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The stress responsive gene myocyte stress 1 (ms1) has been implicated in the initiation of cardiac and skeletal muscle hypertrophy. Upregulation of ms1 is observed shortly after stress induction in rat hearts before the development of left ventricular hypertrophy. The protein sequence of MS1 is highly conserved across many species including the fruit fly model organism Drosophila melanogaster, with high levels of homology found in the actin binding domain. Here, the potential Drosophila homologue of ms1 (dms1, designated CG3630) has been investigated to determine the function and the level of functional homology to mammalian ms1. The role of this gene in the Drosophila heart and somatic muscle has been examined by manipulating the expression of dms1. Knockdown of dms1 affects somatic muscle function, reducing the ability of flies to climb and decreasing their daily activity levels. Overexpression of dms1 appears to increase climbing ability, but does not affect daily activity, suggesting that muscle strength is increased but energy is not. Expression of mouse MS1 in Drosophila muscle appears to have a similar effect to dms1 on muscle function, but under starvation conditions may increase longevity, whereas dms1 does not. This suggests a fundamental difference in function between mammalian and Drosophila ms1 in metabolism. The results of this project revealed an important role for dms1 in the function of Drosophila muscle, and have contributed to the development of a fruit fly model for research into this gene.

The present studies have demonstrated that alpha1 -adrenoceptors play an important role in the ischaemia/reoxygenation-induced injury of the human atrial myocardium. They have shown that stimulation of alpha1-adrenoceptors with phenylephrine protects against injury whereas their blockade with prazosin is detrimental, both effects obtained in a dose-dependent manner. They have also shown that the effect of the stimulation or blockade of alpha1-adrenoceptors depends on the time of administration so that 1-adrenoceptors' stimulation is protective when given prior to ischaemia but detrimental when giving during ischaemia. On the contrary, alpha1-adrenoceptors' blockade is beneficial during ischaemia, detrimental during reoxygenation but has no significant effect prior to ischaemia. It appears that similar maximal protection can be obtained with alpha1-stimulation prior to ischaemia and with alpha1-blockade during ischaemia although the combination of the two does not induce additional protection. Furthermore, the protective effect of alpha1-stimulation prior to ischaemia is as potent as ischaemic preconditioning (IP). In this thesis, I have also demonstrated that protection with pharmacological preconditioning by activation of alpha1-adrenoreceptors or adenosine receptors is identical to that of IP in the human myocardium.;These studies have provided novel information to understand the underlying mechanism of protection by preconditioning of the human myocardium. They have shown that mitoKATP channels, PKC and p38MAPK are an integral part of the cellular signal transduction involved in this cardioprotection in which mitoKATP channels are placed upstream and p38MAPK is placed downstream of PKC.;The abolition of the ability of the human myocardium to be protected by ischaemic and pharmacological preconditioning without exacerbating the susceptibility to ischaemic injury when nicorandil, a mitoKATP channel opener and nitric oxide donor, was administered clinically was unexpected. I demonstrated that the likely cause of the failure to precondition the myocardium of patients on nicrorandil is the unresponsiveness of the mitoKATP channels since protection cannot be obtained with diazoxide, a specific mitoKchannel opener, but can be elicited by activation of PKC and p38MAPK that are downstream of mitoKATP channels in the signalling transduction cascade of preconditioning.;The sulfonylureas glibenclamide and gliclazide, that block KATP channels, have distinctive effects on IP. Thus, although glibenclamide abolished the protective effect of preconditioning even at 0.1 muM, gliclazide did at a higher concentration 30muM. The cardioprotection induced by diazoxide, which open mitoKATP channels was also abrogated by glibenclamide. However glibenclamide did not block the protective effect of activation of PKC and p38MAPK.

In addition to its central role in excitation-contraction coupling (ECCl, calcium is heavily implicated in the genesis of arrhythmias, including atrial fibrillation (AF). In contrast to ventricular myocytes, there is comparatively little quantitative information on Ca 2 + handling in atrial cells. A thorough appreciation of physiological Ca2 + handling is necessary to understand fully the pathological significance of changes leading to AF. The aim of this thesis was to perform a quantitative assessment of atrial Ca 2 + handling and report the effects of angiotensin-lion Ca2 +- current. Differences in ultrastructure between atrial and ventricular cells are likely to be significant. The extensiveness of t-tubules was investigated in rabbit and pig atrial cells in comparison with ventricular cells. No t-tubules were observed in rabbit atrial cells, however, a subset of pig atrial cells showed a t-tubule network, albeit less extensive than that of ventricular myocytes. Ca2 + transients in rabbit atrial cells were spatio-temporally non-uniform, initiated at the cell edge and spreading to the cell centre. The importance of CICR in atrial cells was highlighted by the reduced transient amplitude in the absence of SR and the high contribution of the SR to Ca2 + removal. The centripetal spread was not greatly affected by increasing Ca2 + influx, or SR ci+ release, possibly due to the buffering power of atrial cells, which was greater than that of ventricular cells. Surprisingly ECC gain was not significantly different between atrial and ventricular cells, however it is suggested that differences in stimulus underlie this result. Data presented here highlight that not only the differences in ultrastructure, but also fundamental ci+ handling properties are responsible for atrial-ventricu lar tissue differences. Initial experiments to investigate the effects of angiotensin-I I suggest that this does not have a significant impact on ci+ influx, however further work to fully assess its effects in atrial cells is required.

It is known that different point mutations in α-tropomyosin (Tm) can cause either hypertrophic (HCM) or dilated (DCM) cardiomyopathy. Both of these serious pathologies have a distinct phenotype with unknown mechanisms of development. Biochemical in vitro studies provide valuable information for exploring downstream consequences of cardiomyopathy mutations in sarcomeric proteins leading to cardiac remodelling and consequent heart failure. Tm is a linear a-helical coil-coiled dimer involved in calcium dependent regulation of muscle contraction. Prior to this work, the effects of mutations in Tm on regulation of muscle contraction have been made exclusively with Tm mutant homodimers. However, individuals with a heterozygous background may express mutant and WT proteins in a 1 : 1 ratio which can assembly into a mixture of αα, αα* and α*α* Tm dimers. We found that presence of mutation has little effect on dimer formation between the mutant and the WT monomers, therefore theoretical ratio of the Tm dimers in vivo may be 1 : 2 : 1, respectively. This assumption would make the heterodimer predominant. The properties of in vitro assembled Tm heterodimers carrying HCM (WT-D175N and WT-E180G) and DCM (WT-E40K, WT-E54K, and WT-D230N) causing mutations were examined including thermal stability, flexibility, actin affinity, calcium regulation of 51 biding, and calcium regulation of myofibril force. We showed that various properties of the heterodimers can be similar to those of the wild-type (thermal stability of reduced WTD175N; actin affinity of WT-E40K, and WT-D175N; ΔpCa of WT-E40K, WT-E54K, WT-E180G, WT-D230N), similar of those to the mutant homodimer (ΔpCa of WT-D175N, flexibility of WT-D175N and WT-E180G), intermediate between the two (actin affinity of WT-E180G), or different from both (thermal stability of reduced WT-E40K, WT-E54K, WT-E180G, and WTD230N; actin affinity ofWT-E40, WT-E54K, and WT-D230N; ΔpCa of D175N). The results demonstrate that the properties of Tm heterodimers cannot be predicted from the interpolation of the WT and mutant homodimer data. The distinct properties of heterodimers establish that it will be important to define if the pathogenic agent is a homodimer, a heterodimer or both for each known cardiomyopathy mutation in Tm.

Μοντελοποίηση του Δυναμικού Δράσης (Action Potential) σε καρδιακά κύτταρα για περιπτώσεις γενετικής βλάβης στα ιοντικά κανάλια Νατρίου και Καλίου

Previous work has indicated that in failing heart muscle the defect in contractility is primarily due to altered troponin function. Troponin has been isolated from non-failing (donor) and end-stage failing human heart muscle. Using in vitro motility assays we have found an increase in Ca2+ -sensitivity (EC50 decreased 2.6-fold) and a 13% reduction in cross-bridge turnover rate for troponin from failing heart muscle. In vitro motility analysis of thin filaments containing a replaced unphosphorylated troponin I resulted in a failing phenotype of a reduced sliding speed and an increased calcium sensitivity. When replaced with a PKA phosphorylated troponin I, motility parameters reverted to a pattern indistinguishable from non-failing troponin. Measurement of total phosphorylation using the Pro-Q Diamond phosphoprotein gel stain showed troponin T phosphorylation was 3.05 ± 0.20 mol Pi / mol protein in non- failing and 3.11 ± 0.47 in failing heart troponin. Troponin I phosphorylation level was 2.25 ± 0.31 mol Pi / mol protein in non-failing and 0.38 ± 0.16 in failing. Measurement of phosphorylation at troponin I serine 23/24 was determined using a phospho-specific antibody and yielded 1.14 ± 0.09 mol Pi / mol in non-failing and 0.19 ± 0.04 in failing. The difference between Pro-Q and serine 23/24 measurements is ascribed to phosphorylation at PKC sites on troponin. It was 1.11 ± 0.23 mol Pi / mol protein in non- failing heart and 0.18 ± 0.16 in failing. Thus the level of phosphorylation of troponin I in failing heart is substantially reduced at both PKA-specific and PKC-specific sites. It has been reported that PKC activity is increased in failing hearts and therefore, it is possible that phosphatase activity may be increased. Using recombinant human cardiac troponin I phosphorylated with PKA and [y32p]_ATP as a substrate we found substantial troponin I-specific phosphatase activity in washed myofibrils from human heart. We observed that the activity in myofibrils from failing heart was 19.3 ± 7.4% higher than from non-failing heart and that the dephosphorylating ability is predominantly type PP2a as it was 95% inhibited by 2nM okadaic acid. In conclusion, we showed that decreased troponin I phosphorylation in failing heart was correlated with higher Ca2+ -sensitivity and lower cross-bridge turnover rate and that troponin I is dephosphorylated at PKA specific sites in end-stage heart failure and could account for the contractile defect seen. We also studied HCM heart muscle obtained by surgical myectomy and several animal models of heart failure. It appears that this relationship between troponin function and phosphorylation does not apply in several of these cases.

Gap junctions are clusters of transmembrane channels, composed of connexins (Cx), that facilitate electrical and chemical communication between the cytoplasmic compartments of contiguous cells. Three connexins are expressed in cardiac myocytes, Cx40, Cx43 and Cx45. Targeted deletion of these connexin genes in mice results in cardiac malformations and conduction abnormalities. From this background, the question arises as to whether connexins play a role in human congenital heart disease. Atrial and ventricular tissue samples were studied from patients undergoing cardiac surgery. Immunoconfocal microscopy and western blot analysis of atrial tissue revealed that expression of CX40 and CX43 in children and adults with congenital heart disease is the same as that in the normal adult atrium, irrespective of the underlying malformation. Normal adult ventricular working myocytes express only CX43. Study of control ventricular samples in children confirmed that, as in adults, CX43 only is expressed. However, immunoconfocal microscopy of samples from patients with right ventricular outflow obstruction (tetralogy of Fallot and double chambered right ventricle) revealed that in addition to CX43, CX40 is also highly expressed. Expression is heterogeneous and CX40 is eo-localised with CX43. Quantitative western blot analysis showed that up to 10% of the total connexin expressed in these samples is CX40. As patients re-operated following previous repair (with markedly different underlying haemodynamics) still demonstrated high CX40 expression in the working myocardium, these results suggest that a lack of normal CX40 repression during development leads to heart malformations . . Gap junction channels formed from each connexin isofonn have distinctive biophysical properties. Connexin eo-expression further alters these properties. To investigate the functional consequences of the connexin eo-expression patterns observed in the ventricular samples, in vitro cell models were used. Intercellular communication was assessed using cell-to-cell Lucifer Yellow dye transfer in an inducible RLE cell line and conducjion properties were studied in an atrial myocyte (HL-l) cell line. The findings are consistent with the idea that altered function arising from abnormal embryonic connexin expression is a contributor to some types of human cardiac malformation.

Myosin Binding Protein-C (MyBP-C) is a multi-domain protein located on the thick filaments of striated muscle. Mutations in the gene encoding the cardiac isoform of the protein, cMyBP-C, are known to cause hypertrophic cardiomyopathy (HCM). cMyBP-C is composed of 11 globular domains, eight of which have homology to Igl, the other three to fibronectin III. The N-terminal region of the protein has been assigned a role in the regulation of muscle contraction through its interaction with the Subfragment-2 (S2) portion of myosin.