Christopher M. Loughrey

Calcium/calmodulin-dependent protein kinase IIdelta associates with the ryanodine receptor complex and regulates channel function in rabbit heart

Cardiac ryanodine receptors (RyR2s) play a critical role in excitation-contraction coupling by providing a pathway for the release of Ca(2+) from the sarcoplasmic reticulum into the cytosol. RyR2s exist as macromolecular complexes that are regulated via binding of Ca(2+) and protein phosphorylation/dephosphorylation. The present study examined the association of endogenous CaMKII (calcium/calmodulin-dependent protein kinase II) with the RyR2 complex and whether this enzyme could modulate RyR2 function in isolated rabbit ventricular myocardium. Endogenous phosphorylation of RyR2 was verified using phosphorylation site-specific antibodies. Co-immunoprecipitation studies established that RyR2 was physically associated with CaMKIIdelta. Quantitative assessment of RyR2 protein was performed by [(3)H]ryanodine binding to RyR2 immunoprecipitates. Parallel kinase assays allowed the endogenous CaMKII activity associated with these immunoprecipitates to be expressed relative to the amount of RyR2. The activity of RyR2 in isolated cardiac myocytes was measured in two ways: (i) RyR2-mediated Ca(2+) release (Ca(2+) sparks) using confocal microscopy and (ii) Ca(2+)-sensitive [(3)H]ryanodine binding. These studies were performed in the presence and absence of AIP (autocamtide-2-related inhibitory peptide), a highly specific inhibitor of CaMKII. At 1 microM AIP Ca(2+) spark duration, frequency and width were decreased significantly. Similarly, 1 microM AIP decreased [(3)H]ryanodine binding. At 5 microM AIP, a more profound inhibition of Ca(2+) sparks and a decrease in [(3)H]ryanodine binding was observed. Separate measurements showed that AIP (1-5 microM) did not affect sarcoplasmic reticulum Ca(2+)-ATPase-mediated Ca(2+) uptake. These results suggest the existence of an endogenous CaMKIIdelta that associates directly with RyR2 and specifically modulates RyR2 activity.

Effects of Adenovirus-Mediated Sorcin Overexpression on Excitation-Contraction Coupling in Isolated Rabbit Cardiomyocytes

&NA; To evaluate the effect of sorcin on cardiac excitation‐contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad‐sorcin). A &bgr;‐galactosidase adenovirus (Ad‐LacZ) was used as a control. Fractional shortening in response to 1‐Hz field stimulation (at 37°C) was significantly reduced in Ad‐sorcin‐transfected myocytes compared with control myocytes (2.10±0.05% [n=311] versus 2.42±0.06% [n=312], respectively; P<0.001). Action potential duration (at 20°C) was significantly less in the Ad‐sorcin group (458±22 ms, n=11) compared with the control group (520±19 ms, n=10; P<0.05). In voltage‐clamped, fura 2‐loaded myocytes (20°C), a reduced peak‐systolic and end‐diastolic [Ca2+]i was observed after Ad‐sorcin transfection. L‐type Ca2+ current amplitude and time course were unaffected. Caffeine‐induced Ca2+ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na+‐Ca2+ exchanger (NCX) current revealed a significantly lower SR Ca2+ content and faster Ca2+‐extrusion kinetics in Ad‐sorcin‐transfected cells. Higher NCX activity after Ad‐sorcin transfection was confirmed by measuring the NCX current‐voltage relationship. &bgr;‐Escin‐permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on Ca2+ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca2+] using a confocal microscope. Under these conditions, caffeine‐mediated SR Ca2+ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in sorcin‐overexpressing myocytes. In summary, sorcin overexpression in rabbit cardiomyocytes decreased Ca2+‐transient amplitude predominately by lowering SR Ca2+ content via increased NCX activity. The effect of sorcin overexpression on Ca2+ sparks indicates an effect on the ryanodine receptor that may also influence excitation‐contraction coupling. (Circ Res. 2003;93:132‐139.)

Characterization of a Range of Fura Dyes with Two-Photon Excitation

Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca(2+)-free and Ca(2+)-bound forms of the dyes were measured in free solution and expressed in terms of the two-photon fluorescence cross section (Goppert-Meyer units). The Fura dyes displayed the same Ca(2+)-free TPE spectrum in the intracellular volume of permeabilized and intact cardiomyocytes. Fluorescence measurements over a range of laser powers confirmed the TPE of both Ca(2+)-free and Ca(2+)-bound forms of the dyes. Single-wavelength excitation at 810 nM was used to determine the effective dissociation constants (K(eff)) and dynamic ranges (R(f)) of Fura-2, -4F, -6F, -FF, and Furaptra dyes (K(eff) = 181 +/- 52 nM, 1.16 +/- 0.016 micro M, 5.18 +/- 0.3 micro M, 19.2 +/- 1 micro M, and 58.5 +/- 2 micro M; and R(f) = 22.4 +/- 3.8, 12.2 +/- 0.34, 6.3 +/- 0.17, 16.1 +/- 2.8, and 25.4 +/- 4, respectively). Single-wavelength excitation of intracellular Fura-4F resolved diastolic and peak [Ca(2+)] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 micro M) extracellular [Ca(2+)]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca(2+) imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.

Over‐expression of FK506‐binding protein FKBP12.6 alters excitation–contraction coupling in adult rabbit cardiomyocytes

This study investigated the function of FK506‐binding protein (FKBP12.6) using adenoviral‐mediated gene transfer to over‐express FKBP12.6 (Ad‐FKBP12.6) in adult rabbit ventricular cardiomyocytes. Infection with a β‐galactosidase‐expressing adenovirus (Ad‐LacZ) was used as a control. Peak‐systolic intracellular [Ca2+] (measured with Fura‐2) was higher in the Ad‐FKBP12.6 group compared to Ad‐LacZ (1 Hz field stimulation at 37°C). The amplitude of caffeine‐induced Ca2+ release was also greater, indicating a higher SR Ca2+ content in the Ad‐FKBP12.6 group. Voltage clamp experiments indicated that FKBP12.6 over‐expression did not change L‐type Ca2+ current amplitude or Ca2+ efflux rates via the Na+–Ca2+ exchanger. Ca2+ transients comparable to those after Ad‐FKBP12.6 transfection could be obtained by enhancing SR Ca2+ content of Ad‐LacZ infected cells with periods of high frequency stimulation. Line‐scan confocal microscopy (Fluo‐3 fluorescence) of intact cardiomyocytes stimulated at 0.5 Hz (20−21°C) revealed a higher degree of synchronicity of SR Ca2+ release and fewer non‐responsive Ca2+ release sites in the Ad‐FKBP12.6 group compared to control. Ca2+ spark morphology was measured in β‐escin‐permeabilized cardiomyocytes at a free [Ca2+]i of 150 nm. The average values of the spark parameters (amplitude, duration, width and frequency) were reduced in the Ad‐FKBP12.6 group. Increasing [Ca2+]i to 400 nm caused coherent propagating Ca2+ waves in the Ad‐FKBP12.6 group but only limited Ca2+ release events were recorded in the control group. These data indicate that FKBP12.6 over‐expression enhances Ca2+ transient amplitude predominately by increasing SR Ca2+ content. Moreover, there is also evidence that FKBP12.6 can enhance the coupling between SR Ca2+ release sites independently of SR content.

Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics.

The Ca(2+) dissociation constant (K(d)) of Fluo-3 was determined using confocal fluorescence microscopy in two different situations: (i) within the cytosol of a permeabilised cardiomyocyte; and (ii) in an intact cardiomyocyte after incubation with the acetoxymethyl ester form of Fluo-3 (AM). Measurements were made on isolated rabbit ventricular cardiomyocytes after permeabilisation by a brief treatment with beta-escin (0.1mg/ml) and equilibration with 10 microM Fluo-3. The K(d) of Fluo-3 within the cytosol was not significantly different from that in free solution (558 +/- 15 nM, n=6). Over a range of cytoplasmic [Ca(2+)], the minimum [Ca(2+)] values between Ca(2+) waves was relatively constant despite changes in wave frequency. After loading intact cardiomyocytes with Fluo-3 by incubation with the -AM, spontaneous Ca(2+) waves were produced by incubation with strophanthidin (10 microM). By assuming a common minimum [Ca(2+)] in permeabilised and intact cells, the intracellular K(d) of Fluo-3 in intact myocytes was estimated to be 898 +/-64 nM (n=6). Application of this K(d) to fluorescence records shows that Ca(2+) waves in intact cells have similar amplitudes to those in permeabilised cells. Stimulation of cardiac myocytes at 0.5 Hz in the absence of strophanthidin (room temperature) resulted in a Ca(2+) transient with a maximum and minimum [Ca(2+)] of 1190 +/- 200 and 158 +/- 30 nM (n=11), respectively.

Two candidates at the heart of dysfunction: the ryanodine receptor and calcium/calmodulin protein kinase II as potential targets for therapeutic intervention-An in vivo perspective

At the start of a new decade (2011), heart failure and sudden cardiac death are still leading causes of mortality worldwide. There is a very obvious need for improved treatment strategies. Research over the past decade has focused on understanding and realising the therapeutic potential of molecular mechanisms that underlie the pathophysiology of cardiac dysfunction. There is now recognition that cell- and gene-based therapies could prove beneficial if aimed at the appropriate molecular targets. Two cardiac proteins that have received considerable attention over the last decade, have been identified as possible therapeutic targets. The cardiac sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor) and calcium/calmodulin dependent kinase II (CaMKIIδ) can act independently and in partnership, to regulate cardiac Ca(2+) handling. CaMKIIδ, by the very nature of its core function as a kinase, also modulates cardiac function globally, promoting effects on gene transcription and modulating inflammatory and proliferative responses, all events that are associated with both the functional and dysfunctional heart. In vivo approaches using genetic and pharmacologic strategies have revealed the prominent role of both proteins in cardiac dysfunction. More excitingly, they have also shown the potential for cardioprotection that modulation at the level of each protein can have. Translating these effects to the human heart is in its infancy. Whether intervention at these targets could result in clinical application is unknown at present, however current in vivo research has proved invaluable in revealing the potential that targeting of RyR and CaMKIIδ could have in limiting cardiac dysfunction.

The Relationship between Intracellular [Ca2+] and Ca2+ Wave Characteristics in Permeabilised Cardiomyocytes from the Rabbit

Spontaneous sarcoplasmic reticulum (SR) Ca2+ release and propagated intracellular Ca2+ waves are a consequence of cellular Ca2+ overload in cardiomyocytes. We examined the relationship between average intracellular [Ca2+] and Ca2+ wave characteristics. The amplitude, time course and propagation velocity of Ca2+ waves were measured using line‐scan confocal imaging of β‐escin‐permeabilised cardiomyocytes perfused with 10 μM Fluo‐3 or Fluo‐5F. Spontaneous Ca2+ waves were evident at cellular [Ca2+] > 200 nM. Peak [Ca2+] during a wave was 2.0–2.2 μM; the minimum [Ca2+] between waves was 120–160 nM; wave frequency was ≈0.1 Hz. Raising mean cellular [Ca2+] caused increases in all three parameters, particularly Ca2+ wave frequency. Increases in the rate of SR Ca2+ release and Ca2+ uptake were observed at higher cellular [Ca2+], indicating calcium‐sensitive regulation of these processes. At extracellular [Ca2+] > 2 μM, the mean [Ca2+] inside the permeabilised cell did not increase above 2 μM. This extracellular‐intracellular Ca2+ gradient could be maintained for periods of up to 5 min before the cardiomyocyte developed a sustained and irreversible hypercontraction. Inclusion of mitochondrial inhibitors (2 μM carbonyl cyanide m‐chlorophenylhydrazone and 2 μM oligomycin) while perfusing with > 2 μM Ca2+ abolished the extracellular‐intracellular Ca2+ gradient through the generation of Ca2+ waves with a higher peak [Ca2+] compared to control conditions. Under these conditions, cardiomyocytes rapidly (< 2 min) developed a sustained and irreversible contraction. These results suggest that mitochondrial Ca2+ uptake acts to delay an increase in [Ca2+] by blunting the peak of the Ca2+ wave.

Overexpression of FK-506–Binding Protein 12.0 Modulates Excitation–Contraction Coupling in Adult Rabbit Ventricular Cardiomyocytes

The effect of the 12-kDa isoform of FK-506–binding protein (FKBP)12.0 on cardiac excitation–contraction coupling was studied in adult rabbit ventricular myocytes after transfection with a recombinant adenovirus coding for human FKBP12.0 (Ad-FKBP12.0). Western blots confirmed overexpression (by 2.6±0.4 fold, n=5). FKBP12.0 association with rabbit cardiac ryanodine receptor (RyR2) was not detected by immunoprecipitation. However, glutathione S-transferase pull-down experiments indicated FKBP12.0–RyR2 binding to proteins isolated from human and rabbit but not dog myocardium. Voltage-clamp experiments indicated no effects of FKBP12.0 overexpression on L-type Ca2+ current (ICa,L) or Ca2+ efflux rates via the Na+/Ca2+ exchanger. Ca2+ transient amplitude was also not significantly different. However, sarcoplasmic reticulum Ca2+ load was ≈25% higher in myocytes in the Ad-FKBP12.0 group. The reduced ability of ICa,L to initiate sarcoplasmic reticulum Ca2+ release was observed over a range of values of sarcoplasmic reticulum Ca2+ content, indicating that overexpression of FKBP12.0 reduces the sensitivity of RyR2 to Ca2+. Ca2+ spark morphology was measured in β-escin–permeabilized cardiomyocytes. Ca2+ spark amplitude and duration were significantly increased, whereas frequency was decreased in cells overexpressing FKBP12.0. These changes were accompanied by an increased sarcoplasmic reticulum Ca2+ content. In summary, the effects of FKBP12.0 overexpression on intact and permeabilized cells were similar to those of tetracaine, a drug known to reduce RyR2 Ca2+ sensitivity and distinctly different from the effects of overexpression of the FKBP12.6 isomer. In conclusion, FKBP12.0-RyR2 interaction can regulate the gain of excitation–contraction coupling.

Exclusive expression of MeCP2 in the nervous system distinguishes between brain and peripheral Rett syndrome-like phenotypes.

Abstract Rett syndrome (RTT) is a severe genetic disorder resulting from mutations in the X-linked MECP2 gene. MeCP2 protein is highly expressed in the nervous system and deficiency in the mouse central nervous system alone recapitulates many features of the disorder. This suggests that RTT is primarily a neurological disorder, although the protein is reportedly widely expressed throughout the body. To determine whether aspects of the RTT phenotype that originate in non-neuronal tissues might have been overlooked, we generated mice in which Mecp2 remains at near normal levels in the nervous system, but is severely depleted elsewhere. Comparison of these mice with wild type and globally MeCP2-deficient mice showed that the majority of RTT-associated behavioural, sensorimotor, gait and autonomic (respiratory and cardiac) phenotypes are absent. Specific peripheral phenotypes were observed, however, most notably hypo-activity, exercise fatigue and bone abnormalities. Our results confirm that the brain should be the primary target for potential RTT therapies, but also strongly suggest that some less extreme but clinically significant aspects of the disorder arise independently of defects in the nervous system.

Trypanosoma brucei cathepsin-L increases arrhythmogenic sarcoplasmic reticulum-mediated calcium release in rat cardiomyocytes

Aims African trypanosomiasis, caused by Trypanosoma brucei species, leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, the cardiac pathogenesis remains unknown. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter cardiac function independent of a systemic inflammatory/immune response. Methods and results Using confocal imaging, T. brucei and T. brucei culture media (supernatant) caused an increased frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves) in isolated adult rat ventricular cardiomyocytes. Studies utilising inhibitors, recombinant protein and RNAi all demonstrated that this altered SR function was due to T. brucei cathepsin-L (TbCatL). Separate experiments revealed that TbCatL induced a 10–15% increase of SERCA activity but reduced SR Ca2+ content, suggesting a concomitant increased SR-mediated Ca2+ leak. This conclusion was supported by data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide, highlighting a role for CaMKII in the TbCatL action on SR function. Isolated Langendorff perfused whole heart experiments confirmed that supernatant caused an increased number of arrhythmic events. Conclusion These data demonstrate for the first time that African trypanosomes alter cardiac function independent of a systemic immune response, via a mechanism involving extracellular cathepsin-L-mediated changes in SR function.