Peter Razeghi

Return to the fetal gene program protects the stressed heart: a strong hypothesis

A common feature of the hemodynamically or metabolically stressed heart is the return to a pattern of fetal metabolism. A hallmark of fetal metabolism is the predominance of carbohydrates as substrates for energy provision in a relatively hypoxic environment. When the normal heart is exposed to an oxygen rich environment after birth, energy substrate metabolism is rapidly switched to oxidation of fatty acids. This switch goes along with the expression of “adult” isoforms of metabolic enzymes and other proteins. However, the heart retains the ability to return to the “fetal” gene program. Specifically, the fetal gene program is predominant in a variety of pathophysiologic conditions including hypoxia, ischemia, hypertrophy, and atrophy. A common feature of all of these conditions is extensive remodeling, a decrease in the rate of aerobic metabolism in the cardiomyocyte, and an increase in cardiac efficiency. The adaptation is associated with a whole program of cell survival under stress. The adaptive mechanisms are prominently developed in hibernating myocardium, but they are also a feature of the failing heart muscle. We propose that in failing heart muscle at a certain point the fetal gene program is no longer sufficient to support cardiac structure and function. The exact mechanisms underlying the transition from adaptation to cardiomyocyte dysfunction are still not completely understood.

Linking Gene Expression to Function: Metabolic Flexibility in the Normal and Diseased Heart

Abstract: Metabolism transfers energy from substrates to ATP. As a “metabolic omnivore,” the normal heart adapts to changes in the environment by switching from one substrate to another. We propose that this flexibility is lost in the maladapted, diseased heart. Both adaptation and maladaptation are the results of metabolic signals that regulate transcription of key cardiac regulatory genes. We propose that metabolic remodeling precedes, initiates, and sustains functional and structural remodeling. The process of metabolic remodeling then becomes a target for pharmacological intervention restoring metabolic flexibility and normal contractile function of the heart.

Downregulation of metabolic gene expression in failing human heart before and after mechanical unloading.

Background: We have previously shown that several metabolic genes are downregulated in the failing human heart. We now tested the hypothesis that mechanical unloading might reverse this process. Methods: Clinical data and myocardial tissue were collected from 14 failing hearts paired for the time of implantation and explantation of a left ventricular assist device (LVAD) and compared to 10 non-failing hearts. Transcript levels for key regulators of energy metabolism and for atrial natriuretic factor (ANF) were measured by real-time quantitative RT-PCR. Results: The expression of the glucose transporter 1 and 4 (GLUT1, GLUT4), muscle carnitine palmitoyl transferase-1 (mCPT-1), and uncoupling protein 3 (UCP3) were downregulated by up to 80% in the failing heart. Although LVAD treatment improved clinical markers of heart failure (decrease of left ventricular diastolic dimension and normalization of serum sodium), only UCP3 expression reversed to non-failing transcript levels following mechanical unloading. Conclusions: LVAD treatment only partially reverses depressed metabolic gene expression in the failing human heart. Reversal of depressed UCP3 expression may be an important mechanism for reducing the formation of oxygen-derived free radicals. Further studies are necessary to define the effects of mechanical unloading on post-transcriptional mechanisms.

Atrophic Remodeling of the Heart In Vivo Simultaneously Activates Pathways of Protein Synthesis and Degradation

Background—Mechanical unloading of the heart results in atrophic remodeling. In skeletal muscle, atrophy is associated with inactivation of the mammalian target of rapamycin (mTOR) pathway and upregulation of critical components of the ubiquitin proteosome proteolytic (UPP) pathway. The hypothesis is that mechanical unloading of the mammalian heart has differential effects on pathways of protein synthesis and degradation. Methods and Results—In a model of atrophic remodeling induced by heterotopic transplantation of the rat heart, we measured gene transcription, protein expression, polyubiquitin content, and regulators of the mTOR pathway at 2, 4, 7, and 28 days. In atrophic hearts, there was an increase in polyubiquitin content that peaked at 7 days and decreased by 28 days. Furthermore, gene and protein expression of UbcH2, a ubiquitin conjugating enzyme, was also increased early in the course of unloading. Transcript levels of TNF-&agr;, a known regulator of UbcH2-dependent ubiquitin conjugating activity, were upregulated early and transiently in the atrophying rat heart. Unexpectedly, p70S6K and 4EBP1, downstream components of mTOR, were activated in atrophic rat heart. This activation was independent of Akt, a known upstream regulator of mTOR. Rapamycin treatment of the unloaded rat hearts inhibited the activation of p70S6K and 4EBP1 and subsequently augmented atrophy in these hearts compared with vehicle-treated, unloaded hearts. Conclusions—Atrophy of the heart, secondary to mechanical unloading, is associated with early activation of the UPP. The simultaneous activation of the mTOR pathway suggests active remodeling, involving both protein synthesis and degradation.

Degree of cardiac fibrosis and hypertrophy at time of implantation predicts myocardial improvement during left ventricular assist device support

BACKGROUND There have been increasing reports of cardiac improvement in heart failure patients supported by left ventricular assist devices (LVADs i.e.), including a number of patients who have tolerated removal of the device without the benefit of cardiac transplant. In the current study, we retrospectively investigated echocardiographic and histologic changes in patients supported by LVADs (n = 18). The goal of our study was to determine if the degree of cardiac fibrosis and myocyte size in pre-implant biopsies could predict myocardial improvement as assessed by improvements in ejection fraction (EF) during LVAD support. METHODS We determined total collagen content in myocardial biopsy specimens by a semi-quantitative analysis of positive Picro-Sirius Red-stained areas and myocyte size measurements by computerized edge detection software. RESULTS During LVAD support, 9 of the 18 patients (Group A) were distinguished by significant improvement in ejection fraction (pre <20% vs unloaded 34 +/- 5%). In addition, Group A patients had significantly less fibrosis and smaller myocytes than their Group B counterparts, whose EF did not improve. There was an inverse correlation between pre-implant biopsy collagen levels and myocyte size with increases in EF during LVAD unloading. CONCLUSIONS We found that the patients who demonstrated the greatest improvements in EF during support had less fibrosis and smaller myocytes at the time of device implantation. We propose that tissue profiling a patients pre-implant biopsy for fibrosis and myocyte size may allow stratification in Stage IV heart failure and may predict myocardial improvement during LVAD support.

Proposed Regulation of Gene Expression by Glucose in Rodent Heart

Background During pressure overload-induced hypertrophy, unloading-induced atrophy, and diabetes mellitus, the heart induces ‘fetal’ genes (e.g. myosin heavy chain β; mhcβ). Hypothesis We propose that altered glucose homeostasis within the cardiomyocyte acts as a central mechanism for the regulation of gene expression in response to environmental stresses. The evidence is as follows. Methods and Results Forced glucose uptake both ex vivo and in vivo results in mhc isoform switching. Restricting dietary glucose prevents mhc isoform switching in hearts of both GLUT1-Tg mice and rats subjected to pressure overload-induced hypertrophy. Thus, glucose availability correlates with mhc isoform switching under all conditions investigated. A potential mechanism by which glucose affects gene expression is through O-linked glycosylation of specific transcription factors. Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the flux generating step in UDP-N-acetylglucosamine biosynthesis, the rate determining metabolite in protein glycosylation. Ascending aortic constriction increased intracellular levels of UDP-N-acetylglucosamine, and the expression of gfat2, but not gfat1, in the rat heart. Conclusions Collectively, the results strongly suggest glucose-regulated gene expression in the heart, and the involvement of glucose metabolites in isoform switching of sarcomeric proteins characteristic for the fetal gene program.

Atrophic Remodeling of the Transplanted Rat Heart

We have previously shown that the common feature of both pressure overload-induced hypertrophy and atrophy is a reactivation of the fetal gene program. Although gene expression profiles and signal transduction pathways in pressure overload hypertrophy have been well studied, little is known about the mechanisms underlying atrophic remodeling of the unloaded heart. Here, we induced atrophic remodeling by heterotopic transplantation of the rat heart. The activity parameters of three signal transduction pathways important in hypertrophy, i.e. mitogen-activated protein (MAP) kinase, mammalian target of rapamycin (mTOR), and Janus kinase/signal transducers and activators of transcription (JAK/STAT), were interrogated. Gene expression of upstream stimuli – insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) – and metabolic correlates, i.e. peroxisome proliferator-activated receptor-α (PPARα) and PPARα-regulated genes, of these pathways were also measured. In addition, we measured transcript levels of genes known to regulate skeletal muscle atrophy, all of which are negatively regulated by IGF-1 (Mafbx/Atrogin-1, MuRF-1). Atrophic remodeling of the heart was associated with increased expression of IGF-1 and FGF-2. Transcript levels of the nuclear receptor PPARα were decreased, as were the levels of PPARα-regulated genes. Furthermore, there was phosphorylation of ERK1, STAT3, and p70S6K with unloading. Consistent with the increase in IGF-1, we found a decrease in Mafbx/Atrogin-1 and MuRF-1 transcript levels. Rapamycin administration at 0.8 mg/kg/day for 7 days resulted in enhanced atrophy and attenuated the phosphorylation of ERK1, STAT3, and p70S6K without altering gene expression. We conclude that there is significant crosstalk between the mTOR, MAP kinase, and JAK/STAT signaling cascades. Furthermore, ubiquitin ligases, known to be essential for skeletal muscle atrophy, decrease in unloading-induced cardiac atrophy.

Hypoxia-induced switches of myosin heavy chain iso-gene expression in rat heart

Cardiac hypertrophy and atrophy increase expression of fetal iso-genes. A common factor is a decrease in cellular oxygen tension. To test the hypothesis that hypoxia changes cardiac MHC iso-gene expression Wistar rats were exposed to 24 and 48 h of hypobaric hypoxia (11% oxygen) and mRNA was isolated from the left ventricle. In addition, neonatal rat cardiomyocytes were incubated for up to 48 h in a hypoxic chamber. Transcript levels of MHCalpha (adult isoform), MHCbeta (fetal isoform), and Nkx2.5, the earliest known marker for cardiogenesis, were measured by real-time quantitative RT-PCR and normalized to levels of 18S rRNA. Expression of the transcription factor Nkx2.5 increased with hypoxia. Hypoxia decreased MHCalpha and increased MHCbeta transcript levels, both in vivo and in vitro. We conclude that hypoxia per se induces a pattern of isoform gene expression associated with early cardiac development.

Reverse remodeling of the failing human heart with mechanical unloading. Emerging concepts and unanswered questions

Anecdotal and clinical evidence suggests that mechanical unloading may restore cardiac function by inducing changes in the biological properties of the failing heart. This challenges the notion that the progression of end-stage heart failure is an irreversible process ending in either death of the patient or transplantation. Although it is still not clear how mechanical unloading of the failing heart improves cardiac function, the process likely involves adaptive responses of cardiac myocytes on the cellular, extracellular, and subcellular levels.

Mechanical Unloading of the Failing Human Heart Fails to Activate the Protein Kinase B/Akt/Glycogen Synthase Kinase-3β Survival Pathway

Background: Left ventricular assist device (LVAD) support of the failing human heart improves myocyte function and increases cell survival. One potential mechanism underlying this phenomenon is activation of the protein kinase B (PKB)/Akt/glycogen synthase kinase-3beta (GSK-3β) survival pathway. Methods and Results: Left ventricular tissue was obtained both at the time of implantation and explantation of the LVAD (n = 11). Six patients were diagnosed with idiopathic dilated cardiomyopathy, 4 patients with ischemic cardiomyopathy and 1 patient with peripartum cardiomyopathy. The mean duration of LVAD support was 205 ± 35 days. Myocyte diameter and phosphorylation of ERK were used as indices for reverse remodeling. Transcript levels of genes required for the activation of PKB/Akt (insulin-like growth factor-1, insulin receptor substrate-1) were measured by quantitative RT-PCR. In addition, we measured the relative activity of PKB/Akt and GSK-3β, and assayed for molecular and histological indices of PKB/Akt activation (cyclooxygenase mRNA levels and glycogen levels). Myocyte diameter and phosphorylation of ERK decreased with LVAD support. In contrast, none of the components of the PKB/Akt/GSK-3β pathway changed significantly with mechanical unloading. Conclusion: The PKB/Akt/GSK-3β pathway is not activated during LVAD support. Other signaling pathways must be responsible for the improvement of cellular function and cell survival during LVAD support.