Abstract-Phosphorylation of cardiac myofibrils by cAMP-dependent protein kinase (PKA) can increase the intrinsic rate of myofibrillar relaxation, which may contribute to the shortening of the cardiac twitch during ␤-adrenoceptor stimulation. However, it is not known whether the acceleration of myofibrillar relaxation is due to phosphorylation of troponin I (TnI) or of myosin binding protein-C (MyBP-C). To distinguish between these possibilities, we used transgenic mice that overexpress the nonphosphorylatable, slow skeletal isoform of TnI in the myocardium and do not express the normal, phosphorylatable cardiac TnI. The intrinsic rate of relaxation of myofibrils from wild-type and transgenic mice was measured using flash photolysis of diazo-2 to rapidly decrease the [Ca 2ϩ ] within skinned muscles from the mouse ventricles. Incubation with PKA nearly doubled the intrinsic rate of myofibrillar relaxation in muscles from wild-type mice (relaxation half-time fell from Ϸ150 to Ϸ90 ms at 22°C) but had no effect on the relaxation rate of muscles from the transgenic mice. In parallel studies with intact muscles, we assessed crossbridge kinetics indirectly by determining f min (the frequency for minimum dynamic stiffness) during tetanic contractions. Stimulation of ␤-adrenoceptors with isoproterenol increased f min from 1.9 to 3.1 Hz in muscles from wild-type mice but had no effect on f min in muscles from transgenic mice. We conclude that the acceleration of myofibrillar relaxation rate by PKA is due to phosphorylation of TnI, rather than MyBP-C, and that this may be due, at least in part, to faster crossbridge cycle kinetics. . This increase of relaxation rate is important for proper pump function, because it allows adequate time for diastolic filling of the ventricles despite the raised heart rate during sympathetic stimulation. The activation of ␤ 1 -adrenoceptors stimulates the cAMP/protein kinase A (PKA) pathway, and the faster relaxation of the myocardial cells is partly due to an enhanced reuptake of Ca 2ϩ into the sarcoplasmic reticulum (SR) as a result of phosphorylation of phospholamban by PKA. 1 In addition, PKA phosphorylates the cardiac myofibrils during ␤-stimulation. [2][3][4] This may lead to an acceleration of the intrinsic rate of myofibrillar relaxation, thereby contributing to the abbreviation of the twitch. Using flash photolysis of the caged chelator of Ca 2ϩ , diazo-2, to rapidly decrease Ca 2ϩ concentration inside skinned fibers, Zhang et al 5 reported that PKA accelerated relaxation in pig skinned muscles. However, a later study by Johns et al 6 found no effect in similar experiments using guinea pig skinned muscles. Recent work with intact mouse muscles has suggested that ␤-stimulation can produce an SR-independent, presumably myofibril-mediated, acceleration of relaxation that is seen in isometric but not isotonic contractions. 4 Assuming that phosphorylation does increase the relaxation rate of cardiac myofibrils, how might this be produced? It is known that phosphorylation of troponin I (...

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.

There is evidence that multi-site phosphorylation of cardiac troponin I (cTnI) by protein kinase C is important in both long-and short-term regulation of cardiac function. To determine the specific functional effects of these phosphorylation sites (Ser-43, Ser-45, and Thr-144), we measured tension and sliding speed of thin filaments in reconstituted preparations in which endogenous cTnI was replaced with cTnI phosphorylated by protein kinase C-⑀ or mutated to cTnI-S43E/S45E/T144E, cTnI-S43E/S45E, or cTnI-T144E. We used detergentskinned mouse cardiac fiber bundles to measure changes in Ca 2؉ -dependence of force. Compared with controls, fibers reconstituted with phosphorylated cTnI, cTnI-S43E/S45E/T144E, or cTnI-S43E/S45E were desensitized to Ca 2؉ , and maximum tension was as much as 27% lower, whereas fibers reconstituted with cTnI-T144E showed no change. In the in vitro motility assay actin filaments regulated by troponin complexes containing phosphorylated cTnI or cTnI-S43E/S45E/T144E showed both a decrease in Ca 2؉ sensitivity and maximum sliding speed compared with controls, whereas filaments regulated by cTnI-S43E/S45E showed only decreased maximum sliding speed and filaments regulated by cTnI-T144E demonstrated only desensitization to Ca 2؉ . Our results demonstrate novel site specificity of effects of PKC phosphorylation on cTnI function and emphasize the complexity of modulation of the actin-myosin interaction by specific changes in the thin filament.

Myosin heavy chain (MHC) isoforms alpha and beta have intrinsically different ATP hydrolysis activities (ATPase) and therefore cross-bridge cycling rates in solution. There is considerable evidence of altered MHC expression in rodent cardiac disease models; however, the effect of incremental beta-MHC expression over a wide range on the rate of high-strain, isometric cross-bridge cycling is yet to be ascertained. We treated male rats with 6-propyl-2-thiouracil (PTU; 0.8 g/l in drinking water) for short intervals (6, 11, 16, and 21 days) to generate cardiac MHC patterns in transition from predominantly alpha-MHC to predominantly beta-MHC. Steady-state calcium-dependent tension development and tension-dependent ATP consumption (tension cost; proportional to cross-bridge cycling) were measured in chemically permeabilized (skinned) right ventricular muscles at 20 degrees C. To assess dynamic cross-bridge cycling kinetics, the rate of force redevelopment (ktr) was determined after rapid release-restretch of fully activated muscles. MHC isoform content in each experimental muscle was measured by SDS-PAGE and densitometry. alpha-MHC content decreased significantly and progressively with length of PTU treatment [68 +/- 5%, 58 +/- 4%, 37 +/- 4%, and 27 +/- 6% for 6, 11, 16, and 21 days, respectively; P < 0.001 (ANOVA)]. Tension cost decreased, linearly, with decreased alpha-MHC content [6.7 +/- 0.4, 5.6 +/- 0.5, 4.0 +/- 0.4, and 3.9 +/- 0.3 ATPase/tension for 6, 11, 16, and 21 days, respectively; P < 0.001 (ANOVA)]. Likewise, ktr was significantly and progressively depressed with length of PTU treatment [11.1 +/- 0.6, 9.1 +/- 0.5, 8.2 +/- 0.7, and 6.2 +/- 0.3 s(-1) for 6, 11, 16, and 21 days, respectively; P < 0.05 (ANOVA)] Thus cross-bridge cycling, under high strain, for alpha-MHC is three times higher than for beta-MHC. Furthermore, under isometric conditions, alpha-MHC and beta-MHC cross bridges hydrolyze ATP independently of one another.

Smooth muscle myosin heavy chain (SMHC) isoforms, SM1 and SM2, are the products of alternative splicing from a single gene (P. Babij and M. Periasamy. J. Mol. Biol. 210: 673-679, 1989). We have previously shown that this splicing occurs at the 3'-end of the mRNA, resulting in proteins that differ at the carboxyterminal (R. Nagai, M. Kuro-o, P. Babij, and M. Periasamy. J. Biol. Chem. 264: 9734-9737, 1989). In the present study we demonstrate that additional SMHC isoform diversity occurs in the globular head region by isolating and characterizing two distinct rat SMHC cDNA (SMHC-11 = SM1B and SMHC-5 = SM1A). Sequence comparison of the two clones reveals that they are completely identical in their coding regions, except at the region encoding the 25/50 kDa junction of the myosin head, where the SM1B isoform contains an additional seven amino acids. This divergent region is located adjacent to the Mg(2+)-ATPase site, and differences in this region may be of functional importance. Ribonuclease protection analysis demonstrates that the corresponding SM1B and SM1A mRNA messages are coexpressed in all smooth muscle tissues; however, the proportion of the two mRNA present differs significantly between tissues. The SM1A-type mRNA predominates in most smooth muscle tissues, with the exception of intestine and urinary bladder, which contain greater proportions of the SM1B message. The differential distribution of these two isoforms may provide important clues toward understanding differences in smooth muscle contractile properties.