To assess the specific functions of the cardiac isoform of troponin I (cTnI), we produced transgenic mice that expressed slow skeletal troponin I (ssTnI) specifically in cardiomyocytes. Cardiomyocytes from these mice displayed quantitative replacement of cTnI with transgene‐encoded ssTnI. The ssTnI transgenic mice were viable and fertile and did not display increased mortality or detectable cardiovascular histopathology. They exhibited normal ventricular weights and heart rates. Permeabilized transgenic cardiomyocytes demonstrated an increased Ca2+ sensitivity of tension and a lack of contractile responsiveness to cAMP‐dependent protein kinase (PKA). Isolated cardiomyocytes from transgenic mice had normal velocities of unloaded shortening but unlike wild‐type controls exhibited no enhancement of the velocity of shortening in response to treatment with isoprenaline. Transgenic cardiomyocytes exhibited greater extents of shortening than non‐transgenic cardiomyocytes at baseline and after treatment with isoprenaline. The rates of rise of intracellular [Ca2+] and the peak amplitudes of the intracellular [Ca2+] transients were similar in transgenic and wild‐type myocytes. However, the half‐time of intracellular [Ca2+] decay was significantly greater in the transgenic myocytes. This change in decay of intracellular [Ca2+] was correlated with an increase in the re‐lengthening time of the transgenic cells. These changes in cardiomyocyte function in vitro were manifested in vivo as impaired diastolic function both at baseline and after stimulation with isoprenaline. Thus, cTnI has important roles in regulating the Ca2+ sensitivity of cardiac myofibrils and controlling cardiomyocyte relaxation and cardiac diastolic function. cTnI is also required for the normal responsiveness of cardiomyocytes to β‐adrenergic receptor stimulation.

We compared mechanical activity and Ca2+ transients of ventricular myocytes isolated from wild-type and phospholamban (PLB)-deficient mouse hearts in control conditions and during beta-adrenergic stimulation. Compared with wild-type controls, cells isolated from PLB-deficient mouse hearts showed 1) a 2-fold increase in extent of cell shortening, 2) a 3-fold increase in maximal shortening velocity, and 3) a 3.4-fold increase in maximal relengthening velocity. PLB-deficient myocytes also demonstrated significant increases in the peak amplitude of the fura 2 fluorescence ratio and the rates of rising and falling phases of the Ca2+ transient. The fura 2 diastolic ratios were similar in both groups, suggesting no change in intracellular Ca2+ during diastole. In PLB-deficient myocytes, 0.05 microM isoproterenol induced an increase in the twitch amplitude by 152 +/- 11% (n = 6) compared with 290 +/- 31% (n = 6) in wild-type cells. Maximal shortening velocity was increased by 183 +/- 10% (n = 6) in PLB-deficient myocytes, compared with 398 +/- 62% (n = 6) in wild-type cells. The isoproterenol-induced increase in maximum relengthening velocity was increased by 168 +/- 8% (n = 6) in PLB-deficient cells compared with 445 +/- 71% (n = 6) in wild-type myocytes. In both groups, these changes in contractile parameters were accompanied by changes in the Ca2+ transient. Our results indicate that phosphorylation of sites other than PLB may play an important role in regulation of contraction-relaxation dynamics of heart cells responding to beta-adrenergic stimulation.

We studied how the nitric oxide (NO*) donor 3-morpholinosydnonimine (SIN-1) alters the response to beta-adrenergic stimulation in cardiac rat myocytes. We found that SIN-1 decreases the positive inotropic effect of isoproterenol (Iso) and decreases the extent of both cell shortening and Ca2+ transient. These effects of SIN-1 were associated with an increased intracellular concentration of cGMP, a decreased intracellular concentration of cAMP, and a reduction in the levels of phosphorylation of phospholamban (PLB) and troponin I (TnI). The guanylyl cyclase inhibitor 1H-8-bromo-1,2,4-oxadiazolo (3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) was not able to prevent the SIN-1-induced reduction of phosphorylation levels of PLB and TnI. However, the effects of SIN-1 were abolished in the presence of superoxide dismutase (SOD) or SOD and catalase. These data suggest that, in the presence of Iso, NO-related congeners, rather than NO*, are responsible for SIN-1 effects. Our results provide new insights into the mechanism by which SIN-1 alters the positive inotropic effects of beta-adrenergic stimulation.

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In experiments reported here, we tested the ability of CGP-48506 to reverse the depressed cardiac contractility associated with hypercapnic acidosis in isolated rat cardiac myocytes. CGP-48506 is a cardiotonic agent that directly and specifically promotes the actin-cross-bridge reaction. Myocytes superfused at pH 6.8 demonstrated a significantly reduced extent of cell shortening, but an increase in the peak amplitude of the Ca 2+ transient. Moreover, cells in acidosis showed small, but significant, decreases in time to peak shortening to 50% relaxation. Superfusion of the cells with 3, 7, and 10 micro-molar CGP-48506 restored the inhibited contractility as a function of concentration with no significant effects on the Ca 2+ -transient. Moreover, 10 micro-molar CGP-48506 completely reversed the depressed myocyte contraction associated with an increase in time to peak shortening and time to 50% and 75% relaxation. Our results indicate that the depression of contractility associated with acidosis is due to a reduced myofilament response to Ca 2+ , which can be overcome by agents working downstream from troponin C through a direct effect on the actin-myosin interaction. KeywordsCa 2+ -sensitizer; Ischemia; Cardiotonic; Contractility; Myocyte Shortening; Ca-Transient INTRODUCTIONIn experiments reported here we have tested whether a cardiotonic agent with direct effects on the actin-myosin reaction is able to reverse the effects of acidosis on contraction of isolated cardiac myocytes. Our rationale for these experiments was based on: 1) evidence that the decrease in the response of the myofilaments to increased intracellular Ca 2+ during acidosis is the main mechanism for the decline of force production (1), and 2) the unique properties of CGP-48506 to directly and specifically affect the myofilament response to Ca 2+ with much less of an effect on cardiac relaxation than other agents working through this mechanism (2,3).Although acidosis inhibits the Ca 2+ current, the Ca 2+ pump activity of the sarcoplasmic reticulum (4), and the Na + -Ca 2+ exchange mechanism (5), direct measurements of intracellular Ca 2+ have shown that Ca 2+ delivery to the myofilaments actually becomes Send correspondence to: R. John Solaro PhD, Department of Physiology and Biophysics (M/C 901), College of Medicine, University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, IL 60612-7342, Tel: 312-996-7620, Fax: 312-996-1414 larger during acidosis (1). Apart from displacement of Ca 2+ from buffer sites including troponin C (6), it is known that acidic pH leads to an increase of intracellular Ca 2+ indirectly through changes in cytoplasmic Na + via Na + -H + mechanism (5). This increased Na + in turn induces an elevation of intracellular Ca 2+ via the Na + -Ca 2+ exchange mechanism. The increase in cytoplasmic Ca 2+ leads to increased Ca 2+ loading of SR and hence increases release from SR. Yet, despite this increase in cytoplasmic Ca 2+ , force falls, most likely due to an altered response of the myofilaments to Ca 2+ . Mecha...