Rationale: Increased aortic stiffness, an important feature of many vascular diseases, eg, aging, hypertension, atherosclerosis, and aortic aneurysms, is assumed because of changes in extracellular matrix (ECM).Objective: We tested the hypothesis that the mechanisms also involve intrinsic stiffening of vascular smooth muscle cells (VSMCs). Methods and Results:

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...

Increased vascular stiffness is fundamental to hypertension, and its complications, including atherosclerosis, suggest that therapy should also be directed at vascular stiffness, rather than just the regulation of peripheral vascular resistance. It is currently held that the underlying mechanisms of vascular stiffness in hypertension only involve the extracellular matrix and endothelium. We hypothesized that increased large-artery stiffness in hypertension is partly due to intrinsic mechanical properties of vascular smooth muscle cells. After confirming increased arterial pressure and aortic stiffness in spontaneously hypertensive rats, we found increased elastic stiffness of aortic smooth muscle cells of spontaneously hypertensive rats compared with Wistar-Kyoto normotensive controls using both an engineered aortic tissue model and atomic force microscopy nanoindentation. Additionally, we observed different temporal oscillations in the stiffness of vascular smooth muscle cells derived from hypertensive and control rats, suggesting that a dynamic component to cellular elastic stiffness is altered in hypertension. Treatment with inhibitors of vascular smooth muscle cell cytoskeletal proteins reduced vascular smooth muscle cell stiffness from hypertensive and control rats, suggesting their participation in the mechanism. This is the first study demonstrating that stiffness of individual vascular smooth muscle cells mediates vascular stiffness in hypertension, a novel concept, which may elucidate new therapies for hypertension and for vascular stiffness.