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

Abstract

Background: The heat shock protein (HSP) inducer, geranylgeranylacetone (GGA), was previously found to protect against atrial fibrillation (AF) remodeling in experimental model systems. Clinical application of GGA in AF is limited, due to low systemic concentrations owing to the hydrophobic character of GGA.

Objectives: To identify novel HSP-inducing compounds, with improved physicochemical properties, that prevent contractile dysfunction in experimental model systems for AF.

Methods: Eighty-one GGA-derivatives were synthesized and explored for their HSP-inducing properties by assessment of HSP expression in HL-1 cardiomyocytes pretreated with or without a mild heat shock (HS), followed by incubation with 10 µM GGA or GGA-derivative. Subsequently, the most potent HSP-inducers were tested for preservation of calcium transient (CaT) amplitudes or heart wall contraction in pretreated tachypaced HL-1 cardiomyocytes (with or without HSPB1 siRNA) and Drosophilas, respectively. Finally, CaT recovery in tachypaced HL-1 cardiomyocytes posttreated with GGA or protective GGA-derivatives was determined.

Results: Thirty GGA-derivatives significantly induced HSPA1A expression after HS, and seven showed exceeding HSPA1A expression compared to GGA. GGA and nine GGA-derivatives protected significantly from tachypacing (TP)-induced CaT loss, which was abrogated by HSPB1 suppression. GGA and four potent GGA-derivatives protected against heart wall dysfunction after TP compared to non-paced control Drosophilas. Of these compounds, GGA and three GGA-derivatives induced a significant restoration from CaT loss after TP of HL-1 cardiomyocytes.

Conclusion: We identified novel GGA-derivatives with improved physicochemical properties compared to GGA. GGA-derivatives, particularly GGA^*-59, boost HSP expression resulting in prevention and restoration from TP-induced remodeling, substantiating their role as novel therapeutics in clinical AF.

Keywords: Drosophila; atrial fibrillation; geranylger anylacetone; heat shock protein; proteostasis.

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.