Screening of novel HSP-inducing compounds to conserve cardiomyocyte function in experimental atrial fibrillation

Figure 1. Design and synthesis of a compound library based upon GGA/GA. Note: The east side of the molecule to GA was truncated, variations were made at the east and west sides of the molecule, and several bio-isosters of the central keto moiety were prepared. Abbreviations: GA, geranylacetone; GGA, geranylgeranylacetone.

Figure 2. GGA and GGA-derivatives induce HSPA1A expression in HL-1 cardiomyocytes pretreated with GGA and GGA-derivatives upon a mild HS. Notes: (A) Representative example of a Western blot for HSPA1A and GAPDH of HL-1 cardiomyocytes pretreated with DMSO (Control), 10 µM GGA and 10 µM GGA*-19, -20, -21, -27, -31, -59, -60, -61, and -67 upon a mild HS. (B) Quantified Western blot results for HSPA1A relative to GAPDH for GGA and GGA-derivatives. Mean ± SEM, * is P<0.05, ** is P<0.01, *** is P<0.001, and **** is P<0.0001 compared to control cardiomyocytes and # is P<0.05 and ## is P<0.01 compared to GGA. Abbreviations: DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GGA, geranylgeranylacetone; HS, heat shock.

Figure 3. GGA and GGA-derivatives protect against CaT loss in HL-1 cardiomyocytes. Notes: Compared to normal-paced controls, TP induces a loss in CaT amplitude which is protected by pretreatment with GGA and GGA-derivatives. (A) Illustrations of CaT tracers of control HL-1 cardiomyocytes (paced at 1 Hz) and of HL-1 cardiomyocytes after 8 hours of TP (4.5 Hz) pretreated with DMSO (TP), 10 µM GGA, or 10 µM GGA*-18, -59, or -60. (B) Quantified CaT amplitude of HL-1 cardiomyocytes treated with DMSO (Control), 10 µM GGA or 10 µM GGA-derivatives. Nine GGA-derivatives, namely GGA*-18, -26, -28, -31, -32, -57, -58, -59, and -60, show significant protection against CaT loss. **** is P<0.0001 compared to normal-paced control, # is P<0.05, ## is P<0.01, and #### is P<0.0001 compared to tachypaced control. The first control is a normal-paced control, treated with DMSO, as indicated by the line above the column with ‘Con’ above it. The second control is a tachypaced control, treated with DMSO, indicated with the line above the columns of tachypaced cells with ‘TP’ above it. Abbreviations: CaT, calcium transient; Con, control; DMSO, dimethylsulfoxide; GGA, geranylgeranylacetone; TP, tachypacing.

Figure 4. siRNA against HSPB1 abrogated the protective effect of the GGA-derivatives in HL-1 cardiomyocytes. Notes: (A) Successful suppression of HSPB1 expression in HL-1 cardiomyocytes. (B) CaT tracers of control HL-1 cardiomyocytes (paced at 1 Hz) and of HL-1 cardiomyocytes after 8 hours of TP (4.5 Hz) pretreated with DMSO (Control), 10 µM GGA or 10 µM GGA*-26 or 10 µM GGA*-28 show, that HSPB1 knock down abrogate the protective effects of the compounds. (C) Quantified CaT amplitude of HL-1 cardiomyocytes pretreated with DMSO (Control), 10 µM GGA or 10 µM GGA*-26 or 10 µM GGA*-28 with/without siRNA against HSPB1. *** is P<0.001 compared to normal-paced control cardiomyocytes and # is P<0.05 compared to tachypaced control cardiomyocytes. The first control is a normal-paced, DMSO treated control, indicated by the line above with ‘Con’. The second control is a tachypaced, DMSO treated, control, indicated by the line above the bars with ‘TP’. Abbreviations: CaT, calcium transient; Con, control; DMSO, dimethylsulfoxide; GGA, geranylgeranylacetone; TP, tachypacing.

Figure 5. GGA and GGA-derivatives protect against contractile dysfunction in Drosophila. Notes: TP (5 Hz) induces significant heart wall contractile dysfunction in Drosophila. Drosophila prepupae pretreated with 100 µM GGA, 100 µM GGA*-31, -58, -59, or -60 were protected against TP-induced contractile dysfunction, while GGA*-16, -18, -28, or -57 were not protective. Mean ± SEM, **** is P<0.0001 compared to nonpaced Control prepupae, # is P<0.05, ## is P<0.01 and #### is P<0.0001 compared to tachypaced control prepupae. The first control is a nonpaced control, treated with DMSO. The second control is a tachypaced control, treated with DMSO. Abbreviations: Con, control; GGA, geranylgeranylacetone; TP, tachypacing.

Figure 6. GGA and GGA*-31, -59, and -60 accelerate restoration of CaT loss in HL-1 atrial cardiomyocytes after 24 hours posttreatment, compared to the nontreated cardiomyocytes. Notes: (A) CaT of Control, TP, TP with 24 hours recovery and TP with 24 hours recovery in combination with posttreatment with 10 µM GGA or GGA-derivatives. (B) Quantified data revealing that GGA and GGA*-31, -59, and -60 accelerate restoration of CaT loss in HL-1 atrial cardiomyocytes after 24 hours posttreatment. **** is P<0.0001 compared to control cardiomyocytes, # is P<0.05; #### is P<0.0001 compared to tachypaced cardiomyocytes that recovered for 24 hours and † is P<0.05 and †††† is P<0.0001 compared to GGA*-59. The first control is a normal-paced control, treated with DMSO, indicated by the line above it with ‘Con’. The second control is a tachypaced control, treated with DMSO, indicated by the line above the bars with ‘TP’. Abbreviations: CaT, calcium transient; Con, control; GGA, geranylgeranylacetone; TP, tachypacing.

Figure 7. GGA*-59 enhances HSF1 hyperphosphorylation and subsequent HSPA1A boosting in HL-1 cardiomyocytes. Notes: (A) GGA*-59 enhances hyperphosphorylation of HSF1, shown by decreased mobility of HSF1 in the gel. (B) GGA*-59 enhances the ratio of hyperphosphorylated HSF1/HSF1 (quantified higher band/lower band). (C) Representative Western blot showing HSPA1A expression levels for the conditions as indicated. (D) GGA*-59 treatment enhances HSPA1A protein expression levels after 6 hours treatment, compared to nontreated heat shocked control cardiomyocytes. * is P<0.05 compared to Control, # is P<0.05 and #### is P<0.0001 compared to nontreated cardiomyocytes at 1 hour and 6 hours, respectively. Abbreviations: GGA, geranylgeranylacetone; HSF1, heat shock factor 1; HS, heat shock.