Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.

Abstract-We recently showed that phosphoinositide-3-kinase-␥-deficient (PI3K␥ Ϫ/Ϫ ) mice have enhanced cardiac contractility attributable to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca 2ϩ content and release but not L-type Ca 2ϩ current (I Ca,L ), demonstrating PI3K␥ locally regulates cAMP levels in cardiomyocytes. Because phosphodiesterases (PDEs) can contribute to cAMP compartmentation, we examined whether the PDE activity was altered by PI3K␥ ablation. Selective inhibition of PDE3 or PDE4 in wild-type (WT) cardiomyocytes elevated Ca 2ϩ transients, SR Ca 2ϩ content, and phospholamban phosphorylation (PLN-PO 4 ) by similar amounts to levels observed in untreated PI3K␥

Targeted deletion of cytoskeletal muscle LIM protein (MLP) in mice consistently leads to dilated cardiomyopathy (DCM) after one or more months. However, next to nothing is known at present about the mechanisms of this process. We investigated whether diastolic performance including passive mechanics and systolic behavior are altered in 2-week-old MLP knockout (MLPKO) mice, in which heart size, fractional shortening and ejection fraction are still normal. Right ventricular trabeculae were isolated from 2-week-old MLPKO and wildtype mice and placed in an apparatus that allowed force measurements and sarcomere length measurements using laser diffraction. During a twitch from the unloaded state at 1 Hz, MLPKO muscles relengthened to slack length more slowly than controls, although the corresponding force relaxation time was unchanged. Active developed stress at a diastolic sarcomere length of 2.00 microm was preserved in MLPKO trabeculae over a wide range of pacing frequencies. Force relaxation under the same conditions was consistently prolonged compared with wildtype controls, whereas time to peak and maximum rate of force generation were not significantly altered. Ca2+ content of the sarcoplasmic reticulum (SR) and the quantities of Ca2+ handling proteins were similar in both genotypes. In summary, young MLPKO mice revealed substantial alterations in passive myocardial properties and relaxation time, but not in most systolic characteristics. These results indicate that the progression to heart failure in the MLPKO model may be driven by diastolic myocardial dysfunction and abnormal passive properties rather than systolic dysfunction.

Connective Tissue Growth Factor (CTGF, CCN2) is considered to play an important role in cardiac remodelling. We studied whether stretch is a primary stimulus to induce CTGF expression in vivo in rabbit heart, and in vitro in isolated cardiomyocytes and fibroblasts. Twenty weeks of combined volume and pressure overload resulted in eccentric left ventricular (LV) hypertrophy, with increased LV internal diameter (+36 %) and LV weight (+53 %). Myocardial CTGF mRNA and protein levels were substantially increased in the overloaded animals. In isolated adult rabbit cardiomyocytes, cyclic stretch strongly induced CTGF mRNA expression (2.9-fold at 48 h), whereas in cardiac fibroblasts CTGF-induction was transient and modest (1.4-fold after 4 h). Conditioned medium from stretched fibroblasts induced CTGF mRNA expression in non-stretched cardiomyocytes (2.3-fold at 48 h). Our findings indicate that stretch is an important primary trigger for CTGF-induction in the overloaded heart.

Brief periods of ventricular pacing during the early reperfusion phase (pacing-induced postconditioning, PPC) have been shown to reduce infarct size as measured after 2 h of reperfusion. In this study, we investigated (1) whether PPC leads to maintained reduction in infarct size, (2) whether abnormal mechanical load due to asynchronous activation is the trigger for PPC and (3) the signaling pathways that are involved in PPC. Rabbit hearts were subjected to 30 min of coronary occlusion in vivo, followed by 6 weeks of reperfusion. PPC consisted of ten 30-s intervals of left ventricular (LV) pacing, starting at reperfusion. PPC reduced infarct size (TTC staining) normalized to area at risk, from 49.0 ± 3.3% in control to 22.9 ± 5.7% in PPC rabbits. In isolated ejecting rabbit hearts, replacing LV pacing by biventricular pacing abolished the protective effect of PPC, whereas ten 30-s periods of high preload provided a protective effect similar to PPC. The protective effect of PPC was neither affected by the adenosine receptor blocker 8-SPT nor by the angiotensin II receptor blocker candesartan, but was abrogated by the cytoskeletal microtubule-disrupting agent colchicine. Blockers of the mitochondrial KATP channel (5HD), PKC (chelerythrine) and PI3-kinase (wortmannin) all abrogated the protection provided by PPC. In the in situ pig heart, PPC reduced infarct size from 35 ± 4 to 16 ± 12%, a protection which was abolished by the stretch-activated channel blocker gadolinium. No infarct size reduction was achieved if PPC application was delayed by 5 min or if only five pacing cycles were used. The present study indicates that (1) PPC permanently reduces myocardial injury, (2) abnormal mechanical loading is a more likely trigger for PPC than electrical stimulation or G-coupled receptor stimulation and (3) PPC may share downstream pathways with other modes of cardioprotection.

The health effects of the endocrine disruptor Bisphenol A (BPA) led to its partial replacement with Bisphenol S (BPS) in several products including food containers, toys, and thermal paper receipts. The acute effects of BPS on myocardial contractility are unknown. We perfused mouse hearts from both sexes for 15 min with physiologically relevant doses of BPS or BPA. In females BPS (1 nM) decreased left ventricular systolic pressure by 5 min, whereas BPA (1 nM) effects were delayed to 10 min. BPS effects in male mice were attenuated. In both sexes ER-β antagonism abolished the effects of BPS. Cardiac myofilament function was not impacted by BPS or BPA in either sex, although there were sex-dependent differences in troponin I phosphorylation. BPS increased phospholamban phosphorylation at S16 only in female hearts, whereas BPA reduced phosphorylation in both sexes. BPA decreased phospholamban phosphorylation at T17 in both sexes while BPS caused dephosphorylation only in females. This is the first study to compare sex differences in the acute myocardial response to physiologically relevant levels of BPS and BPA, and demonstrates a rapid ability of both to depress heart function. This study raises concerns about the safety of BPS as a replacement for BPA.

Ventricular myocytes are continuously exposed to fluid shear in vivo by relative movement of laminar sheets and adjacent cells. Preliminary observations have shown that neonatal myocytes respond to fluid shear by increasing their beating rate, which could have an arrhythmogenic effect under elevated shear conditions. The objective of this study is to investigate the characteristics of the fluid shear response in cultured myocytes and to study selected potential mechanisms. Cultured neonatal rat ventricular myocytes that were spontaneously beating were subjected to low shear rates (5-50/s) in a fluid flow chamber using standard culture medium. The beating rate was measured from digital microscopic recordings. The myocytes reacted to low shear rates by a graded and reversible increase in their spontaneous beating rate of up to 500%. The response to shear was substantially attenuated in the presence of the beta-adrenergic agonist isoproterenol (by 86+/-8%), as well as after incubation with integrin-blocking RGD peptides (by 92+/-8%). The results suggest that the beta-adrenergic signaling pathway and integrin activation, which are known to interact, may play an important role in the response mechanism.

The β 3 -adrenoceptors (β 3 -ARs) have been identified and characterized in the human heart. Specific β 3 -AR stimulation, unlike β 1 -AR or β 2 -AR stimulation, decreases cardiac contractility, partly via the G i -NO pathway. However, the precise role of cardiac β 3 -ARs is not yet completely understood. Indeed, under normal conditions, the β 3 -AR response is present only to a very low degree in rats and mice. Therefore, we evaluated whether β 3 -ARs were present and functional in rabbit ventricular cardiomyocytes, and whether the rabbit could serve as a relevant model for the study of cardiac β 3 -ARs. We used RT-PCR and Western blot to measure the β 3 -AR transcripts and protein levels in rabbit ventricular cardiomyocytes. We also analysed the effect of β 3 -AR stimulation using isoproterenol in combination with nadolol or SR 58611A on cardiomyocyte shortening, Ca 2+ transient, L-type Ca 2+ current (I Ca,L ), delayed rectifier potassium current (I Ks ) and action potential duration (APD). For the first time, we show that β 3 -ARs are expressed in rabbit ventricular cardiomyocytes. The mRNA and protein sequences present a high homology to those of rat and human β 3 -ARs. Furthermore, β 3 -AR stimulation decreases cardiomyocyte shortening, Ca 2+ transient and I Ca,L amplitudes, via a G i -NO pathway. Importantly, β 3 -AR stimulation enhances I Ks amplitude and shortens the APD. Taken together, our results indicate that the rabbit provides a relevant model, easily used in laboratories, to study the roles of cardiac β 3 -ARs in physiological conditions.