Maintenance Of Cardiac Function Research Articles

PINK1-mediated mitophagy attenuates pathological cardiac hypertrophy by suppressing the mtDNA release-activated cGAS-STING pathway.

Sterile inflammation is implicated in the development of heart failure (HF). Mitochondria plays important roles in triggering and maintaining inflammation. Mitophagy is important for regulation of mitochondrial quality and maintenance of cardiac function under pressure overload. The association of mitophagy with inflammation in HF is largely unclear. As PINK1 is a central mediator of mitophagy, our objective was to investigate its involvement in cardiac hypertrophy, and the effect of PINK1-mediated mitophagy on cGAS-STING activation during cardiac hypertrophy. PINK1 knockout and cardiac-specific PINK1-overexpressing transgenic mice were created and subsequently subjected to transverse aortic constriction (TAC) surgery. In order to explore whether PINK1 regulates STING-mediated inflammation during HF, PINK1/STING (stimulator of interferon genes) double-knockout mice were created. Pressure overload was induced by TAC. Our findings indicate a significantly decline in PINK1 expression in TAC-induced hypertrophy. Cardiac hypertrophic stimuli caused the release of mitochondrial DNA (mtDNA) into the cytosol, activating the cGAS-STING signaling, which in turn initiated cardiac inflammation and promoted the progression of cardiac hypertrophy. PINK1 deficiency inhibited mitophagy activity, promoted mtDNA release, and then drove the overactivation of cGAS-STING signaling, exacerbating cardiac hypertrophy. Conversely, cardiac-specific PINK1 overexpression protected against hypertrophy thorough inhibition of the cGAS-STING signaling. Double-knockout mice revealed that the effects of PINK1 on hypertrophy were dependent on STING. Our findings suggest that PINK1-mediated mitophagy plays a protective role in pressure overload-induced cardiac hypertrophy via inhibiting the mtDNA-cGAS-STING pathway.

Abstract Tu087: Knockout of TIGAR Rescues Cardiac Dysfunction in SIRT3 Knockout Mice

Introduction: Heart failure (HF) is the leading cause of death in the United States, despite advances in treatment, it maintains the highest disease mortality rate in the country. Previously it has been established that Sirtuin 3 (SIRT3), a mitochondrial NAD+- dependent deacetylase, and the Tp53-induced glycolysis and apoptosis regulator (TIGAR) are associated with the development of HF. SIRT3 plays a key role in the maintenance of cardiac function. Decreases in SIRT3 are associated with human aging and diabetes, whereas knockout results in cardiac dysfunction in mice. We have shown: (1) overexpression of SIRT3 downregulates TIGAR and attenuates diabetic cardiomyopathy; (2) knockout of TIGAR enhances myocardial glycolysis and reduces pathological remodeling while maintaining cardiac function in pressure-overload-induced heart failure. To date, the direct interactions between TIGAR and SIRT3 in the development of HF remain unexplored. Aim: By crossing TIGAR knockout mice (TIGARKO) with SIRT3 knockout (SIRT3KO) mice, this study tested if genetic targeting of TIGAR could rescue cardiac dysfunction in SIRT3KO mice. Methods and Results: Echocardiography was performed on SIRT3KO, TIGARKO, TIGARKO/SIRT3KO, and SIRT3 Control mice. Analysis of cardiac function showed that SIRT3KO mice had significant decreases in coronary flow reserve (CFR), ejection fraction (EF) SIRT3KO (41.52 ± 4.50) vs SIRT3 Control (60.91 ± 4.91, p<0.001) and fractional shortening (FS) SIRT3KO (20.11 ± 2.48) vs SIRT3 Control (32.07 ± 3.37, p<0.001). The diastolic E’/A’ ratio was decreased; IVRT and MPI were significantly increased. Knockout of TIGAR in mice maintained systolic function by increasing EF (62.25 ± 9.84) vs (41.52 ± 4.50, p<0.001), FS (33.33 ± 7.12 vs 20.11 ± 2.48, p<0.05), and CFR, as well as improved diastolic function by decreasing IVRT, MPI, and increasing E’/A’ ratio in SIRT3KO mice. SIRT3KO and TIGARKO mice had a significant increase in weight/tibia length ratio (HW/TL). Interestingly, the knockout of TIGAR significantly reduced LV mass and HW/TL in SIRT3KO mice. Conclusions: The knockout of TIGAR can rescue the HF phenotype of SIRT3KO mice, but the underlying mechanisms require further investigation to develop potential treatments for HF.

Quantitative Analysis of Innate Lymphoid Cells in Patients with ST-Segment Elevation Myocardial Infarction

ABSTRACT Background Acute myocardial infarction (AMI) is one of the principal causes of death in Mexico and worldwide. AMI triggers an acute inflammatory process that induces the activation of different populations of the innate immune system. Innate lymphoid cells (ILCs) are an innate immunity, highly pleiotropic population, which have been observed to participate in tissue repair and polarization of the adaptive immune response. Objective We aimed to analyze the levels of subsets of ILCs in patients with ST-segment elevation myocardial infarction (STEMI), immediately 3 and 6 months post-AMI, and analyze their correlation with clinical parameters. Results We evaluated 29 STEMI patients and 15 healthy controls and analyzed the different subsets of circulating ILCs, immediately 3 and 6 months post-AMI. We observed higher levels of circulating ILCs in STEMI patients compared to control subjects and a significant correlation between ILC levels and cardiac function. We also found increased production of the cytokines interleukin 5 (IL-5) and interleukin 17A (IL-17A), produced by ILC2 cells and by ILC3 cells, respectively, in the STEMI patients. Conclusion This study shows new evidence of the role of ILCs in the pathophysiology of AMI and their possible involvement in the maintenance of cardiac function.

Spatiotemporal development and the regulatory mechanisms of cardiac resident macrophages: Contribution in cardiac development and steady state.

Cardiac resident macrophages (CRMs) are integral components of the heart and play significant roles in cardiac development, steady-state, and injury. Advances in sequencing technology have revealed that CRMs are a highly heterogeneous population, with significant differences in phenotype and function at different developmental stages and locations within the heart. In addition to research focused on diseases, recent years have witnessed a heightened interest in elucidating the involvement of CRMs in heart development and the maintenance of cardiac function. In this review, we primarily concentrated on summarizing the developmental trajectories, both spatial and temporal, of CRMs and their impact on cardiac development and steady-state. Moreover, we discuss the possible factors by which the cardiac microenvironment regulates macrophages from the perspectives of migration, proliferation, and differentiation under physiological conditions. Gaining insight into the spatiotemporal heterogeneity and regulatory mechanisms of CRMs is of paramount importance in comprehending the involvement of macrophages in cardiac development, injury, and repair, and also provides new ideas and therapeutic methods for treating heart diseases.

The dichotomous function of FUNDC1‐dependent mitophagy in doxorubicin cardiotoxicity

Doxorubicin (DOX), an extremely effective and wide‐spectrum antineoplastic anthracycline, has been known for its notorious adverse effect of dose‐dependent dilated cardiotoxicity that culminates in heart failure. The current approach for reducing DOX‐induced cardiotoxicity is to limit the overall cumulative dose of the drug, but at the expense of narrowing the therapeutic window for cancer treatment. Therefore, it is imperative to identify new strategies to protect against DOX‐induced heart damage without compromising its antineoplastic activity. DOX cardiotoxicity is closely associated with mitochondrial injury which is characterized by an early loss of mitochondrial membrane potential followed by dysregulation of mitochondrial quality control mechanisms including mitophagy, a process through which injured mitochondria are degraded by the autophagic pathway. Mitophagy is generally believed to play protective roles under various normal and disease conditions. However, evidence also suggests that mitophagy can become detrimental, leading to cell death under certain conditions. The effect of DOX on mitophagy has been assessed previously, but neither mitophagy activity nor its functional role in DOX cardiotoxicity has been clearly defined. Parkin and FUNDC1 are two well‐established positive regulators of mitophagy. Knockdown of Parkin diminished DOX‐induced cell death, while overexpression of Parkin had the opposite effects, suggesting that DOX cardiotoxicity was mediated, at least in part, by accelerated mitophagy through a Parkin‐dependent pathway. However, the role of FUNDC1‐mediated mitophagy in DOX cardiotoxicity remains unclear. In this study, we investigated the functional role of FUNDC1 in DOX‐induced cardiotoxicity using both FUNDC1 knockout (KO) and transgenic (TG) mice. We hypothesized that knockout of FUNDC1 would alleviate DOX‐induced cardiotoxicity while overexpression of FUNDC1 would exacerbate DOX cardiotoxicity. DOX‐induced cardiac injury was examined and compared with WT and FUNDC1 KO (FKO) or FUNDC1 transgenic (FTG) mice. The Fractional Shortening (FS) was measured by echocardiography. Serum LDH activity and cardiac troponin‐I (cTnI) levels were measured and used as indicators of cardiac tissue damage. For the mitophagy flux assay, mice were treated with lysosomal proteasome inhibitors (25mg/kg pepA and 5mg/kg E64d) for 4 hours and the heart tissues were collected for Western blot analyses. Knockout of FUNDC1 reduced mitophagy in the mouse heart, while overexpression of FUNDC1 accelerated mitophagy flux, confirming FUNDC1 as a positive regulator of mitophagy. Knockout of FUNDC1 attenuated DOX‐induced cardiac functional impairment as shown by improved fractional shortening (FS), supporting the conclusion that FUNDC1‐dependent mitophagy may mediate DOX cardiotoxicity. Surprisingly, however, overexpression of FUNDC1 also alleviated DOX‐induced cardiac injury, suggesting that FUNDC1 may function independently of mitophagy in the maintenance of cardiac function.

Capillaries as a Therapeutic Target for Heart Failure.

Prognosis of heart failure remains poor, and it is urgent to find new therapies for this critical condition. Oxygen and metabolites are delivered through capillaries; therefore, they have critical roles in the maintenance of cardiac function. With aging or age-related disorders, capillary density is reduced in the heart, and the mechanisms involved in these processes were reported to suppress capillarization in this organ. Studies with rodents showed capillary rarefaction has causal roles for promoting pathologies in failing hearts. Drugs used as first-line therapies for heart failure were also shown to enhance the capillary network in the heart. Recently, the approach with senolysis is attracting enthusiasm in aging research. Genetic or pharmacological approaches concluded that the specific depletion of senescent cells, senolysis, led to reverse aging phenotype. Reagents mediating senolysis are described to be senolytics, and these compounds were shown to ameliorate cardiac dysfunction together with enhancement of capillarization in heart failure models. Studies indicate maintenance of the capillary network as critical for inhibition of pathologies in heart failure.

Zyxin protects from hypertension-induced cardiac dysfunction

Arterial hypertension causes left ventricular hypertrophy leading to dilated cardiomyopathy. Following compensatory cardiomyocyte hypertrophy, cardiac dysfunction develops due to loss of cardiomyocytes preceded or paralleled by cardiac fibrosis. Zyxin acts as a mechanotransducer in vascular cells that may promote cardiomyocyte survival. Here, we analyzed cardiac function during experimental hypertension in zyxin knockout (KO) mice. In zyxin KO mice, made hypertensive by way of deoxycorticosterone acetate (DOCA)-salt treatment telemetry recording showed an attenuated rise in systolic blood pressure. Echocardiography indicated a systolic dysfunction, and isolated working heart measurements showed a decrease in systolic elastance. Hearts from hypertensive zyxin KO mice revealed increased apoptosis, fibrosis and an upregulation of active focal adhesion kinase as well as of integrins α5 and β1. Both interstitial and perivascular fibrosis were even more pronounced in zyxin KO mice exposed to angiotensin II instead of DOCA-salt. Stretched microvascular endothelial cells may release collagen 1α2 and TGF-β, which is characteristic for the transition to an intermediate mesenchymal phenotype, and thus spur the transformation of cardiac fibroblasts to myofibroblasts resulting in excessive scar tissue formation in the heart of hypertensive zyxin KO mice. While zyxin KO mice per se do not reveal a cardiac phenotype, this is unmasked upon induction of hypertension and owing to enhanced cardiomyocyte apoptosis and excessive fibrosis causes cardiac dysfunction. Zyxin may thus be important for the maintenance of cardiac function in spite of hypertension.

Untangling the Cooperative Role of Nuclear Receptors in Cardiovascular Physiology and Disease.

The heart is the first organ to acquire its physiological function during development, enabling it to supply the organism with oxygen and nutrients. Given this early commitment, cardiomyocytes were traditionally considered transcriptionally stable cells fully committed to contractile function. However, growing evidence suggests that the maintenance of cardiac function in health and disease depends on transcriptional and epigenetic regulation. Several studies have revealed that the complex transcriptional alterations underlying cardiovascular disease (CVD) manifestations such as myocardial infarction and hypertrophy is mediated by cardiac retinoid X receptors (RXR) and their partners. RXRs are members of the nuclear receptor (NR) superfamily of ligand-activated transcription factors and drive essential biological processes such as ion handling, mitochondrial biogenesis, and glucose and lipid metabolism. RXRs are thus attractive molecular targets for the development of effective pharmacological strategies for CVD treatment and prevention. In this review, we summarize current knowledge of RXR partnership biology in cardiac homeostasis and disease, providing an up-to-date view of the molecular mechanisms and cellular pathways that sustain cardiomyocyte physiology.

Potassium channel Shaker play a protective role against cardiac aging in Drosophila.

Potassium channels, which are the most diverse group of the ion channel family, play an important role in the repolarization of cardiomyocytes. Recent studies showed that potassium channels, such as KCNQ and HERG/eag, play an important role in regulating adult heart function through shaping the action potential and maintaining the rhythm of cardiac contraction. The potassium channel protein Shaker is the first voltage-gated potassium channel found in Drosophila to maintain the electrical excitability of neurons and muscle cells, but its role in adult cardiac function is still unclear. In this study, Drosophila was used as a model to study the role of Shaker channel in the maintenance of cardiac function under stress and aging. The incidence of heart failure was observed in shaker mutant after external electrical pacing, which simulates cardiac stress. Additionally, The cardiac-specific driver hand4.2 Gal4 was used to specifically knock down the expression of the potassium channel shaker in Drosophila. The cardiac parameter was analyzed at 1, 3, 5 weeks of age on cardiac specific knockdown of shaker using Drosophila adult cardiac physiological assay. The results showed that the mutation of shaker gene seriously affect the cardiac function under stress, demonstrated by significant increase in heart failure rate under electrical stimulation. In addition, cardiac specific knockdown of shaker increased the incidence of arrhythmias in Drosophila at the age of 5 weeks. Cardiac-specific knockdown of shaker reduces life span. Therefore, the results of this study suggest a vital role of the potassium channel shaker in maintaining normal cardiac function during aging.

Centruroides margaritatus scorpion complete venom exerts cardiovascular effects through alpha-1 adrenergic receptors

Centruroides margaritatus scorpion stings are common in Colombia. However, the cardiovascular toxicity of the venom has not been clarified. Aim: To study the effect and mechanisms of action of the complete venom of C. margaritatus (CmV) on the murine cardiovascular system. Methods: We evaluated the in vivo effect of CmV LD50 on the mean arterial pressure (MABP), heart rate, and surface electrocardiogram in male adult normotensive Wistar rats. Ex vivo, we evaluated the vascular reactivity of rat aortic rings to increasing concentrations (1 to 60 μg/mL) of CmV using the blockers L-NAME, indomethacin, seratrodast, and prazosin. Results: In the first hour of poisoning, CmV increased the MABP. In the second hour after poisoning, the heart rate decreased as the normalized PR interval and QT corrected increased. After that, cardiovascular shock was demonstrated by a drastic fall in the MABP and signs of cardiac conduction system block. In aortic rings, CmV caused a direct vasoconstrictor effect mediated by alpha-1 adrenergic receptors and counteracted by nitric oxide. Conclusion: The direct vascular and probably the cardiac alpha-1 effects likely explain the transient hypertension and the maintenance of cardiac function, while interval lengthening may be due to K+ channel blockage. Afterwards, the effects of both the alpha-1 pathway and the K+ channel pathway converged, resulting in fatal cardiovascular shock. This knowledge could aid in understanding the dynamics of the effects of the venom and in designing treatments to address its cardiovascular effects.

Aging-associated cardiac dysfunction and leaky gut in heterozygous Akap1 knockout mice

Abstract Background Mitochondrial A-kinase anchoring proteins (mitoAKAPs) encoded by the Akap1 gene are crucially involved in multiple cellular processes, including cardiomyocyte survival and function. There is a correlation between ROS production, intestinal permeability and the composition of the intestinal microbiota, especially during aging. Whether Akap1 deletion affects microbiota composition, intestinal function during senescence is currently unknown. Purpose The purpose of the study was to shed light into the complex interplay between gut permeability and microbiota composition, cardiac function and aging in adult (6-month-old, 6m) and old (24-month-old, 24m) Akap 1 wild type (Akap1+/+) or Akap1 heterozygous knockout mice (Akap1+/−). Methods Cardiac function was non-invasively analyzed by echocardiography in adult and old, Akap1+/+ and Akap1+/− mice. Colon, serum and feces samples were collected after sacrifice in 6m and 24m mice of either genotype. Intestinal barrier permeability was evaluated in colon samples by Occludin (Ocln) and Tight junction protein ZO-1 (Tjp1) mRNA expression analysis. Systemic inflammation was measured by Tumor Necrosis Factor-alpha (TNF-alpha), Lipopolysaccharide (LPS), Interleukin-10 (IL-10) and Interleukin-1 (IL-1) circulating levels. Microbial DNA was extracted from feces samples and gut microbiota composition was evaluated by Illumina Mi-Seq analysis. Bioinformatic analyses were carried out to identify intestinal populations. Results Akap1 partial deletion accelerated the progression of cardiac dysfunction in 24m mice as demonstrated by a significant reduction of fractional shortening in Akap1+/− 24m mice compared to Akap1+/+ 24m (Figure 1A). Colon permeability was impaired in Akap1+/− 24m as shown by reduced Ocln expression (Figure 1B), while circulating TNF-alpha was increased in Akap1+/− 24m (Figure 1C). Next, we analyzed the differences in abundance of all 2042 Operational Taxonomic Units (OTUs) between age-matched Akap1+/+ and Akap1+/−. We identified 10 OTUs differently represented in Akap1+/+ and Akap1+/− 6m mice, while a bigger set of bacterial OTUs (19) were different between Akap1+/+ and Akap1+/− 24m mice. LDA scores of differentially microbiome abundant taxa in Akap1+/+ and Akap1+/− 24m (Figure 1D) showed different assortment in Clostridiales family (Ruminococcus torques specie), Porphyromonadaceae family (Barseniella intestinihominis specie) and Lachnospiraceae genus (Blautia producta specie), bacterial species that have been previously identified in patients with heart failure and involved in anti-inflammatory mechanisms. Conclusions mitoAKAPs play a crucial role in the maintenance of cardiac function and intestinal barrier during aging, since Akap1 partial deletion promotes gut permeability, bacteria translocation and systemic inflammation associated with systolic cardiac dysfunction. Figure 1 Funding Acknowledgement Type of funding source: Other. Main funding source(s): CP was supported by Ministero dell'Istruzione, Università e Ricerca Scientifica grant (2015583WMX) and Programma STAR grant by Federico II University and Compagnia di San Paolo. RP was supported by a research grant provided by the Cardiopath PhD program. LC was supported by 2018-2019 Postdoctoral Fellowship Grants provided by Fondazione Umberto Veronesi.

Tilianin Protects against Ischemia/Reperfusion-Induced Myocardial Injury through the Inhibition of the Ca2+/Calmodulin-Dependent Protein Kinase II-Dependent Apoptotic and Inflammatory Signaling Pathways.

Tilianin is a naturally occurring phenolic compound with a cardioprotective effect against myocardial ischemia/reperfusion injury (MIRI). The aim of our study was to determine the potential targets and mechanism of action of tilianin against cardiac injury induced by MIRI. An in silico docking model was used in this study for binding mode analysis between tilianin and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Oxygen-glucose deprivation/reperfusion- (OGD/R-) injured H9c2 cardiomyocytes and ischemia/reperfusion- (I/R-) injured isolated rat hearts were developed as in vitro and ex vivo models, respectively, which were both treated with tilianin in the absence or presence of a specific CaMKII inhibitor KN93 for target verification and mechanistic exploration. Results demonstrated the ability of tilianin to facilitater the recovery of OGD/R-induced cardiomyocyte injury and the maintenance of cardiac function in I/R-injured hearts. Tilianin interacted with CaMKIIδ with an efficient binding performance, a favorable binding score, and restraining p-CaMKII and ox-CaMKII expression in cardiomyocytes injured by MIRI. Importantly, inhibition of CaMKII abolished tilianin-mediated recovery of OGD/R-induced cardiomyocyte injury and maintenance of cardiac function in I/R-injured hearts, accompanied by the disability to protect mitochondrial function. Furthermore, the protective effects of tilianin towards mitochondrion-associated proapoptotic and antiapoptotic protein counterbalance and c-Jun N-terminal kinase (JNK)/nuclear factor- (NF-) κB-related inflammation suppression were both abolished after pharmacological inhibition of CaMKII. Our investigation indicated that the inhibition of CaMKII-mediated mitochondrial apoptosis and JNK/NF-κB inflammation might be considered as a pivotal mechanism used by tilianin to exert its protective effects on MIRI cardiac damage.

Isoliquiritigenin attenuates diabetic cardiomyopathy via inhibition of hyperglycemia-induced inflammatory response and oxidative stress

Inflammation and oxidative stress play essential roles in the occurrence and progression of diabetic cardiomyopathy (DCM). Isoliquiritigenin (ISL), a natural chalcone, exhibits strong anti-inflammatory and antioxidant activities. In this study, we aimed to investigate the protective effects of ISL on DCM using high glucose (HG)-challenged cultured cardiomyocytes and streptozotocin (STZ)-induced diabetic mice. Embryonic rat heart-derived H9c2 cells challenged with a high concentration of glucose were used to evaluate the anti-inflammatory and antioxidant effects of ISL. STZ-induced diabetic mice were used to study the effects of ISL in DCM in vivo. Furthermore, cardiac fibrosis, hypertrophy, and apoptosis were explored both in vitro and in vivo. ISL effectively inhibited HG-induced hypertrophy, fibrosis, and apoptosis probably by alleviating the inflammatory response and oxidative stress in H9c2 cells. Results from in vivo experiments showed that ISL exhibited anti-inflammatory and antioxidant stress activities that were characterized by the attenuation of cardiac hypertrophy, fibrosis, and apoptosis, which resulted in the maintenance of cardiac function. The protective effects of ISL against inflammation and oxidative stress were mediated by the inhibition of mitogen-activated protein kinases (MAPKs) and induction of nuclear factor-erythroid 2 related factor 2 (Nrf2) signaling pathway, respectively. Our results provided compelling evidence that ISL, by virtue of neutralizing excessive inflammatory response and oxidative stress, could be a promising agent in the treatment of DCM. Targeting the MAPKs and Nrf2 signaling pathway might be an effective therapeutic strategy for the prevention and treatment of DCM.

Mitochondrial Ca2+ regulation in the etiology of heart failure: physiological and pathophysiological implications.

Heart failure (HF) represents one of the leading causes of cardiovascular diseases with high rates of hospitalization, morbidity and mortality worldwide. Ample evidence has consolidated a crucial role for mitochondrial injury in the progression of HF. It is well established that mitochondrial Ca2+ participates in the regulation of a wide variety of biological processes, including oxidative phosphorylation, ATP synthesis, reactive oxygen species (ROS) generation, mitochondrial dynamics and mitophagy. Nonetheless, mitochondrial Ca2+ overload stimulates mitochondrial permeability transition pore (mPTP) opening and mitochondrial swelling, resulting in mitochondrial injury, apoptosis, cardiac remodeling, and ultimately development of HF. Moreover, mitochondria possess a series of Ca2+ transport influx and efflux channels, to buffer Ca2+ in the cytoplasm. Interaction at mitochondria-associated endoplasmic reticulum membranes (MAMs) may also participate in the regulation of mitochondrial Ca2+ homeostasis and plays an essential role in the progression of HF. Here, we provide an overview of regulation of mitochondrial Ca2+ homeostasis in maintenance of cardiac function, in an effort to identify novel therapeutic strategies for the management of HF.

Chronic Sympathetic Hyperactivity Triggers Electrophysiological Remodeling and Disrupts Excitation-Contraction Coupling in Heart

The sympathetic nervous system is essential for maintenance of cardiac function via activation of post-junctional adrenergic receptors. Prolonged adrenergic receptor activation, however, has deleterious long-term effects leading to hypertrophy and the development of heart failure. Here we investigate the effect of chronic adrenergic receptors activation on excitation-contraction coupling (ECC) in ventricular cardiomyocytes from a previously characterized mouse model of chronic sympathetic hyperactivity, which are genetically deficient in the adrenoceptor α2A and α2C genes (ARDKO). When compared to wild-type (WT) cardiomyocytes, ARDKO displayed reduced fractional shortening (~33%) and slower relaxation (~20%). Furthermore, ARDKO cells exhibited several electrophysiological changes such as action potential (AP) prolongation (~50%), reduced L-type calcium channel (LCC) current (~33%), reduced outward potassium (K+) currents (~30%), and increased sodium/calcium exchanger (NCX) activity (~52%). Consistent with reduced contractility and calcium (Ca2+) currents, the cytosolic Ca2+ ([Ca2+]i) transient from ARDKO animals was smaller and decayed slower. Importantly, no changes were observed in membrane resting potential, AP amplitude, or the inward K+ current. Finally, we modified our existing cardiac ECC computational model to account for changes in the ARDKO heart. Simulations suggest that cellular changes in the ARDKO heart resulted in variable and dyssynchronous Ca2+-induced Ca2+ release therefore altering [Ca2+]i transient dynamics and reducing force generation. In conclusion, chronic sympathetic hyperactivity impairs ECC by changing the density of several ionic currents (and thus AP repolarization) causing altered Ca2+ dynamics and contractile activity. This demonstrates the important role of ECC remodeling in the cardiac dysfunction secondary to chronic sympathetic activity.

Loss of Functional Non‐Coding RNA Diversity in Diabetic Cardiac Mitochondria

Individuals with diabetes mellitus are predisposed to cardiovascular complications, but no effective predictive or therapeutic measure is in place to prevent these morbidities. Maintenance of cardiac function is heavily dependent on mitochondrial bioenergetics, making the regulation of mitochondria crucial in understanding cardiac pathologies. Both declining cardiovascular health as well as diminished mitochondrial efficiency have been separately correlated with disruption of non‐coding RNA (ncRNA) presence and activity. The recent detection of ncRNA within mitochondria by our lab and others has led us to investigate whether or not certain disease states could dysregulate ncRNA distribution and function inside the organelle. The objective of this study was to identify ncRNAs impacted within the mitochondrion by type 2 diabetes mellitus in human patients and evaluate the downstream effects caused by their dysregulation. Cytoplasm and mitochondria from human atrial appendages in type 2 diabetic (n=7) and non‐diabetic (n=7) patients were isolated and RNA purified for high‐throughput sequencing. In silico analyses of differentially expressed ncRNA revealed potential functions relevant to mitochondrial bioenergetics and cardiac dysfunction, primarily through targeting of mitochondrial and cytoplasmic mRNA but additionally for interactions between ncRNAs themselves. Approximately 20 tRNAs, 6 rRNAs, 447 lncRNAs, and 154 miRNAs were identified through mitochondrial sequencing, using a mean count value greater than 50 as a cutoff for baseline expression. Of these, all 6 rRNAs as well as 41 lncRNAs and 140 miRNAs were significantly downregulated in expression in type 2 diabetics compared to non‐diabetics. In contrast, 37 lncRNAs were significantly increased relative to non‐diabetic patients. The majority of lncRNAs and miRNAs were determined to have RNA‐binding properties, either for mRNAs or other non‐coding RNAs, based on predictive software and databases such as LncBase, NPInter, and miRcode. The mRNAs identified as targets for dysregulated miRNAs were correlated with regulation of proteins such as insulin, Akt, ERK1/2, and similarly metabolically‐crucial factors according to Ingenuity Pathway Analysis, implying cytoplasmic activity despite being found in the mitochondrion. Additionally, various cellular pathways influencing growth and viability were identified to be potentially impacted by miRNA dysregulation. Overall this study reveals a loss of non‐coding RNA diversity within type 2 diabetic cardiac mitochondria, which could contribute to mitochondrial dysfunction and ultimately cardiovascular morbidities associated with type 2 diabetes mellitus. These findings may also support ncRNA as therapeutic and diagnostic targets for type 2 diabetic cardiac complications.Support or Funding InformationThis work was supported by: The National Heart, Lung, and Blood Institute [R01 HL‐128485] (JMH), American Heart Association [AHA‐17PRE33660333] (QAH), West Virginia IDeA Network of Biomedical Research WV‐INBRE support by National Institute of Health Grant [P20GM103434], and the Community Foundation for the Ohio Valley Whipkey Trust.

Cardiomyocyte Specific Deletion of ADAR1 Causes Severe Cardiac Dysfunction and Increased Lethality.

Background: Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA-editing enzyme that is involved in several functions including the deamination of adenosine to inosine, RNA interference (RNAi) mechanisms and microRNA (miRNA) processing, rendering ADAR1 essential for life.Methods and Results: To investigate whether maintenance of ADAR1 expression is required for normal myocardial homeostasis, we bypassed the early embryonic lethality of ADAR1-null mice through the use of a tamoxifen-inducible Cre recombinase under the control of the cardiac-specific α-myosin heavy chain promoter (αMHC). Targeted ADAR1 deletion in adult mice caused a significant increase in lethality accompanied by severe ventricular remodeling and quick and spontaneous cardiac dysfunction, induction of stress markers and overall reduced expression of miRNAs. Administration of a selective inhibitor of the unfolded protein response (UPR) stress significantly blunted the deleterious effects and improved cardiac function thereby prolonging animal survival. In vitro restoring miR-199a-5p levels in cardiomyocytes lacking ADAR1 diminished UPR activation and concomitant apoptosis.Conclusions: Our findings demonstrate an essential role for ADAR1 in cardiomyocyte survival and maintenance of cardiac function through a mechanism that integrates ADAR1 dependent miRNA processing and the suppression of UPR stress.

Fibroblast transdifferentiation promotes conversion of M1 macrophages and replenishment of cardiac resident macrophages following cardiac injury in mice.

Resident cardiac macrophages play important roles in homeostasis, maintenance of cardiac function, and tissue repair. After cardiac injury, monocytes infiltrate the tissue, undergo phenotypic and functional changes, and are involved in inflammatory injury and functional remodelling. However, the fate of cardiac infiltrating/polarized macrophages and the relationship between these cells and resident cardiac macrophage replenishment following injury remain unclear. Our results showed that angiotensin II induces cardiac fibroblast transdifferentiation into cardiac myofibroblasts (MFBs). In cocultures with MFBs and murine macrophages, the MFBs promoted macrophage polarization to M1 phenotype, followed by selective apoptosis, which was associated with TNF/TNFR1 axis and independent of NO production. Surprisingly, after 36 h of coculture, the surviving macrophages were converted to M2 phenotype and settled in heart, which was dependent on leptin produced by MFBs or polarized macrophages via the PI3K or Akt pathway. CCR2+ CD45.2+ cells adoptively transferred into CD45.1+ mice with viral myocarditis, differentiated into CD45.2+ CCR2+ CX3CR1+ M2 cells during the resolution of inflammation and settled within the heart. Our data highlight a novel mechanism related to the renewal or replenishment of cardiac resident macrophages following cardiac injury; and suggest that transdifferentiation of cardiac fibroblasts may promote the resolution of inflammation.

Developmental plasticity of cardiac anoxia-tolerance in juvenile common snapping turtles ( Chelydra serpentina).

For some species of ectothermic vertebrates, early exposure to hypoxia during embryonic development improves hypoxia-tolerance later in life. However, the cellular mechanisms underlying this phenomenon are largely unknown. Given that hypoxic survival is critically dependent on the maintenance of cardiac function, we tested the hypothesis that developmental hypoxia alters cardiomyocyte physiology in a manner that protects the heart from hypoxic stress. To test this hypothesis, we studied the common snapping turtle, which routinely experiences chronic developmental hypoxia and exploits hypoxic environments in adulthood. We isolated cardiomyocytes from juvenile turtles that embryonically developed in either normoxia (21% O2) or hypoxia (10% O2), and subjected them to simulated anoxia and reoxygenation, while simultaneously measuring intracellular Ca2+, pH and reactive oxygen species (ROS) production. Our results suggest developmental hypoxia improves cardiomyocyte anoxia-tolerance of juvenile turtles, which is supported by enhanced myofilament Ca2+-sensitivity and a superior ability to suppress ROS production. Maintenance of low ROS levels during anoxia might limit oxidative damage and a greater sensitivity to Ca2+ could provide a mechanism to maintain contractile force. Our study suggests developmental hypoxia has long-lasting effects on turtle cardiomyocyte function, which might prime their physiology for exploiting hypoxic environments.

Conditioned Medium from Mesenchymal Stem Cells Protects Vascular Grafts of Brain-Dead Rats against In Vitro Ischemia/Reperfusion Injury

Purpose The endothelium has a pivotal role in the maintenance of cardiac function after heart transplantation, mainly by controlling the coronary circulation. Brain death (BD) as well as ischemia/reperfusion (IR) injury compromise vascular endothelium and thereby myocardial protection. Beneficial effects of conditioned medium (CM) from mesenchymal stem cells (MSCs) against IR injury have been demonstrated. We hypothesized that physiological saline supplemented CM could protect vascular grafts of BD rats from IR injury. Methods Rat MSCs derived CM was used for conservation purposes. We detected 28 factors involved in inflammation, oxidative stress and apoptosis. Donors were either subjected to sham operation or BD by inflation of a subdural balloon for 5.5h. Then, thoracic aortic rings from BD rats were isolated and immediately mounted after preparation in an organ bath chamber (BD group) or underwent 24h cold ischemic preservation in physiological saline supplemented either with a vehicle (BD-IR group) or CM (BD-IR+CM group). Immunohistochemical staining for myeloperoxidase (MPO), nitrotyrosine (NT), caspase-8, -9, and -12 was performed. Results Characterization of BD donors: Compared to the sham group, BD leads to significantly impaired hemodynamic parameters, higher aortic MPO, NT, caspase-8, -9, and -12 contents. Organ bath experiments: The preservation of BD-IR aortic rings with CM significantly improved IR-induced decreased maximum endothelium dependent vasorelaxation (Rmax) to acetylcholine compared to the vehicle treated BD-IR group (Rmax to acetylcholine: BD 81±2 vs BD-IR 50±3 vs BD-IR+CM 72±2%, p Conclusion Endothelial dysfunction during IR injury is improved by preservation of vascular grafts from BD rats with CM. This protective effect may partly arise from a reduction of inflammation and nitro oxidative stress responses, and inhibition of caspase-8 and -9 mediated apoptosis.