Dehydration, with and without heat, in kangaroos from mesic and arid habitats: different thermal responses including varying patterns in heterothermy in the field and laboratory.

Field data showing the daily patterns in body temperature (T(b)) of kangaroos in hot, arid conditions, with and without water, indicate the use of adaptive heterothermy, i.e. large variation in T(b). However, daily T(b) variation was greater in the Eastern Grey Kangaroo (Macropus giganteus), a species of mesic origin, than in the desert-adapted Red Kangaroo (Macropus rufus). The nature of such responses was studied by an examination of their thermal adjustments to dehydration in thermoneutral temperatures (25 degrees C) and at high temperature (45 degrees C) via the use of tame, habituated animals in a climate chamber. At the same level of dehydration M. rufus was less impacted, in that its T(b) changed less than that for M. giganteus while it evaporated significantly less water. At a T(a) of 45 degrees C with water restriction T(b) reached 38.9 +/- 0.3 degrees C in M. rufus compared with 40.2 +/- 0.4 degrees C for M. giganteus. The ability of M. rufus to reduce dry conductance in the heat while dehydrated was central to its superior thermal control. While M. giganteus showed more heterothermy, i.e. its T(b) varied more, this seemed due to a lower tolerance of dehydration in concert with a strong thermal challenge. The benefits of heterothermy to M. giganteus were also limited because of thermal (Q(10)) effects on metabolic heat production and evaporative heat loss. The impacts of T(b) on heat production were such that low morning T(b)'s seen in the field may be associated with energy saving, as well as water saving. Kangaroos respond to dehydration and heat similarly to many ungulates, and it is apparent that the accepted notions about adaptive heterothermy in large desert mammals may need revisiting.

Myocardial release of nitric oxide during ischaemia and reperfusion: effects of L-arginine and hypercholesterolaemia.

AIMS: Nitric oxide (NO) may modulate myocardial ischaemia/reperfusion (I/R) injury, but effects of hypercholesterolaemia on myocardial NO release during I/R are unknown. METHODS: A NO-specific carbon fibre electrode continuously measured coronary sinus [NO] during 60 min low-flow ischaemia (1 ml/min) and 60 min free reperfusion (I/R) in isolated rabbit hearts. Experimental groups (n=7 per group) were control, L-arginine supplement (200 microM), N-nitro-L-arginine methyl ester (L-NAME) treatment (8 microM) and hypercholesterolaemic. RESULTS: During early I, NO release decreased markedly in control (-1356+/-286 pmol/min/g) and L-arginine (-1972+/-172) groups, but less in L-NAME (-441+/-89) and hypercholesterolaemic (-602+/-164) groups (both p<0.01 vs. controls). No increase in NO release during I was seen in any group. After R, NO release increased above baseline in control (+2333+/-591 pmol/min/g) and L-arginine (+1048+/-278) groups and hypercholesterolaemic (+1100+/-478) (p<0.05 vs. pre-ischaemia each group). There was little increase in NO release in the L-NAME group (+436+/-247 pmol/min/g, p<0.05 vs. controls). In each group, myocardial NO release declined towards pre-ischaemic levels during 60 min R. Hearts treated with L-arginine had similar NO release but better functional recovery than controls (p<0.01). Treatment with L-NAME was also associated with better functional recovery than in controls or hypercholesterolaemic hearts. CONCLUSION: Myocardial NO release declines rapidly during ischaemia, but increases above baseline during early reperfusion. Improved function after L-arginine treatment appears to be independent of effects upon NO release. Hypercholesterolaemia is associated with reduced myocardial NO release, under both baseline conditions and during ischaemia and reperfusion.

Proteomics of ischemia and reperfusion injuries in rabbit myocardium with and without intervention by an oxygen-free radical scavenger.

A brief period of ischemia followed by timely reperfusion may lead to prolonged, yet reversible, contractile dysfunction (myocardial stunning). Damage to the myocardium occurs not only during ischemia, but also during reperfusion, where a massive release of oxygen-free radicals (OFR) occurs. We have previously utilized 2-DE and MS to define 57 protein spot changes during brief ischemia/reperfusion (15 min ischemia, 60 min reperfusion; 15I/60R) injury in a rabbit model (White, M. Y., Cordwell, S. J., McCarron, H. C. K., Prasan, A. M. et al., Proteomics 2005, 5, 1395-1410) and shown that the majority of these occur because of physical and/or chemical PTMs. In this study, we subjected rabbit myocardium to 15I/60R in the presence of the OFR scavenger N-(2-mercaptopropionyl) glycine (MPG). Thirty-seven of 57 protein spots altered during 15I/60R remained at control levels in the presence of MPG (15I/60R + MPG). Changes to contractile proteins, including myosin light chain 2 (MLC-2) and troponin C (TnC), were prevented by the addition of MPG. To further investigate the individual effects of ischemia and reperfusion, we generated 2-DE gels from rabbit myocardium subjected to brief ischemia alone (15I/0R), and observed alterations of 33 protein spots, including 18/20 seen in both 15I/60R-treated and 15I/60R + MPG-treated tissue. The tissue was also subjected to ischemia in the presence of MPG (15I/0R + MPG), and 21 spot changes, representing 14 protein variants, remained altered despite the presence of the OFR scavenger. These ischemia-specific proteins comprised those involved in energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin). We conclude that contractile dysfunction associated with myocardial stunning is predominantly caused by OFR damage at the onset of reperfusion, but that OFR-independent damage also occurs during ischemia. These ischemia-specific protein modifications may be indicative of early myocardial injury.

Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems.

Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein.

Modifications of myosin-regulatory light chain correlate with function of stunned myocardium.

The precise molecular basis for myocardial stunning remains unresolved, but protein damage within the myofibril is a likely mechanism. We used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to identify protein modifications in stunned myocardium. In isolated, perfused rabbit hearts, low-flow ischemia (1 ml/min) and reperfusion resulted in impaired left-ventricular function (rate-pressure product (RPP) after 15-min ischemia: 65 +/- 5% pre-ischemia). We have characterised the sequence of ventricular myosin-regulatory light chain (MLC-2, 18 kDa) in rabbit myocardium and identified two non-phosphorylated (P(1) and P(2)) and two phosphorylated (P(3) and P(4) at Ser-14) isoelectric point variants. MS revealed that the acidic isoelectric point post-translational modification of P(1) and P(3), resulting in P(2) and P(4) respectively, was due to deamidation of asparagine to aspartate at residue 13, adjacent to Ser-14 phosphorylation site. After 15-min ischemia and reperfusion, a 15-kDa MLC-2 fragment was detected (MLC-2(14-165)), resulting from N-terminal cleavage between Asn/Asp-13 and Ser-14 of non-phosphorylated MLC-2, which accounted for 9.8% of visible non-phosphorylated MLC-2. Subsequent 2-DE of subcellular fractions showed that the fragment was lost from the myofilament. Treatment with an OH radical scavenger, N-(2-mercaptopropionyl) glycine (MPG, 3 mmol/l), preserved contractile function (RPP: 106 +/- 9% pre-ischemia) and prevented cleavage of MLC-2. Proteolytic damage to MLC-2, related to presence of OH radicals during reperfusion, correlates with myocardial stunning and may contribute to impaired contractility.

Duration of ischaemia determines matrix metalloproteinase-2 activation in the reperfused rabbit heart.

It has been hypothesised that activation of matrix metalloproteinase-2 (MMP-2) contributes to reversible myocardial dysfunction (stunning) following short-term ischaemia and reperfusion. Gelatin zymography was used to measure release of both pro-MMP-2 (72 kDa) and MMP-2 (62 kDa), into the coronary effluent from isolated, perfused rabbit hearts during 90 min aerobic perfusion (control), or low-flow ischaemia (15 or 60 min at 1 mL/min), followed by 60 min reperfusion. In controls, pro-MMP-2 was detected in the coronary effluent throughout the first 30 min of aerobic perfusion, but MMP-2 was not detected. In contrast, MMP-2 was detected in the coronary effluent during reperfusion after both 15 and 60 min ischaemia. However, while left ventricular systolic function was impaired after both 15 min and 60 min ischaemia, a significant increase in the release of MMP-2 was only detected in hearts following 60 min ischaemia. The dissociation between mechanical function and MMP-2 levels suggest that MMP-2 does not contribute to myocardial stunning in this model, but may contribute to myocardial dysfunction following prolonged ischaemia.

Effect of treatment on ventricular function and troponin I proteolysis in reperfused myocardium.

Effects of ischemia time and treatment interventions upon troponin I (TnI) proteolysis and function of reperfused myocardium were examined in isolated, perfused rabbit hearts. Hearts were randomized to 90 min aerobic perfusion, 15 min low-flow (1 ml/min) ischemia (I) and 60 min reperfusion (R) or 60 min low-flow I and 60 min R. Hearts subject to 60 min I and 60 min R received either no treatment, l -arginine treatment, or treatment with oxygen free radical (OFR) scavengers (mercapto-proponyl-glycine, catalase and superoxide dismutase). Hearts from cholesterol-fed rabbits were also studied after 60 min I and R. Isovolumic LV pressure and heart rate were recorded throughout and Western analysis of ventricular myocardium, using 3 specific antibodies, detected intact TnI (29 kDa) and TnI fragment (25 kDa). Hearts subject to 15 min I had minimal irreversible injury (TTC negative region=0.6+/-0.4% LV) but hearts subject to 60 min I had more extensive injury (TTC negative=40.7+/-5.8% LV). Recovery of rate-pressure product after 15 min I and 60 min R (56+/-9% of baseline) was better than after 60 min I and 60 min R (23+/-9%, P<0.01). Both l -arginine and OFR scavengers were associated with better recovery of function after 60 min I, (66+/-7% and 72+/-3% of baseline respectively, P<0.01 v no treatment) but cholesterol hearts had poor recovery after 60 min I (37+/-8%). The 25 kDa TnI (% total TnI immunoreactivity) was 8.7+/-0.9% in controls, 10.0+/-1.6% after 15 min I and 60 min R, and 17.4+/-2.4% after 60 min I and 60 min R (P<0.01 v controls and 15 min I). The proportion of 25 kDa TnI was increased in all hearts after 60 min I and did not change with treatment (l -arginine 16.8+/-1.8%, OFR scavengers 16.0+/-3.2%, cholesterol 14.0+/-1.9%). There was no relation between proportion of 25 kDa TnI and recovery of function. Samples from freshly excised rabbit hearts and human right atria also had 25 kDa TnI (relative intensities 8.5+/-2.3% and 5.1+/-2.6% respectively). Although TnI fragmentation increases after prolonged ischemia and reperfusion, the functional recovery of stunned myocardium is independent of degree of TnI fragmentation.