Marie-Louise Ward

Stretch-activated channels in the heart: Contributions to length-dependence and to cardiomyopathy

The stretch-induced increase in force production of ventricular muscle is biphasic. An abrupt increase in force coincides with the stretch, which is then followed by a slower response that develops over minutes (the slow force response or SFR). The SFR is accompanied by a slow increase in the magnitude of the intracellular Ca2+ transient, but the stretch-dependent mechanisms that give rise to this remain controversial. We characterized the SFR using right ventricular trabeculae from mouse hearts. Application of three different blockers of stretch-activated non-selective cation channels (SAC NSC) reduced the magnitude of the SFR 60s after stretch (400 microM streptomycin: from 86+/-25% to 38+/-14%, P<0.01, n=9; 10 microM GdCl3: from 65+/-21%, to 12+/-7%, P<0.01, n=7; 10 microM GsMTx-4 from 122+/-40% to 15+/-8%, P<0.05, n=6). Streptomycin also decreased the increase in Ca2+ transient amplitude 60s after the stretch from 43.5+/-12.7% to 5.7+/-3.5% (P<0.05, n=4), and reduced the stretch-dependent increase in intracellular Ca2+ in quiescent muscles when stretched. The transient receptor potential, canonical channels TRPC1 and TRPC6 are mechano-sensitive, non-selective cation channels. They are expressed in mouse ventricular muscle, and could therefore be responsible for stretch-dependent influx of Na+ and/or Ca2+ during the SFR. Expression of TRPC1 was investigated in the mdx heart, a mouse model of Duchennes muscular dystrophy. Resting Ca2+ was raised in isolated myocytes from old mdx animals, which was blocked by application of SAC blockers. Expression of TRPC1 was increased in the older mdx animals, which have developed a dilated cardiomyopathy, and might therefore contribute to the dilated cardiomyopathy.

Reduced contraction strength with increased intracellular [Ca2+] in left ventricular trabeculae from failing rat hearts

Intracellular calcium ([Ca2+]i) and isometric force were measured in left ventricular (LV) trabeculae from spontaneously hypertensive rats (SHR) with failing hearts and normotensive Wistar‐Kyoto (WKY) controls. At a physiological stimulation frequency (5 Hz), and at 37 °C, the peak stress of SHR trabeculae was significantly (P≤; 0.05) reduced compared to WKY (8 ± 1 mN mm−2(n= 8)vs. 21 ± 5 mN mm−2(n= 8), respectively). No differences between strains in either the time‐to‐peak stress, or the time from peak to 50 % relaxation were detected. Measurements using fura‐2 showed that in the SHR both the peak of the Ca2+ transient and the resting [Ca2+]i were increased compared to WKY (peak: 0.69 ± 0.08 vs. 0.51 ± 0.08 μm (P≤ 0.1) and resting: 0.19 ± 0.02 vs. 0.09 ± 0.02 μm (P≤ 0.05), SHR vs. WKY, respectively). The decay of the Ca2+ transient was prolonged in SHR, with time constants of: 0.063 ± 0.002 vs. 0.052 ± 0.003 s (SHR vs. WKY, respectively). Similar results were obtained at 1 Hz stimulation, and for [Ca2+]o between 0.5 and 5 mm. The decay of the caffeine‐evoked Ca2+ transient was slower in SHR (9.8 ± 0.7 s (n= 8)vs. 7.7 ± 0.2 s (n= 8) in WKY), but this difference was removed by use of the SL Ca2+‐ATPase inhibitor carboxyeosin. Histological examination of transverse sections showed that the fractional content of perimysial collagen was increased in SHR compared to WKY (18.0 ± 4.6 % (n= 10)vs. 2.9 ± 0.9 % (n= 11) SHR vs. WKY, respectively). Our results show that differences in the amplitude and the time course of the Ca2+ transient between SHR and WKY do not explain the reduced contractile performance of SHR myocardium per se. Rather, we suggest that, in this animal model of heart failure, contractile function is compromised by increased collagen, and its three‐dimensional organisation, and not by reduced availability of intracellular Ca2+.

Altered Calcium Homeostasis Does Not Explain the Contractile Deficit of Diabetic Cardiomyopathy

OBJECTIVE—This study examines the extent to which the contractile deficit of diabetic cardiomyopathy is due to altered Ca2+ homeostasis. RESEARCH DESIGN AND METHODS—Measurements of isometric force and intracellular calcium ([Ca2+]i, using fura-2/AM) were made in left ventricular (LV) trabeculae from rats with streptozotocin-induced diabetes and age-matched siblings. RESULTS—At 1.5 mmol/l [Ca2+]o, 37°C, and 5-Hz stimulation frequency, peak stress was depressed in diabetic rats (10 ± 1 vs. 17 ± 2 mN/mm2 in controls; P < 0.05) with a slower time to peak stress (77 ± 3 vs. 67 ± 2 ms; P < 0.01) and time to 90% relaxation (76 ± 7 vs. 56 ± 3 ms; P < 0.05). No difference was found between groups for either resting or peak Ca2+, but the Ca2+ transient was slower in time to peak (39 ± 2 vs. 34 ± 1 ms) and decay (time constant, 61 ± 3 vs. 49 ± 3 ms). Diabetic rats had a longer LV action potential (APD50, 98 ± 5 vs. 62 ± 5 ms; P < 0.0001). Western blotting showed that diabetic rats had a reduced expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a, with no difference in expression of the Na+/Ca2+ exchanger. Immunohistochemistry of LV free wall showed that type I collagen was increased in diabetic rats (diabetic 7.1 ± 0.1%, control 12.7 ± 0.1%; P < 0.01), and F-actin content reduced (diabetic 56.9 ± 0.6%; control 61.7 ± 0.4%; P < 0.0001) with a disrupted structure. CONCLUSIONS—We find no evidence to support the idea that altered Ca2+ homeostasis underlies the contractile deficit of diabetic cardiomyopathy. The slower action potential and reduced SERCA2a expression can explain the slower Ca2+ transient kinetics in diabetic rats but not the contractile deficit. Instead, we suggest that the observed LV remodeling may play a crucial role.

Protection of the heart by treatment with a divalent-copper-selective chelator reveals a novel mechanism underlying cardiomyopathy in diabetic rats

BackgroundIntracellular calcium (Ca2+) coordinates the cardiac contraction cycle and is dysregulated in diabetic cardiomyopathy. Treatment with triethylenetetramine (TETA), a divalent-copper-selective chelator, improves cardiac structure and function in patients and rats with diabetic cardiomyopathy, but the molecular basis of this action is uncertain. Here, we used TETA to probe potential linkages between left-ventricular (LV) copper and Ca2+ homeostasis, and cardiac function and structure in diabetic cardiomyopathy.MethodsWe treated streptozotocin-diabetic rats with a TETA-dosage known to ameliorate LV hypertrophy in patients with diabetic cardiomyopathy. Drug treatment was begun either one (preventative protocol) or eight (restorative protocol) weeks after diabetes induction and continued thereafter for seven or eight weeks, respectively. Total copper content of the LV wall was determined, and simultaneous measurements of intracellular calcium concentrations and isometric contraction were made in LV trabeculae isolated from control, diabetic and TETA-treated diabetic rats.ResultsTotal myocardial copper levels became deficient in untreated diabetes but were normalized by TETA-treatment. Cardiac contractility was markedly depressed by diabetes but TETA prevented this effect. Neither diabetes nor TETA exerted significant effects on peak or resting [Ca2+]i. However, diabetic rats showed extensive cardiac remodelling and decreased myofibrillar calcium sensitivity, consistent with observed increases in phosphorylation of troponin I, whereas these changes were all prevented by TETA.ConclusionsDiabetes causes cardiomyopathy through a copper-mediated mechanism that incorporates myocardial copper deficiency, whereas TETA treatment prevents this response and maintains the integrity of cardiac structure and myofibrillar calcium sensitivity. Altered calcium homeostasis may not be the primary defect in diabetic cardiomyopathy. Rather, a newly-described copper-mediated mechanism may cause this disease.

Mechanisms underlying the impaired contractility of diabetic cardiomyopathy

Cardiac dysfunction is a well-known consequence of diabetes, with sustained hyperglycaemia leading to the development of a cardiomyopathy that is independent of cardiovascular disease or hypertension. Animal models of diabetes are commonly used to study the pathophysiology of diabetic cardiomyopathy, with the hope that increased knowledge will lead ultimately to better therapeutic strategies being developed. At physiological temperature, left ventricular trabeculae isolated from the streptozotocin rat model of type 1 diabetes showed decreased stress and prolonged relaxation, but with no evidence that decreased contractility was a result of altered myocardial Ca(2+) handling. Although sarcoplasmic reticulum (SR) Ca(2+) reuptake appeared slower in diabetic trabeculae, it was offset by an increase in action-potential duration, thereby maintaining SR Ca(2+) content and favouring increased contraction force. Frequency analysis of t-tubule distribution by confocal imaging of ventricular tissue labeled with wheat germ agglutinin or ryanodine receptor antibodies showed a reduced T-power for diabetic tissue, but the differences were minor in comparison to other models of heart failure. The contractile dysfunction appeared to be the result of disrupted F-actin in conjunction with the increased type I collagen, with decreased myofilament Ca(2+) sensitivity contributing to the slowed relaxation.

Energetics of stress production in isolated cardiac trabeculae from the rat

The heat liberated upon stress production in isolated cardiac muscle provides insights into the complex thermodynamic processes underlying mechanical contraction. To that end, we simultaneously measured the heat and stress (force per cross-sectional area) production of cardiac trabeculae from rats using a flow-through micromechanocalorimeter. In a flowing stream of O(2)-equilibrated Tyrode solution (∼22°C), the stress and heat production of actively contracting trabeculae were varied by 1) altering stimulus frequency (0.2-4 Hz) at optimal muscle length (L(o)), 2) reducing muscle length below L(o) at 0.2 and 2 Hz, and 3) changing extracellular Ca(2+) concentrations ([Ca(2+)](o); 1 and 2 mM). Linear regression lines were adequate to fit the active heat-stress data. The active heat-stress relationships were independent of stimulus frequency and muscle length but were dependent on [Ca(2+)](o), having greater intercepts at 2 mM [Ca(2+)](o) than at 1 mM [Ca(2+)](o) (3.5 and 2.0 kJ·m(-3)·twitch(-1), respectively). The slopes among the heat-stress relationships did not differ. At the highest experimental stimulus frequency, pronounced elevation of diastolic Ca(2+) resulted in incomplete twitch relaxation. The resulting increase of diastolic stress, which occurred with negligible metabolic energy expenditure, subsequently diminished due to the time-dependent loss of myofilament Ca(2+)-sensitivity.

Myocardial twitch duration and the dependence of oxygen consumption on pressure-volume area: experiments and modelling

•  The energy expenditure of the heart is linearly related to its work performance, as measured by its development of pressure–volume area. •  We have explored the basis of this phenomenon both experimentally (by measuring the heat production of isolated ventricular tissue undergoing cyclic contraction and relaxation) and theoretically (using mathematical modelling). •  We provide the first evidence that the heat production of isolated trabeculae undergoing fixed‐end contractions varies linearly with force–length area, and confirm that twitch duration increases progressively with muscle length. •  Mathematical modelling reveals that length‐dependent prolongation of the twitch reflects length‐ (or, equivalently, force‐) dependent binding of Ca2+ to troponin‐C, together with Ca2+‐dependent crossbridge cooperativity. •  Mathematical modelling further reveals that the apparent linear dependence of heat production on force–length area is remarkably robust against departures from the linearity of length‐dependent twitch duration.

Mechanisms of reduced contractility in an animal model of hypertensive heart failure

1. Alterations in intracellular Ca2+ homeostasis have frequently been implicated as underlying the contractile dysfunction of failing hearts. Contraction in cardiac muscle is due to a balance between sarcolemmal (SL) and sarcoplasmic reticulum (SR) Ca2+ transport, which has been studied in single cells and small tissue samples. However, many studies have not used physiological temperatures and pacing rates, and this could be problematic given different temperature dependencies and kinetics for transport processes.

Stretch-activated channels in the heart: contribution to cardiac performance

Stretch-activated ion channels are widely expressed in most cell types and play an important role in a variety of normal cell functions, including volume regulation and length detection. In the heart, transduction of mechanical energy into cellular responses is an essential component of cardiac function. The heart is passively stretched, and actively shortens in every cardiac cycle; in addition, longer-term changes in volume occur during exercise, and in diseases such as heart failure. In this article, we discuss the importance of stretch-activated ion channels as mechano-transducers in the heart, with emphasis on their contribution to the regulation of contractile performance. As well, the role of stretch-activated channels in modifying the electrical activity of the heart is also discussed.

Does the intercept of the heat–stress relation provide an accurate estimate of cardiac activation heat?

The heat of activation of cardiac muscle reflects the metabolic cost of restoring ionic homeostasis following a contraction. The accuracy of its measurement depends critically on the abolition of crossbridge cycling. We abolished crossbridge activity in isolated rat ventricular trabeculae by use of blebbistatin, an agent that selectively inhibits myosin II ATPase. We found cardiac activation heat to be muscle length independent and to account for 15–20% of total heat production at body temperature. We conclude that it can be accurately estimated at minimal muscle length.