Background-Ischemia-induced cardiomyopathy usually is accompanied by elevated left ventricular end-diastolic pressure, which follows from increased myocardial stiffness resulting from upregulated collagen expression. In addition to collagen, a main determinant of stiffness is titin, whose role in ischemia-induced left ventricular stiffening was studied here. Human heart sarcomeres coexpress 2 principal titin isoforms, a more compliant N2BA isoform and a stiffer N2B isoform. In comparison, normal rat hearts express almost no N2BA titin. Methods and Results-Gel electrophoresis and immunoblotting were used to determine the N2BA-to-N2B titin isoform ratio in nonischemic human hearts and nonnecrotic left ventricle of coronary artery disease (CAD) patients. The average N2BA-to-N2B ratio was 47:53 in severely diseased CAD transplanted hearts and 32:68 in nonischemic transplants. In normal donor hearts and donor hearts with CAD background, relative N2BA titin content was Ϸ30%. The titin isoform shift in CAD transplant hearts coincided with a high degree of modifications of cardiac troponin I, probably indicating increased preload. Immunofluorescence microscopy on CAD transplant specimens showed a regular cross-striated arrangement of titin and increased expression of collagen and desmin. Force measurements on isolated myofibrils revealed reduced passive-tension levels in sarcomeres of CAD hearts with high left ventricular end-diastolic pressure compared with sarcomeres of normal hearts. In a rat model of ischemia-induced myocardial infarction (left anterior descending coronary artery ligature), 43% of animals, but only 14% of sham-operated animals, showed a distinct N2BA titin band on gels. Conclusions-A titin isoform switch was observed in chronically ischemic human hearts showing extensive remodeling, which necessitated cardiac transplantation. The shift, also confirmed in rat hearts, caused reduced titin-derived myofibrillar stiffness. Titin modifications in long-term ischemic myocardium could impair the ability of the heart to use the Frank-Starling mechanism.

In the pathogenesis of dilated cardiomyopathy, cytoskeletal proteins play an important role. In this study, we analyzed titin expression in left ventricles of 19 control human donors and 9 severely diseased (nonischemic) dilated cardiomyopathy (DCM) transplant-patients, using gel-electrophoresis, immunoblotting, and quantitative RT-PCR. Both human-heart groups coexpressed smaller (approximately 3 MDa) N2B-isoform and longer (3.20 to 3.35 MDa) N2BA-isoforms, but the average N2BA:N2B-protein ratio was shifted from approximately 30:70 in controls to 42:58 in DCM hearts, due mainly to increased expression of N2BA-isoforms >3.30 MDa. Titin per unit tissue was decreased in some DCM hearts. The titin-binding protein obscurin also underwent isoform-shifting in DCM. Quantitative RT-PCR revealed a 47% reduction in total-titin mRNA levels in DCM compared with control hearts, but no differences in N2B, all-N2BA, and individual-N2BA transcripts. The reduction in total-titin transcripts followed from a decreased area occupied by myocytes and increased connective tissue in DCM hearts, as detected by histological analysis. Force measurements on isolated cardiomyofibrils showed that sarcomeric passive tension was reduced on average by 25% to 30% in DCM, a reduction readily predictable with a model of wormlike-chain titin elasticity. Passive-tension measurements on human-heart fiber bundles, before and after titin proteolysis, revealed a much-reduced relative contribution of titin to total passive stiffness in DCM. Results suggested that the titin-isoform shift in DCM depresses the proportion of titin-based stiffness by approximately 10%. We conclude that a lower-than-normal proportion of titin-based stiffness in end-stage failing hearts results partly from loss of titin and increased fibrosis, partly from titin-isoform shift. The titin-isoform shift may be beneficial for myocardial diastolic function, but could impair the contractile performance in systole.

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) were described, this malignancy is clinically still treated as a single disease. Here, we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Individuals bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or shRNA-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4 alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and is highly expressed in several additional malignancies. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance.

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The elastic section of the giant muscle protein titin contains many immunoglobulin-like domains, which have been shown by single-molecule mechanical studies to unfold and refold upon stretch-release. Here we asked whether the mechanical properties of Ig domains and/or other titin regions could be responsible for the viscoelasticity of nonactivated skeletal-muscle sarcomeres, particularly for stress relaxation and force hysteresis. We show that isolated psoas myofibrils respond to a stretch-hold protocol with a characteristic force decay that becomes more pronounced following stretch to above 2.6-microm sarcomere length. The force decay was readily reproducible by a Monte Carlo simulation taking into account both the kinetics of Ig-domain unfolding and the worm-like-chain model of entropic elasticity used to describe titin's elastic behavior. The modeling indicated that the force decay is explainable by the unfolding of only a very small number of Ig domains per titin molecule. The simulation also predicted that a unique sequence in titin, the PEVK domain, may undergo minor structural changes during sarcomere extension. Myofibrils subjected to 1-Hz cycles of stretch-release exhibited distinct hysteresis that persisted during repetitive measurements. Quick stretch-release protocols, in which variable pauses were introduced after the release, revealed a two-exponential time course of hysteresis recovery. The rate constants of recovery compared well with the refolding rates of Ig-like or fibronectin-like domains measured by single-protein mechanical analysis. These findings suggest that in the sarcomere, titin's Ig-domain regions may act as entropic springs capable of adjusting their contour length in response to a stretch.