Differential expression of sarcolipin protein during muscle development and cardiac pathophysiology

Abstract

Sarcolipin (SLN) is a small molecular weight sarcoplasmic reticulum (SR) membrane protein expressed both in cardiac and skeletal muscle tissues. Recent studies using transgenic mouse models have demonstrated that SLN is an important regulator of cardiac SR Ca2+ ATPase 2a (SERCA2a). However, there is a paucity of information regarding the SLN protein expression in small versus larger mammals and its regulation during development and cardiac pathophysiology. Therefore, the major goal of this study was to generate an SLN specific antibody and perform detailed analyses of SLN protein expression during muscle development and in the diseased myocardium. The important findings of the present study are: (i) in small mammals, SLN expression is predominant in the atria but low in the ventricle and in skeletal muscle tissues, whereas in large mammals, SLN is quite abundant in skeletal muscle tissues than the atria, (ii) SLN and SERCA2a are co-expressed in all striated muscle tissues studied except ventricle and co-ordinately regulated during muscle development and (iii) SLN protein levels are approximately 3 fold upregulated in the atria of heart failure dogs and approximately 30% decreased in the atria of hearts prone to myocardial ischemia. In addition we found that in the phospholamban null atria, SLN protein levels are upregulated.

Figure 1

(A) Western blot analysis of sarcolipin (SLN) and phospholamban (PLB) in the total homogenate (Total), SR enriched microsomal fractions (SR) and post microsomal fractions (PM) from rat atria and ventricle. Bacterial extract containing rat and human SLN were used as control. 1 μg of each fraction were used. (B) Two different concentrations (1 and 2μg) of total homogenates from atria (A) and ventricle (V) of non-transgenic (NTG) and SLN- transgenic (TG) mice were resolved on a 16% tricine gel and immuno probed with SLN-CTAb. NF-SLN= N-terminally Flagged SLN

Figure 2

Expression analysis of SLN, PLB and SERCA2a proteins in striated muscle tissues of rodents (Panel A-mouse and rat) and larger mammals (Panel B- rabbit and dog). Total homogenates from atria (A), ventricle (V), diaphragm (D), soleus (S), quadriceps (Q), gastrocnemius (G) tongue (T) and EDL (E) from different species were resolved on polyacrylamide gel electrophoresis and probed with SLN-CTAb, PLB and SERCA2a specific antibodies as described in “methods” section. LA- left atria, RA-right atria, LV-left ventricle and RV-right ventricle. Data are representative of two independent experiments.

Figure 2

Expression analysis of SLN, PLB and SERCA2a proteins in striated muscle tissues of rodents (Panel A-mouse and rat) and larger mammals (Panel B- rabbit and dog). Total homogenates from atria (A), ventricle (V), diaphragm (D), soleus (S), quadriceps (Q), gastrocnemius (G) tongue (T) and EDL (E) from different species were resolved on polyacrylamide gel electrophoresis and probed with SLN-CTAb, PLB and SERCA2a specific antibodies as described in “methods” section. LA- left atria, RA-right atria, LV-left ventricle and RV-right ventricle. Data are representative of two independent experiments.

Figure 3

Western blot analysis of SLN, SERCA2a, PLB and SERCA1a proteins during muscle development. Two micrograms of total homogenates of atria, ventricular (Panel A), fast-twitch quadriceps muscle and tongue (Panel B) from rat embryos of different days of postcoitum (dpc) and from neonatal rats were separated on polyacrylamide gel electrophoresis and immunoprobed with specific antibodies. Data are representative of two independent experiments.

Figure 3

Western blot analysis of SLN, SERCA2a, PLB and SERCA1a proteins during muscle development. Two micrograms of total homogenates of atria, ventricular (Panel A), fast-twitch quadriceps muscle and tongue (Panel B) from rat embryos of different days of postcoitum (dpc) and from neonatal rats were separated on polyacrylamide gel electrophoresis and immunoprobed with specific antibodies. Data are representative of two independent experiments.

Figure 4

Quantitation of SLN in the atria of heart failure and ischemic injured dog models. (A) Representative Western blots showing the expression of SLN, SERCA2a and PLB in the atria of tachypacing induced heart failure dog model. SLN is upregulated in the heart failure (HF) atria. SERCA2a and PLB levels are unchanged between control (Ctr) and HF groups. Bar diagram showing the fold induction of SLN in HF atria. (B) Representative Western blots showing the protein levels of SLN, SERCA2a, PLB and CSQ in ischemic injured but either susceptible (S) or resistant (R) to ventricular fibrillation and exercise trained susceptible dogs (S/ExT). SLN expression is significantly downregulated in the atria of susceptible (S) and resistant groups but its level restored in the S/ExT groups to the levels of control atria (Ctr). SERCA2a, PLB and CSQ levels are unchanged between the groups. Bar diagram showing the percent expression of SLN in different groups. * indicates values significantly different from control groups and ** indicates values not significantly different from control groups.

Figure 4

Quantitation of SLN in the atria of heart failure and ischemic injured dog models. (A) Representative Western blots showing the expression of SLN, SERCA2a and PLB in the atria of tachypacing induced heart failure dog model. SLN is upregulated in the heart failure (HF) atria. SERCA2a and PLB levels are unchanged between control (Ctr) and HF groups. Bar diagram showing the fold induction of SLN in HF atria. (B) Representative Western blots showing the protein levels of SLN, SERCA2a, PLB and CSQ in ischemic injured but either susceptible (S) or resistant (R) to ventricular fibrillation and exercise trained susceptible dogs (S/ExT). SLN expression is significantly downregulated in the atria of susceptible (S) and resistant groups but its level restored in the S/ExT groups to the levels of control atria (Ctr). SERCA2a, PLB and CSQ levels are unchanged between the groups. Bar diagram showing the percent expression of SLN in different groups. * indicates values significantly different from control groups and ** indicates values not significantly different from control groups.

Figure 5

Increased SLN protein expression in plb-/- atria. Representative Western blots showing the expression of SLN and SERCA2a in the atria of plb-/- mice. SLN protein levels are 52% more in the plb-/- atria. SERCA2a levels are unchanged between WT and plb-/- atria. Bar diagram showing the percent expression of SLN in WT and plb-/- atria. * indicates values significantly different from WT atria.