unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca 2ϩ concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca 2ϩ sensitivity (pCa50) was significantly lower in cTnI-DD (pCa50: 5.39 Ϯ 0.01) compared with cTnI-AA (pCa50: 5.50 Ϯ 0.01), cTnI-AD (pCa50: 5.48 Ϯ 0.01), and cTnI-DA (pCa50: 5.51 Ϯ 0.01) at ϳ70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa50 with cTnI-DD content was observed, saturating at ϳ55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca 2ϩ sensitivity, which is maximal at ϳ55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the DISCUSSION in this article. myofilament function; protein phosphorylation; cardiomyocyte; troponin I DURING STRESS AND EXERCISE, sympathetic activation of the heart increases heart rate and stroke volume to meet the demands of the body. This is mediated via the stimulation of ␤ 1 -adrenergic receptors, which leads to the activation of a downstream kinase, PKA. PKA enhances cardiomyocyte contraction and relaxation by phosphorylation of proteins involved in Ca 2ϩ handling and myofilament proteins such as cardiac troponin (cTn)I, cardiac myosin-binding protein-C (cMyBP-C), and titin (for reviews, see Refs. 1 and 37).PKA-mediated phosphorylation of myofilament proteins is thought to exert a positive lusitropic effect, which enables the heart to relax more rapidly when heart rate increases. This positive lusitropic effect may be induced by a decrease in myofilament Ca 2ϩ sensitivity (32, 35, 51) and by enhanced cross-bridge cycling kinetics (11,20,33). It is well established (mainly from studies in rodents) that phosphorylation of cTnI at the PKA sites Ser 23 and Ser 24 leads to a decrease in myofilament Ca 2ϩ sensitivity, through a conformational change of the troponin complex. This structural change reduces the affinity of Ca 2ϩ binding to cTnC (16,30). The role of phosphorylation of cTnI at the PKA sites as a regulator of cross-bridge cycling is less clear. Some studies (11,20,36) have reported an increase in cross-bridge kinetics via phosphorylation of cTnI. However, others (7, 33) have attributed an increase in cross-bridge kinetics to phosphorylation of cMyBP-C independent of cTnI phosphorylation, whereas several studies (8,15,17,44) did not find an effect of PKA on cross-bridge kinetics at all. In the present study, we aimed to study the effect of site-specific phosphorylation of cTnI on myofilament Ca 2ϩ sensitivity and cross-bridge kinetics in human cardiomyocytes since insights into the functional effects of cTnI phosphorylation and the relation between the level of phosphorylation and the functional effec...

A major drawback to doxorubicin as a cancer-treating drug is cardiac toxicity. To understand the mechanism of doxorubicin cardiac toxicity and the potent synergic effect seen when doxorubicin is combined with anti-ErbB2 (trastuzumab), we developed an in vivo rat model that exhibits progressive dose-dependent cardiac damage and loss of cardiac function after doxorubicin treatment. The hearts of these animals respond to doxorubicin damage by increasing levels of ErbB2 and the ErbB family ligand, neuregulin 1B, and by activating the downstream Akt signaling pathway. These increases in ErbB2 protein levels are not due to increased ErbB2 mRNA, however, suggesting post-transcriptional mechanisms for regulating this protein in the heart. Accordingly, levels of heat shock protein 90 (HSP90), a known ErbB2 protein stabilizer and chaperone, are increased by doxorubicin treatment, and coimmunoprecipitation reveals binding of HSP90 to ErbB2. Isolated cardiomyocytes are more susceptible to doxorubicin after treatment with HSP90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin, suggesting that the HSP90 is protective during doxorubicin treatment. Thus, our results provide one plausible mechanism for the susceptibility of the heart to anti-ErbB2 therapy post-doxorubicin therapy in subclinical and clinical conditions. Additionally, these results suggest that further testing is needed for HSP90 inhibitors under various conditions in the heart. [Cancer Res 2007;67(4):1436-41]

The aim of this study was to evaluate whether technetium 99m hydrazinonicotinamide (99mTc-HYNIC)-annexin V single-photon emission computed tomography (SPECT) would detect dose-dependent doxorubicin (DOX)-mediated cell death in the heart compared with functional echocardiography. Adult female Sprague-Dawley rats were treated with DOX (cumulative dose of 15 or 7.5 mg/kg) or saline (n = 7) and monitored by echocardiography. Rats were injected with 7 to 8 mCi 99mTc-HYNIC-annexin V and imaged 1 hour postinjection using a small animal dual-head SPECT/computed tomography (CT) system with multipinhole technology. Two regions of interest were drawn in the myocardium and soft tissue regions to calculate the cardiac uptake ratio (CUR) of reconstructed images. Myocardium and blood were harvested for radioactivity measurements or TUNEL assay. Biodistribution of 99mTc-HYNIC-annexin V uptake, CUR from SPECT/CT fused cardiac images, and TUNEL of myocardium demonstrated a dose-dependent toxicity response, with the cumulative 15 mg/kg DOX treatment showing the greatest degree of cell death. In contrast, echocardiography detected functional deficits only at the highest DOX dose. In vivo molecular imaging of DOX-induced cardiac toxicity with 99mTc-HYNIC-annexin V detects dose-dependent cell death before ventricular deficits are observed with echocardiography. 99mTc-HYNIC-annexin V SPECT-based molecular imaging may provide an attractive new technique for assessing early changes in myocardial function in patients undergoing DOX therapy.