W. Glenn L. Kerrick

Triadins Modulate Intracellular Ca2+ Homeostasis but Are Not Essential for Excitation-Contraction Coupling in Skeletal Muscle

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca2+ imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca2+ transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca2+.[3H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca2+ activation or Ca2+ and Mg2+ inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca2+ homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.

F110I and R278C Troponin T Mutations That Cause Familial Hypertrophic Cardiomyopathy Affect Muscle Contraction in Transgenic Mice and Reconstituted Human Cardiac Fibers

We have studied the physiological effects of the troponin T (TnT) F110I and R278C mutations associated with familial hypertrophic cardiomyopathy (FHC) in humans. Three to four-month-old transgenic (Tg) mice expressing F110I-TnT and R278C-TnT did not develop significant hypertrophy or ventricular fibrosis even after chronic exercise challenge. The F110I mutation impaired acute exercise tolerance, whereas R278C did not. Skinned papillary muscle fibers from transgenic mice expressing F110I-TnT demonstrated increased Ca2+ sensitivity of force and ATPase activity, and likewise an increased Ca2+ sensitivity of force was observed in F110I-TnT-reconstituted human cardiac muscle preparations. In contrast, no changes in force or the ATPase-pCa dependencies were observed in transgenic R278C fibers or in human fibers reconstituted with the R278C-TnT mutant. The maximal level of force development was dramatically decreased in both transgenic mice. However, the maximal ATPase was not different (R278C-TnT) or only slightly less (F110I-TnT) than that of non-Tg and WT-Tg littermates. Consequently, their ratios of ATPase/force (energy cost) at all Ca2+ concentrations were dramatically higher compared with non-Tg and WT-Tg fibers. This increase in energy cost most likely results from a decrease in force per myosin cross-bridge, because forcing all cross-bridges into the force generating state by substitution of MgADP for MgATP in maximum contracting solutions resulted in the same increase in maximal force (15%) in all transgenic and non-transgenic preparations. The combination of increased Ca2+ sensitivity and energy cost in the F110I hearts may be responsible for the greater severity of this phenotype compared with the R278C mutation.

Malignant familial hypertrophic cardiomyopathy D166V mutation in the ventricular myosin regulatory light chain causes profound effects in skinned and intact papillary muscle fibers from transgenic mice

Transgenic (Tg) mice expressing ~95% of the D166V (aspartic acid to valine) mutation in the ventricular myosin regulatory light chain (RLC) shown to cause a malignant familial hypertrophic cardiomyopathy (FHC) phenotype were generated, and the skinned and intact papillary muscle fibers from the Tg‐D166V mice were examined using a Guth muscle research system. A large increase in the Ca2+ sensitivity of force and ATPase (ΔpCa50>0.25) and a significant decrease in maximal force and ATPase were observed in skinned muscle fibers from Tg‐D166V mice compared with control mice. The cross‐bridge dissociation rate g was dramatically decreased, whereas the energy cost (ATPase/ force) was slightly increased in Tg‐D166V fibers compared with controls. The calculated average force per D166V cross‐bridge was also reduced. Intact papillary muscle data demonstrated prolonged force transients with no change in calcium transients in Tg‐D166V fibers compared with control fibers. Histopathological examination revealed fibrotic lesions in the hearts of the older D166V mice. Our results suggest that a charge effect of the D166V mutation and/or a mutation‐dependent decrease in RLC phosphorylation could initiate the slower kinetics of the D166V cross‐bridges and ultimately affect the regulation of cardiac muscle contraction. Profound cellular changes observed in Tg‐D166V myocardium when placed in vivo could trigger a series of pathological responses and result in poor prognosis for D166V‐positive patients.— Kerrick, W. G. L., Kazmierczak, K., Xu, Y., Wang, Y., Szczesna‐Cordary, D. Malignant familial hypertrophic cardiomyopathy D166V mutation in the ventricular myosin regulatory light chain causes profound effects in skinned and intact papillary muscle fibers from transgenic mice. FASEB J. 23, 855–865 (2009)

Functional consequences of the human cardiac troponin I hypertrophic cardiomyopathy mutation R145G in transgenic mice.

In this study, we addressed the functional consequences of the human cardiac troponin I (hcTnI) hypertrophic cardiomyopathy R145G mutation in transgenic mice. Simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca2+-activated force without changes in the maximal ATPase activity and an increase in the Ca2+ sensitivity of both ATPase and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state), showing that changes in Ca2+ sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared with Tg-WT fibers. The addition of 3 mm 2,3-butanedione monoxime at pCa 9.0 showed that there was ∼2-4% of force generating cross-bridges attached in Tg-R145G fibers compared with less than 1.0% in Tg-WT fibers, suggesting that the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca2+] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared with Tg-WT. These results suggest that the phenotype of hypertrophic cardiomyopathy is most likely caused by the compensatory mechanisms in the cardiovascular system that are activated by 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge, 2) slowed relaxation (diastolic dysfunction) caused by prolonged [Ca2+] and force transients, and 3) an inability of the cardiac TnI to completely inhibit activation in the absence of Ca2+ in Tg-R145G mice.

The effect of pH on the Ca2+ affinity of the Ca2+ regulatory sites of skeletal and cardiac troponin C in skinned muscle fibres

It is known that intracellular pH drops rapidly after the onset of ischemia in cardiac muscle and may play some role in the rapid drop in force that ensues. It is also known that α1- adrenoceptor agonists alkalinize intracellular pH by stimulating Na+/H+ exchange and may represent a mechanism which facilitates recovery of intracellular pH from acidosis. Lowering or raising pH shifts the Ca2+ dependence of force development in muscle fibres to higher or lower free Ca2+ concentrations, respectively, yet the precise mechanism is unknown. To investigate this phenomenon we have used skinned skeletal or cardiac muscle fibres whose endogenous troponin C (TnC) has been replaced with chicken skeletal TnC labelled with DANZ (STnCDANZ) or recombinant cardiac TnC labelled with IAANS (CTnC3(C84)IAANS), respectively. The fluorescence of the STnCDANZ or CTnC3(C84)IAANS was enhanced by Ca2+ binding to the Ca2+-specific (regulatory) site(s) of STnC or CTnC when incorporated into skinned fibres, and was measured simultaneously with force. When the pH was changed from 7.0 to 6.5 or 7.5 the shift in the Ca2+ dependence of force paralleled the shift in fluorescence. Since the force and fluorescence shift in parallel as the pH is lowered or raised, it can be concluded that these changes in Ca2+ sensitivity are caused by a decrease or increase, respectively, in the Ca2+ affinity of the Ca2+- specific site(s) of TnC. Since lowering or raising the pH also resulted in lower or higher, respectively, maximal Ca2+ activated force while maximal fluorescence remained unchanged, it is possible that H+ may act indirectly, as well, by reducing or increasing, respectively, the number or type of crossbridges attached to actin and thereby alter the crossbridge induced depression or elevation, respectively of the observed TnC Ca2+ affinity. Experiments with 2,3-butanedione monoxime, however, where force-generating crossbridges were greatly reduced, indicated that the pH effect may be primarily related to a direct change in the Ca2+ affinity to the regulatory sites of TnC

The role of the N-terminus of the myosin essential light chain in cardiac muscle contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Delta43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Delta43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Delta43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Delta43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Delta43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.

MgADP− increases maximum tension and Ca2+ sensitivity in skinned rabbit soleus fibers

Increasing concentrations of MgADP− or MgCDP− in the millimolar range cause an increase in the maximum Ca2+-activated tension that a skinned rabbit soleus muscle fiber can develop in the presence of 2 mM MgATP2− or MgCTP2 respectively. In contrast, the maximal Ca2+-activated ATPase activity of the fiber decreases in the presence of MgADP−. As the nucleoside diphosphate (MgADP− or MgCDP−) concentration is increased, the Ca2+ concentration required for half-maximal activation of tension is reduced.MgADP− has a similar effect on the Ca2+ concentration required to half-maximally activate the fiber ATPase. The effects on tension are due to magnesium nucleoside diphosphate and not some other form of nucleoside diphosphate since the effects occur at both low (pMg 4) and control (pMg 3) Mg2+ concentrations. Cooperativity, as judged by the Hill “n” value relating isometric tension and Ca2+, is less in the presence of 5 mM MgADP− as compared to a control (no added MgADP−) “n” value. Increasing concentrations of inorganic phosphate (Pi) in the millimolar range decrease maximum Ca2+-activated tension, and increase the concentration of Ca2+ required to half-maximally activate tension, effects opposite to those of MgADP. These data are consistent with the hypothesis that cooperative interactions between actin and myosin can affect the affinity of troponin for Ca2+.

Myosin regulatory light chain E22K mutation results in decreased cardiac intracellular calcium and force transients

The glutamic acid to lysine mutation at the 22nd amino acid residue (E22K) in the human cardiac myosin regulatory light chain (RLC) gene causes familial hypertrophic cardiomyopathy (FHC) with a phenotype of midventricular obstruction and septal hypertrophy. Our recent histopathology results have shown that the hearts of transgenic E22K mice (Tg‐E22K) resemble those of human patients, demonstrating enlarged interventricular septa and papillary muscles. In this study, we show no effect of the E22K mutation on the kinetics of mutated myosin in its ATP‐powered interaction with fluorescently labeled single actin filaments compared to nontransgenic or trans‐genic wild‐type (Tg‐WT) control mice. Likewise, no change in cross‐bridge dissociation rates (gapp) was observed in freshly skinned papillary muscle fibers. In contrast, maximal force and ATPase were decreased ~20% in Tg‐E22K skinned papillary muscle fibers and intracellular [Ca2+] and force transients were significantly decreased in intact papillary muscle fibers from Tg‐E22K compared to Tg‐WT mice. Moreover, energy metabolism measured in isolated working Tg‐E22K mouse hearts perfused under conditions of physiologically relevant levels of metabolic demand was similar in Tg‐E22K and control hearts before and after 20 min of no‐flow ischemia. Our results suggest that the patho‐logical response observed in the E22K myocardium might be triggered by mutation induced changes in the properties of the RLC Ca2+‐Mg2+ site, the state of the Ca2+/Mg2+ occupancy and consequently the Ca2+ buffering ability of the RLC. By decreasing the affinity of the RLC for Ca2+, the E22K mutation most likely promotes a Mg2+‐saturated RLC producing less force and ATPase than the Ca2+‐saturated RLC of WT fibers. Decreased Ca2+ binding may also lead to faster Ca2+ dissociation kinetics in Tg‐E22K intact fibers resulting in decreased duration and amplitude of [Ca2+] and force transients. These changes when placed in vivo would result in higher workloads and consequently cardiac hypertrophy.— Szczesna‐Cordary, D., Jones, M., Moore, J. R., Watt, J., Kerrick, W. G. L., Xu, Y., Wang, Y., Wagg, C., Lopaschuk, G. D. Myosin regulatory light chain E22K mutation results in decreased cardiac intra‐cellular calcium and force transients. FASEB J. 21, 3974–3985 (2007)

Activation of striated muscle: nearest-neighbor regulatory-unit and cross-bridge influence on myofilament kinetics.

We have formulated a three-compartment model of muscle activation that includes both strong cross-bridge (XB) and Ca(2+)-activated regulatory-unit (RU) mediated nearest-neighbor cooperative influences. The model is based on the tight coupling premise--that XB retain activating Ca(2+) on the thin filament. Using global non-linear least-squares, the model produced excellent fits to experimental steady-state force-pCa and ATPase-pCa data from skinned rat soleus fibers. In terms of the model, nearest-neighbor influences over the range of Ca(2+) required for activation cause the Ca(2+) dissociation rate from regulatory-units (k(off)) to decrease and the cross-bridge association rate (f) to increase each more than ten-fold. Moreover, the rate variations occur in separate Ca(2+) regimes. The energy of activation governing f is strongly influenced by both neighboring RU and XB. In contrast, the energy of activation governing k(off) is less affected by neighboring XB than by neighboring RU. Nearest-neighbor cooperative influences provide both an overall sensitization to Ca(2+) and the well-known steep response of force to free Ca(2+). The apparent sensitivity for Ca(2+)-activation of force and ATPase is a function of cross-bridge kinetic rates. The model and derived parameter set produce simulated behavior in qualitative agreement with steady-state experiments reported in the literature for partial TnC replacement, increased [P(i)], increased [ADP], and MalNEt-S1 addition. The model is an initial attempt to construct a general theory of striated muscle activation-one that can be consistently used to interpret data from various types of muscle manipulation experiments.

The apparent rate constant for the dissociation of force generating myosin crossbridges from actin decreases during Ca2+ activation of skinned muscle fibres.

SummaryThe effect of Ca2+ activation on the apparent rate constant governing the dissociation of force generating myosin cross-bridges was studied in skinned rabbit adductor magnus fibres (fast-twitch) at 21±1 °C. Simultaneous measurements of Ca2+-activated isometric force and ATPase activity were conducted in parallel with simultaneous measurements of DANZ-labelled troponin C (TnCDANZ) fluorescence and isometric force in fibres whose endogenous troponin C had been partially replaced with TnCDANZ. The Ca2+ activation of isometric force occurred at approximately two times higher Ca2+ concentration than did actomyosin ATPase activity at 2.0 mM MgATP. Since increases in both TnCDANZ fluorescence and ATPase activity occurred over approximately the same Ca2+ concentration range at substantially lower concentrations of Ca2+ than did force, this data suggests that the TnCDANZ fluorescence is associated with the Ca2+ activation of myosin crossbridge turnover (ATPase) rather than force. According to the model of Huxley (1957) and assuming the hydrolysis of one molecule of ATP per cycle of the crossbridge, the apparent rate constantgapp for the dissociation of force generating myosin crossbridges is proportional to the actomyosin ATPase/isometric force ratio. This measure ofgapp shows approximately a fivefold decrease during Ca2+ activation of isometric force. This change ingapp is responsible for separation of the Ca2+ sensitivity of the normalized ATPase activity and isometric force curves. If the MgATP concentration is reduced to 0.5 mM, the change ingapp is reduced and consequently the difference in Ca2+ sensitivity between normalized steady state ATPase and force is also reduced.