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Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types., Graphical abstract, D’Antonio et al. characterize associations between GWAS signals for COVID-19 disease and eQTLs in 69 human tissues to identify causal variants and their underlying molecular mechanisms. They show that diverse symptoms and disease severity of COVID-19 are associated with variants affecting gene expression in a wide variety of tissues.

Chondroitin sulfate proteoglycans (CSPGs), up-regulated in and around the lesion after traumatic spinal cord injury (SCI), are key extracellular matrix inhibitory molecules that limit axon growth and consequent recovery of function. CSPG-mediated inhibition occurs via interactions with axonal receptors, including leukocyte common antigen- related (LAR) phosphatase. We tested the effects of a novel LAR inhibitory peptide in rats after hemisection at cervical level 2, a SCI model in which bulbospinal inspiratory neural circuitry originating in the medullary rostral ventral respiratory group (rVRG) becomes disconnected from phrenic motor neuron (PhMN) targets in cervical spinal cord, resulting in persistent partial-to-complete diaphragm paralysis. LAR peptide was delivered by a soaked gelfoam, which was placed directly over the injury site immediately after C2 hemisection and replaced at 1 week post-injury. Axotomized rVRG axons originating in ipsilateral medulla or spared rVRG fibers originating in contralateral medulla were separately assessed by anterograde tracing via AAV2-mCherry injection into rVRG. At 8 weeks post-hemisection, LAR peptide significantly improved ipsilateral hemidiaphragm function, as assessed in vivo with electromyography recordings. LAR peptide promoted robust regeneration of ipsilateral-originating rVRG axons into and through the lesion site and into intact caudal spinal cord to reach PhMNs located at C3-C5 levels. Furthermore, regenerating rVRG axons re-established putative monosynaptic connections with their PhMNs targets. In addition, LAR peptide stimulated robust sprouting of both modulatory serotonergic axons and contralateral-originating rVRG fibers within the PhMN pool ipsilateral/caudal to the hemisection. Our study demonstrates that targeting LAR-based axon growth inhibition promotes multiple forms of respiratory neural circuit plasticity and provides a new peptide-based therapeutic strategy to ameliorate the devastating respiratory consequences of SCI.

Populations of neural stem cells (NSCs) reside in a number of defined niches in the adult central nervous system (CNS) where they continually give rise to mature cell types throughout life, including newly born neurons. In addition to the prototypical niches of the subventricular zone (SVZ) and subgranular zone (SGZ) of the hippocampal dentate gyrus, novel stem cell niches that are also neurogenic have recently been identified in multiple midline structures, including circumventricular organs (CVOs) of the brain. These resident NSCs serve as a homeostatic source of new neurons and glial cells under intact physiological conditions. Importantly, they may also have the potential for reparative processes in pathological states such as traumatic spinal cord injury (SCI) and traumatic brain injury (TBI). As the response in these novel CVO stem cell niches has been characterized after stroke but not following SCI or TBI, we quantitatively assessed cell proliferation and the neuronal and glial lineage fate of resident NSCs in three CVO nuclei—area postrema (AP), median eminence (ME), and subfornical organ (SFO) —in rat models of cervical contusion-type SCI and controlled cortical impact (CCI)-induced TBI. Using bromodeoxyuridine (BrdU) labeling of proliferating cells, we find that TBI significantly enhanced proliferation in AP, ME, and SFO, whereas cervical SCI had no effects at early or chronic time-points post-injury. In addition, SCI did not alter NSC differentiation profile into doublecortin-positive neuroblasts, GFAP-expressing astrocytes, or Olig2-labeled cells of the oligodendrocyte lineage within AP, ME, or SFO at both time-points. In contrast, CCI induced a pronounced increase in Sox2- and doublecortin-labeled cells in the AP and Iba1-labeled microglia in the SFO. Lastly, plasma derived from CCI animals significantly increased NSC expansion in an in vitro neurosphere assay, whereas plasma from SCI animals did not exert such an effect, suggesting that signaling factors present in blood may be relevant to stimulating CVO niches after CNS injury and may explain the differential in vivo effects of SCI and TBI on the novel stem cell niches.

The overall goal of our research is to establish a preformed molecular guidance pathway to direct the growth of dopaminergic axons from embryonic ventral mesencephalon (VM), tissue placed within the substantia nigra (SN), into the striatum to reconstruct the nigrostriatal pathway in a hemi-Parkinson's disease rat model. Guidance pathways were prepared by injecting lentivirus encoding either GFP or a combination of glial-cell-line-derived neurotrophic factor (GDNF) with either GDNF family receptor α1 (GFRα1) or netrin1. In another cohort of animals, adeno-associated virus (AAV) encoding brain-derived neurotrophic factor (BDNF) was injected within the striatum after guidance pathway formation. GDNF combined with either GFRα1 or netrin significantly increased growth of dopaminergic axons out of transplants and along the pathway, resulting in a significant reduction in the number of amphetamine-induced rotations. Retrograde tract tracing showed that the dopaminergic axons innervating the striatum were from A9 neurons within the transplant. Increased dopaminergic innervation of the striatum and improved behavioral recovery were observed with the addition of BDNF. Preformed guidance pathways using a combination of GDNF and netrin1 can be used to reconstruct the nigrostriatal pathway and improve motor recovery.

Stem/progenitor cell transplantation delivery of astrocytes is a potentially powerful strategy for spinal cord injury (SCI). Axon extension into SCI lesions that occur spontaneously or in response to experimental manipulations is often observed along endogenous astrocyte "bridges," suggesting that augmenting this response via astrocyte lineage transplantation can enhance axon regrowth. Given the importance of respiratory dysfunction post-SCI, we transplanted glial-restricted precursors (GRPs)-a class of lineage-restricted astrocyte progenitors-into the C2 hemisection model and evaluated effects on diaphragm function and the growth response of descending rostral ventral respiratory group (rVRG) axons that innervate phrenic motor neurons (PhMNs). GRPs survived long term and efficiently differentiated into astrocytes in injured spinal cord. GRPs promoted significant recovery of diaphragm electromyography amplitudes and stimulated robust regeneration of injured rVRG axons. Although rVRG fibers extended across the lesion, no regrowing axons re-entered caudal spinal cord to reinnervate PhMNs, suggesting that this regeneration response-although impressive-was not responsible for recovery. Within ipsilateral C3-5 ventral horn (PhMN location), GRPs induced substantial sprouting of spared fibers originating in contralateral rVRG and 5-HT axons that are important for regulating PhMN excitability; this sprouting was likely involved in functional effects of GRPs. Finally, GRPs reduced the macrophage response (which plays a key role in inducing axon retraction and limiting regrowth) both within the hemisection and at intact caudal spinal cord surrounding PhMNs. These findings demonstrate that astrocyte progenitor transplantation promotes significant plasticity of rVRG-PhMN circuitry and restoration of diaphragm function and suggest that these effects may be in part through immunomodulation., National Institute of Neurological Disorders and Stroke. Grant Number: 2R01NS079702‐06 (to A.C.L.) Paralyzed Veterans of America Research Foundation. Grant Numbers: Grant 476686 (to A.C.L), 476686 NINDS. Grant Number: 2R01NS079702‐06

Damage to respiratory neural circuitry and consequent loss of diaphragm function is a major cause of morbidity and mortality in individuals suffering from traumatic cervical spinal cord injury (SCI). Repair of CNS axons after SCI remains a therapeutic challenge, despite current efforts. SCI disrupts inspiratory signals originating in the rostral ventral respiratory group (rVRG) of the medulla from their phrenic motor neuron (PhMN) targets, resulting in loss of diaphragm function. Using a rat model of cervical hemisection SCI, we aimed to restore rVRG-PhMN-diaphragm circuitry by stimulating regeneration of injured rVRG axons via targeted induction of Rheb (ras homolog enriched in brain), a signaling molecule that regulates neuronal-intrinsic axon growth potential. Following C2 hemisection, we performed intra-rVRG injection of an adeno-associated virus serotype-2 (AAV2) vector that drives expression of a constitutively-active form of Rheb (cRheb). rVRG neuron-specific cRheb expression robustly increased mTOR pathway activity within the transduced rVRG neuron population ipsilateral to the hemisection, as assessed by levels of phosphorylated ribosomal S6 kinase. By co-injecting our novel AAV2-mCherry/WGA anterograde/trans-synaptic axonal tracer into rVRG, we found that cRheb expression promoted regeneration of injured rVRG axons into the lesion site, while we observed no rVRG axon regrowth with AAV2-GFP control. AAV2-cRheb also significantly reduced rVRG axonal dieback within the intact spinal cord rostral to the lesion. However, cRheb expression did not promote any recovery of ipsilateral hemi-diaphragm function, as assessed by inspiratory electromyography (EMG) burst amplitudes. This lack of functional recovery was likely because regrowing rVRG fibers did not extend back into the caudal spinal cord to synaptically reinnervate PhMNs that we retrogradely-labeled with cholera toxin B from the ipsilateral hemi-diaphragm. Our findings demonstrate that enhancing neuronal-intrinsic axon growth capacity can promote regeneration of injured bulbospinal respiratory axons after SCI, but this strategy may need to be combined with other manipulations to achieve reconnection of damaged neural circuitry and ultimately recovery of diaphragm function.

Downregulation in the astroglial glutamate transporter EAAT2 in amyotrophic lateral sclerosis (ALS) patients and mutant SOD1 mouse models of ALS is believed to contribute to the death of motor neurons by excitotoxicity. We previously reported that caspase-3 cleaves EAAT2 at a unique cleavage consensus site located in its c-terminus domain, a proteolytic cleavage that also occurs in vivo in the mutant SOD1 mouse model of ALS and leads to accumulation of a sumoylated EAAT2 C-terminus fragment (CTE-SUMO1) beginning around onset of disease. CTE-SUMO1 accumulates in PML nuclear bodies of astrocytes and causes them to alter their mature phenotypes and secrete factors toxic to motor neurons. Here, we report that mutating the caspase-3 consensus site in the EAAT2 sequence with an aspartate to asparagine mutation (D504N), thereby inhibiting caspase-3 cleavage of EAAT2, confers protection to the SOD1-G93A mouse. EAAT2-D504N knock-in mutant mice were generated and crossed with SOD1-G93A mice to assess the in vivo pathogenic relevance for ALS symptoms of EAAT2 cleavage. The mutation did not affect normal EAAT2 function nor non-ALS mice. In agreement with the timing of CTE-SUMO1 accumulation, while onset of disease was not affected, the mutation caused an extension in progression time, a delay in the development of hindlimb and forelimb muscle weakness, and a significant increase in the lifespan of SOD1-G93A mice.

Different experimental and clinical strategies have been used to promote survival of transplanted embryonic ventral mesencephalic (VM) neurons. However, few studies have focused on the long-distance growth of dopaminergic axons from VM transplants. The aim of this study is to identify some of the growth and guidance factors that support directed long-distance growth of dopaminergic axons from VM transplants. Lentivirus encoding either glial cell line-derived neurotrophic factor (GDNF) or netrin-1, or a combination of lenti-GDNF with either lenti-GDNF family receptor α1 (GFRα-1) or lenti-netrin-1 was injected to form a gradient along the corpus callosum. Two weeks later, a piece of embryonic day 14 VM tissue was transplanted into the corpus callosum adjacent to the low end of the gradient. Results showed that tyrosine hydroxylase (TH(+)) axons grew a very short distance from the VM transplants in control groups, with few axons reaching the midline. In GDNF or netrin-1 expressing groups, more TH(+) axons grew out of transplants and reached the midline. Pathways co-expressing GDNF with either GFRα-1 or netrin-1 showed significantly increased axonal outgrowth. Interestingly, only the GDNF/netrin-1 combination resulted in the majority of axons reaching the distal target (80%), whereas along the GDNF/GFRα-1 pathway only 20% of the axons leaving the transplant reached the distal target. This technique of long-distance axon guidance may prove to be a useful strategy in reconstructing damaged neuronal circuits, such as the nigrostriatal pathway in Parkinson's disease.

We have recently reported that α2β1 and α1β1 isozymes of Na+/K+-ATPase (NKA) are localized in the caveolae whereas only the α1β1 isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α2 isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α1 isoform of NKA. To investigate the mechanism of regulation of α2 isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na+; and (iii) even after phosphorylation by PKC, PLM still remains associated with the α2 isoform of NKA.

Aims We sought to identify, purify and partially characterize a protein inhibitor of Na + /K + -ATPase in cytosol of pulmonary artery smooth muscle. Main methods (i) By spectrophotometric assay, we identified an inhibitor of Na + /K + -ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na + /K + -ATPase α 2 β 1 and α 1 β 1 isozymes for determining some characteristics of the inhibitor. Key findings We identified a novel endogenous protein inhibitor of Na + /K + -ATPase having an apparent mol mass of ∼ 70 kDa in the cytosolic fraction of the smooth muscle. The IC 50 value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the α 2 β 1 and α 1 β 1 isozymes of the Na + /K + -ATPase; (ii) it interacted reversibly to the E 1 site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na + /K + -ATPase exists as (αβ) 2 diprotomer. Significance The inhibitor binds to the Na + /K + -ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where α 2 is more sensitive than α 1 .

The Na+/K+-ATPase is a membrane bound P-type ATPase that exchanges Na+ and K+ ions across the plasma membrane in expense of ATP hydrolysis. It maintains for electrical excitability, neurotransmitter transport, volume regulation, and other vital cellular functions. The four isoforms of the Na+/K+-ATPase α-subunit have a cell-specific and developmentally regulated expression pattern. There are different isoforms of the Na+/K+-ATPase expressed in neurons and glial cells. Alterations of Na+/K+-ATPase activity due to global loss or isoform specific mutational effect are evident in different neurological disorders. Mutations in the ATP1A3 gene which encodes Na+/K+-ATPase α3 subunits, cause Rapid-Onset of Dystonia Parkinsonism (RDP), a rare movement disorder characterized by sudden onset of dystonic spasms and slowness of movement. It is evident that Na+/K+-ATPase α2 subunits are upregulated in spinal cord of sporadic and familial amyotrophic lateral sclerosis (ALS) patients. The α2-Na+/K+-ATPases are also enriched in astrocytes expressing mutant superoxide dismutase 1 (SOD1), which causes familial ALS. Here we focused to understand the physiological and molecular fundamentals associated with dysfunction of Na+/K+-ATPase in RDP and ALS.

Treatment of microsomes (preferably enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with the O2*- -generating system (hypoxanthine (HPX) plus xanthine oxidase (XO)), markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and also enhanced Ca2+ ATPase activity and ATP-dependent Ca2+ uptake. Pretreatment with superoxide dismutase (SOD) and tissue inhibitor of metalloproteinase (TIMP-2) (50 microg ml(-1)), preserved the increase in MMP-2 activity, Ca2+ ATPase activity and also ATP-dependent Ca2+ uptake in the microsomes. In contrast, Na+-dependent Ca2+ uptake in the microsomes was found to be inhibited by the O2*- - generating system. Additionally, O2*- -induced inhibition of Na+-dependent Ca2+ uptake was reversed by SOD and TIMP-2 (50 microg ml(-1)). Electron microscopy revealed that treatment with the O2*- -generating system did not cause any noticeable damage to the microsomes. O2*- -induced changes in MMP-2 activity, ATP-dependent Ca2+ uptake and Na+-dependent Ca2+ uptake, were not reversed upon pretreatment of the microsomes with a low dose (5 microg ml(-1)) of TIMP-2 which, on the contrary, reversed MMP-2 (1 microg ml(-1))-mediated alteration on these parameters. The inhibition of Na+-dependent Ca2+ uptake by O2*- and MMP-2, overpowered the stimulation of ATP-dependent Ca2+ uptake in the microsomes. Treatment of TIMP-2 (5 microg ml(-1)) with the O2*- -generating system abolished the inhibitory effect of TIMP-2 (5 microg ml(-1)) on MMP-2 (1 microg ml(-1)) (measured by (14)C-gelatin degradation). Overall, the present study suggests that O2*- inactivated TIMP-2, the ambient inhibitor of MMP-2, leading to activation of the ambient proteinase, MMP-2, which subsequently stimulated Ca2+ ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na+-dependent Ca2+ uptake, resulting in a marked decrease in Ca2+ uptake in the smooth muscle microsomes.

The cytoplasmic Ca2+clearance rate affects neuronal excitability, plasticity, and synaptic transmission. Here, we examined the modulation of the plasma membrane Ca2+ATPase (PMCA) by tyrosine kinases. In rat sensory neurons grown in culture, the PMCA was under tonic inhibition by a member of the Src family of tyrosine kinases (SFKs). Ca2+clearance accelerated in the presence of selective tyrosine kinase inhibitors. Tonic inhibition of the PMCA was attenuated in cells expressing a dominant-negative construct or shRNA directed to message for the SFKs Lck or Fyn, but not Src. SFKs did not appear to phosphorylate the PMCA directly but instead activated focal adhesion kinase (FAK). Expression of constitutively active FAK enhanced and dominant-negative or shRNA knockdown of FAK attenuated tonic inhibition. Antisense knockdown of PMCA isoform 4 removed tonic inhibition of Ca2+clearance, indicating that FAK acts on PMCA4. The hyaluronan receptor CD44 activates SFK-FAK signaling cascades and is expressed in sensory neurons. Treating neurons with a CD44-blocking antibody or short hyaluronan oligosaccharides, which are produced during injury and displace macromolecular hyaluronan from CD44, attenuated tonic PMCA inhibition. Ca2+-activated K+channels mediate a slow afterhyperpolarization in sensory neurons that was inhibited by tyrosine kinase inhibitors and enhanced by knockdown of PMCA4. Thus, we describe a novel kinase cascade in sensory neurons that enables the extracellular matrix to alter Ca2+signals by modulating PMCA-mediated Ca2+clearance. This signaling pathway may influence the excitability of sensory neurons following injury.

We identified alpha(2), alpha(1), and beta(1) isoforms of Na(+)/K(+)-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C(12)E(8), whereas C(12)E(8) was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase elicited higher E1Na-E2 K transition compared with that of the C(12)E(8)- and Triton X-100-purified enzyme. The rate of Na(+) efflux in DHPC-DOPC-reconstituted isozyme was higher compared to the C(12)E(8)-DOPC- and Triton X100-DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase possessed more organized secondary structure compared to the C(12)E(8)- and Triton X-100-purified isozyme.

Aims We sought to determine the mechanisms of an increase in Ca 2+ level in caveolae vesicles in pulmonary smooth muscle plasma membrane during Na + /K + -ATPase inhibition by ouabain. Main methods The caveolae vesicles isolated by density gradient centrifugation were characterized by electron microscopic and immunologic studies and determined ouabain induced increase in Na + and Ca 2+ levels in the vesicles with fluorescent probes, SBFI-AM and Fura2-AM, respectively. Key findings We identified the α 2 β 1 and α 1 β 1 isozymes of Na + /K + -ATPase in caveolae vesicles, and only the α 1 β 1 isozyme in noncaveolae fraction of the plasma membrane. The α 2 -isoform contributes solely to the enzyme inhibition in the caveolae vesicles at 40 nM ouabain. Methylisobutylamiloride (Na + /H + -exchange inhibitor) and tetrodotoxin (voltage-gated Na + -channel inhibitor) pretreatment prevented ouabain induced increase in Na + and Ca 2+ levels. Ouabain induced increase in Ca 2+ level was markedly, but not completely, inhibited by KB-R7943 (reverse-mode Na + /Ca 2+ -exchange inhibitor) and verapamil (L-type Ca 2+ -channel inhibitor). However, pretreatment with tetrodotoxin in conjunction with KB-R7943 and verapamil blunted ouabain induced increase in Ca 2+ level in the caveolae vesicles, indicating that apart from Na + /Ca + -exchanger and L-type Ca 2+ -channels, “slip-mode conductance” of Na + channels could also be involved in this scenario. Significance Inhibition of α 2 isoform of Na + /K + -ATPase by ouabain plays a crucial role in modulating the Ca 2+ influx regulatory components in the caveolae microdomain for marked increase in (Ca 2+ ) i in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.

Ca(2+) is a major intracellular messenger and nature has evolved multiple mechanisms to regulate free intracellular (Ca(2+))(i) level in situ. The Ca(2+) signal inducing contraction in cardiac muscle originates from two sources. Ca(2+) enters the cell through voltage dependent Ca(2+) channels. This Ca(2+) binds to and activates Ca(2+) release channels (ryanodine receptors) of the sarcoplasmic reticulum (SR) through a Ca(2+) induced Ca(2+) release (CICR) process. Entry of Ca(2+) with each contraction requires an equal amount of Ca(2+) extrusion within a single heartbeat to maintain Ca(2+) homeostasis and to ensure relaxation. Cardiac Ca(2+) extrusion mechanisms are mainly contributed by Na(+)/Ca(2+) exchanger and ATP dependent Ca(2+) pump (Ca(2+)-ATPase). These transport systems are important determinants of (Ca(2+))(i) level and cardiac contractility. Altered intracellular Ca(2+) handling importantly contributes to impaired contractility in heart failure. Chronic hyperactivity of the beta-adrenergic signaling pathway results in PKA-hyperphosphorylation of the cardiac RyR/intracellular Ca(2+) release channels. Numerous signaling molecules have been implicated in the development of hypertrophy and failure, including the beta-adrenergic receptor, protein kinase C, Gq, and the down stream effectors such as mitogen activated protein kinases pathways, and the Ca(2+) regulated phosphatase calcineurin. A number of signaling pathways have now been identified that may be key regulators of changes in myocardial structure and function in response to mutations in structural components of the cardiomyocytes. Myocardial structure and signal transduction are now merging into a common field of research that will lead to a more complete understanding of the molecular mechanisms that underlie heart diseases. Recent progress in molecular cardiology makes it possible to envision a new therapeutic approach to heart failure (HF), targeting key molecules involved in intracellular Ca(2+) handling such as RyR, SERCA2a, and PLN. Controlling these molecular functions by different agents have been found to be beneficial in some experimental conditions.