Remote ischaemic preconditioning-evoked intracellular signalling pathways in cardiac maturation and disease

© 2013 Dr. Norman Yu Hui LiawBackground & Aims: Children with congenital heart defects are burdened with additional stress on the myocardium, which is exacerbated by cardiopulmonary bypass (CPB) and cardiac surgery. Ischaemic preconditioning (IPC) induced by brief, intermittent periods of ischaemia and reperfusion (IR) on the coronary vasculature can activate intrinsic protective mechanisms. However, remote IPC (RIPC) induced by inflating and deflating a standard blood-pressure cuff attached to a limb is a practical, non-invasive and clinically applicable model for protection against sustained ischaemic injury. RIPC regulates phosphorylation of key intracellular proteins that are recruited by IR and propagate signalling for metabolic control of the heart. Agonism of G-protein coupled receptors promotes protein signalling via key kinases such as Akt, p38MAPK, GSK3β, and HSP27 amongst other important effectors for cell survival against IR injury. However, most RIPC studies have focussed on adult myocardium. There is a dearth of such studies in immature myocardium. Thus, the aims of this Thesis were to: • test the efficacy of RIPC in a double-blind randomised trial in patients undergoing cardiac surgery for tetralogy of Fallot (ToF) and to measure key signalling protein kinases; • determine whether these signalling pathways are developed in murine neonatal hearts, and to compare their activation and ability to functionally recover after sublethal IR compared to adults; • determine the efficacy of RIPC in immature (4 weeks) and adult (12 weeks) hearts to functionally recover and express kinase signalling proteins after sublethal IR; • examine the effects of chronic hypoxia and fentanyl on immature cardiomyocytes on modulating kinase signalling proteins; • characterise the ability for advanced age (72 weeks) murine hearts to functionally recover and express kinase signalling proteins after sublethal IR. Methods: Limb RIPC was induced by four 5 min periods of inflation (ischaemia) and deflation (reperfusion) of a pressure cuff. IR occurred in ToF neonates during cardiac surgery utilising CPB, and in murine hearts during Langendorff-mode isolated heart perfusion. Resected right ventricular outflow tract myocardium from neonates and murine heart protein homogenates were assayed for protein expression by western immunoblotting. Effects of chronic hypoxia and fentanyl were studied in immature cardiomyocytes differentiated from murine P19 cells. Results: The major findings from this Thesis are: • remotely preconditioned ToF neonates did not have different expression of pro-survival signalling proteins relative to sham controls, which had a high proportion of phospho-kinase activation masking the effects of RIPC; • complex proteomic changes and a greater ability to functionally recover from sublethal IR were evident in immature murine hearts relative to the adult; • remotely preconditioned immature murine hearts had improved left ventricular (LV) functional recovery and an increase in the total available pool of protein kinases available for phosphorylation; • immature cardiomyocytes exposed to chronic hypoxia had an increase in total abundance of protein kinases available for phosphorylation; hypoxia blunted the effect of the opioid receptor agonist fentanyl; • advanced age hearts had impaired LV functional recovery, with a greater propensity for apoptosis and equitable levels of necrosis and autophagy, relative to adults. Conclusions: A protective role exists for RIPC in immature murine hearts exposed to IR despite undetectable protection in RIPC neonates undergoing ToF surgery. An extrapolation of these findings indicates confounders related to cyanotic disease progression (adaptation to chronic hypoxia) and pharmacological intervention may limit protection by RIPC in the surgical setting

Adaptations in Protein Expression and Regulated Activity of Pyruvate Dehydrogenase Multienzyme Complex in Human Systolic Heart Failure

Pyruvate dehydrogenase (PDH) complex, a multienzyme complex at the nexus of glycolytic and Krebs cycles, provides acetyl-CoA to the Krebs cycle and NADH to complex I thus supporting a critical role in mitochondrial energy production and cellular survival. PDH activity is regulated by pyruvate dehydrogenase phosphatases (PDP1, PDP2), pyruvate dehydrogenase kinases (PDK 1-4), and mitochondrial pyruvate carriers (MPC1, MPC2). As NADH-dependent oxidative phosphorylation is diminished in systolic heart failure, we tested whether the left ventricular myocardium (LV) from end-stage systolic adult heart failure patients (n=26) exhibits altered expression of PDH complex subunits, PDK, MPC, PDP, and PDH complex activity, compared to LV from nonfailing donor hearts (n=21). Compared to nonfailing LV, PDH activity and relative expression levels of E2, E3bp, E1α, and E1β subunits were greater in LV failure. PDK4, MPC1, and MPC2 expressions were decreased in failing LV, whereas PDP1, PDP2, PDK1, and PDK2 expressions did not differ between nonfailing and failing LV. In order to examine PDK4 further, donor human LV cardiomyocytes were induced in culture to hypertrophy with 0.1 μM angiotensin II and treated with PDK inhibitors (0.2 mM dichloroacetate, or 5 mM pyruvate) or activators (0.6 mM NADH plus 50 μM acetyl CoA). In isolated hypertrophic cardiomyocytes in vitro, PDK activators and inhibitors increased and decreased PDK4, respectively. In conclusion, in end-stage failing hearts, greater expression of PDH proteins and decreased expression of PDK4, MPC1, and MPC2 were evident with higher rates of PDH activity. These adaptations support sustained capacity for PDH to facilitate glucose metabolism in the face of other failing bioenergetic pathways

Postnatal shifts in ischemic tolerance and cell survival signaling in murine myocardium

The immature heart is known to be resistant to ischemia-reperfusion (I/R) injury; however, key proteins engaged in phospho-dependent signaling pathways crucial to cell survival are not yet defined. Our goal was to determine the postnatal changes in myocardial tolerance to I/R, including baseline expression of key proteins governing I/R tolerance and their phosphorylation during I/R. Hearts from male C57Bl/6 mice (neonates, 2, 4, 8, and 12 wk of age, n = 6/group) were assayed for survival signaling/effectors [Akt, p38MAPK, glycogen synthase kinase-3 beta (GSK-3 beta), heat shock protein 27 (HSP27), connexin-43, hypoxiainducible factor-1 alpha (HIF-1 alpha), and caveolin-3] and regulators of apoptosis (Bax and Bcl-2) and autophagy (LC3B, Parkin, and Beclin1). The effect of I/R on ventricular function was measured in isolated perfused hearts from immature (4 wk) and adult (12 wk) mice. The neonatal myocardium exhibits a large pool of inactive Akt; high phospho-activation of p38MAPK, HSP27 and connexin-43; phosphoinhibition of GSK-3 beta; and high expression of caveolin-3, HIF-1 alpha, LC3B, Beclin1, Bax, and Bcl-2. Immature hearts sustained less dysfunction and infarction following I/R than adults. Emergence of I/R intolerance in adult vs. immature hearts was associated with complex proteomic changes: decreased expression of Akt, Bax, and Bcl-2; increased GSK-3 beta, connexin-43, HIF-1 alpha, LC3B, and Bax: Bcl-2; enhanced postischemic HIF-1 alpha, caveolin-3, Bax, and Bcl-2; and greater postischemic GSK-3 beta and HSP27 phosphorylation. Neonatal myocardial stress resistance reflects high expression of prosurvival and autophagy proteins and apoptotic regulators. Notably, there is high phosphorylation of GSK-3 beta, p38MAPK, and HSP27 and low phosphorylation of Akt (high Akt "reserve"). Subsequent maturation-related reductions in I/R tolerance are associated with reductions in Akt, Bcl-2, LC3B, and Beclin1, despite increased expression and reduced phospho-inhibition of GSK-3 bet

Regenerative potential of epicardium-derived extracellular vesicles mediated by conserved miRNA transfer

AIMS: After a myocardial infarction, the adult human heart lacks sufficient regenerative capacity to restore lost tissue, leading to heart failure progression. Finding novel ways to reprogram adult cardiomyocytes into a regenerative state is a major therapeutic goal. The epicardium, the outermost layer of the heart, contributes cardiovascular cell types to the forming heart and is a source of trophic signals to promote heart muscle growth during embryonic development. The epicardium is also essential for heart regeneration in zebrafish and neonatal mice and can be reactivated after injury in adult hearts to improve outcome. A recently identified mechanism of cell–cell communication and signalling is that mediated by extracellular vesicles (EVs). Here, we aimed to investigate epicardial signalling via EV release in response to cardiac injury and as a means to optimize cardiac repair and regeneration. METHODS AND RESULTS: We isolated epicardial EVs from mouse and human sources and targeted the cardiomyocyte population. Epicardial EVs enhanced proliferation in H9C2 cells and in primary neonatal murine cardiomyocytes in vitro and promoted cell cycle re-entry when injected into the injured area of infarcted neonatal hearts. These EVs also enhanced regeneration in cryoinjured engineered human myocardium (EHM) as a novel model of human myocardial injury. Deep RNA-sequencing of epicardial EV cargo revealed conserved microRNAs (miRs) between human and mouse epicardial-derived exosomes, and the effects on cell cycle re-entry were recapitulated by administration of cargo miR-30a, miR-100, miR-27a, and miR-30e to human stem cell-derived cardiomyocytes and cryoinjured EHM constructs. CONCLUSION: Here, we describe the first characterization of epicardial EV secretion, which can signal to promote proliferation of cardiomyocytes in infarcted mouse hearts and in a human model of myocardial injury, resulting in enhanced contractile function. Analysis of exosome cargo in mouse and human identified conserved pro-regenerative miRs, which in combination recapitulated the therapeutic effects of promoting cardiomyocyte proliferation

Catecholamine-dependent β-adrenergic signaling in a pluripotent stem cell model of takotsubo cardiomyopathy

BACKGROUND: Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction and is associated with life-threating complications in the acute phase. The underlying disease mechanism in TTS is still unknown. A genetic basis has been suggested to be involved in the pathogenesis. OBJECTIVES: The aims of the study were to establish an in vitro induced pluripotent stem cell (iPSC) model of TTS, to test the hypothesis of altered β-adrenergic signaling in TTS iPSC-cardiomyocytes (CMs), and to explore whether genetic susceptibility underlies the pathophysiology of TTS. METHODS: Somatic cells of patients with TTS and control subjects were reprogrammed to iPSCs and differentiated into CMs. Three-month-old CMs were subjected to catecholamine stimulation to simulate neurohumoral overstimulation. We investigated β-adrenergic signaling and TTS cardiomyocyte function. RESULTS. Enhanced β-adrenergic signaling in TTS-iPSC-CMs under catecholamine-induced stress increased expression of the cardiac stress marker NR4A1; cyclic adenosine monophosphate levels; and cyclic adenosine monophosphate-dependent protein kinase A-mediated hyperphosphorylation of RYR2-S2808, PLN-S16, TNI-S23/24, and Cav1.2-S1928, and leads to a reduced calcium time to transient 50% decay. These cellular catecholamine-dependent responses were mainly mediated by β-adrenoceptor signaling in TTS. Engineered heart muscles from TTS-iPSC-CMs showed an impaired force of contraction and a higher sensitivity to isoprenaline-stimulated inotropy compared with control subjects. In addition, altered electrical activity and increased lipid accumulation were detected in catecholamine-treated TTS-iPSC-CMs, and were confirmed by differentially expressed lipid transporters CD36 and CPT1C. Furthermore, we uncovered genetic variants in different key regulators of cardiac function. CONCLUSIONS. Enhanced β-adrenergic signaling and higher sensitivity to catecholamine-induced toxicity were identified as mechanisms associated with the TTS phenotype. (International Takotsubo Registry [InterTAK Registry] [InterTAK]; NCT01947621)

KLF15-Wnt–Dependent Cardiac Reprogramming Up-Regulates SHISA3 in the Mammalian Heart

BACKGROUND The combination of cardiomyocyte (CM) and vascular cell (VC) fetal reprogramming upon stress culminates in end-stage heart failure (HF) by mechanisms that are not fully understood. Previous studies suggest KLF15 as a key regulator of CM hypertrophy. OBJECTIVES This study aimed to characterize the impact of KLF15-dependent cardiac transcriptional networks leading to HF progression, amenable to therapeutic intervention in the adult heart. METHODS Transcriptomic bioinformatics, phenotyping of Klf15 knockout mice, Wnt-signaling-modulated hearts, and pressure overload and myocardial ischemia models were applied. Human KLF15 knockout embryonic stem cells, engineered human myocardium, and human samples were used to validate the relevance of the identified mechanisms. RESULTS The authors identified a sequential, postnatal transcriptional repression mediated by KLF15 of pathways implicated in pathological tissue remodeling, including distinct Wnt-pathways that control CM fetal reprogramming and VC remodeling. The authors further uncovered a vascular program induced by a cellular crosstalk initiated by CM, characterized by a reduction of KLF15 and a concomitant activation of Wnt-dependent transcriptional signaling. Within this program, a so-far uncharacterized cardiac player, SHISA3, primarily expressed in VCs in fetal hearts and pathological remodeling was identified. Importantly, the KLF15 and Wnt codependent SHISA3 regulation was demonstrated to be conserved in mouse and human models. CONCLUSIONS The authors unraveled a network interplay defined by KLF15-Wnt dynamics controlling CM and VC homeostasis in the postnatal heart and demonstrated its potential as a cardiac-specific therapeutic target in HF. Within this network, they identified SHISA3 as a novel, evolutionarily conserved VC marker involved in pathological remodeling in HF. (C) 2019 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation