Novel therapeutic cav1.3-C-terminus normalizes L-type calcium current and ejection fraction in ischemia-induced murine heart failure

Figures

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  Fig. 1.. (A,B) Representative echocardiograms from Sham and LAD ligation-induced HF mice. (C) Echocardiographic measurements comparing WT Sham (n = 11) and WT LAD ligation-induced HF (n = 15) mice at day 15. (D,E) Masson’s trichome staining in sham and LAD-induced HF mice (n = 1 each) at day 15 post LAD ligation. (F) Cav1.3 mRNA re-expression in the left ventricles of HF mice (LAD1 and LAD2 in triplicate) relative to sham (Sham1 and Sham2 in triplicate) control hearts at day 15. (G) Western blot showing Cav1.3 protein expression in HF mice (LAD1 and LAD2) vs sham controls (Sham1 and Sham2) at day 15 post LAD ligation. (*) p < 0.01; (**) p < 0.005. Abbreviations used: LAD – left anterior descending coronary artery; HF – heart failure; IVS – intraventricular septum; LVDD – left ventricular diameter in diastole; LVDS – left ventricular diameter in systole; LVEF – left ventricular ejection fraction; LVFS – left ventricular fractional shortening; PW – Left ventricular posterior wall.

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  Fig. 2.. L-type calcium currents (ICaL) in cardiomyocytes from untreated Sham (A) and AAV9-Cav1.3-C-terminus-treated (B) Sham mice. The corresponding I–V curves are shown in (C; n = 4 each group). (*) p < 0.01. Abbreviations used: AAV9 - adeno-associated virus serotype 9.

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  Fig. 3.. AAV9-Cav1.3-C-terminus increased L-type calcium current (ICaL) densities. (A–D) representative ICaL traces and (E) Comparison of ICaL densities in ventricular myocytes from the specified conditions (4 mice each group except WT HF PBS with 3 mice; n represents number of cells). (F) LVEF in WT HF and Cav1.3 HF mice treated with AAV9-C-terminus (C-term); n = 6 each group. (*) p < 0.01; (**) p < 0.001; (***) p < 0.0001. Abbreviations used: AAV9 - adeno-associated virus serotype 9; HF – heart failure; WT – wild type; LVEF – left ventricular ejection fraction.

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  Fig. 4.. Optical mapping of arrhythmias in WT HF mice. (A) Optical voltage signal from WT HF mouse treated with PBS (red) demonstrating bigeminy (black box) after premature simulation (2Hz S1, 140 ms S2). (B) Optical voltage signal from WT HF mouse treated with PBS (red) demonstrating a repeating pattern of 1 sinus beat followed by 3 ectopic triplets (black box) after premature stimulation (2Hz S1, 170 ms S2). (C) Optical voltage signal from WT HF mouse treated with AAV9-Cav1.3-C-terminus (green) demonstrating premature stimulation and recovery into normal sinus rhythm (5Hz S1, 50 ms S1, 40 ms S3, 40 ms S4). (D) Optical voltage signal from WT Sham (black) demonstrating premature stimulation and recovery into normal sinus rhythm (4Hz S1, 80 ms S2, 60 ms S3, 60 ms S4). Abbreviations used: AAV9 - adeno-associated virus serotype 9; HF – heart failure; WT – wild type; LVEF – left ventricular ejection fraction; PBS – phosphate-buffered saline; S2–S4 – premature stimuli.

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  Fig. 5.. RNA-seq of transcriptome changes at day 45 (30 days post treatment) between HF treated mice with PBS (n = 3) and HF mice treated with AAV9-C-terminus (n = 3). (A) Significant DEGs in untreated (PBS) and treated (AAV9-Cav1.3-C-terminus) groups. (B,C) Volcano plots showing upregulated (red) and downregulated (blue) genes. (D) Venn diagram indicating overlap of DEGs. (E) Overlapping GO categories. (F) Overlapping upregulated DEGs; (G) DEGs related to ion channels and calcium handling proteins. Significance was determined at p < 0.05 and Log2 fold change >+1.5 and <−1.5. Abbreviations used: HF – heart failure; PBS – phosphate-buffered saline; DEGs – differentially expressed genes; GO – gene ontology.