Hypertrophic cardiomyopathy (HCM) is a genetic disease caused by mutations in sarcomeric proteins. Mutations in sarcomeric proteins that give rise to cardiomyopathies produce abnormalities in the biophysical properties of the sarcomere that are propagated beyond the cardiac myocyte. In this study, we evaluated the hypothesis that early desensitization of myofilament Ca2+ sensitivity in the TnT-R92Q mouse model, achieved through the introduction of pseudo-phosphorylated TnI (TnI-S23,24D or TnI-DD), may delay the progression of the HCM phenotype in cardiac myocyte and endothelial cellular compartments of the heart. We studied non-transgenic mice, transgenic (TG) mice expressing TnT-R92Q (TnT-R92Q), TG mice expressing TnI-DD, and double transgenic mice expressing TnT-R92Q and TnI-DD at 28 days and 16 weeks of age. Experiments reported here demonstrate that expression of TnI-DD in the TnT-R92Q HCM mouse model results in partial normalization of myofilament Ca2+ sensitivity, improved cardiac morphology and function, reduced fibrosis, normalization of YAP signaling in endothelial cells, but a lack of normalization of coronary flow parameters. The novelty of the approach reported here highlights that although small corrections made to offset the sarcomeric defect may not fully or immediately resolve the disease’s pathophysiologic state, they can lessen the severity of HCM. Our findings further support the concept that early desensitization of myofilaments to Ca2+ in HCM, mainly when arising from mutations in thin filament proteins, represents a promising avenue for developing new therapeutic drugs.

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 with > 40% reduction in MLC2a phosphorylation. 12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. 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, with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. 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 regulator of atrial contractility in AF.

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 ± 18.81 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.

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.

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 μ m 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.

The hypothesis tested is that unloading of mechanical forces affects acetylation, ubiquitination, and phosphorylation of the actin-binding protein α-actinin located in the Z-disc. We applied targeted proteomics to interrogate the post-translational modification changes during loading and unloading. Cell morphology and post-translational modifications were determined in a mechanical intervention consisting of 1 Hz cyclic strain for 24 hr (loaded) followed by 6 hr rest (unloaded) of cultured neonatal rat ventricular myocytes (NRVMs). This was compared to a chemical intervention consisting of treating NRVMs with the myosin inhibitor Mavacamten (1 μM, 6hr) or activator Omecamtiv Mecarbil (0.5 μM, 6hr). Quantitative immunofluorescence showed both chemical and mechanical loading increased α-actinin content while Mavacamten decreased the α-actinin content. Mass spectrometry analysis of affinity-purified α-actinin revealed that the mechanical intervention led to increased levels of post-translational modifications compared with the chemical interventions. Specifically, α-actinin ubiquitination increased with mechanical loading-unloading; acetylation decreased with mechanical loading-unloading and increased with mechanical loading; and phosphorylation remained unchanged with mechanical loading-unloading but increased with mechanical loading. Fluorescence recovery after photobleaching (FRAP) experiments demonstrated that Mavacamten increased the dynamics of overexpressed YFP-tagged α-actinin and a GFP-tagged CapZ in NRVMs when compared to Omecamtiv Mecarbil treatments and controls. Overall, the results suggest a link between sarcomere homeostasis and mechanical forces via mechanisms involving acetylation, phosphorylation and ubiquitination of α-actinin and a second Z-disc protein, CapZ. These findings could have consequences for cardiac heart disease with abnormal sarcomeric proteostasis. Funded by NIH grants HL151825 (CS) and HL62426 (RJS, BR, and CMW).

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.

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.

Introduction: The production of functional proteins is a multistep process whereby key maturation and regulatory processes occur co-translationally by coupling mRNA translation and nascent polypeptide chain maturation on the ribosome. However, limited data exist regarding co-translational regulation in the cells of the intact cardiovascular system. To overcome this limitation, we adapted the translating ribosome affinity purification (TRAP) approach using the HA-tagged ribosomal protein L22 reporter mouse (RiboTag) crossed with an inducible endothelial-specific Cdh5creERT2 mouse ( RiboTag EC ). Hypothesis: We hypothesized this model would permit the simultaneous immunoprecipitation (IP) of mRNA and nascent proteins for RNAseq and proteomic analysis from the coronary endothelium. Methods: Mice were injected with E. coli lipopolysaccharide (LPS) (6mg/Kg, i.p. 12 hours) to induce sepsis and perturb endothelial cell function. Hearts were homogenized, a small amount (~10%) was used for RNA-Seq and proteomics input controls (IN), and the remainder was used to IP ribosomal bound polyA+ mRNA and nascent proteins. Eluted proteins were digested, labeled with isobaric tags for quantification, and analyzed by MS3 with a Thermo Scientific Fusion Lumos Orbitrap. Results: Principal components analysis of the RNAseq data showed clear separation of the IP (endothelial cells) and IN (whole heart) transcriptomes between control and LPS groups. Transcripts characteristic of endothelial cells (EC) were enriched, whereas markers characteristic of cardiomyocytes, fibroblasts, and smooth muscle cells were depleted. Overall, >7000 proteins were identified, and initial analysis showed similar depletion and enrichment of proteins in the control and LPS hearts. Alignment of transcript and protein datasets was used for bioinformatic analysis. A hierarchical view of one of the top networks in which concordant expression of both EC transcripts and proteins identified interferon-gamma (IFNG) as an upstream signal in the pathogenesis of sepsis. Conclusions: Our “functional translatome” approach is a new discovery platform for a deeper predictive analysis of functionally relevant signaling networks and disease pathways in the intact cardiovascular system.

Introduction: Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 6.1 million . AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light chain phosphorylation in AF. Objective We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12CPP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

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.

A dominant mechanism of sudden cardiac death in the young is the progression of maladaptive responses to genes encoding proteins linked to hypertrophic cardiomyopathy. Most are mutant sarcomere proteins that trigger the progression by imposing a biophysical defect on the dynamics and levels of myofilament tension generation. We discuss approaches for personalized treatments that are indicated by recent advanced understanding of the progression.

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

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.

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.

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>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.