Brandon J. Biesiadecki

The rates of Ca2+ dissociation and cross-bridge detachment from ventricular myofibrils as reported by a fluorescent cardiac Troponin C

Background: The rate-limiting step of cardiac muscle relaxation is not completely understood. Results: We were able to measure two proposed rate-limiting steps of relaxation in ventricular myofibrils. Conclusion: The rate of Ca2+ dissociation from troponin C may be rate-limiting during myofilament inactivation under physiological conditions. Significance: Strategies that target both troponin C and myosin will be needed to treat diastolic dysfunction. The rate-limiting step of cardiac muscle relaxation has been proposed to reside in the myofilament. Both the rates of cross-bridge detachment and Ca2+ dissociation from troponin C (TnC) have been hypothesized to rate-limit myofilament inactivation. In this study we used a fluorescent TnC to measure both the rate of Ca2+ dissociation from TnC and the rate of cross-bridge detachment from several different species of ventricular myofibrils. The fluorescently labeled TnC was sensitive to both Ca2+ dissociation and cross-bridge detachment at low Ca2+ (presence of EGTA), allowing for a direct comparison between the two proposed rates of myofilament inactivation. Unlike Ca2+ dissociation from TnC, cross-bridge detachment varied in myofibrils from different species and was rate-limited by ADP release. At subphysiological temperatures (<20 °C), the rate of Ca2+ dissociation from TnC was faster than the rate of cross-bridge detachment in the presence of ADP. These results support the hypothesis that cross-bridge detachment rate-limits relaxation. However, Ca2+ dissociation from TnC was not as temperature-sensitive as cross-bridge detachment. At a near physiological temperature (35 °C) and ADP, the rate of cross-bridge detachment may actually be faster than the rate of Ca2+ dissociation. This provides evidence that there may not be a simple, single rate-limiting step of myofilament inactivation.

Engineered troponin C constructs correct disease-related cardiac myofilament calcium sensitivity.

Background: Improved myofilament Ca2+ sensitivity alleviates defects in thin filament bearing disease-causing mutations. Results: By engineering the cardiac muscle Ca2+ sensor troponin C, aberrant myofilament Ca2+ sensitivity can be corrected in vitro. Conclusion: Engineered TnC provides a novel and versatile avenue to reset disease-related myofilament Ca2+ sensitivity. Significance: Engineered TnC could be a new therapeutic strategy for cardiac muscle diseases. Aberrant myofilament Ca2+ sensitivity is commonly observed with multiple cardiac diseases, especially familial cardiomyopathies. Although the etiology of the cardiomyopathies remains unclear, improving cardiac muscle Ca2+ sensitivity through either pharmacological or genetic approaches shows promise of alleviating the disease-related symptoms. Due to its central role as the Ca2+ sensor for cardiac muscle contraction, troponin C (TnC) stands out as an obvious and versatile target to reset disease-associated myofilament Ca2+ sensitivity back to normal. To test the hypothesis that aberrant myofilament Ca2+ sensitivity and its related function can be corrected through rationally engineered TnC constructs, three thin filament protein modifications representing different proteins (troponin I or troponin T), modifications (missense mutation, deletion, or truncation), and disease subtypes (familial or acquired) were studied. A fluorescent TnC was utilized to measure Ca2+ binding to TnC in the physiologically relevant biochemical model system of reconstituted thin filaments. Consistent with the pathophysiology, the restrictive cardiomyopathy mutation, troponin I R192H, and ischemia-induced truncation of troponin I (residues 1–192) increased the Ca2+ sensitivity of TnC on the thin filament, whereas the dilated cardiomyopathy mutation, troponin T ΔK210, decreased the Ca2+ sensitivity of TnC on the thin filament. Rationally engineered TnC constructs corrected the abnormal Ca2+ sensitivities of the thin filament, reconstituted actomyosin ATPase activity, and force generation in skinned trabeculae. Thus, the present study provides a novel and versatile therapeutic strategy to restore diseased cardiac muscle Ca2+ sensitivity.

Dissociation of Calcium Transients and Force Development following a Change in Stimulation Frequency in Isolated Rabbit Myocardium.

As the heart transitions from one exercise intensity to another, changes in cardiac output occur, which are modulated by alterations in force development and calcium handling. Although the steady-state force-calcium relationship at various heart rates is well investigated, regulation of these processes during transitions in heart rate is poorly understood. In isolated right ventricular muscle preparations from the rabbit, we investigated the beat-to-beat alterations in force and calcium during the transition from one stimulation frequency to another, using contractile assessments and confocal microscopy. We show that a change in steady-state conditions occurs in multiple phases: a rapid phase, which is characterized by a fast change in force production mirrored by a change in calcium transient amplitude, and a slow phase, which follows the rapid phase and occurs as the muscle proceeds to stabilize at the new frequency. This second/late phase is characterized by a quantitative dissociation between the calcium transient amplitude and developed force. Twitch timing kinetics, such as time to peak tension and 50% relaxation rate, reached steady-state well before force development and calcium transient amplitude. The dynamic relationship between force and calcium upon a switch in stimulation frequency unveils the dynamic involvement of myofilament-based properties in frequency-dependent activation.

Staurosporine inhibits frequency-dependent myofilament desensitization in intact rabbit cardiac trabeculae.

Myofilament calcium sensitivity decreases with frequency in intact healthy rabbit trabeculae and associates with Troponin I and Myosin light chain-2 phosphorylation. We here tested whether serine-threonine kinase activity is primarily responsible for this frequency-dependent modulations of myofilament calcium sensitivity. Right ventricular trabeculae were isolated from New Zealand White rabbit hearts and iontophoretically loaded with bis-fura-2. Twitch force-calcium relationships and steady state force-calcium relationships were measured at frequencies of 1 and 4 Hz at 37 °C. Staurosporine (100 nM), a nonspecific serine-threonine kinase inhibitor, or vehicle (DMSO) was included in the superfusion solution before and during the contractures. Staurosporine had no frequency-dependent effect on force development, kinetics, calcium transient amplitude, or rate of calcium transient decline. The shift in the pCa50 of the force-calcium relationship was significant from 6.05 ± 0.04 at 1 Hz versus 5.88 ± 0.06 at 4 Hz under control conditions (vehicle, P < 0.001) but not in presence of staurosporine (5.89 ± 0.08 at 1 Hz versus 5.94 ± 0.07 at 4 Hz, P = NS). Phosphoprotein analysis (Pro-Q Diamond stain) confirmed that staurosporine significantly blunted the frequency-dependent phosphorylation at Troponin I and Myosin light chain-2. We conclude that frequency-dependent modulation of calcium sensitivity is mediated through a kinase-specific effect involving phosphorylation of myofilament proteins.

Adenosine-Induced Atrial FibrillationClinical Perspective

Background: Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels (IK,Ado). Methods: We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). Results: Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10–100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P<0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P<0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10–100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P<0.01) and GIRK4 (1.7±0.8-fold; P<0.05) protein expression than lateral/posterior LA. Conclusions: This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine.Background: Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels ( I K,Ado). Methods: We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). Results: Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10–100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P <0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P <0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10–100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P <0.01) and GIRK4 (1.7±0.8-fold; P <0.05) protein expression than lateral/posterior LA. Conclusions: This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. # Clinical Perspective {#article-title-40}