 5) a decreased ISO response and diastolic dysfunction, and 6) an activation of UPS in the heart. Previously, we and others have demonstrated that overexpression of NF-wildtype SLN in mouse heart decreased the cardiac SERCA pump affinity for Ca2+ and the contractile function. Although one of these mouse models develops mild ventricular hypertrophy, both TG mouse models did not show any atrial pathology. On the other hand, the cardiac specific expression of SLNT5A results in severe atrial structural remodeling including collagen accumulation and cell necrosis, followed by atrial enlargement. These changes are progressive upon aging. Structural remodeling is also apparent in the TG ventricles but to a lesser extent in comparison with that of atria. In the TG atria, SLNT5A replaces the endogenous protein without altering the total SLN content. We also made similar observations in the atria of TG mice expressing NF-SLN , in which the NF-SLN replaces the endogenous SLN to maintain the total SLN content. Thus, the atrial remodeling observed in the SLNT5A TG mouse model could be due to the functional consequences of mutant SLN rather than non-specific effect of transgene expression. At this juncture, it is also of interest to note that the cardiac specific overexpression of PLN impaired the atrial contractility but did not cause any atrial pathology. To determine the effect of SLNT5A on SERCA activity and Ca2+ transient.
