This study indicates that LVAD therapy does not reverse many of the transcriptional changes associated with heart failure and increased the expression of PDK4, a key regulator of glycolysis. Heart failure is a complex disease process where transcriptional changes contribute to contractile dysfunction, arrhythmias, and ultimately cell death [20]. These ongoing changes in gene expression likely contribute to poor rates of myocardial recovery post-LVAD therapy and ongoing issues with symptomatic heart failure as well as arrhythmias. LVADs are currently being utilized as destination therapy, bridge to transplantation or bridge to recovery. Despite the device indication only 1% of patients are being explanted for recovery of cardiac function suggesting new therapies or approaches are necessary to achieve this goal [21]. Long periods of changes in gene expression could be related to ongoing changes in metabolism or continued DNA methylation.
The main finding at this study is that LVAD therapy increases PDK4 expression. The heart consumes more energy than any other organ and is capable of metabolizing carbohydrates, fatty acids and amino acids in order to meet its needs. Measured metabolites of glucose metabolism, amino acids, creatinine and citric acid cycle intermediates are diminished in heart failure contributing to an overall energy depleted state [6]. Many of these metabolites are normalized with LVAD therapy along with improvements in hemoglobin A1C (HbA1c), fasting plasma glucose, and daily insulin requirements [22]. Unfortunately, abnormalities the citric acid cycle persist post-LVAD likely contributing to ongoing contractile dysfunction [6, 23]. In heart failure there is a switch from fatty acids (β-oxidation) to carbohydrates (glycolysis) as a fuel preference [24]. PDK4 is a key regulator of glucose metabolism and it’s expression is elevated in heart failure and worsened by LVAD therapy in the current study [25]. These changes would contribute to an ongoing preference for glycolysis over β-oxidation in heart failure and post-LVAD hearts. Both overexpression and under-expression of PDK4 can contribute to contractile dysfunction. For example, deficiency and mutation in PDK4 gene leads to apoptosis and dilated cardiomyopathy in Doberman Pinschers [26, 27], whereas PDK4 overexpression perturbs metabolism and worsens calcineurin-induced cardiomyopathy [28]. PDK expression is regulated by numerous ligands including insulin, epinephrine, and adiponectin, along with nuclear hormone receptors such as peroxisome proliferator-activated, glucocorticoid, estrogen, and thyroid receptors [29].
PER1 expression is decreased in heart failure and increased significantly in post-LVAD compared to pre-LVAD conditions (Figure 1). PER1 is a clock gene and involved in rhythmic expression in human hearts [30]. PER1 also controls blood pressure and normalizes renal sodium transport protein levels [31]. Circadian clock proteins have potential effects on myocardial gene expression, metabolism, and function within hearts [32]. Circadian rhythm signaling was the only major pathway in IPA analysis when compared between post- and pre-LVAD hearts. Increased expression of PER1 was insufficient to alter the overall transcriptional profile of heart failure in these LVAD patients.
Oxidative stress contributes to numerous different diseases including diabetes and heart failure through activation of stress pathways involving serine/threonine kinases which have a negative effect on insulin signaling [33]. Excessive levels of ROS and free radicals can cause DNA damage, modify proteins and cause cellular injury. Excess generation of these reactive molecules can arise from several sources including mitochondria, NAD(P)H oxidase, xanthine oxidase and uncoupled nitric oxide synthase [8]. In this study, LVAD therapy did not impact levels of free radicals, as measured by EPR Spectrosopy. These chronic increases in free radicals likely contribute to a further decline in myocardial function and ongoing changes in myocardial gene expression. Pathway analysis suggests many of these changes are attributable to alterations in DNA methylation. Our results showed that inhibition of micro RNA21 (mir21) was also significant on pathway analysis in the post-LVAD population with a Z-score of -2.00. Mir21 plays a vital role in vascular smooth muscle cell proliferation and apoptosis, cardiac cell growth and death, and cardiac fibroblast functions. Mir21 targeted Phosphatase and tensin homolog (PTEN), Programmed cell death protein 4 (PDCD4), Protein sprouty homolog 1 (SPRY1) and Sprouty homolog 2(SPRY2) and effects on their expression and eventually that influences on cardiovascular system [34]. Other groups have also demonstrated an increase in oxidative stress and abnormalities in DNA post-LVAD therapy [7, 35]. Mondal et al also demonstrated that patients post-LVAD had higher levels of ROS, DNA damage in leukocytes and abnormalities in DNA repair. There are several possible explanations as to why LVAD therapy does not improve oxidative stress. Many patients with LVADs have ongoing issues with heart failure, arrhythmias, anemia due to hemolysis as well as inflammation from surgery or infections. While medicines can have antioxidant effects and aid myocardial recovery, guideline directed medical therapy is often poorly tolerated in patients with advanced heart failure, and poorly utilized after LVAD therapy. We previously have found only 1/3 of patients are on beta-blockers three months post-LVAD [36]. New approaches may be necessary to metabolically reprogram the heart after LVAD therapy in order to improve myocardial energetics and facilitate recovery.
Previous transcriptional studies utilizing oligonucleotide microarrays have shown that LVAD placement induces significant down regulation of myocardial and inflammatory gene expression including brain natriuretic peptide, collagen, dystrophin, interleukin 8, metaloprotein and tumor necrosis factor α (TNFα) [37]. Several recent studies have demonstrated that LVAD support results in alterations in gene expression, including (TNFα) [38, 39], Her2/neu, Her4 [27, 39] and glucose transporter 1 and 4 [40]. Further, Chen et al have shown that Endothelial nitric oxide synthase (eNOS) and Dimethylarginine Dimethylaminohydrolase 1 (DDAH1) expression are significantly increased after LVAD support compared to pre-LVAD [41]. A recent study demonstrated a significant decrease in the expression of genes that boost a healthy immune response that was partially recovered after 6 months of LVAD support [42] and proteins involved in cytoskeleton and mitochondrial energy metabolisms significantly downregulated post-LVAD [43].
This work was supported by a pilot grant to explore transcriptional changes in association of LVAD therapy along with changes to oxidative species. We plan additional studies with the next generation of left ventricular assist devices along with newer medical therapy to hopefully further aid recovery in this population.
Limitations
This study has several limitations. Only a targeted panel of genes previously demonstrated to be abnormal in heart failure were evaluated and much broader transcriptional changes are known to occur with LVAD therapy. Our analysis was confined to genes we previously have associated with contractile dysfunction in heart failure to limit false discovery. In addition, this study is limited to translational data and proteomics and metabolic studies are needed to further understand the biology of these changes. Our sample size was limited to 14 as part of a pilot study and patients were predominantly male Caucasians so our results may not be generalizable to the broader population. The scope of the current study does not allow for in depth bioinformatics of protein interactions and numerous confounders potentially exist. LVAD therapy continues to evolve and the next generation of LVADs is less thrombotic and has shown an improvement in clinical outcomes [44]. New pumps however, while more hemocompatible with patients, have not yet demonstrated improvements in myocardial recovery or remission from heart failure. Finally, the medical therapy of these patients was not standardized and it is not feasible to adjust for the confounding effects of medications on gene expression or oxidative stress. Further studies are needed to better understand the mechanisms of cardiac recovery with LVAD therapy, interact action with newer devices, and risk factors for non-responders.