Troponin I Phosphorylation Research Articles

Aging Reduces Cardiac Contractility by Altering Cardiac Regulatory and Contractile Proteins

Aging is associated with an increased incidence of cardiac dysfunction. We hypothesized that aging associated changes in cardiac regulatory and contractile proteins produce contractile dysfunction. We assessed regulatory and contractile protein expression as well as contractility in left ventricular cardiac muscle from Fischer F-344 rats at 6-, 18-, and 24-month of age. Age-related changes in the expression of β-MHC as well as the expression and phosphorylation of troponin I (TnI) and cardiac myosin binding C protein (MyBP-C) were assessed by Western blot. Aging was associated with an increase in the expression of β-MHC and the phosphorylation of both TnI at Ser23/24 and MyBP-C. Mechanical properties were assessed in Triton-X permeabilized anterior papillary muscle from the left ventricle from both 6- and 24-month old rats. In papillary muscle of 24-month old rats, there was a decrease in maximum Ca2+ activated force, Ca2+ sensitivity of force and the rate constant for force recovery after quick release (ktr). Additionally, cardiomyocytes were isolated from the left ventricle at 6-, 18-, and 24-month of age using a modified Langendorff system. Cardiomyocytes were then loaded with fura-2 AM to measure cytosolic calcium ([Ca2+]cyt) responses to electrical field stimulation (0.5 Hz). [Ca2+]cyt and contractile (sarcomere length shortening) responses to stimulation were simultaneously measured using an IonOptix system. In older cardiomyocytes, the amplitude of evoked [Ca2+]cyt responses was reduced while there was no difference in the kinetics of the [Ca2+]cyt transient. The extent of sarcomere length shortening as well as the rates of both shortening and relaxation were reduced. These results demonstrate that aging is associated with changes in the expression and phosphorylation of contractile and regulatory proteins, which produce changes in cardiac contractility. Supported by a Mayo CV Prospective Grant (FVB). 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.

Differential sex-dependent susceptibility to diastolic dysfunction and arrhythmia in cardiomyocytes from obese diabetic HFpEF model.

Sex-differences in heart failure with preserved ejection fraction (HFpEF) are important, but key mechanisms involved are incompletely understood. While animal models can inform about sex-dependent cellular and molecular changes, many previous preclinical HFpEF models have failed to recapitulate sex-dependent characteristics of human HFpEF. We tested for sex-differences in HFpEF using a two-hit mouse model (leptin receptor-deficient db/db mice plus aldosterone infusion for 4 weeks; db/db+Aldo). We performed echocardiography, electrophysiology, intracellular Ca2+ imaging, and protein analysis. Female HFpEF mice exhibited more severe diastolic dysfunction in line with increased titin N2B isoform expression and PEVK element phosphorylation, and reduced troponin-I phosphorylation. Female HFpEF mice had lower BNP levels than males despite similar comorbidity burden (obesity, diabetes) and cardiac hypertrophy in both sexes. Male HFpEF mice were more susceptible to cardiac alternans. Male HFpEF cardiomyocytes (versus female) exhibited higher diastolic [Ca2+], slower Ca2+ transient decay, reduced L-type Ca2+ current, more pronounced enhancement of the late Na+ current, and increased short-term variability of action potential duration (APD). However, male and female HFpEF myocytes showed similar downregulation of inward rectifier and transient outward K+ currents, APD prolongation, and frequency of delayed afterdepolarizations. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed all pathological APD changes in HFpEF in both sexes, and empagliflozin pretreatment mimicked these effects of CaMKII inhibition. Vericiguat had only slight benefits, and these effects were larger in HFpEF females. We conclude that the db/db+Aldo preclinical HFpEF murine model recapitulates key sex-specific mechanisms in HFpEF and provides mechanistic insights into impaired excitation-contraction coupling and sex-dependent differential arrhythmia susceptibility in HFpEF with potential therapeutic implications. In male HFpEF myocytes, altered Ca2+ handling and electrophysiology aligned with diastolic dysfunction and arrhythmias, while worse diastolic dysfunction in females may depend more on altered myofilaments properties.

Troponin I Tyrosine Phosphorylation Beneficially Accelerates Diastolic Function.

A healthy heart is able to modify its function and increase relaxation through post-translational modifications of myofilament proteins. While there are known examples of serine/threonine kinases directly phosphorylating myofilament proteins to modify heart function, the roles of tyrosine (Y) phosphorylation to directly modify heart function have not been demonstrated. The myofilament protein TnI (troponin I) is the inhibitory subunit of the troponin complex and is a key regulator of cardiac contraction and relaxation. We previously demonstrated that TnI-Y26 phosphorylation decreases calcium-sensitive force development and accelerates calcium dissociation, suggesting a novel role for tyrosine kinase-mediated TnI-Y26 phosphorylation to regulate cardiac relaxation. Therefore, we hypothesize that increasing TnI-Y26 phosphorylation will increase cardiac relaxation in vivo and be beneficial during pathological diastolic dysfunction. The signaling pathway involved in TnI-Y26 phosphorylation was predicted in silico and validated by tyrosine kinase activation and inhibition in primary adult murine cardiomyocytes. To investigate how TnI-Y26 phosphorylation affects cardiac muscle, structure, and function in vivo, we developed a novel TnI-Y26 phosphorylation-mimetic mouse that was subjected to echocardiography, pressure-volume loop hemodynamics, and myofibril mechanical studies. TnI-Y26 phosphorylation-mimetic mice were further subjected to the nephrectomy/DOCA (deoxycorticosterone acetate) model of diastolic dysfunction to investigate the effects of increased TnI-Y26 phosphorylation in disease. Src tyrosine kinase is sufficient to phosphorylate TnI-Y26 in cardiomyocytes. TnI-Y26 phosphorylation accelerates in vivo relaxation without detrimental structural or systolic impairment. In a mouse model of diastolic dysfunction, TnI-Y26 phosphorylation is beneficial and protects against the development of disease. We have demonstrated that tyrosine kinase phosphorylation of TnI is a novel mechanism to directly and beneficially accelerate myocardial relaxation in vivo.

Diastolic Cardiomyopathy Secondary to Experimentally Induced Exacerbated Emphysema.

Chronic obstructive pulmonary disease (COPD) is a clinical entity of increasing significance. COPD involves abnormalities of the airways and, in emphysema, parenchymal pulmonary destruction. Cardiovascular disease has emerged as a significant comorbidity to COPD. Heart failure with preserved ejection fraction (HFpEF) appears to be particularly associated with COPD-emphysema. Traditional treatments have shown limited efficacy in improving COPD-associated HFpEF. This lack of therapeutic efficacy highlights the need to identify potential mechanisms that link COPD-emphysema to HFpEF. Therefore, we aimed to study the delayed cardiac physiological impacts in a rat model with acute exacerbated emphysema. Emphysema was induced by four weekly 4 units elastase (ELA) intratracheal pulmonary instillations and exacerbation by one final additional lipolysaccharide (LPS) instillation in male Wistar rats. At 5 weeks after the ELA and LPS exposure, invivo and ex vivo pulmonary and cardiac measurements were performed. Experimental exacerbated emphysema resulted in decreased pulmonary function and exercise intolerance. Histological analysis revealed parenchymal pulmonary destruction without signs of inflammation or cardiac fibrosis. In vivo cardiac functional analysis revealed diastolic dysfunction and tachycardia. Ex vivo analysis revealed a cellular cardiomyopathy with decreased myofilament Ca2+ sensitivity, cross-bridge cycling kinetics, and increased adrenergic PKA (protein kinase A)-dependent phosphorylation of troponin-I. Experimental exacerbated emphysema was associated with exercise intolerance that appeared to be secondary to increased β-adrenergic tone and subsequent cardiac myofilament dysfunction. A β1-receptor antagonist treatment (bisoprolol) started 24 hours after ELA-LPS instillation prevented invivo and ex vivo diastolic dysfunction. These results suggest that novel treatment strategies targeted to the cardiac myofilament may be beneficial to combat exacerbated emphysema-associated HFpEF.

Cardiac Protein Kinase D1 ablation alters the myocytes β-adrenergic response

β-adrenergic (β-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute β-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte β-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal β-adrenergic responses in Ca2+ handling.

The lack of Troponin I Ser-23/24 phosphorylation is detrimental to in vivo cardiac function and exacerbates cardiac disease

Troponin I (TnI) is a key regulator of cardiac contraction and relaxation with TnI Ser-23/24 phosphorylation serving as a myofilament mechanism to modulate cardiac function. Basal cardiac TnI Ser-23/24 phosphorylation is high such that both increased and decreased TnI phosphorylation may modulate cardiac function. While the effects of increasing TnI Ser-23/24 phosphorylation on heart function are well established, the effects of decreasing TnI Ser-23/24 phosphorylation are not clear. To understand the in vivo role of decreased TnI Ser-23/24 phosphorylation, mice expressing TnI with Ser-23/24 mutated to alanine (TnI S23/24A) that lack the ability to be phosphorylated at these residues were subjected to echocardiography and pressure-volume hemodynamic measurements in the absence or presence of physiological (pacing increasing heart rate or adrenergic stimulation) or pathological (transverse aortic constriction (TAC)) stress. In the absence of pathological stress, the lack of TnI Ser-23/24 phosphorylation impaired systolic and diastolic function. TnI S23/24A mice also had an impaired systolic and diastolic response upon stimulation increased heart rate and an impaired adrenergic response upon dobutamine infusion. Following pathological cardiac stress induced by TAC, TnI S23/24A mice had a greater increase in ventricular mass, worse diastolic function, and impaired systolic and diastolic function upon increasing heart rate. These findings demonstrate that mice lacking the ability to phosphorylate TnI at Ser-23/24 have impaired in vivo systolic and diastolic cardiac function, a blunted cardiac reserve and a worse response to pathological stress supporting decreased TnI Ser23/24 phosphorylation is a modulator of these processes in vivo.

Suppression of lusitropy as a disease mechanism in cardiomyopathies.

In cardiac muscle the action of adrenaline on β1 receptors of heart muscle cells is essential to adjust cardiac output to the body's needs. Adrenergic activation leads to enhanced contractility (inotropy), faster heart rate (chronotropy) and faster relaxation (lusitropy), mainly through activation of protein kinase A (PKA). Efficient enhancement of heart output under stress requires all of these responses to work together. Lusitropy is essential for shortening the heartbeat when heart rate increases. It therefore follows that, if the lusitropic response is not present, heart function under stress will be compromised. Current literature suggests that lusitropy is primarily achieved due to PKA phosphorylation of troponin I (TnI) and phospholamban (PLB). It has been well documented that PKA-induced phosphorylation of TnI releases Ca2+ from troponin C faster and increases the rate of cardiac muscle relaxation, while phosphorylation of PLB increases SERCA activity, speeding up Ca2+ removal from the cytoplasm. In this review we consider the current scientific evidences for the connection between suppression of lusitropy and cardiac dysfunction in the context of mutations in phospholamban and thin filament proteins that are associated with cardiomyopathies. We will discuss what advances have been made into understanding the physiological mechanism of lusitropy due to TnI and PLB phosphorylation and its suppression by mutations and we will evaluate the evidence whether lack of lusitropy is sufficient to cause cardiomyopathy, and under what circumstances, and consider the range of pathologies associated with loss of lusitropy. Finally, we will discuss whether suppressed lusitropy due to mutations in thin filament proteins can be therapeutically restored.

More evident roles of nNOS for the regulation of Ca2+‐sensitivity and SERCA activity in the right than the left ventricular cardiomyocytes of rats

It is known that neuronal NOS (nNOS, NOS1) regulates the intracellular Ca2+ ([Ca²⁺]i) and contractility of cardiomyocytes, which was investigated only in the left ventricular myocytes (LVCM) while not in the right ventricle (RVCM). In the paced cardiomyocytes, we have recently reported more relaxed diastolic sarcomere length (SLD) and smaller shortening (∆SL) while the amplitude of Ca²⁺ transient (∆[Ca²⁺]i) were not different (Jeon et al., 2021). Here we compared the expression and the functional roles of nNOS in the RVCMs and LVCMs from Sprague‐Dawley rats. The total expression and the phosphorylated Ser1417 ratio of nNOS were not different between RVCM and LVCM. However, the treatment with nNOS‐specific inhibitor, S‐methyl‐L‐thiocitrulline (SMTC, 100 nM), induced more prominent augmentation of ∆SL along with faster contraction while paradoxical faster relaxation and decreased ∆[Ca²⁺]i in RVCM. The hysteresis curve between ∆SL and ∆[Ca²⁺]i­ showed more prominent leftward shift, i.e. sensitization of the myofilaments in RVCM by SMTC. Consistently, the speed of contraction became faster, and the longer SLD of RVCM became similar to that of RVCM by the treatment with SMTC. Intriguingly, the SMTC treatment accelerated the Ca2+ removal by SERCA in RVCM, which appears to be consistent with the decreased ∆[Ca²⁺]i and faster relaxation. As for the decreased myofilament calcium sensitivity through sGC‐cGMP‐PKG pathwy, altered phosphorylation of troponin I (TnI) was suggested. In fact, the SMTC treatment decreased the TnI phosphorylation in RVCMs, while not LVCMs. Immunoblotting revealed higher expression of nNOSβ and its phosphorylated form in the myofilament fraction from RVCM than LVCM. In summary, the nNOS‐dependent longer SLD and the paradoxical negative inotropic effect despite the inhibitory effect on SERCA might be mediated by the myofilament‐associated nNOSβ decreasing Ca2+‐sensitivity through TnI phosphorylation. Interestingly, we also found that the treatment with omecamtiv mecarbil, a recently proposed cardiac myosin activator, induced more prominent inotropic effect in RVCM, which might imply a larger functional reservoir for the RV contraction.

Progressive Reduction in Right Ventricular Contractile Function Attributable to Altered Actin Expression in an Aging Mouse Model of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited genetic disorder of desmosomal dysfunction, and PKP2 (plakophilin-2) has been reported to be the most common disease-causing gene when mutation-positive. In the early concealed phase, the ACM heart is at high risk of sudden cardiac death before cardiac remodeling occurs because of mistargeted ion channels and altered Ca2+ handling. However, the results of pathogenic PKP2 variants on myocyte contraction in ACM pathogenesis remain unknown. We studied the outcomes of a human truncating variant of PKP2 on myocyte contraction using a novel knock-in mouse model with insertion of thymidine in exon 5 of Pkp2, which mimics a familial case of ACM (PKP2-L404fsX5). We used serial echocardiography, electrocardiography, blood pressure measurements, histology, cardiomyocyte contraction, intracellular calcium measurements, and gene and protein expression studies. Serial echocardiography of Pkp2 heterozygous (Pkp2-Het) mice revealed progressive failure of the right ventricle (RV) in animals older than 3 months. By contrast, left ventricular function remained normal. ECGs of 6-month-old anesthetized Pkp2-Het mice showed normal baseline heart rates and QRS complexes. Cardiac responses to β-adrenergic agonist isoproterenol (2 mg/kg) plus caffeine (120 mg/kg) were also normal. However, adrenergic stimulation enhanced the susceptibility of Pkp2-Het hearts to tachyarrhythmia and sudden cardiac death. Histological staining showed no significant fibrosis or adipocyte infiltration in the RVs and left ventricles of 6- and 12-month-old Pkp2-Het hearts. Contractility assessment of isolated myocytes demonstrated progressively reduced Pkp2-Het RV cardiomyocyte function consistent with RV failure measured by echocardiography. However, aging Pkp2-Het and control RV myocytes loaded with intracellular Ca2+ indicator Fura-2 showed comparable Ca2+ transients. Western blotting of Pkp2-RV homogenates revealed a 40% decrease in actin, whereas actin immunoprecipitation followed by a 2,4-dinitrophenylhydrazine staining showed doubled oxidation level. This correlated with a 39% increase in troponin-I phosphorylation. In contrast, Pkp2-Het left ventricular myocytes had normal contraction, actin expression and oxidation, and troponin-I phosphorylation. Last, Western blotting of cardiac biopsies revealed that actin expression was 40% decreased in RVs of patients with end-stage ACM. During the early concealed phase of ACM, reduced actin expression drives loss of RV myocyte contraction, contributing to progressive RV dysfunction.

NAD + Redox Imbalance Exacerbates Diabetic Cardiomyopathy via Enhanced Troponin I Phosphorylation, Impaired Energetics and Prolonged Cardiac Relaxation

Diabetes is a major risk factor of heart failure. Diabetes and heart failure have been linked to NAD redox imbalance, whose role in the progression of diabetic cardiomyopathy (DCM) is far from established. Streptozotocin (STZ)-induced diabetes in C57BL/6 mice for 16 weeks promoted declines in systolic and diastolic function and lowered cardiac NAD/NADH ratio. We employed cardiac-specific Ndufs4-KO mice (cKO), which exhibit lowered cardiac NAD/NADH ratio with normal baseline function and energetics, to determine the role of NAD redox imbalance in DCM. Plasma metabolomic analysis showed similar diabetic stress in diabetic control and cKO mice. Systolic (fractional shortening) and diastolic (E’/A’ ratio; isovolumic relaxation time, IVRT) dysfunctions were further exacerbated in diabetic cKO mice in both male and female cohorts. Collagen levels and transcript analyses of fibrosis and extracellular matrix-dependent pathways did not show changes in diabetic cKO hearts, suggesting that the exacerbated cardiac dysfunction was likely due to cardiomyocyte dysfunction. We next analyzed protein acetylation-dependent pathways to account for the exacerbated decline of function in diabetic cKO hearts. NAD redox imbalance in diabetic cKO hearts promoted protein acetylation, including SOD2 acetylation (SOD2-K68Ac). This SOD2 acetylation inhibits its antioxidant function and we observed elevated protein oxidation levels in diabetic cKO hearts. To gain further insights how NAD redox imbalance may regulate cardiomyocyte function, phosphorylation levels of myosin binding protein C (MyBPC) and troponin I (TnI) were examined. MyBPC phosphorylation at S282 is suppressed in failing hearts and remained unchanged in diabetic cKO hearts. TnI phosphorylation at S150 (TnI-S150Pi) increases myofilament calcium sensitivity and prolongs calcium dissociation, while TnI-S23/24Pi decreases calcium sensitivity and accelerates calcium dissociation. In diabetic cKO hearts, levels of TnI-S150Pi were elevated while TnI-S23/24Pi remained unchanged. TnI-S150Pi is sensitive to AMPK, the master energy sensor, and we found that ATP levels decreased, and AMP/ATP ratio increased in diabetic cKO hearts. Elevation of cardiac NAD levels by nicotinamide phosphoribosyltransferase (NAMPT) normalized NAD redox balance and alleviated systolic and diastolic dysfunction. Importantly, elevated TnI-S150Pi, exacerbated diastolic dysfunction and prolonged IVRT in diabetic cKO hearts were all restored by NAMPT over-expression. The data collectively suggest that NAD redox imbalance promotes impairments in cellular energetics and diastolic function via enhancing TnI-S150Pi, and is a critical mediator of the progression of DCM.

How to say NO to the Right vs. Left heart E‐C coupling

There are constitutive NO synthase isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS) in myocardium, regulating the contractility and calcium dynamics. Physiological and anatomical differences between the right (RV) and left ventricle (LV) are evident. Although there is a lot of study of the regulatory roles of NOS in LV myocytes (LVCMs), only a few study in RV myocytes (RVCMs). Here we compared the roles of nNOS and eNOS in the RVCMs and LVCMs isolated by using Langendorff perfusion system from S-D rats. The sarcomere shortening (∆SL) and increased amount of calcium transient (∆[Ca²⁺]i) paced by 2 Hz field stimulation were simultaneously measured using the Ionoptix system. An nNOS-specific inhibitor, S-methyl-L-thiocitrulline (SMTC), and the non-specific NOS inhibitor, L-NG-Nitroarginine methyl ester (L-NAME) were used to dissect the roles of NOS isoforms. RVCMs showed smaller ∆[Ca²⁺]i than LVCMs. The treatment with SMTC and L-NAME significantly decreased the ∆[Ca²⁺]i of RVCMs, while only slightly decreased in LVCMs. Perplexingly, the ∆SLof RVCMs was increased by SMTC, whilenot altered in LVCMs. The mismatched changes in ∆SL and ∆[Ca²⁺]i between RVCMs and LVCMs suggested differential changes in the calcium sensitivity of contraction. In fact, the Ca2+ sensitivity was increased by SMTC in RVCMs while not in LVCMs. In addition, the diastolic sarcomere length (SLD) of RVCMs were decreased by SMTC and L-NAME while not in LVCMs. As for the decreased myofilament calcium sensitivity through sGC-cGMP-PKG pathway, altered phosphorylation of troponin I (TnI) was suggested. In fact, the SMTC treatment decreased the phosphorylated TnI in RVCMs, while not LVCMs. Finally, differential distribution of splice variants of nNOS were supposed; the phosphorylated nNOSβ was higher in the myofilament from RVCMs than LVCMs. Taken together, we firstly provide evidence that the inhibition of nNOSβ in RVCMs increases myofilament calcium sensitivity through decreased phosphorylation of TnI.

Abstract 433: Regulatory and Elastic Protein Modifications in the Myocardium of Hibernating 13-lined Ground Squirrels

The 13-lined ground squirrel exhibits remarkable cardiac adaptation during hibernation, periods of torpor being interrupted by repeated inter-bout arousal (IBA). During IBA heart rate rises from 2-4 bpm to as great as 300 bpm. This rise in heart rate occurs over just 5 hours and remains elevated for 12-24 hours before returning to the lower heart rate. With such rapid and marked changes in heart rate, and thus filling time, myocardial stiffness and relaxation must be regulated within a short timeframe. The objective of this work was to establish post-translational modifications (PTMs) in two proteins critical to myocardial stiffness and relaxation; titin and cardiac troponin I (TnI). It was specifically hypothesized that phosphorylation during IBA would be similar to that of summer tissue and distinct from tissue isolated during torpor. Left ventricular tissue (summer, n=9; torpor, n=10; IBA, n=7) was solubilized and separated by either 2-12% gradient SDS-Page (titin) or 12.5% SDS-PAGE (TnI). Total titin phosphorylation was measured via Pro-Q Diamond Phosphoprotein/SYPRO Ruby staining. PKA-specific TnI phosphorylation was quantified via western blotting, using a primary antibody specific to Ser 23,24. While there was a tendency toward decreased total titin phosphorylation in IBA, the difference was not significant. In contrast, there were significant group effects in PKA-specific TnI phosphorylation. Phospho-TnI/Total TnI ratios were lower in torpor when compared to summer (0.22±0.12 vs. 0.68±0.,34, p<0.05). While there was no significant difference between summer and IBA (0.62±0.35), the difference between torpor and IBA failed to reach significance. This data supports the hypothesis that rapid changes in heart rates are associated with changes in cardiac troponin-I phosphorylation, a modification that contributes to rapid rates of relaxation. Further dissection of site specific titin phosphorylation will be required to assess the extent to which PTM modify titin associated stiffness. Results from this work may have significant implications in our understanding of altered compliance in human heart failure.

Roles of NAD Metabolism in Diabetic Cardiomyopathy

Diabetes is a major risk factor of heart failure. Diabetes and heart failure have been linked to altered NAD metabolism, whose roles in the progression of diabetic cardiomyopathy (DC) are far from established. We induced diabetes in wild type C57BL6N mice (WT) by streptozotocin (STZ) injections. WT mice with chronic diabetic stress (fasting glucose ~600 mg/dl) for 16 weeks had gradual declines in systolic and diastolic function and lowered NAD/NADH ratio, while acute STZ treatment (1‐day) did not affect cardiac function and NAD pool. To determine whether NAD redox imbalance promotes DC, we employed cardiac‐specific Ndufs4‐KO mice (cKO), which exhibit lowered cardiac NAD/NADH ratio without overt dysfunction. While insulin depletion and hyperglycemia were similar in diabetic control and cKO mice stressed by the same diabetogenic protocol, systolic and diastolic dysfunctions were accelerated in diabetic cKO mice. We next determined how NAD metabolism is altered to accelerate DC. In addition to the NAD redox balance, NAD metabolism also involves metabolites and enzymes in the NAD consumption and synthesis pathways, coordinating the homeostasis of NAD. Targeted analyses of transcripts and metabolites involved in these pathways were performed using qPCR and quantitative LC‐MS/MS analyses. Of 22 genes measured, we identified that Nmrk mRNA levels were up‐regulated in diabetic cKO hearts, compared to diabetic control hearts. Of thirteen cardiac NAD metabolite levels surveyed, product metabolites of NMRK enzymatic reaction (i.e. NMN or NAMN) showed decreases in levels in diabetic cKO hearts. The transcript and metabolite data suggest a role of NMRK in DC. Histologically, cardiac fibrosis levels of diabetic control and cKO hearts were slightly elevated compared to non‐diabetic controls, but they were not different. The results suggest that the accelerated decline of cardiac function in diabetic cKO hearts is not be due to altered extracellular matrix environment. We next analyzed acetylation‐dependent pathways to account for the accelerated decline of function in diabetic cKO hearts. NAD redox imbalance in diabetic cKO hearts promoted protein acetylation, including SOD2 acetylation (SOD2‐K68Ac). SOD2 acetylation inhibits its antioxidant function and we observed elevated protein oxidation levels in diabetic cKO hearts. To gain further insights how NAD redox imbalance may regulate cardiomyocyte function, phosphorylation levels of myosin binding protein C (MyBPC) and troponin I (TnI) were examined. In diabetic cKO hearts, TnI phosphorylation was elevated while MyBPC phosphorylation remained unchanged, suggesting a regulation of cardiomyocyte contraction/relaxation by NAD‐dependent mechanisms. To normalize NAD redox imbalance, we elevated NAD levels in diabetic cKO mice with cardiac‐specific NAMPT overexpression. Cardiac NAMPT expression slowed the accelerated decline of systolic and diastolic functions in diabetic cKO hearts. Our results support that altered NAD metabolism is a critical determinant for the progression of DC and warrants further investigations.Support or Funding Information1. Scientist Development Grant, AHA2. Seed Funding, Presbyterian Health Foundation of Oklahoma City 1.

Pressure Overload Is Associated With Low Levels of Troponin I and Myosin Binding Protein C Phosphorylation in the Hearts of Patients With Aortic Stenosis.

In previous studies of septal heart muscle from HCM patients with hypertrophic obstructive cardiomyopathy (HOCM, LVOT gradient 50–120 mmHg) we found that the level of phosphorylation of troponin I (TnI) and myosin binding protein C (MyBP-C) was extremely low yet samples from hearts with HCM or DCM mutations that did not have pressure overload were similar to donor heart controls. We therefore investigated heart muscle samples taken from patients undergoing valve replacement for aortic stenosis, since they have pressure overload that is unrelated to inherited cardiomyopathy. Thirteen muscle samples from septum and from free wall were analyzed (LVOT gradients 30–100 mmHg) The levels of TnI and MyBP-C phosphorylation were determined in muscle myofibrils by separating phosphospecies using phosphate affinity SDS-PAGE and detecting with TnI and MyBP-C specific antibodies. TnI was predominantly monophosphorylated and total phosphorylation was 0.85 ± 0.03 molsPi/mol TnI. This phosphorylation level was significantly different (p < 0.0001) from both donor heart TnI (1.6 ± 0.06 molsPi/mol TnI) and HOCM heart TnI (0.19 ± 0.04 molsPi/mol TnI). MyBP-C is phosphorylated at up to four sites. In donor heart the 4P and 3P species predominate but in the pressure overload samples the 4P species was much reduced and 3P and 1P species predominated. Total phosphorylation was 2.0 ± 0.2 molsPi/mol MyBP-C (n = 8) compared with 3.4 ± 0.07 (n = 21) in donor heart and 1.1 ± 0.1 (n = 10) in HOCM heart. We conclude that pressure overload may be associated with substantial dephosphorylation of troponin I and MyBP-C.

Cardiac troponin-I phosphorylation underlies myocardial contractile dysfunction induced by hypothermia rewarming.

Rewarming the intact heart after a period of hypothermia is associated with reduced myocardial contractility, decreased Ca2+ sensitivity, and increased cardiac troponin-I (cTnI) phosphorylation. We hypothesized that hypothermia/rewarming (H/R) induces left ventricular (LV) contractile dysfunction due to phosphorylation of cTnI at Ser23/24. To test this hypothesis, the response of wild-type mice (n = 7) to H/R was compared with transgenic (TG) mice expressing slow skeletal TnI (TG-ssTnI; n = 7) that lacks the Ser23/24 phosphorylation sites. Hypothermia was induced by surface cooling and maintained at 23-25°C for 3 h. Subsequently, the animals were rewarmed to 37°C. LV systolic and diastolic function was assessed using a 1.4 F pressure-volume Millar catheter introduced via the right carotid artery. At baseline conditions, there were no significant differences in LV systolic function between wild-type and TG-ssTnI mice, whereas measurements of diastolic function [isovolumic relaxation constant (τ) and end-diastolic pressure-volume relationship (EDPVR)] were significantly (P < 0.05) reduced in TG-ssTnI animals. Immediately after rewarming, significant differences between groups were found in cardiac output (CO; wild-type 6.6 ± 0.7 vs. TG-ssTnI 8.8 ± 0.7 mL/min), stroke work (SW; wild-type 796 ± 112 vs. TG-ssTnI 1208 ± 67 mmHg/μL), and the preload recruited stroke work (PRSW; wild-type 38.3 ± 4.9 vs. TG-ssTnI 68.8 ± 8.2 mmHg). However, EDPVR and τ returned to control levels within 1 h in both groups. We conclude that H/R-induced LV systolic dysfunction results from phosphorylation of cTnI at Ser23/24.NEW & NOTEWORTHY Rewarming following a period of accidental hypothermia leads to a form of acute cardiac failure (rewarming shock), which is in part due to reduced sensitivity to Ca2+ activation of myocardial contraction. The results of the present study support the hypothesis that rewarming shock is due to phosphorylation of cardiac troponin I.

Abstract 900: Troponin I Tyrosine 26 Phosphorylation Accelerates i n vivo Myocardial Relaxation

Heart failure results in depressed cardiac systolic contraction and diastolic relaxation, both of which limit the heart’s ability to pump blood. Currently there is no therapy to accelerate the detrimental slowed relaxation in heart failure. A fundamental mechanism of the heart to modulate cardiac relaxation is through serine/threonine kinase-mediated phosphorylation of the contractile regulatory protein troponin I (TnI). Non-receptor tyrosine-specific kinases are expressed in the heart and their activity is altered in disease; yet, the role of tyrosine kinase-mediated phosphorylation to directly modulate cardiac relaxation has not been described. TnI is phosphorylated at tyrosine 26 (Tyr26) in the heart and we previously demonstrated TnI Tyr26 phosphorylation accelerates myofilament deactivation. TnI Tyr26 phosphorylation is therefore the first tyrosine phosphorylation identified to directly modulate cardiac myofilament function, however the effects of tyrosine phosphorylation on in vivo function of the heart are unknown. To determine the effect of tyrosine phosphorylation on heart function, we generated a TnI Tyr26 phosphorylation (Tg Y26E) mouse. At the muscle level, myofibrils from Tg Y26E mice exhibit accelerated myofibril relaxation. Echocardiography and pressure-volume hemodynamic measurements demonstrate Tg Y26E mice have enhanced diastolic function evident in accelerated myocardial relaxation, without depressed systolic function or altered morphology. As a whole these data support the phosphorylation of TnI at Tyr26 as a novel tyrosine signaling mechanism to accelerate in vivo diastolic function without depressing systolic contraction.

Cardiac troponins may be irreversibly modified by glycation: novel potential mechanisms of cardiac performance modulation

Dynamic movements of the cardiac troponin complex are an important component of the cardiac cycle. Whether cardiac troponins are subjected to irreversible advanced glycation end-product (AGE) modification is unknown. This study interrogated human and rat cardiac troponin-C, troponin-I and troponin-T to identify endogenous AGE modifications using mass spectrometry (LC-MS/MS). AGE modifications were detected on two amino acid residues of human troponin-C (Lys6, Lys39), thirteen troponin-I residues (Lys36, Lys50, Lys58, Arg79, Lys117, Lys120, Lys131, Arg148, Arg162, Lys164, Lys183, Lys193, Arg204), and three troponin-T residues (Lys107, Lys125, Lys227). AGE modifications of three corresponding troponin-I residues (Lys58, Lys120, Lys194) and two corresponding troponin-T residues (Lys107, Lys227) were confirmed in cardiac tissue extracts from an experimental rodent diabetic model. Additionally, novel human troponin-I phosphorylation sites were detected (Thr119, Thr123). Accelerated AGE modification of troponin-C was evident in vitro with hexose sugar exposure. This study provides the first demonstration of the occurrence of cardiac troponin complex AGE-modifications. These irreversible AGE modifications are situated in regions of the troponin complex known to be important in myofilament relaxation, and may be of particular pathological importance in the pro-glycation environment of diabetic cardiomyopathy.

Molecular Defects in Cardiac Myofilament Ca2+-Regulation Due to Cardiomyopathy-Linked Mutations Can Be Reversed by Small Molecules Binding to Troponin.

The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca2+ regulation via troponin. HCM is usually linked to higher myofilament Ca2+-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca2+-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested). We have labeled this property “re-coupling.” The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca2+-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19) compared to 2.0 ± 0.24-fold (n = 7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitizers, indicating these properties are independent; moreover, re-coupling ability could be lost with small changes of compound structure, indicating the possibility of specificity. Small molecules that can re-couple may have therapeutic potential.HIGHLIGHTS - Inherited cardiomyopathies are common diseases that are currently untreatable at a fundamental level and therefore finding a small molecule treatment is highly desirable.- We have identified a molecular level dysfunction common to nearly all mutations: uncoupling of the relationship between troponin I phosphorylation and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline.- We have identified a new class of drugs that are capable of both reducing Ca2+-sensitivity and/or recouping the relationship between troponin I phosphorylation and Ca2+-sensitivity.- The re-coupling phenomenon can be explained on the basis of a single mechanism that is testable.- Measurements with a wide range of small molecules of varying structures can indicate the critical molecular features required for recoupling and allows the prediction of other potential re-couplers.

Abstract 223: Troponin I Tyrosine Phosphorylation Modulation of Cardiac Function

Heart failure results in depressed contraction and slowed relaxation, both of which limit heart function and contribute to the progression of heart disease. Currently there is no chronic therapy to accelerate relaxation and reverse the diastolic dysfunction present in heart failure. Myocardial relaxation is regulated by serine/threonine phosphorylation of key regulatory proteins. Tyrosine (Tyr) specific kinases are expressed in the heart but the Tyr phosphorylation of regulatory proteins to modulate heart function has not been demonstrated. To investigate the effects of Tyr kinase phosphorylation on cardiac contraction we employed a novel cell penetrating peptide to deliver a direct Tyr kinase activator into isolated adult myocytes. Results demonstrate Tyr kinases activation increases Tyr phosphorylation of the regulatory protein troponin I (TnI) at Tyr26. We have demonstrated that TnI Tyr26 phosphorylation is beneficial to cardiac health by decreasing calcium sensitivity and accelerating myofilament deactivation (key determinants in accelerating myocardial relaxation) and that TnI Tyr26 phosphorylation undergoes functional integration with TnI Ser23/24 resulting in further accelerated calcium dissociation (accelerated relaxation) without further decreased calcium sensitivity (no further depression of contraction). We now demonstrate TnI Tyr26 also undergoes novel signaling integration with TnI Ser23/24 phosphorylation increasing the rate of Tyr kinase mediated Tyr26 phosphorylation. For the first time we demonstrate tyrosine kinase phosphorylation of TnI at Tyr26 modulates cardiac function resulting in accelerated relaxation. Increasing TnI Tyr26 phosphorylation may therefore serve as a novel targeted mechanism for future therapeutic development to accelerate depressed myocardial relaxation and improve diastolic dysfunction in heart failure.

Simvastatin Promotes Cardiac Myocyte Relaxation in Association with Phosphorylation of Troponin I

The number of people taking statins is set to increase across the globe due to recent changes in prescription guidelines. For example, half the US population over 40 is now eligible for these drugs, whether they have high serum cholesterol or not. With such development in policy comes a stronger need for understanding statins’ myriad of effects. Surprisingly little is known about possible direct actions of statins on cardiac myocytes, although claims of a direct myocardial toxicity have been made. Here, we determine the impact of simvastatin administration (40 mg/kg/day) for 2 weeks in normocholesterolemic rats on cardiac myocyte contractile function and identify an underlying mechanism. Under basal conditions, statin treatment increased the time to half (t0.5) relaxation without any effect on the magnitude of shortening, or the magnitude/kinetics of the [Ca2+]i transient. Enhanced myocyte lusitropy could be explained by a corresponding increase in phosphorylation of troponin I (TnI) at Ser23,24. Statin treatment increased expression of eNOS and Ser1177 phosphorylated eNOS, decreased expression of the NOS-inhibitory proteins caveolins 1 and 3, and increased (P = 0.06) NO metabolites, consistent with enhanced NO production. It is well-established that NO stimulates protein kinase G, one of the effectors of TnI phosphorylation at Ser23,24. Trends for parallel changes in phospho-TnI, phospho-eNOS and caveolin 1 expression were seen in atrial muscle from patients taking statins. Our data are consistent with a mechanism whereby chronic statin treatment enhances TnI phosphorylation and myocyte lusitropy through increased NO bioavailability. We see no evidence of impaired function with statin treatment; the changes we document at the level of the cardiac myocyte should facilitate diastolic filling and cardiac performance.