Protein Kinase C , but Not PKC or PKC , Regulates Contractility and Heart Failure Susceptibility: Implications for Ruboxistaurin as a Novel Therapeutic Approach

Children's Hospital Medical Center, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave, University of Cincinnati, Cincinnati, OH 45229-3039, USA.
Circulation Research (Impact Factor: 11.02). 07/2009; 105(2):194-200. DOI: 10.1161/CIRCRESAHA.109.195313
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Protein kinase (PK)Calpha, PKCbeta, and PKCgamma comprise the conventional PKC isoform subfamily, which is thought to regulate cardiac disease responsiveness. Indeed, mice lacking the gene for PKCalpha show enhanced cardiac contractility and reduced susceptibility to heart failure. Recent data also suggest that inhibition of conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 enhances heart function and antagonizes failure, although the isoform responsible for these effects is unknown. Here, we investigated mice lacking PKCalpha, PKCbeta, and PKCgamma for effects on cardiac contractility and heart failure susceptibility. PKCalpha(-/-) mice, but not PKCbetagamma(-/-) mice, showed increased cardiac contractility, myocyte cellular contractility, Ca(2+) transients, and sarcoplasmic reticulum Ca(2+) load. PKCalpha(-/-) mice were less susceptible to heart failure following long-term pressure-overload stimulation or 4 weeks after myocardial infarction injury, whereas PKCbetagamma(-/-) mice showed more severe failure. Infusion of ruboxistaurin (LY333531), an orally available PKCalpha/beta/gamma inhibitor, increased cardiac contractility in wild-type and PKCbetagamma(-/-) mice, but not in PKCalpha(-/-) mice. More importantly, ruboxistaurin prevented death in wild-type mice throughout 10 weeks of pressure-overload stimulation, reduced ventricular dilation, enhanced ventricular performance, reduced fibrosis, and reduced pulmonary edema comparable to or better than metoprolol treatment. Ruboxistaurin was also administered to PKCbetagamma(-/-) mice subjected to pressure overload, resulting in less death and heart failure, implicating PKCalpha as the primary target of this drug in mitigating heart disease. As an aside, PKCalphabetagamma triple-null mice showed no defect in cardiac hypertrophy following pressure-overload stimulation. In conclusion, PKCalpha functions distinctly from PKCbeta and PKCgamma in regulating cardiac contractility and heart failure, and broad-acting PKC inhibitors such as ruboxistaurin could represent a novel therapeutic approach in treating human heart failure.

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    • "Further, PKCδ (PRKCD) was shown to bind to Cx43 (Niger et al., 2010). PKCs are considered a therapeutic target due to the expression of multiple PKCs in the heart and their expression changes and contribution to heart diseases (Liu et al., 2009; Palaniyandi et al., 2009). The cGMP dependent Protein kinase G (cGK, PKG, PRKG) was also reported to phosphorylate connexins and modulate their expression (Kwak et al., 1995; Patel et al., 2006; Joshi et al., 2012). "
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    ABSTRACT: Gap junction communication (GJC) mediated by connexins is critical for heart function. To gain insight into the causal relationship of molecular mechanisms of disease pathology, it is important to understand which mechanisms contribute to impairment of gap junctional communication. Here, we present an update on the known modulators of connexins, including various interaction partners, kinases, and signaling cascades. This gap junction network (GJN) can serve as a blueprint for data mining approaches exploring the growing number of publicly available data sets from experimental and clinical studies.
    Frontiers in Physiology 02/2014; 5:82. DOI:10.3389/fphys.2014.00082 · 3.53 Impact Factor
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    • "A study by Liu et al. found that PKCα knockout mice exhibit increased myocyte contractility, and are less susceptible to heart failure. In contrast, PKCβγ knockout mice have the same susceptibility as wild type mice, which suggests that the PKCα isoform is the primary regulator of cardiac contractility and susceptibility to heart failure[73]. Administration of specific PKCα/β/γ inhibitors, Ro-32-0432, Ro-31-8220 orruboxistaurin (LY333531), can protect against heart failure in wide type mice, but not in PKCα knockout mice. "
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    ABSTRACT: NADPH oxidase5 (Nox5) is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular complications of diabetes and other diseases with increased ROS production.
    PLoS ONE 02/2014; 9(2):e88405. DOI:10.1371/journal.pone.0088405 · 3.23 Impact Factor
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    • "PKCα is another protein that is regulated by the microtubule cytoskeleton [25], [26] that is believed to play a role in pathological hypertrophy [27]. PKCα expression was most abundant in the Free fraction, but also was observed at moderate levels in MT and membrane associated fractions (Figure 6A), and increased significantly in response to TAC in both WT and MACF1 KO hearts. "
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    ABSTRACT: Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r(2) = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload.
    PLoS ONE 09/2013; 8(9):e73887. DOI:10.1371/journal.pone.0073887 · 3.23 Impact Factor
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