Retrospective Study of Intercalated Disk Defects Associated with Dilated Cardiomyopathy, Atrial Thrombosis, and Heart Failure in BALB/c Mice Deficient in IL4 Receptor α

An increased incidence of dilated cardiomyopathy and atrial thrombosis was noted in a breeding colony of BALB/c mice deficient in IL4 receptor α. The condition affected mice of both sexes and of various ages, and extensive testing (microbiology, serology, histopathology) failed to ascertain the cause. Transmission electron microscopy of heart samples showed structural defects in the myocardial intercalated disks, characterized by unorganized and heavily convoluted arrangement with lower density and less prominent desmosomes and adherens junctions, widening of the intercellular space, myofibrillar lysis adjacent to intercalated disks, occasional sarcomere lysis with marked myofiber degeneration, vacuolation, accumulation of cell debris, and myelin figures. The intercalated disk contains cell adhesion molecules that form cell junctions, allowing contraction coupling of cardiomyocytes and the electrical and mechanical connection between cardiac fibers. Thus, defects at this level result in poor myocardial contraction, intracardiac blood stagnation, and consequently cardiac dilation with clinical signs of heart failure. The background strain or, potentially, the Cre–loxP-mediated recombination system used to create these mice may have contributed to the elevated incidence of cardiomyopathy and atrial thrombosis in this colony. Due to the backcrossing breeding scheme used, we cannot discount the emergence and colonywide dissemination of a spontaneous mutation that affects the intercalated disk. This report underscores the importance of carefully monitoring genetically modified mice colonies for unexpected phenotypes that may result from spontaneous or unintended mutations or enhanced strain background pathology.

P6453Activation of necroptotic pathway by downregulated caspase-8 expression is associated with progression of left ventricular remodeling in nonischemic dilated cardiomyopathy

Background Necroptosis, a form of programmed necrosis, has been suggested to be involved in the pathogenesis of various pathological conditions including heart failure. Protein expression of caspase-8, an endogenous inhibitor of necroptosis, is reported to be downregulated in human failing hearts, but its clinical significance remains unclear. Methods Endomyocardial biopsy specimens were obtained from patients with nonischemic dilated cardiomyopathy (n=57, 56.2±14.5 years old, 70% male). The area stained with antibodies against caspase-8 and phospho-MLKL-Ser358 was calculated using an image analyzer, and fibrotic and cardiomyocyte areas were determined by Masson's Trichrome staining. Using a level of median caspase-8 expression (6.04% of the area of the myocardium with caspase-8 signal), patients were classified into a high caspase-8 expression group (H-cas8) and a low caspase-8 expression group (L-cas8). Results Caspase-8 signals were detected in cytoplasm and intercalated disks of cardiomyocytes. Patients in the L-cas8 group was younger (51.3±13.1 vs. 61.2±14.3 years old) and had larger left ventricular end-diastolic volume (LVEDV: 174±49 vs. 131±41 ml), larger left ventricular end-systolic volume (LVESV: 123±51 vs. 87±39 ml), and higher ratio of mitral peak velocity of early filling to late diastolic filling (E/A: 1.94±1.48 vs. 1.12±0.66) compared with the H-cas8 group. Caspase-8 expression level was positively correlated with age (r=0.34, p=0.01) and negatively correlated with LVEDV (r=−0.47, p<0.01), LVESV (r=−0.40, p<0.01), and E/A (r=−0.39, p<0.01) in simple linear regression analysis. The extent of myocardial fibrosis was not correlated with caspase-8 expression level. Multiple regression analysis indicated that LVEDV, LVESV, and E/A were independent explanatory factors of caspase-8 expression level after adjusting age and sex. Phospho-MLKL signals, an index of activation of necroptotic pathway, were frequently observed in cytoplasm, intercalated disks, and nuclei in the L-cas8 group but not in the H-cas8 group. Conclusion Lower caspase-8 expression in cardiomyocytes was associated with increased phosphorylation of MLKL and larger left ventricular volume, suggesting that downregulated caspase-8 may contribute to progression of myocardial remodeling via activation of MLKL in human dilated cardiomyopathy.

TRPA1 channel contributes to myocardial ischemia-reperfusion injury

Myocardial ischemia-reperfusion (I/R) results in the generation of free radicals, accumulation of lipid peroxidation-derived unsaturated aldehydes, variable angina (pain), and infarction. The transient receptor potential ankyrin 1 (TRPA1) mediates pain signaling and is activated by unsaturated aldehydes, including acrolein and 4-hydroxynonenal. The contribution of TRPA1 (a Ca2+-permeable channel) to I/R-induced myocardial injury is unknown. We tested the hypothesis that cardiac TRPA1 confers myocyte sensitivity to aldehyde accumulation and promotes I/R injury. Although basal cardiovascular function in TRPA1-null mice was similar to that in wild-type (WT) mice, infarct size was significantly smaller in TRPA1-null mice than in WT mice (34.1 ± 9.3 vs. 14.3 ± 9.9% of the risk region, n = 8 and 7, respectively, P < 0.05), despite a similar I/R-induced area at risk (40.3 ±8.4% and 42.2 ± 11.3% for WT and TRPA1-null mice, respectively) after myocardial I/R (30 min of ischemia followed by 24 h of reperfusion) in situ. Positive TRPA1 immunofluorescence was present in murine and human hearts and was colocalized with connexin43 at intercalated disks in isolated murine cardiomyocytes. Cardiomyocyte TRPA1 was confirmed by quantitative RT-PCR, DNA sequencing, Western blot analysis, and electrophysiology. A role of TRPA1 in cardiomyocyte toxicity was demonstrated in isolated cardiomyocytes exposed to acrolein, an I/R-associated toxin that induces Ca2+ accumulation and hypercontraction, effects significantly blunted by HC-030031, a TRPA1 antagonist. Protection induced by HC-030031 was quantitatively equivalent to that induced by SN-6, a Na+/Ca2+ exchange inhibitor, further supporting a role of Ca2+ overload in acrolein-induced cardiomyocyte toxicity. These data indicate that cardiac TRPA1 activation likely contributes to I/R injury and, thus, that TRPA1 may be a novel therapeutic target for decreasing myocardial I/R injury. NEW & NOTEWORTHY Transient receptor potential ankyrin 1 (TRPA1) activation mediates increased blood flow, edema, and pain reception, yet its role in myocardial ischemia-reperfusion (I/R) injury is unknown. Genetic ablation of TRPA1 significantly decreased myocardial infarction after I/R in mice. Functional TRPA1 in cardiomyocytes was enriched in intercalated disks and contributed to acrolein-induced Ca2+ overload and hypercontraction. These data indicate that I/R activation of TRPA1 worsens myocardial infarction; TRPA1 may be a potential target to mitigate I/R injury.

ZO-1 determines adherens and gap junction localization at intercalated disks

The disruption of the spatial order of electromechanical junctions at myocyte-intercalated disks (ICDs) is a poorly understood characteristic of many cardiac disease states. Here, in vitro and in vivo evidence is provided that zonula occludens-1 (ZO-1) regulates the organization of gap junctions (GJs) and adherens junctions (AJs) at ICDs. We investigated the contribution of ZO-1 to cell-cell junction localization by expressing a dominant-negative ZO-1 construct (DN-ZO-1) in rat ventricular myocytes (VMs). The expression of DN-ZO-1 in cultured neonatal VMs for 72 h reduced the interaction of ZO-1 and N-cadherin, as assayed by colocalization and coimmunoprecipitation, prompting cytoplasmic internalization of AJ and GJ proteins. DN-ZO-1 expression in adult VMs in vivo also reduced N-cadherin colocalization with ZO-1, a phenomenon not observed when the connexin-43 (Cx43)-ZO-1 interaction was disrupted using a mimetic of the ZO-1-binding ligand from Cx43. DN-ZO-1-infected VMs demonstrated large GJs at the ICD periphery and showed a loss of focal ZO-1 concentrations along plaque edges facing the disk interior. Additionally, there was breakdown of the characteristic ICD pattern of small interior and large peripheral GJs. Continuous DN-ZO-1 expression in VMs over postnatal development reduced ICD-associated Cx43 GJs and increased lateralized and cytoplasmic Cx43. We conclude that ZO-1 regulation of GJ localization is via an association with the N-cadherin multiprotein complex and that this is a key determinant of stable localization of both AJs and GJs at the ICD.