Control of myocardial PKC: A novel paradigm and the roleof CapZQian Zhong, Jenna Usprech, W. Glen Pyle. Dept. of Bio-medical Sciences, University of Guelph, Guelph, Ontario,Canada

Protein kinase C (PKC) is a putative cause of heart failure.We found that reduced CapZ protein disrupts PKC regulation ofmyofilaments through an unknown mechanism. TraditionallyPKC activation occurs at cell membranes followed by sub-cellular translocation. We propose a PKC signalling modelwhere activation occurs at the myofilaments. To test our model,immunoblot and confocal microscopy examined PKC activa-tion co-factors in cardiac myofilaments. Phospholipase C β(PLCβ1), phosphatidyl-inositol-4,5-bisphosphate (PIP2), andreceptor for activated C-kinase 1 (RACK1) were found, but notPLCβ3. CapZ removal decreased myofilament PIP2 levels,suggesting that CapZ extraction blocks PKC signalling byremoving a key cofactor. To test the physiological relevance ofour model we used endothelin-1 (ET) and phenylephrine (PHE)to activate PKC. Both PHE and ET decreased myofilamentPIP2, without altering PLCβ1 or RACK1. ET and PHE causedGqα to translocate to the myofilaments. To determine if Gqαtriggers myofilament PKC activation, we exposed myofila-ments to purified Gqα in amounts seen after PHE or ET treat-ment. Myofilament PIP2 decreased, while PLCβ1 and RACK1levels were unchanged. Together these data are the first to showa compliment of PKC activation co-factors associated withcardiac myofilaments. We also report the novel finding that Gqαtranslocates to myofilaments after membrane receptor stimula-tion. The results support our model of a local PKC signallingsystem in association with myocardial contractile elements.

Keywords: Myofilaments; Actin capping protein/CapZ; Proteinkinase C (PKC)

doi:10.1016/j.yjmcc.2007.03.123

The pregnancy hormone relaxin increases myofilamentforce through a PKC-dependent pathwayPhilip Wood, W. Glen Pyle. Dept. of Biomedical Sciences,University of Guelph, Guelph, Ontario, Canada

During pregnancy maternal cardiac output increases con-comitant with increased plasma levels of the hormone relaxin(RLX). RLX treatment of isolated rat hearts increases myo-cardial contractility. The myocardial mechanisms and intracel-lular signalling pathways activated by RLX are unknown.Given the central role of cardiac myofilaments in determiningcontractility, we examined the effects of RLX on murine cardiacmyofilaments. Left ventricular papillary muscles were exposedto 30 ng/mL H2-RLX for 10 min. H2RLX is the predominantisoform in humans, and is analogous to the major circulatingisoform in mice. The dose is the minimal amount that producesmaximal effects on cardiac output. After treatment of myo-cardium, myofilaments were isolated and function examined.

Maximum isometric force was not affected by RLX, but myo-filament Ca2+ sensitivity of force was increased. ActomyosinATPase activity was not affected in any way. Previous studiesexamining the effects of RLX in other non-cardiac tissues haveimplicated protein kinase C (PKC) as an intracellular mediator.The PKC inhibitors chelerythrine or bisindolmaleimide bothabolished the RLX-dependent increase in myofilament Ca2+

sensitivity of force. This study is the first to investigate RLX'sintracellular mechanisms of action in the heart. Our data suggestthat RLX increases myocardial contractility by enhancingmyofilament Ca2+ sensitivity of force through a PKC-depen-dent pathway, without requiring a concomitant increase inmyofilament energy consumption.

Keywords: Myofilaments; Relaxin (RLX); Protein kinase C(PKC)

doi:10.1016/j.yjmcc.2007.03.124

Hyperglycemia induced cell growth and gene expression viathe serum response element through PKCβ, RhoA andRho-kinaseKai Ishiko, Tsuyoshi Sakoda, Takahumi Akagami,Mitsumasa Ohyanagi. Dept of Internal Med., Hyogo Collegeof Med., Japan

Postprangial hyperglycemia has been shown the importantrole on the development of cardiovascular diseases. Chronichyperglycemia has been reported to enhance the vasoconstrictorresponse by Rho-kinase and PKC. Furthermore, an oral PKCβselective inhibitor has been reported to be an effective therapyfor diabetes. We have reported Phenylephrine enhanced thevasoconstrictor response in a spontaneous diabetes rat model.However, the mechanism of these reactions is still not wellunderstood. We therefore examined the effect of hyperglycemiaon the cell growth and gene expression of rat aortic smooth-muscle cells (RASMCs). Hyperglycemia accelerated the growthof RASMCs in a concentration-dependent manner. Furthermore,the c-fos gene expression was also increased by hyperglycemia.The deletion analysis revealed the c-fos serum response element(SRE) accounts for the c-fos expression. PKCβ, RhoA, and Rho-kinase (Rho-K) were involved in this signal transduction path-way. Furthermore, PKCβ-induced SRE activation was inhibitedby RhoA and Rho-kinase. These results indicate RhoA and Rho-K are downstream molecules of PKCβ. Phenylephrine inducedSRE activation. Hyperglycemia also augmented phenylephrine-induced SRE activation. Fluvastatin inhibited hyperglycemia-augmented these reactions by inhibiting RhoA. Furthermore,dominant negative PKCβ also inhibited these reactions. Statinand PKCβ inhibitor might be effective for the treatment of hy-perglycemia-induced cardiovascular events.

Keywords: Diabetes mellitus; PKC; Statin

doi:10.1016/j.yjmcc.2007.03.125

S44 ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S37–S54