<p> Peroxynitrite is formed in blood vessels upon reaction of superoxide anion with nitric oxide (NO). It can oxidize proteins and thiols and nitrosylate free or protein bound thiols and tyrosine residues, thereby producing vascular dysfunction. Peroxynitrite therefore, may contribute to hypertension and cardiovascular diseases. We investigated the in vitro effects of commercially available peroxynitrite. De-endothelialized rings from the left descending coronary artery of pig were treated with peroxynitrite for 30 min, washed and then contracted with cyclopiazonic acid (CPA) or by membrane depolarization with KCl. Tissues pre-treated with peroxynitrite showed inhibition of the CPA-induced contraction with an IC50 of ≈100 uM but there was no effect on KCl-induced contraction. Peroxynitrite is stable only at alkaline pH and it may decompose to form superoxide and NO. However, including superoxide dismutase + catalase along with peroxynitrite did not change its effect.</p> <p> Next, we used the same protocol to compare the effects of peroxynitrite and NO generating agents: 3-morpholino sydnonimine (SIN-1), s-nitroso-N-acetylpenicilliamine (SNAP), sodium nitroprusside (SNP) and spermine nonoate. The effectiveness of these agents to inhibit the CPA-induced contraction was SNAP > spermine nonoate ≥ SIN-1 > SNP. SNAP was the most effective in inhibiting the KCl-induced contraction with spermine nononoate being less effective and SIN-1 and SNP not producing any significant inhibition. We further investigated the effect of SNAP. Catalase, superoxide dismutase or CPTIO (a NO scavenger) did not prevent the effects of SNAP on the KCl or the CPA-induced contractions. The guanylate cyclase inhibitor ODQ, partially reversed the effects of only low concentrations of SNAP. Thus, pretreatment with NO generating agents such as SNAP and spermine NONOate appear to be more effective in inhibiting the contraction of the pig coronary artery than with peroxynitrite or the peroxynitrite generating agent SIN-1. Since SIN-1, SNAP, SNP and NONOates produce different amounts of peroxynitrite, nitric oxide and S-nitrosylation products, their effects may be used to delineate the molecular basis of the actions of peroxynitrite and NO on the arterial function.</p> / Thesis / Master of Science (MSc)

Yes / Superoxide anions have been associated with many aspects of cardiovascular disease. Menadione is a superoxide anion donor that alters the heart’s electrical and mechanical functions. The aim of this study was to demonstrate simultaneous changes in intracellular Ca2+ ([Ca2+]i) and mechanical activity in intact adult cardiac myocytes, and mechanical activity and electrical activity in isolated whole hearts in order to provide greater insight into the mechanisms associated with the detrimental effects of menadione on the myocardium. Isolated hearts from adult male Wistar rats (n = 11, 200–250 g) were Langendorff perfused at 38°C with a Krebs–Henseleit solution. A saline-filled balloon was placed in the left ventricle (LV) in order to measure diastolic and developed pressure. Monophasic action potentials were simultaneously recorded from the epicardial surface. External stimulation at 5 Hz and intrinsic pacing were used throughout a 10 min control period and 30 min exposure to 50 μM menadione. Single LV myocytes (n = 7 from n = 4 animals) were loaded with the Ca2+-indicator Fura4-AM, stimulated at 1 Hz and exposed to 50 μM menadione. Myocyte length was simultaneously measured with [Ca2+]i using a video edge detection system. In isolated hearts, exposure to menadione significantly decreased contractility and action potential duration (with a similar time course); intrinsic heart rate and rhythmicity. Diastolic pressure was significantly increased. In single adult myocytes, menadione caused a significant increase in diastolic [Ca2+]i and a decrease in resting cell length and led to spontaneous release of [Ca2+]i. We conclude that the effects of menadione upon electrical and mechanical activity of the heart are at least in part a consequence of dysregulation of [Ca2+]i handling and the subsequent increase in diastolic [Ca2+] alterations in [Ca2+]i are consistent with the generation of delayed after depolarization arrhythmias.

Irx5 is a homeodomain transcription factor that negatively regulates cardiac fast transient outward K+ currents (Ito,f) via the KV4.2 gene and is thereby a major determinant of the transmural repolarization gradient. While Ito,f is invariably reduced in heart disease and changes in Ito,f can modulate both cardiac contractility and hypertrophy, less is known about a functional role of Irx5, and its relationship with Ito,f, in the normal and diseased heart. Here I show that Irx5 plays crucial roles in the regulation of cardiac contractility and proper adaptive hypertrophy. Specifically, Irx5-deficient (Irx5-/-) hearts had reduced cardiac contractility and lacked the normal regional difference in excitation-contraction with decreased action potential duration, Ca2+ transients and myocyte shortening in sub-endocardial, but not sub-epicardial, myocytes. In addition, Irx5-/- mice showed less cardiac hypertrophy, but increased interstitial fibrosis and greater contractility impairment following pressure overload. A defect in hypertrophic responses in Irx5-/- myocardium was confirmed in cultured neonatal mouse ventricular myocytes, exposed to norepinephrine while being restored with Irx5 replacement. Interestingly, studies using mice virtually lacking Ito,f (i.e. KV4.2-deficient) showed that reduced contractility in Irx5-/- mice was completely restored by loss of KV4.2, whereas hypertrophic responses to pressure-overload in hearts remained impaired when both Irx5 and Ito,f were absent. These findings suggest that Irx5 regulates cardiac contractility in an Ito,f-dependent manner while affecting hypertrophy independent of Ito,f. On the other hand, Irx5-ablation attenuated calcineurin (Cn)-induced hypertrophy in hearts and cultured cardiomyocytes, suggesting that the effect of Irx5 on hypertrophy involves the Cn-NFAT signalling cascade. Biochemical assessments further revealed that Irx5 can positively mediate Cn-NFAT activities as well as Nfatc3 and Gata4 expression, and interacts with Nfatc3 and Gata4, suggesting the formation of a transcription complex for hypertrophic gene regulation. Taken together, these studies have identified Irx5 as a vital cardiac transcription factor, important for contractile function of the heart by regulating Ito,f, and compensatory hypertrophic response to biomechanical stress in the heart by affecting the Cn-NFAT (and Gata4) signaling pathway.

Thesis (M. Sc.)--York University, 2000. Graduate Programme in Kinesiology and Health Sciences. / Typescript. Includes bibliographical references (leaves 31-46, 67-72). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ59171.

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

Following contractile activity, fast twitch skeletal muscle exhibits increases in submaximal force known as potentiation. Although there is no consensus on the purpose of potentiation, it is known to enhance power during rapid dynamic contractions and counteract the early stages of peripheral fatigue. Potentiation is primarily attributed to phosphorylation of the myosin regulatory light chain (RLC) through a calcium-mediated process which results in increased calcium-sensitivity of crossbridge formation. However, there is a growing body of evidence showing that potentiation can be achieved in the absence of RLC phosphorylation, albeit to a lesser degree. A secondary characteristic of the potentiated contraction is an acceleration of relaxation properties, which could be teleologically beneficial to enhance the cycling rate of rapid motions (e.g. running). However, accelerated relaxation is inconsistent with elevations in calcium-sensitivity as this would tend to slow the time course and slow relaxation. Therefore there are multiple mechanisms involved in potentiation, some of which enhance crossbridge formation, and some of which enhance crossbridge detachment. A possible explanation for these events involves contraction-induced changes in the intracellular cytosolic calcium signal that triggers muscle contraction. For example, elevations in submaximal force could be achieved by increasing the amplitude of the calcium signal while enhanced relaxation speed could be achieved by a shorter duration of the calcium signal. Thus the main objective of this thesis was to investigate the contribution of changes in cytosolic Ca²⁺ to force potentiation. To achieve this objective, intact lumbrical muscles were extracted from the hind feet of C57BL/6 mice for use as the experimental model. The first study in this thesis examined cytosolic calcium signals during posttetanic potentiation using high (AM-fura-2 and AM-indo-1) and low (AM-furaptra) affinity calcium-sensitive fluorescent indicators to monitor resting and peak calcium respectively, both before and after a potentiating stimulation protocol of 2.5 s of 20 Hz stimulation at 37^oC. This protocol resulted in an immediate 17±3% increase in twitch force (n=10; P<0.05), though this potentiation dissipated quickly, lasting only 30 s. Resting cytosolic Ca²⁺ was also increased following the potentiating stimulus as indicated by increases of 11.1 ± 1.3% and 8.1 ± 1.3% in the fura-2 and indo-1 fluorescence ratios respectively. Like the force potentiation, these increases were short lived, lasting 20-30 s. No changes were detected in either the amplitude or kinetics of the Ca²⁺ transients following the potentiating stimulus. Western blotting analysis of the myosin heavy chain isoforms which determine the contractile phenotype of lumbrical muscle revealed predominance of fast type IIX fibres, while immunohistochemical analysis of proteins important for relaxation, namely parvalbumin, sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) 1a and SERCA2a, revealed that the expression of these proteins in lumbrical moderated those found in the soleus (slow) and EDL (fast) archetypes. Surprisingly, despite the fast phenotype of the lumbrical, it exhibited low expression of the skeletal muscle isoform of myosin light chain kinase, the enzyme responsible for phosphorylating the myosin RLC, and high expression of myosin targeting phosphatase subunit 2, the enzyme responsible for dephosphorylating the myosin RLC. These data were corroborated by a complete lack of myosin RLC phosphorylation in either the rested or potentiated states. It was thus concluded that elevations in resting cytosolic calcium concentration, in the absence of changes in the intracellular calcium transient and RLC phosphorylation, can potentiate twitch force. The next objective of this thesis was to determine if there are changes in the cytosolic calcium transient during staircase potentiation, defined as a stepwise increase in twitch force during low frequency stimulation (<10 Hz). Staircase potentiation has been repeatedly demonstrated to exhibit more robust potentiation than posttetanic potentiation in the absence of RLC phosphorylation. It was hypothesized that while the calcium transient is not altered during posttetanic potentiation, it may be an important potentiating factor in staircase due to the lower rest intervals between successive contractions. The effects of temperature on the intracellular calcium transient during staircase potentiation were also examined as part of this investigation. Here, lumbricals were loaded with AM- furaptra and then subjected to stimulation at 8 Hz for 8.0 s to induce staircase potentiation at either 30 or 37^oC. This stimulation protocol resulted in a 26.8 ± 3.2 % increase in twitch force at 37^oC (P<0.05) and a 6.8 ± 1.9 % decrease in twitch force at 30^oC (P<0.05) at the 8 s mark. Both the peak amplitude and the calcium-time integral of the calcium transient decreased during the first 2.0 s of the protocol (P<0.05), however these decreases were greater at 30^oC than 37^oC (P<0.05 amplitude; P=0.09 area). While peak amplitude remained low throughout the duration of the protocol, the calcium-time integral began to increase after the 2 s time point (P<0.05), a change reflective of the progressive increases in the 50% decay time and full width at half maximum of the calcium transient (P<0.05). Regression analysis of raw furaptra fluorescence ratios revealed a progressive decline in the peak amplitude of the calcium transients throughout the protocol which was not present at 37^oC. The increases in the duration of the calcium transient were mirrored by increases in the half relaxation time of the twitch contractions at both 30 and 37^oC, which had initially been reduced by ~20 and 9 % at 30 and 37^oC during the first 2 s of the protocol. Therefore the degree of staircase potentiation depends, in part, on the magnitude of the decline in the amplitude and the degree of slowing of the cytosolic calcium transient. The declines in calcium transient amplitude noted above occurred simultaneously with increased rates of relaxation and abbreviated contraction times. To determine if there was a causal relationship between the reduced amplitude and the faster contractions, AM-furaptra-loaded lumbrical muscles were stimulated at 8 Hz for 2 s in the presence and absence of caffeine, an agonist of the calcium release channel. Caffeine treatment attenuated the decline of the calcium transient amplitude (P<0.05), and was associated with greater potentiation at 37^oC (P<0.05), and attenuated force loss at 30^oC (P<0.05). Despite the increases in calcium and force, the relaxation times and rates of relaxation exhibited a greater acceleration following caffeine treatment (P<0.05). Therefore the relaxation-enhancing factor during potentiated twitches cannot be attributed to the calcium transient, and must be localized to changes on the myofilament. The case for inorganic phosphate as the effector is made. Similar to the findings of the posttetanic potentiation study, the resting cytosolic calcium concentration was elevated during staircase potentiation, as revealed by fura-2 ratio signals. The largest increase occurring immediately following the first twitch of the protocol. This coincided with the largest increases in force potentiation at both 30 and 37^oC. This finding is in accordance with the initial conclusion that elevations in resting calcium can enhance twitch force and contribute to potentiation, though the mechanism of action is unclear. One possibility is that increases in resting calcium, sub-threshold for force production, can enhance the number of attached but non-force producing crossbridges, thereby accelerating the transition of crossbridges to force-producing states upon calcium-release following stimulation. To test this hypothesis, the resting stiffness, a measure of crossbridge attachment, of lumbrical muscles was examined before and after a potentiating stimulus of 20 Hz 2.5 s. Resting stiffness was assessed using sinusoidal length oscillations, ~0.5 nm per half sarcomere in amplitude and ranging in frequency from 10-200 Hz. Subsequent analysis revealed decreases in the elastic stiffness (P<0.05) that lasted for ~20 s which were greater in magnitude (P<0.05) than increases in viscous stiffness which only lasted for ~5 s. This finding is consistent with the disappearance of short range elastic component (SREC) upon stretch or muscle activation which is commonly attributed to a population of stable, bound crossbridges in resting muscle. Subsequent analysis using imposed length changes to eliminate the SREC prior to contraction had no effect on the amplitude or duration of a subsequent twitch or tetanic contraction, and the changes in elastic and viscous stiffness of resting muscle were identical whether SREC was ablated by a contraction or imposed length change. Therefore it appears that potentiation occurs without an associated increase in bound crossbridges at rest, and may actually occur with fewer bound crossbridges at rest than the unpotentiated state. The lack of effect may be related to the relaxation-enhancing factor discussed above, and be an important feature of skeletal muscle serving to protect against damage via an involuntary eccentric contraction. This thesis describes potentiation as a complex and important biological function which is the sum of factors that serve to enhance and oppose force production.

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

No description available.

Irx5 is a homeodomain transcription factor that negatively regulates cardiac fast transient outward K+ currents (Ito,f) via the KV4.2 gene and is thereby a major determinant of the transmural repolarization gradient. While Ito,f is invariably reduced in heart disease and changes in Ito,f can modulate both cardiac contractility and hypertrophy, less is known about a functional role of Irx5, and its relationship with Ito,f, in the normal and diseased heart. Here I show that Irx5 plays crucial roles in the regulation of cardiac contractility and proper adaptive hypertrophy. Specifically, Irx5-deficient (Irx5-/-) hearts had reduced cardiac contractility and lacked the normal regional difference in excitation-contraction with decreased action potential duration, Ca2+ transients and myocyte shortening in sub-endocardial, but not sub-epicardial, myocytes. In addition, Irx5-/- mice showed less cardiac hypertrophy, but increased interstitial fibrosis and greater contractility impairment following pressure overload. A defect in hypertrophic responses in Irx5-/- myocardium was confirmed in cultured neonatal mouse ventricular myocytes, exposed to norepinephrine while being restored with Irx5 replacement. Interestingly, studies using mice virtually lacking Ito,f (i.e. KV4.2-deficient) showed that reduced contractility in Irx5-/- mice was completely restored by loss of KV4.2, whereas hypertrophic responses to pressure-overload in hearts remained impaired when both Irx5 and Ito,f were absent. These findings suggest that Irx5 regulates cardiac contractility in an Ito,f-dependent manner while affecting hypertrophy independent of Ito,f. On the other hand, Irx5-ablation attenuated calcineurin (Cn)-induced hypertrophy in hearts and cultured cardiomyocytes, suggesting that the effect of Irx5 on hypertrophy involves the Cn-NFAT signalling cascade. Biochemical assessments further revealed that Irx5 can positively mediate Cn-NFAT activities as well as Nfatc3 and Gata4 expression, and interacts with Nfatc3 and Gata4, suggesting the formation of a transcription complex for hypertrophic gene regulation. Taken together, these studies have identified Irx5 as a vital cardiac transcription factor, important for contractile function of the heart by regulating Ito,f, and compensatory hypertrophic response to biomechanical stress in the heart by affecting the Cn-NFAT (and Gata4) signaling pathway.

The general purpose of this thesis was to investigate new cardiac resynchronization therapy (CRT) optimization techniques and to assess their reliability when compared to invasive measurements of left ventricular contractility (LV dP/dtmax).We first assessed whether cardiac output (CO) measured by trans-thoracic impedance cardiography could correctly identify the optimal interventricular (VV) pacing interval while using invasive measurements of LV dP/dtmax as reference. We did not find any significant statistical correlation between the two optimizing methods when their corresponding optimal VV intervals were compared. We also tested the hypothesis that measurements of right ventricular contractility (RV dP/dtmax) could be used to guide VV delay optimization in CRT. The comparison of optimal VV intervals obtained from the left and right ventricular dP/dtmax did not show a statistically significant correlation; however, a positive correlation was found when broader VV intervals were evaluated and we concluded that this finding deserves further investigation. An interesting alternative for CRT optimization is the use of device integrated algorithms or sensors capable to adapt the CRT settings to the current needs of the individual patient. In this respect we investigated the use of cardiogenic impedance (CI) measurements obtained through the CRT-D device as a method for CRT optimization with invasive measurements of LV dP/dtmax as a reference. Our results showed that CI could be measured through the device after implantation and that a patient-specific impedance-based prediction model was capable to accurately predict the optimal AV and VV delays. To follow up on these positive results we re-evaluated the patient-specific impedance-based prediction models 24 hours post implantation and investigated the possibility of calibrating them using parameters derived from non-invasive measurements of arterial pressure obtained by finger pelthysmography at implantation.The results showed that the patient-specific impedance-based prediction models did not perform as well on the follow-up data as they did on the data from implantation day and that they correlated poorly with plethysmographic parameters. Our studies suggest that novel methods for CRT optimization should be thoroughly evaluated and compared to established measures of left ventricular function prior to introduction into clinical practice.