Online Supplementarymaterial

Online supplementarymaterial

Supplemental methods

Supplemental Table 1: Human amyloidogenic light chain proteins examined.

Abbreviations: control light chain proteins (Con-LC); amyloidogenic light chain proteins (AL-LC), multiple myeloma (MM), AL primary amyloidosis (AL), female (F), male (M)

Supplemental Table 2: Heart tissue -patient characteristics.

Abbreviations: AL primary amyloidosis (AL), female (F), male (M)

Supplementary methods

In vivo adenoviral gene transfer

Mice were anesthetized with 2% isofluorane vaporized with 100% O2 and ventilated using a rodent ventilator (Harvard Apparatus) at a respiration rate of 120bpm. In vivo gene delivery was conducted using similar protocols as published previously [2, 3, 5, 6]. The chest was opened from the left side through the third intercostal space. 30 L of adenovirus solution containing 1×109 virus was injected into the left ventricular cavity via a 0.5 mL syringe with a 28.5 gauge needle. During the injection, the aortic and pulmonary arteries were identified and cross-clamped distal to the opening of the coronary artery for 10 seconds to allow the adenovirus solution to circulate through the coronary arteries and perfuse the heart before entering circulation. The animals were extubated and warmed to37°C.

Non-invasive transthoracic echocardiography

Echocardiography was performed to monitor cardiac function longitudinally prior to, and 1 or 2 weeks following in vivoadenoviral gene transfer using a Vevo2100 system (VisualSonics) as per the methods described previously [1, 4].

Fig.S1 STC1 overexpression via adenovirus in cardiomyocytes. Titration of Adeno-STC1 multiplicity of infection (MOI) was performed to achieve a 2-fold increase in STC1 protein expression compared to untreated conditions. a Representative immunoblotting and quantitation of STC1 expression. b GFP expression was titrated to match that in STC1 treated cells. c Representative images and western blot of GFP expression is shown.

Fig.S2Preparation of fish STC1 mRNA and confirmation of overexpression of STC1 in zebrafish. To delineate whether STC1 is sufficient to impair cardiac function and induce cell death in vivo, we employed transient expression of STC1 by injecting STC1 mRNA that was transcribed in vitrousing mMESSAGEmMACHINE kit. As a control, STC mRNA in the reverse order (anti-sense STC1) was also synthesized. aAgarose gel electrophoresis analysis of synthesized mRNA. Only single band around 1kb was seen in both groups. bPonceau staining demonstrated equal loading forimmunoblotting against STC1 for protein expression.

Fig.S3Characterization of STC1 overexpressing zebrafish. a-bCardiac cell death 5 days following mRNA injection was determined by TUNEL staining of isolated hearts. Representative images are shown on left and quantification shown on right. Scale bar= 20 m.

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Fig. S4Overexpression of STC1 induces contractile dysfunction and cell death in a mouse model via adenovirus in vivo gene transfer. aImmunoblotting were performed to confirm cardiac STC1 protein expression in mice two weeks following Adeno-STC1 or Adeno-GFP cardiac infection. b Serial assessment of cardiac contractile function by transthoracic echocardiography (percent fractional shortening [FS %]) in mice expressing STC1 or GFP. c-dBax/Bcl-2 protein ratio and caspase 3/7 activity were measured in heart lysates from mice with cardiac expression of STC1 or GFP. * p<0.05 between indicated groups. (N=6-10 for mice echocardiography and N=6 for cell death assay)

Fig. S5STC1 protein expression is not induced in other cells types in response to AL-LC stimulation. STC1 protein expression was quantified in rat cardiac fibroblasts and human cardiac microvascular endothelial cells treated with AL-LC via immunoblotting for STC1 protein expression. a Immunoblotting analysis of STC1 induced by AL-LC treatment in cardiac fibroblasts. b Immunoblotting analysis of STC1 induced by AL-LC in human cardiac microvascular endothelial cells. N=3 each group.

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