Cardiac Myocyte Protein Research Articles

The E3 ubiquitin-protein ligase synoviolin (Syvn1/Hrd1) promotes adaptive decreases in cardiac myocyte protein synthesis via eIF2α/ATF4 pathway activation

As the heart undergoes pathologic hypertrophy in response to many disease conditions, an imbalance between protein synthesis and degradation in cardiac myocytes, due to the increases in protein synthesis relative to protein degradation, happens, which challenges protein folding and endoplasmic reticulum (ER) proteostasis. This leads to ER stress and the activation of the ER stress response. Synoviolin (Syvn1), also known as HMG-CoA reductase degradation protein 1 (Hrd1), is an ER-localized protein that is upregulated in cardiac myocytes during ER stress. We previously found that Syvn1 mediates the ubiquitylation and consequent degradation of toxic misfolded proteins and preserved cardiac function in a model of pressure overload-induced cardiac pathology. The objective of this study was to examine whether Syvn1 is involved in regulating protein synthesis, and if so, what its function is during hypertrophic growth of cardiac myocytes. Given that cardiac hypertrophy is caused by increases in protein synthesis, we hypothesized that Syvn1 provides an adaptive response in part through regulation of protein synthesis and consequent decrease in cardiac hypertrophic response. We examined the effects of Syvn1 on ventricular myocyte growth in vitro and found that overexpression of Syvn1 decreased cardiac myocyte surface area as well as protein synthesis when treated with phenylephrine (PE), a treatment which normally leads to increased protein synthesis and increased cardiac myocyte size, mimicking pathological cardiac hypertrophy. We next focused on identifying the mechanism of Syvn1-mediated growth inhibition and found that increases in Syvn1 lead to activation of the PERK (protein kinase RNA (PKR)-like ER kinase) branch of the ER stress response, which leads to increased phosphorylation of the α-subunit of translation initiation factor 2 (eIF2α) at Ser51, thereby inhibiting global translational initiation. PERK activation also results in selective translation of a subset of mRNAs, including activating transcription factor 4 (ATF4), which activates the transcription of a wide range of genes involved in adaptation to stress conditions. We found increased ATF4 levels and increased ATF4 target gene expression with Syvn1 overexpression during PE treatment, compared to control PE treatment. Inhibition of the PERK pathway blocked Syvn1-mediated decrease in cardiac myocyte size and protein synthesis as well as PERK activation and ATF4 target gene induction. Additionally, we found that an adeno-associated virus encoding Syvn1, administered in a therapeutically-relevant manner after the onset of pathological cardiac hypertrophy induced by transverse-aortic constriction in mice, blunted pathological remodeling and preserved cardiac function. We posit that the Syvn1/eIF2α/ATF4 pathway constitutes a newly-discovered mechanism for balancing the proteome in cardiac myocytes. Here we found that Syvn1 upregulation blunts maladaptive increase in cardiac myocyte size during pathologic hypertrophy. In conclusion, these findings suggest that Syvn1 is a critical regulator of cardiac myocyte protein synthesis and subsequent growth and that increases in Syvn1 lead to the activation of the PERK branch of the ER stress response, which results in inhibition of global protein synthesis, and activation of ATF4. This work was supported by the National Institutes of Health (R01HL157027) and the University of Arizona Health Sciences Career Development Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Linggui Zhugan Decoction activates the SIRT1-AMPK-PGC1α signaling pathway to improve mitochondrial and oxidative damage in rats with chronic heart failure caused by myocardial infarction.

Objective: To investigate the effects of Linggui Zhugan Decoction on mitochondrial and oxidative damage in rats with chronic heart failure after myocardial infarction and the related mechanisms. Methods: Chronic heart failure after myocardial infarction was established by coronary artery ligation. Heart failure rats were randomly divided into three groups: Model group (n = 11), Linggui Zhugan Decoction group (n = 12), and captopril group (n = 11). Rats whose coronary arteries were only threaded and not ligated were sham group (n = 11). Cardiac function, superoxide dismutase (SOD), malondialdehyde (MDA) contents, soluble growth-stimulating expression factor (ST2), and N-terminal B-type brain natriuretic peptide precursor (NTproBNP) levels were analyzed after treatment. Moreover, the level of mitochondrial membrane potential was detected by JC-1 staining, the ultrastructural of myocardial mitochondria were observed by transmission electron microscopy. The related signal pathway of silent information regulator factor 2-related enzyme 1 (SIRT1), adenylate activated protein kinase (AMPK), phosphorylated adenylate activated protein kinase (p-AMPK), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is an important pathway to regulate mitochondrial energy metabolism, and to initiate mitochondrial biogenesis. The expression level was detected by Western blot and reverse transcription to explore the mechanism of the decoction. Results: Compared with the model rats, Linggui Zhugan Decoction significantly improved cardiac function (p < 0.05), reduced MDA production (p < 0.01), increased SOD activity (p < 0.05), reduced ST-2(p < 0.01), and NT-proBNP(p < 0.05) levels, increased mitochondrial membrane potential, and improved mitochondria function. In addition, Linggui Zhugan Decoction upregulated the expression of SIRT1, p-AMPK, PGC-1α protein, and mRNA in cardiac myocytes. Conclusion: Linggui Zhugan Decoction can improve the cardiac function of heart failure rats by enhancing myocardial antioxidant capacity and protecting the mitochondrial function, the mechanism is related to activating SIRT1/AMPK/PGC-1α signaling pathway.

The prevalence and associated factors of myocardial involvement in Duchenne muscular dystrophy patients in the first decade of life.

The prevalence and associated factors of myocardial involvement in Duchenne muscular dystrophy patients in the first decade of life.

Blood prestin levels in COVID-19 patients.

Many studies have found that viral infections affect different tissues, including the inner ear. Coronavirus disease 2019 (COVID-19), a viral infection, is a significant health problem worldwide. Prestin is a motor protein with important functions both in the outer hair cells of the inner ear and in cardiac tissue. In addition, prestin is promising as an early biomarker in the detection of ototoxicity. To determine the severity of infection in COVID-19 patients and to determine whether other tissues are affected by the infection, lactate dehydrogenase (LDH), C-reactive protein (CRP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase MB (CK-MB), biochemical markers such as ferritin and D-dimer are used. This study aimed to compare prestin levels in patients with COVID-19 and healthy volunteers. In blood samples taken from 45 patients diagnosed with COVID-19 and 40 healthy volunteers, prestin levels were determined with the kit that used an enzyme-linked immunosorbent assay method and was commercially available. At the same time, LDH, CRP, ALT, AST, CK-MB, ferritin, and D-dimer levels were also detected in both patients and healthy control groups and correlations with prestin levels were examined. The main result of our study is that serum prestin levels in COVID-19 patients are significantly higher than in healthy controls ( p < 0.001). In addition, a statistically significant strong positive correlation was found between prestin-LDL ( r = 0.537, p = 0.001), prestin-CRP ( r = 0.654, p = 0.001), and prestin-D-dimer ( r = 0.659, p = 0.001). The levels of prestin, a motor protein in inner ear outer hair cells and cardiac myocytes, were found to be higher in COVID-19 patients than in healthy volunteers. It also showed a positive correlation with CRP and D-dimer. This may be associated with systemic dysfunction.

Striated muscle MLCP is regulated by MYPT2 to limit baseline cardiac myosin phosphorylation.

Cardiac myosin is a hexameric protein comprised of two protamers that each have a heavy chain, a regulatory light chain (RLC), and an essential light chain. Constitutive phosphorylation of cardiac myosin in beating hearts at 0.4 mol phosphate/mol RLC is necessary for normal function. This level is maintained by balanced activities of the cardiac myosin light chain kinase (cMLCK) and myosin light chain phosphatase (MLCP). The canonical MLCP is well-studied in smooth muscles, and is defined as a trimeric holoenzyme comprised of a myosin target regulatory subunit MYPT1, a type-1 catalytic subunit PP1cβ, and an accessory protein M21. In striated muscles, the dominantly expressed regulatory subunit is MYPT2. There is limited information on the localization and regulatory contributions of MYPT2. Studies of regulation of cardiac myosin dephosphorylation is hindered by the fact that myosin dephosphorylation occurs rapidly in isolated cardiac myocytes, and cultured “cardiac” cell lines do not express the regulatory proteins at levels comparable to those of freshly isolated cardiac myocytes. We generated a floxed MYPT2 mouse line and conditionally knocked out nearly all detectable MYPT2 protein in cardiac myocytes, and used this model to determine that: 1) MYPT2 localizes to myosin in vivo, 2) MYPT1 expression does not compensate for the reduction in MYPT2 in the cardiac muscle, 3) the cardiac MLCP holoenzyme expression level is partly regulated by stabilization of the catalytic subunit by MYPT2, 4) without MYPT2-regulated MLCP activity, baseline cardiac RLC phosphorylation is increased to 0.6 mol phosphate/mol RLC, and 5) MYPT2 knockout animals have reduced responses to pressure-overload induced cardiac hypertrophy. These findings help to fill knowledge-gaps about the fundamental properties of the cardiac muscle MLCP.

A-Kinase Anchoring Proteins in Cardiac Myocytes and Their Roles in Regulating Calcium Cycling.

The rate of calcium cycling and calcium transient amplitude are critical determinants for the efficient contraction and relaxation of the heart. Calcium-handling proteins in the cardiac myocyte are altered in heart failure, and restoring the proper function of those proteins is an effective potential therapeutic strategy. The calcium-handling proteins or their regulators are phosphorylated by a cAMP-dependent kinase (PKA), and thereby their activity is regulated. A-Kinase Anchoring Proteins (AKAPs) play a seminal role in orchestrating PKA and cAMP regulators in calcium handling and contractile machinery. This cAMP/PKA orchestration is crucial for the increased force and rate of contraction and relaxation of the heart in response to fight-or-flight. Knockout models and the few available preclinical models proved that the efficient targeting of AKAPs offers potential therapies tailor-made for improving defective calcium cycling. In this review, we highlight important studies that identified AKAPs and their regulatory roles in cardiac myocyte calcium cycling in health and disease.

Protective role of protein Klotho biomarker in acute heart failure

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): This work was supported by a grant from the Institute of Medical Sciences, Univeristy of Opole, Poland Background Klotho is an antiaging protein with pleiotropic actions that exerts organ protection. There is evidence that Klotho protein acts as a human aging-suppression molecule and correlates with life span, coronary artery disease, atherosclerosis, and osteoporosis. In laboratory models of damage, an increased expression of Klotho protein in human cardiac myocytes and release of Klotho protein into the extracellular space suggests a potential compensatory and cardioprotective effect. The aim of the present study was to assess changes of Klotho protein levels during an episode of acute heart failure (AHF) as well as its long-term prognostic utility in population of AHF patients compared to other biomarkers. Methods We analysed 112 consecutive patients admitted to ICCU between June 2019 and January 2021 due to hight-risk AHF. Blood samples were collected on admission, at discharge as well as at the 30-day. Patients were then followed-up for all-cause mortality and rehospitalizations due to heart failure. Results Patients (N=112; median age 68 years, 75% men, median left ventricular ejection fraction 30% [IQR 20-38%]) had median admission levels of Klotho protein = 670 pg/ml [501-851], FGF-23 = 1278 pg/ml [283-4429], NT-proBNP = 5361 ng/l [3019-13182], and GDF-15 = 4582 pg/ml [3028-9081]. Klotho protein decreased by 19% from admission to discharge and then increased by 13% from discharge to 30-day (P&amp;lt;0.001). During median 478 days follow-up 40 patients (36%) died or were hospitalised due to heart failure - they were older, more often with new onset AHF, of ischaemic aetiology and diabetics. Patients with lower levels of Klotho protein (in the 1st as well as in the 2nd quartile) had higher mortality (Figure). ROC analyses showed Klotho had comparable predictive value of unfavourable prognosis as other biomarkers (GDF-15, NT-proBNP). Conclusion The levels of Klotho protein in patients with AHF are higher compared to healthy population and they show dynamics dependent on the clinical state what indicates the utility of Klotho as a typical biomarker. Lower values of Klotho protein in AHF patients are associated with higher mortality which may suggests protective effect of higher Klotho levels.

PO-617-06 BIDIRECTIONAL NEAREST NEIGHBOR COLOCALIZATION IDENTIFIES CARDIOMYOCYTE MICROTRANSLATOMES PRODUCING CALMODULIN AND ITS EFFECTOR TARGETS

Calmodulin (CaM), which regulates myriad proteins in cardiac myocytes, features translational redundancy with three encoding genes (CALM1-3). Mutations in CaM result in calmodulinopathies, characterized by arrhythmia and myopathy. However, the specific roles of the 3 CaM genes and the structural underpinnings of their function remain unclear.

Loss of cardiac myosin light chain kinase contributes to contractile dysfunction in right ventricular pressure overload.

Nearly 1 in every 100 children born have a congenital heart defect. Many of these defects primarily affect the right heart causing pressure overload of the right ventricle (RV). The RV maintains function by adapting to the increased pressure; however, many of these adaptations eventually lead to RV hypertrophy and failure. In this study, we aim to identify the cellular and molecular mechanisms of these adaptions. We utilized a surgical animal model of pulmonary artery banding (PAB) in juvenile rats that has been shown to accurately recapitulate the physiology of right ventricular pressure overload in young hearts. Using this model, we examined changes in cardiac myocyte protein expression as a result of pressure overload with mass spectrometry 4 weeks post‐banding. We found pressure overload of the RV induced significant downregulation of cardiac myosin light chain kinase (cMLCK). Single myocyte calcium and contractility recordings showed impaired contraction and relaxation in PAB RV myocytes, consistent with the loss of cMLCK. In the PAB myocytes, calcium transients were of smaller amplitude and decayed at a slower rate compared to controls. We also identified miR‐200c, which has been shown to regulate cMLCK expression, as upregulated in the RV in response to pressure overload. These results indicate the loss of cMLCK is a critical maladaptation of the RV to pressure overload and represents a novel target for therapeutic approaches to treat RV hypertrophy and failure associated with congenital heart defects.

Abstract 14114: Role of Toxic Protein Sequestration by Aggregates in Cardiac Myocytes in Ischemic Cardiomyopathy

Introduction: Proteotoxic aggregation causes cardiomyopathy (CMP) associated with rare genetic mutations. The role of protein aggregates in ischemic cardiomyopathy is unclear. Hypothesis: Cardiac myocyte protein aggregation is deleterious in cardiomyopathy. Methods: Human (ischemic) and murine (ischemic and proteotoxic) CMP models were profiled by immunohistochemistry (IHC) and proteomic characterization of the detergent-insoluble fraction. Aggregates were evaluated by immunocytochemistry in p62 deficient neonatal mouse cardiac myocytes (NMCMs) with adenoviral transduction of mutant alpha-B crystallin (CRYABR120G). Mice with cardiac myocyte selective p62 deficiency (p62ckO) were generated and characterized (echocardiography, myocardial structure, autophagic flux, mitophagy, and biochemical analyses). p62cKO and floxed controls were subjected to closed-chest ischemia (90 minutes) or sham followed by reperfusion (IR) and evaluated 4 weeks later. Cardiac selective (AAV9-cTnT) reconstitution of p62, its aggregation-deficient K7R mutant or GFP, was performed. Wild-type mice transduced with a fungal disaggregase (AAV9-Hsp104 or control) were modeled for IR as above. Results: Protein aggregation increases in ischemic CMP, but not controls, with increased insoluble CRYAB with phosphorylation at serine 59 (pS59CRYAB). p62-deficient NMCMs are unable to form aggregates following CRYABR120G transduction, indicating the necessity of p62 in protein aggregation. p62cKO mice show preserved baseline cardiac structure/function, without significantly decreased mitophagy or autophagic flux. Compared to controls, p62cKO mice (post-IR) show infarct expansion and impaired LV remodeling at 4 weeks, associated with decreased sequestration of pS59CRYAB in insoluble fraction. Reconstitution with AAV9 p62, but not aggregation-deficient K7R, shows partial functional rescue after IR. Finally, disruption of protein aggregation by AAV9 Hsp104 (but not controls) results in decreased sequestration of pS59CRYAB, and decreased function post-IR. Conclusions: Contrary to the hypothesis, preventing or disrupting protein aggregation, associated with decreased pS59CRYAB sequestration, is maladaptive in ischemic cardiomyopathy.

Distributed synthesis of sarcolemmal and sarcoplasmic reticulum membrane proteins in cardiac myocytes

It is widely assumed that synthesis of membrane proteins, particularly in the heart, follows the classical secretory pathway with mRNA translation occurring in perinuclear regions followed by protein trafficking to sites of deployment. However, this view is based on studies conducted in less-specialized cells, and has not been experimentally addressed in cardiac myocytes. Therefore, we undertook direct experimental investigation of protein synthesis in cardiac tissue and isolated myocytes using single-molecule visualization techniques and a novel proximity-ligated in situ hybridization approach for visualizing ribosome-associated mRNA molecules for a specific protein species, indicative of translation sites. We identify here, for the first time, that the molecular machinery for membrane protein synthesis occurs throughout the cardiac myocyte, and enables distributed synthesis of membrane proteins within sub-cellular niches where the synthesized protein functions using local mRNA pools trafficked, in part, by microtubules. We also observed cell-wide distribution of membrane protein mRNA in myocardial tissue from both non-failing and hypertrophied (failing) human hearts, demonstrating an evolutionarily conserved distributed mechanism from mouse to human. Our results identify previously unanticipated aspects of local control of cardiac myocyte biology and highlight local protein synthesis in cardiac myocytes as an important potential determinant of the heart’s biology in health and disease.

Mitochondrial A-kinase anchoring proteins in cardiac ventricular myocytes.

Compartmentation of cAMP signaling is a critical factor for maintaining the integrity of receptor‐specific responses in cardiac myocytes. This phenomenon relies on various factors limiting cAMP diffusion. Our previous work in adult rat ventricular myocytes (ARVMs) indicates that PKA regulatory subunits anchored to the outer membrane of mitochondria play a key role in buffering the movement of cytosolic cAMP. PKA can be targeted to discrete subcellular locations through the interaction of both type I and type II regulatory subunits with A‐kinase anchoring proteins (AKAPs). The purpose of this study is to identify which AKAPs and PKA regulatory subunit isoforms are associated with mitochondria in ARVMs. Quantitative PCR data demonstrate that mRNA for dual specific AKAP1 and 2 (D‐AKAP1 & D‐AKAP2), acyl‐CoA‐binding domain‐containing 3 (ACBD3), optic atrophy 1 (OPA1) are most abundant, while Rab32, WAVE‐1, and sphingosine kinase type 1 interacting protein (SPHKAP) were barely detectable. Biochemical and immunocytochemical analysis suggests that D‐AKAP1, D‐AKAP2, and ACBD3 are the predominant mitochondrial AKAPs exposed to the cytosolic compartment in these cells. Furthermore, we show that both type I and type II regulatory subunits of PKA are associated with mitochondria. Taken together, these data suggest that D‐AKAP1, D‐AKAP2, and ACBD3 may be responsible for tethering both type I and type II PKA regulatory subunits to the outer mitochondrial membrane in ARVMs. In addition to regulating PKA‐dependent mitochondrial function, these AKAPs may play an important role by buffering the movement of cAMP necessary for compartmentation.

Local Synthesis of Sarcolemma and Sarcoplasmic Reticulum Membrane Proteins in Cardiac Myocytes

Classically, cardiac sarcolemma (SL) and sarcoplasmic reticulum (SR) membrane proteins are thought to be synthesized and processed on the rough ER, in the perinuclear region and then transported to locations of employment in the SR and SL. However, this view is largely based on studies in non-myocyte cell types. Therefore, we investigated the localization and regulation of synthesis of several key sarcolemmal SL and SR membrane proteins in adult cardiac myocytes. in contrast to the canonical view, in mouse ventricular myocytes we identified synthesis machinery and translation for such proteins, namely SERCA2a, RyR2, Cav1.2 and Nav1.5 localized not only at the perinuclear area, but spread throughout the entire cell volume, particularly in the vicinity of surface/T-tubule and SR membranes. inhibition of microtubular trafficking by colchicine resulted in redistribution of the mRNA signal for these proteins towards the nuclei, consistent with microtubule trafficking providing a dedicated pool of mRNA for local protein translation and synthesis. Active translation of SERCA2a mRNA throughout the cardiac myocyte space was confirmed through visualization of active translation sites using a novel in-situ hybridization assay, which we developed. Collectively, these results suggest that synthesis of SR and SL proteins in cardiomyocytes occurs locally at sites of utilization from dedicated, local mRNA pools.

Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

The contraction and relaxation of the heart is controlled by stimulation of the β1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of β1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

Effect of Yixinshu Capsules on calcium regulatory protein in cardiac myocytes of rats with heart failure

To explore the molecular mechanism of Yixinshu Capsules(YXS) in restoring cardiac function in rats with heart failure(HF) from the perspective of calmodulin in cardiac myocytes on the basis of determining the therapeutic effect of YXS on HF. The SD rats were subjected to the surgery of ligating the anterior descending branch of the left coronary artery for 4 weeks to established myocardial ischemia-induced heart failure animal model. Then the rats were randomly divided into Sham operation group(Sham, saline), model group(HF, saline), high dose YXS group(HF+YXS-H, 1 600 mg·kg~(-1)·d~(-1)), low dose YXS group(HF+YXS-L, 800 mg·kg~(-1)·d~(-1)) and positive drug valsartan group(HF+VST, 8 mg·kg~(-1)·d~(-1)). After continuous intragastric administration for 6 weeks, the rats were sacrificed and myocardial tissue was collected. Real time quantitative polymerase chain reaction(RT-PCR) and Western blot were used to detect the expression of genes and proteins related to calcium regulation in cardiomyocytes. RESULTS:: showed that as compared with the model group, YXS increased the transcription level of Atp2 a2, Ryr2, CACNA1 C and PRKACA, and increased the expression levels of P-Ryr2, CACNA1 C and SERCA2 a, while decreased the level of NCX1.On the other hand, YXS treatment significantly decreased the RIP3 level and the phosphorylation of its substrate CaMKⅡ protein, and enhanced the phosphorylation expression of PLB. In summary, YXS therapy could regulate the expression of genes and proteins related to calcium regulation in cardiomyocytes, decrease RIP3 and the phosphorylation of CaMKⅡ protein, increase the phosphorylation of PLB at Ser16, and increase the expression of SERCA2 a protein, suggesting that YXS may regulate myocardial calcium homeostasis through CaMKⅡ/PLB/SERCA2 a pathway, to improve the ability of calcium uptake in sarcoplasmic reticulum and stabilize intracellular free Ca~(2+), so as to improve the cardiac function in rats with heart failure. Our study revealed the possible mechanism of YXS in the treatment of heart failure, especially from the perspective of intervention of calmodulin, promoting the clinical application of YXS.

Abstract P049: Circadian Clock Dysfunction Impairs Mitochondrial Fitness And Contributes To Myocardial Ischemic Injury

Cardiac function is highly reliant on mitochondrial oxidative metabolism and fitness. The circadian clock is critically linked to vital physiological process including mitochondrial fission, fusion and quality control mechanisms. However, little is known of how the circadian clock regulates these vital processes in the heart. Herein, we identified a putative circadian Clock - mitochondrial interactome that gates an adaptive stress response for cell viability during myocardial ischemia reperfusion (I-R) injury. We show that Clock transcriptionally coordinates expression of mitochondrial dynamic fusion and fission, bioenergetic and quality control proteins in cardiac myocytes. Transcriptome and gene ontology mapping revealed Clock defective hearts subjected to I-R exhibited major transcriptional deficits in several key survival processes including mitochondrial dynamics, bioenergetics and autophagy that were reduced further following I-R. Gain of function of Clock activity re-established gene transcription of mitochondrial respiratory complex activity, quality control mechanisms and cell viability. Collectively, our data show that mitochondrial fitness and cell survival is mutually dependent upon and obligatorily linked to transcription of the circadian Clock gene in cardiac myocytes. Our data suggest the functional loss of Clock activity predisposes cardiac myocytes to metabolic catastrophe. Hence, therapeutic strategies designed to preserve circadian clock activity in the hearts may prove beneficial in reducing morbidity and mortality following ischemia -related pathologies such as myocardial infarction.

Estrogen and calcium handling proteins: new discoveries and mechanisms in cardiovascular diseases.

Estrogen deficiency is considered to be an important factor leading to cardiovascular diseases (CVDs). Indeed, the prevalence of CVDs in postmenopausal women exceeds that of premenopausal women and men of the same age. Recent research findings provide evidence that estrogen plays a pivotal role in the regulation of calcium homeostasis and therefore fine-tunes normal cardiomyocyte contraction and relaxation processes. Disruption of calcium homeostasis is closely associated with the pathological mechanism of CVDs. Thus, this paper maps out and summarizes the effects and mechanisms of estrogen on calcium handling proteins in cardiac myocytes, including L-type Ca2+ channel, the sarcoplasmic reticulum Ca2+ release channel named ryanodine receptor, sarco(endo)plasmic reticulum Ca2+-ATPase, and sodium-calcium exchanger. In so doing, we provide theoretical and experimental evidence for the successful design of estrogen-based prevention and treatment therapies for CVDs.

Divergent off-target effects of RSK N-terminal and C-terminal kinase inhibitors in cardiac myocytes

P90 ribosomal S6 kinases (RSK) are ubiquitously expressed and regulate responses to neurohumoral stimulation. To study the role of RSK signalling on cardiac myocyte function and protein phosphorylation, pharmacological RSK inhibitors were tested. Here, the ATP competitive N-terminal kinase domain-targeting compounds D1870 and SL0101 and the allosteric C-terminal kinase domain-targeting FMK were evaluated regarding their ability to modulate cardiac myocyte protein phosphorylation. Exposure to D1870 and SL0101 significantly enhanced phospholamban (PLN) Ser16 and cardiac troponin I (cTnI) Ser22/23 phosphorylation in response to D1870 and SL0101 upon exposure to phenylephrine (PE) that activates RSK. In contrast, FMK pretreatment significantly reduced phosphorylation of both proteins in response to PE. D1870-mediated enhancement of PLN Ser16 phosphorylation was also observed after exposure to isoprenaline or noradrenaline (NA) stimuli that do not activate RSK. Inhibition of β-adrenoceptors by atenolol or cAMP-dependent protein kinase (PKA) by H89 prevented the D1870-mediated increase in PLN phosphorylation, suggesting that PKA is the kinase responsible for the observed phosphorylation. Assessment of changes in cAMP formation by FRET measurements revealed increased cAMP formation in vicinity to PLN after exposure to D1870 and SL0101. D1870 inhibited phosphodiesterase activity similarly as established PDE inhibitors rolipram or 3-isobutyl-1-methylxanthine. Assessment of catecholamine-mediated force development in rat ventricular muscle strips revealed significantly reduced EC50 for NA after D1870 pretreatment (DMSO/NA: 2.33 μmol/L vs. D1870/NA: 1.30 μmol/L). The data reveal enhanced cardiac protein phosphorylation by D1870 and SL0101 that was not detectable in response to FMK. This disparate effect might be attributed to off-target inhibition of PDEs with impact on muscle function as demonstrated for D1870.

DNA Damage Response/TP53 Pathway Is Activated and Contributes to the Pathogenesis of Dilated Cardiomyopathy Associated With LMNA (Lamin A/C) Mutations.

Mutations in the LMNA gene, encoding LMNA (lamin A/C), are responsible for laminopathies. Dilated cardiomyopathy (DCM) is a major cause of mortality and morbidity in laminopathies. To gain insights into the molecular pathogenesis of DCM in laminopathies. We generated a tet-off bigenic mice expressing either a WT (wild type) or a mutant LMNA (D300N) protein in cardiac myocytes. LMNAD300N mutation is associated with DCM in progeroid syndromes. Expression of LMNAD300N led to severe myocardial fibrosis, apoptosis, cardiac dysfunction, and premature death. Administration of doxycycline suppressed LMNAD300N expression and prevented the phenotype. Whole-heart RNA sequencing in 2-week-old WT and LMNAD300N mice led to identification of ≈6000 differentially expressed genes. Gene Set Enrichment and Hallmark Pathway analyses predicted activation of E2F (E2F transcription factor), DNA damage response, TP53 (tumor protein 53), NFκB (nuclear factor κB), and TGFβ (transforming growth factor-β) pathways, which were validated by Western blotting, quantitative polymerase chain reaction of selected targets, and immunofluorescence staining. Differentially expressed genes involved cell death, cell cycle regulation, inflammation, and epithelial-mesenchymal differentiation. RNA sequencing of human hearts with DCM associated with defined LMNA pathogenic variants corroborated activation of the DNA damage response/TP53 pathway in the heart. Increased expression of CDKN2A (cyclin-dependent kinase inhibitor 2A)-a downstream target of E2F pathway and an activator of TP53-provided a plausible mechanism for activation of the TP53 pathway. To determine pathogenic role of TP53 pathway in DCM, Tp53 gene was conditionally deleted in cardiac myocytes in mice expressing the LMNAD300N protein. Deletion of Tp53 partially rescued myocardial fibrosis, apoptosis, proliferation of nonmyocyte cells, left ventricular dilatation and dysfunction, and slightly improved survival. Cardiac myocyte-specific expression of LMNAD300N, associated with DCM, led to pathogenic activation of the E2F/DNA damage response/TP53 pathway in the heart and induction of myocardial fibrosis, apoptosis, cardiac dysfunction, and premature death. The findings denote the E2F/DNA damage response/TP53 axis as a responsible mechanism for DCM in laminopathies and as a potential intervention target.

Cardiac Hypertrophy is Positively Regulated by MicroRNA‑24 in Rats

Background:MicroRNA-24 (miR-24) plays an important role in heart failure by reducing the efficiency of myocardial excitation-contraction coupling. Prolonged cardiac hypertrophy may lead to heart failure, but little is known about the role of miR-24 in cardiac hypertrophy. This study aimed to preliminarily investigate the function of miR-24 and its mechanisms in cardiac hypertrophy.Methods:Twelve Sprague-Dawley rats with a body weight of 50 ± 5 g were recruited and randomly divided into two groups: a transverse aortic constriction (TAC) group and a sham surgery group. Hypertrophy index was measured and calculated by echocardiography and hematoxylin and eosin staining. TargetScans algorithm-based prediction was used to search for the targets of miR-24, which was subsequently confirmed by a real-time polymerase chain reaction and luciferase assay. Immunofluorescence labeling was used to measure the cell surface area, and 3H-leucine incorporation was used to detect the synthesis of total protein in neonatal rat cardiac myocytes (NRCMs) with the overexpression of miR-24. In addition, flow cytometry was performed to observe the alteration in the cell cycle. Statistical analysis was carried out with GraphPad Prism v5.0 and SPSS 19.0. A two-sided P < 0.05 was considered as the threshold for significance.Results:The expression of miR-24 was abnormally increased in TAC rat cardiac tissue (t = −2.938, P < 0.05). TargetScans algorithm-based prediction demonstrated that CDKN1B (p27, Kip1), a cell cycle regulator, was a putative target of miR-24, and was confirmed by luciferase assay. The expression of p27 was decreased in TAC rat cardiac tissue (t = 2.896, P < 0.05). The overexpression of miR-24 in NRCMs led to the decreased expression of p27 (t = 4.400, P < 0.01), and decreased G0/G1 arrest in cell cycle and cardiomyocyte hypertrophy.Conclusion:MiR-24 promotes cardiac hypertrophy partly by affecting the cell cycle through down-regulation of p27 expression.