Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited genetic myocardial disease characterized by fibrofatty replacement of the myocardium and a predisposition to cardiac arrhythmias and sudden death. We evaluated the cardiomyopathy gene titin (TTN) as a candidate ARVC gene because of its proximity to an ARVC locus at position 2q32 and the connection of the titin protein to the transitional junction at intercalated disks. Methods and Results All 312 titin exons known to be expressed in human cardiac titin and the complete 3’ untranslated region were sequenced in 38 ARVC families. Eight unique TTN variants were detected in 7 families including a prominent Thr2896Ile mutation that showed complete segregation with the ARVC phenotype in one large family. The Thr2896IIe mutation maps within a highly conserved immunoglobulin-like fold (Ig10 domain), located in titin’s spring region. Native gel electrophoresis, NMR, intrinsic fluorescence, and proteolysis assays of wildtype and mutant Ig10 domains revealed that the Thr2896IIe exchange reduces the structural stability and increases the propensity towards degradation of the Ig10 domain. The phenotype of TTN variant carriers was characterized by history of sudden death (5/7 families), progressive myocardial dysfunction causing death or heart transplant (8/14 cases), frequent conduction disease (11/14), and incomplete penetrance (86%). Conclusions Our data provide evidence that titin mutations can cause ARVC, a finding that further expands the origin of the disease beyond desmosomal proteins. Structural impairment of the titin spring is a likely cause of ARVC and constitutes a novel mechanism underlying myocardial remodeling and sudden cardiac death.

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 PKC␣ 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 PKC␣ targets the proline -glutamate -valinelysine (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 PKC␣ (S11878 and S12022); when these 2 sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned left ventricular myocardium revealed that PKC␣ significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that PKC␣ 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 PKC␣ phosphorylation. Conclusions: PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness. (Circ Res. 2009;105:631-638.)Key Words: connectin Ⅲ diastole Ⅲ passive stiffness Ⅲ posttranslational modification P osttranslational modifications of contractile and regulatory proteins profoundly alter myocardial function during normal physiological adaptations as well as during pathological processes. Two key mediators of many diverse physiological and pathological responses are the ␤-adrenergic pathway that results in activation of protein kinase A (PKA) and the ␣1-adrenergic pathway that results in activation of protein kinase C (PKC). Many cardiac proteins can be phosphorylated by both PKA and PKC and, interestingly, this can have either similar or disparate effects, with interplay among the phosphorylation sites (for recent review with original citations, see 1 ). In this study we focused on phosphorylation of the giant protein titin.Titin, the largest protein known, is the third most abundant myofilament of striated muscle (after myosin and actin) and spans the half sarcomeric distance from Z-disk to M-line. 2 In the I-band region of the sarcomere titin is extensible and functions as a molecular spring that develops force in sarcomeres stretched beyond their slack length (Ϸ1.9 m). This force largely determines the passive tension of the cardiac myocyte and, together with co...