Rationale : Protein kinase C (PKC) regulates contractility of cardiac muscle cells by phosphorylating thin- and thick- filament-based proteins. Myocardial sarcomeres also contain a third myofilament, titin, and it is unknown whether titin can be phosphorylated by PKC and whether it affects passive tension. Objective : The purpose of this study was to examine the effect of PKC on titin phosphorylation and titin-based passive tension. Methods and Results : Phosphorylation assays with PKCα revealed that titin is phosphorylated in skinned myocardial tissues; this effect is exacerbated by pretreating with protein phosphatase 1. In vitro phosphorylation of recombinant protein representing titin’s spring elements showed that PKCα targets the proline – glutamate – valine – lysine (PEVK) spring element. Furthermore, mass spectrometry in combination with site-directed mutagenesis identified 2 highly conserved sites in the PEVK region that are phosphorylated by PKCα (S11878 and S12022); when these 2 sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned left ventricular myocardium revealed that PKCα significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that PKCα decreases the PEVK persistence length (from 1.20 nm to 0.55 nm), without altering the contour length, and using a serially-linked wormlike chain model we show that this increases titin-based passive force with a sarcomere length dependence that is similar to that measured in skinned myocardium after PKCα phosphorylation. Conclusions : PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness.

Background—: Experimentally upregulating compliant titins has been suggested as a therapeutic for lowering pathological diastolic stiffness levels. However, how increasing titin compliance impacts global cardiac function requires in-depth study. We investigate the effect of upregulating compliant titins in a novel mouse model with a genetically altered titin splicing factor; integrative approaches were used from intact cardiomyocyte mechanics to pressure-volume analysis and Doppler echocardiography. Methods and Results—: Compliant titins were upregulated through deletion of the RNA Recognition Motif of the splicing factor RBM20 ( Rbm20 ^ΔRRM mice). A genome-wide exon expression analysis and a candidate approach revealed that the phenotype is likely to be dominated by greatly increased lengths of titin’s spring elements. At both cardiomyocyte and left ventricular chamber levels, diastolic stiffness was reduced in heterozygous (+/−) Rbm20 ^ΔRRM mice with a further reduction in homozygous (−/−) mice at only the intact myocyte level. Fibrosis was present in only −/− Rbm20 ^ΔRRM hearts. The Frank-Starling Mechanism was reduced in a graded fashion in Rbm20 ^ΔRRM mice, at both the cardiomyocyte and left ventricular chamber levels. Exercise tests revealed an increase in exercise capacity in +/− mice. Conclusions—: Titin is not only important in diastolic but also in systolic cardiac function. Upregulating compliant titins reduces diastolic chamber stiffness owing to the increased compliance of myocytes, but it depresses end-systolic elastance; under conditions of exercise, the beneficial effects on diastolic function dominate. Therapeutic manipulation of the RBM20-based splicing system might be able to minimize effects on fibrosis and systolic function while improving the diastolic function in patients with heart failure.

Nemaline myopathy (NM) is the most common non-dystrophic congenital myopathy. Clinically the most important feature of NM is muscle weakness; however, the mechanisms underlying this weakness are poorly understood. Here, we studied the muscular phenotype of NM patients with a well-defined nebulin mutation (NM-NEB), using a multidisciplinary approach to study thin filament length regulation and muscle contractile performance. SDS–PAGE and western blotting revealed greatly reduced nebulin levels in skeletal muscle of NM-NEB patients, with the most prominent reduction at nebulin’s N-terminal end. Muscle mechanical studies indicated ∼60% reduced force generating capacity of NM-NEB muscle and a leftward-shift of the force–sarcomere length relation in NM-NEB muscle fibers. This indicates that the mechanism for the force reduction is likely to include shorter and non-uniform thin filament lengths in NM-NEB muscle compared with control muscle. Immunofluorescence confocal microscopy and electron microscopy studies indicated that average thin filament length is reduced from ∼1.3 µm in control muscle to ∼0.75 µm in NM-NEB muscle. Thus, the present study is the first to show a distinct genotype-functional phenotype correlation in patients with NM due to a nebulin mutation, and provides evidence for the notion that dysregulated thin filament length contributes to muscle weakness in NM patients with nebulin mutations. Furthermore, a striking similarity between the contractile and structural phenotypes of nebulin-deficient mouse muscle and human NM-NEB muscle was observed, indicating that the nebulin knockout model is well suited for elucidating the functional basis of muscle weakness in NM and for the development of treatment strategies.

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.