Myocardial stretch produces an increase in developed force (DF) that occurs in two phases: the first (rapidly occurring) is generally attributed to an increase in myofilament calcium responsiveness and the second (gradually developing) to an increase in [Ca(2+)](i). Rat ventricular trabeculae were stretched from approximately 88% to approximately 98% of L(max), and the second force phase was analyzed. Intracellular pH, [Na(+)](i), and Ca(2+) transients were measured by epifluorescence with BCECF-AM, SBFI-AM, and fura-2, respectively. After stretch, DF increased by 1.94+/-0.2 g/mm(2) (P<0.01, n = 4), with the second phase accounting for 28+/-2% of the total increase (P<0.001, n = 4). During this phase, SBFI(340/380) ratio increased from 0.73+/-0.01 to 0.76+/-0.01 (P<0.05, n = 5) with an estimated [Na(+)](i) rise of approximately 6 mmol/L. [Ca(2+)](i) transient, expressed as fura-2(340/380) ratio, increased by 9.2+/-3.6% (P<0.05, n = 5). The increase in [Na(+)](i) was blocked by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). The second phase in force and the increases in [Na(+)](i) and [Ca(2+)](i) transient were blunted by AT(1) or ET(A) blockade. Our data indicate that the second force phase and the increase in [Ca(2+)](i) transient after stretch result from activation of the Na(+)/H(+) exchanger (NHE) increasing [Na(+)](i) and leading to a secondary increase in [Ca(2+)](i) transient. This reflects an autocrine-paracrine mechanism whereby stretch triggers the release of angiotensin II, which in turn releases endothelin and activates the NHE through ET(A) receptors.

This study was designed to gain additional insight into the mechanism of the slow force response (SFR) to stretch of cardiac muscle. SFR and changes in intracellular Na(+) concentration ([Na(+)](i)) were assessed in cat papillary muscles stretched from 92% to approximately 98% of L(max). The SFR was 120+/-0.6% (n=5) of the rapid initial phase and coincided with an increase in [Na(+)](i). The SFR was markedly depressed by Na(+)-H(+) exchanger inhibition, AT(1) receptor blockade, nonselective endothelin-receptor blockade and selective ET(A)-receptor blockade, extracellular Na(+) removal, and inhibition of the reverse mode of the Na(+)-Ca(2+) exchange by KB-R7943. KB-R7943 prevented the SFR but not the increase in [Na(+)](i). Inhibition of endothelin-converting enzyme activity by phosphoramidon suppressed both the SFR and the increase in [Na(+)](i). The SFR and the increase in [Na(+)](i) after stretch were both present in muscles with their endothelium (vascular and endocardial) made functionally inactive by Triton X-100. In these muscles, phosphoramidon also suppressed the SFR and the increase in [Na(+)](i). The data provide evidence that the last step of the autocrine-paracrine mechanism leading to the SFR to stretch is Ca(2+) entry through the reverse mode of Na(+)-Ca(2+) exchange.

Myocardial stunning is characterized by decreased myofilament Ca sup 2+ responsiveness. To investigate the molecular basis of stunned myocardium, we performed PAGE and Western immunoblot analysis of the contractile proteins. Isolated rat hearts were retrogradely perfused at 37 degrees C for either 50 minutes (control group) or for 10 minutes, followed by 20-minute global ischemia and 20-minute reperfusion (stunned group), or for 20-minute ischemia without reflow. Another group consisted of hearts subjected to 20-minute ischemia in which stunning was mitigated by 10-minute reperfusion with low Ca 2 +/low pH solution. Myocardial tissue samples subjected to PAGE revealed no obvious differences among groups. Western immunoblots for actin, tropomyosin, troponin C, troponin T, myosin light chain-1, and myosin light chain-2 showed highly selective recognition of the appropriate full-length molecular weight bands in all groups. Troponin I (TnI) Western blots revealed an additional band ([nearly =]26 kD, compared with 32 kD for the full-length protein) in stunned myocardial samples only. In parallel experiments, skinned trabeculae were treated with calpain I for 20 minutes; Western blots showed a TnI degradation pattern similar to that observed in stunned myocardium. Such TnI degradation was prevented by calpastatin, a naturally occurring calpain inhibitor. The results show that (1) TnI is partially and selectively degraded in stunned myocardium; (2) this degradation could be prevented by low Ca sup 2+/low pH reperfusion. which also prevented the contractile dysfunction of stunning; and (3) calpain I could similarly degrade TnI, supporting the idea that Ca 2 +-dependent myofilament proteolysis underlies myocardial stunning.

When the length of the myocardium is increased, a biphasic response to stretch occurs involving an initial rapid increase in force followed by a delayed slow increase called the slow force response (SFR). Confirming previous findings involving angiotensin II in the SFR, it was blunted by AT1 receptor blockade (losartan). The SFR was accompanied by an increase in reactive oxygen species (ROS) of ∼30% and in intracellular Na + concentration ([Na + ] i ) of ∼2.5 mmol l −1 over basal detected by H 2 DCFDA and SBFI fluorescence, respectively. Abolition of ROS by 2-mercapto-propionyl-glycine (MPG) and EUK8 suppressed the increase in [Na + ] i and the SFR, which were also blunted by Na + /H + exchanger (NHE-1) inhibition (HOE642). NADPH oxidase inhibition (apocynin or DPI) or blockade of the ATP-sensitive mitochondrial potassium channels (5HD or glybenclamide) suppressed both the SFR and the increase in [Na + ] i after stretch, suggesting that endogenous angiotensin II activated NADPH oxidase leading to ROS release by the ATP-sensitive mitochondrial potassium channels, which promoted NHE-1 activation. Supporting the notion of ROS-mediated NHE-1 activation, stretch increased the ERK1/2 and p90rsk kinases phosphorylation, effect that was cancelled by losartan. In agreement, the SFR was cancelled by inhibiting the ERK1/2 signalling pathway with PD98059. Angiotensin II at a dose that mimics the SFR (1 nmol l −1 ) induced an increase in ·O 2 − production of ∼30-40% detected by lucigenin in cardiac slices, an effect that was blunted by losartan, MPG, apocynin, 5HD and glybenclamide. Taken together the data suggest a pivotal role of mitochondrial ROS in the genesis of the SFR to stretch.

Myocardial stretch elicits a rapid increase in developed force, which is mainly caused by an increase in myofilament calcium sensitivity (Frank-Starling mechanism). Over the ensuing 10-15 min, a second gradual increase in force takes place. This slow force response to stretch is known to be the result of an increase in the calcium transient amplitude and constitutes the in vitro equivalent of the Anrep effect described 100 years ago in the intact heart. In the present review, we will update and discuss what is known about the Anrep effect as the mechanical counterpart of autocrine/paracrine mechanisms involved in its genesis. The chain of events triggered by myocardial stretch comprises 1) release of angiotensin II, 2) release of endothelin, 3) activation of the mineralocorticoid receptor, 4) transactivation of the epidermal growth factor receptor, 5) increased formation of mitochondria reactive oxygen species, 6) activation of redox-sensitive kinases upstream myocardial Na(+)/H(+) exchanger (NHE1), 7) NHE1 activation, 8) increase in intracellular Na(+) concentration, and 9) increase in Ca(2+) transient amplitude through the Na(+)/Ca(2+) exchanger. We will present the experimental evidence supporting each of the signaling steps leading to the Anrep effect and its blunting by silencing NHE1 expression with a specific small hairpin interference RNA injected into the ventricular wall.

Excitation‐contraction coupling in mouse cardiac muscle remains poorly characterized, despite the fact that the mouse is the mammalian species of choice for genetic manipulation. In this study, we characterized the relationship between internal calcium concentration ([Ca2+]i) and contraction in intact mouse ventricular muscle loaded with fura‐2 salt at 20–22°C. Both Ca2+ transient amplitude and twitch force increased monotonically as external Ca2+ concentration ([Ca2+]o) was increased up to 8.0 mm, with no changes in diastolic levels or in the times to peak of either Ca2+ transients or force. The decay of Ca2+ transients was accelerated as [Ca2+]o increased, while relaxation was prolonged. Both Ca2+ transient amplitude and twitch force increased as stimulation rate increased from 0.2 to 4 Hz, but the increase in force was much greater than the underlying increase in [Ca2+]i. The steady‐state force‐[Ca2+]i relationship revealed an [Ca2+]i required for 50% of maximal activation (Ca50) of 0.95 ± 0.08 μm, a Hill coefficient of 9.9 ± 2.6, and a maximal Ca2+‐activated force (Fmax) of 60 ± 5 mN mm−2. Unlike rat ventricular myocardium, mouse cardiac muscle resists supraphysiological [Ca2+]o. The strong positive force‐frequency relationship in mouse cardiac muscle, with increases of force disproportionate to the increases in Ca2+ transients, suggests frequency‐dependent ‘sensitization’ of the myofilaments. During steady‐state activation, mouse muscle exhibits decreased Ca2+ responsiveness relative to other species, but high co‐operativity. These physiological features of mouse cardiac muscle merit consideration when interpreting the phenotypic consequences of genetic manipulations

Myocardial stretch elicits a biphasic contractile response: the Frank-Starling mechanism followed by the slow force response (SFR) or Anrep effect. In this study we hypothesized that the SFR depends on epidermal growth factor receptor (EGFR) transactivation after the myocardial stretch-induced angiotensin II (Ang II)/endothelin (ET) release. Experiments were performed in isolated cat papillary muscles stretched from 92 to 98% of the length at which maximal twitch force was developed (L max ). The SFR was 123 ± 1% of the immediate rapid phase (n = 6, P < 0.05) and was blunted by preventing EGFR transactivation with the Src-kinase inhibitor PP1 (99 ± 2%, n = 4), matrix metalloproteinase inhibitor MMPI (108 ± 4%, n = 11), the EGFR blocker AG1478 (98 ± 2%, n = 6) or the mitochondrial transition pore blocker clyclosporine (99 ± 3%, n = 6). Stretch increased ERK1/2 phosphorylation by 196 ± 17% of control (n = 7, P < 0.05), an effect that was prevented by PP1 (124 ± 22%, n = 7) and AG1478 (131 ± 17%, n = 4). In myocardial slices, Ang II (which enhances ET mRNA) or endothelin-1 (ET-1)-induced increase in O 2 − production (146 ± 14%, n = 9, and 191 ± 17%, n = 13, of control, respectively, P < 0.05) was cancelled by AG1478 (94 ± 5%, n = 12, and 98 ± 15%, n = 8, respectively) or PP1 (100 ± 4%, n = 6, and 99 ± 8%, n = 3, respectively). EGF increased O 2 − production by 149 ± 4% of control (n = 9, P < 0.05), an effect cancelled by inhibiting NADPH oxidase with apocynin (110 ± 6% n = 7), mKATP channels with 5-hydroxydecanoic acid (5-HD; 105 ± 5%, n = 8), the respiratory chain with rotenone (110 ± 7%, n = 7) or the mitochondrial permeability transition pore with cyclosporine (111 ± 10%, n = 6). EGF increased ERK1/2 phosphorylation (136 ± 8% of control, n = 9, P < 0.05), which was blunted by 5-HD (97 ± 5%, n = 4), suggesting that ERK1/2 activation is downstream of mitochondrial oxidative stress. Finally, stretch increased Ser703 Na + /H + exchanger-1 (NHE-1) phosphorylation by 172 ± 24% of control (n = 4, P < 0.05), an effect that was cancelled by AG1478 (94 ± 17%, n = 4). In conclusion, our data show for the first time that EGFR transactivation is crucial in the chain of events leading to the Anrep effect.

No abstract