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Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. There are numerous studies reporting developmental cardiac toxicity in multiple fish species due to PAH exposure. However, there are relatively few instances where the effects of acute PAH exposure in adult fish have been characterized. Furthermore, the majority of experiments comparing PAH toxicity with exposure route in adult fish focus on CYP1A gene expression or enzyme activity, while there is a lack of information about the possible pathophysiological effects. Therefore, the overall objective of this thesis was to characterize the sublethal effects of benzo[a]pyrene (BaP), a prototypical PAH, on adult zebrafish (Danio rerio) cardiorespiratory function and fitness following acute exposure by two different routes. In the first experiment, adult zebrafish were intraperitoneally (i.p.) injected twice (one injection/24 hr) with increasing concentrations of BaP (0.1, 10, and 1000 μg/kg) and compared to corresponding dimethylsulfoxide (DMSO) controls. In a second set of experiments, adult zebrafish were aqueously exposed to BaP (static, renewal at 24 hr; 16.2 and 162 μg/L) and compared to DMSO controls. Following 48 hr exposure, one group of fish (n=10/treatment group) were subjected to swimming performance tests to assess critical swimming speed (Ucrit), oxygen consumption rate (MO2), cost of transport (COT), standard metabolic rate (SMR), active metabolic rate (AMR), and factorial aerobic scope (F-AS). Another group of fish (n=12/treatment group) were subjected to echocardiography following 48 hr BaP exposure to evaluate cardiac function. Following echocardiography analysis, samples were collected for parent compound (BaP) body burden and CYP1A mRNA induction analysis. 48 hr BaP injection resulted in significant sublethal effects on adult zebrafish cardiorespiratory function. Oxygen consumption (MO2) was increased at three swimming speeds in injected BaP groups compared to control. In contrast, aqueously BaP-exposed fish showed increased MO2 only at the single lowest swim speed. COT was also similarly increased for both exposure routes. SMR was elevated with both exposure routes, while AMR remained unchanged. This resulted in a significant decrease in F-AS for all treatment groups compared to corresponding controls with both exposure routes. Cardiac function was significantly affected by both routes of BaP exposure. Ventricular heart rate was significantly decreased in BaP-exposed fish, both injected and aqueously-exposed. However, stroke volume was decreased only in fish aqueously exposed to BaP, which resulted in significantly reduced cardiac output with that exposure route. In contrast, the ratio of atrial to ventricular heart rate (AV ratio) was increased only in fish i.p. injected with BaP, indicating the possibility of cardiac arrhythmias occurring. Analysis of BaP body burdens in fish tissue allowed for identification of an overlapping dose group between exposure routes, through which comparisons of cardiotoxicity were then made. This comparison revealed slight differences in cardiotoxicity between exposure routes. BaP-injected fish suffered from more severe bradycardia than aqueously exposed fish. Furthermore, cytochrome P4501A (CYP1A) mRNA levels in liver and heart tissue showed more significant increases in injected fish, while skeletal muscle CYP1A was increased only following aqueous exposure. In conclusion, acute BaP exposure caused metabolic alterations and impaired cardiorespiratory function in adult zebrafish regardless of exposure route. Interestingly, the primary mechanism behind these effects appeared to differ slightly with exposure route. These results suggest that acute BaP exposure may have negative effects on adult fish survivability in the environment. Overall, this work provides valuable insight into the pathophysiogical consequences of acute PAH exposure in adult stage fish.

Previous research has suggested that emotional expression is important for psychological adjustment to disease (e.g., Stanton et al., 2000). Indeed, experimentally prescribed emotional disclosure (traditionally, expressive writing) in the context of illness has been shown to provide benefits for mental and physical health (Pennebaker, 1993). However, the experimentally prescribed disclosure in previous research has typically been asocial, akin to writing in a diary. In contrast, the present research, by experimentally manipulating the intended audience of one’s disclosure, examined the effect of addressing one’s emotional disclosure to specific types of listener, namely a therapist or one’s spouse. Cardiac couples in which one partner had a recent cardiac event took part in the current study. First, partners completed pre-study characteristics questionnaires. Next, in a lab session, partners (in separate rooms) were randomly assigned to one of three conditions: (1) they disclosed their thoughts and feelings about the cardiac event as though speaking to their partner; (2) they disclosed their thoughts and feelings about the event as though speaking to a therapist; or (3) in a non-disclosure, control condition, they spoke about a neutral topic. Partners then engaged in a dyadic discussion about each partner’s experiences, thoughts and feelings about the cardiac event. Finally, a one-month follow-up measure assessed their relational outcomes since participation. It was hypothesized that the partner-oriented condition would lead to better outcomes than the therapist-oriented condition, and that disclosing overall would be more beneficial than non-disclosure. The study also examined the influence of pre-study characteristics on in-lab and follow-up outcomes, with the hypothesis being that participants doing less well initially (i.e., higher on psychological distress, Type D personality, emotional suppression, and lower on mindfulness, cognitive reappraisal, relationship satisfaction, and perceived social support) would experience relatively more benefits from participation than those initially doing well. In addition, the study investigated whether type of event (unexpected and sudden vs. planned and more gradual) would differentially influence measures throughout the study, with the hypothesis being that couples who experienced a sudden event (MI) would be more distressed and therefore benefit more from participation than those who went through a planned procedure. Hypotheses were partially supported. Although the manipulation of disclosing to one’s partner vs. a therapist did not elicit many differences, one important and novel finding emerged regarding the orientation of disclosures: partner-disclosures yielded a more communal orientation whereas therapist-disclosures yielded a more self-focused orientation. The importance of patients’ disclosure orientation (me vs. we) was revealed when it emerged that greater communal focus led to better outcomes for both partners, but greater self-focus led to less positive outcomes for spouses. Compared to non-disclosure, disclosure generally was found to provide greater benefits for relational communication as well as marital satisfaction at follow-up. Consistent with predictions, participants who seemed most in need at pre-study (i.e., greater distress, Type D personality, emotional suppression, and less marital satisfaction and perceived support) experienced better outcomes than those who were initially well. Finally, couples who went through a sudden event were found to be more in need and benefited more than those whose event was planned, and this was especially pronounced in the effects on the spouses. Directions for future research and implications for clinical practice were discussed. For example, in addition to highlighting the value of emotional disclosure in the context of serious illness, the findings identified characteristics of cardiac couples who may be more in need of communication interventions and drew attention to important, relatively unmet needs in the patients’ spouses.

Autophagy is a process by which cells re-cycle organelles and macromolecular proteins during cellular stress. Defects in the regulation of autophagy have been associated with various human pathologies including heart failure. In the heart tumor suppressor p53 protein is known to promote apoptotic and autophagic cell death. We found p53 over-expression increased endogenous protein level of the hypoxia-inducible Bcl-2 death gene Bnip3 which leads to loss of mitochondrial membrane potential (ΔΨm). This was accompanied by autophagic flux and cell death. Conversely, loss of function of Bnip3 in cardiac myocytes or Bnip3-/- mouse embryonic fibroblasts prevented mitochondrial targeting of p53 and autophagic cell death. These data provide the first evidence for the dual regulation of autophagic cell death of cardiac myocytes by p53 that is mutually dependent on Bnip3 activation. Hence, our findings may explain how autophagy and cell death are dually regulated during cardiac stress conditions where p53 is activated.

Scorpion toxins specific for Na+ and K+ channels, have been studied extensively but relatively little has been done on Ca2+ channel toxins. Toxins in the venom of only two South African scorpions P. transvaalicus and P. granulatus have been found to interact with Ca2+ channels. Kurtoxin isolated from the venom of P. transvaalicus inhibits the T and L-type neuronal Ca2+ channels, whereas KLI and KLII (Kurtoxin-like peptide I and II), isolated from P. granulatus, inhibits T-type Ca2+ channel activity in mouse male germ cells. In this study the effects of fractions isolated from the venom of P. granularus on Cca2+ channels in rat ventricular myocytes were investigated by means of the whole-cell patch clamp technique. Fractions of P. granulatus crude venom were isolated with Sephadex G50 columns (fraction I-IV). Fraction III (PgIII) showed a voltage dependent increase of the inward Ca2+ current and influenced the channel kinetics by shifting the voltage dependence of activation towards more hyperpolarizing membrane potentials and decreased the rate of inactivation and deactivation. The time of the current to reach peak was also delayed. PgIII was further separated by HPLC in an attempt to identify the subfraction/s responsible for the agonistic effect. Subfraction I had an agonistic effect similar to PgIII, whereas subfraction II and III, decreased the Ca2+ current. The observed agonistic effect has not been described in the literature. The identification of new peptide structures with unique functions are important in the field of toxin research. Peptides that target Ca2+ channels can be valuable tools to characterize Ca2+ channels. Ca2+ channels in the heart are implicated in a number of pathological disorders like angina, ischemia, some arrhythmias and hypertension. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

The incidence of cardiovascular disease has reached epidemic proportions in North America. Specifically, myocardial infarctions (MI) are a major contributor to heart failure which greatly influences morbidity and mortality rates in developed nations. In the post-MI heart, cardiac fibroblasts migrate to the damaged area, convert to myofibroblasts and contribute to infarct scar contraction. As well, cardiac myofibroblasts are hypersynthetic for matrix components eg, collagen, and de novo production of fibrillar collagens lessens the chance for acute scar rupture. TGF-1 is important in the initiation of cardiac healing and fibrosis. Canonical TGF-1 signaling occurs with the activation of receptor-operated Smads (R-Smads) including Smad3. The current study addresses the question of whether Smad3 and/or myocardin influence myofibroblast contractility. We believe that myocardin is a Smad3 binding partner and cofactor and thus contributes to Smad associated healing and fibrotic events in the heart. In mesenchyme-derived cells, myocardin exists as a nuclear protein and is a cardiac and smooth muscle specific transcriptional coactivator of serum response factor (SRF). This transcription factor has been shown to bind to Smad3 in COS-7 cells (a green monkey kidney fibroblast-like cell line) and we suggest that it may contribute to fibroproliferative events. Precisely how Smad3/myocardin facilitates post-MI wound healing and/or contributes to inappropriate post-MI fibrosis is unknown. Very little work has been done to address myocardin expression in cardiac ventricular myofibroblasts. While a number of previous studies address TGF-β/Smad signaling in cardiac myofibroblasts, none have addressed the effects of overexpressed Smad3 on cellular contractility and collagen secretion. As Smad3 and its endogenous inhibitor Smad eg, I-Smad7, contribute significantly to TGF-β signaling in myofibroblasts, we rationalize that they must be important in the regulation of many fibroproliferative processes. Our goals were first to measure/determine myocardin expression in primary ventricular myofibroblasts; second, to explore a putative interaction between Smad3 and myocardin; third to examine a possible link between TGF-β1 stimulation, myocardin and Smad3. Finally, we sought to examine the effect of overexpressed Smad3, Smad7 and myocardin on contractility and collagen production. These experiments were conducted by using RT-PCR, co-immunoprecipitation, adenoviral overexpression of Smad3, Smad7 and myocardin, Western blot analysis, collagen gel deformation assays (contractility studies) and finally, Pro-collagen 1 N-terminal Peptide (P1NP) secretion as a measure of mature collagen production. We document the novel expression of myocardin in ventricular myofibroblasts and provide evidence that myocardin may serve as a Smad3 cofactor in cardiac myofibroblasts. Further, myocardin overexpression is linked to increased contractility in myofibroblasts compared to LacZ infected controls, and that TGF-β1 acutely stimulated myocardin expression followed by a dramatic reduction 1 hour thereafter. Overexpressed Smad3 alone led to increased contractility in primary ventricular myofibroblasts. Thus the effect of increasing myocardin expression had a comparable effect to that of increased Smad3 alone with this endpoint. Finally, overexpression of both Smad3 and myocardin in the presence of TGF-β1 led to an additive stimulation of contractility in cells when compared to the effect of TGF-β1 stimulation alone. Overexpressed Smad7 alone was associated with decreased secretion of type I collagen when compared to the control; when cells overexpressing Smad7 are stimulated with TGF-β1, collagen secretion is dramatically reduced when compared to cells treated with TGF-β1. In an addition series of experiments we addressed reverse mode NCX1 function as a means of Ca2+ entry to the cytosol of myofibroblasts upon their excitation. We have previously shown the stimulatory effect of TGF-β1 on myofibroblast contractility, and we now report that overexpression of Smad3 alone led to increased mRNA expression of NCX1. Thus it is possible that TGF-β1 signaling via Smad3 may influence Ca2+ movements and thus contractile performance in ventricular myofibroblasts.

Aims: Non-responder rates for CRT vary from 11 to 46 %. Retrospective data imply a better outcome with stimulation of the latest contracting LV region. Our study analyzed feasibility, safety and clinical outcome of prospectively planned LV lead placement at the site of latest mechanical activation. Methods: Thirty-eight heart failure patients with CRT indication were assessed by 3D TEE and rotation angiography of the coronary sinus. Both images were merged into a single 3D-model to identify CS target veins close to the site of latest mechanical activation. Subsequently LV lead deployment was attempted at the desired target position. Patients were clinically and echocardiographically evaluated at baseline, after 3 and 6 months. Results: The area of latest mechanical activation covered 6 ± 2 segments (38 ± 13 % of LV surface) and was found lateral in 24/37 (65 %), anterior in 11/37 (30 %), inferior in 2/37 (5 %) and septal in 1/37 (3 %) patients. In 36/37 (97 %) patients an appropriate target vein was identified and successful implantation could be performed in 34/37 (92%) patients. Among those patients clinical and echocardiographic response was observed in 91 % and 81 %, respectively. Conclusions: Individualized lead placement at the latest contracting LV site can be performed safely and successfully in the majority of patients. Initial clinical outcome data are encouraging. Identification of target sites requires multimodality integration between LV wall motion data and CS anatomy. Future developments need to improve those technologies and require randomized data on clinical outcome parameters.

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Left ventricular (LV) filling increases during exercise, but left atrial (LA) phasic function and its contribution to LV filling is poorly understood. Sixteen endurance-trained middle-aged males were studied at rest and during light (LE) and moderate (ME) intensity cycle-ergometry. Atrioventricular-plane displacement (AVPD) increased from rest to LE (from 14±2 mm to 18±2 mm, p<0.01), but did not increase further at ME. LA reservoir volume increased from rest to LE (from 32±8 mL to 40±10 mL, p<0.01). LA passive contribution increased at LE (from 21±5 mL to 27±8 mL, p<0.01), while LA active contribution increased from rest only at ME (from 12±5 mL to 23±9 mL, p<0.01). AVPD, and thus the longitudinal shortening of LV systole, contributes to LA filling primarily during LE, but is a limited mechanism beyond LE. These data suggest that LV filling appears to shift to a reliance on conduit function to increase LV filling at ME.