Abstract
Heart failure (HF) as a result of myocardial infarction (MI) is a major cause of fatality worldwide. However, the cause of cardiac dysfunction succeeding MI has not been elucidated at a sarcomeric level. Thus, studying the alterations within the sarcomere is necessary to gain insights on the fundamental mechansims leading to HF and potentially uncover appropriate therapeutic targets. Since existing research portrays regulatory light chains (RLC) to be mediators of cardiac muscle contraction in both human and animal models, its role was further explored In this study, a detailed characterisation of the physiological changes (i.e., isometric force, calcium sensitivity and sarcomeric protein phosphorylation) was assessed in an MI mouse model, between 2D (2 days) and 28D post-MI, and the changes were related to the phosphorylation status of RLCs. MI mouse models were created via complete ligation of left anterior descending (LAD) coronary artery. Left ventricular (LV) papillary muscles were isolated and permeabilised for isometric force and Ca2+ sensitivity measurement, while the LV myocardium was used to assay sarcomeric proteins’ (RLC, troponin I (TnI) and myosin binding protein-C (MyBP-C)) phosphorylation levels and enzyme (myosin light chain kinase (MLCK), zipper interacting protein kinase (ZIPK) and myosin phosphatase target subunit 2 (MYPT2)) expression levels. Finally, the potential for improving the contractility of diseased cardiac papillary fibres via the enhancement of RLC phosphorylation levels was investigated by employing RLC exchange methods, in vitro. RLC phosphorylation and isometric force potentiation were enhanced in the compensatory phase and decreased in the decompensatory phase of HF failure progression, respectively. There was no significant time-lag between the changes in RLC phosphorylation and isometric force during HF progression, suggesting that changes in RLC phosphorylation immediately affect force generation. Additionally, the in vitro increase in RLC phosphorylation levels in 14D post-MI muscle segments (decompensatory stage) enhanced its force of isometric contraction, substantiating its potential in HF treatment. Longitudinal observation unveils potential mechanisms involving MyBP-C and key enzymes regulating RLC phosphorylation, such as MLCK and MYPT2 (subunit of MLCP), during HF progression. This study primarily demonstrates that RLC phosphorylation is a key sarcomeric protein modification modulating cardiac function. This substantiates the possibility of using RLCs and their associated enzymes to treat HF.
Highlights
Heart failure (HF) is a clinical syndrome where the ability of the heart to pump blood to the rest of the body is compromised
The results collectively showed that the hearts concurrently displayed characteristics of hypertrophic and dilated cardiomyopathy and that the hearts failed with time
C57BL/6 is useful for studying HF progression, as it displays characteristics of a failing heart by 28D post-myocardial infarction (MI) [38,39], while rats take longer at 20–32 weeks post-MI [42], where MI is induced by coronary artery ligation
Summary
Heart failure (HF) is a clinical syndrome where the ability of the heart to pump blood to the rest of the body is compromised. At 2D post-MI, both RLC and TnI phosphorylation decreased significantly below sham control levels with maximal isometric force, while phosphorylation levels of MyBP-C increased above sham levels (Figure 4E). The RLC phosphorylation level increases in the compensatory phase and decreases in the decom‐ pensTathoerycoprhraeslae.tiTonnI,boentwtheeenotthheer hchanandg, sehsoowf sthneorpeasrpteiccutilvarecshaarncogme ienriTcnpI rpohtoeisnphpohroyslap‐horyltaitoinonlelveevlse.lsThaendreissuoltms esutrgicgefsotracne winatesrraenlatliyosnesdhiupsbinegtwSepeenaRrmLCana’nsdcoMryreBlPa‐tCionphtoespt.hTohr‐ere is aylsattrioonng(Faingdurnee4gFa)t,iavlethcoourgrehlaittsiounndbeertlwyienegnmMeychBaPn-iCsmpshaorsepuhnokrnyolawtino.nTahnedloinsgoimtuedtirnicalforce (pr r=of−ile0.a9l4s)o, swhhoiwchs tihsasttathtiestMicyalBlyP‐sCigpnhifioscpahnotr(yTlaabtiloenSp2)a.ttern presents a progression that is opposite to the isometric force profile (Figure 4E), which is contrary to the current ob‐ 2s.e5r.vMatoiodnusl.atTihone pofrothteiPnhpohsopshporhyolraytiloantioLnevlelvoeflsRaLrCe sIunmdumceasriRsedcoivnerTyaobflethSe1C. ZIPK expression levels of sham populations, on the other hand, seemed to increase very gradually from the timepoint of 2D post-MI, the differences among the means of all timepoints were not statistically significant (according to ANOVA test). The expression levels of all three enzymes increased at the 28D post-MI timepoint, corresponding to a significant decrease in RLC phosphorylation. To isolate the roles of the individual enzymes, other forms of study models (e.g., causative models such as gene knockout) must be employed
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