Ubiquitin-mediated protein degradation is necessary for both increased ventricular mass and survival signaling for compensated hypertrophy in pressure-overloaded (PO) myocardium. Another molecular keystone involved in the hypertrophic growth process is the mammalian target of rapamycin (mTOR), which forms two distinct functional complexes: mTORC1 that activates p70S6 kinase-1 to enhance protein synthesis and mTORC2 that activates Akt to promote cell survival. Independent studies in animal models show that rapamycin treatment that alters mTOR complexes also reduces hypertrophic growth and increases lifespan by an unknown mechanism. We tested whether the ubiquitin-mediated regulation of growth and survival in hypertrophic myocardium is linked to the mTOR pathway. For in vivo studies, right ventricle PO in rats was conducted by pulmonary artery banding; the normally loaded left ventricle served as an internal control. Rapamycin (0.75 mg/kg per day) or vehicle alone was administered intraperitoneally for 3 days or 2 wk. Immunoblot and immunofluorescence imaging showed that the level of ubiquitylated proteins in cardiomyocytes that increased following 48 h of PO was enhanced by rapamycin. Rapamycin pretreatment also significantly increased PO-induced Akt phosphorylation at S473, a finding confirmed in cardiomyocytes in vitro to be downstream of mTORC2. Analysis of prosurvival signaling in vivo showed that rapamycin increased PO-induced degradation of phosphorylated inhibitor of κB, enhanced expression of cellular inhibitor of apoptosis protein 1, and decreased active caspase-3. Long-term rapamycin treatment in 2-wk PO myocardium blunted hypertrophy, improved contractile function, and reduced caspase-3 and calpain activation. These data indicate potential cardioprotective benefits of rapamycin in PO hypertrophy.

To compensate for hemodynamic overload of the heart, an event which stretches the myocardium, growth and survival signaling are activated in cardiac muscle cells (cardiomyocytes). Integrins serve as the signaling receptors of cardiomyocytes responsible for mechanotransduction toward intracellular signaling. The main integrin heterodimers on the cardiomyocyte surface are α 5 β 1 and α v β 3, and elimination of either β 1 or β 3 integrins impedes pressure-induced hypertrophic signaling and leads to increased mortality. The growth signaling pathways downstream of β 1 and β 3 integrins are well characterized. However, new integrin pathways responsible for inhibiting apoptosis induced by hemodynamic overload are emerging. β 1 and β 3 integrins activate differential survival signaling, yet both integrins initiate survival signaling downstream of ubiquitination and the kinase pathway including phosphoinositol-3-kinase (PI3K)/Akt. Further characterization of these integrin-signaling mechanisms may lead to drug targets to prevent decompensation to heart failure.

Current U.S. requirements for testing cell substrates used in production of human biological products for contamination with bovine and porcine viruses are U.S. Department of Agriculture (USDA) 9CFR tests for bovine serum or porcine trypsin. 9CFR requires testing of bovine serum for seven specific viruses in six families (immunofluorescence) and at least 2 additional families non-specifically (cytopathicity and hemadsorption). 9CFR testing of porcine trypsin is for porcine parvovirus. Recent contaminations suggest these tests may not be sufficient. Assay sensitivity was not the issue for these contaminations that were caused by viruses/virus families not represented in the 9CFR screen. A detailed literature search was undertaken to determine which viruses that infect cattle or swine or bovine or porcine cells in culture also have human host range [ability to infect humans or human cells in culture] and to predict their detection by the currently used 9CFR procedures. There are more viruses of potential risk to biological products manufactured using bovine or porcine raw materials than are likely to be detected by 9CFR testing procedures; even within families, not all members would necessarily be detected. Testing gaps and alternative methodologies should be evaluated to continue to ensure safe, high quality human biologicals.

Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development of various hypertrophic responses, including cardiomyocyte survival. mTOR forms two independent complexes, mTORC1 and mTORC2, by associating with common and distinct cellular proteins. Both complexes are sensitive to a pharmacological inhibitor, torin1, although only mTORC1 is inhibited by rapamycin. Since mTORC2 is known to mediate the activation of a prosurvival kinase, Akt, we analyzed whether mTORC2 directly mediates Akt activation or whether it requires the participation of another prosurvival kinase, PKC ε (epsilon isoform of protein kinase-C). Our studies reveal that treatment of adult feline cardiomyocytes in vitro with insulin results in Akt phosphorylation at S473 for its activation which could be augmented with rapamycin but blocked by torin1. Silencing the expression of Rictor (rapamycin-insensitive companion of mTOR), an mTORC2 component, with a sh-RNA in cardiomyocytes lowers both insulin-stimulated Akt and PKC ε phosphorylation. Furthermore, phosphorylation of PKC ε and Akt at the critical S729 and S473 sites respectively was blocked by torin1 or Rictor knockdown but not by rapamycin, indicating that the phosphorylation at these specific sites occurs downstream of mTORC2. Additionally, expression of DN-PKC ε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKC ε in the Akt activation. Biochemical analyses also revealed that PKC ε was part of Rictor but not Raptor (a binding partner and component of mTORC1). Together, these studies demonstrate that mTORC2 mediates prosurvival signaling in adult cardiomyocytes where PKC ε functions downstream of mTORC2 leading to Akt activation.

2'-Modified inosine analogs have been synthesized from 6-chloropurine riboside via 6-dimethylaminopurine or 6-benzyloxypurine intermediates. The dimethylaminopurine intermediate was obtained via an unusually facile dimethylamine transfer from dimethylformamide. Graphical Abstract:

Mammalian target of rapamycin (mTOR) has been shown to influence hypertrophic growth of the myocardium through two unique complexes, mTOR complexed with Raptor (mTORC1) and mTOR complexed to Rictor (mTORC2) with downstream activation of S6K1 and AKT respectively. Previously, we characterized the role of novel PKC isoforms (epsilon and delta) on mTORC1-dependent activation of S6K1 downstream of both PI3K-dependent and independent pathways in adult cardiomyocytes. PKC epsilon (PKCε) has been implicated in AKT activation during ischemia-reperfusion injury and has been co-immunoprecipitated in transgenic PKCε overexpressing murine myocardium. In the present study, we explored the role of PKCε on PI3K-dependent AKT S473 phosphorylation. As such, insulin stimulated pAKT S473 was significantly reduced by pretreatment with wortmannin, a selective PI3K inhibitor. However, insulin stimulated activation of AKT was not reduced by pretreatment with either Gö6976 blockade of the classical PKC isoforms (PKC alpha/beta) or U0126 blockade of MEK. Thus, insulin stimulated activation of AKT is mediated primarily through a PI3K-dependent pathway independent of the classical PKC isoforms. To begin to evaluate the role of the novel PKCs, we utilized adenoviral expression of dominant negative PKCε (dnPKCε). After 48 hours of expression of dnPKCε, insulin stimulated pAKT S473 was significantly reduced as compared to B-gal infection controls. Next we identified for the first time in isolated cardiomyocytes a direct link between PKCε and AKT through immunoprecipitation of a unique signaling complex comprised of PKCε bound to mTORC2 and pAKT (S473) only during insulin stimulation. These data establish for the first time in adult isolated cardiomyocytes: i) the necessity of PKCε during insulin stimulated PI3K-dependent AKT activation and ii) a signaling complex containing mTOR, Rictor, PKCε, and pAKT that forms during insulin stimulation. Given the importance of AKT in physiologic growth and survival, these studies indicate that PKCε plays a pivotal role during AKT activation and for the first time displays a direct link between PKCε, pAKT and mTORC2 in isolated cardiomyocytes.

2′‐Modified inosine analogues (V) and (X) are synthesized from 6‐chloropurine riboside (I) via 6‐(dimethylamino)purine (IV) or 6‐(benzyloxy)purine (VIII) intermediates.