Excitation-contraction Coupling Research Articles

The Developmental Implications of Muscle-Targeted Magnetic Mitohormesis: A Human Health and Longevity Perspective.

Muscle function reflects muscular mitochondrial status, which, in turn, is an adaptive response to physical activity, representing improvements in energy production for de novo biosynthesis or metabolic efficiency. Differences in muscle performance are manifestations of the expression of distinct contractile-protein isoforms and of mitochondrial-energy substrate utilization. Powerful contractures require immediate energy production from carbohydrates outside the mitochondria that exhaust rapidly. Sustained muscle contractions require aerobic energy production from fatty acids by the mitochondria that is slower and produces less force. These two patterns of muscle force generation are broadly classified as glycolytic or oxidative, respectively, and require disparate levels of increased contractile or mitochondrial protein production, respectively, to be effectively executed. Glycolytic muscle, hence, tends towards fibre hypertrophy, whereas oxidative fibres are more disposed towards increased mitochondrial content and efficiency, rather than hypertrophy. Although developmentally predetermined muscle classes exist, a degree of functional plasticity persists across all muscles post-birth that can be modulated by exercise and generally results in an increase in the oxidative character of muscle. Oxidative muscle is most strongly correlated with organismal metabolic balance and longevity because of the propensity of oxidative muscle for fatty-acid oxidation and associated anti-inflammatory ramifications which occur at the expense of glycolytic-muscle development and hypertrophy. This muscle-class size disparity is often at odds with common expectations that muscle mass should scale positively with improved health and longevity. Brief magnetic-field activation of the muscle mitochondrial pool has been shown to recapitulate key aspects of the oxidative-muscle phenotype with similar metabolic hallmarks. This review discusses the common genetic cascades invoked by endurance exercise and magnetic-field therapy and the potential physiological differences with regards to human health and longevity. Future human studies examining the physiological consequences of magnetic-field therapy are warranted.

Cardiomyocytes from female compared to male mice have larger ryanodine receptor clusters and higher calcium spark frequency.

Sex differences in cardiac physiology are receiving increased attention as it has become clear that men and women have different aetiologies of cardiac disease and require different treatments. There are experimental data suggesting that male cardiomyocytes exhibit larger Ca2+ transients due to larger Ca2+ sparks and a higher excitation-contraction coupling gain; in addition, they exhibit a larger response to adrenergic stimulation with isoprenaline (ISO). Here, we studied whether there are sex differences relating to structural organization of the transverse tubular network and ryanodine receptors (RyRs). Surprisingly, we found that female cardiomyocytes exhibited a higher spark frequency in a range of spark magnitudes. While overall RyR expression and phosphorylation were the same, female cardiomyocytes had larger but fewer RyR clusters. The density of transverse t-tubules was the same, but male cardiomyocytes had more longitudinal t-tubules. The Ca2+ transients were similar in male and female cardiomyocytes under control conditions and in the presence of ISO. The synchrony of the Ca2+ transients was similar between sexes as well. Overall, our data suggest subtle sex differences in the Ca2+ influx and efflux pathways and their response to ISO, but these differences are balanced, resulting in similar Ca2+ transients in field-stimulated male and female cardiomyocytes. The higher spark frequency in female cardiomyocytes is related to the organization of RyRs into larger, but fewer clusters. KEY POINTS: During a heartbeat, the force of contraction depends on the amplitude of the calcium transient, which in turn depends on the amount of calcium released as calcium sparks through ryanodine receptors in the sarcoplasmic reticulum. Previous studies suggest that cardiomyocytes from male compared to female mice exhibit larger calcium sparks, larger sarcoplasmic reticulum calcium release and greater response to adrenergic stimulation triggering a fight-or-flight response. In contrast, we show that cardiomyocytes from female mice have a higher spark frequency during adrenergic stimulation and similar spark morphology. The higher spark frequency is related to the organization of ryanodine receptors into fewer, but larger clusters in female compared to male mouse cardiomyocytes. Despite subtle sex differences in cardiomyocyte structure and calcium fluxes, the differences are balanced, leading to similar calcium transients in cardiomyocytes from male and female mice.

Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B.

Purine-rich element binding protein B (Purβ) is a single-stranded DNA (ssDNA) and RNA-binding protein that functions as a transcriptional repressor of genes encoding certain muscle-restricted contractile proteins in the setting of cellular stress or tissue injury. A prior report from our laboratory implicated specific basic amino acid residues in the physical and functional interaction of Purβ with the smooth muscle-α actin gene (Acta2) promoter. Independent structural analysis of fruit fly Purα uncovered a role for several aromatic residues in the binding of this related protein to ssDNA. Herein, we examine the functional importance of a comparable set of hydrophobic residues that are positionally conserved in the repeat I (Y59), II (F155), and III (F256) domains of murine Purβ. Site-directed Y/F to alanine substitutions were engineered, and the resultant Purβ point mutants were tested in various biochemical and cell-based assays. None of the mutations affected the cellular expression, structural stability, or dimerization capacity of Purβ. However, the Y59A and F155A mutants demonstrated weaker Acta2 repressor activity in transfected fibroblasts and reduced binding affinity for the purine-rich strand of an Acta2 cis-regulatory element in vitro. Mutation of Y59 and F155 also altered the multisite binding properties of Purβ for ssDNA and diminished the interaction of Purβ with Y-box binding protein 1, a co-repressor of Acta2. Collectively, these findings suggest that some of the same aromatic residues, which govern the specific and high-affinity binding of Purβ to ssDNA, also mediate certain heterotypic protein interactions underlying the Acta2 repressor function of Purβ.

The inter-chamber differences in the contractile function between left and right atrial cardiomyocytes in atrial fibrillation in rats.

The left and right atria (LA, RA) work under different mechanical and metabolic environments that may cause an intrinsic inter-chamber diversity in structure and functional properties between atrial cardiomyocytes (CM) in norm and provoke their different responsiveness to pathological conditions. In this study, we assessed a LA vs. RA difference in CM contractility in paroxysmal atrial fibrillation (AF) and underlying mechanisms. We investigated the contractile function of single isolated CM from LA and RA using a 7-day acetylcholine (ACh)-CaCl2 AF model in rats. We compared auxotonic force, sarcomere length dynamics, cytosolic calcium ([Ca2+]i) transients, intracellular ROS and NO production in LA and RA CM, and analyzed the phosphorylation levels of contractile proteins and actin-myosin interaction using an in vitro motility assay. AF resulted in more prominent structural and functional changes in LA myocardium, reducing sarcomere shortening amplitude, and velocity of sarcomere relengthening in mechanically non-loaded LA CM, which was associated with the increased ROS production, decreased NO production, reduced myofibrillar content, and decreased phosphorylation of cardiac myosin binding protein C and troponin I. However, in mechanically loaded CM, AF depressed the auxotonic force amplitude and kinetics in RA CM, while force characteristics were preserved in LA CM. Thus, inter-atrial differences are increased in paroxysmal AF and affected by the mechanical load that may contribute to the maintenance and progression of AF.

Abstract P3072: Effects Of Antiviral Innate Immune Activation On Cytokine Secretion And Function Of Human Ipsc-derived Cardiomyocytes

Introduction: Acute and long-term cardiovascular complications of COVID-19 have been reported, ranging from arrhythmias, ischemic heart disease, and heart failure to cerebrovascular and cardiometabolic disorders, yet the underlying molecular mechanisms are not well understood. Here, we examine the effects of stimulating the double-stranded RNA (dsRNA) pattern recognition receptor pathway, which is strongly induced to activate interferon-stimulated gene (ISG) expression in the heart during COVID-19, on excitation-contraction coupling and mitochondrial function. While providing antiviral protection, we hypothesize that, even in the absence of cardiac viral replication, this pathway might contribute to COVID-19-related cardiac dysfunction. Results: We used the viral dsRNA mimetic polyinosinic: polycytidylic (PIC) to induce a type I interferon response in iPSC-CM or A549 (lung epithelial) cells. In iPSC-CM, PIC induced robust protein expression of STAT1/pSTAT1, OAS2, OAS3, IRF7, pIRF7, IRF9 and MX1. A marked increase in cytokine protein secretion was also observed, with significant increases in CXCL-10 (~160 fold), IL6, CCL5 (~20-fold) IL8 (15-fold), and CXCL1 (~10-fold), and a 2-5-fold increase in ~30 other cytokines. Negative effects on excitation-contraction coupling were observed after 72 hours of PIC treatment, manifested as a 25% reduction in iPSC-CM Ca2+ transient amplitude. Microelectrode array analysis of iPSC-CM monolayers revealed electrophysiological dysfunction: PIC treatment increased cardiac field potential duration and spike amplitude, but reduced beat periods. A549 cells also showed increases in interferon pathway genes (STAT1, pSTAT1, IRF3, pIRF3, OAS1,2,3 and IFNb1), along with increased rates of RNA degradation after PIC treatment, consistent with activation of the OAS/RNAseL dsRNA sensor pathway. PIC-treated A549 cells also showed a large decrease in oxidative phosphorylation (maximal respiration and spare respiratory capacity), which was prevented by the mitochondrial antioxidant, mitoTEMPO. Conclusions: Activation of the viral dsRNA innate immune sensor pathway, even in the absence of active cardiac viral replication, might contribute to mitochondrial and cardiac dysfunction in COVID-19.

Abstract P3040: Development Of A Protein Synthesis Network At The Sarcoplasmic Reticulum In Adult Cardiomyocytes

Introduction: The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. In striated muscle cells, a specialized membrane system known as the sarcoplasmic reticulum (SR) sequesters Ca 2+ and carries key functions in excitation-contraction coupling. Textbooks often define the SR as the ER of muscle, but surprisingly, the biochemical and physiological relationship between the ER and SR and even whether they are distinct membrane networks or refer to the same structure remain unknown. Methods: To characterize the relationship between the ER and SR, we applied biochemical and imaging techniques, including immunofluorescence and election microscopy, fluorescence in situ hybridization, and STimulated Emission Depletion (STED) microscopy to ventricular myocytes, which allowed us to follow the differentiation of these membrane compartments in developing cardiomyocytes. Results: Using localization and expression levels of SERCA2, RyR2, KDEL and different markers of ribosomes, mRNA, and the secretory pathway, we found that the membrane compartment that exists in the early postnatal stage, resembles mostly the ER, and this membrane system decreases in postnatal development. The SR is the main membrane network that exists in the adult cardiomyocytes, replacing the ER, in all but a small perinuclear region. Both sites which appear to be interconnected, perform specialized tasks, with protein secretion through the classical ER-Golgi secretory pathway originating mainly from the perinuclear ER and Ca 2+ -dependent excitation-contraction coupling originating exclusively from the SR. Other functions, such as Ca 2+ intake or posttranslational protein processing occur in both compartments. Importantly, we show that in adult cardiomyocytes ribosomes are not only attached to the ER but also to the SR, where they actively perform mRNA translation and are major sites of protein synthesis. Conclusion: The ER and SR are two distinct coexisting membrane networks in cardiomyocytes that undergo molecular differentiation and structural organization during cardiac muscle development. Significant amounts of protein synthesis occur in adult cardiomyocytes at the SR and may serve localized mRNA translation near the sarcomere.

Abstract P2001: Deficiency Of Claudin-5 Leads To Myocardial Atrophy And Dilation In Mouse Heart With Severe Mitochondrial Dysfunction And Electrical Dissociation

Background: Cardiac overexpression of Claudin-5 (Cldn5) exerts protection against myocardial ischemia and reperfusion injury. However, whether cardiac deficiency of Cldn5 affects cardiac function is still unclear. Here we investigated the role of Cldn5 in mouse heart by means of cardiac Cldn5 knockdown to better understand the intracellular function of Cldn5 in cardio-protection. Methods and Results: Cldn5 shRNA adeno-associated virus (AAV) was used to knockdown Cldn5 in the left ventricle of male C57BL/6J mice by intramyocardial injection. Four weeks post AAV injection, echocardiography and electrocardiography were performed to measure cardiac function. We found 25% of mice died in Cldn5 shRNA AAV injection group. The residual mice were terminated followed by left ventricles isolation for proteomics analysis, cytokine analysis, WGA staining, Masson-trichrome staining and transmission electron microscope (TEM) observation. The signal pathway markers of apoptosis, autophagy and lipid peroxidation were also detected by western blot. The protein level of Cldn5 in left ventricles was decreased by 75% in shRNA AAV injection group accompanied by changes of bcl-2, bax, LC3II, p62, Cox2 and GPX4. The fraction shortening of the heart was decreased by 23.5% with ventricular dilation and ST elevation. We also observed the increase of IL-1beta, TNF alpha, BNP, CT and cTnI in myocardium. WGA staining showed the area of cross section of single cardiomyocyte was decreased and Masson staining showed the collagen deposition was increased in myocardium. The TEM indicated Cldn5 knockdown induced mitochondrial fission and depletion. Finally, the proteomics analysis demonstrated that Cldn5 knockdown severely disturbed ATP metabolic regulation, mitochondrial permeability transition pore opening, calcium ion transport, cardiac contraction, endoplasmic reticulum stress response and antioxidant activity in the AAV injected myocardium. Conclusion: We demonstrated for the first time the deficiency of Cldn5 in myocardium caused myocardial atrophy and dilation with deteriorative heart function. The mechanism of Cldn5 deficiency-induced cardiac dysfunction is associated with severe mitochondrial dysfunction and excitation-contraction coupling.

Abstract P3133: Role Of Myofilament-associatedNeuronal NOSFor Ca 2+ Desensitization Via Troponin I Phosphorylation In The Right Ventricular Cardiomyocytes Of Rats

This study aimed to compare the role of neuronal nitric oxide synthase (nNOS, NOS1) in the excitation-contraction coupling and myofilament Ca 2+ -sensitivity between left ventricular cardiomyocytes (LVCMs) and right ventricular cardiomyocytes (RVCMs). Previous research had only investigated the effects of nNOS on LVCMs, but our study focused on RVCMs which have slightly larger diastolic sarcomere length (SL D ) and smaller sarcomere shortening (ΔSL) than LVCMs. Immunoblot study showed that the total expression and phosphorylated Ser 1417 ratio of nNOS were not different between RVCMs and LVCMs. However, treating RVCMs with the nNOS-specific inhibitor, S-methyl-L-thiocitrulline (SMTC), resulted in more prominent augmentation of ΔSL and shortened SL D in RVCMs, along with a paradoxical decrease of the amplitude of Ca 2+ transient (Δ[Ca 2+ ] i ) due to a more significant inhibition of L-type Ca 2+ current in RVCMs. The Ca 2+ -contraction loop showed a more leftward shift in the RVCMs by nNOS inhibition, indicating Ca 2+ desensitization of the myofilaments by the intrinsic nNOS. Previous studies offered troponin I (cTnI) phosphorylation as the regulatory target of myofilament Ca 2+ -sensitivity through the sGC-cGMP-PKG pathway downstream to NO. The SMTC treatment decreased cTnI phosphorylation in the RVCMs, while not in the LVCMs, suggesting that nNOS plays a more prominent role in myofilament Ca 2+ desensitization in RVCMs by targeting cTnI. Additionally, the higher phosphorylation of nNOSβ, the isoform of nNOS expressed in the myofilaments, was detected in the myofilament of RVCMs than LVCMs. This study is the first to demonstrate the more significant role of nNOS in RVCMs in regulating myofilament Ca 2+ desensitization through cTnI.

Abstract P1018: Peptidyl-prolyl Cis-trans Isomerase-like 4 Is A Novel Regulator Of Smooth Muscle Cell Remodeling

Introduction: Smooth muscle cell (SMC) remodeling is a complex and multifaceted process that plays an essential role in the maintenance and function of various organ systems in the body. In response to physiological and pathological stimuli, smooth muscle cells exhibit remarkable plasticity, undergoing morphological and functional changes that can impact vascular tone. Dysregulation of SMC remodeling can contribute to the development and progression of numerous pathological conditions, such as hypertension and atherosclerosis. However, the role of SMC remodeling in intracranial aneurysm (IA) formation has yet to be explored. Objective-Method: In this study, we aimed to elucidate the role of PPIL4, an IA-implicated gene, in SMC remodeling using a targeted experimental approach. We performed immunostaining and bulk RNA sequencing (RNA-seq) in human primary brain vascular smooth muscle cells (HPBVSMC) treated with lentiviral shRNA and open-reading-frame (ORF) particles to individually downregulate and overexpress PPIL4, respectively. Results: We found that PPIL4 downregulation results in significant upregulation of the genes encoding contractile proteins, including ACTA2, CNN1, TNNT1, and TNNT2, in HPBVSMC and leads to a significantly increased cell size, favoring a contractile SMC phenotype. Conversely, overexpressing PPIL4 using ORF-lentiviral particles caused downregulation of contractile proteins, increased expression of MMP1 and MMP3, and promoted a synthetic phenotype in SMCs. Furthermore, PPIL4 overexpression caused cell elongation and spindle-like cell formation in HPBVSMC. Conclusion: In conclusion, our findings highlight the critical role of PPIL4 in modulating SMC remodeling, suggesting a potential mechanistic link between PPIL4 dysregulation, SMC remodeling, and IA formation, which warrants further investigation.

Abstract P3130: Preventing Site-specific Calpain Cleavage Of Junctophilin-2 Protects Against Stress-induced E-c Coupling Dysfunction And Heart Failure Development In Mice

In heart failure (HF), excitation-contraction (E-C) coupling processes become disrupted, resulting in abnormal Ca 2+ homeostasis that triggers cardiac dysfunction. Junctophilin-2 (JP2) is an essential E-C coupling constituent for maintaining cardiomyocyte integrity and cardiac function both at baseline and in response to stress. We previously showed that calpain-mediated proteolytic cleavage of JP2 at arginine 565 / threonine 566 (R 565 /T 566 ) is key to its downregulation in the diseased heart. However, it remains unclear whether preventing site-specific proteolysis of JP2 can prevent cardiac remodeling and HF progression following stress. To investigate this, calpain-resistant mutant JP2 knockin mice (JP2 CR ) were generated by deleting residues 563-568 spanning the primary calpain cleavage site. Studies in cultured cardiomyocytes treated with isoproterenol (ISO) showed that JP2 CR is resistant to stress-induced loss of JP2 integrity and T-tubule disorganization compared to wild-type (WT) cardiomyocytes. Further, in vivo studies demonstrate JP2 CR mice are more resistant to pressure overload stress having significantly less abnormal Ca 2+ homeostasis, cardiac dysfunction, hypertrophy, lung edema and fibrosis relative to WT mice. Western blot analysis revealed JP2 CR hearts have preserved expression levels of several key E-C coupling proteins, including RyR2, SERCA2a and Ca V 1.2. RNA-Sequencing analysis further revealed attenuated pressure overload induced hypertrophic and heart failure-related transcriptional reprogramming in JP2 CR hearts. These findings indicate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain-cleavage of JP2 offers cardiac protection against stress-induced hypertrophy and heart failure. Our data indicate that preventing JP2 cleavage by gene therapy approaches may hold great therapeutic potential for treating heart failure.

Abstract P3128: Synapse-associated Protein 97 Is Necessary For Enhancing Cardiac Contractility Via Beta1 Adrenoceptor-neuronal Nitric Oxide Synthase Pathway And Guanosine3',5'-cyclic Monophosphate Signaling

Objective: Synapse-associated protein 97 (SAP97) is a scaffold protein widely expressed in the brain, heart, and kidney. SAP97 has been suggested function in the regulation of cardiac contractility. SAP97 directly binds to cardiac beta1 adrenceptor. Our recent studies have shown that increased cAMP and cGMP levels are involved in enhancing excitation-contraction (E-C) coupling after sympathetic nervous system activation. We aim to study the role of SAP97 in beta1 adrenoceptor-induced increases in cyclic nucleotides and cardiac E-C coupling. Results and Methods: Cardiac function in mice with cardiomyocyte-specific deletion of the SAP97 (SAP97-cKO) was comparable to that in flox/flox control littermates. Ligand-induced cGMP signal in freshly isolated adult ventricular myocytes (AVMs) using FRET biosensor Gi500. The cGMP responsiveness caused by β1AR agonist as dobutamine was reduced in SAP97-cKO versus f/f cells. No significant difference between the groups using nonselective beta-adrenergic stimulation isoproterenol (ISO). In comparison, the dobutamine-induced cAMP levels were not reduced in SAP97-cKO when compared to f/f controls. The dobutamine-induced ejection fraction in hearts and sarcomere shortening in AVMs were significantly attenuated in SAP97-cKO hearts and AVMs when compared to f/f controls, respectively. On the other hand, no difference was observed between f/f and cKO AVMs and hearts after stimulation by ISO. To further examine the LTCC activity, whole-cell currents (ICa) were recorded from freshly isolated AVMs. The results showed that the LTCC current was attenuated in SAP97-cKO AVMs than cKO controls when exposed to dobutamine. These results show SAP97 is necessary for increasing intracellular cGMP level, LTCC current, and cardiac E-C coupling and contractility after β1AR stimulation. Conclusion: SAP97 plays a crucial role in β 1 AR signaling, which is necessary for triggering E-C coupling through cAMP and cGMP pathways.

Abstract P3131: SIRTUIN5 Modulates Na + /Ca 2+ Handling Through Oxidative Stress Dependent Mechanism In Mouse Heart

Mutations in cardiac sodium channel Na V 1.5 (encoded by SCN5A) can cause inherited or acquired arrhythmias, including ~20% of cases of Brugada Syndrome (BrS) and inherited conduction disease. The peak Na V 1.5 current (I Na, Peak ) and the late current (I Na, Late ) govern the fast upstroke and plateau of the cardiac action potential, respectively. Ca 2+ ions impact excitation-contraction coupling, and imbalance in Na + /Ca 2+ homeostasis can cause electrical disturbance. BrS is characterized by a right bundle branch block pattern on EKG, arrhythmias, and sudden cardiac death. Recently, potential mutations in SIRT5, including P114T-SIRT5, were identified in small families with BrS. SIRT5 is a member of the Sirtuin family, acts as NAD + -dependent deACYLase.We explored the potential role of SIRT5 in BrS using heterologous expression systems and homozygous P114T-SIRT5 knock-in (P114T-KI) mice. SIRT5 co-immunoprecipitates with Na V 1.5 in HEK293 cells. Live-cell staining using DCFDA or mitoSOX showed that P114T-KI hearts had increased basal reactive oxygen species (ROS) and were more sensitive to oxidative stress induced by H 2 O 2 than wild-type littermates. P114T mice displayed Ca 2+ -mishandling as measured by Ca 2+ imaging in perfused hearts, increased I Na, Late , prolonged QTc, and arrhythmias. Notably, treatment with mitochondrial ROS scavenger mitotempo mitigated the aberrant Ca 2+ handling and arrhythmia in P114T mice.Together, these suggest that P114T-SIRT5 causes Na + /Ca 2+ mishandling-induced arrhythmias in ROS-dependent manner, highlighting potential mechanisms underlying BrS. They pave the way for using SIRT5 or its activators as novel anti-arrhythmic therapies.

Abstract P3195: Mitochondrial CaMKII Drives Cardiometabolic Dysfunction In Heart Failure

Background: Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is an established negative regulator of cardiac injury. Both the expression and activity levels of CaMKII delta, the primary cardiac isoform of this kinase, are elevated in models of heart failure (HF) such as ischemia-reperfusion (I/R) injury and following myocardial infarction (MI). This upregulation is due in part to CaMKII’s role in the regulation of excitation-contraction coupling, apoptosis, activation of hypertrophic programming, arrhythmias and pro-inflammatory signaling. Recently, a pool of mitochondrial CaMKII (mtCaMKII) has been identified; we have demonstrated that there is an observed increase in mitochondrial CaMKII activation with left ventricular dilation acutely following injury in a mouse model of MI. This deleterious post-MI phenotype is rescued with genetic mitochondrial CaMKII inhibition; conversely, mice with myocardial mitochondrial CaMKII overexpression present with a severe dilated cardiomyopathy phenotype, alterations in electron transport chain (ETC) activity, and decreased ATP production. Objective: To date, the molecular mechanisms by which CaMKII regulates mitochondrial energetics are unknown and present a novel therapeutic target for HF which we are currently studying. Methods: As we have identified changes in the activity of enzymes, particularly complexes I and II, in the mitochondrial ETC as well as TCA cycle in response to increased CaMKII levels or activity, we are in the process of assessing changes in the mitochondrial CaMKII interactome between uninjured and failing hearts. Utilizing liquid chromatography-mass spectrometry (LCMS) and proteomics analysis of both scaffolding interactions alongside a novel ATP analogue labeling technique, we have mapped novel mitochondrial proteins which interact with mtCaMKII. Significance: The identification of previously unknown mitochondrial pathways involving CaMKII may uncover promising pharmacological targets for cardiovascular therapeutics, as this kinase is a critical player in the progression of HF.

Abstract P3145: Enhancing Cardiac Cav1.2 Ion Channel Function By Targeted Deubiquitination

Ventricular excitation-contraction coupling relies on precise spatial arrangement of voltage-gated calcium channels CaV1.2 within T-tubular dyadic junctions proximal to ryanodine receptors (RyR2) that gate Ca2+ release from the sarcoplasmic reticulum. Impaired CaV1.2 trafficking is linked to devastating cardiovascular diseases such as arrythmias and cardiac hypertrophy. Ubiquitination is an important regulatory mechanism that controls ion channel surface trafficking and turnover. Emerging evidence hint at a prominent role for CaV1.2 ubiquitination to underlie heart failure disease progression, that suppresses CaV1.2 currents (ICa,L), resulting in harmful compensatory mechanisms. Targeted protein stabilization (TPS) is a burgeoning therapeutic approach for undruggable targets. We hypothesized that harnessing deubiquitinases using TPS can counteract pathological ubiquitination of CaV1.2, overcoming the need for hormonal remodeling and lead to novel treatment. To this end, we sought to develop engineered deubiquitinases (enDUBs) that can increase functional expression of CaV1.2 channels in the heart. The catalytic domain of ubiquitin specific protease 21 (USP21) fused to nb.F3 (F3-USP21) robustly increased CaV1.2 currents reconstituted in HEK293 cells as well as endogenous ICa,L in mammalian ventricular cardiomyocytes. This increase was within the physiological range of the impact of β-adrenergic stimulation of CaV1.2 and did not alter ventricular action potential duration. The mechanism of nbF3-USP21-induced increase in ICa,L is augmented CaV1.2 surface density. Reassuringly, this approach did not adversely interfere with the correct placement of CaV1.2 channels at dyadic junctions as cardiomyocytes expressing nb.F3-USP21 displayed normal colocalization of CaV1.2 with RyR2 at T-tubules. In conclusion, our study provides evidence that targeting deubiquitinases using TPS could be a viable therapeutic approach for rescuing CaV1.2 functional expression and these findings may pave the way for future development of TPS-based therapies for cardiovascular diseases such as arrhythmias and cardiac hypertrophy.

Abstract P2102: The Basic Helix-loop-helix Transcription Factor Tcf21 Regulates Fibroblast Cell States In The Adult Murine Heart

Objective: Transcription factor 21 (Tcf21) is a basic helix-loop-helix protein that is essential for normal mesodermal organogenesis and its loss is incompatible with life. In the developing heart, epicardial progenitor cells differentiate to either vascular smooth muscle cells or fibroblasts, the latter of which is dependent on Tcf21 expression. In the adult heart, Tcf21 is expressed by the majority of fibroblasts but is downregulated in response to fibrotic stimuli as the fibroblast differentiates to a myofibroblast. In atherosclerosis, smooth muscle cells that undergo phenotypic modulation towards a fibroblast-like cell acquire expression of Tcf21. These findings led to the hypothesis that Tcf21 could be a regulator of fibroblast cell-fate in the adult mammalian heart and contribute to cardiac fibrosis. Methods and Results: To test this hypothesis, we generated tamoxifen-inducible genetic mouse models to study cardiac fibroblast Tcf21 deletion and overexpression in vivo . Acute and long-term Tcf21 ablation in the adult heart did not promote differentiation to myofibroblasts. After long-term ablation, no differences were detected on cardiac interstitial cell populations, cardiac structure or function and extracellular matrix deposition. In contrast, in response to fibrotic stimuli, overexpression of Tcf21 suppressed differentiation to myofibroblasts with reduced expression of contractile proteins. This was accompanied by reduced cardiac fibrosis. Conclusions: The expression of Tcf21 in the healthy fibroblast is not required to maintain a ‘resting’ fibroblast cell-state. In disease, Tcf21 functions as a suppressor of fibroblast differentiation to the contractile myofibroblast and its overexpression reduces the fibrotic burden on the heart.

Non-invasive electromechanical assessment during atrial fibrillation identifies underlying atrial myopathy alterations with early prognostic value

Electromechanical characterization during atrial fibrillation (AF) remains a significant gap in the understanding of AF-related atrial myopathy. This study reports mechanistic insights into the electromechanical remodeling process associated with AF progression and further demonstrates its prognostic value in the clinic. In pigs, sequential electromechanical assessment during AF progression shows a progressive decrease in mechanical activity and early dissociation from its electrical counterpart. Atrial tissue samples from animals with AF reveal an abnormal increase in cardiomyocytes death and alterations in calcium handling proteins. High-throughput quantitative proteomics and immunoblotting analyses at different stages of AF progression identify downregulation of contractile proteins and progressive increase in atrial fibrosis. Moreover, advanced optical mapping techniques, applied to whole heart preparations during AF, demonstrate that AF-related remodeling decreases the frequency threshold for dissociation between transmembrane voltage signals and intracellular calcium transients compared to healthy controls. Single cell simulations of human atrial cardiomyocytes also confirm the experimental results. In patients, non-invasive assessment of the atrial electromechanical relationship further demonstrate that atrial electromechanical dissociation is an early prognostic indicator for acute and long-term rhythm control.

Abstract P1025: Hsp70 Co-chaperones Regulate Sarcomeric Proteostasis In Hypertrophic Cardiomyopathy

Significance: Recent Genome Wide Association Studies (GWAS) identified non-sarcomeric variants in hypertrophic cardiomyopathy (HCM) with three of the top 10 variant loci in or near genes that encode for co-chaperones of HSP70: BAG3, DNAJC18 and HSPB7. These variants are also present in dilated cardiomyopathy (DCM), but have an opposing effect on cardiac function, leading us to hypothesize that GWAS variants in HSP70 co-chaperones can affect the proteostasis of contractile proteins in the HCM heart. As a first step, we determined the effects each HSP70 co-chaperone knockdown (KD) had on sarcomeric and z-disc protein expression and the poly-ubiquitinated state of cardiomyocyte-specific proteins. Methods and Results: Transduction of human iPSC-CMs (iPSC-CMs) with adenoviral vectors driving shRNA expression against BAG3, DNAJC18, HSPB7, or scramble (control) yielded transduction efficiencies > 75% and >80% reduction in target transcript 4 days post-transduction. BAG3 KD increased poly-ubiquitinated proteins by 38+/-9% (p<0.001) and reduced steady state expression of sarcomeric and z-disc proteins, including: MyBP-C (p<0.0001), actin (p<0.0001), cardiac troponin I (p<0.05), tropomyosin (TPM1) (p<0.01), myosin light chain 3 (MYL3) (p<0.0001), filamin C (p<0.05), and alpha-actinin-2 (p<0.0001). DNAJC18 KD increased poly-ubiquitinated protein by 66+/-17% (p<0.01) but did not impact sarcomere protein expression. Conversely, HSPB7 KD reduced poly-ubiquitinated proteins by 21+/-7% (p<0.01) and increased protein expression of MyBP-C (p<0.01), TPM1 (p<0.05), and myosin light chain 2 (p<0.05). DNAJC18 KD increased BAG3 and HSPB7 protein expression levels by ~2-fold (p<0.001) without increasing transcript levels, suggesting BAG3 and HSPB7 may be protein clients of DNAJC18. Conclusions: HSP70 co-chaperones that have an established genetic association with human HCM play an important role in cardiomyocyte proteostasis. Multiple sarcomeric and z-disc proteins appear to be clients of BAG3, while BAG3 and HSPB7 themselves may be clients of DNAJC18. Targeting this axis could be therapeutic for patients with HCM that possess genetic variants in sarcomeric proteins.

Vascular smooth muscle cell contractile proteins are better markers of vasa vasorum as compared to endothelial cell proteins

Vascular smooth muscle cell contractile proteins are better markers of vasa vasorum as compared to endothelial cell proteins

ATPase activity of actomyosin and myosin in different types of muscles of intact and irradiated animals

The large-scale use of sources of ionizing radiation and related accidents caused a fundamentally new problem. The body's resistance to the influence of adverse environmental factors and the probability of pathological disorders are significantly determined by the state of adaptive systems. One of the general manifestations of adaptation is a change in motor activity, which is associated, in particular, with changes in the content of contractile proteins, muscle function, and muscle contraction. It is crucial to understand the detailed mechanisms of muscle tissue functioning in the body of intact animals and animals exposed to different doses of radiation because, in this case, preventive and curative/rehabilitative medical measures should be milder for muscle dysfunctions caused by ionizing radiation The purpose of the work is to investigate the effect of different doses of radiation on Mg2+, Ca2+-ATPase and K+-ATPase activity of actomyosin and myosin in the mechanisms of intact and irradiated in different doses of sexually mature animals in order to find out the mechanisms of adaptation of the organism to the influence of stress factor by studying changes in the activity of Mg2+, Ca2+-ATPase and K+-ATPase in the muscle system. The data obtained showed that irradiation at a dose of 0.5 Gy affects Mg2+,Ca2+-ATPase and K+-ATPase activity, however, these changes occur differently. K+-ATPase actomyosin activity in cardiac and skeletal muscles decreased starting from the 1st day of the experiment. Its gradual increase was observed only on the 30th day but still, it was smaller when compared with such an indicator in intact animals. It was proved that the Mg2+, Ca2+-ATPase actomyosin activity in sexually mature animals irradiated at a dose of 1.0 Gy increased 1 day after irradiation in skeletal muscle. In сardiac muscle an acute increase in Mg2+, Ca2+-ATPase actomyosin activity was observed on the 1st day and its decrease - by the 30th day. The conclusion was made that irradiation at a dose of 1.0 Gy is accompanied by skeletal muscle damage, as well as modification of the actomyosin protein complex, which is the major unit of muscle contraction. This leads to changes in its functional activity and is expressed in an increase in K+-ATPase actomyosin activity of muscles sensitive to irradiation. We assume such a mechanism of the effect of ionizing radiation on the actomyosin complex. The authors sure that a decrease in Mg2+, Ca2+-ATP-ase myosin activity as a result of exposure to ionizing radiation at a dose of 1.0 Gy can be caused by a violation of the structure of its active center since the ATPase center of pure myosin is free from interaction with actin, and that is why myosin appears more sensitive to the action of ionizing radiation.

Structural Adaptation of the Excitation-Contraction Coupling Apparatus in Calsequestrin1-Null Mice during Postnatal Development.

The precise arrangement and peculiar interaction of transverse tubule (T-tubule) and sarcoplasmic reticulum (SR) membranes efficiently guarantee adequate contractile properties of skeletal muscle fibers. Fast muscle fibers from mice lacking calsequestrin 1 (CASQ1) are characterized by the profound ultrastructural remodeling of T-tubule/SR junctions. This study investigates the role of CASQ1, an essential component of calcium release units (CRUs), in the postnatal development of muscle fibers. By using CASQ1-knockout mice, we examined the maturation of CRUs and the involvement of different junctional proteins in the juxtaposition of the membrane system. Our morphological investigation of both wild-type (WT) and CASQ1-null extensor digitorum longus (EDL) fibers, from 1 week to 4 months of age, yielded noteworthy findings. Firstly, we observed that the absence of CASQ1 hindered the full maturation of CRUs, despite the correct localization of key junctional components (ryanodine receptor, dihydropyridine receptor, and triadin) to the junctional SR in adult animals. Furthermore, analysis of protein expression profiles related to T-tubule biogenesis and organization (junctophilin 1, amphiphysin 2, caveolin 3, and mitsugumin 29) demonstrated delayed progression in their expression during postnatal development in the absence of CASQ1, suggesting the impaired maturation of CRUs. The absence of CASQ1 directly impacts the proper assembly of CRUs during development and influences the expression and coordination of other proteins involved in T-tubule biogenesis and organization.