Dynamic patterns of ventricular remodeling and apoptosis in hearts unloaded by heterotopic transplantation

Abstract

Aims Mechanical unloading of failing hearts can trigger functional recovery, but results in progressive atrophy and possibly detrimental adaptation. In an unbiased approach, we examined dynamic effects of unloading duration on molecular markers indicative of myocardial damage, hypothesizing that potential recovery may be improved by optimized unloading time. Methods Heterotopically transplanted normal rat hearts were harvested at 3, 8, 15, 30 and 60 days. 47 genes were analyzed using TaqMan-based microarray, western blot and immunohistochemistry. Results In parallel with marked atrophy (22% to 64% volume loss at 3 resp. 60 days), expression of myosin-heavy-chain isoforms (MHC-α/-β) was characteristically switched in a time-dependent manner. Genes involved in tissue remodeling (FGF-2, CTGF, TGFb, IGF-1) were increasingly upregulated with duration of unloading. A distinct pattern was observed for genes involved in generation of contractile force; an indiscriminate early downregulation was followed by a new steady state below normal. For pro-apoptotic transcripts bax, bnip-3, cCasp-6 and -9, mRNA levels demonstrated a slight increase up to 30 days unloading with pronunciation at 60 days. Findings regarding cell death were confirmed on the protein level. Proteasome activity indicated early increase of protein degradation, but decreased below baseline in 60 days-unloaded hearts. Conclusions We identified incrementally increased apoptosis after myocardial unloading of the normal rat heart, which is exacerbated at late time-points (60 days) and inversely related to loss of myocardial mass. Our findings suggest an irreversible detrimental impact of long-term unloading on myocardium that may be precluded by partial reloading and amenable to molecular therapeutic intervention.

Rat Heterotopic Heart Transplantation Model to Investigate Unloading-Induced Myocardial Remodeling

“…In hypertrophic and failing hearts, unloading reverses pathological enlargement of cardiac myocytes and normalizes myocardial mass, while in heterotopically transplanted normal hearts, we find reduction of cellular diameters as well as a relative decrease of myocytes compared with connective tissue, this being termed atrophy ( 19 , 20 ). While the normalization of hypertrophy in the failing unloaded heart can be explained by the disappearance of increased wall stress, which goes along with the initiation of a similar gene expression program in both hypertrophy and after unloading ( 21 ), the atrophy following unloading of a normal rat heart must be an active adaptation to the loss of volume load, which includes autophagy and apoptosis ( 22 ).…”

“…The underlying mechanisms are not clear. It was reported that there is a significant increase in caspase-3 mRNA expression and activity after mechanical unloading in a normal unloaded heart ( 22 , 27 , 28 ). In addition, relatively late expression changes in genes involved in the apoptosis process were found at the protein level.…”

“…Increased protein expression of bax resulted in a higher apoptotic index (bax/bcl-2 ratio) after 30 and 60 days of unloading. Expression of the antiapoptotic protein bcl-2 was only decreased at the mRNA level, but not at the protein level ( 22 ).…”

Hold or fold—Proteins in advanced heart failure and myocardial recovery

“…In AHF, the 20S proteasome abundance and activity are decreased commensurate with accumulation of ubiquitinated proteins, suggesting impairment of proteasome-dependent degradation, and reversal of this has been observed with mechanical unloading [51,52]. Autophagy markers including Beclin-1, Atg5-Atg12 conjugate, and LC3-II, which are increased in hypertrophy, are reduced with mechanical unloading in humans and rodents [52,53]. Collectively, these studies indicate that protein and cellular homeostasis mechanisms may be normalized at the protein level by MCS.…”

Mesenchymal stromal cells improve cardiac function and left ventricular remodeling in a heart transplantation model

“…These models are considered "non-volumeloaded" (NL), and have been utilized in almost 90% of published studies since 2000. 33 Brinks et al 34 identified the kinetics of load-dependent regulation of myocardial composition over time in a pre-clinical model to investigate strategies that potentially enhance myocardial recovery. They demonstrated that the HHT model in rats is relevant for human disease.…”