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Background: Adenosine triphosphate-sensitive potassium (K ATP ) channel openers have been found to be cardioprotective in multiple animal models via an unknown mechanism. Mouse models allow genetic manipulation of K ATP channel components for the investigation of this mechanism. Mouse Langendorff models using 30 min of global ischemia are known to induce measurable myocardial infarction and injury. Prolongation of global ischemia in a mouse Langendorff model could allow the determination of the mechanisms involved in K ATP channel opener cardioprotection., Methods: Mouse hearts (C57BL/6) underwent baseline perfusion with Krebs-Henseleit buffer (30 min), assessment of function using a left ventricular balloon, delivery of test solution, and prolonged global ischemia (90 min). Hearts underwent reperfusion (30 min) and functional assessment. Coronary flow was measured using an inline probe. Test solutions included were as follows: hyperkalemic cardioplegia alone (CPG, n = 11) or with diazoxide (CPG + DZX, n = 12)., Results: Although the CPG + DZX group had greater percent recovery of developed pressure and coronary flow, this was not statistically significant. Following a mean of 74 min (CPG) and 77 min (CPG + DZX), an additional increase in end-diastolic pressure was noted (plateau), which was significantly higher in the CPG group. Similarly, the end-diastolic pressure (at reperfusion and at the end of experiment) was significantly higher in the CPG group., Conclusions: Prolongation of global ischemia demonstrated added benefit when DZX was added to traditional hyperkalemic CPG. This model will allow the investigation of DZX mechanism of cardioprotection following manipulation of targeted K ATP channel components. This model will also allow translation to prolonged ischemic episodes associated with cardiac surgery., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Rationale: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly., Objective: The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis., Methods and Results: RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor β1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor β1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor β1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice ( Txndc5 -/- ) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 ( P <0.001) in Txndc5 -/- versus wild-type mice, respectively., Conclusions: The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against β agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure., (© 2018 American Heart Association, Inc.)

Background: Myocardial, transient, outward currents, I to , have been shown to play pivotal roles in action potential (AP) repolarization and remodeling in animal models. The properties and contribution of I to to left ventricular (LV) repolarization in the human heart, however, are poorly defined., Methods and Results: Whole-cell, voltage-clamp recordings, acquired at physiological (35°C to 37°C) temperatures, from myocytes isolated from the LV of nonfailing human hearts identified 2 distinct transient currents, I to,fast ( I to,f ) and I to,slow ( I to,s ), with significantly ( P <0.0001) different rates of recovery from inactivation and pharmacological sensitives: I to,f recovers in ≈10 ms, 100× faster than I to,s , and is selectively blocked by the Kv4 channel toxin, SNX-482. Current-clamp experiments revealed regional differences in AP waveforms, notably a phase 1 notch in LV subepicardial myocytes. Dynamic clamp-mediated addition/removal of modeled human ventricular I to,f , resulted in hyperpolarization or depolarization, respectively, of the notch potential, whereas slowing the rate of I to,f inactivation resulted in AP collapse. AP-clamp experiments demonstrated that changes in notch potentials modified the time course and amplitudes of voltage-gated Ca 2+ currents, I Ca . In failing LV subepicardial myocytes, I to,f was reduced and I to,s was increased, notch and plateau potentials were depolarized ( P <0.0001) and AP durations were prolonged ( P <0.001)., Conclusions: I to,f and I to,s are differentially expressed in nonfailing human LV, contributing to regional heterogeneities in AP waveforms. I to,f regulates notch and plateau potentials and modulates the time course and amplitude of I Ca . Slowing I to,f inactivation results in dramatic AP shortening. Remodeling of I to,f in failing human LV subepicardial myocytes attenuates transmural differences in AP waveforms., (© 2018 American Heart Association, Inc.)

Background: The adenosine triphosphate-sensitive potassium (K ATP ) channel opener diazoxide (DZX) prevents myocyte volume derangement and reduced contractility secondary to stress. K ATP channels are composed of pore-forming (Kir6.1 or Kir6.2) and regulatory (sulfonylurea receptor, SUR1 or SUR2) subunits. Gain of function (GOF) of Kir6.1 subunits has been implicated in cardiac pathology in Cantu syndrome in humans (cardiomegaly, lymphedema, and pericardial effusions). We hypothesized that GOF of Kir6.1 subunits would result in altered myocyte response to stress., Materials and Methods: Isolated cardiac myocytes from wild type (WT) and transgenic Kir6.1GOF mice were exposed to Tyrode's physiologic solution for 20 min, test solution (Tyrode's or stress [hyperkalemic cardioplegia {CPG, known myocyte stress}] +/- K ATP channel opener DZX), followed by Tyrode's for 20 min. Myocyte volume and contractility were measured and compared., Results: WT myocytes demonstrated significant swelling in response to stress, but significantly less swelling was seen in Kir6.1GOF myocytes. DZX prevented swelling secondary to CPG in WT but resulted in a nonsignificant reduction in swelling in Kir6.1GOF myocytes. Both WT and Kir6.1GOF myocytes demonstrated a reduction in contractility during stress, although this was only significant in Kir6.1GOF myocytes. DZX was not associated with an improvement in contractility in Kir6.1GOF myocytes following stress., Conclusions: Similar to previous results in Kir6.1(-/-) myocytes, Kir6.1GOF myocytes demonstrate resistance (less volume derangement) to stress of cardioplegia. Understanding the role of Kir6.1 in myocyte response to stress may aid in the treatment of patients with Cantu syndrome and warrants further investigation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: ATP-sensitive potassium (K(ATP)) channel openers provide cardioprotection in multiple models. Ion flux at an unidentified mitochondrial K(ATP) channel has been proposed as the mechanism. The renal outer medullary kidney potassium channel subunit, potassium inward rectifying (Kir)1.1, has been implicated as a mitochondrial channel pore-forming subunit. We hypothesized that subunit Kir1.1 is involved in cardioprotection (maintenance of volume homeostasis and contractility) of the K(ATP) channel opener diazoxide (DZX) during stress (exposure to hyperkalemic cardioplegia [CPG]) at the myocyte and mitochondrial levels., Methods and Results: Kir subunit inhibitor Tertiapin Q (TPN-Q) was utilized to evaluate response to stress. Mouse ventricular mitochondrial volume was measured in the following groups: isolation buffer; 200 μmol/L of ATP; 100 μmol/L of DZX+200 μmol/L of ATP; or 100 μmol/L of DZX+200 μmol/L of ATP+TPN-Q (500 or 100 nmol/L). Myocytes were exposed to Tyrode's solution (5 minutes), test solution (Tyrode's, cardioplegia [CPG], CPG+DZX, CPG+DZX+TPN-Q, Tyrode's+TPN-Q, or CPG+TPN-Q), N=12 for all (10 minutes); followed by Tyrode's (5 minutes). Volumes were compared. TPN-Q, with or without DZX, did not alter mitochondrial or myocyte volume. Stress (CPG) resulted in myocyte swelling and reduced contractility that was prevented by DZX. TPN-Q prevented the cardioprotection afforded by DZX (volume homeostasis and maintenance of contractility)., Conclusions: TPN-Q inhibited myocyte cardioprotection provided by DZX during stress; however, it did not alter mitochondrial volume. Because TPN-Q inhibits Kir1.1, Kir3.1, and Kir3.4, these data support that any of these Kir subunits could be involved in the cardioprotection afforded by diazoxide. However, these data suggest that mitochondrial swelling by diazoxide does not involve Kir1.1, 3.1, or 3.4., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: The sarcolemmal adenosine triphosphate-sensitive potassium channel (sK(ATP)), composed primarily of potassium inward rectifier (Kir) 6.2 and sulfonylurea receptor 2A subunits, has been implicated in cardiac myocyte volume regulation during stress; however, it is not involved in cardioprotection by the adenosine triphosphate-sensitive potassium channel (K(ATP)) channel opener diazoxide (7-chloro-3-methyl-1,2,4-benzothiadiazine-1,1-dioxide [DZX]). Paradoxically, the sK(ATP) channel subunit Kir6.2 is necessary for detrimental myocyte swelling secondary to stress. The Kir6.1 subunit can also contribute to K(ATP) channels in the heart, and we hypothesized that this subunit might play a role in myocyte volume regulation in response to stress., Study Design: Isolated cardiac myocytes from either wild-type mice or mice lacking the Kir6.1 subunit (Kir6.1[-/-]) were exposed to control Tyrode's solution (TYR) for 20 minutes, test solution (TYR, hypothermic hyperkalemic cardioplegia [CPG], or CPG + K(ATP) channel opener DZX [CPG + DZX]) for 20 minutes, followed by TYR for 20 minutes. Myocyte volume and contractility were measured and analyzed., Results: Both wild-type and Kir6.1(-/-) myocytes demonstrated substantial swelling during exposure to stress (CPG), which was prevented by DZX. Wild-type myocytes also demonstrated reduced contractility during stress of CPG that was prevented by DZX. However, Kir6.1(-/-) myocytes did not show reduced contractility during stress., Conclusions: These data indicate that K(ATP) channel subunit Kir6.1 is not necessary for DZX's maintenance of cell volume during the stress of CPG. The absence of Kir6.1 does not affect the contractile properties of myocytes during stress, suggesting the absence of Kir6.1 improves myocyte tolerance to stress via an unknown mechanism., (Copyright © 2015 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: The concept that pore-forming Kir6.2 and regulatory SUR2A subunits form cardiac ATP-sensitive potassium (K(ATP)) channels is challenged by recent reports that SUR1 is predominant in mouse atrial K(ATP) channels., Objective: To assess SUR subunit composition of K(ATP) channels and consequence of K(ATP) activation for action potential duration (APD) in dog hearts., Methods: Patch-clamp techniques were used on isolated dog cardiomyocytes to investigate K(ATP) channel properties. Dynamic current clamp, by injection of a linear K(+) conductance to simulate activation of the native current, was used to study the consequences of K(ATP) activation on APD., Results: Metabolic inhibitor (MI)-activated current was not significantly different from pinacidil (SUR2A-specific)-activated current, and both currents were larger than diazoxide (SUR1-specific)-activated current in both the atrium and the ventricle. Mean K(ATP) conductance (activated by MI) did not differ significantly between chambers, although, within the ventricle, both MI-induced and pinacidil-induced currents tended to decrease from the epicardium to the endocardium. Dynamic current-clamp results indicate that myocytes with longer baseline APDs are more susceptible to injected K(ATP) current, a result reproduced in silico by using a canine action potential model (Hund-Rudy) to simulate epicardial and endocardial myocytes., Conclusions: Even a small fraction of K(ATP) activation significantly shortens APD in a manner that depends on existing heterogeneity in K(ATP) current and APD., (© 2013 Heart Rhythm Society. All rights reserved.)

Background: Cardiac myocytes demonstrate significant swelling and associated reduced contractility in response to stress that is prevented by the ATP-sensitive potassium channel opener, diazoxide (DZX) via an unknown mechanism. One proposed mechanism of cardioprotection is mitochondrial matrix swelling. To establish the relationship between mitochondrial and cellular volume during stress, this study examined the effect of DZX on mitochondrial volume., Methods and Results: Isolated mouse mitochondria were exposed to the following solutions: Tyrode, isolation buffer, cardioplegia (CPG)±DZX±ATP-sensitive potassium channel inhibitor, 5-hydroxydecanoate, and metabolic inhibition (MI) ± DZX ± 5-hydroxydecanoate. Mitochondrial volume was measured. DZX resulted in significant mitochondrial swelling (P<0.0001 versus Tyrode). MI and CPG resulted in significant mitochondrial swelling compared with baseline volume. The addition of DZX did not alter the response of mitochondrial volume to CPG (P=0.912) but increased swelling in response to MI (P=0.036). The addition of 5-hydroxydecanoate to MI + DZX or CPG+DZX significantly reduced mitochondrial swelling (P<0.003 MI+DZX versus MI + DZX + 5HD; P<0.001 CPG+DZX versus CPG + DZX + 5HD)., Conclusions: Both cellular and mitochondrial volume increased during exposure to MI and CPG. DZX did not alter mitochondrial volume during CPG; however, it was associated with an increase in mitochondrial volume during MI. 5-Hydroxydecanoate reduced mitochondrial volume during exposure to both stresses with DZX, supporting a role for a mitochondrial ATP-sensitive potassium channel in the mechanism of cardioprotection by DZX.

Background: Diazoxide maintains myocyte volume and contractility during stress via an unknown mechanism. The mechanism of action may involve an undefined (genotype unknown) mitochondrial ATP-sensitive potassium channel and is dependent on the ATP-sensitive potassium channel subunit sulfonylurea type 1 receptor (SUR1). The ATP-sensitive potassium channel openers have been shown to inhibit succinate dehydrogenase (SDH) and a gene for a portion of SDH has been found in the SUR intron. Diazoxide may be cardioprotective via inhibition of SDH, which can form part of an ATP-sensitive potassium channel or share its genetic material. This study investigated the role of inhibition of SDH by diazoxide and its relationship to the SUR1 subunit., Study Design: Mitochondria were isolated from wild-type and SUR1 knockout mice. Succinate dehydrogenase activity was measured by spectrophotometric analysis of 2,6-dichloroindophenol reduction for 20 minutes as the relative change in absorbance over time. Mitochondria were treated with succinate (20 mM), succinate + 1% dimethylsulfoxide, succinate + malonate (8 mM) (competitive inhibitor of SDH), or succinate + diazoxide (100 μM)., Results: Both malonate and diazoxide inhibit SDH activity in mitochondria of wild-type mice and in mice lacking the SUR1 subunit (p < 0.05 vs control)., Conclusions: The ability of DZX to inhibit SDH persists even after deletion of the SUR1 gene. Therefore, the enzyme complex SDH is not dependent on the SUR1 gene. The inhibition of SDH by DZX can play a role in the cardioprotection afforded by DZX; however, this role is independent of the ATP-sensitive potassium channel subunit SUR1., (Copyright © 2013 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: The adenosine triphosphate-sensitive potassium (KATP) channel opener, diazoxide, preserves myocyte volume homeostasis and contractility during stress via an unknown mechanism. Pharmacologic overlap has been suggested between succinate dehydrogenase (SDH) activity and KATP channel modulators. Diazoxide may be cardioprotective due to the inhibition of SDH which may form a portion of the mitochondrial KATP channel. To determine the role of inhibition of SDH in diazoxide's cardioprotection, this study utilized glutathione to prevent the inhibition of SDH., Methods: SDH activity was measured in isolated mitochondria exposed to succinate (control), malonate (inhibitor of succinate dehydrogenase), diazoxide, and varying concentrations of glutathione alone or in combination with diazoxide. Enzyme activity was measured by spectrophotometric analysis. To evaluate myocyte volume and contractility, cardiac myocytes were superfused with Tyrode's physiologic solution (Tyrode's) (20 minutes), followed by test solution (20 minutes), including Tyrode's, hyperkalemic cardioplegia (stress), cardioplegia + diazoxide, cardioplegia + diazoxide + glutathione, or glutathione alone; followed by Tyrode's (20 minutes). Myocyte volume and contractility were recorded using image grabbing software., Results: Both malonate and diazoxide inhibited succinate dehydrogenase. Glutathione prevented the inhibition of succinate dehydrogenase by diazoxide in a dose-dependent manner. The addition of diazoxide prevented the detrimental myocyte swelling due to cardioplegia alone and this benefit was lost with the addition of glutathione. However, glutathione elicited an independent cardioprotective effect on myocyte contractility., Conclusions: The ability of diazoxide to provide beneficial myocyte homeostasis during stress involves the inhibition of succinate dehydrogenase, which may also involve the opening of a purported mitochondrial adenosine triphosphate sensitive potassium channel., (Copyright © 2013 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: The purpose of this investigation was to characterize differential right atrial (RA) and ventricular (RV) molecular changes in Ca(2+)-handling proteins consequent to RV pressure overload and hypertrophy in two common, yet distinct models of pulmonary hypertension: dehydromonocrotaline (DMCT) toxicity and pulmonary artery (PA) banding., Methods: A total of 18 dogs underwent sternotomy in four groups: (1) DMCT toxicity (n = 5), (2) mild PA banding over 10 wk to match the RV pressure rise with DMCT (n = 5); (3) progressive PA banding to generate severe RV overload (n = 4); and (4) sternotomy only (n = 4)., Results: In the right ventricle, with DMCT, there was no change in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) or phospholamban (PLB), but we saw a trend toward down-regulation of phosphorylated PLB at serine-16 (p[Ser-16]PLB) (P = 0.07). Similarly, with mild PA banding, there was no change in SERCA or PLB, but p(Ser-16)PLB was down-regulated by 74% (P < 0.001). With severe PA banding, there was no change in PLB, but SERCA fell by 57% and p(Ser-16)PLB fell by 67% (P < 0.001). In the right atrium, with DMCT, there were no significant changes. With both mild and severe PA banding, p(Ser-16)PLB fell (P < 0.001), but SERCA and PLB did not change., Conclusions: Perturbations in Ca(2+)-handling proteins depend on the degree of RV pressure overload and the model used to mimic the RV effects of pulmonary hypertension. They are similar, but blunted, in the atrium compared with the ventricle., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.

In this paper, we examine how variations in normal tissue can influence disease classification of Raman spectra. Raman spectra from normal areas may be affected by previous disease or proximity to areas of dysplasia. Spectra were acquired in vivo from 172 patients and classified into five tissue categories: true normal (no history of disease), previous disease normal (history of disease, current normal diagnosis), adjacent normal (disease on cervix, spectra acquired from visually normal area), low grade, and high grade. Taking into account the various "normal" states of the tissue before statistical analysis led to a disease classification accuracy of 97%. These results indicate that abnormal changes significantly affect Raman spectra, even when areas are histopathologically normal. The sensitivity of Raman spectroscopy to subtle biochemical differences must be considered in order to successfully implement it in a clinical setting for diagnosing cervical dysplasia and cancer.

Connexin43 (Cx43) is a major cardiac gap junction channel protein required for normal electrical and contractile activity. Gap junction channel assembly, function, and turnover are regulated by phosphorylation under both normal and disease conditions. The carboxyl terminus (CT) of Cx43 contains numerous amino acid residues that are phosphorylated by protein kinases. However, our knowledge of the specific residues and kinases involved is incomplete. The objective of this study was to identify amino acid residues in the Cx43-CT that are targets of the multifunctional protein kinase, Ca(2+)/calmodulin protein kinase II (CaMKII), an enzyme known to play critical roles in Ca(2+) homeostasis, transcription, apoptosis, and ischemic heart disease. We subjected fusion protein containing the Cx43-CT to phosphorylation by CaMKII in vitro, digestion with Lys-C and trypsin followed by enrichment for phosphorylated peptides using TiO(2), and analysis in an LTQ XL Orbitrap with collision-induced dissociation and electron transfer dissociation. We deduced the sites of modification by interpreting tandem spectra from these "orthogonal" methods of gas phase peptide fragmentation. We have identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts.

Background: We have recently shown that native murine ventricular fibroblasts express both connexin43 (Cx43) and Cx45, and that the level of Cx43 expression influences intercellular coupling and cell proliferation. Relatively little is known, however, about how myocardial infarction (MI) influences expression of Cx43, or how altered Cx43 expression may affect fibroblast function post-MI. Fibroblasts are critical for infarct healing and post-infarct ventricular remodeling. They can couple electrically with cardiac myocytes and influence myocardial activation patterns. Thus, Cx43 remodeling and the level of intercellular communication in fibroblasts expressed in the infarcted heart were the subject of the present investigation., Methods: Fibroblasts were isolated from both infarct scar and remote, noninfarcted regions of murine hearts 6 d after coronary ligation. Expression levels of Cx43, α-smooth muscle actin and N-cadherin were quantified by immunoblotting. Gap junctional intercellular communication was quantified by Lucifer yellow dye transfer., Results and Conclusions: Fibroblasts isolated from infarcted hearts exhibited marked up-regulation of Cx43 protein expression and enhanced intercellular coupling. Exogenous administration of transforming growth factor-β (TGF-β) to fibroblast cultures from normal, non-operated hearts produced comparable up-regulation of Cx43, suggesting that increased intercellular communication between fibroblasts in infarct and peri-infarct regions may be secondary to activation of a TGF-β pathway. Unlike cardiac myocytes that down-regulate Cx43, presumably to limit intercellular transmission of biochemical mediators of ischemic injury, fibroblasts may up-regulate Cx43 to maintain electrical and metabolic coupling at a time when intercellular communication is compromised., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In addition to mediating cell-to-cell electrical coupling, gap junctions are important in tissue repair, wound healing, and scar formation. The expression and distribution of connexin43 (Cx43), the major gap junction protein expressed in the heart, are altered substantially after myocardial infarction (MI); however, the effects of Cx43 remodeling on wound healing and the attendant ventricular dysfunction are incompletely understood. Cx43-deficient and wild-type mice were subjected to proximal ligation of the anterior descending coronary artery and followed for 6 days or 4 wk to test the hypothesis that reduced expression of Cx43 influences wound healing, fibrosis, and ventricular remodeling after MI. We quantified the progression of infarct healing by measuring neutrophil expression, collagen content, and myofibroblast expression. We found significantly reduced transformation of fibroblasts to myofibroblasts at 6 days and significantly reduced collagen deposition both in the infarct at 6 days and at 4 wk in the noninfarcted region of Cx43-deficient mice. As expected, transforming growth factor (TGF)-beta, a profibrotic cytokine, was dramatically upregulated in MI hearts, but its phosphorylated comediator (pSmad) was significantly downregulated in the nuclei of Cx43-deficient hearts post-MI, suggesting that downstream signaling of TGF-beta is diminished substantially in Cx43-deficient hearts. This diminution in profibrotic TGF-beta signaling resulted in the attenuation of adverse structural remodeling as assessed by echocardiography. These findings suggest that efforts to enhance the expression of Cx43 to maintain intercellular coupling or reduce susceptibility to arrhythmias should be met with caution until the role of Cx43 in infarct healing is fully understood.

Objective: The objective of the study was to characterize the variations in normal cervical spectra because of menopausal status and location within the menstrual cycle. Using the information obtained, the accuracy of Raman spectroscopy to diagnose low-grade squamous intraepithelial lesion (LGSIL) will be improved., Study Design: A total of 133 patients undergoing either colposcopy or Papanicolaou smear were recruited from either Vanderbilt University or Tri-State Women's Health. Raman spectra were collected from both normal and diseased areas. The data were processed and analyzed using a multiclass discrimination and classification algorithm to determine whether the spectra were correctly classified., Results: Stratifying the data by menopausal state resulted in correctly classifying LGSIL 97% of the time (from 74%)., Conclusion: This study brings Raman spectroscopy one step closer to clinical use by improving the sensitivity to differentiate LGSIL from normal.

Raman spectroscopy has the potential to differentiate among the various stages leading to high-grade cervical cancer such as normal, squamous metaplasia, and low-grade cancer. For Raman spectroscopy to successfully differentiate among the stages, an applicable statistical method must be developed. Algorithms like linear discriminant analysis (LDA) are incapable of differentiating among three or more types of tissues. We developed a novel statistical method combining the method of maximum representation and discrimination feature (MRDF) to extract diagnostic information with sparse multinomial logistic regression (SMLR) to classify spectra based on nonlinear features for multiclass analysis of Raman spectra. We found that high-grade spectra classified correctly 95% of the time; low-grade data classified correctly 74% of the time, improving sensitivity from 92 to 98% and specificity from 81 to 96% suggesting that MRDF with SMLR is a more appropriate technique for categorizing Raman spectra. SMLR also outputs a posterior probability to evaluate the algorithm's accuracy. This combined method holds promise to diagnose subtle changes leading to cervical cancer.

Cervical cancer is the second most common malignancy among women worldwide, with over 490 000 cases diagnosed and 274 000 deaths each year. Although current screening methods have dramatically reduced cervical cancer incidence and mortality in developed countries, a "See and Treat" method would be preferred, especially in developing countries. Results from our previous work have suggested that Raman spectroscopy can be used to detect cervical precancers; however, with a classification accuracy of 88%, it was not clinically applicable. In this paper, we describe how incorporating a woman's hormonal status, particularly the point in menstrual cycle and menopausal state, into our previously developed classification algorithm improves the accuracy of our method to 94%. The results of this paper bring Raman spectroscopy one step closer to being utilized in a clinical setting to diagnose cervical dysplasia., (((c) 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).)

Little is known about connexin expression and function in murine cardiac fibroblasts. The authors isolated native ventricular fibroblasts from adult mice and determined that although they expressed both connexin43 (Cx43) and connexin45 (Cx45), the relative abundance of Cx45 was greater than that of Cx43 in fibroblasts compared to myocytes, and the electrophoretic mobility of both Cx43 and Cx45 differed in fibroblasts and in myocytes. Increasing Cx43 expression by adenoviral infection increased intercellular coupling, whereas decreasing Cx43 expression by genetic ablation decreased coupling. Interestingly, increasing Cx43 expression reduced fibroblast proliferation, whereas decreasing Cx43 expression increased proliferation. These data demonstrate that native fibroblasts isolated from the mouse heart exhibit intercellular coupling via gap junctions containing both Cx43 and Cx45. Fibroblast proliferation is inversely related to the expression level of Cx43. Thus, connexin expression and remodeling is likely to alter fibroblast function, maintenance of the extracellular matrix, and ventricular remodeling in both normal and diseased hearts.

A common goal of most Raman spectroscopists is the acquisition of clear Raman spectra in short integration times. It is paramount, then, that the hardware used in a Raman system be optimized for throughput, signal-to-noise ratio, and resolution. To this end, we performed a systematic comparison of two Raman spectrographs: the widely used Kaiser HoloSpec and a relatively new commercial offering, the Raman Explorer by Headwall Photonics. Both strong and weak Raman scattering samples were measured using various launching conditions. When resolution-matched, the throughputs of both spectrographs were found to be roughly similar over the central range of the fingerprint region (approximately 800 to 1140 cm(-1), using 785 nm illumination), with the Raman Explorer demonstrating a slight throughput advantage outside this range. Other factors are also considered such that end users may better select the optimum spectrograph for their particular application.

Epithelial cancers, including those of the skin and cervix, are the most common type of cancers in humans. Many recent studies have attempted to use Raman spectroscopy to diagnose these cancers. In this paper, Raman spectral markers related to the temporal and spatial effects of cervical and skin cancers are examined through four separate but related studies. Results from a clinical cervix study show that previous disease has a significant effect on the Raman signatures of the cervix, which allow for near 100% classification for discriminating previous disease versus a true normal. A Raman microspectroscopy study showed that Raman can detect changes due to adjacent regions of dysplasia or HPV that cannot be detected histologically, while a clinical skin study showed that Raman spectra may be detecting malignancy associated changes in tissues surrounding nonmelanoma skin cancers. Finally, results of an organotypic raft culture study provided support for both the skin and the in vitro cervix results. These studies add to the growing body of evidence that optical spectroscopy, in this case Raman spectral markers, can be used to detect subtle temporal and spatial effects in tissue near cancerous sites that go otherwise undetected by conventional histology.

Atrial tissue expresses both connexin 40 (Cx40) and 43 (Cx43) proteins. To assess the relative roles of Cx40 and Cx43 in atrial electrical propagation, we synthesized cultured strands of atrial myocytes derived from mice with genetic deficiency in Cx40 or Cx43 expression and measured propagation velocity (PV) by high-resolution optical mapping of voltage-sensitive dye fluorescence. The amount of Cx40 and/or Cx43 in gap junctions was measured by immunohistochemistry and total or sarcolemmal Cx43 or Cx40 protein by immunoblotting. Progressive genetic reduction in Cx43 expression decreased PV from 34+/-6 cm/sec in Cx43(+/+) to 30+/-8 cm/sec in Cx43(+/-) and 19+/-11 cm/sec in Cx43(-/-) cultures. Concomitantly, the cell area occupied by Cx40 immunosignal in gap junctions decreased from 2.0+/-1.6% in Cx43(+/+) to 1.7+/-0.5% in Cx43(+/-) and 1.0+/-0.2% in Cx43(-/-) strands. In contrast, progressive genetic reduction in Cx40 expression increased PV from 30+/-2 cm/sec in Cx40(+/+) to 40+/-7 cm/sec in Cx40(+/-) and 45+/-10 cm/sec in Cx40(-/-) cultures. Concomitantly, the cell area occupied by Cx43 immunosignal in gap junctions increased from 1.2+/-0.9% in Cx40(+/+) to 2.8+/-1.4% in Cx40(+/-) and 3.1+/-0.6% in Cx40(-/-) cultures. In accordance with the immunostaining results, immunoblots of the Triton X-100-insoluble fraction revealed an increase of Cx43 in gap junctions in extracts from Cx40-ablated atria, whereas total cellular Cx43 remained unchanged. Our results suggest that the relative abundance of Cx43 and Cx40 is an important determinant of atrial impulse propagation in neonatal hearts, whereby dominance of Cx40 decreases and dominance of Cx43 increases local propagation velocity.

Ovarian cancer is the fifth leading cause of cancer death in women, in part because of the limited knowledge about early stage disease. We develop a novel rat model of ovarian cancer and perform a pilot study to examine the harvested ovaries with complementary optical imaging modalities. Rats are exposed to repeated daily dosing (20 days) with 4-vinylcyclohexene diepoxide (VCD) to cause early ovarian failure (model for postmenopause), and ovaries are directly exposed to 7,12-dimethylbenz(a)anthracene (DMBA) to cause abnormal ovarian proliferation and neoplasia. Harvested ovaries are examined with optical coherence tomography (OCT) and light-induced fluorescence (LIF) at one, three, and five months post-DMBA treatment. VCD causes complete ovarian follicle depletion within 8 months after onset of dosing. DMBA induces abnormal size, cysts, and neoplastic changes. OCT successfully visualizes normal and abnormal structures (e.g., cysts, bursa, follicular remnant degeneration) and the LIF spectra show statistically significant changes in the ratio of average emission intensity at 390:450 nm between VCD-treated ovaries and both normal cycling and neoplastic DMBA-treated ovaries. Overall, this pilot study demonstrates the feasibility of both the novel animal model for ovarian cancer and the ability of optical imaging techniques to visualize ovarian function and health.

Optical coherence tomography (OCT) is an imaging modality capable of acquiring cross-sectional images of tissue using back-reflected light. Conventional OCT images have a resolution of 10-15 microm, and are thus best suited for visualizing tissue layers and structures. OCT images of collagen (with and without endothelial cells) have no resolvable features and may appear to simply show an exponential decrease in intensity with depth. However, examination of these images reveals that they display a characteristic repetitive structure due to speckle. The purpose of this study is to evaluate the application of statistical and spectral texture analysis techniques for differentiating living and non-living tissue phantoms containing various sizes and distributions of scatterers based on speckle content in OCT images. Statistically significant differences between texture parameters and excellent classification rates were obtained when comparing various endothelial cell concentrations ranging from 0 cells/ml to 25 million cells/ml. Statistically significant results and excellent classification rates were also obtained using various sizes of microspheres with concentrations ranging from 0 microspheres/ml to 500 million microspheres/ml. This study has shown that texture analysis of OCT images may be capable of differentiating tissue phantoms containing various sizes and distributions of scatterers.

Introduction: Electrophysiologic heterogeneity across the ventricular wall is a result of differential transmural expression of various ion channel proteins that underlie the different action potential waveforms observed in epicardial, midmyocardial, and endocardial regions. Cardiac connexins mediate cell-to-cell communication, are critical for normal impulse propagation, and play a role in electrophysiologic remodeling in disease states. However, little is known about the transmural distribution of cardiac gap junction proteins., Methods and Results: Connexin expression in epicardium, midmyocardium, and endocardium was assessed immunohistochemically in mouse and rat hearts. The total connexin protein content within different ventricular regions was measured by immunoblotting. Connexin43 is twice as abundant in midmyocardium and endocardium compared with epicardium in the mouse but not in the rat. Connexin45 is expressed equally across the left ventricular wall., Conclusion: Epicardial myocytes express significantly less Cx43 and therefore may be less well coupled than midmyocardial and endocardial myocytes. A transmural gradient of connexin43 expression across the left ventricular free wall likely results in differences in the stoichiometry of connexins expressed in different regions of the heart.

Objective: Adult ventricular myocytes express two gap junction channel proteins: connexin43 (Cx43) and connexin45 (Cx45). Cx43-deficient mice exhibit slow ventricular epicardial conduction, suggesting that Cx43 plays an important role in intercellular coupling in the ventricle. Cx45 is much less abundant than Cx43 in working ventricular myocytes. Its role in ventricular conduction has not been defined, nor is it known whether expression or distribution of Cx45 is altered in Cx43-deficient mice. The present study was undertaken to determine (1) whether expression of Cx45 is upregulated and (2) whether gap junction structure and distribution are altered in Cx43-deficient mice., Methods: Ventricular tissue from neonatal Cx43(+/+), Cx43(+/-) and Cx43(-/-) and adult Cx43(+/+) and Cx43(+/-) mice was analyzed by immunoblotting and confocal immunofluorescence microscopy., Results: Total Cx45 protein abundance measured by immunoblotting was not different in Cx43-deficient or null hearts compared to wild-type control hearts. However, the amount and distribution of Cx45 immunoreactive signal measured by quantitative confocal analysis were markedly reduced in both Cx43(+/-) and Cx43(-/-) hearts., Conclusion: Although the total content of Cx45 is not upregulated in Cx43-deficient hearts, the localization of Cx45 to cardiac gap junctions depends on the expression level of Cx43 and is dramatically altered in mice that express no Cx43.

Long-chain acylcarnitines increase intracellular Ca2+ (Ca2+i) and induce electrophysiologic alterations that likely contribute to the genesis of malignant ventricular arrhythmias induced during myocardial ischemia. The mechanisms by which long-chain acylcarnitines increase Ca2+i are not known, although it occurs in the presence of Ca2+ channel blockade and inhibition of Na+/Ca2+ exchange. Long-chain acylcarnitines activate Ca2+ release channels from skeletal muscle sarcoplasmic reticulum (SR), but their effect on cardiac SR is unclear. To test the hypothesis that long-chain acylcarnitines increase Ca2+i from the SR, SR-enriched membrane fractions were prepared from rabbit left ventricular myocardium using sucrose density-gradient centrifugation and characterized by marker enzyme analysis. 45Ca2+ efflux was assessed in the presence or absence of long-chain acylcarnitines. Palmitoylcarnitine and stearoylcarnitine produced concentration-dependent efflux of 45Ca2+, whereas shorter chain acylcarnitines, palmitate, and palmitoyl-coenzyme A did not. Pretreatment of cardiac SR vesicles with ryanodine did not prevent palmitoylcarnitine-induced Ca2+ release. In addition, palmitoylcarnitine did not influence specific [3H]ryanodine binding, suggesting a mechanism independent of alterations in ryanodine receptor/Ca2+ release channel binding. In summary, long-chain acylcarnitines enhance Ca2+ release from cardiac SR vesicles and may thereby mobilize Ca2+i to induce electrophysiologic derangements under conditions, such as ischemia, in which these amphiphiles accumulate.

Introduction: Dynamic interplay between active and passive electrical properties of cardiac myocytes is based on interrelationships between various channels responsible for depolarizing and repolarizing ionic currents and intercellular conductances. Mice with targeted disruption of the connexin43 (Cx43) gene have hearts completely devoid of Cx43, the principal gap junctional protein expressed in mammalian hearts., Methods and Results: To determine whether cardiac myocytes that develop in an abnormal environment of reduced intercellular coupling have altered active membrane properties, we studied whole cell action potentials, Na+ channel currents, and Na+ channel expression and distribution via immunoblotting and confocal immunofluorescence in neonatal ventricular myocytes isolated from Cx43 wild-type, heterozygous, and homozygous null hearts. Action potential morphology, peak Na+ current, activation and inactivation kinetics, and Na+ channel protein expression and distribution were not different among myocytes isolated from wild-type, heterozygous, or null hearts. Active membrane properties and Na+ channel activity were completely normal in Cx43-deficient myocytes isolated from hearts that have been shown to exhibit markedly reduced Cx43 expression, gap junction number, and epicardial conduction delay., Conclusion: Despite a genetic inability to produce Cx43 and a developmental history that culminates in marked gross cardiac morphologic abnormalities, premature death, and myocardial inexcitability ex vivo, cardiac Na+ channel distribution and function appear to be normal in Cx43 null hearts. Although intimate structural and functional interrelationships have been described between ion channels and gap junction channels, expression and function of Na+ channels is not affected by the absence of Cx43.

To investigate the potential influence on one analogue of carnitine on the electrophysiologic derangements elicited by myocardial ischemia and subsequent reperfusion, we evaluated whether increasing concentrations of propionylcarnitine would interact with carnitine acyltransferase I and thereby decrease the accumulation of long-chain acylcarnitines during hypoxia in isolated adult canine myocytes. Propionylcarnitine (1-100 microM) did not alter the sixfold reversible increase in long-chain acylcarnitines elicited by 10 minutes of hypoxia. Likewise, propionylcarnitine did not alter the reversal of the accumulation of long-chain acylcarnitines associated with reoxygenation of hypoxic myocytes. To assess whether analogues of carnitine could influence the development or reversal of the electrophysiologic derangements induced by hypoxia in adult canine epicardial tissue, selected concentrations of propionylcarnitine (1 microM to 10 mM) were administered prior to and during 15 minutes of hypoxic perfusion at 35 degrees C followed by 5-20 minutes of reoxygenation. Continuous intracellular transmembrane action potentials were recorded with glass microelectrodes. Administration of propionylcarnitine prior to and during hypoxia did not alter the electrophysiologic derangements elicited by hypoxia or subsequent reoxygenation. Therefore, propionylcarnitine does not influence the activity of carnitine acyltransferase I and does not alter the accumulation of long-chain acylcarnitines during hypoxia. Although propionylcarnitine may protect ischemic myocardium by enhancing the recovery of contractile function during reperfusion, propionylcarnitine does not attenuate any of the electrophysiologic alterations observed during hypoxia or subsequent reoxygenation in isolated tissue.

To elucidate the mechanisms responsible for the increase in alpha 1-adrenergic receptors during ischemia in vivo, we developed a procedure for measuring alpha 1-adrenergic receptors in isolated, calcium-tolerant adult canine myocytes. Specific [3H]prazosin binding was rapid, saturable, reversible, and demonstrated the expected order of potency and stereospecificity for the alpha 1-adrenergic receptor. Myocytes exposed to 30 minutes of hypoxia at 25 degrees C or only 10 minutes at 37 degrees C exhibited a twofold to threefold increase in the number of alpha 1-adrenergic receptors with no significant change in receptor affinity. This hypoxia-induced increase in receptor number was reversible by 10 minutes of reoxygenation at 37 degrees C. In contrast, more prolonged hypoxia of 80 minutes or hypotonic shock actually decreased receptor number below normoxic, control values. The concentration of long-chain acylcarnitines in myocytes also increased threefold on exposure to 30 minutes of hypoxia. Sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA, 10 microM), a potent inhibitor of carnitine acyltransferase I, not only abolished the accumulation of long-chain acylcarnitines but also the increase in alpha 1-adrenergic receptor number induced by 30 minutes of hypoxia. Likewise, incubation of normoxic cells with exogenous palmitoyl carnitine (1 microM) for 10 minutes also increased alpha 1-adrenergic receptor number in the presence or absence of POCA. Thus, hypoxia results in an increase in alpha 1-adrenergic receptors associated with an increase in endogenous long-chain acylcarnitines. Furthermore, inhibition of carnitine acyltransferase I prevents not only the sarcolemmal accumulation of long-chain acylcarnitines but also the exposure of the alpha 1-adrenergic receptor, indicating that accumulation of endogenous long-chain acylcarnitines is critical to the hypoxia-induced increase in alpha 1-adrenergic receptors on adult myocytes.