Brinks et al., Appendix A - page 10 CSQ NCX SERCA2 Normal AdGFP AdS100A1 Human heart HFCMs ICM Legend: Abnormal expression of calcium handling proteins in HFCMs. Representative Western Blots for calsequestrin (CSQ), sodium calcium exchanger (NCX) and sarcoplasmic reticulum Ca-ATPase (SERCA2) demonstrate persistent abnormalities in NCX and SERCA2 protein levels in HFCMs despite normalized S100A1 proteins levels after 24 hours. Experiments have been performed in samples from 5 different HFCM isolations with similar results (data not shown).

A B C 60 50 40 (ATP nmol/mg protein×min) SR Ca2+ATPase activity 30 20 Brinks et al., Appendix B - page 11 P < 0.05 A 60 50 Legend A: S100A1 gene transfer improves enzymatic SR Ca2+ ATPase (SERCA2a) activity in HFCMs. Ca2+ dependent SERCA2 function determined in microsomal preparations from control and S100A1-expressing HFCMs yielded enhanced Ca2+ ATPase activity. Results shown are from 3 different cell isolations. SERCA2a activity was assessed at pCa 6.2 as thapsigargin-sensitive fraction and given as ATP consumption in nmol per mg microsomal protein and min. 40 (ATP nmol/mg protein×min) SR Ca2+ATPase activity 30 20 Ad-control Ad-S100A1 10 HFCMs B C anti-SERCA 2 AB + blocking peptide anti-SERCA2 AB IP: SERCA2 IB: S100A1 EGTA Ca2+ + vehicle + hrS100A1 (0.1M) IB pellet: SERCA2 IB supernatant: SERCA2 IB pellet: S100A1 Legend B: Ca2+ dependent interaction of human S100A1 protein with human SERCA2a. Representative Western Blots depict a greater amount of the Ca2+ sensor protein co-precipitating with SERCA2 in the presence of Ca2+ (300 nM free Ca2+) in SR vesicle preparations derived from failing human cardiomyocytes treated with recombinant human S100A1 protein. EGTA (1 mM) prevents the interaction. Experiments have been performed in samples from 4 different HFCM isolations with similar results (data not shown). Legend C: Control experiment revealing specificity of SERCA2/S100A1 co-immunoprecipitation with the polyclonal goat anti-SERCA2 antibody. The corresponding blocking peptide was pre-incubated with the anti-SERCA2 antibody (5-fold excess) which prevented SERCA2 immunoprecipitation (upper panel). As expected, the remaining supernatant of the anti-SERCA2a/blocking peptide group exhibited a greater SERCA2 signal (middle panel) and absence of co-precipitating S100A1 protein (lower panel).

Ca2+ leak (Fluo 3 emission 530 nm) Brinks et al., Appendix C – page 12 A control + TG Legend A: S100A1 reduces SR Ca2+ leak in SR vesicle preparations from failing human myocardium. Representative Fluo-3 tracings of SR vesicle preparations from human failing hearts show abrogated Ca2+ leak due to recombinant S100A1 protein treatment (lower tracing) compared with control buffer (upper tracing). Results are representative of 3 different SR vesicle preparations (data not shown). 10 SR Ca2+ leak Ca2+ leak (Fluo 3 emission 530 nm) arbitrary units hrS100A1 (0.1M) + TG 10 SR Ca2+ leak 150 sec IgG1 isotype control anti-RyR2 B C IP: RyR2 EGTA Ca2+ control hrS100A1 (0.1M) IB pellet: RyR2 IB: S100A1 IB supernatant: RyR2 IB pellet: S100A1 Legend B: Ca2+ dependent interaction of human S100A1 protein with human RyR2. Representative Western Blots depict a greater amount of the Ca2+ sensor protein co-precipitating with RyR2 in the presence of Ca2+ (300 nM free Ca2+) in SR vesicle preparations derived from failing human cardiomyocytes treated with recombinant human S100A1 protein. EGTA (1 mM) prevented the interaction. Experiments have been performed in samples from 3 different HFCM isolations with similar results (data not shown). Legend C: Control experiment revealing specificity of RyR2/S100A1 co-immunoprecipitation with the monoclonal IgG1 anti-RyR2 antibody. The control isotype specific IgG1 antibody did not precipitate RyR2 as shown by the missing RyR2 signal in the pellet (upper panel). As expected, the remaining supernatant exhibited a much stronger RyR2 signal (middle panel). Note that the pellet of IgG1 treated RyR2-free homogenates does not contain S100A1 (lower panel) indicating specificity of S100A1 co-precipitation with RyR2.

A B C Brinks et al., Appendix D – page 13 CytC CSQ S100A1 S100A1 HS3 CytC CytC α-actinin Adcontrol AdS100A1 control S100A1 (0.1 µM) pre-column lysate post-column lysate Legend: Efficacy of human mitochondria isolation and enhanced mitochondrial S100A1 protein content due to AdS100A1 and recombinant S100A1 protein treatment. A, representative Western blots showing efficacy of the mitochondrial isolation protocol. In the pre-column lysate (right, prior to anti-TOM22 magnetic bead purification) consisting of whole cardiac tissue homogenate, markers for mitochondria (cytochrom C; CytC), sarcoplasmic reticulum (calsequestrin; CSQ), sarcomer (α-actinin) and nuclei (histone 3, HS3) are highly abundant. Post-column eluates (left, after anti-TOM22 magnetic bead purification) are free of CSQ, HS3 and α-actinin but enriched in the mitochondrial marker CytC. B, representative Western blots demonstrating enhanced mitochondrial S100A1 protein content after adenoviral overexpression of human S100A1 protein for 24 hours in isolated mitochondria from control and S100A1-expressing HFCMs. C, show representative Western blots of augmented S100A1 content in isolated mitochondria from failing human myocardial tissue that were (pre)incubated with human recombinant S100A1 protein (0.1 µM) for 60 min. CytC served as loading control in B and C. Similar results were obtained with 2 different cellular and tissue preparations (data not shown).

Brinks et al., Appendix E – page 14 Ad control Ad control + Ca2+ 20 40 60 80 100 101 102 103 104 20 40 60 80 100 101 102 103 104 M2 M2 CCCP 51.37% 89.22% 20 40 60 80 100 101 102 103 104 M1 M1 Counts M2 20 40 60 80 100 101 102 103 104 Ad S100A1 20 40 60 80 100 101 102 103 104 Ad S100A1 + Ca2+ 97.36% M1 M2 M2 12.43% 25.78% M1 M1 Rh123-FL1 Legend: S100A1 prevents depolarization of mitochondrial membrane potential (ΔΨm). ΔΨm was determined using rhodamine123 (Rh123) in isolated mitochondria derived from control and S100A1-expressing human failing cardiomyocytes. Histogram plots show distribution of depolarized vs. hyperpolarized mitochondria under markers M1 and M2, respectively. Note that S100A1-treated human failing cardiomyocytes revealed a smaller percentage of depolarized mitochondria than control treated cells both under basal conditions and calcium overload (CaCl2: 100 µM). Results are representative for a total of three different isolations. The uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 10 µM) was used as a negative control for depolarized ΔΨm.

Mitochondrial swelling in % of control + Pi Brinks et al., Appendix F – page 15 A B P < 0.01 Ad S100A1 Ad control Ad S100A1 120 P < 0.01 * Ad S100A1 + Ca2+ 100 * OD Abs520 = 0.2nm Ad control 80 P < 0.01 Ad S100A1 + Pi 60 40 Ad control + Ca2+ 20 Time = 3 min Ad control + Pi Swelling Buffer Mitochondrial swelling in % of control + Pi Ca2+ Pi P < 0.01 S100A1 control 120 # S100A1 protein + Ca2+ 100 P < 0.01 OD Abs520 = 0.2nm S100A1 protein + Pi 80 60 control + Ca2+ 40 Time = 3 min control + Pi 20 Ca2+ Pi Legend: S100A1 attenuates mitochondrial membrane permeability transition (mitochondrial swelling). A, linear graph represents representative traces of swelling (decrease in optical density, OD) in isolated mitochondria from control (dashed lines) and S100A1 expressing (solid lines) human failing cardiomyocytes (upper panel) during Pi (KH2PO4: 2mM) and calcium overload (CaCl2: 100 µM) treatment. The lower panel shows representative results for isolated mitochondria from failing human myocardial tissue (pre)treated either with control buffer (dashed lines) or human recombinant S100A1 protein (0.1µM, 60 min, solid lines). B, statistical analysis from 3 different mitochondrial preparations depict S100A1-mediated protection from Pi and calcium induced membrane permeability transition. *P<0.05 control buffer vs. buffer calcium and buffer Pi; # P<0.05 control calcium vs. buffer Pi (n=3).

Systolic Heart Failure (HF) S100A1 HF Gene Therapy Brinks et al., Appendix G – page 16 Proposed model for therapeutic effects of S100A1 in failing human cardiomyocytes Systolic Heart Failure (HF) S100A1 HF Gene Therapy S100A1 Downregulation Ca2+ Ca2+ S100A1 Recovery extracellular [Ca2+] Contraction [Ca2+] Contraction LLC NCX intracellular 1. Ca transient amplitude 1. Ca transient amplitude 200 ms 200 ms - Decreased Ca transient amplitude 2. SR Ca leak 2. SR Ca leak - Enhanced Ca transient amplitude - Prolonged Ca transient decay - Accelerated Ca transient decay - Enhanced diastolic [Ca] S100A1 S100A1 - Decreased diastolic [Ca] RyR2 Ca2+ Ca2+ Ca2+ Ca2+ S100A1 Ca2+ S100A1 Ca2+ Ca2+ Ca2+ 3. diastolic [Ca] Ca2+ 3. diastolic [Ca] Myofilaments Ca2+ Ca2+ 4. SR Ca load Ca2+ 4. SR Ca load 5. PCr/ATP 5. PCr/ATP Mitochondria Ca2+ Ca2+ MPT 6. Pi Pi MPT 6. PLB PLB S100A1 S100A1 S100A1 S100A1 SERCA2 SERCA2 7. Electron flow and ΔΨm SR Ca resequestration SR Ca resequestration 8. 8. 7. Electron flow and ΔΨm Legend: Therapeutic actions of S100A1 gene therapy in human failing cardiomyocytes (right) summarized and opposed to pathophysiological alterations in untreated failing cells (1.-8.) with diminished S100A1 expression (left). The figure was modified from Kraus et al. (2009) “S100A1 in cardiovascular health and disease: closing the gap between basic science and clinical therapy.” J Mol Cell Cardiol;47:445-55.