PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility. METHODS: Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies. RESULTS: In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (P=2.0×10-2; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (P=1.4×10-6; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9×10-2 and 6.7×10-3, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (P=5.0×10-6; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (P=1.8×10-2 and 4.1×10-2, respectively; n=6, 6, and 5). CONCLUSIONS: Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Three-dimensional spatial quantitative analysis of cardiac lymphatics in the mouse heart.

OBJECTIVE: Three-dimensional (3D) microscopy and image data analysis are necessary for studying the morphology of cardiac lymphatic vessels (LyVs) and their association with other cell types. We aimed to develop a methodology for 3D multiplexed lightsheet microscopy and highly sensitive and quantitative image analysis to identify pathological remodeling in the 3D morphology of LyVs in young adult mouse hearts with familial hypertrophic cardiomyopathy (HCM). METHODS: We developed a 3D lightsheet microscopy workflow providing a quick turn-around (as few as 5-6 days), multiplex fluorescence detection, and preservation of LyV structure and epitope markers. Hearts from non-transgenic and transgenic (TG) HCM mice were arrested in diastole, retrograde perfused, immunolabeled, optically cleared, and imaged. We built an image-processing pipeline to quantify LyV morphological parameters at the chamber and branch levels. RESULTS: Chamber-specific pathological alterations of LyVs were identified, and significant changes were seen in the right atrium (RA). TG hearts had a higher volume percent of ER-TR7+ fibroblasts and reticular fibers. In the RA, we found associations between ER-TR7+ volume percent and both LyV segment density and median diameter. CONCLUSIONS: This workflow and study enabled multi-scale analysis of pathological changes in cardiac LyVs of young adult mice, inviting ideas for research on LyVs in cardiac disease.

Mechanisms of Pathogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant R278C+/- During Development.

Hypertrophic cardiomyopathy (HCM) is one of the most common heritable cardiovascular diseases and variants of TNNT2 (cardiac troponin T) are linked to increased risk of sudden cardiac arrest despite causing limited hypertrophy. In this study, a TNNT2 variant, R278C+/-, was generated in both human cardiac recombinant/reconstituted thin filaments (hcRTF) and human- induced pluripotent stem cells (hiPSCs) to investigate the mechanisms by which the R278C+/- variant affects cardiomyocytes at the proteomic and functional levels. The results of proteomics analysis showed a significant upregulation of markers of cardiac hypertrophy and remodeling in R278C+/- vs. the isogenic control. Functional measurements showed that R278C+/- variant enhances the myofilament sensitivity to Ca2+, increases the kinetics of contraction, and causes arrhythmia at frequencies >75 bpm. This study uniquely shows the profound impact of the TNNT2 R278C+/- variant on the cardiomyocyte proteomic profile, cardiac electrical and contractile function in the early stages of cardiac development.

Cardiomyocyte external mechanical unloading activates modifications of α-actinin differently from sarcomere-originated unloading.

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano-signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano-signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1 µm mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α-actinin by 6 h. In myosin inhibition, this was accompanied by redistribution of intracellular poly-ubiquitin K48 to the cellular periphery relative to the poly-ubiquitin K48 reservoir at the I-band. Moreover, loading and unloading of the cellular substrate led to a three-fold increase in post-translational modifications (PTMs) when compared to the myosin-specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve extracellular signal-regulated kinase 1/2 and focal adhesion kinase activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α-actinin to increase its propensity to bind F-actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α-actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α-actinin are discussed with respect to cardiac atrophy and heart failure.

A perspective on Notch signalling in progression and arrhythmogenesis in familial hypertrophic and dilated cardiomyopathies.

In this perspective, we discussed emerging data indicating a role for Notch signalling in inherited disorders of the heart failure with focus on hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) linked to variants of genes encoding mutant proteins of the sarcomere. We recently reported an upregulation of elements in the Notch signalling cascade in cardiomyocytes derived from human inducible pluripotent stem cells expressing a TNNT2 variant encoding cardiac troponin T (cTnT-I79N+/-), which induces hypertrophy, remodelling, abnormalities in excitation-contraction coupling and electrical instabilities (Shafaattalab S et al. 2021 Front. Cell Dev. Biol. 9, 787581. (doi:10.3389/fcell.2021.787581)). Our search of the literature revealed the novelty of this finding and stimulated us to discuss potential connections between the Notch signalling pathway and familial cardiomyopathies. Our considerations focused on the potential role of these interactions in arrhythmias, microvascular ischaemia, and fibrosis. This finding underscored a need to consider the role of Notch signalling in familial cardiomyopathies which are trigged by sarcomere mutations engaging mechano-signalling pathways for which there is evidence of a role for Notch signalling with crosstalk with Hippo signalling. Our discussion included a role for both cardiac myocytes and non-cardiac myocytes in progression of HCM and DCM. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Myosin Light Chain Dephosphorylation by PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

Background: Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods: Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results: In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls ( P =2.0×10 -2 , n=12,12 in each group) with > 40% reduction in MLC2a phosphorylation ( P =1.4×10 -6 , n=12,12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF ( P =2.9×10 -2 and 6.7×10 -3 respectively, n=8,8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls ( P =5.0×10 -6 , n=12,8,12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls ( P =1.8×10 -2 and 4.1×10 -2 respectively, n= 6,6,5). Conclusions: AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

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/ß 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 ± 18.81 mm/s, NTG: 338.7 ± 28.07 mm/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.

Three-dimensional spatial quantitative analysis of cardiac lymphatics in the mouse heart.

Objective: 3D microscopy and image data analysis are necessary for studying the morphology of cardiac lymphatic vessels (LyVs) and association with other cell types. We aimed to develop a methodology for 3D multiplexed lightsheet microscopy and highly sensitive and quantitative image analysis to identify pathological remodeling in the 3D morphology of LyVs in young adult mouse hearts with familial hypertrophic cardiomyopathy (HCM). Methods: We developed a 3D lightsheet microscopy workflow providing a quick turn-around (as few as 5-6 days), multiplex fluorescence detection, and preservation of LyV structure and epitope markers. Hearts from non-transgenic (NTG) and transgenic (TG) HCM mice were arrested in diastole, retrograde perfused, immunolabeled, optically cleared, and imaged. We built an image processing pipeline to quantify LyV morphological parameters at the chamber and branch levels. Results: Chamber-specific pathological alterations of LyVs were identified, but most significantly in the right atrium (RA). TG hearts had a higher volume fraction of ER-TR7 + fibroblasts and reticular fibers. In the RA, we found associations between ER-TR7 + volume fraction and both LyV segment density and median diameter. Conclusions: This workflow and study enabled multi-scale analysis of pathological changes in cardiac LyVs of young adult mice, inviting ideas for research on LyVs in cardiac disease.

Mechano-growth factor E-domain modulates cardiac contractile function through 14-3-3 protein interactomes.

In the heart, alternative splicing of the igf-I gene produces two isoforms: IGF-IEa and IGF-IEc, (Mechano-growth factor, MGF). The sequence divergence between their E-domain regions suggests differential isoform function. To define the biological actions of MGF's E-domain, we performed in silico analysis of the unique C-terminal sequence and identified a phosphorylation consensus site residing within a putative 14-3-3 binding motif. To test the functional significance of Ser 18 phosphorylation, phospho-mimetic (S/E18) and phospho-null (S/A18) peptides were delivered to mice at different doses for 2 weeks. Cardiovascular function was measured using echocardiography and a pressure-volume catheter. At the lowest (2.25 mg/kg/day) and highest (9 mg/kg/day) doses, the peptides produced a depression in systolic and diastolic parameters. However, at 4.5 mg/kg/day the peptides produced opposing effects on cardiac function. Fractional shortening analysis also showed a similar trend, but with no significant change in cardiac geometry. Microarray analysis discovered 21 genes (FDR p < 0.01), that were expressed accordant with the opposing effects on contractile function at 4.5 mg/kg/day, with the nuclear receptor subfamily 4 group A member 2 (Nr4a2) identified as a potential target of peptide regulation. Testing the regulation of the Nr4a family, showed the E-domain peptides modulate Nr4a gene expression following membrane depolarization with KCl in vitro. To determine the potential role of 14-3-3 proteins, we examined 14-3-3 isoform expression and distribution. 14-3-3γ localized to the myofilaments in neonatal cardiac myocytes, the cardiac myocytes and myofilament extracts from the adult heart. Thermal shift analysis of recombinant 14-3-3γ protein showed the S/A18 peptide destabilized 14-3-3γ folding. Also, the S/A18 peptide significantly inhibited 14-3-3γ's ability to interact with myosin binding protein C (MYPC3) and phospholamban (PLN) in heart lysates from dobutamine injected mice. Conversely, the S/E18 peptide showed no effect on 14-3-3γ stability, did not inhibit 14-3-3γ's interaction with PLN but did inhibit the interaction with MYPC3. Replacing the glutamic acid with a phosphate group on Ser 18 (pSer18), significantly increased 14-3-3γ protein stability. We conclude that the state of Ser 18 phosphorylation within the 14-3-3 binding motif of MGF's E-domain, modulates protein-protein interactions within the 14-3-3γ interactome, which includes proteins involved in the regulation of contractile function.

The effect of Mg2+ on Ca2+ binding to cardiac troponin C in hypertrophic cardiomyopathy associated TNNC1 variants.

Cardiac troponin C (cTnC) is the critical Ca2+ -sensing component of the troponin complex. Binding of Ca2+ to cTnC triggers a cascade of conformational changes within the myofilament that culminate in force production. Hypertrophic cardiomyopathy (HCM)-associated TNNC1 variants generally induce a greater degree and duration of Ca2+ binding, which may underly the hypertrophic phenotype. Regulation of contraction has long been thought to occur exclusively through Ca2+ binding to site II of cTnC. However, work by several groups including ours suggest that Mg2+ , which is several orders of magnitude more abundant in the cell than Ca2+ , may compete for binding to the same cTnC regulatory site. We previously used isothermal titration calorimetry (ITC) to demonstrate that physiological concentrations of Mg2+ may decrease site II Ca2+ -binding in both N-terminal and full-length cTnC. Here, we explore the binding of Ca2+ and Mg2+ to cTnC harbouring a series of TNNC1 variants thought to be causal in HCM. ITC and thermodynamic integration (TI) simulations show that A8V, L29Q and A31S elevate the affinity for both Ca2+ and Mg2+ . Further, L48Q, Q50R and C84Y that are adjacent to the EF hand binding motif of site II have a more significant effect on affinity and the thermodynamics of the binding interaction. To the best of our knowledge, this work is the first to explore the role of Mg2+ in modifying the Ca2+ affinity of cTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg2+ both at baseline and when elevated on modifying the Ca2+ binding properties of cTnC and the subsequent conformational changes which precede cardiac contraction.

Widely cited publications of Michael Bárány in 1964 and 1967 as tipping points in understanding myosin molecular motors.

In 1964 Michael Bárány and colleagues published a paper ((M. Bárány, E. Gaetjens, K. Bárány, Karp E. Arch Biochem Biophys 106(1964)280-93. http://10.1016/0003-9861(64)90,189-4)) that has been one of the most cited papers in Archives of Biochemistry and Biophysics. This was followed in 1967 by another most cited paper (M. Bárány. J Gen Physiol 50(1967)197-218. https://doi.org/10.1085/jgp.50.6.197). I have commemorated these achievements as tipping points in the understanding of myosin motors in muscle function. Tipping points are generally defined as a temporal point in which a series of progressive advances (in this case the understanding of the relations between myosin ATP hydrolysis and muscle function) inspire more expansive, wide-ranging, significant changes. I first concisely summarize the background against which the papers came to publication as well as the unimaginable personal challenges faced by Michael and Kate Bárány. A final section summarizes the impact of these publications as key steps in the progression of contemporary understanding of diverse control of myosin ATPase activity with focus on the thick filaments in cardiac homeostasis, disorders, and as targets for therapeutic applications in translational investigations.

Truncation of the N-terminus of cardiac troponin I initiates adaptive remodeling of the myocardial proteosome via phosphorylation of mechano-sensitive signaling pathways.

The cardiac isoform of troponin I has a unique N-terminal extension (~ 1-30 amino acids), which contributes to the modulation of cardiac contraction and relaxation. Hearts of various species including humans produce a truncated variant of cardiac troponin I (cTnI-ND) deleting the first ~ 30 amino acids as an adaption in pathophysiological conditions. In this study, we investigated the impact of cTnI-ND chronic expression in transgenic mouse hearts compared to wildtype (WT) controls (biological n = 8 in each group). We aimed to determine the global phosphorylation effects of cTnI-ND on the cardiac proteome, thereby determining the signaling pathways that have an impact on cardiac function. The samples were digested and isobarically labeled and equally mixed for relative quantification via nanoLC-MS/MS. The peptides were then enriched for phospho-peptides and bioinformatic analysis was done with Ingenuity Pathway Analysis (IPA). We found approximately 77% replacement of the endogenous intact cTnI with cTnI-ND in the transgenic mouse hearts with 1674 phospho-proteins and 2971 non-modified proteins. There were 73 significantly altered phospho-proteins; bioinformatic analysis identified the top canonical pathways as associated with integrin, protein kinase A, RhoA, and actin cytoskeleton signaling. Among the 73 phospho-proteins compared to controls cTnI-ND hearts demonstrated a significant decrease in paxillin and YAP1, which are known to play a role in cell mechano-sensing pathways. Our data indicate that cTnI-ND modifications in the sarcomere are sufficient to initiate changes in the phospho-signaling profile that may underly the chronic-adaptive response associated with cTnI cleavage in response to stressors by modifying mechano-sensitive signaling pathways.

Effects of Sarcomere Activators and Inhibitors Targeting Myosin Cross-Bridges on Ca2+-Activation of Mature and Immature Mouse Cardiac Myofilaments.

We tested the hypothesis that isoform shifts in sarcomeres of the immature heart modify the effect of cardiac myosin-directed sarcomere inhibitors and activators. Omecamtiv mecarbil (OM) activates tension and is in clinical trials for the treatment of adult acute and chronic heart failure. Mavacamten (Mava) inhibits tension and is in clinical trials to relieve hypercontractility and outflow obstruction in advanced genetic hypertrophic cardiomyopathy (HCM), which is often linked to mutations in sarcomeric proteins. To address the effect of these agents in developing sarcomeres, we isolated heart fiber bundles, extracted membranes with Triton X-100, and measured tension developed over a range of Ca2+ concentrations with and without OM or Mava treatment. We made measurements in fiber bundles from hearts of adult nontransgenic (NTG) controls expressing cardiac troponin I (cTnI), and from hearts of transgenic (TG-ssTnI) mice expressing the fetal/neonatal form, slow skeletal troponin I (ssTnI). We also compared fibers from 7- and 14-day-old NTG mice expressing ssTnI and cTnI. These studies were repeated with 7- and 14-day-old transgenic mice (TG-cTnT-R92Q) expressing a mutant form of cardiac troponin T (cTnT) linked to HCM. OM increased Ca2+-sensitivity and decreased cooperative activation in both ssTnI- and cTnI-regulated myofilaments with a similar effect: reducing submaximal tension in immature and mature myofilaments. Although Mava decreased tension similarly in cTnI- and ssTnI-regulated myofilaments controlled either by cTnT or cTnT-R92Q, its effect involved a depressed Ca2+-sensitivity in the mature cTnT-R92 myofilaments. Our data demonstrate an influence of myosin and thin-filament associated proteins on the actions of myosin-directed agents such as OM and Mava. SIGNIFICANCE STATEMENT: The effects of myosin-targeted activators and inhibitors on Ca2+-activated tension in developing cardiac sarcomeres presented here provide novel, ex vivo evidence as to their actions in early-stage cardiac disorders. These studies advance understanding of the molecular mechanisms of these agents, which are important in preclinical studies employing sarcomere Ca2+-response as a screening approach. The data also inform the use of commonly immature cardiac myocytes generated from human-inducible pluripotent stem cells in screening for sarcomere activators and inhibitors.

Implications of S-glutathionylation of sarcomere proteins in cardiac disorders, therapies, and diagnosis.

The discovery that cardiac sarcomere proteins are substrates for S-glutathionylation and that this post-translational modification correlates strongly with diastolic dysfunction led to new concepts regarding how levels of oxidative stress affect the heartbeat. Major sarcomere proteins for which there is evidence of S-glutathionylation include cardiac myosin binding protein C (cMyBP-C), actin, cardiac troponin I (cTnI) and titin. Our hypothesis is that these S-glutathionylated proteins are significant factors in acquired and familial disorders of the heart; and, when released into the serum, provide novel biomarkers. We consider the molecular mechanisms for these effects in the context of recent revelations of how these proteins control cardiac dynamics in close collaboration with Ca2+ fluxes. These revelations were made using powerful approaches and technologies that were focused on thin filaments, thick filaments, and titin filaments. Here we integrate their regulatory processes in the sarcomere as modulated mainly by neuro-humoral control of phosphorylation inasmuch evidence indicates that S-glutathionylation and protein phosphorylation, promoting increased dynamics and modifying the Frank-Starling relation, may be mutually exclusive. Earlier studies demonstrated that in addition to cTnI as a well-established biomarker for cardiac disorders, serum levels of cMyBP-C are also a biomarker for cardiac disorders. We describe recent studies approaching the question of whether serum levels of S-glutathionylated-cMyBP-C could be employed as an important clinical tool in patient stratification, early diagnosis in at risk patients before HFpEF, determination of progression, effectiveness of therapeutic approaches, and as a guide in developing future therapies.

Mechanisms of troponin release into serum in cardiac injury associated with COVID-19 patients.

Serum levels of thin filament proteins, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) employing high sensitivity antibodies provide a state-of-the art determination of cardiac myocyte injury in COVID-19 patients. Although there is now sufficient evidence of the value of these determinations in patients infected with SARS-CoV-2, mechanisms of their release have not been considered in depth. We summarize the importance of these mechanisms with emphasis on their relation to prognosis, stratification, and treatment of COVID-19 patients. Apart from frank necrotic cell death, there are other mechanisms of myocyte injury leading to membrane fragility that provoke release of cTnT and cTnI. We discuss a rationale for understanding these mechanisms in COVID-19 patients with co-morbidities associated with myocyte injury such as heart failure, hypertension, arrythmias, diabetes, and inflammation. We describe how understanding these significant aspects of these mechanisms in the promotion of angiotensin signaling by SARS-CoV-2 can affect treatment options in the context of individualized therapies. Moreover, with likely omic data related to serum troponins and with the identification of elevations of serum troponins now more broadly detected employing high sensitivity antibodies, we think it is important to consider molecular mechanisms of elevations in serum troponin as an element in clinical decisions and as a critical aspect of development of new therapies.

Negative inotropic mechanisms of ß-cardiotoxin in cardiomyocytes by depression of myofilament ATPase activity without activation of the classical ß-adrenergic pathway.

Beta-cardiotoxin (ß-CTX) from the king cobra venom (Ophiophagus hannah) was previously proposed as a novel ß-adrenergic blocker. However, the involvement of ß-adrenergic signaling by this compound has never been elucidated. The objectives of this study were to investigate the underlying mechanisms of ß-CTX as a ß-blocker and its association with the ß-adrenergic pathway. The effects of ß-CTX on isolated cardiac myocyte functions, calcium homeostasis, the phosphorylation level of targeted proteins, and the myofibrillar ATPase activity were studied. Healthy Sprague Dawley rats were used for cardiomyocytes isolation. Like propranolol, ß-CTX attenuated the cardiomyocyte inotropy and calcium transient alterations as induced by isoproterenol stimulation. In contrast, these effects were not observed in forskolin-treated cells. Interestingly, cardiomyocytes treated with ß-CTX showed no changes in phosphorylation level at any PKA-targeted sites in the myofilaments as demonstrated in Western blot analysis. The skinned fibers study revealed no change in myofilament kinetics by ß-CTX. However, this protein exhibited the direct inhibition of myofibrillar ATPase activity with calcium de-sensitization of the enzyme. In summary, the negative inotropic mechanism of ß-CTX was discovered. ß-CTX exhibits an atypical ß-blocker mechanism. These properties of ß-CTX may benefit in developing a novel agent aid to treat hypertrophic cardiomyopathy.

NH2-Terminal Cleavage of Cardiac Troponin I Signals Adaptive Response to Cardiac Stressors.

Cardiac sarcomeres express a variant of troponin I (cTnI) that contains a unique N-terminal extension of ~30 amino acids with regulatory phosphorylation sites. The extension is important in the control of myofilament response to Ca2+, which contributes to the neuro-humoral regulation of the dynamics of cardiac contraction and relaxation. Hearts of various species including humans express a stress-induced truncated variant of cardiac troponin I (cTnI-ND) missing the first ~30 amino acids and functionally mimicking the phosphorylated state of cTnI. Studies have demonstrated that upregulation of cTnI-ND potentially represents a homeostatic mechanism as well as an adaptive response in pathophysiology including ischemia/reperfusion injury, beta adrenergic maladaptive activation, and aging. We present evidence showing that cTnI-ND can modify the trigger for hypertrophic cardiomyopathy (HCM) by reducing the Ca2+ sensitivity of myofilaments from hearts with an E180G mutation in α-tropomyosin. Induction of this truncation may represent a therapeutic approach to modifying Ca2+-responses in hearts with hypercontractility or heat failure with preserved ejection fraction.

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

Experiments reported here tested the hypothesis that ß-arrestin-2 is an important element in the preservation of cardiac function during aging. We tested this hypothesis by aging ß-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 ß-arrestin-2 signaling. ß-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 ß-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 ß-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 ß-arrestin-2 as an important signaling mechanism that preserves cardiac function during aging.