Abstract
Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control the length of titin. We hypothesized that together with diastolic [Ca2+], titin‐based stretch pre‐activates cardiomyocytes during diastole and is a major determinant of force production in the subsequent contraction. Through this mechanism titin would play an important role in active force development and length‐dependent activation. Mutations in the splicing factor RNA binding motif protein 20 (RBM20) result in expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work loops that mimic the cardiac cycle in wild‐type (WT) and heterozygous (HET) RBM20‐deficient rats. In addition, we studied the role of diastolic [Ca2+] in membrane‐permeabilized WT and HET cardiomyocytes. Intact cardiomyocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT) at low pacing frequencies, but this difference disappeared at high pacing frequencies. Length‐dependent activation (force–sarcomere length relationship) was blunted in HET cardiomyocytes, but the force–end‐diastolic force relationship was not different between HET and WT cardiomyocytes. To delineate the effects of diastolic [Ca2+] and titin pre‐activation on force generation, measurements were performed in detergent‐permeabilized cardiomyocytes. Cardiac twitches were simulated by transiently exposing permeabilized cardiomyocytes to 2 µm Ca2+. Increasing diastolic [Ca2+] from 1 to 80 nm increased force development twofold in WT. Higher diastolic [Ca2+] was needed in HET. These findings are consistent with our hypothesis that pre‐activation increases active force development. Highly compliant titin allows cells to function at higher diastolic [Ca2+].
Highlights
During exercise and increased stress, cardiac work is adjusted to meet the increased demands of the body
Because phosphorylation of Cardiac troponin I (cTnI) and PLN is an important regulator of cardiac contractility, we studied their phosphorylation levels
It was later identified that mutations in the splicing factor RNA binding motif protein 20 (RBM20) caused these compliant titin molecules (Guo et al 2012) and that mutations in the RBM20 gene can cause dilated cardiomyopathy (Brauch et al 2009; Guo et al 2012)
Summary
During exercise and increased stress, cardiac work is adjusted to meet the increased demands of the body. Mutations in RBM20 have been identified as a cause for dilated cardiomyopathy in patients, and were associated with expression of highly compliant ‘giant’ titin isoforms (Brauch et al 2009; Guo et al 2012; Beqqali et al 2016). While large compliant titin isoforms reduce passive tension (Greaser et al 2008; Patel et al 2012; Methawasin et al 2014) and may improve cardiac relaxation (Methawasin et al 2014), expression of giant titin isoforms has been associated with reductions in maximal force (Patel et al 2012; Mateja et al 2013; Methawasin et al 2014) and length-dependent activation (Patel et al 2012; Methawasin et al 2014). Having a large compliant titin isoform might confer some benefits, as exercise capacity was increased in heterozygote RBM20 mutant mice (Methawasin et al 2014)
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