Myosin-binding protein H-like regulates myosin-binding protein distribution and function in atrial cardiomyocytes.

Mutations in atrial-enriched genes can cause a primary atrial myopathy that can contribute to overall cardiovascular dysfunction. MYBPHL encodes myosin-binding protein H-like (MyBP-HL), an atrial sarcomere protein that shares domain homology with the carboxy-terminus of cardiac myosin-binding protein-C (cMyBP-C). The function of MyBP-HL and the relationship between MyBP-HL and cMyBP-C is unknown. To decipher the roles of MyBP-HL, we used structured illumination microscopy, immuno-electron microscopy, and mass spectrometry to establish the localization and stoichiometry of MyBP-HL. We found levels of cMyBP-C, a major regulator of myosin function, were half as abundant compared to levels in the ventricle. In genetic mouse models, loss of MyBP-HL doubled cMyBP-C abundance in the atria, and loss of cMyBP-C doubled MyBP-HL abundance in the atria. Structured illumination microscopy showed that both proteins colocalize in the C-zone of the A-band, with MyBP-HL enriched closer to the M-line. Immuno-electron microscopy of mouse atria showed MyBP-HL strongly localized 161 nm from the M-line, consistent with localization to the third 43 nm repeat of myosin heads. Both cMyBP-C and MyBP-HL had less-defined sarcomere localization in the atria compared to ventricle, yet areas with the expected 43 nm repeat distance were observed for both proteins. Isometric force measurements taken from control and Mybphl null single atrial myofibrils revealed that loss of Mybphl accelerated the linear phase of relaxation. These findings support a mechanism where MyBP-HL regulates cMyBP-C abundance to alter the kinetics of sarcomere relaxation in atrial sarcomeres.

BAG3 localizes to mitochondria in cardiac fibroblasts and regulates mitophagy.

The co-chaperone Bcl2-associated athanogene 3 (BAG3) is a central node in protein quality control in the heart. In humans and animal models, decreased BAG3 expression is associated with cardiac dysfunction and dilated cardiomyopathy. Although previous studies focused on BAG3 in cardiomyocytes, cardiac fibroblasts are also critical drivers of pathologic remodeling. Yet, the role of BAG3 in cardiac fibroblasts is almost completely unexplored. Here, we show that BAG3 is expressed in primary rat neonatal cardiac fibroblasts and preferentially localizes to mitochondria. Knockdown of BAG3 reduces mitophagy and enhances fibroblast activation, which is associated with fibrotic remodeling. Heat shock protein 70 (Hsp70) is a critical binding partner for BAG3 and inhibiting this interaction in fibroblasts using the drug JG-98 decreased autophagy, decreased mitofusin-2 expression, and disrupted mitochondrial morphology. Together, these data indicate that BAG3 is expressed in cardiac fibroblasts, where it facilitates mitophagy and promotes fibroblast quiescence. This suggests that depressed BAG3 levels in heart failure may exacerbate fibrotic pathology, thus contributing to myocardial dysfunction through sarcomere-independent pathways.NEW & NOTEWORTHY We report BAG3's localization to mitochondria and its role in mitophagy for the first time in primary ventricular cardiac fibroblasts. We have also collected the first evidence showing that loss of BAG3 increases cardiac fibroblast activation into myofibroblasts, which are major drivers of cardiac fibrosis and pathological remodeling during heart disease.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance, often treated via electrical cardioversion. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs, suggesting that defects in contractility occur in AFib and are revealed upon restoration of rhythm. This project aims to define the contractile remodeling that occurs in AFib. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. Atrial cardiomyocytes showed reduced force of contraction, decreased resting tension, and increased calcium sensitivity in skinned single cardiomyocyte studies. These alterations correlated with degradation of myofilament proteins including myosin heavy chain altering force of contraction, titin altering resting tension, and TnI altering calcium sensitivity. We measured degradation of other myofilament proteins including cMyBP-C and actininshowing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. These results provide an understanding of the contractile remodeling that occurs in AFib and provide insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.

GSK-3ß Localizes to the Cardiac Z-Disc to Maintain Length Dependent Activation.

BACKGROUND: Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3ß (glycogen synthase kinase 3ß) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3ß's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. METHODS: Inducible cardiomyocyte-specific GSK-3ß knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. RESULTS: Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3ß. Agreeing with the localization of its targets, GSK-3ß that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3ß in neonatal rat ventricular cardiomyocytes. One of GSK-3ß's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3ß. Last, GSK-3ß myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. CONCLUSIONS: We identified a novel mechanism by which GSK-3ß localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3ß levels were reduced in patients with heart failure, indicating z-disc localized GSK-3ß is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

Myofilament glycation in diabetes reduces contractility by inhibiting tropomyosin movement, is rescued by cMyBPC domains.

Diabetes doubles the risk of developing heart failure (HF). As the prevalence of diabetes grows, so will HF unless the mechanisms connecting these diseases can be identified. Methylglyoxal (MG) is a glycolysis by-product that forms irreversible modifications on lysine and arginine, called glycation. We previously found that myofilament MG glycation causes sarcomere contractile dysfunction and is increased in patients with diabetes and HF. The aim of this study was to discover the molecular mechanisms by which MG glycation of myofilament proteins cause sarcomere dysfunction and to identify therapeutic avenues to compensate. In humans with type 2 diabetes without HF, we found increased glycation of sarcomeric actin compared to non-diabetics and it correlated with decreased calcium sensitivity. Depressed calcium sensitivity is pathogenic for HF, therefore myofilament glycation represents a promising therapeutic target to inhibit the development of HF in diabetics. To identify possible therapeutic targets, we further defined the molecular actions of myofilament glycation. Skinned myocytes exposed to 100 µM MG exhibited decreased calcium sensitivity, maximal calcium-activated force, and crossbridge kinetics. Replicating MG's functional affects using a computer simulation of sarcomere function predicted simultaneous decreases in tropomyosin's blocked-to-closed rate transition and crossbridge duty cycle were consistent with all experimental findings. Stopped-flow experiments and ATPase activity confirmed MG decreased the blocked-to-closed transition rate. Currently, no therapeutics target tropomyosin, so as proof-of-principal, we used a n-terminal peptide of myosin-binding protein C, previously shown to alter tropomyosin's position on actin. C0C2 completely rescued MG-induced calcium desensitization, suggesting a possible treatment for diabetic HF.

Pharmacological inhibition of BAG3-HSP70 with the proposed cancer therapeutic JG-98 is toxic for cardiomyocytes.

The co-chaperone Bcl2-associated athanogene-3 (BAG3) maintains cellular protein quality control through the regulation of heat shock protein 70 (HSP70). Cancer cells manipulate BAG3-HSP70-regulated pathways for tumor initiation and proliferation, which has led to the development of promising small molecule therapies, such as JG-98, which inhibit the BAG3-HSP70 interaction and mitigate tumor growth. However, it is not known how these broad therapies impact cardiomyocytes, where the BAG3-HSP70 complex is a key regulator of protein turnover and contractility. Here, we show that JG-98 exposure is toxic in neonatal rat ventricular myocytes (NRVMs). Using immunofluorescence microscopy to assess cell death, we found that apoptosis increased in NRVMs treated with JG-98 doses as low as 10 nM. JG-98 treatment also reduced autophagy flux and altered expression of BAG3 and several binding partners involved in BAG3-dependent autophagy, including SYNPO2 and HSPB8. We next assessed protein half-life with disruption of the BAG3-HSP70 complex by treating with JG-98 in the presence of cycloheximide and found BAG3, HSPB5, and HSPB8 half-lives were reduced, indicating that complex formation with HSP70 is important for their stability. Next, we assessed sarcomere structure using super-resolution microscopy and found that disrupting the interaction with HSP70 leads to sarcomere structural disintegration. To determine whether the effects of JG-98 could be mitigated by pharmacological autophagy induction, we cotreated NRVMs with rapamycin, which partially reduced the extent of apoptosis and sarcomere disarray. Finally, we investigated whether the effects of JG-98 extended to skeletal myocytes using C2C12 myotubes and found again increased apoptosis and reduced autophagic flux. Together, our data suggest that nonspecific targeting of the BAG3-HSP70 complex to treat cancer may be detrimental for cardiac and skeletal myocytes.

BAG3 expression and sarcomere localization in the human heart are linked to HSF-1 and are differentially affected by sex and disease.

Mutations to the sarcomere-localized cochaperone protein Bcl2-associated athanogene 3 (BAG3) are associated with dilated cardiomyopathy (DCM) and display greater penetrance in male patients. Decreased protein expression of BAG3 is also associated with nongenetic heart failure; however, the factors regulating cardiac BAG3 expression are unknown. Using left ventricular (LV) tissue from nonfailing and DCM human samples, we found that whole LV BAG3 expression was not significantly impacted by DCM or sex; however, myofilament localized BAG3 was significantly decreased in males with DCM. Females with DCM displayed no changes in BAG3 compared with nonfailing. This sex difference appears to be estrogen independent, as estrogen treatment in ovariectomized female rats had no impact on BAG3 expression. BAG3 gene expression in noncardiac cells is primarily regulated by the heat shock transcription factor-1 (HSF-1). We show whole LV HSF-1 expression and nuclear localized/active HSF-1 each displayed a striking positive correlation with whole LV BAG3 expression. We further found that HSF-1 localizes to the sarcomere Z-disc in cardiomyocytes and that this myofilament-associated HSF-1 pool decreases in heart failure. The decrease of HSF-1 was more pronounced in male patients and tightly correlated with myofilament BAG3 expression. Together our findings indicate that cardiac BAG3 expression and myofilament localization are differentially impacted by sex and disease and are linked to HSF-1.NEW & NOTEWORTHY Myofilament BAG3 expression decreases in male patients with nonischemic DCM but is preserved in female patients with DCM. BAG3 expression in the human heart is tightly linked to HSF-1 expression and nuclear translocation. HSF-1 localizes to the sarcomere Z-disc in the human heart. HSF-1 expression in the myofilament fraction decreases in male patients with DCM and positively correlates with myofilament BAG3.

Reperfused vs. nonreperfused myocardial infarction: when to use which model.

There is a lack of understanding in the cardiac remodeling field regarding the use of nonreperfused myocardial infarction (MI) and reperfused MI in animal models of MI. This Perspectives summarizes the consensus of the authors regarding how to select the optimum model for your experiments and is a part of ongoing efforts to establish rigor and reproducibility in cardiac physiology research.

Guidelines for in vivo mouse models of myocardial infarction.

Despite significant improvements in reperfusion strategies, acute coronary syndromes all too often culminate in a myocardial infarction (MI). The consequent MI can, in turn, lead to remodeling of the left ventricle (LV), the development of LV dysfunction, and ultimately progression to heart failure (HF). Accordingly, an improved understanding of the underlying mechanisms of MI remodeling and progression to HF is necessary. One common approach to examine MI pathology is with murine models that recapitulate components of the clinical context of acute coronary syndrome and subsequent MI. We evaluated the different approaches used to produce MI in mouse models and identified opportunities to consolidate methods, recognizing that reperfused and nonreperfused MI yield different responses. The overall goal in compiling this consensus statement is to unify best practices regarding mouse MI models to improve interpretation and allow comparative examination across studies and laboratories. These guidelines will help to establish rigor and reproducibility and provide increased potential for clinical translation.

Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease.

Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Under construction: The dynamic assembly, maintenance, and degradation of the cardiac sarcomere.

The sarcomere is the basic contractile unit of striated muscle and is a highly ordered protein complex with the actin and myosin filaments at its core. Assembling the sarcomere constituents into this organized structure in development, and with muscle growth as new sarcomeres are built, is a complex process coordinated by numerous factors. Once assembled, the sarcomere requires constant maintenance as its continuous contraction is accompanied by elevated mechanical, thermal, and oxidative stress, which predispose proteins to misfolding and toxic aggregation. To prevent protein misfolding and maintain sarcomere integrity, the sarcomere is monitored by an assortment of protein quality control (PQC) mechanisms. The need for effective PQC is heightened in cardiomyocytes which are terminally differentiated and must survive for many years while preserving optimal mechanical output. To prevent toxic protein aggregation, molecular chaperones stabilize denatured sarcomere proteins and promote their refolding. However, when old and misfolded proteins cannot be salvaged by chaperones, they must be recycled via degradation pathways: the calpain and ubiquitin-proteasome systems, which operate under basal conditions, and the stress-responsive autophagy-lysosome pathway. Mutations to and deficiency of the molecular chaperones and associated factors charged with sarcomere maintenance commonly lead to sarcomere structural disarray and the progression of heart disease, highlighting the necessity of effective sarcomere PQC for maintaining cardiac function. This review focuses on the dynamic regulation of assembly and turnover at the sarcomere with an emphasis on the chaperones involved in these processes and describes the alterations to chaperones - through mutations and deficient expression - implicated in disease progression to heart failure.

Intact myocardial preparations reveal intrinsic transmural heterogeneity in cardiac mechanics.

Determining transmural mechanical properties in the heart provides a foundation to understand physiological and pathophysiological cardiac mechanics. Although work on mechanical characterisation has begun in isolated cells and permeabilised samples, the mechanical profile of living individual cardiac layers has not been examined. Myocardial slices are 300 µm-thin sections of heart tissue with preserved cellular stoichiometry, extracellular matrix, and structural architecture. This allows for cardiac mechanics assays in the context of an intact in vitro organotypic preparation. In slices obtained from the subendocardium, midmyocardium and subepicardium of rats, a distinct pattern in transmural contractility is found that is different from that observed in other models. Slices from the epicardium and midmyocardium had a higher active tension and passive tension than the endocardium upon stretch. Differences in total myocyte area coverage, and aspect ratio between layers underlined the functional readouts, while no differences were found in total sarcomeric protein and phosphoprotein between layers. Such intrinsic heterogeneity may orchestrate the normal pumping of the heart in the presence of transmural strain and sarcomere length gradients in the in vivo heart.

Deletion of cardiac polycystin 2/PC2 results in increased SR calcium release and blunted adrenergic reserve.

Transient receptor potential proteins (TRPs) act as nonselective cation channels. Of the TRP channels, PC2 (also known as polycystin 2) is localized to the sarcoplasmic reticulum (SR); however, its contribution to calcium-induced calcium release and overall cardiac function in the heart is poorly understood. The goal of this study was to characterize the effect of cardiac-specific PC2 deletion in adult cardiomyocytes and in response to chronic ß-adrenergic challenge. We used a temporally inducible model to specifically delete PC2 from cardiomyocytes (Pkd2 KO) and characterized calcium and contractile dynamics in single cells. We found enhanced intracellular calcium release after Pkd2 KO, and near super-resolution microscopy analysis suggested this was due to close localization of PC2 to the ryanodine receptor. At the organ level, speckle-tracking echocardiographical analysis showed increased dyssynchrony in the Pkd2 KO mice. In response to chronic adrenergic stimulus, cardiomyocytes from the Pkd2 KO had no reserve ß-adrenergic calcium responses and significantly attenuated wall motion in the whole heart. Biochemically, without adrenergic stimulus, there was an overall increase in PKA phosphorylated targets in the Pkd2 KO mouse, which decreased following chronic adrenergic stimulus. Taken together, our results suggest that cardiac-specific PC2 limits SR calcium release by affecting the PKA phosphorylation status of the ryanodine receptor, and the effects of PC2 loss are exacerbated upon adrenergic challenge.NEW & NOTEWORTHY Our goal was to characterize the role of the transient receptor potential channel polycystin 2 (PC2) in cardiomyocytes following adult-onset deletion. Loss of PC2 resulted in decreased cardiac shortening and cardiac dyssynchrony and diminished adrenergic reserve. These results suggest that cardiac-specific PC2 modulates intracellular calcium signaling and contributes to the maintenance of adrenergic pathways.

Right Ventricular Myofilament Functional Differences in Humans With Systemic Sclerosis-Associated Versus Idiopathic Pulmonary Arterial Hypertension.

BACKGROUND: Patients with systemic sclerosis (SSc)-associated pulmonary arterial hypertension (PAH) have a far worse prognosis than those with idiopathic PAH (IPAH). In the intact heart, SSc-PAH exhibits depressed rest and reserve right ventricular (RV) contractility compared with IPAH. We tested whether this disparity involves underlying differences in myofilament function. METHODS: Cardiac myocytes were isolated from RV septal endomyocardial biopsies from patients with SSc-PAH, IPAH, or SSc with exertional dyspnea but no resting PAH (SSc-d); control RV septal tissue was obtained from nondiseased donor hearts (6-7 per group). Isolated myocyte passive length-tension and developed tension-calcium relationships were determined and correlated with in vivo RV function and reserve. RV septal fibrosis was also examined. RESULTS: Myocyte passive stiffness from length-tension relations was similarly increased in IPAH and SSc-PAH compared with control, although SSc-PAH biopsies had more interstitial fibrosis. More striking disparities were found between active force-calcium relations. Compared with controls, maximal calcium-activated force (Fmax) was 28% higher in IPAH but 37% lower in SSc-PAH. Fmax in SSc-d was intermediate between control and SSc-PAH. The calcium concentration required for half-maximal force (EC50) was similar between control, IPAH, and SSc-d but lower in SSc-PAH. This disparity disappeared in myocytes incubated with the active catalytic subunit of protein kinase A. Myocyte Fmax directly correlated with in vivo RV contractility assessed by end-systolic elastance (R2 =0.46, P=0.002) and change in end-systolic elastance with exercise (R2 =0.49, P=0.008) and was inversely related with exercise-induced chamber dilation (R2 =0.63, P<0.002), which also was a marker of depressed contractile reserve. CONCLUSIONS: A primary defect in human SSc-PAH resides in depressed sarcomere function, whereas this is enhanced in IPAH. These disparities correlate with in vivo RV contractility and contractile reserve and are consistent with worse clinical outcomes in SSc-PAH. The existence of sarcomere disease before the development of resting PAH in patients with SSc-d suggests that earlier identification and intervention may prove useful.

Estrogen but not testosterone preserves myofilament function from doxorubicin-induced cardiotoxicity by reducing oxidative modifications.

Here, we aimed to explore sex differences and the impact of sex hormones on cardiac contractile properties in doxorubicin (DOX)-induced cardiotoxicity. Male and female Sprague-Dawley rats were subjected to sham surgery or gonadectomy and then treated or untreated with DOX (2 mg/kg) every other week for 10 wk. Estrogen preserved maximum active tension (Tmax) with DOX exposure, whereas progesterone and testosterone did not. The effects of sex hormones and DOX correlated with both altered myosin heavy chain isoform expression and myofilament protein oxidation, suggesting both as possible mechanisms. However, acute treatment with oxidative stress (H2O2) or a reducing agent (DTT) indicated that the effects on Tmax were mediated by reversible myofilament oxidative modifications and not only changes in myosin heavy chain isoforms. There were also sex differences in the DOX impact on myofilament Ca2+ sensitivity. DOX increased Ca2+ sensitivity in male rats only in the absence of testosterone and in female rats only in the presence of estrogen. Conversely, DOX decreased Ca2+ sensitivity in female rats in the absence of estrogen. In most instances, this mechanism was through altered phosphorylation of troponin I at Ser23/Ser24. However, there was an additional DOX-induced, estrogen-dependent, irreversible (by DTT) mechanism that altered Ca2+ sensitivity. Our data demonstrate sex differences in cardiac contractile responses to chronic DOX treatment. We conclude that estrogen protects against chronic DOX treatment in the heart, preserving myofilament function. NEW & NOTEWORTHY We identified sex differences in cardiotoxic effects of chronic doxorubicin (DOX) exposure on myofilament function. Estrogen, but not testosterone, decreases DOX-induced oxidative modifications on myofilaments to preserve maximum active tension. In rats, DOX exposure increased Ca2+ sensitivity in the presence of estrogen but decreased Ca2+ sensitivity in the absence of estrogen. In male rats, the DOX-induced shift in Ca2+ sensitivity involved troponin I phosphorylation; in female rats, this was through an estrogen-dependent mechanism.

Phospho-Proteomic Analysis of Cardiac Dyssynchrony and Resynchronization Therapy.

Cardiac dyssynchrony arises from conduction abnormalities during heart failure and worsens morbidity and mortality. Cardiac resynchronization therapy (CRT) re-coordinates contraction using bi-ventricular pacing, but the cellular and molecular mechanisms involved remain largely unknown. The aim is to determine how dyssynchronous heart failure (HFdys ) alters the phospho-proteome and how CRT interacts with this unique phospho-proteome by analyzing Ser/Thr and Tyr phosphorylation. Phospho-enriched myocardium from dog models of Control, HFdys , and CRT is analyzed via MS. There were 209 regulated phospho-sites among 1761 identified sites. Compared to Con and CRT, HFdys is hyper-phosphorylated and tyrosine phosphorylation is more likely to be involved in signaling that increased with HFdys and was exacerbated by CRT. For each regulated site, the most-likely targeting-kinase is predicted, and CK2 is highly specific for sites that are "fixed" by CRT, suggesting activation of CK2 signaling occurs in HFdys that is reversed by CRT, which is supported by western blot analysis. These data elucidate signaling networks and kinases that may be involved and deserve further study. Importantly, a possible role for CK2 modulation in CRT has been identified. This may be harnessed in the future therapeutically to compliment CRT, improving its clinical effects.