Epitranscriptomic regulation in fasting hearts: implications for cardiac health.

Cardiac tolerance to ischaemia can be increased by dietary interventions such as fasting, which is associated with significant changes in myocardial gene expression. Among the possible mechanisms of how gene expression may be altered are epigenetic modifications of RNA - epitranscriptomics. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two of the most prevalent modifications in mRNA. These methylations are reversible and regulated by proteins called writers, erasers, readers, and m6A-repelled proteins. We analysed 33 of these epitranscriptomic regulators in rat hearts after cardioprotective 3-day fasting using RT-qPCR, Western blot, and targeted proteomic analysis. We found that the most of these regulators were changed on mRNA or protein levels in fasting hearts, including up-regulation of both demethylases - FTO and ALKBH5. In accordance, decreased methylation (m6A+m6Am) levels were detected in cardiac total RNA after fasting. We also identified altered methylation levels in Nox4 and Hdac1 transcripts, both of which play a role in the cytoprotective action of ketone bodies produced during fasting. Furthermore, we investigated the impact of inhibiting demethylases ALKBH5 and FTO in adult rat primary cardiomyocytes (AVCMs). Our findings indicate that inhibiting these demethylases reduced the hypoxic tolerance of AVCMs isolated from fasting rats. This study showed that the complex epitranscriptomic machinery around m6A and m6Am modifications is regulated in the fasting hearts and might play an important role in cardiac adaptation to fasting, a well-known cardioprotective intervention.

Dysregulation of hypoxia-inducible factor 1α in the sympathetic nervous system accelerates diabetic cardiomyopathy.

BACKGROUND: An altered sympathetic nervous system is implicated in many cardiac pathologies, ranging from sudden infant death syndrome to common diseases of adulthood such as hypertension, myocardial ischemia, cardiac arrhythmias, myocardial infarction, and heart failure. Although the mechanisms responsible for disruption of this well-organized system are the subject of intensive investigations, the exact processes controlling the cardiac sympathetic nervous system are still not fully understood. A conditional knockout of the Hif1a gene was reported to affect the development of sympathetic ganglia and sympathetic innervation of the heart. This study characterized how the combination of HIF-1α deficiency and streptozotocin (STZ)-induced diabetes affects the cardiac sympathetic nervous system and heart function of adult animals. METHODS: Molecular characteristics of Hif1a deficient sympathetic neurons were identified by RNA sequencing. Diabetes was induced in Hif1a knockout and control mice by low doses of STZ treatment. Heart function was assessed by echocardiography. Mechanisms involved in adverse structural remodeling of the myocardium, i.e. advanced glycation end products, fibrosis, cell death, and inflammation, was assessed by immunohistological analyses. RESULTS: We demonstrated that the deletion of Hif1a alters the transcriptome of sympathetic neurons, and that diabetic mice with the Hif1a-deficient sympathetic system have significant systolic dysfunction, worsened cardiac sympathetic innervation, and structural remodeling of the myocardium. CONCLUSIONS: We provide evidence that the combination of diabetes and the Hif1a deficient sympathetic nervous system results in compromised cardiac performance and accelerated adverse myocardial remodeling, associated with the progression of diabetic cardiomyopathy.

HIF-1α is required for development of the sympathetic nervous system.

The molecular mechanisms regulating sympathetic innervation of the heart during embryogenesis and its importance for cardiac development and function remain to be fully elucidated. We generated mice in which conditional knockout (CKO) of the Hif1a gene encoding the transcription factor hypoxia-inducible factor 1α (HIF-1α) is mediated by an Islet1-Cre transgene expressed in the cardiac outflow tract, right ventricle and atrium, pharyngeal mesoderm, peripheral neurons, and hindlimbs. These Hif1aCKO mice demonstrate significantly decreased perinatal survival and impaired left ventricular function. The absence of HIF-1α impaired the survival and proliferation of preganglionic and postganglionic neurons of the sympathetic system, respectively. These defects resulted in hypoplasia of the sympathetic ganglion chain and decreased sympathetic innervation of the Hif1aCKO heart, which was associated with decreased cardiac contractility. The number of chromaffin cells in the adrenal medulla was also decreased, indicating a broad dependence on HIF-1α for development of the sympathetic nervous system.

Excess ischemic tachyarrhythmias trigger protection against myocardial infarction in hypertensive rats.

Increased level of C-reactive protein (CRP) is a risk factor for cardiovascular diseases, including myocardial infarction and hypertension. Here, we analyzed the effects of CRP overexpression on cardiac susceptibility to ischemia/reperfusion (I/R) injury in adult spontaneously hypertensive rats (SHR) expressing human CRP transgene (SHR-CRP). Using an in vivo model of coronary artery occlusion, we found that transgenic expression of CRP predisposed SHR-CRP to repeated and prolonged ventricular tachyarrhythmias. Excessive ischemic arrhythmias in SHR-CRP led to a significant reduction in infarct size (IS) compared with SHR. The proarrhythmic phenotype in SHR-CRP was associated with altered heart and plasma eicosanoids, myocardial composition of fatty acids (FAs) in phospholipids, and autonomic nervous system imbalance before ischemia. To explain unexpected IS-limiting effect in SHR-CRP, we performed metabolomic analysis of plasma before and after ischemia. We also determined cardiac ischemic tolerance in hearts subjected to remote ischemic perconditioning (RIPer) and in hearts ex vivo. Acute ischemia in SHR-CRP markedly increased plasma levels of multiple potent cardioprotective molecules that could reduce IS at reperfusion. RIPer provided IS-limiting effect in SHR that was comparable with myocardial infarction observed in naïve SHR-CRP. In hearts ex vivo, IS did not differ between the strains, suggesting that extra-cardiac factors play a crucial role in protection. Our study shows that transgenic expression of human CRP predisposes SHR-CRP to excess ischemic ventricular tachyarrhythmias associated with a drop of pump function that triggers myocardial salvage against lethal I/R injury likely mediated by protective substances released to blood from hypoxic organs and tissue at reperfusion.

Renal Sympathetic Denervation Attenuates Congestive Heart Failure in Angiotensin II-Dependent Hypertension: Studies with Ren-2 Transgenic Hypertensive Rats with Aortocaval Fistula.

OBJECTIVE: We examined if renal denervation (RDN) attenuates the progression of aortocaval fistula (ACF)-induced heart failure or improves renal hemodynamics in Ren-2 transgenic rats (TGR), a model of angiotensin II (ANG II)-dependent hypertension. METHODS: Bilateral RDN was performed 1 week after creation of ACF. The animals studied were ACF TGR and sham-operated controls, and both groups were subjected to RDN or sham denervation. In separate groups, renal artery blood flow (RBF) responses were determined to intrarenal ANG II (2 and 8 ng), norepinephrine (NE) (20 and 40 ng) and acetylcholine (Ach) (10 and 40 ng) 3 weeks after ACF creation. RESULTS: In nondenervated ACF TGR, the final survival rate was 10 versus 50% in RDN rats. RBF was significantly lower in ACF TGR than in sham-operated TGR (6.2 ± 0.3 vs. 9.7 ± 0.5 mL min-1 g-1, p < 0.05), the levels unaffected by RDN. Both doses of ANG II decreased RBF more in ACF TGR than in sham-operated TGR (-19 ± 3 vs. -9 ± 2% and -47 ± 3 vs. -22 ± 2%, p < 0.05 in both cases). RDN did not alter RBF responses to the lower dose, but increased it to the higher dose of ANG II in sham-operated as well as in ACF TGR. NE comparably decreased RBF in ACF TGR and sham-operated TGR, and RDN increased RBF responsiveness. Intrarenal Ach increased RBF significantly more in ACF TGR than in sham-operated TGR (29 ± 3 vs. 17 ± 3%, p < 0.05), the changes unaffected by RDN. ACF creation induced marked bilateral cardiac hypertrophy and lung congestion, both attenuated by RDN. In sham-operated but not in ACF TGR, RDN significantly decreased mean arterial pressure. CONCLUSION: The results show that RDN significantly improved survival rate in ACF TGR; however, this beneficial effect was not associated with improvement of reduced RBF or with attenuation of exaggerated renal vascular responsiveness to ANG II.

Programmed Cell Death in the Left and Right Ventricle of the Late Phase of Post-Infarction Heart Failure.

While necroptosis has been shown to contribute to the pathogenesis of post-infarction heart failure (HF), the role of autophagy remains unclear. Likewise, linkage between these two cell death modalities has not been sufficiently investigated. HF was induced by 60-min left coronary occlusion in adult Wistar rats and heart function was assessed 6 weeks later followed by immunoblotting analysis of necroptotic and autophagic proteins in both the left (LV) and right ventricle (RV). HF had no effect on RIP1 and RIP3 expression. PhosphoSer229-RIP3, acting as a pro-necroptotic signal, was increased in LV while deceased in RV of failing hearts. Total MLKL was elevated in RV only. Decrease in pSer555-ULK1, increase in pSer473-Akt and no significant elevation in beclin-1 and LC3-II/I ratio indicated rather a lowered rate of autophagy in LV. No beclin-1 upregulation and decreased LC3 processing also suggested the inhibition of both autophagosome formation and maturation in RV of failing hearts. In contrast, p89 PARP1 fragment, a marker of executed apoptosis, was increased in RV only. This is the first study showing a different signaling in ventricles of the late phase of post-infarction HF, highlighting necroptosis itself rather than its linkage with autophagy in LV, and apoptosis in RV.

Different signalling in infarcted and non-infarcted areas of rat failing hearts: A role of necroptosis and inflammation.

Necroptosis has been recognized in heart failure (HF). In this study, we investigated detailed necroptotic signalling in infarcted and non-infarcted areas separately and its mechanistic link with main features of HF. Post-infarction HF in rats was induced by left coronary occlusion (60 minutes) followed by 42-day reperfusion. Heart function was assessed echocardiographically. Molecular signalling and proposed mechanisms (oxidative stress, collagen deposition and inflammation) were investigated in whole hearts and in subcellular fractions when appropriate. In post-infarction failing hearts, TNF and pSer229-RIP3 levels were comparably increased in both infarcted and non-infarcted areas. Its cytotoxic downstream molecule p-MLKL, indicating necroptosis execution, was detected in infarcted area. In non-infarcted area, despite increased pSer229-RIP3, p-MLKL was present in neither whole cells nor the cell membrane known to be associated with necroptosis execution. Likewise, increased membrane lipoperoxidation and NOX2 levels unlikely promoted pro-necroptotic environment in non-infarcted area. Collagen deposition and the inflammatory csp-1-IL-1ß axis were active in both areas of failing hearts, while being more pronounced in infarcted tissue. Although apoptotic proteins were differently expressed in infarcted and non-infarcted tissue, apoptosis was found to play an insignificant role. p-MLKL-driven necroptosis and inflammation while inflammation only (without necroptotic cell death) seem to underlie fibrotic healing and progressive injury in infarcted and non-infarcted areas of failing hearts, respectively. Upregulation of pSer229-RIP3 in both HF areas suggests that this kinase, associated with both necroptosis and inflammation, is likely to play a dual role in HF progression.

In vitro and in vivo investigation of cardiotoxicity associated with anticancer proteasome inhibitors and their combination with anthracycline.

Although proteasome inhibitors (PIs) are modern targeted anticancer drugs, they have been associated with a certain risk of cardiotoxicity and heart failure (HF). Recently, PIs have been combined with anthracyclines (ANTs) to further boost their anticancer efficacy. However, this raised concerns regarding cardiac safety, which were further supported by several in vitro studies on immature cardiomyocytes. In the present study, we investigated the toxicity of clinically used PIs alone (bortezomib (BTZ), carfilzomib (CFZ)) as well as their combinations with an ANT (daunorubicin (DAU)) in both neonatal and adult ventricular cardiomyocytes (NVCMs and AVCMs) and in a chronic rabbit model of DAU-induced HF. Using NVCMs, we found significant cytotoxicity of both PIs around their maximum plasma concentration (cmax) as well as significant augmentation of DAU cytotoxicity. In AVCMs, BTZ did not induce significant cytotoxicity in therapeutic concentrations, whereas the toxicity of CFZ was significant and more profound. Importantly, neither PI significantly augmented the cardiotoxicity of DAU despite even more profound proteasome-inhibitory activity in AVCMs compared with NVCMs. Furthermore, in young adult rabbits, no significant augmentation of chronic ANT cardiotoxicity was noted with respect to any functional, morphological, biochemical or molecular parameter under study, despite significant inhibition of myocardial proteasome activity. Our experimental data show that combination of PIs with ANTs is not accompanied by an exaggerated risk of cardiotoxicity and HF in young adult animal cardiomyocytes and hearts.

Epoxyeicosatrienoic acid analog EET-B attenuates post-myocardial infarction remodeling in spontaneously hypertensive rats.

Epoxyeicosatrienoic acids (EETs) and their synthetic analogs have cardiovascular protective effects. Here, we investigated the action of a novel EET analog EET-B on the progression of post-myocardial infarction (MI) heart failure in spontaneously hypertensive rats (SHR). Adult male SHR were divided into vehicle- and EET-B (10 mg/kg/day; p.o., 9 weeks)-treated groups. After 2 weeks of treatment, rats were subjected to 30-min left coronary artery occlusion or sham operation. Systolic blood pressure (SBP) and echocardiography (ECHO) measurements were performed at the beginning of study, 4 days before, and 7 weeks after MI. At the end of the study, tissue samples were collected for histological and biochemical analyses. We demonstrated that EET-B treatment did not affect blood pressure and cardiac parameters in SHR prior to MI. Fractional shortening (FS) was decreased to 18.4 ± 1.0% in vehicle-treated MI rats compared with corresponding sham (30.6 ± 1.0%) 7 weeks following MI induction. In infarcted SHR hearts, EET-B treatment improved FS (23.7 ± 0.7%), markedly increased heme oxygenase-1 (HO-1) immunopositivity in cardiomyocytes and reduced cardiac inflammation and fibrosis (by 13 and 19%, respectively). In conclusion, these findings suggest that EET analog EET-B has beneficial therapeutic actions to reduce cardiac remodeling in SHR subjected to MI.

Selection of optimal reference genes for gene expression studies in chronically hypoxic rat heart.

Adaptation to chronic hypoxia renders the heart more tolerant to ischemia/reperfusion injury. To evaluate changes in gene expression after adaptation to chronic hypoxia by RT-qPCR, it is essential to select suitable reference genes. In a chronically hypoxic rat model, no specific reference genes have been identified in the myocardium. This study aimed to select the best reference genes in the left (LV) and right (RV) ventricles of chronically hypoxic and normoxic rats. Sprague-Dawley rats were adapted to continuous normobaric hypoxia (CNH; 12% O2 or 10% O2) for 3 weeks. The expression levels of candidate genes were assessed by RT-qPCR. The stability of genes was evaluated by NormFinder, geNorm and BestKeeper algorithms. The best five reference genes in the LV were Top1, Nupl2, Rplp1, Ywhaz, Hprt1 for the milder CNH and Top1, Ywhaz, Sdha, Nupl2, Tomm22 for the stronger CNH. In the RV, the top five genes were Hprt1, Nupl2, Gapdh, Top1, Rplp1 for the milder CNH and Tomm22, Gapdh, Hprt1, Nupl2, Top1 for the stronger CNH. This study provides validation of reference genes in LV and RV of CNH rats and shows that suitable reference genes differ in the two ventricles and depend on experimental protocol.

Enhanced Renal Vascular Responsiveness to Angiotensin II and Norepinephrine: A Unique Feature of Female Rats with Congestive Heart Failure.

BACKGROUND/AIMS: We found recently that the aortocaval fistula (ACF)-induced heart failure (HF) results in higher mortality in female than in male rats. Possibly, the development of renal dysfunction in the females, unlike in males, is associated with altered renal vascular responsiveness to angiotensin II (ANG II). METHODS: Five or 20 weeks after ACF creation (compensated and decompensated HF, respectively), we assessed renal blood flow (RBF) responses to intrarenal administration of ANG II, norepinephrine (NE), and acetylcholine (Ach) in female ACF and sham-operated rats. RESULTS: In ACF females, ANG II decreased RBF more than in healthy animals, unlike with earlier published data in male ACF rats that responded similarly. Also, NE decreased RBF more in female ACF rats, whereas Ach increased RBF to the same extent in female ACF and sham-operated rats. RBF responses to intravenous administration of NE and Ach were almost identical in female and male ACF rats. CONCLUSION: Female ACF rats studied at the onset of HF decompensation reveal, in contrast to male rats, enhanced renal vascular responsiveness to both NE and ANG II. When associated with the demonstrated increased intrarenal ANG II and NE concentrations, such hyperresponsiveness might promote the development of renal dysfunction and accelerate HF decompensation.

Altered Renal Vascular Responsiveness to Vasoactive Agents in Rats with Angiotensin II-Dependent Hypertension and Congestive Heart Failure.

OBJECTIVE: We evaluated the hypothesis that the development of renal dysfunction and congestive heart failure (CHF) caused by volume overload in rats with angiotensin II (ANG II)-dependent hypertension is associated with altered renal vascular responsiveness to ANG II and to epoxyeicosatrienoic acids (EETs). METHODS: Ren-2 transgenic rats (TGRs) were used as a model of ANG II-dependent hypertension. CHF was induced by volume overload achieved by the creation of the aorto-caval fistula (ACF). Renal blood flow (RBF) responses were determined to renal arterial administration of ANG II, native 11,12-EET, an analog of 14,15-EETs (EET-A), norepinephrine (NE), acetylcholine (Ach) and bradykinin (Bk) in healthy (i.e., sham-operated) TGR and ACF TGR (5 weeks after ACF creation). RESULTS: Selective intrarenal administration of neither vasoactive drug altered mean arterial pressure in any group. Administration of ANG II caused greater decreases in RBF in ACF TGR than in sham-operated TGR, whereas after administration of NE the respective decreases were comparable in the 2 groups. Administration of Ach and Bk elicited significantly higher RBF increases in ACF TGR as compared with sham-operated TGR. In contrast, administration of 11,12-EET and EET-A caused significantly smaller RBF increases in ACF TGR than in sham-operated TGR. CONCLUSION: The findings show that 5 weeks after creation of ACF, the TGR exhibit exaggerated renal vasoconstrictor responses to ANG II and reduced renal vasodilatory responses to EETs, suggesting that both these alterations might play an important role in the development of renal dysfunction in this model of CHF.

Mitochondrial genome modulates myocardial Akt/Glut/HK salvage pathway in spontaneously hypertensive rats adapted to chronic hypoxia.

Recently we have shown that adaptation to continuous normobaric hypoxia (CNH) decreases myocardial ischemia/reperfusion injury in spontaneously hypertensive rats (SHR) and in a conplastic strain (SHR-mtBN). The protective effect was stronger in the latter group characterized by a selective replacement of the SHR mitochondrial genome with that of a more ischemia-resistant Brown Norway strain. The aim of the present study was to examine the possible involvement of the hypoxia inducible factor (HIF)-dependent pathway of the protein kinase B/glucose transporters/hexokinase (Akt/GLUT/HK) in this mitochondrial genome-related difference of the cardioprotective phenotype. Adult male rats were exposed for 3 wk to CNH ([Formula: see text] 0.1). The expression of dominant isoforms of Akt, GLUT, and HK in left ventricular myocardium was determined by real-time RT-PCR and Western blotting. Subcellular localization of GLUTs was assessed by quantitative immunofluorescence. Whereas adaptation to hypoxia markedly upregulated protein expression of HK2, GLUT1, and GLUT4 in both rat strains, Akt2 protein level was significantly increased in SHR-mtBN only. Interestingly, a higher content of HK2 was revealed in the sarcoplasmic reticulum-enriched fraction in SHR-mtBN after CNH. The increased activity of HK determined in the mitochondrial fraction after CNH in both strains suggested an increase of HK association with mitochondria. Interestingly, HIF1a mRNA increased and HIF2a mRNA decreased after CNH, the former effect being more pronounced in SHR-mtBN than in SHR. Pleiotropic effects of upregulated Akt2 along with HK translocation to mitochondria and mitochondria-associated membranes can potentially contribute to a stronger CNH-afforded cardioprotection in SHR-mtBN compared with progenitor SHR.

Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice.

TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at ∼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.

Infarct size-limiting effect of epoxyeicosatrienoic acid analog EET-B is mediated by hypoxia-inducible factor-1α via downregulation of prolyl hydroxylase 3.

Epoxyeicosatrienoic acids (EETs) decrease cardiac ischemia-reperfusion injury; however, the mechanism of their protective effect remains elusive. Here, we investigated the cardioprotective action of a novel EET analog, EET-B, in reperfusion and the role of hypoxia-inducible factor (HIF)-1α in such action of EET-B. Adult male rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion. Administration of 14,15-EET (2.5 mg/kg) or EET-B (2.5 mg/kg) 5 min before reperfusion reduced infarct size expressed as a percentage of the area at risk from 64.3 ± 1.3% in control to 42.6 ± 1.9% and 46.0 ± 1.6%, respectively, and their coadministration did not provide any stronger effect. The 14,15-EET antagonist 14,15-epoxyeicosa-5( Z)-enoic acid (2.5 mg/kg) inhibited the infarct size-limiting effect of EET-B (62.5 ± 1.1%). Similarly, the HIF-1α inhibitors 2-methoxyestradiol (2.5 mg/kg) and acriflavine (2 mg/kg) completely abolished the cardioprotective effect of EET-B. In a separate set of experiments, the immunoreactivity of HIF-1α and its degrading enzyme prolyl hydroxylase domain protein 3 (PHD3) were analyzed in the ischemic areas and nonischemic septa. At the end of ischemia, the HIF-1α immunogenic signal markedly increased in the ischemic area compared with the septum (10.31 ± 0.78% vs. 0.34 ± 0.08%). After 20 min and 2 h of reperfusion, HIF-1α immunoreactivity decreased to 2.40 ± 0.48% and 1.85 ± 0.43%, respectively, in the controls. EET-B blunted the decrease of HIF-1α immunoreactivity (7.80 ± 0.69% and 6.44 ± 1.37%, respectively) and significantly reduced PHD3 immunogenic signal in ischemic tissue after reperfusion. In conclusion, EET-B provides an infarct size-limiting effect at reperfusion that is mediated by HIF-1α and downregulation of its degrading enzyme PHD3. NEW & NOTEWORTHY The present study shows that EET-B is an effective agonistic 14,15-epoxyeicosatrienoic acid analog, and its administration before reperfusion markedly reduced myocardial infarction in rats. Most importantly, we demonstrate that increased hypoxia-inducible factor-1α levels play a role in cardioprotection mediated by EET-B in reperfusion likely by mechanisms including downregulation of the hypoxia-inducible factor -1α-degrading enzyme prolyl hydroxylase domain protein 3.

Adverse effects of Hif1a mutation and maternal diabetes on the offspring heart.

BACKGROUND: Epidemiological studies show that maternal diabetes predisposes offspring to cardiovascular and metabolic disorders. However, the precise mechanisms for the underlying penetrance and disease predisposition remain poorly understood. We examined whether hypoxia-inducible factor 1 alpha, in combination with exposure to a diabetic intrauterine environment, influences the function and molecular structure of the adult offspring heart. METHODS AND RESULTS: In a mouse model, we demonstrated that haploinsufficient (Hif1a+/-) offspring from a diabetic pregnancy developed left ventricle dysfunction at 12 weeks of age, as manifested by decreased fractional shortening and structural remodeling of the myocardium. Transcriptional profiling by RNA-seq revealed significant transcriptome changes in the left ventricle of diabetes-exposed Hif1a+/- offspring associated with development, metabolism, apoptosis, and blood vessel physiology. In contrast, both wild type and Hif1a+/- offspring from diabetic pregnancies showed changes in immune system processes and inflammatory responses. Immunohistochemical analyses demonstrated that the combination of haploinsufficiency of Hif1a and exposure to maternal diabetes resulted in impaired macrophage infiltration, increased levels of advanced glycation end products, and changes in vascular homeostasis in the adult offspring heart. CONCLUSIONS: Together our findings provide evidence that a global reduction in Hif1a gene dosage increases predisposition of the offspring exposed to maternal diabetes to cardiac dysfunction, and also underscore Hif1a as a critical factor in the fetal programming of adult cardiovascular disease.

Proteomic analysis of cardiac ventricles: baso-apical differences.

The heart is characterized by a remarkable degree of heterogeneity. Since different cardiac pathologies affect different cardiac regions, it is important to understand molecular mechanisms by which these parts respond to pathological stimuli. In addition to already described left ventricular (LV)/right ventricular (RV) and transmural differences, possible baso-apical heterogeneity has to be taken into consideration. The aim of our study has been, therefore, to compare proteomes in the apical and basal parts of the rat RV and LV. Two-dimensional electrophoresis was used for the proteomic analysis. The major result of this study has revealed for the first time significant baso-apical differences in concentration of several proteins, both in the LV and RV. As far as the LV is concerned, five proteins had higher concentration in the apical compared to basal part of the ventricle. Three of them are mitochondrial and belong to the "metabolism and energy pathways" (myofibrillar creatine kinase M-type, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase). Myosin light chain 3 is a contractile protein and HSP60 belongs to heat shock proteins. In the RV, higher concentration in the apical part was observed in two mitochondrial proteins (creatine kinase S-type and proton pumping NADH:ubiquinone oxidoreductase). The described changes were more pronounced in the LV, which is subjected to higher workload. However, in both chambers was the concentration of proteins markedly higher in the apical than that in basal part, which corresponds to the higher energetic demand and contractile activity of these segments of both ventricles.

The Role of Renal Vascular Reactivity in the Development of Renal Dysfunction in Compensated and Decompensated Congestive Heart Failure.

BACKGROUND/AIMS: Reduction of renal blood flow (RBF) is commonly thought to be a causative factor of renal dysfunction in congestive heart failure (CHF), but the exact mechanism of the renal hypoperfusion is not clear. Apart from the activation of neurohormonal systems controlling intrarenal vascular tone, the cause might be altered reactivity of the renal vasculature to endogenous vasoactive agents. METHODS: To evaluate the role of this mechanism, we assessed by an ultrasonic transient-time flow probe maximum RBF responses to renal artery infusion of angiotensin II (ANG II), norepinephrine (NE) and acetylcholine (Ach) in healthy male rats and animals with compensated and decompensated CHF. CHF was induced by volume overload achieved by the creation of the aorto-caval fistula (ACF) in Hannover Sprague-Dawley rats. RESULTS: Maximum responses in RBF to ANG II were similar in rats studied five weeks (compensated phase) and 20 weeks (decompensated phase) after ACF creation when compared to sham-operated rats. On the other hand, NE elicited larger maximum decreases in RBF in rats with CHF (five and 20 weeks post-ACF) than in sham-operated controls. We observed greater maximum vasodilatory responses to Ach only in rats with a compensated stage of CHF (five weeks post-ACF). CONCLUSION: Greater renal vasoconstrictor responsiveness to ANG II or reduced renal vasodilatation in response to Ach do not play a decisive role in the development of renal dysfunction in ACF rats with compensated and decompensated CHF. On the other hand, exaggerated renal vascular responsiveness to NE may be here a contributing causative factor, active in either CHF phase.

Selective replacement of mitochondrial DNA increases the cardioprotective effect of chronic continuous hypoxia in spontaneously hypertensive rats.

Mitochondria play an essential role in improved cardiac ischaemic tolerance conferred by adaptation to chronic hypoxia. In the present study, we analysed the effects of continuous normobaric hypoxia (CNH) on mitochondrial functions, including the sensitivity of the mitochondrial permeability transition pore (MPTP) to opening, and infarct size (IS) in hearts of spontaneously hypertensive rats (SHR) and the conplastic SHR-mtBN strain, characterized by the selective replacement of the mitochondrial genome of SHR with that of the more ischaemia-resistant brown Norway (BN) strain. Rats were adapted to CNH (10% O2, 3 weeks) or kept at room air as normoxic controls. In the left ventricular mitochondria, respiration and cytochrome c oxidase (COX) activity were measured using an Oxygraph-2k and the sensitivity of MPTP opening was assessed spectrophotometrically as Ca2+-induced swelling. Myocardial infarction was analysed in anaesthetized open-chest rats subjected to 20 min of coronary artery occlusion and 3 h of reperfusion. The IS reached 68±3.0% and 65±5% of the area at risk in normoxic SHR and SHR-mtBN strains, respectively. CNH significantly decreased myocardial infarction to 46±3% in SHR. In hypoxic SHR-mtBN strain, IS reached 33±2% and was significantly smaller compared with hypoxic SHR. Mitochondria isolated from hypoxic hearts of both strains had increased detergent-stimulated COX activity and were less sensitive to MPTP opening. The maximum swelling rate was significantly lower in hypoxic SHR-mtBN strain compared with hypoxic SHR, and positively correlated with myocardial infarction in all experimental groups. In conclusion, the mitochondrial genome of SHR modulates the IS-limiting effect of adaptation to CNH by affecting mitochondrial energetics and MPTP sensitivity to opening.

Adaptation to chronic continuous hypoxia potentiates Akt/HK2 anti-apoptotic pathway during brief myocardial ischemia/reperfusion insult.

Adaptation to chronic hypoxia represents a potential cardioprotective intervention reducing the extent of acute ischemia/reperfusion (I/R) injury, which is a major cause of death worldwide. The main objective of this study was to investigate the anti-apoptotic Akt/hexokinase 2 (HK2) pathway in hypoxic hearts subjected to I/R insult. Hearts isolated from male Wistar rats exposed either to continuous normobaric hypoxia (CNH; 10% O2) or to room air for 3 weeks were perfused according to Langendorff and subjected to 10 min of no-flow ischemia and 10 min of reperfusion. The hearts were collected either after ischemia or after reperfusion and used for protein analyses and quantitative fluorescence microscopy. The CNH resulted in increased levels of HK1 and HK2 proteins and the total HK activity after ischemia compared to corresponding normoxic group. Similarly, CNH hearts exhibited increased ischemic level of Akt protein phosphorylated on Ser473. The CNH also strengthened the interaction of HK2 with mitochondria and prevented downregulation of mitochondrial creatine kinase after reperfusion. The Bax/Bcl-2 ratio was significantly lower after I/R in CNH hearts than in normoxic ones, suggesting a lower probability of apoptosis. In conclusion, the Akt/HK2 pathway is likely to play a role in the development of a cardioprotective phenotype of CNH by preventing the detachment of HK2 from mitochondria at reperfusion period and decreases the Bax/Bcl-2 ratio during I/R insult, thereby lowering the probability of apoptosis activation in the mitochondrial compartment.