L-type Ca2+ channel function is linked to dystrophin expression in mammalian muscle

Background

In dystrophic mdx skeletal muscle, aberrant Ca²⁺ homeostasis and fibre degeneration are found. The absence of dystrophin in models of Duchenne muscular dystrophy (DMD) has been connected to altered ion channel properties e.g. impaired L-type Ca²⁺ currents. In regenerating mdx muscle, ‘revertant’ fibres restore dystrophin expression. Their functionality involving DHPR-Ca²⁺-channels is elusive.

Methods and Results

We developed a novel ‘in-situ’ confocal immuno-fluorescence and imaging technique that allows, for the first time, quantitative subcellular dystrophin-DHPR colocalization in individual, non-fixed, muscle fibres. Tubular DHPR signals alternated with second harmonic generation signals originating from myosin. Dystrophin-DHPR colocalization was substantial in wt fibres, but diminished in most mdx fibres. Mini-dystrophin (MinD) expressing fibres successfully restored colocalization. Interestingly, in some aged mdx fibres, colocalization was similar to wt fibres. Most mdx fibres showed very weak membrane dystrophin staining and were classified ‘mdx-like’. Some mdx fibres, however, had strong ‘wt-like’ dystrophin signals and were identified as ‘revertants’. Split mdx fibres were mostly ‘mdx-like’ and are not generally ‘revertants’. Correlations between membrane dystrophin and DHPR colocalization suggest a restored putative link in ‘revertants’. Using the two-micro-electrode-voltage clamp technique, Ca²⁺-current amplitudes (i_max) showed very similar behaviours: reduced amplitudes in most aged mdx fibres (as seen exclusively in young mdx mice) and a few mdx fibres, most likely ‘revertants’, with amplitudes similar to wt or MinD fibres. Ca²⁺ current activation curves were similar in ‘wt-like’ and ‘mdx-like’ aged mdx fibres and are not the cause for the differences in current amplitudes. i_max amplitudes were fully restored in MinD fibres.

Conclusions

We present evidence for a direct/indirect DHPR-dystrophin interaction present in wt, MinD and ‘revertant’ mdx fibres but absent in remaining mdx fibres. Our imaging technique reliably detects single isolated ‘revertant’ fibres that could be used for subsequent physiological experiments to study mechanisms and therapy concepts in DMD.     

Diapocynin,a Dimer of the NADPH Oxidase Inhibitor Apocynin,Reduces ROS Production and Prevents Force Loss in Eccentrically Contracting Dystrophic Muscle

Elevation of intracellular Ca²⁺, excessive ROS production and increased phospholipase A₂ activity contribute to the pathology in dystrophin-deficient muscle. Moreover, Ca²⁺, ROS and phospholipase A₂, in particular iPLA₂, are thought to potentiate each other in positive feedback loops. NADPH oxidases (NOX) have been considered as a major source of ROS in muscle and have been reported to be overexpressed in muscles of mdx mice. We report here on our investigations regarding the effect of diapocynin, a dimer of the commonly used NOX inhibitor apocynin, on the activity of iPLA₂, Ca²⁺ handling and ROS generation in dystrophic myotubes. We also examined the effects of diapocynin on force production and recovery ability of isolated EDL muscles exposed to eccentric contractions in vitro, a damaging procedure to which dystrophic muscle is extremely sensitive. In dystrophic myotubes, diapocynin inhibited ROS production, abolished iPLA₂ activity and reduced Ca²⁺ influx through stretch-activated and store-operated channels, two major pathways responsible for excessive Ca²⁺ entry in dystrophic muscle. Diapocynin also prevented force loss induced by eccentric contractions of mdx muscle close to the value of wild-type muscle and reduced membrane damage as seen by Procion orange dye uptake. These findings support the central role played by NOX-ROS in the pathogenic cascade leading to muscular dystrophy and suggest diapocynin as an effective NOX inhibitor that might be helpful for future therapeutic approaches.     

Inhibitory control over Ca(2+) sparks via mechanosensitive channels is disrupted in dystrophin deficient muscle but restored by mini-dystrophin expression

Background

In dystrophic skeletal muscle, osmotic stimuli somehow relieve inhibitory control of dihydropyridine receptors (DHPR) on spontaneous sarcoplasmic reticulum elementary Ca²⁺ release events (ECRE) in high Ca²⁺ external environments. Such ‘uncontrolled’ Ca²⁺ sparks were suggested to act as dystrophic signals. They may be related to mechanosensitive pathways but the mechanisms are elusive. Also, it is not known whether truncated dystrophins can correct the dystrophic disinhibition.

Methodology/Principal Findings

We recorded ECRE activity in single intact fibers from adult wt, mdx and mini-dystrophin expressing mice (MinD) under resting isotonic conditions and following hyper-/hypo-osmolar external shock using confocal microscopy and imaging techniques. Isotonic ECRE frequencies were small in wt and MinD fibers, but were markedly increased in mdx fibers. Osmotic challenge dramatically increased ECRE activity in mdx fibers. Sustained osmotic challenge induced marked exponential ECRE activity adaptation that was three times faster in mdx compared to wt and MinD fibers. Rising external Ca²⁺ concentrations amplified osmotic ECRE responses. The eliminated ECRE suppression in intact osmotically stressed mdx fibers was completely and reversibly resuscitated by streptomycine (200 µM), spider peptide GsMTx-4 (5 µM) and Gd³⁺ (20 µM) that block unspecific, specific cationic and Ca²⁺ selective mechanosensitive channels (MsC), respectively. ECRE morphology was not substantially altered by membrane stress. During hyperosmotic challenge, membrane potentials were polarised and a putative depolarisation through aberrant MsC negligible excluding direct activation of ECRE through tubular depolarisation.

Conclusions/Significance

Dystrophin suppresses spontaneous ECRE activity by control of mechanosensitive pathways which are suggested to interact with the inhibitory DHPR loop to the ryanodine receptor. MsC-related disinhibition prevails in dystrophic muscle and can be resuscitated by transgenic mini-dystrophin expression. Our results have important implications for the pathophysiology of DMD where abnormal MsC in dystrophic muscle confer disruption of microdomain Ca²⁺ homeostasis. MsC blockers should have considerable therapeutic potential if more muscle specific compounds can be found.     

FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro

We previously observed that disruption of FK506‐binding protein 12.6 (FKBP12.6) gene resulted in cardiac hypertrophy in male mice. Studies showed that overexpression of FKBP12.6 attenuated thoracic aortic constriction (TAC)‐induced cardiac hypertrophy in mice, whereas the adenovirus‐mediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes, indicating that the role of FKBP12.6 in cardiac hypertrophy is still controversial. In this study, we aimed to investigate the roles and mechanisms of FKBP12.6 in angiotensin II (AngII)‐induced cardiac hypertrophy using various transgenic mouse models in vivo and in vitro. FKBP12.6 knockout (FKBP12.6^?/?) mice and cardiac‐specific FKBP12.6 overexpressing (FKBP12.6 TG) mice were infused with AngII (1500 ng/kg/min) for 14 days subcutaneously by implantation of an osmotic mini‐pump. The results showed that FKBP12.6 deficiency aggravated AngII‐induced cardiac hypertrophy, while cardiac‐specific overexpression of FKBP12.6 prevented hearts from the hypertrophic response to AngII stimulation in mice. Consistent with the results in vivo, overexpression of FKBP12.6 in H9c2 cells significantly repressed the AngII‐induced cardiomyocyte hypertrophy, seen as reductions in the cell sizes and the expressions of hypertrophic genes. Furthermore, we demonstrated that the protection of FKBP12.6 on AngII‐induced cardiac hypertrophy was involved in reducing the concentration of intracellular Ca²⁺ ([Ca²⁺]i), in which the protein significantly inhibited the key Ca²⁺/calmodulin‐dependent signalling pathways such as calcineurin/cardiac form of nuclear factor of activated T cells 4 (NFATc4), calmodulin kinaseII (CaMKII)/MEF‐2, AKT/Glycogen synthase kinase 3β (GSK3β)/NFATc4 and AKT/mTOR signalling pathways. Our study demonstrated that FKBP12.6 protects heart from AngII‐induced cardiac hypertrophy through inhibiting Ca²⁺/calmodulin‐mediated signalling pathways.     

Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

There is substantial evidence indicating that disruption of Ca²⁺ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca²⁺ deregulation and the Ca²⁺ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca²⁺ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca²⁺ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca²⁺ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca²⁺ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca²⁺ store contributes to the disrupted Ca²⁺ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.     

Depolarization-induced contraction and SR function in mechanically skinned muscle fibers from dystrophic mdx mice

Dystrophin is absent in muscle fibers of patients with Duchenne muscular dystrophy (DMD) and in muscle fibers from the mdx mouse, an animal model of DMD. Disrupted excitation-contraction (E-C) coupling has been postulated to be a functional consequence of the lack of dystrophin, although the evidence for this is not entirely clear. We used mechanically skinned fibers (with a sealed transverse tubular system) prepared from fast extensor digitorum longus muscles of wild-type control and dystrophic mdx mice to test the hypothesis that dystrophin deficiency would affect the depolarization-induced contractile response (DICR) and sarcoplasmic reticulum (SR) function. DICR was similar in muscle fibers from mdx and control mice, indicating normal voltage regulation of Ca2+ release. Nevertheless, rundown of DICR (<50% of initial) was reached more rapidly in fibers from mdx than control mice [control: 32 +/- 5 depolarizations (n = 14 fibers) vs. mdx: 18 +/- 1 depolarizations (n = 7) before rundown, P < 0.05]. The repriming rate for DICRs was decreased in fibers from mdx mice, with lower submaximal DICR observed after 5, 10, and 20 s of repriming compared with fibers from control mice (P < 0.05). SR Ca2+ reloading was not different in fibers from control and mdx mice, and no difference was observed in SR Ca2+ leak. Caffeine (2-7 mM)-induced contraction was diminished in fibers from mdx mice compared with control (P < 0.05), indicating depressed SR Ca2+ release channel activity. Our findings indicate that fast fibers from mdx mice exhibit some impairment in the events mediating E-C coupling and SR Ca2+ release channel activity.     

Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

Maternal Nutrient Restriction Alters Ca2+ Handling Properties and Contractile Function of Isolated Left Ventricle Bundles in Male But Not Female Juvenile Rats

Intrauterine growth restriction (IUGR), defined as a birth weight below the 10th centile, may be caused by maternal undernutrition, with evidence that IUGR offspring have an increased risk of cardiovascular disease (CVD) in adulthood. Calcium ions (Ca²⁺) are an integral messenger for several steps associated with excitation-contraction coupling (ECC); the cascade of events from the initiation of an action potential at the surface membrane, to contraction of the cardiomyocyte. Any changes in Ca²⁺ storage and release from the sarcoplasmic reticulum (SR), or sensitivity of the contractile apparatus to Ca²⁺ may underlie the mechanism linking IUGR to an increased risk of CVD. This study aimed to explore the effects of maternal nutrient restriction on cardiac function, including Ca²⁺ handling by the SR and force development by the contractile apparatus. Juvenile Long Evans hooded rats born to Control (C) and nutrient restricted (NR) dams were anaesthetized for collection of the heart at 10–12 weeks of age. Left ventricular bundles from male NR offspring displayed increased maximum Ca²⁺-activated force, and decreased protein content of troponin I (cTnI) compared to C males. Furthermore, male NR offspring showed a reduction in rate of rise of the caffeine-induced Ca²⁺ force response and a decrease in the protein content of ryanodine receptor (RYR2). These physiological and biochemical findings observed in males were not evident in female offspring. These findings illustrate a sex-specific effect of maternal NR on cardiac development, and also highlight a possible mechanism for the development of hypertension and hypertrophy in male NR offspring.     

Dynamic responses of the glutathione system to acute oxidative stress in dystrophic mouse (mdx) muscles

The precise mechanisms underlying skeletal muscle damage in Duchenne muscular dystrophy (DMD) remain ill-defined. Functional ischemia during muscle activation, with subsequent reperfusion during rest, has been documented. Therefore, one possibility is the presence of increased oxidative stress. We applied a model of acute hindlimb ischemia/reperfusion (I/R) in mdx mice (genetic homolog of DMD) to evaluate dynamic in vivo responses of dystrophic muscles to this form of oxidative stress. Before the application of I/R, mdx muscles showed: 1) decreased levels of total glutathione (GSH) with an increased oxidized (GSSG)-to-reduced (GSH) glutathione ratio; 2) greater activity of the GSH-metabolizing enzymes glutathione peroxidase (GPx) and glutathione reductase; and 3) lower activity levels of NADP-linked isocitrate dehydrogenase (ICDH) and aconitase, two metabolic enzymes that are sensitive to inactivation by oxidative stress and also implicated in GSH regeneration. Interestingly, nondystrophic muscles subjected to I/R exhibited similar changes in total glutathione, GSSG/GSH, GPx, ICDH, and aconitase. In contrast, all of the above remained stable in mdx muscles subjected to I/R. Taken together, these results suggest that mdx muscles are chronically subjected to increased oxidative stress, leading to adaptive changes that attempt to protect (although only in part) the dystrophic muscles from acute I/R-induced oxidative stress. In addition, mdx muscles show significant impairment of the redox-sensitive metabolic enzymes ICDH and aconitase, which may further contribute to contractile dysfunction in dystrophic muscles.     

Dietary interaction of high fat and marginal copper deficiency on cardiac contractile function

Objective: High‐fat and marginally copper‐deficient diets impair heart function, leading to cardiac hypertrophy, increased lipid droplet volume, and compromised contractile function, resembling lipotoxic cardiac dysfunction. However, the combined effect of the two on cardiac function is unknown. This study was designed to examine the interaction between high‐fat and marginally copper‐deficient diets on cardiomyocyte contractile function. Research Methods and Procedures: Weanling male rats were fed diets incorporating a low‐ or high‐fat diet (10% or 45% of kcal from fat, respectively) with adequate (6 mg/kg) or marginally deficient (1.5 mg/kg) copper content for 12 weeks. Contractile function was determined with an IonOptix system including peak shortening (PS), time‐to‐PS, time‐to‐90% relengthening, maximal velocity of shortening/relengthening, and intracellular Ca²⁺ ([Ca²⁺]_I) rise and decay. Results: Neither dietary treatment affected blood pressure or glucose levels, although the high‐fat diet elicited obesity and glucose intolerance. Both diets depressed PS, maximal velocity of shortening/relengthening, and intracellular Ca²⁺ ([Ca²⁺]_I) rise and prolonged time‐to‐90% relengthening and Ca²⁺ decay without an additive effect between the two. Ca²⁺ sensitivity, apoptosis, lipid peroxidation, nitrosative damage, tissue ceramide, and triglyceride levels were unaffected by either diet or in combination. Phospholamban (PLB) but not sarco(endo)plasmic reticulum Ca²⁺‐ATPase was increased by both diets. Endothelial NO synthase was depressed with concurrent treatments. The electron transport chain was unaffected, although mitochondrial aconitase activity was inhibited by the high‐fat diet. Discussion: These data suggest that high‐fat and marginally copper deficient diets impaired cardiomyocyte contractile function and [Ca²⁺]_i homeostasis, possibly through a similar mechanism, without obvious lipotoxicity, nitrosative damage, and apoptosis.     

The effect of Sirt1 deficiency on Ca2+ and Na+ regulation in mouse ventricular myocytes

This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca²⁺ and Na⁺ regulation, leading to cardiac dysfunction and arrhythmogenesis. We used mice with cardiac‐specific Sirt1 knockout (Sirt1^?/?). Sirt1^flox/flox mice were served as control. Sirt1^?/? mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca²⁺ transient amplitudes and sarcoplasmic reticulum (SR) Ca²⁺ stores, and increased SR Ca²⁺ leak were shown in the Sirt1^?/? mice. Electrophysiological measurements were performed using patch‐clamp method. While L‐type Ca²⁺ current (I_{Ca, L}) was smaller in Sirt1^?/? myocytes, reverse‐mode Na⁺/Ca²⁺ exchanger (NCX) current was larger compared with those in control myocytes. Late Na⁺ current (I_{Na, L}) was enhanced in the Sirt1^?/? mice, alongside with elevated cytosolic Na⁺ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1^?/? mice. Sirt1^?/? cardiomyocytes showed down‐regulation of L‐type Ca²⁺ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), but up‐regulation of Ca²⁺/calmodulin‐dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca²⁺ and Na⁺ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.     

Cardiac functions and taurine's actions at different extracellular calcium concentrations in forced swimming stress-loaded rats

Modulation of the sinus rate and contractile force by taurine at different extracellular Ca²⁺ concentrations ([Ca²⁺]_o) was examined using rat right atria loaded with forced swimming stress. Serum concentration of corticosterone profoundly increased in stress-loaded rats as compared with native rats. The taurine level in serum also increased in stress-loaded rats, but was not changed in the different heart tissues and aorta. Heat-shock protein (HSP72) was detectable in cardiac muscles and in the lumen of cardiac blood vessels of stress-loaded rats using a monoclonal antibody. Increasing [Ca²⁺]_o (from 0.9 to 3.6 mM) enhanced the sinus rate and contractile force in a [Ca²⁺]_o-dependent fashion in native rats, but not in stress-loaded rats. Taurine (1–20 mM) caused a negative chronotropic and inotropic effect in a concentration-dependent manner. At 1.8 mM [Ca²⁺]_o, the negative chronotropic effect of taurine (10–20 mM) was attenuated in stress-loaded rats as compared with native rats. These results indicate that swimming stress causes a release of taurine into the serum and reduces the sensitivity to [Ca²⁺]_o. Taurine administration might, in part, exhibit the protective actions on acute stress-induced responses.     

Cardiac mechanical efficiency is preserved in primary cardiac hypertrophy despite impaired mechanical function

Increased heart size is a major risk factor for heart failure and premature mortality. Although abnormal heart growth subsequent to hypertension often accompanies disturbances in mechano-energetics and cardiac efficiency, it remains uncertain whether hypertrophy is their primary driver. In this study, we aimed to investigate the direct association between cardiac hypertrophy and cardiac mechano-energetics using isolated left-ventricular trabeculae from a rat model of primary cardiac hypertrophy and its control. We evaluated energy expenditure (heat output) and mechanical performance (force length work production) simultaneously at a range of preloads and afterloads in a microcalorimeter, we determined energy expenditure related to cross-bridge cycling and Ca²⁺ cycling (activation heat), and we quantified energy efficiency. Rats with cardiac hypertrophy exhibited increased cardiomyocyte length and width. Their trabeculae showed mechanical impairment, evidenced by lower force production, extent and kinetics of shortening, and work output. Lower force was associated with lower energy expenditure related to Ca²⁺ cycling and to cross-bridge cycling. However, despite these changes, both mechanical and cross-bridge energy efficiency were unchanged. Our results show that cardiac hypertrophy is associated with impaired contractile performance and with preservation of energy efficiency. These findings provide direction for future investigations targeting metabolic and Ca²⁺ disturbances underlying cardiac mechanical and energetic impairment in primary cardiac hypertrophy.     

Cathepsin K knockout alleviates aging‐induced cardiac dysfunction

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload‐induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age‐dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca²⁺ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura‐2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24‐month‐old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross‐sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca²⁺ release compared to young (6‐month‐old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged‐cathepsin K knockout mice compared to their wild‐type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age‐induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis‐inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin‐induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age‐related decline in cardiac function via suppressing caspase‐dependent and caspase‐independent apoptosis.     

Double knockout of Akt2 and AMPK accentuates high fat diet-induced cardiac anomalies through a cGAS-STING-mediated mechanism

High fat diet intake contributes to undesired cardiac geometric and functional changes although the underlying mechanism remains elusive. Akt and AMPK govern to cardiac homeostasis. This study examined the impact of deletion of Akt2 (main cardiac isoform of Akt) and AMPKα2 on high fat diet intake-induced cardiac remodeling and contractile anomalies and mechanisms involved. Cardiac geometry, contractile, and intracellular Ca²⁺ properties were evaluated using echocardiography, IonOptix® edge-detection and fura-2 techniques in wild-type (WT) and Akt2-AMPK double knockout (DKO) mice receiving low fat (LF) or high fat (HF) diet for 4 months. Our results revealed that fat diet intake elicit obesity, cardiac remodeling (hypertrophy, LV mass, LVESD, and cross-sectional area), contractile dysfunction (fractional shortening, peak shortening, maximal velocity of shortening/relengthening, time-to-90% relengthening, and intracellular Ca²⁺ handling), ultrastructural disarray, apoptosis, O₂⁻, inflammation, dampened autophagy and mitophagy. Although DKO did not affect these parameters, it accentuated high fat diet-induced cardiac remodeling and contractile anomalies. High fat intake upregulated levels of cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and STING phosphorylation while suppressing phosphorylation of ULK1 (Ser⁷⁵⁷ and Ser⁷⁷⁷), with a more pronounced effect in DKO mice. In vitro data revealed that inhibition of cGAS and STING using PF-06928215 and Astin C negated palmitic acid-induced cardiomyocyte contractile dysfunction. Biological function analysis for all differentially expressed genes (DEGs) depicted that gene ontology terms associated with Akt and AMPK signaling processes were notably changed in high fat-fed hearts. Our data indicate that Akt2-AMPK ablation accentuated high fat diet-induced cardiac anomalies possibly through a cGAS-STING-mechanism.     

IP3 receptor blockade restores autophagy and mitochondrial function in skeletal muscle fibers of dystrophic mice

Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive destruction of muscle fibers associated with altered Ca²⁺ homeostasis. We have previously shown that the IP₃ receptor (IP₃R) plays a role in elevating basal cytoplasmic Ca²⁺ and that pharmacological blockade of IP₃R restores muscle function. Moreover, we have shown that the IP₃R pathway negatively regulates autophagy by controlling mitochondrial Ca²⁺ levels. Nevertheless, it remains unclear whether IP₃R is involved in abnormal mitochondrial Ca²⁺ levels, mitochondrial dynamics, or autophagy and mitophagy observed in adult DMD skeletal muscle. Here, we show that the elevated basal autophagy and autophagic flux levels were normalized when IP₃R was downregulated in mdx fibers. Pharmacological blockade of IP₃R in mdx fibers restored both increased mitochondrial Ca²⁺ levels and mitochondrial membrane potential under resting conditions. Interestingly, mdx mitochondria changed from a fission to an elongated state after IP₃R knockdown, and the elevated mitophagy levels in mdx fibers were normalized. To our knowledge, this is the first study associating IP₃R1 activity with changes in autophagy, mitochondrial Ca²⁺ levels, mitochondrial membrane potential, mitochondrial dynamics, and mitophagy in adult mouse skeletal muscle. Moreover, these results suggest that increased IP₃R activity in mdx fibers plays an important role in the pathophysiology of DMD. Overall, these results lead us to propose the use of specific IP₃R blockers as a new pharmacological treatment for DMD, given their ability to restore both autophagy/mitophagy and mitochondrial function.     

Stretch-activated calcium channel protein TRPC1 is correlated with the different degrees of the dystrophic phenotype in mdx mice

In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin is related to enhanced calcium influx and muscle degeneration. Stretch-activated channels (SACs) might be directly involved in the pathology of DMD, and transient receptor potential cation channels have been proposed as likely candidates of SACs. We investigated the levels of transient receptor potential canonical channel 1 (TRPC1) and the effects of streptomycin, a SAC blocker, in muscles showing different degrees of the dystrophic phenotype. Mdx mice (18 days old, n = 16) received daily intraperitoneal injections of streptomycin (182 mg/kg body wt) for 18 days, followed by removal of the diaphragm, sternomastoid (STN), biceps brachii, and tibialis anterior muscles. Control mdx mice (n = 37) were injected with saline. Western blot analysis showed higher levels of TRPC1 in diaphragm muscle compared with STN and limb muscles. Streptomycin reduced creatine kinase and prevented exercise-induced increases of total calcium and Evans blue dye uptake in diaphragm and in STN muscles. It is suggested that different levels of the stretch-activated calcium channel protein TRPC1 may contribute to the different degrees of the dystrophic phenotype seen in mdx mice. Early treatment designed to regulate the activity of these channels may ameliorate the progression of dystrophy in the most affected muscle, the diaphragm.     

Ablation of cardiac myosin–binding protein-C accelerates contractile kinetics in engineered cardiac tissue

Hypertrophic cardiomyopathy (HCM) caused by mutations in cardiac myosin–binding protein-C (cMyBP-C) is a heterogenous disease in which the phenotypic presentation is influenced by genetic, environmental, and developmental factors. Though mouse models have been used extensively to study the contractile effects of cMyBP-C ablation, early postnatal hypertrophic and dilatory remodeling may overshadow primary contractile defects. The use of a murine engineered cardiac tissue (mECT) model of cMyBP-C ablation in the present study permits delineation of the primary contractile kinetic abnormalities in an intact tissue model under mechanical loading conditions in the absence of confounding remodeling events. We generated mechanically integrated mECT using isolated postnatal day 1 mouse cardiac cells from both wild-type (WT) and cMyBP-C–null hearts. After culturing for 1 wk to establish coordinated spontaneous contraction, we measured twitch force and Ca²⁺ transients at 37°C during pacing at 6 and 9 Hz, with and without dobutamine. Compared with WT, the cMyBP-C–null mECT demonstrated faster late contraction kinetics and significantly faster early relaxation kinetics with no difference in Ca²⁺ transient kinetics. Strikingly, the ability of cMyBP-C–null mECT to increase contractile kinetics in response to adrenergic stimulation and increased pacing frequency were severely impaired. We conclude that cMyBP-C ablation results in constitutively accelerated contractile kinetics with preserved peak force with minimal contractile kinetic reserve. These functional abnormalities precede the development of the hypertrophic phenotype and do not result from alterations in Ca²⁺ transient kinetics, suggesting that alterations in contractile velocity may serve as the primary functional trigger for the development of hypertrophy in this model of HCM. Our findings strongly support a mechanism in which cMyBP-C functions as a physiological brake on contraction by positioning myosin heads away from the thin filament, a constraint which is removed upon adrenergic stimulation or cMyBP-C ablation.