Low-dose T₃ replacement restores depressed cardiac T₃ levels, preserves coronary microvasculature and attenuates cardiac dysfunction in experimental diabetes mellitus

Abstract

Thyroid dysfunction is common in individuals with diabetes mellitus (DM) and may contribute to the associated cardiac dysfunction. However, little is known about the extent and pathophysiological consequences of low thyroid conditions on the heart in DM. DM was induced in adult female Sprague Dawley (SD) rats by injection of nicotinamide (N; 200 mg/kg) followed by streptozotocin (STZ; 65 mg/kg). One month after STZ/N, rats were randomized to the following groups (N = 10/group): STZ/N or STZ/N + 0.03 μg/mL T3; age-matched vehicle-treated rats served as nondiabetic controls (C). After 2 months of T3 treatment (3 months post-DM induction), left ventricular (LV) function was assessed by echocardiography and LV pressure measurements. Despite normal serum thyroid hormone (TH) levels, STZ/N treatment resulted in reductions in myocardial tissue content of THs (T3 and T4: 39% and 17% reduction versus C, respectively). Tissue hypothyroidism in the DM hearts was associated with increased DIO3 deiodinase (which converts THs to inactive metabolites) altered TH transporter expression, reexpression of the fetal gene phenotype, reduced arteriolar resistance vessel density, and diminished cardiac function. Low-dose T3 replacement largely restored cardiac tissue TH levels (T3 and T4: 43% and 10% increase versus STZ/N, respectively), improved cardiac function, reversed fetal gene expression and preserved the arteriolar resistance vessel network without causing overt symptoms of hyperthyroidism. We conclude that cardiac dysfunction in chronic DM may be associated with tissue hypothyroidism despite normal serum TH levels. Low-dose T3 replacement appears to be a safe and effective adjunct therapy to attenuate and/or reverse cardiac remodeling and dysfunction induced by experimental DM.

Figure 1

Serum and tissue THs. (A) Serum FT₃, free triiodothyronine in serum. (B) Serum FT₄, free thyroxine in serum. (C) TSH, thyroid stimulating hormone in serum. (D) Cardiac T₃, triiodothyronine in cardiac tissue. (E) Cardiac T₄, thyroxine in cardiac tissue. (F) Cardiac T₃:T₄ ratio, ratio of intracardiac T₃ to T₄. Values represented as means (SD) for groups with ≥3 samples and means (raw values) for groups with two samples; n = 2–3 pooled samples/ group for D–F. Each pooled LV sample within an experimental group consisted of pooled LV tissue from 2 to 4 hearts (6 to 8 hearts/group); ^†p < 0.05 versus STZ/N. Statistical analysis was only performed on A–C.

Figure 2

T₃ treatment attenuates DM-induced hemodynamic dysfunction and significantly improves parameters of cardiac relaxation. Values are means (SD). (A) HR, heart rate. (B) LVSP, LV systolic pressure. (C) LVEDP, LV end-diastolic pressure. (D) Tau, time constant of LV isovolumic relaxation. (E) dP/dT Max, maximal rate of pressure development. (F) dP/dT, Maximal rate of pressure decline. n, Number of animals per group. *p < 0.05 versus control; ^†p < 0.05 versus STZ/N.

Figure 3

Loss of small arteriolar resistance vessels in the diabetic myocardium is prevented with T₃ treatment. Values represent means (SD). (A) Representative images of small arteriolar resistance vessels used for arteriolar numeric (ND) and length density (LD) quantification (scale bar = 50 μm). (B) Representative images of myocardial capillaries used for capillary numeric density and length quantification (scale bar = 20 μm). (C) Arteriolar ND, arteriolar numeric density quantification. (D) Arteriolar LD, arteriolar length density quantification. (E) Capillary ND & Length, capillary numeric density and length quantification. Arteriolar ND, arteriolar LD, capillary ND, capillary length were calculated using previously described methods (34). (F) Absolute gene expression of cardiac regulators of vascular function. n = 5–8/group; IB4, Isolectin-B4; α-SMA, α-smooth muscle actin; TR-β, thyroid receptor β; VEGF-A, vascular endothelial growth factor A; eNOS, endothelial nitric oxide synthase; bFGF; basic fibroblast growth factor; MdK, midkine; HIF1α, hypoxia inducible factor 1-α; SMO, smoothened homolog (Drosophila); Angpt2, angiopoietin-2; *p < 0.05 versus control; ^†p < 0.05 versus STZ/N.

Figure 4

Diabetes leads to reexpression of fetal genes in adult cardiac tissue. (A) MHC isoform mRNA distribution shown as % of total MHC mRNAs as measured by qPCR. (B) Protein expression of β-MHC isoform and total MHC detected by Western blot. Expression represented as ratio of β-MHC to total MHC expression. (C) Cardiac protein expression of sarcoplasmic reticulum proteins detected by Western blot. (D) Quantitative summary of normalized protein expression. MHC, myosin heavy chain; SERCA2a, sarcoplasmic/endoplasmic reticulum calcium ATPase 2; PLB, phospholamban; p-PLB, phosphorylated phospholamban (site of phosphorylation); n = 4–8/group. *p < 0.05 versus control; ^†p < 0.05 versus STZ/N.

Figure 5

LV Deiodinase Expression. (A) LV DIO3 staining in control and diabetic hearts at 2× magnification. (B) DIO3 IHC protein quantification. Values represented as the percentage of DIO3 staining versus the total tissue area. (C) DIO2, type 2 deiodinase mRNA expression. (D) DIO3, type 3 deiodinase mRNA expression. Gene expression values represented as means (SD). Gene expression was normalized using the housekeeping genes cyclophilin A and Rplp1; n = 3–5/group. *p < 0.05 versus control; ^†p < 0.05 versus STZ/N.

Figure 6

Expression of cardiac thyroid hormone transporters. Gene expression values represented as means (SD). Gene expression was normalized using the housekeeping genes cyclophilin A and Rplp1. (A) MCT, monocarboxylate transporters; LAT, large neutral amino acid transporters. (B) 4F2hc, 4F2 cell surface antigen heavy chain; FAT, fatty acid translocase; SLCO, solute carrier organic anion transporter family. n = 5/group; *p < 0.05 versus control; ^†p < 0.05 versus STZ/N.