Mitochondrial and energetic cardiac phenotype in hypothyroid rat. Relevance to heart failure

Abstract

Changes in thyroid status are associated with profound alterations in biochemical and physiological functioning of cardiac muscle, although its impact on cardiac energy metabolism is still debated. Similarities between the changes in cardiac gene expression in pathological hypertrophy leading to heart failure and hypothyroidism prompted scientists to suggest a role for thyroid hormone status in the development of metabolic and functional alterations in this disease. We thus investigated the effects of hypothyroidism on cardiac energy metabolism. Hypothyroid state (HYPO) was induced by thyroidectomy and propyl-thio-uracyl in male rats for 3 weeks. We examined the effects of hypothyroid state on oxidative capacity and mitochondrial substrate utilization by measuring oxygen consumption of saponin permeabilized cardiac fibers, mitochondrial biogenesis by reverse transcription polymerase chain reaction and energy metabolism, and energy transfer enzymes by spectrophotometry. The results show that maximal oxidative capacity of the myocardium was decreased from 24.9 +/- 0.9 in control (CT) to 19.3 +/- 0.7 micromol O(2) min(-1) g dry weight(-1) in HYPO. However, protein content and messenger RNA (mRNA) of PGC-1alpha and mRNA of its transcription cascade that is thought to control mitochondrial content in normal myocardium and heart failure, were unchanged in HYPO. Mitochondrial utilization of glycerol-3P (-70%), malate (-45%), and octanoate (-24%) but not pyruvate was decreased in HYPO. Moreover, the creatine kinase system and energy transfer were hardly affected in HYPO. Besides, hypothyroidism decreased the activation of other signaling pathways like p38 mitogen-activated protein kinases, AMP-activated protein kinase, and calcineurin. These results show that cellular hypothyroidism can hardly account for the specific energetic alterations of heart failure.

Figure 1. Effects of hypothyroidism on cardiac oxidative capacity

A. Maximal respiration rates with glutamate and malate (respiration from complex I, Vglu/mal), succinate (respiration from complex II, Vsuc), TMPD/ascorbate (respiration from complex IV, V_TMPD) or with malate+glutamate+glycerol-3P+octanoate (Vmax substrates) were obtained in the presence of 2 mM ADP. They were all decreased in hypothyroid rats (CT n=8, HYPO n=8). B. Protein content of complexes of the respiratory chain (Cx I, II, III, V); upper part, mean values normalized to β-actin; lower part, representative Western blots. C: protein content of PGC-1α; upper part, mean values normalized to β-actin; lower part, representative Western blot of PGC-1α (upper lane) and β-actin (lower lane). * p<0.05, ** p<0.01, *** p<0.001 versus CT.

Figure 2. Effects of hypothyroidism on substrate utilization by mitochondria

A. Respiration rates were obtained by cumulative addition of glycerol-3P (G3P, 4mM), malate (4 mM), octanoyl-carnitine (0.4 mM), and pyruvate (1 mM). The increments in respiration rates are plotted to show the substrate preference of control and hypothyroid cardiac fibers (CT n=16, HYPO n=8). B. Enzymatic activity of total and mitochondrial MDH. C. mRNA expression of proteins of the β-oxidation pathway. PPAR: peroxisome proliferator activated receptor; MCAD: middle chain AcylCoA dehydrogenase; mCPTI: muscle carnitine palmitoyl transferase I. * p<0.05, ** p<0.01, *** p<0.001 versus CT.

Figure 3. Effects of hypothyroidism on AMPK and p38 MAPK phosphorylation

A. upper part, mean values of AMPKα1 and α2 protein content and of the ratio of phosphorylated over total AMPK; lower part, representative Western blot showing the decrease in phosphorylated AMPK, without changes in total AMPK or α1 and α2 subunits. B. upper part, mean values of phosphorylated p38 MAPK over total p38; lower part, representative Western blot showing the decrease in phosphorylated p38 without significant changes in total p38. * p<0.05 versus CT.

Figure 4. Effects of hypothyroidism on calcineurin pathway

A. Western blot analysis of calcineurin content, normalized to β-actin. B. Real-time quantitative RT-PCR analysis of mRNA content of modulatory calcineurin interacting protein 1 (MCIP1) and 2 (MCIP2), normalized to cyclophilin A and expressed as arbitrary units (CT n=8, HYPO n=8). * p<0.05 versus CT.