Rat Ventricular Cardiomyocytes Research Articles

Cholinergic action of spleen‐derived cardioprotective factors

IntroductionThere is an ambivalent relationship between the spleen and the heart in myocardial infarction (Circ Res 124; 2019; 26‐8). Sympathetic activation of the spleen initially increases myocardial inflammatory infiltration but resolves it later by initiating a myocardial healing response. Vagal activation of the spleen induces the release of cardioprotective factors: Perfusion of an isolated rat spleen with the vagomimetic carbachol resulted in the release of factors which then reduced infarct size (IS) in an isolated perfused rat heart.AimTo characterize cardioprotection of spleen‐derived factors, with a focus on their cholinergic action.MethodsRat spleens and hearts were isolated and buffer‐perfused at constant pressure. Isolated spleens were either challenged by infusion of saline (as control) or the vagomimetic carbachol (500 pmol/L), and splenic effluate (SEff; 100 mL) was sampled after saline infusion (SEffsaline) or carbachol infusion (SEffcarbachol), respectively. Cell‐free SEff was prepared by centrifugation and filtration. SEffsaline or SEffcarbachol was infused into isolated hearts which were then subjected to 30 min global ischemia and 120 min reperfusion (I/R). In subsets of experiments, the hearts were pretreated with the nicotinic receptor antagonist hexamethonium (50 µmol/L) or the muscarinic receptor antagonist atropine (100 pmol/L). IS was demarcated by triphenyltetrazolium chloride staining and calculated as percent of ventricular mass. To study the relevance of spleen‐derived protection on the cardiomyocyte level, adult rat ventricular cardiomyocytes were prepared. Cells were incubated for 30 minutes with SEffsaline or SEffcarbachol, respectively, and then subjected to 30 min hypoxia/ 5 min reoxygenation (H/R) or exposed to SEffsaline or SEffcarbachol for 35 minutes as time control (TC). Since nicotinic and muscarinic receptors are also expressed on cardiomyocytes, all experiments were repeated after pretreatment with hexamethonium (1 µmol/L) or atropine (100 nmol/L). Cardiomyocyte viability was quantified before (baseline) and after H/R or TC, as the percent fraction of rod‐shaped, unstained (5% trypan blue) cells over all cells. The estimated final concentration of carbachol in the SEffcarbachol of ≤10 pmol/L and the SEffsaline did neither reduce IS in isolated perfused rat hearts nor improve cardiomyocyte viability in isolated rat cardiomyocytes.ResultsWith SEffsaline, IS was 34±7%. With SEffcarbachol, IS was reduced to 19±8%. Hexamethonium or atropine had no impact on IS per se in hearts with SEffsaline (33±8%; 34±6%), but attenuated the cardioprotection by SEffcarbachol (27±10%; 26±10%; Figure A). The viability of cardiomyocytes subjected to H/R was better preserved with SEffcarbachol than with SEffsaline or saline alone. Again, this protection was attenuated by hexamethonium or atropine (Figure B, insert).ConclusionThe cardioprotection by spleen‐derived factors, which were released in response to the vagomimetic carbachol, is mediated through nicotinic and muscarinic receptors within the heart. Apparently, this protection does not need to involve neuronal structures within the myocardium – nicotinic and muscarinic receptors on cardiomyocytes per se mediate protection.

Combination of Sildenafil and Ba2+ at a Low Concentration Show a Significant Synergistic Inhibition of Inward Rectifier Potassium Current Resulting in Action Potential Prolongation.

Sildenafil (Viagra) is a vasodilator mainly used in the treatment of erectile dysfunction. Atrial or ventricular fibrillation may rarely occur as a side effect during sildenafil therapy. Although changes in inward rectifier potassium currents including I K1 are known to contribute to the pathogenesis of fibrillation, the effect of sildenafil on I K1 has not been studied. In experiments, Ba2+ is used as a specific inhibitor of I K1 at high concentrations (usually 100 µM). Being an environmental contaminant, it is also present in the human body; Ba2+ plasmatic concentrations up to 1.5 µM are usually reported in the general population. This study was primarily aimed to investigate changes of I K1 induced by sildenafil in a wide range of concentrations (0.1–100 µM). Additionally, the effect of combination of sildenafil and Ba2+ at selected clinically-relevant concentrations was tested, at 0.1 µM both on I K1 and on the action potential duration (APD). Experiments were performed by the whole-cell patch-clamp technique on enzymatically isolated rat ventricular cardiomyocytes, mostly at 23°C with the exception of APD measurements which were performed at 37°C as well. Sildenafil caused a significant, reversible, and concentration-dependent inhibition of I K1 that did not differ at −50 and −110 mV. Simultaneous application of sildenafil and Ba2+ at 0.1 µM revealed a massive inhibition of both inward and outward components of I K1 (this synergy was missing at other tested combinations). The combined effect at 0.1 µM (45.7 ± 5.7 and 43.0 ± 6.9% inhibition at −50 and −110 mV, respectively) was significantly higher than a simple sum of almost negligible effects of the individual substances and it led to a significant prolongation of APD at both 23 and 37°C. To our knowledge, similar potentiation of the drug-channel interaction has not been described. The observed massive inhibition of I K1 induced by a combined action of the vasodilator sildenafil and environmental contaminant Ba2+ at a low concentration and resulting in a significant APD prolongation may contribute to the genesis of arrhythmias observed in some patients treated with sildenafil.

The role of activation of two different sGC binding sites by NO-dependent and NO-independent mechanisms in the regulation of SACs in rat ventricular cardiomyocytes.

The mechanoelectrical feedback (MEF) mechanism in the heart that plays a significant role in the occurrence of arrhythmias, involves cation flux through cation nonselective stretch‐activated channels (SACs). It is well known that nitric oxide (NO) can act as a regulator of MEF. Here we addressed the possibility of SAC’s regulation along NO‐dependent and NO‐independent pathways, as well as the possibility of S‐nitrosylation of SACs. In freshly isolated rat ventricular cardiomyocytes, using the patch‐clamp method in whole‐cell configuration, inward nonselective stretch‐activated cation current ISAC was recorded through SACs, which occurs during dosed cell stretching. NO donor SNAP, α1‐subunit of sGC activator BAY41‐2272, sGC blocker ODQ, PKG blocker KT5823, PKG activator 8Br‐cGMP, and S‐nitrosylation blocker ascorbic acid, were employed. We concluded that the physiological concentration of NO in the cell is a necessary condition for the functioning of SACs. An increase in NO due to SNAP in an unstretched cell causes the appearance of a Gd3+‐sensitive nonselective cation current, an analog of ISAC , while in a stretched cell it eliminates ISAC . The NO‐independent pathway of sGC activation of α subunit, triggered by BAY41‐2272, is also important for the regulation of SACs. Since S‐nitrosylation inhibitor completely abolishes ISAC , this mechanism occurs. The application of BAY41‐2272 cannot induce ISAC in a nonstretched cell; however, the addition of SNAP on its background activates SACs, rather due to S‐nitrosylation.ODQ eliminates ISAC , but SNAP added on the background of stretch increases ISAC in addition to ODQ. This may be a result of the lack of NO as a result of inhibition of NOS by metabolically modified ODQ. KT5823 reduces PKG activity and reduces SACs phosphorylation, leading to an increase in ISAC . 8Br‐cGMP reduces ISAC by activating PKG and its phosphorylation. These results demonstrate a significant contribution of S‐nitrosylation to the regulation of SACs.

Gabapentinoid-induced peripheral edema and acute heart failure: A translational study combining pharmacovigilance data and in vitro animal experiments

IntroductionGabapentinoids are ligands of the α2-δ subunit of voltage-gated calcium channels (Cav) that have been associated with a risk of peripheral edema and acute heart failure in connection with a potentially dual mechanism, vascular and cardiac. Objectives & methodsAll cases of peripheral edema or heart failure involving gabapentin or pregabalin reported to the French Pharmacovigilance Centers between January 1, 1994 and April 30, 2020 were included to describe their onset patterns (e.g., time to onset). Based on these data, we investigated the impact of gabapentinoids on the myogenic tone of rat third-order mesenteric arteries and on the electrophysiological properties of rat ventricular cardiomyocytes. ResultsA total of 58 reports were included (gabapentin n = 5, pregabalin n = 53). The female-to-male ratio was 4:1 and the median age was 77 years (IQR 57–85, range 32–95). The median time to onset were 23 days (IQR 10–54) and 17 days (IQR 3–30) for non-cardiogenic edema and acute heart failure, respectively. Cardiogenic and non-cardiogenic peripheral edema occurred frequently after a dose escalation (27/45, 60%), and the course was rapidly favorable after discontinuation of gabapentinoid (median 7 days, IQR 5–13). On rat mesenteric arteries, gabapentinoids significantly decreased the myogenic tone to the same extent as verapamil and nifedipine. Acute application of gabapentinoids had no significant effect on Cav1.2 currents of ventricular cardiomyocytes. ConclusionGabapentinoids can cause concentration-dependent peripheral edema of early onset. The primary mechanism of non-cardiogenic peripheral edema is vasodilatory edema secondary to altered myogenic tone, independent of Cav1.2 blockade under the experimental conditions tested.

Discordant Ca2+ release in cardiac myocytes: characterization and susceptibility to pharmacological RyR2 modulation.

Systolic Ca2+ transients are shaped by the concerted summation of Ca2+ sparks across cardiomyocytes. At high pacing rates, alterations of excitation-contraction coupling manifest as pro-arrhythmic Ca2+ alternans that can be classified as concordant or discordant. Discordance is ascribed to out-of-phase alternation of local Ca2+ release across the cell, although the triggers and consequences of this phenomenon remain unclear. Rat ventricular cardiomyocytes were paced at increasing rates. A discordance index (SD of local alternans ratios) was developed to quantify discordance in confocal recordings of Ca2+ transients. Index values were significantly increased by rapid pacing, and negatively correlated with Ca2+ transient amplitude change, indicating that discordance is an important contributor to the negative Ca2+ transient-frequency relationship. In addition, the largest local calcium transient in two consecutive transients was measured to build a potential "best release" profile, which quantitatively confirmed discordance-induced Ca2+ release impairment (DICRI). Diastolic Ca2+ homeostasis was also observed to be disrupted by discordance, as late Ca2+ release events elicited instability of resting Ca2+ levels. Finally, the effects of two RyR2 inhibitors (VK-II-86 and dantrolene) were tested. While both compounds inhibited Ca2+ wave generation, only VK-II-86 augmented subcellular discordance. Discordant Ca2+ release is a quantifiable phenomenon, sensitive to pacing frequency, and impairs both systolic and diastolic Ca2+ homeostasis. Interestingly, RyR2 inhibition can induce discordance, which should be considered when evaluating pharmacological RyR2 modulators for clinical use.

Cordycepin alleviates myocardial ischemia/reperfusion injury by enhancing autophagy via AMPK-mTOR pathway.

To estimate the cardioprotective mechanism of cordycepin on myocardial ischemia/reperfusion (I/R) injury. The left anterior descending artery of mice was ligated transiently to establish the myocardial I/R model. TTC/Evans Blue staining and TUNEL assay were performed to quantify the infarct size and apoptosis index. The cardiac function was evaluated by echocardiography. Neonatal rat ventricular cardiomyocytes (NRVCs) underwent hypoxia and reoxygenation (H/R). MTS and LDH were detected to measured cell viability and necrosis respectively. The results suggested that cordycepin could markedly decrease apoptosis, reduce infarct size, and improve cardiac function in mice subjected to I/R injury, alongside with enhanced autophagy. In NRVCs, cordycepin treatment obviously reduced ROS production. In addition, cordycepin partly promoted autophagy in the context of H/R injury by regulating AMPK/mTOR pathway. Our data demonstrated that cordycepin exerts cardio-protective effect and promotes cardiac functional recovery following myocardial I/R by enhancing autophagy via AMPK-mTOR signaling pathway.

GSK-3β Localizes to the Cardiac Z-Disc to Maintain Length Dependent Activation.

Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. Inducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3β. Agreeing with the localization of its targets, GSK-3β that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. We identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

Insulin-induced recovery in KCNQ1/KCNE1-current accelerates the ventricular action potential repolarization in insulin-resistant aged-rats via affecting β3-adrenergic receptors

Cardiac aging, including cardiometabolic disturbances, is defined as a progressive and intrinsic decline in heart function, which is also a combination of metabolic dysfunctions mainly characterized by insulin resistance and long-QT, via prolonged late-phase repolarizations of ventricular action potentials (APs). So, we hypothesized a contribution of dysfunctional KCNQ1/KCNE1-channels to these abnormal electrical activities in elderly mammal hearts and positive responses to insulin application. Furthermore, taken into consideration β3-AR activation role in insulin-resistant hearts, we aimed to investigate the possible correlations between dysfunctional KCNQ1/KCNE1-channels, β3-AR activation, and the effect of insulin application on these correlations in insulin-resistant aged rat (24-month-old) ventricular cardiomyocytes.

Sympathetic Stimulation Upregulates the Ca2+ Channel Subunit, CaVα2δ1, via the β1 and ERK 1/2 Pathway in Neonatal Ventricular Cardiomyocytes.

Intracellular Ca2+ overload secondary to chronic hemodynamic stimuli promotes the recruitment of Ca2+-dependent signaling implicated in cardiomyocyte hypertrophy. The present study tested the hypothesis that sympathetic-mediated hypertrophy of neonatal rat ventricular cardiomyocytes (NRVMs) translated to an increase in calcium influx secondary to the upregulation of CaV1.2 channel subunits. Confocal imaging of norepinephrine (NE)-treated NRVMs revealed a hypertrophic response compared to untreated NRVMs. L-type CaV1.2 peak current density was increased 4-fold following a 24-h stimulation with NE. NE-treated NRVMs exhibited a significant upregulation of CaVα2δ1 and CaVβ3 protein levels without significant changes of CaVα1C and CaVβ2 protein levels. Pre-treatment with the β1-blocker metoprolol failed to inhibit hypertrophy or CaVβ3 upregulation whereas CaVα2δ1 protein levels were significantly reduced. NE promoted the phosphorylation of ERK 1/2, and the response was attenuated by the β1-blocker. U0126 pre-treatment suppressed NE-induced ERK1/2 phosphorylation but failed to attenuate hypertrophy. U0126 inhibition of ERK1/2 phosphorylation prevented NE-mediated upregulation of CaVα2δ1, whereas CaVβ3 protein levels remained elevated. Thus, β1-adrenergic receptor-mediated recruitment of the ERK1/2 plays a seminal role in the upregulation of CaVα2δ1 in NRVMs independent of the concomitant hypertrophic response. However, the upregulation of CaVβ3 protein levels may be directly dependent on the hypertrophic response of NRVMs.

HOPX Plays a Critical Role in Antiretroviral Drugs Induced Epigenetic Modification and Cardiac Hypertrophy.

People living with HIV (PLWH) have to take an antiretroviral therapy (ART) for life and show noncommunicable illnesses such as chronic inflammation, immune activation, and multiorgan dysregulation. Recent studies suggest that long-term use of ART induces comorbid conditions and is one of the leading causes of heart failure in PLWH. However, the molecular mechanism of antiretroviral drugs (ARVs) induced heart failure is unclear. To determine the mechanism of ARVs induced cardiac dysfunction, we performed global transcriptomic profiling of ARVs treated neonatal rat ventricular cardiomyocytes in culture. Differentially expressed genes were identified by RNA-sequencing. Our data show that ARVs treatment causes upregulation of several biological functions associated with cardiotoxicity, hypertrophy, and heart failure. Global gene expression data were validated in cardiac tissue isolated from HIV patients having a history of ART. Interestingly, we found that homeodomain-only protein homeobox (HOPX) expression was significantly increased in cardiomyocytes treated with ARVs and in the heart tissue of HIV patients. Furthermore, we found that HOPX plays a crucial role in ARVs mediated cellular hypertrophy. Mechanistically, we found that HOPX plays a critical role in epigenetic regulation, through deacetylation of histone, while the HDAC inhibitor, Trichostatin A, can restore the acetylation level of histone 3 in the presence of ARVs.

Abstract 9815: Role for Btg1 and Btg2 in Postnatal Cardiomyocyte Cell Cycle Arrest and Maturation

Background: Adult mammalian cardiomyocytes (CM) are predominantly post-mitotic and cannot proliferatively repair the heart following myocardial infarction (MI). Overexpression of the transcription factor Tbx20 in adult mouse CMs promotes proliferative cardiac repair post-MI, via mechanisms including direct repression of anti-proliferative genes p21 , Meis1 , and Btg2 . Btg2 (B-cell translocation gene 2), a tumor suppressor and transcriptional co-regulator, exhibits high structural and functional similarity with Btg1. However, both Btg1 and Btg2 (Btg1/2) are virtually uncharacterized in the heart. Here, we investigate the role of Btg1/2 in postnatal cardiac maturation. Methods and Results: By immunostaining in embryonic, neonatal, and adult C57BL/6 mouse hearts, the highest expression of Btg1/2 was observed in late fetal and early neonatal ventricles, concurrent with upregulation of other CM cell cycle inhibitors. In neonatal mouse CMs in vitro, siRNA-mediated loss of Btg2 leads to increased CM proliferation. In vivo , Btg1/2 constitutive single- and double- knockout (SKO and DKO respectively) mice exhibit normal heart weight-to-body weight ratios compared to age-matched wildtype (WT) controls, at postnatal day (P)7, P30, and 1 year after birth. Interestingly, at P7, DKO mice have significantly higher CM mitotic activity, as indicated by pHH3 staining, compared to WT. In addition, DKO mice also exhibit significantly smaller CM cross-sectional area at P7 compared to WT. However, by P15, CM mitotic activity and cell size are comparable between WT and Btg1/2 KO mice. Currently, siRNA-mediated knockdown of Btg1/2 in neonatal rat ventricular cardiomyocyte cultures and RNAseq studies are being performed, to assess the transcriptional regulatory roles of Btg1/2 in rodent CMs. Conclusions: Here, we highlight two novel regulators of postnatal CM maturation, Btg1 and Btg2, which are upregulated coincident with CM mitotic arrest in mice. Similar to p21 and Meis1, Btg1/2 depletion in mice induces a brief period of increased CM proliferative activity before onset of CM cell cycle arrest. Our results provide evidence for Btg1/2 working in tandem with other cardiac transcription factors and cell cycle regulators, to control CM mitotic arrest after birth.

Abstract 14308: Lamotrigine Inhibits the Inward Fast Sodium Current (I Na ) in Epileptic Rat Cardiomyocytes

Introduction: Lamotrigine (LTG) is widely prescribed to treat neurological disorders such as migraine, neurophatic pain, and epilepsy. Recently, LTG has been linked to Brugada síndrome (BrS) ECG associated with a reduction in sodium current (I Na ) amplitude. Experimental studies have revealed that LTG blocks voltage gated sodium channels (VGSC). Hypothesis: Therefore, disrupting the depolarizing fast sodium inward current is a potential cause for arrhythmias. Recently our laboratory has shown that epilepsy induces molecular remodeling of the cardiac ventricle characterized by neuronal sodium channels overexpression, so it would be very plausible that LTG can cause important alterations on I Na in epileptic individuals. Methods: We used Patch-Clamp technique to test the effect of LTG on I Na in sham and epileptic rat ventricular cardiomyocytes. Results: We found that LTG inhibited I Na in a single dose-response manner with an IC 50 =326.35μM in sham rats. In epileptic rat cardiomyocytes the inhibitory effect was biphasic way with an IC 50 (1)=8.09 μM, which is close to the therapeutic range (between 12 and 50 μM), and a IC 50 (2)=333.66 μM. Moreover, LTG also altered sodium channels availability in biphasic manner with Vh(1)=-76.36 ± 0.31 mV and Vh(2)=-121.42 ± 1.38 mV, suggesting that LTG reduced the number of ion channels contributing to I Na . Conclusions: These findings may explain in part how LTG induce a Brugada pattern on the ECG of epileptic patients.

Abstract 14243: The Apelin Mimetic Peptide (2Nal) Partially Inhibits the Transient Outward Potassium Current (Ito) in Rat Cardiomyocytes; a Potential Pharmacological Agent Against Brugada Syndrome

Introduction: Brugada syndrome (BrS) is an inherited disease characterized by ventricular cardiac arrhythmias leading to sudden cardiac death. One potential cause for arrhythmias is a disruption of the balance between the depolarizing fast sodium inward current (I Na ) and its opposing transient outward currents (I to ) favoring a faster repolarization during phase one of the action potential (AP). Hypothesis: BrS is mainly associated with a reduction in I Na amplitude caused by dominant mutations in the gene SCN5A encoding cardiac sodium channels. However, recent studies have shown that gain-of-function mutations in KCND3, the gene encoding Kv4.3 channels, increase I to amplitude. Therefore, targeting I to to modulate the balance of current during phase one of the AP represents a promising therapeutic approach to BrS, Methods: We used Patch-Clamp technique to test the effect of 2Nal, a newly synthesized peptide derived from apelin, on I to in rat ventricular cardiomyocytes. Apelin is an endogenous neurotransmitter with strong inotropic effects on cardiac cells. Results: We found that apelin had no effect on I to . In contrast, 2Nal maximally inhibited 46.9% ± 3.4 (n=9) of I to with an IC 50 =0.29 nM but did not change its kinetics, thus indicating that 2Nal acts as a partial blocker. The voltage dependence of potassium channels availability was not modified, suggesting that 2Nal simply reduced the conductance of each channel contributing to I to . Conclusions: Because of its partial antagonist effect on ITo, 2Nal provide a safety margin that could be potentially useful for the treatment of Brugada syndrome.

Abstract 11598: Post-Developmental Deletion of Cardiomyocytes PR Domain Containing 16 (prdm16) Causes Pathological Hypertrophy

Despite recent medical advances, heart failure (HF) remains to be the leading cause of mortality worldwide. Delineating the underlying molecular mechanisms involved in the pathogenesis of HF is critical for the development of new therapies. The transcription factor PRDM16 is involved in cell fate determination and differentiation. We previously showed that PRDM16 variants/deletion resulted in left ventricular non-compaction cardiomyopathy in humans and mice indicating its involvement in cardiac development. Here, we investigated the role of PRDM16 in adult cardiac physiology. We showed that patients with HF exhibited a significant reduction in cardiac PRDM16 mRNA expression compared to control individuals. These results were substantiated by the significant attenuation of Prdm16 mRNA 2 weeks post- transverse aortic constriction (TAC) in mice when compared to age matched sham controls. Additionally, cultured neonatal rat ventricular cardiomyocytes treated with the hypertrophy inducing agent, phenylephrine, demonstrated a suppression of Prdm16 gene expression while fibrotic gene expression was elevated. To directly assess the contribution of PRDM16 to cardiac hypertrophy and HF, we generated mice with tamoxifen-inducible cardiomyocyte-specific Prdm16 deletion (CiPrdm16). CiPrdm16 KO mice developed concentric hypertrophy as evidenced by enhanced heart weight/tibia length, elevated left ventricular mass and increased cardiomyocyte hypertrophy. Initially, ciPrdm16 KO mice exhibited enhanced contractility but progressively developed contractile dysfunction and impaired diastolic function. Consistent with these findings, transcriptomic analysis revealed an increase in protein synthesis pathways in ciPrdm16 KO hearts compared to wild-type controls. Collectively, these data show for the first time that PRDM16 transcriptionally regulates pro-hypertrophic signaling in the adult mouse heart and may contribute to the progression of HF in humans.

SH3-Binding Glutamic Acid Rich-Deficiency Augments Apoptosis in Neonatal Rat Cardiomyocytes.

Congenital heart disease (CHD) is one of the most common birth defects in humans, present in around 40% of newborns with Down’s syndrome (DS). The SH3 domain-binding glutamic acid-rich (SH3BGR) gene, which maps to the DS region, belongs to a gene family encoding a cluster of small thioredoxin-like proteins sharing SH3 domains. Although its expression is confined to the cardiac and skeletal muscle, the physiological role of SH3BGR in the heart is poorly understood. Interestingly, we observed a significant upregulation of SH3BGR in failing hearts of mice and human patients with hypertrophic cardiomyopathy. Along these lines, the overexpression of SH3BGR exhibited a significant increase in the expression of hypertrophic markers (Nppa and Nppb) and increased cell surface area in neonatal rat ventricular cardiomyocytes (NRVCMs), whereas its knockdown attenuated cellular hypertrophy. Mechanistically, using serum response factor (SRF) response element-driven luciferase assays in the presence or the absence of RhoA or its inhibitor, we found that the pro-hypertrophic effects of SH3BGR are mediated via the RhoA–SRF axis. Furthermore, SH3BGR knockdown resulted in the induction of apoptosis and reduced cell viability in NRVCMs via apoptotic Hippo–YAP signaling. Taking these results together, we here show that SH3BGR is vital for maintaining cytoskeletal integrity and cellular viability in NRVCMs through its modulation of the SRF/YAP signaling pathways.

Telomere shortening in heart failure due to excess neurohormonal activation is associated with loss of nuclear TRF-2 and increased telomeric DNA damage and predicts cardiac recovery

Abstract The mechanisms and clinical significance of telomere shortening in heart failure remain elusive. Mammalian cardiomyocyte (CM) regeneration is limited and CM cell division cannot account for telomere shortening. Whether telomere shortening in turn affects cardiac recovery remains unexplored. We induced heart failure by excess neurohormonal activation (NHA), a universal dysregulation in the failing heart. B6 mice were subjected to AngII-infusion, uninephrectomy and high-salt (AngII++) to induce excess NHA for 5 weeks. Cardiac function was assessed by ultrasound. Human iPSC-derived CMs, mouse CMs and rat ventricular CMs (H9C2) were stimulated with AngII. Human cardiac biopsies from patients with heart failure with reduced ejection fraction (HFrEF) were grouped depending on cardiac recovery. Telomere length was quantified by Q-FISH. ROS-induced DNA/RNA-damage was evaluated after staining for 8-oxo-7,8-dihydro-2'-deoxyguanosine, -guanine and – guanosine-residues (8-oxo-dG). H9C2-cells were co-stained for telomeres, telomere repeat binding factor 2 (TRF-2) and 8-oxo-dG, telomere-specific DNA-damage was quantified by automated colocalization analysis. Superoxide (O2-) was quantified by 2-hydroxyethidium (2-HE) using HPLC analysis. Fibrosis was quantified by Sirius red staining. AngII++-mice exhibited left ventricular (LV) hypertrophy (p<0.0001 vs B6) along with reduced LV ejection fraction (p=0.03 vs B6), reduced global longitudinal strain (p<0.001 vs B6) along with significant telomere shortening in CMs isolated from AngII++-mice (p=0.0082 vs B6). Telomere length showed a significant correlation with LV systolic function (r2=0.64, p=0.006). While 8-oxo-dG-staining revealed significantly increased ROS-induced DNA/RNA damage in AngII++-myocardial sections overall (p<0.0001 vs B6), DNA/RNA-damage was highest in areas of fibrotic adverse remodeling congruent with significant telomere shortening (p<0.0122 fibrotic vs non-fibrotic AngII++-myocardium, p<0.0001 vs B6). In all cells tested, NHA resulted in significant telomere shortening. As a potential explanation, CMs stimulated with AngII revealed significantly increased, dose-dependent O2–production (AngII 10 nM p=0.03, AngII 50 nm p=0.0004 vs H9C2) along with significant loss of nucelar TRF2 (AngII nM p=0.0049, Ang II 50 nM p<0.0001 vs H9C2), while colocalization analysis revealed a significantly increased ROS-induced damage to the telomeric DNA (AngII 10/50 nm p<0.05 vs H9C2). HFrEF-patients that had recovered cardiac function (ΔLVEF +5 – 30%) showed significantly longer myocardial telomeres than patients with no recovery/worsening (ΔLVEF −10 – 0%) (p=0.008), overall the telomere signal correlated significantly with ΔLVEF (r2=0.41, p=0.03). Our data provides insides into how excess NHA via increased ROS, loss of nuclear TRF-2 and increased telomeric DNA-damage causes myocardial telomere shortening in heart failure and further that the extent of telomere shortening determines cardiac recovery. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): BMBF 01EO1003

The effect of canakinumab on COVID19-associated cardiomyocyte contractile dysfunction and arrhythmias

Abstract Background Cardiac injury associated with cytokine release occurs in almost 20% of SARS-CoV-2 positive patients during hospitalization and mortality is particularly high in these patients. Cardiac enzyme (e.g. troponin or creatinine kinase (CK)) elevations are a frequently reported finding, indicating myocardial damage and arrhythmias are the cause for ICU transfer in up to 12% of patients. However, the mechanistic role of COVID19 associated cytokine-storm for the concomitant cardiac dysfunction and associated arrhythmias is unclear. In addition, the role of anti-inflammatory therapy approaches to mitigate this cardiac dysfunction remains elusive. Methods We investigated the effects of COVID19-associated inflammatory response on cardiac cellular function as well as its cardiac arrhythmogenic potential in rat and induced pluripotent stem cell derived cardiomyocytes (iPSc-CM). Moreover, we evaluated the therapeutic potential of the IL1-beta antagonist Canakinumab using state of the art in-vitro confocal and ratiometric high-throughput microscopy. Results Isolated rat ventricular cardiomyocytes were exposed to control or COVID19 plasma from intensive care unit patients with severe ARDS and impaired cardiac function (LVEF 41±5%; 1/3 of patients on veno-venous extracorporeal membrane oxygenation; CK 154±43 U/l). Cardiomyocytes showed decreased Ca2+ transient amplitudes and altered baseline Ca2+ concentrations leading to impaired cellular contractile function upon electrical field-stimulation and exposure to patient plasma (n=276 control and 359 COVID19 cells; Fura). In addition, we used iPSc-CM to explore the long-term effect of patient plasma on cardiac electrical and mechanical function in a translational setting (24h incubation; Fluo). In iPSc, spontaneous Ca2+ release events (i.e. Ca2+ waves and Ca2+ sparks) were more likely to occur upon incubation with COVID19 plasma and nuclear as well as cytosolic Ca2+ release were altered. Co-incubation with Canakinumab had no effect on pro-arrhythmogenic Ca2+ release or Ca2+ signaling during excitation-contraction coupling but influenced cellular automaticity upon prolonged electrical stimulation. Conclusion Plasma derived from COVID19 patients exerts acute cardio-depressant and chronic pro-arrhythmogenic effects in rat and iPS-derived cardiomyocytes. Chronic co-incubation with Canakinumab had no beneficial effect on cellular Ca2+ signaling during excitation-contraction coupling. Funding Acknowledgement Type of funding sources: None.

Abstract MP237: Multi-omics Profiling Reveals Stearoylcarnitine As A Novel Biomarker Of Cardiometabolic Disease Development In Rat Offspring Exposed To Gestational Diabetes

Through unknown mechanisms, fetal exposure to gestational diabetes mellitus (GDM) increases the risk for cardiovascular disease development later in life. We hypothesize that fetal exposure to GDM alters offspring cardiomyocyte metabolism and left ventricular (LV) function with age. To induce GDM, female rats were fed a high fat (45% kcal) and sucrose diet prior to mating, throughout pregnancy and lactation. Lean controls received a low fat (10% kcal) diet. Fetal rat ventricular cardiomyocytes (FRVC) were isolated from e20 offspring for U-13C glucose metabolic flux analysis, mitochondrial respiration and calcium handling. The cardiac transcriptome and metabolome were measured in 3-month old offspring. LV morphology and function was assessed in the offspring from e18 to 12-months of age by transthoracic ultrasound. Offspring exposed to GDM exhibited increased LV posterior wall thickness and impaired LV filling across their life course (fetal to 12-months of age; p<0.05). Consistent with the development of diastolic dysfunction in vivo, alterations in calcium flux and sarcoplasmic reticulum-dependent calcium re-uptake (1.5 and 1.6-fold greater, respectively) were observed in FRVC isolated from GDM offspring (p<0.05). When FRVC were treated with isoproterenol, U-13C glucose metabolic flux through glycolysis and the citric acid cycle was reduced in GDM offspring, compared to Lean controls. In 3-month old GDM offspring serum and cardiac metabolomics revealed an altered acylcarnitine profile, with specific elevation in long chain acylcarnitine species including stearoylcarnitine. Alterations in the metabolome corresponded to changes in gene expression patterns identified by RNA-Seq associated with glucose metabolism and fatty acid transport pathways (e.g. Irs2, Slc2a4, Pfkfb2, Pdk4 and Cpt1a). These alterations corresponded with mitochondrial dysfunction, impaired cardiomyocyte metabolic flux and contractility, in concert with LV hypertrophy and diastolic dysfunction in the rat offspring. Multi-omics profiling reveals stearoylcarnitine as a novel biomarker and implicates mitochondrial oxidative metabolism as a mechanism that links early-life GDM exposure to the development of cardiovascular disease later in life.

Abstract P448: Role For Btg1 And Btg2 In Postnatal Cardiomyocyte Cell Cycle Arrest And Maturation

Background: Adult mammalian cardiomyocytes (CM) are predominantly post-mitotic and cannot proliferatively repair the heart following myocardial infarction (MI). Overexpression of the transcription factor Tbx20 in adult mouse CMs promotes proliferative cardiac repair post-MI, via mechanisms including direct repression of anti-proliferative genes p21 , Meis1 , and Btg2 . Btg2 (B-cell translocation gene 2), a tumor suppressor and transcriptional co-regulator, exhibits high structural and functional similarity with Btg1. However, both Btg1 and Btg2 (Btg1/2) are virtually uncharacterized in the heart. Here, we investigate the role of Btg1/2 in postnatal cardiac maturation. Methods and Results: By immunostaining in embryonic, neonatal, and adult C57BL/6 mouse hearts, the highest expression of Btg1/2 was observed in late fetal and early neonatal ventricles, concurrent with upregulation of other CM cell cycle inhibitors. In neonatal mouse CMs in vitro, siRNA-mediated loss of Btg2 leads to increased CM proliferation. In vivo , Btg1/2 constitutive single- and double- knockout (SKO and DKO respectively) mice exhibit normal heart weight-to-body weight ratios compared to age-matched wildtype (WT) controls, at postnatal day (P)7, P30, and 1 year after birth. Interestingly, at P7, DKO mice have significantly higher CM mitotic activity, as indicated by pHH3 staining, compared to WT. In addition, DKO mice also exhibit significantly smaller CM cross-sectional area at P7 compared to WT. However, by P15, CM mitotic activity and cell size are comparable between WT and Btg1/2 KO mice. Currently, siRNA-mediated knockdown of Btg1/2 in neonatal rat ventricular cardiomyocyte cultures and RNAseq studies are being performed, to assess the transcriptional regulatory roles of Btg1/2 in rodent CMs. Conclusions: Here, we highlight two novel regulators of postnatal CM maturation, Btg1 and Btg2, which are upregulated coincident with CM mitotic arrest in mice. Similar to p21 and Meis1, Btg1/2 depletion in mice induces a brief period of increased CM proliferative activity before onset of CM cell cycle arrest. Our results provide evidence for Btg1/2 working in tandem with other cardiac transcription factors and cell cycle regulators, to control CM mitotic arrest after birth.

Inhibition of the NLRP3/IL-1β axis protects against sepsis-induced cardiomyopathy.

BackgroundSeptic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin‐1β (IL‐1β), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL‐1β in sepsis‐induced cardiomyopathy and cardiac atrophy.Methods Male Nlrp3 knockout (KO) and wild‐type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham‐treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL‐1β effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real‐time deformability cytometry (RT‐DC) analysis were used to investigate functional and mechanical effects of IL‐1β on cardiomyocytes.ResultsHeart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 μL vs. 24.6 ± 8.7 μL, P < 0.05; KO sham vs. KO CLP: 28.3 ± 8.1 μL vs. 29.9 ± 9.9 μL, n.s.; P < 0.05 vs. WT CLP) and diastolic (peak E wave velocity: WT sham vs. WT CLP: 750 ± 132 vs. 522 ± 200 mm/s, P < 0.001; KO sham vs. KO CLP: 709 ± 152 vs. 639 ± 165 mm/s, n.s.; P < 0.05 vs. WT CLP) cardiac function and attenuated cardiac (heart weight–tibia length ratio: WT CLP vs. WT sham: −26.6%, P < 0.05; KO CLP vs. KO sham: −3.3%, n.s.; P < 0.05 vs. WT CLP) and cardiomyocyte atrophy in KO mice during sepsis. IonOptix measurements showed that IL‐1β decreased contractility (cell shortening: IL‐1β: −15.4 ± 2.3%, P < 0.001 vs. vehicle, IL‐1RA: −6.1 ± 3.3%, P < 0.05 vs. IL‐1β) and relaxation of adult rat ventricular cardiomyocytes (time‐to‐50% relengthening: IL‐1β: 2071 ± 225 ms, P < 0.001 vs. vehicle, IL‐1RA: 564 ± 247 ms, P < 0.001 vs. IL‐1β), which was attenuated by an IL‐1 receptor antagonist (IL‐1RA). RT‐DC analysis indicated that IL‐1β reduced cardiomyocyte size (P < 0.001) and deformation (P < 0.05). RNA sequencing showed that genes involved in NF‐κB signalling, autophagy and lysosomal protein degradation were enriched in hearts of septic WT but not in septic KO mice. Western blotting and qPCR disclosed that IL‐1β activated NF‐κB and its target genes, caused atrophy and decreased myosin protein in myocytes, which was accompanied by an increased autophagy gene expression. These effects were attenuated by IL‐1RA.ConclusionsIL‐1β causes atrophy, impairs contractility and relaxation and decreases deformation of cardiomyocytes. Because NLRP3/IL‐1β pathway inhibition attenuates cardiac atrophy and cardiomyopathy in sepsis, it could be useful to prevent septic cardiomyopathy.