Geranylgeranylacetone reduces cardiomyocyte stiffness and attenuates diastolic dysfunction in a rat model of cardiometabolic syndrome

Abstract

Titin-dependent stiffening of cardiomyocytes is a significant contributor to left ventricular (LV) diastolic dysfunction in heart failure with preserved LV ejection fraction (HFpEF). Small heat shock proteins (HSPs), such as HSPB5 and HSPB1, protect titin and administration of HSPB5 in vitro lowers cardiomyocyte stiffness in pressure-overload hypertrophy. In humans, oral treatment with geranylgeranylacetone (GGA) increases myocardial HSP expression, but the functional implications are unknown. Our objective was to investigate whether oral GGA treatment lowers cardiomyocyte stiffness and attenuates LV diastolic dysfunction in a rat model of the cardiometabolic syndrome. Twenty-one-week-old male lean (n = 10) and obese (n = 20) ZSF1 rats were studied, and obese rats were randomized to receive GGA (200 mg/kg/day) or vehicle by oral gavage for 4 weeks. Echocardiography and cardiac catheterization were performed before sacrifice at 25 weeks of age. Titin-based stiffness (F_passive ) was determined by force measurements in relaxing solution with 100 nM [Ca²⁺ ] in permeabilized cardiomyocytes at sarcomere lengths (SL) ranging from 1.8 to 2.4 μm. In obese ZSF1 rats, GGA reduced isovolumic relaxation time of the LV without affecting blood pressure, EF or LV weight. In cardiomyocytes, GGA increased myofilament-bound HSPB5 and HSPB1 expression. Vehicle-treated obese rats exhibited higher cardiomyocyte stiffness at all SLs compared to lean rats, while GGA reduced stiffness at SL 2.0 μm. In obese ZSF1 rats, oral GGA treatment improves cardiomyocyte stiffness by increasing myofilament-bound HSPB1 and HSPB5. GGA could represent a potential novel therapy for the early stage of diastolic dysfunction in the cardiometabolic syndrome.

Keywords: cardiomyocyte; diastolic heart failure; ejection fraction; myofilament protein; small heat shock proteins.

FIGURE 1

Oral GGA treatment halts the progression of diastolic dysfunction, measured as isovolumic relaxation time. Upper panel: Representative scans of transmitral pulse wave Doppler in Lean, Obese, and Obese rats treated with GGA as acquired from the apical long‐axis view at 25 weeks. Green cross‐hair cursors indicate the position used to calculate the IVRT. Lower panel: absolute IVRT values in 21‐ and 25‐week‐old lean and obese rats, measured by transthoracic echocardiography (see Methods). Effects of obesity and oral GGA treatment on progression of diastolic dysfunction, measured as the enhancement of IVRT from 21 to 25 weeks (ΔIVRT). Data are expressed as mean ± SEM. n = 8–10 per group at Week 21 and Week 25. A mixed‐model two‐way ANOVA was utilized to determine effects, and a Bonferroni's post hoc test was used to correct for multiple comparisons between 21‐ and 25‐week timepoints.

FIGURE 2

Oral GGA treatment redistributes HSPB5 and HSPB1 from the cytosol to the myofilaments in the left ventricle of obese ZSF1 rats. (a and b) HSPB5 and HSPB1 levels were similar between myocardia of all groups in cytosolic fraction. (c and d) myofilament levels of HSPB5 and HSPB1 were higher in the myocardium of obese ZSF1 rats treated with GGA when compared to vehicle‐treated obese and lean ZSF1 rats. (e and f) There was a redistribution of HSPB5 and HSPB1 to the myofilaments (myofilament expression levels relative to cytosolic expression levels) in the GGA‐treated obese ZSF1 rats compared to lean and vehicle‐treated obese ZSF1 rats, although this was particularly strong for HSPB1. Data are expressed as mean ± SEM, n = 4–5 per group. A one‐way ANOVA with a Bonferroni's post hoc test was utilized to assess differences between the groups. (g and h) Representative images of confocal laser microscopy for HSPB5 (upper panel) and HSPB1 (lower panel) in LV sections from lean, obese and obese ZSF1 rats treated with GGA (200 mg/kg/day). Immunohistochemical visualization of cell membranes (WGA; green), nuclei (DAPI; blue), and HSPB5/HSPB1 (red) was achieved with confocal laser microscopy. Both HSPB5 and HSPB1 immunostaining primarily occurred in the vicinity of the myofilaments for all rats. Greater HSPB5 immunostaining in the vicinity of the myofilaments was observed in both vehicle‐treated and GGA‐treated obese ZSF1 rats, whereas prominent elevation of HSPB1 immunostaining was noted in the GGA‐treated obese ZSF1 rats. n = 4 per group.

FIGURE 3

Oral GGA treatment reduces myofilament stiffness in obese ZSF1 rats. Myofilament stiffness was measured as increases in passive force (F_passive) of isolated, permeabilized cardiomyocytes in response to increases in sarcomere length (SL). (a) SL‐F_passive curves for lean (solid black line; open circles), vehicle‐treated obese (dashed black line; triangles) and GGA‐treated obese (solid black line; diamonds) ZSF1 rats, using a second order polynomial (quadratic function) for curve fitting. F_passive was significantly higher at sarcomere length (SL) 2.0 μm in obese rats compared to lean and GGA‐treated obese rats (b). ***p < 0.001 vs. lean; ^# p = 0.02 vs. obese vehicle. Data are expressed as mean ± SD. N = 4–5 per group, with four cardiomyocytes tested per rat. A one‐way ANOVA with a Bonferroni's post hoc test was utilized to assess differences between the groups.

FIGURE 4

Recombinant human HSPB5 (0.01 mg/mL) and human HSPB1 (0.01 mg/mL) reduce stiffness of permeabilized cardiomyocytes of obese ZSF1 rats and abrogate the effect of oral GGA treatment. Stiffness was measured as the passive force (F_passive). (a and b) SL‐F_passive curves after HSP treatment for lean, vehicle‐treated obese and GGA‐treated obese ZSF1 rats after incubation with recombinant human HSPB5 (a) or human HSPB1 (b). A second order polynomial (quadratic function) was used for curve fitting. For comparison, the F_passive curve of cardiomyocytes isolated from vehicle‐treated obese ZSF1 rats (dashed line, triangles) and the F_passive curve of cardiomyocytes isolated from lean rats are shown in panels a and b. (c–f) comparison of HSP effects on F_passive of cardiomyocytes in vehicle‐ and GGA‐treated rats. Data are expressed as mean ± SD. ^‡ p < 0.0001, ^† p = 0.0001 and *p < 0.05 vs. baseline of the same group. N = 4–5 per group, with four cardiomyocytes tested per rat. Effects of recombinant sHSPs were established using a two‐way ANOVA with a Bonferroni's post hoc test to correct for differences between the groups.

FIGURE 5

Oral GGA treatment reduces the effect of protein kinase A (PKA) on cardiomyocyte stiffness in cardiomyocytes of obese ZSF1 rats. Isolated, permeabilized cardiomyocytes of lean, obese and GGA‐treated obese rats were incubated with exogenous protein kinase A (PKA; 1 unit/μL) and 3′,5′‐cyclic adenosine monophosphate (cAMP; 0.006 mM) over a 40‐min period at an optimal sarcomere length (SL) of 2.2 μm. (a) absolute F_passive in the absence and presence of PKA for lean vehicle‐treated obese and GGA‐treated obese ZSF1 rats. Data are expressed as mean ± SD. n = 4 per group, with 4 cardiomyocytes tested per rat. ^$ p < 0.0001 vs. baseline, ***p < 0.0001 vs. lean, ^### p < 0.0001 vs. Obese GGA. (b) Obesity increases the effect of PKA treatment on cardiomyocyte passive force (ΔF_passive) compared to lean rats, and oral GGA treatment abolishes this difference. Effects of exogenous PKA was determined using a two‐way ANOVA with a Bonferroni's post hoc test to correct for differences between the groups.

FIGURE 6

No differences in the major titin isoforms or the ratio of their expression relative to the total titin pool were observed among the groups. (a) Raw image of titin blot; (b) lane profiles for each of the raw titin blots. The analysis area is depicted by the corresponding colored rectangles in A; (c) expression of the N2BA1 isoform relative to total titin; (d) expression of the N2BA2 isoform relative to total titin; (e) expression of the N2B isoform relative to total titin; (f) the ratio between the total N2BA (N2BA1 + N2BA2) and N2B isoforms. Data are expressed as mean ± SEM. A one‐way ANOVA with a Bonferroni's post hoc test was utilized to assess differences between the groups.