Myofilament Protein Phosphorylation Research Articles

Arrhythmogenic atrial remodeling during pregnancy in mice

BackgroundPregnancy is associated with greater vulnerability to supraventricular tachyarrhythmias. ObjectiveAs the underlying mechanisms remain to be elucidated, we investigated whether pregnancy induces atrial remodeling that might contribute to this. MethodsAtrial electrophysiological and contractile properties were examined in nonpregnant and pregnant (P) mice. Cell shortening and Ca2+ imaging were measured on atrial myocytes. Atrial action potential and ionic currents were recorded using the patch-clamp technique. Atrial messenger RNA and protein expression were analyzed using quantitative polymerase chain reaction and Western blot. ResultsThe P-wave area on the electrocardiogram increased by 50% during pregnancy, suggesting atrial enlargement, confirmed by echocardiography. The atrial myocytes were longer in P mice, adding further evidence to the physiological hypertrophy associated with pregnancy. Echocardiography showed a 50% increase in atrial fractional area change during pregnancy, indicating much stronger contraction. A similar increase in cell shortening was observed in P mice and was associated with a decrease in sarcomere length and changes in myofilament protein phosphorylation. However, pregnancy did not affect L-type Ca2+ current, Ca2+ transients, and SR Ca2+ load. Myocytes from P mice showed twice as many spontaneous contractions and spontaneous diastolic Ca2+ releases. Moreover, pregnancy was associated with a 50% increase in action potential duration, linked to a reduction in the density of the Ca2+-independent transient outward K+ current and the underlying KV4.3 channel. ConclusionDuring pregnancy, atrial tissues undergo substantial remodeling, potentially contributing to the development of supraventricular tachyarrhythmias.

The selective serotonin reuptake inhibitor paroxetine improves right ventricular systolic function in experimental pulmonary hypertension

BackgroundPulmonary hypertension (PH) often leads to right ventricle (RV) failure, a significant cause of morbidity and mortality. Despite advancements in PH management, progression to RV maladaptation and subsequent failure remain a clinical challenge. This study explored the effect of paroxetine, a selective serotonin reuptake inhibitor (SSRI), on RV function in a rat model of PH, hypothesizing that it improves RV function by inhibiting G protein-coupled receptor kinase 2 (GRK2) and altering myofilament protein phosphorylation. MethodsThe Su5416/hypoxia (SuHx) rat model was used to induce PH. Rats were treated with paroxetine and compared to vehicle-treated and control groups. Parameters measured included RV morphology, systolic and diastolic function, myofilament protein phosphorylation, GRK2 activity, and sympathetic nervous system (SNS) markers. ResultsParoxetine treatment significantly improved RV systolic function, evidenced by increased stroke volume, cardiac output, and ejection fraction, without significantly affecting RV hypertrophy, myosin heavy chain/titin isoform switching, or fibrosis. Enhanced phosphorylation of titin and myosin light chain-2 was observed, correlating positively with improved systolic function. Contrary to the hypothesis, improvements occurred independently of GRK2 inhibition or SNS modulation, suggesting an alternate mechanism, potentially involving antioxidant properties of paroxetine. ConclusionParoxetine improves RV systolic function in PH rats, likely through mechanisms beyond GRK2 inhibition, possibly related to its antioxidant effects. This highlights the potential of paroxetine in managing RV dysfunction in PH, warranting further investigation into its detailed mechanisms of action and clinical applicability.

Hemodynamic Assessment in Takotsubo Syndrome

BackgroundTakotsubo syndrome (TTS) is a reversible form of heart failure with incompletely understood pathophysiology. ObjectivesThis study analyzed altered cardiac hemodynamics during TTS to elucidate underlying disease mechanisms. MethodsLeft ventricular (LV) pressure–volume loops were recorded in 24 consecutive patients with TTS and a control population of 20 participants without cardiovascular diseases. ResultsTTS was associated with impaired LV contractility (end-systolic elastance 1.74 mm Hg/mL vs 2.35 mm Hg/mL [P = 0.024]; maximal rate of change in systolic pressure over time 1,533 mm Hg/s vs 1,763 mm Hg/s [P = 0.031]; end-systolic volume at a pressure of 150 mm Hg, 77.3 mL vs 46.4 mL [P = 0.002]); and a shortened systolic period (286 ms vs 343 ms [P < 0.001]). In response, the pressure–volume diagram was shifted rightward with significantly increased LV end-diastolic (P = 0.031) and end-systolic (P < 0.001) volumes, which preserved LV stroke volume (P = 0.370) despite a lower LV ejection fraction (P < 0.001). Diastolic function was characterized by prolonged active relaxation (relaxation constant 69.5 ms vs 45.9 ms [P < 0.001]; minimal rate of change in diastolic pressure –1,457 mm Hg/s vs –2,192 mm Hg/s [P < 0.001]), whereas diastolic stiffness (1/compliance) was not affected during TTS (end-diastolic volume at a pressure of 15 mm Hg, 96.7 mL vs 109.0 mL [P = 0.942]). Mechanical efficiency was significantly reduced in TTS (P < 0.001) considering reduced stroke work (P = 0.001), increased potential energy (P = 0.036), and a similar total pressure–volume area compared with that of control subjects (P = 0.357). ConclusionsTTS is characterized by reduced cardiac contractility, a shortened systolic period, inefficient energetics, and prolonged active relaxation but unaltered diastolic passive stiffness. These findings may suggest decreased phosphorylation of myofilament proteins, which represents a potential therapeutic target in TTS. (Optimized Characterization of Takotsubo Syndrome by Obtaining Pressure Volume Loops [OCTOPUS]; NCT03726528)

Abstract GS108: Troponin I Tyrosine-26 Phosphorylation Improves Relaxation And Is Beneficial During Pathological Diastolic Dysfunction

Diastolic dysfunction contributes to disease in both heart failure with preserved and reduced ejection fraction. There are currently no approved therapies to accelerate impaired relaxation for heart failure patients with diastolic dysfunction. A healthy heart can modify its ability to relax through post-translational modifications (e.g., phosphorylation) of myofilament proteins. The inhibitory subunit of the troponin complex, troponin I (TnI), is a key regulator of cardiac contraction and relaxation and is endogenously phosphorylated at Tyr-26. We previously demonstrated that this novel tyrosine phosphorylation decreases calcium sensitivity and accelerates calcium dissociation from troponin C. We therefore hypothesize that increasing TnI Tyr-26 phosphorylation will accelerate relaxation and be beneficial during pathological diastolic dysfunction. To determine the effects of TnI Tyr-26 phosphorylation in vivo , we generated a phospho-mimetic mouse with TnI Tyr-26 mutated to Glu. Structural and functional echocardiography and hemodynamics measurements demonstrate that TnI Tyr-26 phosphorylation accelerates relaxation and increases diastolic function in vivo . To determine the effect of this TnI Tyr-26 phosphorylation mediated acceleration of diastolic function during disease, we performed unilateral nephrectomy with DOCA-salt (neph/DOCA) to induce diastolic dysfunction. Wild-type mice subjected to neph/DOCA develop left ventricular hypertrophy, enlarged left atria, elevated diastolic pressure, and slowed relaxation, indicative of diastolic dysfunction. In contrast with wild-type, TnI Tyr-26 phosphorylation mice subjected to neph/DOCA display normal diastolic function without any cardiac remodeling. Overall, we demonstrate that TnI Tyr-26 phosphorylation inhibits the development of diastolic dysfunction and is therefore a novel mechanism to accelerate myocardial relaxation in vivo.

Ca2+/calmodulin‐dependent protein kinase II and protein kinase G oxidation contributes to impaired sarcomeric proteins in hypertrophy model

AimsVolume overload (VO) induced hypertrophy is one of the hallmarks to the development of heart diseases. Understanding the compensatory mechanisms involved in this process might help preventing the disease progression.Methods and resultsTherefore, the present study used 2 months old Wistar rats, which underwent an aortocaval fistula to develop VO‐induced hypertrophy. The animals were subdivided into four different groups, two sham operated animals served as age‐matched controls and two groups with aortocaval fistula. Echocardiography was performed prior termination after 4‐ and 8‐month. Functional and molecular changes of several sarcomeric proteins and their signalling pathways involved in the regulation and modulation of cardiomyocyte function were investigated.ResultsThe model was characterized with preserved ejection fraction in all groups and with elevated heart/body weight ratio, left/right ventricular and atrial weight at 4‐ and 8‐month, which indicates VO‐induced hypertrophy. In addition, 8‐months groups showed increased left ventricular internal diameter during diastole, RV internal diameter, stroke volume and velocity‐time index compared with their age‐matched controls. These changes were accompanied by increased Ca2+ sensitivity and titin‐based cardiomyocyte stiffness in 8‐month VO rats compared with other groups. The altered cardiomyocyte mechanics was associated with phosphorylation deficit of sarcomeric proteins cardiac troponin I, myosin binding protein C and titin, also accompanied with impaired signalling pathways involved in phosphorylation of these sarcomeric proteins in 8‐month VO rats compared with age‐matched control group. Impaired protein phosphorylation status and dysregulated signalling pathways were associated with significant alterations in the oxidative status of both kinases CaMKII and PKG explaining by this the elevated Ca2+ sensitivity and titin‐based cardiomyocyte stiffness and perhaps the development of hypertrophy.ConclusionsOur findings showed VO‐induced cardiomyocyte dysfunction via deranged phosphorylation of myofilament proteins and signalling pathways due to increased oxidative state of CaMKII and PKG and this might contribute to the development of hypertrophy.

Age‐ and sex‐specific differences in activation of the cardiac adrenergic cascade

Heart failure (HF) is a costly and devastating public health problem, which differentially impacts male and female patients of all ages across the life‐course. The adrenergic system is dysregulated with HF and is the target of front‐line therapeutics. However, the majority of the work aimed at elucidating the adrenergic signaling response has been conducted in adult males. Consequently, mechanisms of adrenergic signaling that differ by sex and age remain unclear. Here, we set out to identify mechanistic differences in the adrenergic pathway in response to isoproterenol (ISO) stimulation in juvenile (4 weeks), adult (4‐6 months) and aged (18 months) C57Bl6 mice of both sexes. ISO was delivered by mini‐osmotic pump (30 mg/kg/day) for one week. We assessed changes in β‐adrenergic receptor (β‐AR) expression, protein kinase activity, and myofilament protein phosphorylation and function. β1‐AR and β2‐AR protein expression were significantly lower in response to ISO in juvenile males with no difference in expression in juvenile females. In contrast, ISO stimulation in aged males significantly upregulated β1‐AR and β2‐AR protein expression without changes in expression in aged females. Analysis of sarcomeric proteins indicated that phosphorylation of troponin varied in an age‐ and sex‐dependent manner. Phosphorylation of the inhibitory subunit of troponin (TnI) at Ser23/24 was upregulated in response to ISO in females of all ages with no differences in males. These changes in phosphorylation decreased myofilament Ca2+ sensitivity in female hearts in response to ISO. Together, this work suggests that male hearts responded robustly to ISO stimulation as evident by activation throughout the adrenergic cascade while female hearts demonstrated activation of downstream mechanisms at the level of the sarcomere. Ongoing experiments will characterize calcium handling and myofilament mechanics to mechanistically link upstream signals to downstream changes in contractility. These data demonstrate that age and sex are significant biological factors which contribute to adrenergic‐mediated changes in cardiac performance. Understanding these differences is critical for successful therapeutic intervention within the adrenergic signaling cascade for male and female patients of all ages.

Treadmill running increases the calcium sensitivity of myofilaments in diabetic rats.

The cardiovascular benefits of regular exercise are unequivocal, yet patients with type 2 diabetes respond poorly to exercise due to a reduced cardiac reserve. The contractile response of diabetic cardiomyocytes to β-adrenergic stimulation is attenuated, which may result in altered myofilament calcium sensitivity and posttranslational modifications of cardiac troponin I (cTnI). Treadmill running increases myofilament calcium sensitivity in nondiabetic rats, and thus we hypothesized that endurance training would increase calcium sensitivity of diabetic cardiomyocytes and alter site-specific phosphorylation of cTnI. Calcium sensitivity, or pCa50, was measured in Zucker diabetic fatty (ZDF), nondiabetic (nDM), and diabetic (DM) rat hearts after 8 wk of either a sedentary (SED) or progressive treadmill running (TR) intervention. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+]). Specific phospho-sites on cTnI and O-GlcNAcylation were quantified by immunoblot and total protein phosphorylation by fluorescent gel staining (ProQ Diamond). The novel finding in this study was that training increased pCa50 in both DM and nDM cardiomyocytes (P = 0.009). Phosphorylation of cTnI amino acid residues Ser23/24, a crucial protein kinase A site, and Threonine (Thr)144 was lower in DM hearts, but there was no effect of training on site-specific phosphorylation. In addition, total phosphorylation and O-GlcNAcylation levels were not different between SED and TR groups. These findings suggest that regular exercise may benefit the diabetic heart by specifically targeting myofilament contractile function.NEW & NOTEWORTHY We examined the effects of training on the myofilament calcium in diabetic rat hearts. After 8 wk of treadmill running, both nondiabetic and diabetic cardiomyocytes had increased myofilament calcium sensitivity compared with their sedentary counterparts, but there was no effect of training on the phosphorylation or O-GlcNAcylation status of myofilament proteins measured in this study. These data highlight one potential mechanism capable of reversing, in part, reduced cardiac reserve in the diabetic heart.

Abstract 11592: Glycogen Synthase Kinase 3β Localizes to the Z-Disc to Maintain Length Dependent Activation

Glycogen Synthase Kinase 3β (GSK-3β) can modulate myofilament function in vitro . However, it’s in vivo role, mechanism, and translational relevance are unknown, which we investigated here using inducible cardiomyocyte specific GSK-3β KO mice. Compared to tamoxifen-treated GSK-3β fl/fl Cre- mice (Con), skinned myocytes from KO mice had reduced calcium sensitivity at long sarcomere lengths (SL = 2.2 μm), but there were no differences at short SL (~1.9 μm). Thus, myocytes from KO mice did not sensitize to calcium with stretch, a mechanism called length dependent activation (LDA) that underlies the organ-level Frank-Starling law. LDA has been attributed to (1) phosphorylation of myofilament proteins, (2) altered lattice-spacing, and (3) changes to titin’s elastic properties. Using mass spectrometry and small energy x-ray diffraction we ruled out the first two mechanisms, however we did find that GSK-3β KO myocytes had decreased passive tension – indicating the loss of LDA was due to loss of titin as a length sensor. Interestingly, immunofluorescence showed that GSK-3β localized to the z-disc when phosphorylated at Y216 and, via mass spectrometry, phosphorylated primarily z-disc proteins including several sites on the structural protein Ablim-1, which we showed also localizes to the z-disc. These data suggested that GSK-3β’s effect on titin is likely through altering its ability to anchor to the z-disc through targeting of these z-disc proteins. To provide further evidence that GSK-3β is specifically altering LDA, we genetically removed a downstream effecter of LDA, cardiac myosin binding protein-C (cMyBP-C). In vitro treatment with exogenous GSK-3β was able to increase calcium sensitivity at long SLs in both KO and WT mice but had no effect on cMyBPC KO mice or mice lacking the c-terminal domains of cMyBPC that are known to be important for LDA. Lastly, we found that human heart failure patients had less myofilament GSK-3β compared to non-failing patients, and that these same samples had a depressed LDA. This work has identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA and indicates that z-disc localized GSK-3β may be a possible therapeutic target to restore the Frank-Starling mechanism in heart failure patients.

Abstract 11855: Disruption of β-arrestin-2 Signaling Resulted in Impaired Systolic and Diastolic Function During Aging

Introduction: Angiotensin II (ang II) signaling at ang II receptor type 1 (AT1R), which is a G-protein coupled receptor (GPCR), promotes both G-protein signaling as well as β-arrestin-2 signaling. β-arrestin-2 participates in GPCR desensitization, internalization, but also acts a scaffold for adaptive signal transduction that may occur independently or in parallel to G-protein signaling. Hypothesis: We tested the hypothesis that β-arrestin-2 is an important element in the preservation of cardiac function during aging. Methods: We tested this hypothesis by aging β-arrestin-2 knock-out (KO) mice, and wild-type equivalent (WT) to 12-16 months. We performed echocardiography studies, and Western blot analysis. Results: By echocardiography we found that compared to controls aged KO mice exhibited enlarged left atria (2.2±0.1 vs 2.5±0.1 mm) and left ventricular diameters (4.2±0.2 vs 4.5 ±0.1mm) as well as depressed contractility parameters such as s’ (23.2±1.4 vs 21.4±0.8 mm/s) and decreased circumferential fiber shortening velocity (5.5±0.4 vs 4.5±0.2) with preserved ejection fraction (EF&gt;50%). Aged KO also exhibited depressed relaxation parameters such as e’ (29.2±1.4 vs 21.4±0,6 mm/s) and E/e’ (27.4±1.6 vs 35.6±2.0) when compared to WT controls at the same age. Cardiac dysfunction in aged KO mice was correlated with decreased phosphorylation of RLC and increased phosphorylation of cMyBP-C at Ser302. Among other kinases, cMyBP-C phosphorylation at Ser302 is a PKCε target which activation is negatively regulated by β-arrestin-2 signaling. To determine the direct effects of PKCε over-activation on cMyBP-C phosphorylation, we used mice that over-expresses constitutively active PKC ε in the heart. We found that similarly to our aged KO mice, PKCε overexpression model exhibited significantly higher levels of cMyBP-C phosphorylation at Ser302, when aged to 12 - 15 months. Conclusions: Deletion of β-arrestin-2 signaling in KO mice affected aged mice, resulting in changes in the phosphorylation of myofilament proteins consisting of decreased phosphorylation of RLC and increased phosphorylation of cMyBP-C at Ser302 through the unrestricted activity of PKCε. These changes negatively affected systolic and diastolic function in aged KO mice.

Alterations in ACE and ACE2 Activities and Cardiomyocyte Signaling Underlie Improved Myocardial Function in a Rat Model of Repeated Remote Ischemic Conditioning.

Post-ischemic left ventricular (LV) remodeling and its hypothetical prevention by repeated remote ischemic conditioning (rRIC) in male Sprague–Dawley rats were studied. Myocardial infarction (MI) was evoked by permanent ligation of the left anterior descending coronary artery (LAD), and myocardial characteristics were tested in the infarcted anterior and non-infarcted inferior LV regions four and/or six weeks later. rRIC was induced by three cycles of five-minute-long unilateral hind limb ischemia and five minutes of reperfusion on a daily basis for a period of two weeks starting four weeks after LAD occlusion. Sham operated animals served as controls. Echocardiographic examinations and invasive hemodynamic measurements revealed distinct changes in LV systolic function between four and six weeks after MI induction in the absence of rRIC (i.e., LV ejection fraction (LVEF) decreased from 52.8 ± 2.1% to 50 ± 1.6%, mean ± SEM, p < 0.05) and in the presence of rRIC (i.e., LVEF increased from 48.2 ± 4.8% to 55.2 ± 4.1%, p < 0.05). Angiotensin-converting enzyme (ACE) activity was about five times higher in the anterior LV wall at six weeks than that in sham animals. Angiotensin-converting enzyme 2 (ACE2) activity roughly doubled in post-ischemic LVs. These increases in ACE and ACE2 activities were effectively mitigated by rRIC. Ca2+-sensitivities of force production (pCa50) of LV permeabilized cardiomyocytes were increased at six weeks after MI induction together with hypophosphorylation of 1) cardiac troponin I (cTnI) in both LV regions, and 2) cardiac myosin-binding protein C (cMyBP-C) in the anterior wall. rRIC normalized pCa50, cTnI and cMyBP-C phosphorylations. Taken together, post-ischemic LV remodeling involves region-specific alterations in ACE and ACE2 activities together with changes in cardiomyocyte myofilament protein phosphorylation and function. rRIC has the potential to prevent these alterations and to improve LV performance following MI.

B-arrestin-2 Signaling Is Important to Preserve Cardiac Function During Aging.

Experiments reported here tested the hypothesis that β-arrestin-2 is an important element in the preservation of cardiac function during aging. We tested this hypothesis by aging β-arrestin-2 knock-out (KO) mice, and wild-type equivalent (WT) to 12–16months. We developed the rationale for these experiments on the basis that angiotensin II (ang II) signaling at ang II receptor type 1 (AT1R), which is a G-protein coupled receptor (GPCR) promotes both G-protein signaling as well as β-arrestin-2 signaling. β-arrestin-2 participates in GPCR desensitization, internalization, but also acts as a scaffold for adaptive signal transduction that may occur independently or in parallel to G-protein signaling. We have previously reported that biased ligands acting at the AT1R promote β-arrestin-2 signaling increasing cardiac contractility and reducing maladaptations in a mouse model of dilated cardiomyopathy. Although there is evidence that ang II induces maladaptive senescence in the cardiovascular system, a role for β-arrestin-2 signaling has not been studied in aging. By echocardiography, we found that compared to controls aged KO mice exhibited enlarged left atria and left ventricular diameters as well as depressed contractility parameters with preserved ejection fraction. Aged KO also exhibited depressed relaxation parameters when compared to WT controls at the same age. Moreover, cardiac dysfunction in aged KO mice was correlated with alterations in the phosphorylation of myofilament proteins, such as cardiac myosin binding protein-C, and myosin regulatory light chain. Our evidence provides novel insights into a role for β-arrestin-2 as an important signaling mechanism that preserves cardiac function during aging.

Aged Monkeys Fed a High-Fat/High-Sugar Diet Recapitulate Metabolic Disorders and Cardiac Contractile Dysfunction.

Aged nonhuman primate (NHP) models are of great value for studying the pathology of metabolic heart diseases and developing therapeutic strategies. In this study, aged male cynomolgus monkeys were fed a regular diet or a high-fat/high-sugar diet (HFSD) for 8 months. Metabolic disorders were diagnosed by 1H-NMR and serum biochemistry, and cardiac function was evaluated by echocardiography. Our results showed that serum metabolic profiles were altered in aged monkeys fed a HFSD, in line with aortic tissue damage, cardiac remodeling, and contractile dysfunction. This aged monkey model significantly increased expression of proinflammatory cytokines and altered expression and phosphorylation of intracellular signaling proteins in the heart, as compared to aged monkeys on a regular diet. Furthermore, the animals demonstrated increased phosphorylation of cardiac myofilament proteins which are causatively associated with decreased myofilament contractility. We conclude that the aged monkey model fed a HFSD exhibits metabolic disorders and cardiac contractile dysfunction.

Suppression of myofilament cross-bridge kinetic in the heart of orchidectomized rats

AimsThe increased incidence of heart failure with reduced ejection fraction in men compared with women suggests that male sex hormones significantly impact myocardial contractile activation. This study aims to examine associations among molecular alterations, cellular modulations and in vivo cardiac contractile function upon deprivation of testicular hormones. Main methodsMyocardial structure and functions were compared among sham-operated control and twelve-week orchidectomized (ORX) male rats with and without testosterone supplementation. Key findingsEchocardiography and pressure-volume relationships demonstrated a decreased left ventricular ejection fraction compared with sham-operated controls. The percentage of contractility reduction was generally similar to the decrease in tension development detected in both right ventricular trabeculae and skinned isolated left ventricular cardiomyocytes of ORX rats. Reductions in tension cost and the rate constant of tension redevelopment (ktr) in ORX samples suggested a decrease in the rate of cross-bridge formation, reflecting a reduced number of cross-bridges. Slow cross-bridge detachment in ORX rat hearts could result from a shift of myosin heavy chain isoforms towards a slower ATPase activity β-isoform and reductions in the phosphorylation levels of cardiac troponin I and myosin binding protein-C. All the changes in the ORX rat heart, including ejection fractions and myofilament protein expression and phosphorylation, were completed attenuated by a physiological dose of testosterone. SignificanceTestosterone plays a critical role in regulating the mechanical and contractile dynamics of the heart. Deprivation of male sex hormones cause the loss of normal preserved cardiac contractile function leading to a high risk of severe cardiomyopathy progression.

Empagliflozin improves endothelial and cardiomyocyte functionin human heart failure with preserved ejection fraction via reduced pro-inflammatory-oxidative pathways and protein kinase Gα oxidation.

Sodium-glucose-cotransporter-2 inhibitors showed favourable cardiovascular outcomes, but the underlying mechanisms are still elusive. This study investigated the mechanisms of empagliflozin in human and murine heart failure with preserved ejection fraction (HFpEF). The acute mechanisms of empagliflozin were investigated in human myocardium from patients with HFpEF and murine ZDF obese rats, which were treated in vivo. As shown with immunoblots and ELISA, empagliflozin significantly suppressed increased levels of ICAM-1, VCAM-1, TNF-α, and IL-6 in human and murine HFpEF myocardium and attenuated pathological oxidative parameters (H2O2, 3-nitrotyrosine, GSH, lipid peroxide) in both cardiomyocyte cytosol and mitochondria in addition to improved endothelial vasorelaxation. In HFpEF, we found higher oxidative stress-dependent activation of eNOS leading to PKGIα oxidation. Interestingly, immunofluorescence imaging and electron microscopy revealed that oxidized PKG1α in HFpEF appeared as dimers/polymers localized to the outer-membrane of the cardiomyocyte. Empagliflozin reduced oxidative stress/eNOS-dependent PKGIα oxidation and polymerization resulting in a higher fraction of PKGIα monomers, which translocated back to the cytosol. Consequently, diminished NO levels, sGC activity, cGMP concentration, and PKGIα activity in HFpEF increased upon empagliflozin leading to improved phosphorylation of myofilament proteins. In skinned HFpEF cardiomyocytes, empagliflozin improved cardiomyocyte stiffness in an anti-oxidative/PKGIα-dependent manner. Monovariate linear regression analysis confirmed the correlation of oxidative stress and PKGIα polymerization with increased cardiomyocyte stiffness and diastolic dysfunction of the HFpEF patients. Empagliflozin reduces inflammatory and oxidative stress in HFpEF and thereby improves the NO-sGC-cGMP-cascade and PKGIα activity via reduced PKGIα oxidation and polymerization leading to less pathological cardiomyocyte stiffness.

Cardiac function and feed efficiency: Increased right-heart workload in feed inefficient beef cattle

Heart rate (HR) during rest and stress as a beef cattle feed efficiency proxy (residual feed intake; RFI) has potential to reduce environmental impact and feed costs. A broader understanding of the association between the cardiac system and feed efficiency is needed before commercial application of heart rate as a feed efficiency proxy can occur. Objectives of this study were to assess the relationships between cardiovascular function and structure traits, their relationships with RFI and carcass composition, and the variation in RFI they explained. Residual feed intake, overnight HR, transport HR, abattoir HR, low-frequency power (LF), stroke volume indicator (SVI), cardiac output indicator (COI), blood volume (BV), oxygen-carrying capacity measures, heart structure measures, histomorphometry measures, actomyosin Mg2+ATPase activity measures, myofilament protein phosphorylation measures and carcass composition measures were determined in heifers, bulls and steers (n; 165, age; 390 ± 34 days, weight; 526 ± 127 kg). Correlations amongst traits and with RFI were determined and RFI-dependent multiple regression models containing linear and quadratic effects were selected, where a regression coefficient represented the effect of each trait on RFI. Positive and negative effects of HR and LF within overnight, transport and abattoir recordings on RFI were observed. Mean corpuscular volume trended towards a positive effect on RFI (P = 0.09). Carcass composition measures were only correlated with heart structure measures. Blood volume, SVI, COI and myofilament protein phosphorylation measures were not correlated with RFI. Right ventricle thickness had a positive correlation with RFI (P = 0.005) and myocyte width (P = 0.07) and actomyosin Mg2+ATPase activity measures trended toward positive correlations with RFI (P < 0.10). Positive effects of BV (P = 0.01), right ventricle thickness (P = 0.002), myocyte width (P = 0.05), actomyosin Mg2+ATPase activity at submaximal calcium (P = 0.05) and myosin light chain 2 phosphorylation (P = 0.09) on RFI and negative effects of SVI (P = 0.05) on RFI were observed. Heart structure measures explained the largest amount of RFI variation (Adj. R2 = 0.145; P = 0.002). Results indicate that high-RFI cattle have an increased right heart workload, potentially due to differences in activity elicited by higher brain centers, suggesting selection for low-RFI cattle will not negatively impact cardiovascular structure and function.

Abstract 906: Age-Dependent Modifications in Cardiac Myofilament Proteins are Graded by Overall Health, Measured as Frailty, in Aging Male and Female C57BL/6 Mice

Previously, we showed that the age-dependent decline in ventricular function in male mice is proportional to overall health, as quantified with a frailty index (FI) tool. This tool is based on the accumulation of deficits in health across many systems, but not the cardiovascular system per se . Here we investigated whether frailty grades age-dependent modifications in myofilaments in young and aged mice of both sexes. Myofilaments were isolated from ventricles of adult (12-13 mos; n=5 males, 5 females) and aged (22-23 mos; n=6 males, 5 females) mice. FI scores were obtained from all mice. Actomyosin Mg-ATPase activity as a function of increasing calcium concentration and phosphorylation of major myofilament proteins were quantified and compared between groups. Results showed that myofilament calcium sensitivity (EC 50 values) was similar regardless of age or sex and was not affected by FI score. Maximal actomyosin Mg-ATPase activity increased with age in females, but this was not linked to frailty. Hill coefficients declined with age in males only and were lower than females in the older group (p&lt;0.05). However when all mice were examined, Hill coefficients declined as frailty increased (r=0.68, p=0.001). Total phosphorylation levels of myosin-binding protein C (MyBP-C), desmin, tropomyosin and essential myosin light chain (ELC) increased with age, but only in males. Interestingly, phosphorylation of MyBP-C (r=0.49; p=0.03), desmin (r=0.65; p=0.003), tropomyosin (r=0.62; p=0.004) and ELC (r=0.71; p=0.001) were highly correlated with overall health (FI scores). By contrast there was no link between frailty and phosphorylation of actin, troponin T and troponin I. These findings suggest that poor overall health, quantified in an FI, predicts changes in the myofilaments across the life course in both sexes. This may contribute to changes in cardiac contractile function in frail older adults.

Characterizing the influence of chronic hypobaric hypoxia on diaphragmatic myofilament contractile function and phosphorylation in high-altitude deer mice and low-altitude white-footed mice.

Deer mice, Peromyscusmaniculatus, live at high altitudes where limited O2 represents a challenge to maintaining oxygen delivery to tissues. Previous work has demonstrated that hypoxia acclimation of deer mice and low altitude white-footed mice (P. leucopus) increases the force generating capacity of the diaphragm. The mechanism behind this improved contractile function is not known. Within myocytes, the myofilament plays a critical role in setting the rate and level of force production, and its ability to generate force can change in response to changes in physiological conditions. In the current study, we examined how chronic hypobaric hypoxia exposure of deer mice and white-footed mice influences the Ca2+ activation of force generation by skinned diaphragmatic myofilaments, and the phosphorylation of myofilament proteins. Results demonstrate that myofilament force production, and the Ca2+ sensitivity of force generation, were not impacted by acclimation to hypobaric hypoxia, and did not differ between preparations from the two species. The cooperativity of the force-pCa relationship, and the maximal rate of force generation were also the same in the preparations from both species, and not impacted by acclimation. Finally, the relative phosphorylation of TnT, and MLC was lower in deer mice than white-footed mice, but was not affected by acclimation. These results indicate that species differences in diaphragm function, and the increase in force production with hypoxia acclimation, are not due to differences, or changes, in myofilament function. However, it appears that diaphragmatic myofilament function in these species is not affected by chronic hypobaric hypoxia exposure.

Myofilament Remodeling and Function Is More Impaired in Peripartum Cardiomyopathy Compared with Dilated Cardiomyopathy and Ischemic Heart Disease

Peripartum cardiomyopathy (PPCM) and dilated cardiomyopathy (DCM) show similarities in clinical presentation. However, although DCM patients do not recover and slowly deteriorate further, PPCM patients show either a fast cardiac deterioration or complete recovery. The aim of this study was to assess if underlying cellular changes can explain the clinical similarities and differences in the two diseases. We, therefore, assessed sarcomeric protein expression, modification, titin isoform shift, and contractile behavior of cardiomyocytes in heart tissue of PPCM and DCM patients and compared these with nonfailing controls. Heart samples from ischemic heart disease (ISHD) patients served as heart failure control samples. Passive force was only increased in PPCM samples compared with controls, whereas PPCM, DCM, and ISHD samples all showed increased myofilament Ca2+ sensitivity. Length-dependent activation was significantly impaired in PPCM compared with controls, no impairment was observed in ISHD samples, and DCM samples showed an intermediate response. Contractile impairments were caused by impaired protein kinase A (PKA)-mediated phosphorylation because exogenous PKA restored all parameters to control levels. Although DCM samples showed reexpression of EH-myomesin, an isoform usually only expressed in the heart before birth, PPCM and ISHD did not. The lack of EH-myomesin, combined with low PKA-mediated phosphorylation of myofilament proteins and increased compliant titin isoform, may explain the increase in passive force and blunted length-dependent activation of myofilaments in PPCM samples.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced β2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. In hypertrophic rabbit myocytes, selectively enhanced β2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2 adrenergic receptor signaling and restore myocyte contractility in response to β adrenergic stimulation.

Molecule specific effects of PKA-mediated phosphorylation on rat isolated heart and cardiac myofibrillar function

Increased cardiac myocyte contractility by the β-adrenergic system is an important mechanism to elevate cardiac output to meet hemodynamic demands and this process is depressed in failing hearts. While increased contractility involves augmented myoplasmic calcium transients, the myofilaments also adapt to boost the transduction of the calcium signal. Accordingly, ventricular contractility was found to be tightly correlated with PKA-mediated phosphorylation of two myofibrillar proteins, cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), implicating these two proteins as important transducers of hemodynamics to the cardiac sarcomere. Consistent with this, we have previously found that phosphorylation of myofilament proteins by PKA (a downstream signaling molecule of the beta-adrenergic system) increased force, slowed force development rates, sped loaded shortening, and increased power output in rat skinned cardiac myocyte preparations. Here, we sought to define molecule-specific mechanisms by which PKA-mediated phosphorylation regulates these contractile properties. Regarding cTnI, the incorporation of thin filaments with unphosphorylated cTnI decreased isometric force production and these changes were reversed by PKA-mediated phosphorylation in skinned cardiac myocytes. Further, incorporation of unphosphorylated cTnI sped rates of force development, which suggests less cooperative thin filament activation and reduced recruitment of non-cycling cross-bridges into the pool of cycling cross-bridges, a process that would tend to depress both myocyte force and power. Regarding MyBP-C, PKA treatment of slow-twitch skeletal muscle fibers caused phosphorylation of MyBP-C (but not slow skeletal TnI (ssTnI)) and yielded faster loaded shortening velocity and ∼30% increase in power output. These results add novel insight into the molecular specificity by which the β-adrenergic system regulates myofibrillar contractility and how attenuation of PKA-induced phosphorylation of cMyBP-C and cTnI may contribute to ventricular pump failure.