Phospholamban Phosphorylation Research Articles

Effects of the angiotensin-converting enzyme inhibitor captopril on occlusal-disharmony-induced cardiac dysfunction in mice

Occlusal disharmony is known to affect not only the oral cavity environment, but also the autonomic nervous system in the heart. Since the renin-angiotensin system (RAS) inhibitor captopril (Cap) is one of the first-line drugs for preventing cardiac remodeling in patients with heart failure, we hypothesized that Cap might prevent cardiac dysfunction induced by occlusal disharmony. Here, to test this idea, we used our bite-opening (BO) mouse model, which was developed by cementing a suitable appliance onto the mandibular incisor. Mice were divided into four groups: (1) Control, (2) BO, (3) Cap, and (4) BO + Cap. After 2 weeks, we evaluated cardiac function by echocardiography and confirmed that cardiac function was significantly decreased in the BO group compared to the control, while Cap ameliorated the dysfunction. Cardiac fibrosis, myocyte apoptosis and oxidative stress-induced myocardial damage in the BO group were significantly increased versus the control, and these increases were suppressed by Cap. Cardiac dysfunction induced by BO was associated with dual phosphorylation on PKCδ (Tyr-311/Thr-505), leading to activation of CaMKII with increased phosphorylation of RyR2 and phospholamban. Our results suggest that the RAS might play an important role in the development of cardiac diseases induced by occlusal anomalies.

New therapeutic strategy targeting regulation of the SERCA2 complex protects from myocardial ischemia-reperfusion injury

Abstract 7 million people suffer every year from myocardial infarction. There is significant unmet medical need for cardioprotection during MI treatment. The beta-adrenergic receptor/cyclic AMP/protein kinase A (PKA) signalling pathway regulates heart rate and contractility. Activation of cAMP-dependent PKA causes phospholamban (PLB) phosphorylation and its ability to inhibit sarcoplasmic reticulum calcium ATPase (SERCA) is lost. Discrete control of PLB phosphorylation is facilitated by the A-kinase anchoring protein (AKAP) 18g/d, which holds PKA and PLB in close proximity. We are developing a novel drug candidate, named 13M, with a highly selective mechanism of action in cardioprotection currently in pre-clinical phase. Our drug candidate disrupts the AKAP18g/d-PLB protein-protein interaction (PPI) as this may protect from ischemia reperfusion injury (IRI). We used AlphaScreen assay to screen a library of 79,000 small molecular compounds for compounds that disrupted the AKAP18δ-PLB PPI. Following secondary and tertiary screens of sub-libraries, the resulting hits were further characterized and similarity searches carried out to delineate a compound structure for further derivatization. Newly synthesized compounds were further tested in a cell-line based assay to determine compound toxicity. A combination of biochemistry, electrophysiology and heart function experiments demonstrated that the compounds exclusively block the adrenergic effect on SERCA2 without affecting its basal activity. A 40% reduction in infarction size and improved long term cardiac function was observed in an animal disease model for ischemia-reperfusion injury.

Thrombosis in the coronary microvasculature may be responsible for impaired cardiac relaxation and diastolic dysfunction

Abstract Introduction Heart Failure with Preserved Ejection Fraction (HFpEF) is proposed to be caused by endothelial dysfunction in cardiac small vessels. We previously identified Hhipl1,as a gene upregulated in the coronary vasculature of Leptin receptor deficient mice (Leprdb/db) a well-established mouse model of HFpEF. Importantly, Hhipl1 encodes for a decoy receptor of Desert Hedgehog (DHH) which is known to be critical for endothelial integrity. Objective Our objective is to investigate the functional consequences of impaired Hedgehog (HH) signaling in the adult heart in order to identify novel mechanisms underlying the development of diastolic dysfunction. Method To do so, Cdh5-Cre/ERT2, DhhFlox/Flox (DhhECKO) mice and their control littermates were administered with tamoxifen at 8 weeks of age to induce Dhh KO. Their cardiac function, exercise tolerance, and the phenotype of their coronary vasculature were assessed one month later. Results DhhECKO mice presented significantly reduced exercise tolerance, increased end diastolic pressure (EDP) and Tau, with no change in their ejection fraction consistent with diastolic dysfunction. At molecular and cellular level, impaired cardiac relaxation in DhhECKO mice was associated with a significantly decreased phospholamban phosphorylation on Thr17 and an alteration of sarcomeric shortening in ex-vivo. Besides, as expected, DhhECKO mice exhibited phenotypic changes in their coronary vasculature including a prominent pro-thrombotic phenotype (63±6,2 vs 25±5,2 thrombi/mm²; p<0,001) leading to an impaired capillary perfusion and local hypoxia. Notably, antiaggregant therapies (aspirin and clopidogrel) prevented the occurrence of both diastolic dysfunction and exercise intolerance in DhhECKO mice demonstrating for the first time that thrombosis may promote diastolic dysfunction. Importantly, we confirmed the critical role of thrombosis in Leprdb/db mice which also displayed increased cardiac small vessel thrombosis in comparison to control mice. Alike DhhECKO mice, we found that antiaggregants decreased EDP (6,3±0,4 mmHg in aspirin-treated vs 11,3 ± 0,79 in control mice; p=0,001) and improved exercise tolerance in Leprdb/db mice (34 ± 2,52 min in aspirin-treated vs 24 ± 3,55 in control mice; p=0,004). Conclusion Altogether, these results demonstrate that small vessel thrombosis may participate in the pathophysiology of heart failure with preserved ejection fraction.

Abstract 14169: Deletion of P21 Activated Kinase-1 Modulates Energy Homeostasis and Affects Cardiac Function

Introduction: We investigated the consequences of P21-activated kinase-1 (PAK1) deletion on cardiac function, body weight, body composition, and energy balance using a mouse model of global PAK1 knock out. PAK1 is a protein kinase that plays a role in multiple aspects of cardiac health. We hypothesize that PAK1 plays a role in cardiometabolic health including cardiac function and adipose tissue homeostasis. Methods: We used PAK1 -/- in the FVB/N background. We included 10-12-week-old male and female mice. Age and sex-matched FVB/N mice were used as wild-type (WT) controls. Most results included both male and female mice together, as no differences were found between sexes. Ca 2+ kinetics was measured in neonatal cardiomyocytes. Results: Deletion of PAK1 was found to have a significant impact on cardiac function. Specifically, slower Ca 2+ release and reuptake kinetics accompanied with decreased phospholamban phosphorylation was observed in PAK1 -/- mice. These changes affected cardiac systolic (EF, s’) and lusitropy parameters (E/e’, IVRT). Moreover, our findings revealed body weight differences between PAK1 -/- and WT at 12 weeks, accompanied with increased % fat mass and decreased % lean mass in PAK1 -/- , indicating a shift towards adiposity. Further analysis revealed that PAK1 -/- mice had increased weights of white adipose tissue (WAT), and brown adipose tissue (BAT). Moreover, we found increased food intake, increased number of feeding bouts and decreased energy expenditure accompanied with decreased thermogenic markers in subcutaneous WAT and BAT in PAK1 -/- , further contributing to the obesogenic phenotype (see table for results). Conclusions: Deletion of PAK1 affected systolic and diastolic functions, and resulted in increased energy intake, decreased energy expenditure, and increased adiposity. Therefore, targeting PAK1 signaling pathways may represent a promising therapeutic strategy for improving cardiometabolic diseases.

Hypercontractile cardiac phenotype in mice overexpressing the regulatory subunit PR72 of protein phosphatase 2A.

The activity, localization, and substrate specificity of the protein phosphatase 2A (PP2A) heterotrimer are controlled by various regulatory B subunits. PR72 belongs to the B'' gene family and has been shown to be upregulated in human heart failure. However, little is known about the functions of PR72 in the myocardium. To address this issue, we generated a transgenic mouse model with heart-specific overexpression of PP2A-PR72. Biochemical and physiological methods were used to determine contractility, Ca2+ cycling parameters, and protein phosphorylation. A 2.5-fold increase in PR72 expression resulted in moderate cardiac hypertrophy. Maximal ventricular pressure was increased in catheterized transgenic mice (TG) compared to wild-type (WT) littermates. This was accompanied by an increased shortening of sarcomere length and faster relaxation at the single-cell level in TG. In parallel with these findings, the peak amplitude of Ca2+ transients was increased, and the decay in intracellular Ca2+ levels was shortened in TG compared to WT. The changes in Ca2+ cycling in TG were also evident from an increase in the full duration and width at half maximum of Ca2+ sparks. Consistent with the contractile data, phosphorylation of phospholamban at threonine-17 was higher in TG hearts. The lower expression of the Na+/Ca2+ exchanger may also contribute to the hypercontractile state in transgenic myocardium. Our results suggest that PP2A-PR72 plays an important role in regulating cardiac contractile function and Ca2+ cycling, indicating that the upregulation of PR72 in heart failure is an attempt to compensate functionally.

Exosomes secreted by endothelial cells derived from human induced pluripotent stem cells improve recovery from myocardial infarction in mice

BackgroundHuman induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) exhibit the potential to repair the injured heart after myocardial infarction (MI) by promoting neovascularization and cardiomyocyte survival. However, because of the low cellular retention and poor engraftment efficacy, cell therapy of MI is partly mediated by exosomes secreted from the transplanted cells. In this study, we investigated whether exosomes secreted from hiPSC-ECs could become a promising acellular approach to repair the infarcted heart after MI and elucidated the underlying protective mechanism.MethodsThe hiPSC-ECs were differentiated, and exosomes were isolated in vitro. Then, hiPSC-EC exosomes were delivered by intramyocardial injection in a murine MI model in vivo. Echocardiography, combined with hemodynamic measurement, histological examination, Ca2+ transient and cell shortening assessment, and Western blot, was used to determine the protective effects of hiPSC-EC exosomes on the infarcted heart. Furthermore, microRNA sequencing, luciferase activity assay, and microRNA gain–loss function experiments were performed to investigate the enriched microRNA and its role in exosome-mediated effects.ResultsIn vitro, the hiPSC-EC exosomes enhanced intracellular Ca2+ transients, increased ATP content, and improved cell survival to protect cardiomyocytes from oxygen–glucose deprivation injury. Congruously, hiPSC-EC exosome administration in vivo improved the myocardial contractile function and attenuated the harmful left ventricular remodeling after MI without increasing the frequency of arrhythmias. Mechanistically, the hiPSC-EC exosomes notably rescued the protein expression and function of the sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA-2a) and ryanodine receptor 2 (RyR-2) to maintain intracellular Ca2+ homeostasis and increase cardiomyocyte contraction after MI. The microRNA sequencing showed that miR-100-5p was the most abundant microRNA in exosomes. miR-100-5p could target protein phosphatase 1β (PP-1β) to enhance phospholamban (PLB) phosphorylation at Ser16 and subsequent SERCA activity, which contributes to the hiPSC-EC exosome-exerted cytoprotective effects on maintaining intracellular Ca2+ homeostasis and promoting cardiomyocyte survival.ConclusionThe hiPSC-EC exosomes maintain cardiomyocyte Ca2+ homeostasis to improve myocardial recovery after MI, which may provide an acellular therapeutic option for myocardial injury.

5-Methoxytryptophan alleviates atrial structural remodeling in ibrutinib-associated atrial fibrillation

BackgroundIbrutinib is an effective and well-tolerated treatment for B-cell lymphomas but is associated with an increased risk of atrial fibrillation (AF) by altering the structure of the atrium. 5-Methoxytryptophan (5-MTP) inhibits inflammatory and fibrotic processes. This study aimed to determine the effects and mechanisms of 5-MTP on the underlying mechanisms of AF caused by ibrutinib. MethodsThe effect of 5-MTP on ibrutinib-related AF was investigated in male Sprague Dawley rats using echocardiographic, electrophysiological, immunofluorescent, Masson staining, and molecular analyses. RusultsThe ibrutinib+5-MTP group showed (1) a lower incidence and shorter duration of AF and accelerated atrial conduction; (2) a decreased left atrial mass and left atrial diameter; (3) decreased myocardial fibrosis in the left atrium; (4) lower atrial inflammation; (5) increased sarcoplasmic reticulum Ca2+-ATPase 2a protein expression, decreased phosphorylation of phospholamban at Thr17, and decreased sodium/calcium exchanger 1 protein expression and phosphorylation of ryanodine receptor 2 at S2814; and (6) decreased phosphorylation of CaMKII expression. 5-MTP treatment markedly activated the PI3K-Akt signaling. Inhibiting PI3K-Akt signaling significantly reversed the protective effect of 5-MTP on ibrutinib-related AF. ConclusionsThese findings suggest that 5-MTP administration decreases the vulnerability of ibrutinib-related AF mainly caused by ameliorated maladaptive left atrial remodeling and dysregulation of calcium handling proteins. Mechanistically, 5-MTP treatment markedly enhanced the activation of cardiac PI3K-Akt signaling.

Comprehensive Characterization of Endogenous Phospholamban Proteoforms Enabled by Photocleavable Surfactant and Top-down Proteomics.

Top-down mass spectrometry (MS)-based proteomics has become a powerful tool for analyzing intact proteins and their associated post-translational modifications (PTMs). In particular, membrane proteins play critical roles in cellular functions and represent the largest class of drug targets. However, the top-down MS characterization of endogenous membrane proteins remains challenging, mainly due to their intrinsic hydrophobicity and low abundance. Phospholamban (PLN) is a regulatory membrane protein located in the sarcoplasmic reticulum and is essential for regulating cardiac muscle contraction. PLN has diverse combinatorial PTMs, and their dynamic regulation has significant influence on cardiac contractility and disease. Herein, we have developed a rapid and robust top-down proteomics method enabled by a photocleavable anionic surfactant, Azo, for the extraction and comprehensive characterization of endogenous PLN from cardiac tissue. We employed a two-pronged top-down MS approach using an online reversed-phase liquid chromatography tandem MS method on a quadrupole time-of-flight MS and a direct infusion method via an ultrahigh-resolution Fourier-transform ion cyclotron resonance MS. We have comprehensively characterized the sequence and combinatorial PTMs of endogenous human cardiac PLN. We have shown the site-specific localization of phosphorylation to Ser16 and Thr17 by MS/MS for the first time and the localization of S-palmitoylation to Cys36. Moreover, we applied our method to characterize PLN in disease and reported the significant reduction of PLN phosphorylation in human failing hearts with ischemic cardiomyopathy. Taken together, we have developed a streamlined top-down targeted proteomics method for comprehensive characterization of combinatorial PTMs in PLN toward better understanding the role of PLN in cardiac contractility.

Abstract P2071: Mutation-Specific Calmodulin-Kinase II Activation In Hypertrophic Cardiomyopathy

Autophosphorylation of Calmodulin-Kinase IIδ (CaMKIIδ) at Thr-287 leads to autoactivation such that kinase activity is autonomous of Ca 2+ /CaM. In the heart, CaMKIIδ functions to regulate Ca 2+ homeostasis by targeting Ca 2+ handling proteins, including phospholamban (PLB). In sarcomeric hypertrophic cardiomyopathy (HCM), disruption of Ca 2+ homeostasis and aberrant autoactivation of CaMKIIδ has been linked to disease progression. We have previously shown that the pathogenic role of autonomous CaMKIIδ in HCM is mutation-specific. Transgenic mice expressing the HCM mutation R92W in cardiac troponin T (cTnT) exhibited an increase in autonomous CaMKIIδ while cTnT-R92L mice did not. The potentially clinically relevant mechanism(s) underlying this mutation specificity are unresolved. We hypothesize that a potential trigger of autonomous CaMKIIδ is accelerated myofilament Ca 2+ dissociation kinetics. We have previously shown that cTnT-R92W accelerates Ca 2+ dissociation, while cTnT-R92L decelerates Ca 2+ dissociation. Here, we extended our study to include cTnT-I79N and cTnT-R94H to determine if Ca 2+ dissociation rates can be linked to autonomous CaMKIIδ activity and thus provide a potential novel target in HCM. Our data show that cTnT-I79N accelerates Ca 2+ dissociation, and cTnT-R94H decelerates Ca 2+ dissociation, similar to our earlier findings. To determine whether the mutation-specific changes in Ca 2+ dissociation alter CaMKIIδ activity, transgenic mice expressing cTnT-R94H and cTnT-I79N were probed for autophosphorylated CaMKIIδ and Thr-17 phosphorylated PLB. At 2 months, PLB phosphorylation is unchanged in cTnT-R94H and cTnT-I79N mice. At 4 months, cTnT-I79N mice exhibit a trending increase in autonomous CaMKIIδ and cTnT-R94H mice exhibit a modest increase, similar to cTnT-R92W positive controls. These trends are matched by PLB phosphorylation levels. At 6 months, cTnT-R94H mice exhibit normal autonomous CaMKIIδ levels. While future and ongoing work will explore changes in myocellular Ca 2+ homeostasis in more depth, these preliminary data suggest that, unlike cTnT-R94H mice, cTnT-I79N mice exhibit elevated autonomous CaMKIIδ and these mutation-specific differences may underlie observed phenotypic variation in patients.

Abstract P2082: Thrombosis In The Coronary Microvasculature May Be Responsible For Impaired Cardiac Relaxation And Diastolic Dysfunction

Introduction: Heart Failure with Preserved Ejection Fraction (HFpEF) is proposed to be caused by endothelial dysfunction in cardiac small vessels. We previously identified Hhipl1,as a gene upregulated in the coronary vasculature of Leptin receptor deficient mice (Leprdb/db) a well-established mouse model of HFpEF. Importantly, Hhipl1 encodes for a decoy receptor of Desert Hedgehog (DHH) which is known to be critical for endothelial integrity. Objective: Our objective is to investigate the functional consequences of impaired Hedgehog (HH) signaling in the adult heart in order to identify novel mechanisms underlying the development of diastolic dysfunction. Method: Cdh5-Cre/ERT2, DhhFlox/Flox (DhhECKO) mice and their control littermates were administered with tamoxifen at 8 weeks of age to induce Dhh KO. Their cardiac function, exercise tolerance, and the phenotype of their coronary vasculature were assessed one month later. Results: DhhECKO mice presented significantly reduced exercise tolerance, increased end diastolic pressure (EDP) and Tau, with no change in their ejection fraction consistent with diastolic dysfunction. At molecular and cellular level, impaired cardiac relaxation in DhhECKO mice was associated with a significantly decreased phospholamban phosphorylation on Thr17 and an alteration of sarcomeric shortening in ex-vivo. Besides, as expected, DhhECKO mice exhibited phenotypic changes in their coronary vasculature including a prominent pro-thrombotic phenotype leading to an impaired capillary perfusion and local hypoxia. Notably, antiaggregant therapies (aspirin and clopidogrel) prevented the occurrence of both diastolic dysfunction and exercise intolerance in DhhECKO mice demonstrating for the first time that thrombosis may promote diastolic dysfunction. Importantly, we confirmed the critical role of thrombosis in Leprdb/db mice which also displayed increased cardiac small vessel thrombosis in comparison to control mice. Alike DhhECKO mice, we found that antiaggregants decreased EDP and improved exercise tolerance in Leprdb/db mice. Conclusion: Altogether, these results demonstrate that small vessel thrombosis may participate in the pathophysiology of heart failure with preserved ejection fraction.

Ergometrine stimulates histamine H2 receptors in the isolated human atrium

Ergometrine (6aR,9R)-N-((S)-1-hydroxypropan-2-yl)-7-methyl-4,6,6a,7,8,9-hexa-hydro-indolo-[4,3-fg]chinolin-9-carboxamide or lysergide acid β-ethanolamide or ergonovine) activates several types of serotonin and histamine receptors in the animal heart. We thus examined whether ergometrine can activate human serotonin 5-HT4 receptors (h5-HT4R) and/or human histamine H2 receptors (hH2R) in the heart of transgenic mice and/or in the human isolated atrium. Force of contraction or beating rates were studied in electrically stimulated left atrial or spontaneously beating right atrial preparations or spontaneously beating isolated retrogradely perfused hearts (Langendorff setup) of mice with cardiac specific overexpression of the h5-HT4R (5-HT4-TG) or of mice with cardiac specific overexpression of the hH2R (H2-TG) or in electrically stimulated human right atrial preparations obtained during cardiac surgery. Western blots to assess phospholamban (PLB) phosphorylation on serine 16 were performed. Ergometrine exerted concentration- and time-dependent positive inotropic effects and positive chronotropic effects in atrial preparations starting at 0.3 µM and reaching a plateau at 10 µM in H2-TGs (n = 7). This was accompanied by an increase in PLB phosphorylation at serine 16. Ergometrine up 10 µM failed to increase force of contraction in left atrial preparations from 5-HT4-TGs (n = 5). Ten micrometer ergometrine increased the force of contraction in isolated retrogradely perfused spontaneously beating heart preparations (Langendorff setup) from H2-TG but not 5-HT4-TG. In the presence of the phosphodiesterase inhibitor cilostamide (1 µM), ergometrine at 10 µM exerted positive inotropic effects in isolated electrically stimulated human right atrial preparations, obtained during cardiac surgery, and these effects were eliminated by 10 µM of the H2R antagonist cimetidine but not by 10 µM of the 5-HT4R antagonist tropisetron. Furthermore, ergometrine showed binding to human histamine H2 receptors (at 100 µM and 1 mM) using HEK cells in a recombinant expression system (pKi < 4.5, n = 3). In conclusion, we suggest that ergometrine is an agonist at cardiac human H2Rs.

Empagliflozin reduces arrhythmogenic effects in rat neonatal and human iPSC-derived cardiomyocytes and improves cytosolic calcium handling at least partially independent of NHE1

The antidiabetic agent class of sodium-glucose cotransporter 2 (SGLT2) inhibitors confer unprecedented cardiovascular benefits beyond glycemic control, including reducing the risk of fatal ventricular arrhythmias. However, the impact of SGLT2 inhibitors on the electrophysiological properties of cardiomyocytes exposed to stimuli other than hyperglycemia remains elusive. This investigation tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) affects cardiomyocyte electrical activity under hypoxic conditions. Rat neonatal and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes incubated or not with the hypoxia-mimetic agent CoCl2 were treated with EMPA (1 μM) or vehicle for 24 h. Action potential records obtained using intracellular microelectrodes demonstrated that EMPA reduced the action potential duration at 30%, 50%, and 90% repolarization and arrhythmogenic events in rat and human cardiomyocytes under normoxia and hypoxia. Analysis of Ca2+ transients using Fura-2-AM and contractility kinetics showed that EMPA increased Ca2+ transient amplitude and decreased the half-time to recover Ca2+ transients and relaxation time in rat neonatal cardiomyocytes. We also observed that the combination of EMPA with the Na+/H+ exchanger isoform 1 (NHE1) inhibitor cariporide (10 µM) exerted a more pronounced effect on Ca2+ transients and contractility than either EMPA or cariporide alone. Besides, EMPA, but not cariporide, increased phospholamban phosphorylation at serine 16. Collectively, our data reveal that EMPA reduces arrhythmogenic events, decreases the action potential duration in rat neonatal and human cardiomyocytes under normoxic or hypoxic conditions, and improves cytosolic calcium handling at least partially independent of NHE1. Moreover, we provided further evidence that SGLT2 inhibitor-mediated cardioprotection may be partly attributed to its cardiomyocyte electrophysiological effects.

It takes two to tango: cardiac fibroblast-derived NO-induced cGMP enters cardiac myocytes and increases cAMP by inhibiting PDE3

The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing β-receptor-induced contractile responses appears as one of NO/cGMP’s functions in the non-failing heart.

Thrombosis in the coronary microvasculature may be responsible for impaired cardiac relaxation and diastolic dysfunction

Heart failure with preserved ejection fraction (HFpEF) is proposed to be caused by endothelial dysfunction in cardiac small vessels. We previously identified Hhipl1, as a gene upregulated in the coronary vasculature of Leptin receptor deficient mice (Leprdb/db) a well-established mouse model of HFpEF. Importantly, Hhipl1 encodes for a decoy receptor of Desert Hedgehog (DHH) which is known to be critical for endothelial integrity. Our objective is to investigate the functional consequences of impaired Hedgehog (HH) signaling in the adult heart in order to identify novel mechanisms underlying the development of diastolic dysfunction. To do so, Cdh5-Cre/ERT2, DhhFlox/Flox (DhhECKO) mice and their control littermates were administered with tamoxifen at 8 weeks of age to induce Dhh KO. Their cardiac function, exercise tolerance, and the phenotype of their coronary vasculature were assessed one month later. DhhECKO mice presented significantly reduced exercise tolerance, increased end-diastolic pressure (EDP) and Tau, with no change in their ejection fraction consistent with diastolic dysfunction. At molecular and cellular level, impaired cardiac relaxation in DhhECKO mice was associated with a significantly decreased phospholamban phosphorylation on Thr17 and an alteration of sarcomeric shortening in ex-vivo. Besides, as expected, DhhECKO mice exhibited phenotypic changes in their coronary vasculature including a prominent pro-thrombotic phenotype (63 ± 6.2 vs. 25 ± 5.2 thrombi/mm2; P < 0.001) leading to an impaired capillary perfusion and local hypoxia. Notably, antiaggregant therapies (aspirin and clopidogrel) prevented the occurrence of both diastolic dysfunction and exercise intolerance in DhhECKO mice demonstrating for the first time that thrombosis may promote diastolic dysfunction. Importantly, we confirmed the critical role of thrombosis in Leprdb/db mice which also displayed increased cardiac small vessel thrombosis in comparison to control mice. Alike DhhECKO mice, we found that antiaggregants decreased EDP (6.3 ± 0.4 mmHg in aspirin-treated vs. 11.3 ± 0.79 in control mice; P = 0.001) and improved exercise tolerance in Leprdb/db mice (34 ± 2.52 min in aspirin-treated vs. 24 ± 3.55 in control mice; P = 0.004). Altogether, these results demonstrate that small vessel thrombosis may participate in the pathophysiology of heart failure with preserved ejection fraction.

Altered coronary artery function, arteriogenesis and endothelial YAP signaling in postnatal hypertrophic cardiomyopathy.

Introduction: Hypertrophic cardiomyopathy (HCM) is a cardiovascular genetic disease caused largely by sarcomere protein mutations. Gaps in our understanding exist as to how maladaptive sarcomeric biophysical signals are transduced to intra- and extracellular compartments leading to HCM progression. To investigate early HCM progression, we focused on the onset of myofilament dysfunction during neonatal development and examined cardiac dynamics, coronary vascular structure and function, and mechano-transduction signaling in mice harboring a thin-filament HCM mutation. Methods: We studied postnatal days 7-28 (P7-P28) in transgenic (TG) TG-cTnT-R92Q and non-transgenic (NTG) mice using skinned fiber mechanics, echocardiography, biochemistry, histology, and immunohistochemistry. Results: At P7, skinned myofiber bundles exhibited an increased Ca2+-sensitivity (pCa50 TG: 5.97 ± 0.04, NTG: 5.84 ± 0.01) resulting from cTnT-R92Q expression on a background of slow skeletal (fetal) troponin I and α/β myosin heavy chain isoform expression. Despite the transition to adult isoform expressions between P7-P14, the increased Ca2+- sensitivity persisted through P28 with no apparent differences in gross morphology among TG and NTG hearts. At P7 significant diastolic dysfunction was accompanied by coronary flow perturbation (mean diastolic velocity, TG: 222.5 ± 18.81mm/s, NTG: 338.7 ± 28.07mm/s) along with localized fibrosis (TG: 4.36% ± 0.44%, NTG: 2.53% ± 0.47%). Increased phosphorylation of phospholamban (PLN) was also evident indicating abnormalities in Ca2+ homeostasis. By P14 there was a decline in arteriolar cross-sectional area along with an expansion of fibrosis (TG: 9.72% ± 0.73%, NTG: 2.72% ± 0.2%). In comparing mechano-transduction signaling in the coronary arteries, we uncovered an increase in endothelial YAP expression with a decrease in its nuclear to cytosolic ratio at P14 in TG hearts, which was reversed by P28. Conclusion: We conclude that those early mechanisms that presage hypertrophic remodeling in HCM include defective biophysical signals within the sarcomere that drive diastolic dysfunction, impacting coronary flow dynamics, defective arteriogenesis and fibrosis. Changes in mechano-transduction signaling between the different cellular compartments contribute to the pathogenesis of HCM.

Differential changes in cyclic adenosine 3'-5' monophosphate (cAMP) effectors and major Ca2+ handling proteins during diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is associated with differential and time-specific regulation of β-adrenergic receptors and cardiac cyclic nucleotide phosphodiesterases with consequences for total cyclic adenosine 3'-5' monophosphate (cAMP) levels. We aimed to investigate whether these changes are associated with downstream impairments in cAMP and Ca2+ signalling in a type 1 diabetes (T1D)-induced DCM model. T1D was induced in adult male rats by streptozotocin (65 mg/kg) injection. DCM was assessed by cardiac structural and molecular remodelling. We delineated sequential changes affecting the exchange protein (Epac1/2), cAMP-dependent protein kinase A (PKA) and Ca2+ /Calmodulin-dependent kinase II (CaMKII) at 4, 8 and 12 weeks following diabetes, by real-time quantitative PCR and western blot. Expression of Ca2+ ATPase pump (SERCA2a), phospholamban (PLB) and Troponin I (TnI) was also examined. Early upregulation of Epac1 transcripts was noted in diabetic hearts at Week 4, followed by increases in Epac2 mRNA, but not protein levels, at Week 12. Expression of PKA subunits (RI, RIIα and Cα) remained unchanged regardless of the disease stage, whereas CaMKII increased at Week 12 in DCM. Moreover, PLB transcripts were upregulated in diabetic hearts, whereas SERCA2a and TnI gene expression was unchanged irrespective of the disease evolution. PLB phosphorylation at threonine-17 was increased in DCM, whereas phosphorylation of both PLB at serine-16 and TnI at serine-23/24 was unchanged. We show for the first time differential and time-specific regulations in cardiac cAMP effectors and Ca2+ handling proteins, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

Phospholamban pentamerization increases sensitivity and dynamic range of cardiac relaxation.

A key event in the regulation of cardiac contraction and relaxation is the phosphorylation of phospholamban (PLN) that relieves the inhibition of the sarco/endoplasmic Ca2+-ATPase SERCA2a. PLN exists in an equilibrium between monomers and pentamers. While only monomers can inhibit SERCA2a by direct interaction, the functional role of pentamers is still unclear. This study investigates the functional consequences of PLN pentamerization. We generated transgenic mouse models expressing either a PLN mutant that cannot form pentamers (TgAFA-PLN) or wildtype PLN (TgPLN) in a PLN-deficient background. TgAFA-PLN hearts demonstrated 3-fold stronger phosphorylation of monomeric PLN, accelerated Ca2+ cycling of cardiomyocytes and enhanced contraction and relaxation of sarcomeres and whole hearts in vivo. All of these effects were observed under baseline conditions and abrogated upon inhibition of protein kinase A (PKA). Mechanistically, far western kinase assays revealed that PLN pentamers are phosphorylated by PKA directly and independent of any subunit exchange for free monomers. In vitro-phosphorylation of synthetic PLN demonstrated that pentamers even provide a preferred PKA substrate and compete with monomers for the kinase, thereby reducing monomer phosphorylation and maximizing SERCA2a inhibition. However, β-adrenergic stimulation induced strong PLN monomer phosphorylation in TgPLN hearts and sharp acceleration of cardiomyocyte Ca2+ cycling and hemodynamic values that now were indistinguishable from TgAFA-PLN and PLN-KO hearts. The pathophysiological relevance of PLN pentamerization was evaluated using transverse aortic constriction (TAC) to induce left ventricular pressure overload. Compared to TgPLN, TgAFA-PLN mice demonstrated reduced survival after TAC, impaired cardiac hemodynamics, failure to respond to adrenergic stimulation, higher heart weight, and increased myocardial fibrosis. The findings show that PLN pentamerization greatly impacts on SERCA2a activity as it mediates the full range of PLN effects from maximum inhibition to full release of SERCA2a. function. This regulation is important for myocardial adaptation to sustained pressure overload. Pentamerization of PLN adds to the regulation of cardiac contractile function and facilitates myocardial transition to an energy saving mode during resting phases. Thus, PLN pentamers would protect cardiomyocytes from energetic deficits, and they improve stress adaptation of the heart as shown for sustained pressure overload in this study. Strategies that target PLN pentamerization promise therapeutic potential in the treatment of myocardial maladaptation to stress as well as cardiac pathologies associated with altered monomer-to-pentamer ratios, e.g., cardiomyopathies due to PLN mutations, certain types of heart failure, and aged hearts.

ABCB5+ mesenchymal stromal cells therapy protects from hypoxia by restoring Ca2+ homeostasis in vitro and in vivo

BackgroundHypoxia in ischemic disease impairs Ca2+ homeostasis and may promote angiogenesis. The therapeutic efficacy of mesenchymal stromal cells (MSCs) in peripheral arterial occlusive disease is well established, yet its influence on cellular Ca2+ homeostasis remains to be elucidated. We addressed the influence of ATP-binding cassette subfamily B member 5 positive mesenchymal stromal cells (ABCB5+ MSCs) on Ca2+ homeostasis in hypoxic human umbilical vein endothelial cells (HUVECs) in vitro and in vivo.MethodsHypoxia was induced in HUVECs by Cobalt (II) chloride (CoCl2) or Deferoxamine (DFO). Dynamic changes in the cytosolic- and endoplasmic reticulum (ER) Ca2+ and changes in reactive oxygen species were assessed by appropriate fluorescence-based sensors. Metabolic activity, cell migration, and tube formation were assessed by standard assays. Acute-on-chronic ischemia in Apolipoprotein E knock-out (ApoE−/−) mice was performed by double ligation of the right femoral artery (DFLA). ABCB5+ MSC cells were injected into the ischemic limb. Functional recovery after DFLA and histology of gastrocnemius and aorta were assessed.ResultsHypoxia-induced impairment of cytosolic and ER Ca2+ were restored by ABCB5+ MSCs or their conditioned medium. Similar was found for changes in intracellular ROS production, metabolic activity, migratory ability and tube formation. The restoration was paralleled by an increased expression of the Ca2+ transporter Sarco-/endoplasmic reticulum ATPase 2a (SERCA2a) and the phosphorylation of Phospholamban (PLN). In acute-on-chronic ischemia, ABCB5+ MSCs treated mice showed a higher microvascular density, increased SERCA2a expression and PLN phosphorylation relative to untreated controls.ConclusionsABCB5+ MSCs therapy can restore cellular Ca2+ homeostasis, which may beneficially affect the angiogenic function of endothelial cells under hypoxia in vitro and in vivo.

Endothelial cell-specific roles for tetrahydrobiopterin in myocardial function, cardiac hypertrophy, and response to myocardial ischemia-reperfusion injury.

The cofactor tetrahydrobiopterin (BH4) is a critical regulator of nitric oxide synthase (NOS) function and redox signaling, with reduced BH4 implicated in multiple cardiovascular disease states. In the myocardium, augmentation of BH4 levels can impact on cardiomyocyte function, preventing hypertrophy and heart failure. However, the specific role of endothelial cell BH4 biosynthesis in the coronary circulation and its role in cardiac function and the response to ischemia has yet to be elucidated. Endothelial cell-specific Gch1 knockout mice were generated by crossing Gch1fl/fl with Tie2cre mice, generating Gch1fl/flTie2cre mice and littermate controls. GTP cyclohydrolase protein and BH4 levels were reduced in heart tissues from Gch1fl/flTie2cre mice, localized to endothelial cells, with normal cardiomyocyte BH4. Deficiency in coronary endothelial cell BH4 led to NOS uncoupling, decreased NO bioactivity, and increased superoxide and hydrogen peroxide productions in the hearts of Gch1fl/flTie2cre mice. Under physiological conditions, loss of endothelial cell-specific BH4 led to mild cardiac hypertrophy in Gch1fl/flTie2cre hearts. Endothelial cell BH4 loss was also associated with increased neuronal NOS protein, loss of endothelial NOS protein, and increased phospholamban phosphorylation at serine-17 in cardiomyocytes. Loss of cardiac endothelial cell BH4 led to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia-reperfusion injury. Taken together, these studies reveal a specific role for endothelial cell Gch1/BH4 biosynthesis in cardiac function and the response to cardiac ischemia-reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction and ischemia-reperfusion injury.NEW & NOTEWORTHY We demonstrate a critical role for endothelial cell Gch1/BH4 biosynthesis in coronary vascular function and cardiac function. Loss of cardiac endothelial cell BH4 leads to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia/reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction, ischemia injury, and heart failure.

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.