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 Duchenne's 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.

OBJECTIVE-This study examines the extent to which the contractile deficit of diabetic cardiomyopathy is due to altered Ca 2ϩ homeostasis. RESEARCH DESIGN AND METHODS-Measurements of isometric force and intracellular calcium ([Ca2ϩ] 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 [Ca 2ϩ ] o , 37°C, and 5-Hz stimulation frequency, peak stress was depressed in diabetic rats (10 Ϯ 1 vs. 17 Ϯ 2 mN/mm 2 in controls; P Ͻ 0.05) with a slower time to peak stress (77 Ϯ 3 vs. 67 Ϯ 2 ms; P Ͻ 0.01) and time to 90% relaxation (76 Ϯ 7 vs. 56 Ϯ 3 ms; P Ͻ 0.05). No difference was found between groups for either resting or peak Ca 2ϩ , but the Ca 2ϩ transient was slower in time to peak (39 Ϯ 2 vs. 34 Ϯ 1 ms) and decay (time constant, 61 Ϯ 3 vs. 49 Ϯ 3 ms). Diabetic rats had a longer LV action potential (APD 50 , 98 Ϯ 5 vs. 62 Ϯ 5 ms; P Ͻ 0.0001). Western blotting showed that diabetic rats had a reduced expression of sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA)2a, with no difference in expression of the Na ϩ /Ca 2ϩ exchanger. Immunohistochemistry of LV free wall showed that type I collagen was increased in diabetic rats (diabetic 7.1 Ϯ 0.1%, control 12.7 Ϯ 0.1%; P Ͻ 0.01), and F-actin content reduced (diabetic 56.9 Ϯ 0.6%; control 61.7 Ϯ 0.4%; P Ͻ 0.0001) with a disrupted structure.CONCLUSIONS-We find no evidence to support the idea that altered Ca 2ϩ homeostasis underlies the contractile deficit of diabetic cardiomyopathy. The slower action potential and reduced SERCA2a expression can explain the slower Ca 2ϩ transient kinetics in diabetic rats but not the contractile deficit. Instead, we suggest that the observed LV remodeling may play a crucial role. Diabetes 57:2158-2166, 2008 D iabetic cardiomyopathy was first recognized by Rubler et al. (1) in diabetic patients with congestive heart failure but no evidence of coronary atherosclerosis. Prominent defects of diabetic cardiomyopathy include the prolonged duration of contraction and relaxation (Boudina and Abel [2]) and reduced cardiac compliance. The rat with streptozotocin (STZ)-induced type 1 diabetes has been widely used as a model of diabetic myopathy (3). STZ rats manifest a variety of signs of myopathy, including cardiac rhythm disturbances, prolonged contraction and/or slowed relaxation, and decreased contraction strength (4 -6). Defects in intracellular Ca 2ϩ ([Ca 2ϩ ] i ) homeostasis have been implicated in the impaired mechanical performance of the diabetic heart (7,8). However, there is no consensus in results from studies of intracellular Ca 2ϩ homeostasis performed on isolated cardiomyocytes; resting Ca 2ϩ has been shown to be decreased (9 -11), increased (12,13), or unchanged (14,15). Similarly, the amplitude of the Ca 2ϩ transient is reported as decreased (11,16 -18), increased (12), or unchanged (19,20). It is possible that this lack of consensus is due to different degrees of disease (diabetic stage) and/or experimental conditions (e.g., ...

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+.

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.

Activation heat arises from two sources during the contraction of striated muscle. It reflects the metabolic expenditure associated with Ca pumping by the sarcoplasmic reticular Ca -ATPase and Ca translocation by the Na /Ca exchanger coupled to the Na ,K -ATPase. In cardiac preparations, investigators are constrained in estimating its magnitude by reducing muscle length to the point where macroscopic twitch force vanishes. But this experimental protocol has been criticised since, at zero force, the observed heat may be contaminated by residual crossbridge cycling activity. To eliminate this concern, the putative thermal contribution from crossbridge cycling activity must be abolished, at least at minimal muscle length. We achieved this using blebbistatin, a selective inhibitor of myosin II ATPase. Using a microcalorimeter, we measured the force production and heat output, as functions of muscle length, of isolated rat trabeculae from both ventricles contracting isometrically at 5 Hz and at 37°C. In the presence of blebbistatin (15 μmol l ), active force was zero but heat output remained constant, at all muscle lengths. Activation heat measured in the presence of blebbistatin was not different from that estimated from the intercept of the heat-stress relation in its absence. We thus reached two conclusions. First, activation heat is independent of muscle length. Second, residual crossbridge heat is negligible at zero active force; hence, the intercept of the cardiac heat-force relation provides an estimate of activation heat uncontaminated by crossbridge cycling. Both results resolve long-standing disputes in the literature.