WO2015014666A1 - Methods and pharmaceutical compositions for the treatment of ventilator-induced diaphragmatic dysfunction - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of ventilator-induced diaphragmatic dysfunction. In particular, the present invention relates to agents capable of stabilizing the RyR1-calstabin1 interaction and thus capable of reducing SR Ca²⁺ leak via RyR1 channel for use in the treatment of ventilator-induced diaphragmatic dysfunction.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF VENTILATOR-INDUCED DIAPHRAGMATIC DYSFUNCTION

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of ventilator-induced diaphragmatic dysfunction.

BACKGROUND OF THE INVENTION:

The need for artificial respiratory support by mechanical ventilation (MV) is one of the most common reasons for admission to the intensive care unit (ICU). Although it is life saving in the short term, human and animal studies have previously shown that MV results in a progressive reduction in diaphragmatic force-generating capacity, together with diaphragm muscle fiber injury and atrophy. These findings comprise a condition termed Ventilator- Induced Diaphragmatic Dysfunction (VIDD) .VIDD, which is common in ICU settings,, can interfere with the ability to discontinue MV, with a major negative impact on subsequent patient outcomes and subsequently increased health care costs. The precise cellular pathways involved in MV-induced diaphragm weakness remain poorly understood. Animal models strongly suggest that oxidative stress plays a major role in VIDD. Moreover, recent studies have identified mitochondria as an essential source of reactive oxygen species (ROS) implicated in VIDD. Additionally, proteolytic systems such as caspases and calpains activation, which play significant roles in degrading cytoskeletal proteins in muscle and can also be activated by ROS, are suggested to be involved in the development of MV-induced diaphragm muscle fiber atrophy and injury (9, 20-22).

Although many of the processes implicated in VIDD have been associated with increased oxidative stress, the potential role of Ca²⁺ homeostasis disruption in these pathological changes has not been addressed. Maes et al. (Respiratory Research, 2010, 1 1 : 178) discloses that indirect evidence suggests that prolonged controlled mechanical ventilation (CMV) results in an increase in intracellular calcium levels in the diaphragm such that protection against CMV-induced increases in intracellular calcium levels and/or increasing calpastatin binding to calpain could be potential mechanisms by which corticosteroids prevent activation of the protease calpain and protect against VIDD. VIDD has not been known to be linked to excitation-contraction coupling, which refers to a succession of cellular events leading to muscle contraction. Briefly, in skeletal muscle membrane depolarization activates voltage-sensing Ca²⁺ channels in the transverse tubules that in turn activate RyRl, leading to sarcoplasmic reticulum (SR) Ca²⁺ release. The subsequent rise in cytoplasmic Ca²⁺ concentration triggers actin-myosin cross-bridge formation, sarcomere shortening and muscle contraction. RyRl is a homotetrameric macromolecular protein complex that includes four RyRl monomers (~565 kDa each), kinases, a phosphatase (PP1), a phosphodiesterase (PDE4D3), and calmodulin. In addition, the RyRl channel-stabilizing subunit calstabinl (Call or FK506 binding protein 12, FKBP12) is critical to the proper function of the channel. Maladaptive cAMP-dependent protein kinase A (PKA)-mediated phosphorylation and redox-dependent modifications (cysteine-nitrosylation and oxidation) of the RyRl have been linked to a loss of the normal association between calstabinl and the rest of the RyRl complex .. This in turn results in impaired Ca²⁺ handling and contractile dysfunction in conditions as diverse as heart failure, chronic muscle fatigue, muscular dystrophy and ageing.

Accordingly, there is a need for improved treatment of VIDD that this is now provided by the present invention.

SUMMARY OF THE INVENTION:

The present invention relates to an agent capable of stabilizing the RyRl -calstabinl interaction for use in a method for the treatment of ventilator-induced diaphragmatic dysfunction in a subject in need thereof. In particular, these agents are capable of stabilizing the RyRl -calstabinl interaction and thus capable of reducing SR Ca²⁺ leak via RyRl channel. These agents are generally known as Rycals. Specific compounds and general formulae covering the agents that are suitable for this invention are provided in the detailed description.

The present invention also relates to a method for screening a plurality of test substances useful for treatment of ventilator-induced diaphragmatic dysfunction comprising the steps consisting of (a) testing each of the test substances for its ability stabilize the RyRl- calstabinl interaction and/or to reduce SR Ca2+ leak via RyRl channel and (b) and positively selecting the test substances capable of restoring said integrated stress response.

Another embodiment of the invention is a method for the treatment of ventilator- induced diaphragmatic dysfunction in a subject in need thereof which comprises administering to the subject one of the agents disclosed herein that is capable of stabilizing RyRl -calstabinl interaction. The agent may be identified from the screening method disclosed herein. Yet another embodiment of the invention relates to the use of an agent capable of stabilizing RyRl-calstabinl interaction for the treatment of ventilator-induced diaphragmatic dysfunction in a subject in need thereof. The useful agents are those described herein as well as those identified from the screening method disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES:

Figures 1A to 1C show that VIDD is associated with defective RyRl in human diaphragm muscle, with Figure 1A including immunoblots of immunoprecipitated RyRl of human diaphragm samples collected from short term (control) and long term (MV) mechanically ventilated patients, Figure IB including bar graphs that show quantification of the immunoblots of Figure 1A, and Figure 1C including single channel traces of single RyRl incorporated in planar lipid bilayers.

Figures 2A to 2D show that VIDD is associated with defective RyRl in porcine diaphragm muscle, with Figure 1A providing graphs of diaphragmatic contractile function as a function of stimulation frequency; Figure 2B providing single channel traces of single RyRl incorporated in planar lipid bilayers, Figure 2C providing representative immunoblots of immunoprecipitated RyRl of pig diaphragm samples collected from control and mechanically ventilated groups after 72 hours of anesthesia, and Figure 2D providing bar graphs that show quantification of the immunoblots relative to total RyRl immunoprecipitated.

Figure 3 illustrates that VIDD is associated with early impairment of RyRl in murine diaphragm muscle, with Figure 3 A providing representative records of specific diaphragmatic force production measured ex vivo in muscle bundles under isometric conditions in control and mechanically ventilated (MV) mice, Figure 3B showing averaged force - frequency relationships recorded in control and MV mice, Figure 3C providing representative immunoblots of immunoprecipitated RyRl, and Figure 3D providing bar graphs showing the quantification of immunoblots relative to total RyRl immunoprecipitated of murine diaphragm samples.

Figures 4A to 4E show that RyRl stabilization prevents VIDD, with Figure 4A, with Figure 4A providing representative records of specific diaphragmatic force production measured ex vivo at 1, 30 and 100Hz in muscle under isometric conditions in wild-type mice, Figure 4B providing averaged specific force-frequency relationships recorded in various wild-type MV, Figure 4C providing a comparison of mean Ca²⁺ sparks frequency between MV and MV 107, Figure 4D providing representative records of specific diaphragmatic force production in calstabinl knockout mice, and Figure 4E providing averaged specific force- frequency relationships recorded in various mice.

Figure 5Aprovides a graph that illustrates RyRl open probability in human and pig diaphragm muscles in control condition and after MV.

Figure 5B provides bar graphs that show that Ventilation in Continuous Positive Airway Pressure (Cpap) mode does not affect RyRl remodeling.

Figure 5C provides graphs that illustrate that Ventilation in Continuous Positive Airway Pressure (Cpap) mode does not affect RyRl function. Figure 5C provides graphs that show that force frequency relationships of control and call-/- diaphragm muscle in resting condition.

DETAILED DESCRIPTION OF THE INVENTION:

Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure, but the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here the inventors show in human subjects and animal models of VIDD (pigs and mice) that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) calcium release channel/ryanodine receptor (RyRl) in the diaphragm. The RyRl macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated and depleted of the stabilizing subunit calstabinl, following MV. These post-translational modifications of RyRl were mediated by oxidative stress and resulted in abnormal resting SR Ca²⁺ leak and reduced contractile function. Treatment with SI 07, a small molecule drug that stabilizes the RyRl -calstabinl interaction despite these RyRl complex modifications effectively prevented VIDD. Diaphragm dysfunction is common in MV patients and is a major cause of failure to wean patients from artificial respiratory support. This study provides the first evidence of RyRl alterations as a proximal mechanism underlying VIDD and a potential therapeutic target. Accordingly, the present invention relates to a method for the treatment of ventilator- induced diaphragmatic dysfunction in a subject in need thereof comprising by administering to the subject an agent capable of stabilizing the RyRl-calstabinl interaction and thus capable of reducing SR Ca²⁺ leak via RyRl channel.

In some embodiments, the subject needs artificial respiratory support because he suffers from respiratory failure and/or heart failure which can be aggravated by sepsis, metabolic disorder, neuromuscular diseases, or surgery along with post-surgical recovery. Typically, the subject suffers from a disease for which the worsening of the symptoms has led the subject to need artificial respiratory support (i.e. mechanical ventilation). For example, some lung diseases, such as Chronic Obstructive Pulmonary Disease (COPD), pneumonia, sepsis (including severe sepsis and septic shock), Acute Respiratory Distress Syndrome (ARDS), Severe Acute Respiratory Syndrome (SARS) and cystic fibrosis (CF) usually require some form of ventilation assistance in order to clinically improve the subject. In some embodiments the subject suffers from a trauma. Pulmonary dysfunction in trauma patients is multifactorial and may be the result of direct contusion of the lung tissue, lung injury by fractured ribs, loss of chest wall function, fat embolism to the lung from long bone fractures, aspiration of blood or gastric contents and the consequences of the activation of the systemic inflammatory response syndrome (SIRS) of shock, reperfusion, and transfusion therapy.

As used herein, the expression "ventilator-induced diaphragmatic dysfunction" or "VIDD" has its general meaning in the art and refers to the condition wherein diaphragmatic atrophy and contractile dysfunction occur after prolonged controlled mechanical ventilation (Powers SK, Wiggs MP, Sollanek KJ, Smuder AJ. Invited Review: Ventilator-induced diaphragm dysfunction: cause and effect. Am J Physiol Regul Integr Comp Physiol. 2013 Jul 10.). Ventilator-induced diaphragmatic dysfunction may result from prolonged controlled mechanical ventilation (MV), e.g., greater than 12 hours. However, such prolonged MV is not limited to any specific time-length. For example, in some embodiments, prolonged MV includes a time from at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, or 100 hours, to from at least about 1, 10, 20, 50, 75, 100 or greater hours, days, or years. In another embodiment, prolonged MV includes a time from at least about 5, 6, 7, 8, 9 or 10 hours, to from at least about 10, 20 or 50 hours. In some embodiments, prolonged MV is from about at least 10-12 hours to any time disclosed herein that is greater than the 10-12 hour period. As used herein, the terms "treating" or "treatment" or "alleviation" refers to therapeutic treatment, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. A subject is successfully "treated" for ventilator-induced diaphragmatic dysfunction, if after receiving a therapeutic amount of the agent according to the invention, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of ventilator-induced diaphragmatic dysfunction, such as, e.g., a significant reduction in diaphragmatic contractile function, or a decrease of diaphragm atrophy evaluated by CT scan or ultra sound. In a particular embodiment the treatment is a prophylactic treatment. The term "prophylactic treatment" as used herein, refers to any medical or public health procedure whose purpose is to prevent a disease. As used herein, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk of acquiring or developing a given condition, or the reduction or inhibition of the recurrence or said condition in a subject who is not ill, but who has been or may be near a subject with the disease. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean "substantial," which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.

In some embodiments, the agent is administered before MV, immediately after MV initiation, during MV, and/or immediately after MV. In some embodiments, administration of the agent according to the invention is provided at any time during MV.

The agent according to the invention is also suitable for preventing risks associated with ventilator-induced diaphragmatic dysfunction. Risks associated with ventilator dependence include increased discomfort and risk of secondary diseases for the patient (such as pneumonia, pulmonary fibrosis, aspiration, acute renal failure, cardiac arrhythmias, sepsis, vocal fold dysfunction, and acute lung injury secondary to barotrauma or volotrauma), increased morbidity and mortality, high health care costs, and longer treatment duration times. Although patients with chronic ventilator dependency (CVD) comprise only 5% to 10% of patients in intensive care units, they consume approximately 50%> of all ICU resources, as measured in staff time and equipment usage. Specifically, it has been estimated that weaning patients consumed about 41% of total ventilation time in intensive care unit patients. The economic cost of long term MV dependence is enormous. Episodes of long term MV dependency can financially devastate families and health care institutions and are a financial drain on private insurers and government health care resources.

Typically the agents of the invention are named Rycals which are well known in the art (e.g. Andersson DC, Marks AR. Fixing ryanodine receptor Ca leak - a novel therapeutic strategy for contractile failure in heart and skeletal muscle. Drug Discov Today Dis Mech. 2010 Summer;7(2):el51-el57.; Marks AR. Calcium cycling proteins and heart failure: mechanisms and therapeutics. J Clin Invest. 2013 Jan 2;123(l):46-52. doi: 10.1 172/JCI62834. Epub 2013 Jan 2. Review.).

One example of Rycal is S36 which has the formula I:

In certain embodiments, the Rycal is selected from the group consisting of compounds of the general Formula II:

wherein,

- T is O, CH2, NH, or S=(0₂)_n;

- n is 0, 1, or 2;

q is 0, 1, 2, 3, or 4;

each R is independently selected from the group consisting of H, halogen, -OH,

-NH₂, -N0₂, -CN, -CF₃, -OCF₃, -N₃, -S0₃H, -S(=0)₂alkyl, -S(=0)alkyl, -OS(=0)₂CF₃, acyl, -O-acyl, alkyl, alkoxyl, alkylamino, alkylarylamino, alkylthio, cycloalkyl, alkylaryl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, -O- acyl, alkyl, alkoxyl, alkylamino, alkylarylamino, alkylthio, cycloalkyl, alkylaryl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-) arylthio, and (hetero-)arylamino may be optionally substituted;

Ri is selected from the group consisting of H, oxo, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted; R₂ is selected from the group consisting of H, -C(=0)R₅, -C(=S)R6, -S0₂R₇, -P(=0)RsR₉, -(CH₂)m-Rio, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl; wherein each alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl may be optionally substituted and wherein m is 0, 1, 2, 3, or 4;

R₃ is selected from the group consisting of H, -C0₂Y, -C(=0)NHY, acyl, -O-acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted; and wherein Y is selected from the group consisting of H, alkyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl, and wherein each alkyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted;

R4 is selected from the group consisting of H, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted; R₅ is selected from the group consisting of -NR15R16, -(CH₂)zNRi₅Ri6, -NHNR15R16, -NHOH, -ORis, -C(=0)NHNRi₅Ri6, -CO2R15,

-CH₂X, acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted, and wherein z is 1, 2, 3, 4, 5, or 6;

5 is selected from the group consisting of -OR15, -NHNR15R16, -NHOH, -NR15R16, -CH₂X, acyl, alkenyl, alkyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;

R₇ is selected from the group consisting of -OR15, -NR15R16, -NHNR15R16, -NHOH, alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;

Rs and R9 independently are selected from the group consisting of OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;

Rio is selected from the group consisting of -NR15R16, OH, -SO2R11, -NHSO2R11, C(=0)(Ri₂), NHC=0(Ri₂), -OC=0(Ri₂), and -P(=0)Ri₃Ri₄;

R11 , Ri₂, Ri₃, and Ri₄ independently are selected from the group consisting of H, OH, NH₂, -NHNH₂, -NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted; X is selected from the group consisting of halogen, -CN, -C0₂Ri₅, -C(=0)NRi₅Ri₆, -NR15R16, -OR15, -S0₂R₇ and -P(=0)R₈R₉ and

Ri5 and Ri₆ independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, OH, NH₂, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted; and optionally R15 and Ri₆ together with the N to which they are bonded may form a heterocycle which may be substituted; the nitrogen in the benzothiazepine ring may optionally be a quaternary nitrogen;

and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes, and prodrugs thereof, or any combination thereof.

The term "alkyl" as used herein refers to a linear or branched, saturated hydrocarbon and preferably one having from 1 to 6 carbon atoms. Representative alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, and neohexyl. The term "Ci-C₄ alkyl" refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, and isobutyl.

The term "alkenyl" as used herein refers to a linear or branched hydrocarbon and preferably one having from 2 to 6 carbon atoms and having at least one carbon-carbon double bond. In one embodiment, the alkenyl has one or two double bonds. The alkenyl moiety may exist in the E or Z conformation and the compounds of the present invention include both conformations. The term "alkynyl" as used herein refers to a linear or branched hydrocarbon and preferably one having from 2 to 6 carbon atoms and having at least one carbon-carbon triple bond.

The term "aryl" as used herein refers to an aromatic group and preferably one containing 1 to 3 aromatic rings, either fused or linked.

The term "cyclic group" as used herein includes a cycloalkyl group and a heterocyclic group. The term "cycloalkyl group" as used herein refers to a three- to seven-membered saturated or partially unsaturated carbon ring. Any suitable ring position of the cycloalkyl group may be covalently linked to the defined chemical structure. Non- limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The term "halogen" as used herein refers to fluorine, chlorine, bromine, and iodine.

The term "heterocyclic group" or "heterocyclic" or "heterocyclyl" or "heterocyclo" as used herein refers to fully saturated, or partially or fully unsaturated, including aromatic (i.e., "heteroaryl") cyclic groups (for example, 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 16 membered tricyclic ring systems) which have at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3, or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heterocyclic group may be attached to the remainder of the molecule at any heteroatom or carbon atom of the ring or ring system. Non-limiting examples of heterocyclic groups include, but are not limited to, azepanyl, azetidinyl, aziridinyl, dioxolanyl, furanyl, furazanyl, homo piperazinyl, imidazolidinyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, piperazinyl, piperidinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiomorpholinyl, thiophenyl, triazinyl, and triazolyl. Non- limiting examples of bicyclic heterocyclic groups include indolyl, isoindolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, benzo furazanyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] or furo[2,3- b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo- quinazolinyl), triazinylazepinyl, tetrahydroquinolinyl and the like. Non-limiting examples of tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the like.

The term "phenyl" as used herein refers to a substituted or unsubstituted phenyl group.

The aforementioned terms "alkyl," "alkenyl," "alkynyl," "aryl," "acyl," "phenyl," "cyclic group," "cycloalkyl," "heterocyclyl," "heterocyclo," and "heterocycle" may further be optionally substituted with one or more substituents. Non- limiting examples of substituents include but are not limited to one or more of the following groups: hydrogen, halogen, CF₃, OCF₃, cyano, nitro, N₃, oxo, cycloalkyl, alkenyl, alkynyl, heterocycle, aryl, alkylaryl, heteroaryl, OR_*, SR_*,

NR_bR_c, NR_bS(=0)₂Re, NR_bP(=0)2Re, S(=0)2 NR_bRc,

C(=0)ORa, C(=0)Ra, C(=0)NR_bR_c, OC(=0)Ra, OC(=0)NR_bR_c, NR_bC(=0)ORa, NR_dC(=0)NR_bR_c,

wherein Ra is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, alkylaryl, heteroaryl, heterocycle, or aryl; R_b, R_c and R_d are independently hydrogen, alkyl, cycloalkyl, alkylaryl, heteroaryl, heterocycle, aryl, or said R_b and R_c together with the N to which they are bonded optionally form a heterocycle; and R_e is alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylaryl, heteroaryl, heterocycle, or aryl. In the aforementioned representative substitutents, groups such as alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, alkylaryl, heteroaryl, heterocycle and aryl can themselves be optionally substituted.

The term "quaternary nitrogen" refers to a tetravalent positively charged nitrogen atom including, for example, the positively charged nitrogen in a tetraalkylammonium group (e.g., tetramethylammonium, N-methylpyridinium), the positively charged nitrogen in protonated ammonium species (e.g., trimethyl-hydroammonium, N-hydropyridinium), the positively charged nitrogen in amine N-oxides (e.g., N-methyl-morpholine-N-oxide, pyridine-N-oxide), and the positively charged nitrogen in an N-amino-ammonium group (e.g., N- aminopyridinium) .

In other embodiments, the Rycal is selected form the group consisting of compounds of formula Il-k or II-k-1 :

wherein:

- R, R' and R" are independently selected from the group consisting of H, halogen, -OH, -NH₂, -NO₂, -CN, -CF₃, -OCF₃, -N₃, -S0₃H, -S(=0)₂alkyl, -S(=0)alkyl, -OS(=0)₂CF₃, acyl, alkyl, alkoxyl, alkylamino, alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be substituted or unsubstituted; - Ris is selected from the group consisting of H, -NR15R16, -C(=0)NR₁₅R₁₆, -(C=0)ORi₅, -OR15, alkyl, aryl, cycloalkyl, heterocyclyl, and at one labeling group; wherein each alkyl, aryl, cycloalkyl, and heterocyclyl may be substituted or unsubstituted;

and further wherein:

- q is 0, 1, 2, 3, or 4;

- p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

- n is 0, 1, or 2;

-and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes and prodrugs thereof.

In certain embodiments, the present invention provides compounds of formula Il-k, wherein each R is independently selected from the group consisting of H, halogen, -OH, -OMe, -NH₂, -NO2, -CN, -CF₃, -OCF₃, -N₃, -S(=0)₂Ci-C₄alkyl, -S(=0)Ci-C₄alkyl, -S-Ci-C₄alkyl, -OS(=0)₂CF₃, Ph, -NHCH₂Ph, -C(=0)Me, -OC(=0)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In some cases, R is H or is OMe at position 7 of the benzothiazepine ring.

In certain embodiments, the present invention provides compounds of formula II-k-1, wherein R' and R" are independently selected from the group consisting of H, halogen, -OH, -OMe, -NH₂, -N0₂, -CN, -CF₃, -OCF₃, -N₃, -S(=0)₂Ci-C₄alkyl, -S(=0)Ci-C₄alkyl, -S-Ci-C₄alkyl, -OS(=0)₂CF₃, Ph, -NHCH₂Ph, -C(=0)Me, -OC(=0)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In some cases, R' is H or OMe, and R" is H.

In other embodiments, the present invention provides compounds of formula Il-k or II-k-1, wherein Ris is selected from the group consisting of H, -NR15R16,

-OR15, alkyl, aryl, and at one labeling group; and wherein each alkyl and aryl may be substituted or unsubstituted. In some cases, m is 1, and Ris is Ph, C(=0)OMe, C(=0)OH, aminoalkyl, NH₂, NHOH, or NHCbz. In other cases, m is 0, and Ris is C1-C4 alkyl, such as Me, Et, propyl, and butyl. In yet other cases, m is 2, and Ris is pyrrolidine, piperidine, piperazine, or morpholine. In some embodiments, m is 3, 4, 5, 5, 7, or 8, and Ris is a fluorescent labeling group selected from bodipy, dansyl, fluorescein, rhodamine, Texas red, cyanine dyes, pyrene, coumarins, Cascade Blue™, Pacific Blue, Marina Blue, Oregon Green, 4',6-Diamidino-2-phenylindole (DAPI), indopyra dyes, lucifer yellow, propidium iodide, porphyrins, arginine, and variants and derivatives thereof. In certain other embodiments the invention provides compounds of Formula II-o:

wherein:

Re is -(Ci-C₆ alkyl)-phenyl, -(Ci-C₆ alkyl)-C(0)Rb, or substituted or unsubstituted -Ci-C₆ alkyl; and

R_b is -OH or -0-(Ci-C₆ alkyl), and

and wherein the phenyl or substituted alkyl is substituted with one or more of halogen, hydroxyl, -Ci-C₆ alkyl, -0-(Ci-C₆ alkyl), -NH₂, -NH(Ci-C₆ alkyl), -N(Ci-C₆ alkyl)₂, cyano, or dioxolane.

In some embodiments, the Rycal of Formula II, Il-k, II-k-1 or II-o is selected from the group consisting of SI, S2, S3, S4, S5, S6, S7, S9, Sl l, S12, S13, S14, S19, S20, S22, S23, S24, S25, S26, S27, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S68, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, S100, S101, S102, S103, S104, S107, S108, S109, S110, Si l l, S112, S113, S114, S115, S116, S117, S118, S119, S120, S121, S122, S123, S136, S137, S138, S139, S140, S146, S147, S148, S149, S150, S151, S152, S153, S156, S157, S159, S160, S161, S166, S167, S182, S186, S189, S203, S217, S251, S252, S258, S277, S279, S282, S291, S293, S296, S301, S302, S306, S311, S312, S313, S318, S322, S324, S326, S331, S335, S337, S351, S352, S353, S354, S397, S398, S399, S423, S454, S463, S466, S470, S473, S477 and salts thereof. The structures of these agents are provided in the Detailed Description of the International Patent Application WO2012/037105 and as follows:.

Sft3

Representative compounds of Formula II-o include without limitation SI 07, SI 10, 120, and S121. In a particular embodiment, the Rycal is S107 having the formula III:

In certain embodiments, the Rycal is selected from the group of compounds of the general Formula IV:

(IV)

wherein

- n is 0, 1 , 2, 3, or 4;

- X is O, -NR₅ or -C(R₅)₂;

each R is independently selected from the group consisting of Z, R₅, -OR₅, -SR₅, -N(R₅)₂, -NR₅C(=0)OR₅, -C(=0)N(R₅)₂, -C(=0)OR₅, -C(=0)R₅, -OC(=0)R₅, N0₂, CN, -CZ₃, OCZ₃, -N₃, and -P(=0)R₈R₉;

Ri and R₃ are each independently selected from the group consisting of oxo, R₅, -CH₂OR₅, -CH₂OC(=0)R₆, -C(=0)OR₅, -C(=0)NHR₅, -C(=0)R₅, and -OC(=0)R₅;

R₂ is selected from the group consisting of R₅, -(C=0)R6, -(C=S)R6, and (CH₂)_mRio, wherein m is 1 , 2, 3, 4, 5, or 6; or

Rl and R₂ together with the carbon and nitrogen to which they are respectively attached, form an unsubstituted or substituted heterocycle; or

R₂ and R₃ together with the nitrogen and carbon to which they are respectively attached, form an unsubstituted or substituted heterocycle other than a piperazine; or

R₃ and R₄ together with the carbon atoms to which they are respectively attached, form an unsubstituted or substituted cycloalkyl or heterocyclic ring; or R₄ is selected from the group consisting of R₅ and oxo; each R₅ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, alkylaryl, and alkylheteroaryl;

R6 is selected from the group consisting of R5, -(CH₂)bNRi3Ri4, -NR5OR5, -OR5, -C(0)OR₅, -C(=0)NRi₃Ri4, -(CH₂ )_CY, and -C(=0)R₅, wherein b is 0, 1, 2, 3, 4, 5, or 6 and c is 1, 2, 3, 4 or 5;

Rio is selected from the group consisting of R₅, -OR₅, -S0₂Rn, -C(=0)Ri₂, -NH(C=0)Ri2, -0(C=0)Ri2, and -P(=0)R₈R₉;

Rs, R9, R_{1 1} and Ri₂ are independently selected from the group consisting of R₅, -OR₅, and -N(R₅)₂;

- Y is selected from the group consisting of Z, -C0₂R₅, -C(=0)NRi₃Ri4, and -OR5;

Z is a halogen selected from F, CI, Br and I;

Ri₃ and R₁₄ are independently selected from the group consisting of R₅, or Ri₃ and Ri₄ together with the N to which they are bonded may form an unsubstituted or substituted heterocycle; and

wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, alkylaryl, and alkylheteroaryl may be substituted or unsubstituted;

wherein the nitrogen in the benzoxazepine ring may optionally be a quaternary nitrogen;

and all enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes, polymorphs, metabolites, and prodrugs thereof;

In some embodiments, the Rycal of Formula IV is selected from the group consisting of ARM136, ARM137, ARM138, ARM139, ARM140, ARM146, ARM147, ARM148, ARM149, ARM150, ARM151, ARM152, ARM153, ARM156, ARM157, ARM159, ARM160, ARM161, ARM166, ARM167, ARM182, ARM186, ARM187, ARM189, ARM 200, ARM203, ARM 205, ARM217, ARM251, ARM252, ARM258, ARM277, ARM279, ARM282, ARM291, ARM293, ARM296, ARM301, ARM302, ARM306, ARM311, ARM312, ARM313, ARM318, ARM322, ARM324, ARM326, ARM331, ARM335, ARM337, ARM351, ARM352, ARM353, ARM354, ARM397, ARM398, ARM399, ARM423, ARM454, ARM463, ARM466, ARM470, ARM473 and ARM477. The structures of these agents are provided in the Detailed Description of the International Patent Application WO2008/144483 and as follows:

ARM 182

ARM 186

ARM 187

ARM 189

ARM200

ARM203

ARM.205

ARM217

ARM251

ARM252

ARM282

ARM291

ARM293

AR 296

ARM301

ARM302

ARM306

ARM311

ARM313

ARM318

ARM322

ARM324

ARM326

ARM331

ARM337

O

ARM.351 "tr

ARM352

ARM353

ARM354

ARM397

ARM398

ARM399

ARM423

ARJ 477 Typically, the agent according to the invention is administered to the subject in a therapeutically effective amount. As used herein, the terms "effective amount" or "therapeutically effective amount" or "pharmaceutically effective amount" refer to a quantity sufficient to achieve a desired therapeutic (including prophylactic effect), e.g., an amount which results in the prevention of, or a decrease in, ventilator-induced diaphragmatic dysfunction, or symptoms associated therewith. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. A suitable amount of the agents of the invention effective in the subject ranges from about 0.01 mg/kg/day to about 20 mg/kg/day, and/or is an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml. Alternatively, the amount of compounds from the invention ranges from about 10 mg/kg/day to about 20 mg/kg/day. Also included are amounts of from about 0.01 mg/kg/day or 0.05 mg/kg/day to about 5 mg/kg/day or about 10 mg/kg/day which can be administered. The agents of the invention are formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.

Accordingly, a further aspect of the invention relates a pharmaceutical composition comprising at least one agent of the invention in admixture with a pharmaceutically acceptable diluent and/or carrier.

The pharmaceutically-acceptable carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. The pharmaceutically-acceptable carrier employed herein is selected from various organic or inorganic materials that are used as materials for pharmaceutical compositions and which are incorporated as analgesic agents, buffers, binders, disintegrants, diluents, emulsifiers, excipients, extenders, gellants, glidants, skin-penetration enhancers, solubilizers, stabilizers, suspending agents, tonicity agents, vehicles and viscosity-increasing agents. If necessary, pharmaceutical additives, such as antioxidants, aromatics, colorants, flavor- improving agents, preservatives, and sweeteners, are also added. Examples of acceptable pharmaceutical carriers include carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc and water, among others.

The pharmaceutical compositions of the present invention are prepared by methods well-known in the pharmaceutical arts. For example, the agents of the invention are brought into association with a carrier and/or diluent, as a suspension or solution. Optionally, one or more accessory ingredients (e.g., buffers, flavoring agents, surface active agents, and the like) also are added. The choice of carrier is determined by the solubility and chemical nature of the agent, chosen route of administration and standard pharmaceutical practice.

Additionally, the compounds of the present invention are administered to the subject by known procedures including, without limitation, oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, via inhalation or intranasally, vaginally, rectally, and intramuscularly. The agents of the invention are administered parenterally, by epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, subcutaneous or sublingual injection, or by way of catheter.

For oral administration, a formulation of the agents of the invention may be presented as capsules, tablets, powders, granules, or as a suspension or solution. The formulation has conventional additives, such as lactose, mannitol, cornstarch or potato starch. The formulation also is presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, cornstarch or gelatins. Additionally, the formulation is presented with disintegrators, such as cornstarch, potato starch or sodium carboxymethylcellulose. The formulation also is presented with dibasic calcium phosphate anhydrous or sodium starch glycolate. Finally, the formulation is presented with lubricants, such as talc or magnesium stearate.

For parenteral administration (i.e., administration by injection through a route other than the alimentary canal), the agents of the invention are combined with a sterile aqueous solution that is isotonic with the blood of the subject. Such a formulation is prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile. The formulation is presented in unit or multi-dose containers, such as sealed ampoules or vials. The formulation is delivered by any mode of injection, including, without limitation, epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, subcutaneous, or sublingual or by way of catheter into the subject's heart. For transdermal administration, the agents of the invention are combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, JV-methylpyrrolidone and the like, which increase the permeability of the skin to the agents of the invention and permit the compounds to penetrate through the skin and into the bloodstream. The compound/enhancer compositions also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which are dissolved in a solvent, such as methylene chloride, evaporated to the desired viscosity and then applied to backing material to provide a patch.

The composition may be provided in unit dose form such as a tablet, capsule or single- dose vial. Suitable unit doses, i.e., therapeutically effective amounts, can be determined during clinical trials designed appropriately for each of the conditions for which administration of a chosen compound is indicated and will, of course, vary depending on the desired clinical endpoint. The present invention also provides articles of manufacture for treating and preventing disorders, such as cardiac disorders, in a subject. The articles of manufacture comprise a pharmaceutical composition of one or more of the agents of the invention.

A further aspect of the invention relates to a method for screening a plurality of test substances useful for treatment of ventilator-induced diaphragmatic dysfunction comprising the steps consisting of (a) testing each of the test substances for its ability stabilize the RyRl- calstabinl interaction and/or to reduce SR Ca²⁺ leak via RyRl channel and (b) and positively selecting the test substances capable of restoring said integrated stress response.

The screening method of the invention is implemented by any assay well known in the art. Examples of suitable assays and screening methods that may be used to identify new compounds that possess these properties such that they may be useful for the treatment of VIDD are described in the International Patent Publication WO 2008/064264 ("In vivo methods for identifying and screening compounds that modulate calstabin binding to a ryanodine receptor"), WO 2008/140592 ("Radio actively labeled 1 ,4-benzothiazepines and methods of screening for compounds that bind ryanodine receptors") and WO 2004/080283 ("Type 1 ryanodine receptor-based methods"), the entire contents of each of which are hereby incorporated by reference. The selected test substances indirectly decrease the open probability of RyRl when examined under conditions that simulate diastole, by inhibiting the depletion of the stabilizing subunit calstabinl from the RyRl complex and thereby stabilizing the closed state of the channel, particularly PKA-phosphorylated, and/or nityrosylated, and/or oxidized RyRl, and thereby decrease the Ca²⁺ current through such channels under resting conditions when muscles are relaxed. The selected test substance may exert their effects, at least in part, by increasing the affinity with which calstabinl proteins bind to RyRl, and/or by inhibiting a decrease in binding of calstabinl to RyRl, and/or by inhibiting dissociation of calstabinl from RyRl , particularly PKA phosphorylated RyRl .

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

DETAILED DESCRIPTION OF THE FIGURES:

Figures 1A to 1C. VIDD is associated with defective RyRl in human diaphragm muscle. Representative immunoblots (A) of immunoprecipitated RyRl of human diaphragm samples collected from short term (control) and long term (MV) mechanically ventilated patients (see table 5A). (Each blot corresponds to adjacent wells of the same gel). Bar graphs (B) show quantification of immunoblots, relative to total RyRl immunoprecipitated (mean ± SEM, n = 10 and 9 in control and MV respectively, * p<0.05, MV vs control). CysNO, thio- nitrosylation; DNP, 2,4-dinitrophenylhydrazone; P*RyRl, phosphorylated RyRl (at serine 2844). C) Single channel traces of single RyRl incorporated in planar lipid bilayers, corresponding to representative experiments performed with human diaphragm biopsies from control and MV groups. VIDD increases RyRl open probability (P₀), Mean Po was 0.00062 ± 0.00035 in control (ranging from 0 to 0.00635, n=18). After MV, the channels had a higher Po value of 0.01745 ± 0.01259 (ranging from 0 to 0.28482, n=23) but not significantly different from the control (see Fig. 5A). The lack of significance was due to a large variability in MV sample. 78% of the channels had a Po in the same range as the control channels and 22% had a Po significantly higher (0.07982 ± 0.05259). Figures 2 A to 2D. VIDD is associated with defective RyRl in porcine diaphragm muscle.

A) Diaphragmatic contractile function was assessed by in vivo measurements of transdiaphragmatic pressure (Pdi) at t=0 (baseline) and t=72 (72 hours of anesthesia) in control (Control O and control_72) and mechanically ventilated pigs (MV 0 and MV 72), produced by supramaximal stimulation and expressed as a function of stimulation frequency. (*, p<0.05, MV 72 vs. MV 0). B) Single channel traces of single RyRl incorporated in planar lipid bilayers, corresponding to representative experiments performed with porcine diaphragm biopsies from control and MV groups after 72 hours of anesthesia. As for human samples, VIDD increases RyRl open probability (P₀) in pigs. Mean Po was 0.00115±0.00069 (n=12) in control (ranging from 0.00001 to 0.00827). After MV, the channels had a higher Po value of 0.00839±0.00582 (n=13) (ranging from 0.00001 to 0.06789) but not significantly different from the control (see Fig 5 A). The lack of significance was due to a large variability in MV sample. 16% had a Po significantly higher (0.05394 ± 0.01396). This was never observed in control condition and reflected RyR leaky behavior induced by MV in pigs VIDD as in human. C) Representative immunoblots of immunoprecipitated RyRl of pig diaphragm samples collected from control and mechanically ventilated (MV) groups after 72 hours of anesthesia (each blot corresponds to adjacent wells of the same gel). Bar graphs (D) show quantification of immunoblots relative to total RyRl immunoprecipitated (mean ± SEM, n = 5 in both groups, * p<0.05, MV vs. control). CysNO, thio-nitrosylation ; DNP, 2,4- dinitrophenylhydrazone; P*RyRl, phosphorylated RyRl (at serine 2844). In panels B, C and D Control and MV refer to samples collected at t=72. Data are expressed as mean ± SEM.

Figures 3A to 3F. VIDD is associated with early impairment of RyRl in murine diaphragm muscle.

A) Representative records of specific diaphragmatic force production measured ex vivo at 1, 30 and 100Hz in muscle bundles under isometric conditions in control and mechanically ventilated (MV) mice. B) Averaged force - frequency relationships recorded in control (n=8), MV (n=10) and MV treated with trolox (n=8) groups. Representative immunoblots (each blot corresponds to adjacent wells of the same gel) of immunoprecipitated RyRl (C) and bar graphs (D) showing the quantification of immunoblots relative to total RyRl immunoprecipitated of murine diaphragm samples collected from control, MV, and MV-trolox groups. CysNO, thio-nitrosylation ; DNP, 2,4-dinitrophenylhydrazone; P*RyRl, phosphorylated RyRl (at serine 2844). Spontaneous SR Ca²⁺ release events were recorded in fluo-4-loaded permeabilized diaphragm fibers by laser scanning confocal microscopy. E) Representative AF/F line- scan images (1.54 ms per line) recorded in control, after MV and after MV with trolox treatment. F) Mean Ca²⁺ sparks frequency is used as an index of resting SR Ca²⁺ leak. Results are expressed as mean ± SEM (*, p<0.05, MV vs. control; #, p<0.05, MV-trolox vs. MV).

Figures 4A to 4F. RyRl stabilization prevents VIDD.

A) Representative records of specific diaphragmatic force production measured ex vivo at 1, 30 and 100Hz in muscle under isometric conditions in wild-type mice and B) Averaged specific force-frequency relationships recorded in wild-type MV (MV, n=10) and wild-type MV treated with S 107 (MV_S 107, n=5). Spontaneous SR Ca²⁺ release events (Ca²⁺ sparks) were recorded in fluo-4-loaded permeabilized diaphragm fibers by laser scanning confocal microscopy. C) Comparison of mean Ca²⁺ sparks frequency between MV and MV_107, used as an index of resting SR Ca²⁺ leak. Results are expressed as mean ± SEM (*, p<0.05, vs. MV and MV S107). D) Representative records of specific diaphragmatic force production in calstabinl knockout (Call-/-) mice and E) Averaged specific force-frequency relationships recorded in calstabinl knockout MV (Cair^/__MV, n=6) and Cair^/__MV treated with SI 07 (Call ⁷ _MV-S 107, n=6). While SI 07 prevented VIDD in wild type mice, no effect was observed in Call^" mice (19.9±4.5 and 18.9±3 N.cm^"2 in call^"7" and call^"7" -MV

2+ -/- -/- respectively). F) Comparison of mean Ca sparks frequency between Call^" _MV and Call^{" "} _MV-S107. Results are expressed as mean ± SEM.

Figure 5A: RyRl open probability in human and pig diaphragm muscles in control condition and after MV.

In control and MV human samples the Po was 0.00062± 0.00035 (n=18) and 0.01745±0.01259 (n=23). In pigs, similar results were obtained, with Po of 0.00115±0.00069 (n=12) and 0.00839±0.00582 (n=13) in control and MV respectively. Results are expressed as mean ± SEM. Figure 5B: Ventilation in Continuous Positive Airway Pressure (Cpap) mode does not affect RyPvl remodeling.

Bar graphs represent the quantification of immunoblots relative to total RyRl immunoprecipitated. CysNO, thio-nitrosylation; DNP, 2,4-dinitrophenylhydrazone; P*RyRl, phosphorylated RyRl (at serine 2844). Results are expressed as mean ± SEM, n = 10 and 5 in control and CPAP respectively (*, p<0.05 CPAP vs. control).

Figure 5C: Ventilation in Continuous Positive Airway Pressure (Cpap) mode does not affect RyRl function.

Spontaneous SR Ca²⁺ release events were recorded in fluo-4-loaded permeabilized diaphragm fibers by laser scanning confocal microscopy in control and CPAP condition. Mean Ca²⁺ sparks frequency used, as an index of resting SR Ca²⁺ leak was not significantly different between CPAP and control mice. (Results are expressed as mean ± SEM). Figure 5D: Force frequency relationships of control and call-/- diaphragm muscle in resting condition.

Averaged force - frequency relationships recorded in control (n=8) and calstabin deficiency mice (n=4) in basal condition (i.e. non ventilated, -NV). The force production in basal condition is not affected by the lack of calstabin 1 at the age of 10-12 weeks. Results are expressed as mean ± SEM.

EXAMPLES

EXAMPLE 1: MATERIAL AND METHODS

Human model of VIDD

The study in humans was conducted in accordance with the World Medical Association Guidelines for research in humans, and approved by the institutional ethics board of the Montpellier University Hospital (protocol # NCT00786526). All subjects or their surrogates provided written informed consent to participate in the study. All animal experiments in our study followed the guidelines for animal experiments established by the recommendations of the institutional care committee and the recommendations of the Helsinki Declaration. The protocol has been approved by the Animal Care and Use Committee Languedoc-Roussillon and recorded under the reference #CEEA-LR- 12078. The study design, as previously used included 2 groups of subjects: 1) - patients with brain death destined for organ donation, who had received MV for at least 24 hours prior to organ harvest (MV, n=9); and 2) patients anesthetized and supported with MV for 2-3 hours during thoracic surgery for localized lung nodules (Control, n=10). All subjects were required to have undergone MV via an endotracheal tube in fully controlled mode, i.e., without significant spontaneous breathing efforts during the MV period, with a tidal volume (VT) in the average range of 7-8.5 ml/kg of the body weight, Respiratory Rate (RR) from 12 to 24 min, and positive end-expiratory pressure (PEEP) level at 2-5 cm H₂0 .. Diaphragm biopsies (approximately 1 cm³) were obtained from the zone of apposition of the costal diaphragm at the mid-axillary line. In the MV group, the biopsies were obtained before circulatory arrest and removal of other organs. Each biopsy was partitioned and quick frozen in liquid nitrogen and kept at -80°C. Secondarily tissue blocks were prepared as required for RyR biochemical and RyRl functional analysis with single channel recordings in lipid bilayers as detailed in the last methodology paragraph.

Porcine model of VIDD

As previously described in our laboratory, ten piglets (15-20 kg) separated into 2 groups of animals who both were intubated and ventilated for 72 consecutive hours: 1) a group of 5 piglets mechanically ventilated in a totally controlled mode (MV), with a tidal volume (VT) at 10 -12 ml/kg of the body weight, RR from 15 to 30 min, and positive end- expiratory pressure level at 5 cm H₂0. The absence of spontaneous breathing was verified on the ventilator trend graphs, and electromyographic activity of the diaphragm was measured to ensure that no electrical activity was present in the diaphragm; 2) a group of 5 piglets ventilated in a totally spontaneous pediatric mode (Control), setting in phase with ideal body weight of the piglet, inspiratory flow trigger at 0.3 1/min, percentage of mechanical ventilation between 100 and 150%, positive end-expiratory pressure level at 5 cm H₂0, and expiratory trigger at 25% of peak inspiratory flow. Electromyographic activity of the diaphragm was similarly assessed to verify that active piglets were able to breath spontaneously. In both groups, oxygenation was maintained with FI0₂ from 25 to 35% and minute ventilation to assess normocapnia. The two groups received the same care, except for the ventilator mode. In brief, piglets were anesthetized with intravenous pentobarbital sodium (5- 6 mg/kg), intubated with a cuffed endotracheal tube, and mechanically ventilated (Galileo®; Hamilton Medical AG, Rhazuns, Switzerland). Anaesthesia was maintained with continuous intravenous propofol (15-20 mg/kg/h), midazo-lam (0.1- 0.3 mg/h), and ketamine (3-4 mg /kg/ h). The level of sedation was monitored with bispectral index (BIS®; Aspect, Norwood, MA). Heating pads were used as needed to maintain a normal body temperature of 38.5°- 39.5°C. A carotidal arterial catheter (PiCCO®; Pulsion, Munich, Germany) was inserted for the monitoring of heart rate, arterial blood pressure, and cardiac output. Arterial pressure of carbon dioxide levels was checked by using a capnograph (DELTATRAC®; Datex-Ohmeda, Helsinki, Finland) and then verified by arterial blood gases (iSTAT®; Abbott, Abbott Park,IL). Parenteral nutrition was given from the first day (10% glucose solution and 20% amino acids solution, and HYPERAMINE 20%®; Braun, Boulogne Billancourt, France) providing 30-35 kcal/kg/day. All procedures were performed aseptically. The animals received prophylactic intravenous antibiotics three times daily (amoxicillin- clavulanate, 100 mg/kg/day).

In vivo Diaphragm Contractile function in porcine model of VIDD

Diaphragm contractile function was assessed in vivo by measuring transdiaphragmatic

Pressure (Pdi).). In brief, double air-filled balloon-tipped catheters were placed transorally in the stomach and the distal third of the esophagus. Bipolar transvenous pacing catheters were introduced via each internal jugular vein and adjusted to achieve stimulation of the phrenic nerve and subsequent contraction of the diaphragm by supramaximal stimulation at frequencies ranging from 20 to 120 Hz with trains of stimulation of 2 s and 150 ms pulse duration.

Murine model of VIDD

The experimental design has been described in recent study .. In brief, 35 adult male mice (10 to 12 weeks old, 25 to 30g) C57/BL6 mice were separated into five groups. Three groups were intubated with a 22-gauge angiocatheter and mechanically ventilated for 6 consecutive hours using a volume-driven small-animal ventilator (MINIVENT®, Harvard Apparatus, Saint-Laurent, Canada). Tidal volume was established at ΙΟμΙ/mg body weight with a respiratory rate of 150 breaths/min, a positive end-expiratory pressure (PEEP) level from 2 to 4 cm H₂0 and a fraction of inspired oxygen of 0.21. Non-spontaneous ventilation was defined as a lack of diaphragm contractile activity attested by repetitive stereotypical deflections observed in the airway pressure curve.

The mice were divided into five groups. The first group (MV-Trolox) received a priming dose of trolox (0.125 ml of saline containing 5g/l trolox, corresponding to ~20 mg/kg) intravenously (IV) infused over a 5-minute period, 20 minutes before start of MV. During MV, a constant IV infusion of trolox at a rate of 4 mg/kg per hours (~0.025 ml/h) was maintained. The second group of mice (MV-S107) was treated for 7-days before the start of MV, with SI 07 in their drinking water (final concentration, 0.25 mg.ml^"1) as previously reported and received same volume of IV saline 20 minutes before starting and during MV. The mice drank about 3 ml per day (water consumption was variable, and we recorded water bottle and body weight to monitor consumption) for a daily dose of ~0.75 mg (~37.5 mg/kg/day). The third group (MV) received the same volume of IV saline 20 minutes before starting and during MV. The fourth group (CPAP) (n=7) was intubated and treated in an identical manner to the first three groups but breathed spontaneously with a constant positive airway pressure (CPAP) of 3-4 cm H20. For this purpose, the angio-catheter was connected to an air compressor to deliver a high inspiratory flow rate (1 1 min room air) while the expiratory port was placed under a water seal to obtain PEEP. The fifth group (Control, n=7 mice) did not receive any treatment and served as control.

To address the specificity of SI 07 on RyRl dysfunction in VIDD, we similarly ventilated 12 calstabinl deficient mice (10-12 weeks-old), separated into two groups. The first group (Cai ^/_-MV-S017) received SI 07 treatment similarly to the C57BL6 mice, 5-7 days before the start of MV. The second group (Call ' -MV) received the same amount of water volume. All mice had similar body weights (27 ±0.9 g).

All groups mechanically ventilated or under CPAP received the same general care. Mice were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight) and orally intubated with a 22-gauge angiocatheter. General care applied during the experiments also included continuous reheating using a homeothermic blanket (Homeothermic Blanket Control unit, Harvard Apparatus, Saint-Laurent, Canada, set at 35 °C), and hourly intraperitoneal injection of 0.05 ml of Ringers Lactate solution to maintain hemodynamic stability and compensate insensible losses, as well as bladder expression, eye lubrication and passive limb movements.

In vitro Contractile function in murine muscle samples

At the end of the protocol of MV, the entire diaphragm was surgically excised and mice were euthanized, by exsanguination. Isometric contractile properties were assessed as described previously in detail. The excised diaphragm strip was mounted into jacketed tissue bath chambers filled with equilibrated and oxygenated Krebs solution. The muscles were supra-maximally stimulated using square wave pulses (Model S48; Grass Instruments, West Warwick, RI). The force-frequency relationship was determined by sequentially stimulating the muscles for 600 ms at 10, 20, 30, 50, 60, 80, 100 and 120Hz with 1 minute between each stimulation train. After measurement of contractile properties, muscles were measured at Lo (the length at which the muscle produced maximal isometric tension), dried and weighted. For comparative purposes, diaphragmatic force production was normalized for total muscle strip cross-sectional area and expressed in N.cm^"2. The total muscle strip cross-sectional area was determined by dividing muscle weight by its length and tissue density (1.056 g/cm³).

The rest of the diaphragm was partitioned, one part was quick frozen in liquid nitrogen and secondarily used for biochemical analysis, and the other part was used freshly for Ca²⁺ spark measurements. RyRl biochemical analysis

Muscle biopsies were homogenized in 150 μΐ of buffer containing 5% SDS, 5% beta- mercaptoethanol, 10% glycerol, 10 mM EDTA and 50 mM Tris/HCl buffer (pH= 8.0). Each sample was immediately denatured at 90°C for 4 min. After centrifugation (5000 rpm) at 4°C, supernatant protein concentrations were measured in duplicate using the BCA protein assay, equilibrated at the same concentration by dilution with loading buffer and aliquoted at 2 μg/μl. RyRl was immunoprecipitated from 250 μg of homogenate using an anti-RyR antibody (4 μg RyRl -1327) in 0.5 ml of a modified RIPA buffer (50 mM Tris-HCl pH 7.4, 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na₃V0₄, 1% Triton-XlOO, and protease inhibitors) for 1 hr at 4°C. The immune complexes were incubated with protein A Sepharose beads (Amersham Pharmacia) at 4°C for 1 hr and the beads were washed three times with buffer. Proteins were separated on SDS-PAGE gels (4-20%> gradient) and transferred onto nitrocellulose membranes for 2 hr at 200 mA (SemiDry transfer blot, Bio-Rad). To prevent non-specific antibody binding, the membranes were incubated with blocking solution (LICOR Biosciences) and washed with Tris-buffered saline with 0.1 % Tween-20.

Blots were respectively incubated with primary antibody to RyRl (RyRl -1327, an affinity-purified rabbit polyclonal antibody raised against a KLH-conjugated peptide, corresponding to residues 1327-1339 of mouse skeletal RyRl, with an additional cysteine residue added to the amino terminus), and affinity purified with the unconjugated peptide. We also used antibody to calstabinl (1 : 2500 in blocking buffer, LICOR Biosciences); phospho- epitope-specific antibody to human RyR2 phosphorylated on Ser-2808 (1 :5,000), which detects PKA-phosphorylated mouse RyRl (on Ser-2844) and RyR2 (on Ser-2808); antibody to S-nitrosylated cysteine residues (1 : 1000, Sigma). To determine RyRl oxidation, the immunoprecipitate was treated with 2, 4-dinitrophenyl hydrazine, and the derivatized carbonyls were detected using an OxyBlot protein oxidation detection Kit (catalog S7150, Chemicon International Inc.). After three washes, membranes were incubated with infrared- labeled secondary antibodies. Control samples were analyzed on each gel for normalization and total levels of RyRl were not different between groups.

Calcium sparks measurements.

Diaphragm muscles samples were dissected and stored in a HEPES buffered physiological medium (in mM: 119 NaCl, 5 KC1, 1.25 CaCl₂, 1 MgS0₄, 10 glucose, 1.1 mannitol, 10 HEPES, pH 7.4). Muscles were then rapidly placed in a dissecting chamber and the solution exchanged with a relaxing solution (in mM: 140 K-glutamate, 10 HEPES, 10 MgCl₂, 0.1 EGTA, pH 7.0). Bundles of 5 to 10 EDL fibers were manually dissected, mounted as described previously and permeabilized in a relaxing solution containing 0.01% saponin for 30 s. After washing with saponin free solution, the solution was changed to an internal medium for imaging: (in mM) 140 K-glutamate, 5 Na₂ATP, 10 glucose, 10 HEPES, 4.4 MgCl₂, 1.1 EGTA, 0.3 CaCl₂, Fluo-3 0.05 pentapotassium salt (Invitrogen), pH 7.0, for sparks acquisition as previously reported. Potential sparks were empirically identified using an autodetection algorithm. The mean fluorescence (F0) value for the image was calculated by summing and averaging the temporal F at each spatial location, while ignoring potential spark areas. This F0 value was then used to create a smoothing routine, potential spark locations were visualized and analyzed for spatio temporal properties as described previously 9. Image analysis was performed using IDL (v5.5, Research System, Inc.). Statistical comparisons were performed using an ANOVA test with a significance level set at P<0.05 (Sigmastat v3.5).

Sarcoplasmic reticulum vesicle preparation

Diaphragms were homogenized on ice in 300 mM sucrose, 20 mM PIPES (pH 7.0) in the presence of protease inhibitors (Roche) and centrifuged at 8000 rpm (5900 g) for 20 min at 4°C. The following supernatant was ultracentrifuged at 32 000 rpm (100 000 g) for 1 h at 4°C. The final pellet containing microsomal fractions enriched in SR vesicles was resuspended and aliquoted in 300 mM sucrose, 5 mM PIPES (pH 7.0) containing protease inhibitors. Samples were frozen in liquid nitrogen and stored at -80°C.

Single channel data from planar lipid bilayer measurements

Planar lipid bilayers were formed from a 3 : 1 mixture of phosphatidylethanolamine and phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) suspended (30 mg/ml) in decane by painting the lipid/decane solution across a 200-μιη aperture in a side of a polysulfonate cup (Warner Instruments) separating two chambers. The trans chamber (1 ml) representing the intra-SR (luminal) compartment was connected to the headstage input of a bilayer voltage clamp amplifier (BC-525D, Warner Instruments) and the cis chamber (1 ml), representing the cytoplasmic compartment, was held at virtual ground. Solutions in both chambers were as follows: 1 mM EGTA, 250/125 mM HEPES/Tris, 50 mM KC1, 0.64 mM CaCl₂, pH 7.35 as cis solution and 53 mM Ca(OH)₂, 50 mM KCL, 250 mM HEPES, pH 7.35 as trans solution. The concentration of free Ca²⁺ in the cis chamber was calculated with WinMaxC program (version 2.50; www.stanford.edu/~cpatton/maxc.html). SR vesicles were added to the cis side and fusion with the lipid bilayer was induced by making the cis side hyperosmotic by the addition of 400-500 mM KCL After the appearance of potassium and chloride channels, the cis compartment was perfused with the cis solution. Single-channel currents were recorded at 0 mV using a Bilayer Clamp BC-535 amplifier (Warner Instruments), filtered at 1 kHz and digitized at 4 kHz. All experiments were performed at room temperature. Data acquisition was performed by using Digidata 1440 A and Axoscope 10.2 software and the recordings were analysed by using Clampfit 10.2 (Molecular Devices). Open probability was identified by 50% threshold analysis using a least 2 min of continuous record. At the conclusion of each experiment, ryanodine (5μΜ) was added to the cis chamber to confirm channels as RyRs.

References:

1. S. Jaber et al, Rapidly progressive diaphragmatic weakness and injury during mechanical ventilation in humans. Am J Respir Crit Care Med 183, 364 (Feb 1, 2011).

2. S. Jaber et al., Alteration of the piglet diaphragm contractility in vivo and its recovery after acute hypercapnia. Anesthesiology 108, 651 (Apr, 2008).

3. B. Jung et al, Adaptive support ventilation prevents ventilator-induced diaphragmatic dysfunction in piglet: an in vivo and in vitro study. Anesthesiology 112, 1435 (Jun, 2010). 4. S. Mrozek et al, Rapid onset of specific diaphragm weakness in a healthy murine model of ventilator-induced diaphragmatic dysfunction. Anesthesiology 117, 560 (Sep, 2012).

5. J. Fauconnier et al, Ryanodine receptor leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion. Proc Natl Acad Sci U S

A 108, 13258 (Aug 9, 2011).

6. J. Fauconnier et al, Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy. Proc Natl Acad Sci U S A 107, 1559 (Jan 26, 2010).

7. A. M. Bellinger et al, Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med 15, 325 (Mar, 2009).

8. S. Reiken et al, PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure. J Cell Biol 160, 919 (Mar 17, 2003).

9. C. W. Ward et al, Defects in ryanodine receptor calcium release in skeletal muscle from post-myocardial infarct rats. FASEB J 17, 1517 (Aug, 2003).

EXAMPLE 2: LEAKY RYANODINE RECEPTORS CONTRIBUTE TO DIAPHRAGMATIC MUSCLE WEAKNESS DURING MECHANICAL VENTILATION

Results:

Leaky RyRl in diaphragm fibers correlate with VIDD:

To evaluate the remodeling and functional abnormalities of RyRl in the diaphragms of human patients subjected to MV, we obtained diaphragm biopsies from brain-dead organ donor patients who had undergone long term MV (98+65 hours, referred to as MV) before organ harvest; we then compared these specimens with control diaphragm biopsies obtained on short term MV (2.3+0.4 hours, referred to as control) patients during thoracic surgery for removal of solitary lung nodules (see Table 5A for patients description).

Supplementary Table 1: Clinical description of patients.

SR fractions were purified to analyze the biochemical properties of RyRl macro molecular complex (Figs. 1A-1B). RyRl immunoprecipitation after mechanical ventilation revealed a significant increase in S-nitrosylation, oxidation and phosphorylation on Ser-2844 together with calstabinl dissociation from RyRl . These biochemical abnormalities were associated with an increased RyRl open probability (Po) measured in a subgroup of channels incorporated within planar lipid bilayers (Fig. 1C and 5 A), indicative of increased Ca²⁺ leak.

Because one limitation of human patient samples is the potential influence of comorbidities and confounding factors associated with critical illness, we next evaluated RyRl biochemistry and function in diaphragm from healthy pigs anesthetized and mechanically ventilated for 72 hours in comparison to identically anesthetized control pigs. As previously described a significant reduction in diaphragmatic contractile function was noted after MV (Fig. 2A). SR fractions incorporated into planar lipid bilayers demonstrated an RyRl leak similar to that observed in human MV diaphragm samples (Fig. 2B and 5A). This RyRl leak was also associated with remodeling of the RyRl macromolecular complex, characterized again by a significant increase in S-nitrosylation, oxidation and phosphorylation on Ser-2844 and loss of calstabinl from the RyRl complex (Figs. 2C-2D).

Defective RyRl function: an early pathophysiological event in VIDD:

The experiments described above clearly demonstrate that MV may contribute to structural and functional remodeling of RyRl . However, in these two models, histological damage to muscle fibers is present which could conceivably account for both the reduction in diaphragmatic force production and RyRl remodeling. Therefore, to examine very early proximal events in the course of VIDD, we took advantage of a mouse model that exhibits a major significant loss of diaphragmatic force-generating capacity after only 6 hours of MV (Fig. 3A-B). We were thereby able to evaluate RyRl remodeling prior to the onset of histological alterations associated with the later stages of VIDD.

MV-induced diaphragm muscle weakness in mice was associated with significant RyRl remodeling consisting once again of S-nitrosylation, oxidation, Ser-2844 phosphorylation, and calstabinl dissociation from RyRl (Figs. 3C-3D). RyRl functional properties were next evaluated in situ by measuring spontaneous SR Ca²⁺ release events (i.e. Ca²⁺ sparks). Note that a significant increase in spontaneous Ca²⁺ sparks frequency reflects an exaggerated level of RyRl leak. After 6 hours of MV, Ca²⁺ spark frequency was significantly increased in diaphragm fibers (Figs. 3E-3F). It is important to note that this RyRl impairment is due to MV only, since animals that were anesthetized and immobilized for 6 hours and maintained only on a continuous positive airway pressure (CPAP) mode of respiration, and this did not induce VIDD. Furthermore, there was no biochemical remodeling of RyRl (Fig. 5B) and no functional alteration of the channel complex as indicated by similar Ca²⁺ spark frequency of experimental mice as the control mice (Fig. 5C).

Since MV induces oxidative stress in the diaphragm, and antioxidant treatment has been reported to prevent VIDD, a group of mice was continuously injected with trolox, a permeable analog of vitamin E used as an antioxidant scavenger. As previously reported in rats mechanically ventilated for 12 hours, trolox treatment in mice ventilated for 6 hours prevented MV-induced diaphragm muscle weakness (Figs. 3A-3B) along with MV-induced RyRl biochemical remodeling (Figs. 3C-3D) and the associated increase in Ca²⁺ spark frequency (Figs. 3E-3F).

RyRl as a novel therapeutic target in VIDD:

As described above, a pharmacological approach aimed at reducing oxidative stress appears to prevent MV-induced RyRl dysfunction. However, to directly target RyRl and thus prove its role as a major pathophysiological target in VIDD, we treated mechanically ventilated mice with the rycal SI 07. Rycals are small orally available agents known to prevent depletion of calstabinl from the RyRl complex in spite of its PKA phosphorylation, S-nitrosylated and/or oxidation. In wild-type mice, SI 07 prevented the loss of muscle weakness induced by MV (Fig. 4A-B) as well as the previously observed increase in Ca²⁺ spark frequency (Fig. 4C).

To address the specificity of SI 07, experiments were further conducted in calstabinl knockout (Call^"7") mice. At baseline, wild type and Call^"7" mice exhibited similar diaphragmatic muscle specific force production (Fig. 5D). After 6 hours of MV, Call ^"7" mice exhibited diaphragmatic weakness (Figs. 4D-4E) as well as an increase in Ca²⁺ spark frequency (Fig. 4F) that was comparable to that recorded in wild-type mice. However, in Call^"7" mice subjected to MV, SI 07 treatment failed to prevent both the decrease in force production and the increase in Ca²⁺ spark frequency, thus confirming that the beneficial effects of the rycal SI 07 in preventing these manifestations of VIDD were dependent upon the RyRl stabilizer, calstabinl .

Explanation of the significance of the invention: The present work provides the first evidence, consistently found across the three different models of VIDD (humans, pigs, and mice) employed in this study, of impaired regulation and function of diaphragmatic intracellular Ca²⁺ release channels, i.e., ryanodine receptors (RyR). This RyRl dysfunction depends on the MV-induced oxidative stress that has been extensively described. RyRs are highly sensitive to oxidative/nitrosative stress in skeletal muscle as well as in the heart. This occurs in many pathological situations including heart failure, diabetes, and Duchenne muscular dystrophy (DMD)²⁹'³⁸. Postranslational modification of RyRl also progresses with aging and partially accounts for age-dependent muscle weakness. Therefore, as previously reported in skeletal muscle during heart failure and aging, an overly leaky RyRl similar to that observed in the present study may account for a reduction in Ca²⁺ transient and force production, i.e. impaired excitation-contraction coupling, without the need for invoking other muscle pathology such as atrophy or injury. Evidence is provided herein that postranslational modifications of RyRl are associated with a leaky channel. This is observed in the mouse models by evaluating the frequency of spontaneous Ca²⁺ release events (i.e. Ca²⁺ sparks). The term, Ca²⁺ sparks, refers to a local Ca²⁺ release events arising from a single Ca²⁺ release unit (CRU).). Each CRU corresponds to a cluster of RyRs. Depending on the type of muscle and species, a CRU may contain a variable number of RyRs, remaining approximate (from a few tens up to a few hundred). Therefore, by measuring Ca²⁺ sparks in a muscle fiber, we have a direct measurement in situ of the gating behavior of RyRs belonging to a single CRU. In a CRU, RyRs are functionally coupled. The most common mechanism involved in triggering a rapid opening of RyR in a CRU is a calcium-induced calcium release mechanisms. However, a cooperative mechanism involving calstabine has also been proposed to explain coupled RyR gating in a CRU. Thus, posttranslational modifications of RyR may not only affect individual RyR properties but also CRU behavior. To date the threshold of modified channels (i.e. depleted from calstabinl, oxidized, nitrosylated and or phosphorylated) to affect the overall properties of a single CRU has not been characterized. This nevertheless explains the heterogeneity of the leaky behavior measured in RyR incorporated into lipid planar bilayer was heterogeneous. After MV only about 20% of the channels had a Po higher than 0.01 while 80% had a Po lower than 0.001, with a range comparable with control RyR. This leaky behavior was never observed in control condition, which had more homogeneous Po.

Taken together, these findings suggest that excessive SR Ca²⁺ leak due to redox- dependent modifications of the RyRl is a key factor in VIDD pathogenesis that occurs at a very early stage. Trolox was previously reported to prevent VIDD in a rat model. It was also reported that oxidative stress is required for MV-induced activation of calpain and caspase-3 in the diaphragm. Our data demonstrate that trolox also prevents RyR dysfunction induced by MV.

These data suggest that impaired RyRl function is an early proximal event, which precedes histological damage. Furthermore, this early RyRl -dependent defect in intracellular Ca²⁺ homeostasis may be an important mediator of later histological remodeling in VIDD as previously reported in DMD. Indeed, impaired Ca²⁺ signaling may in part be responsible for the activation of Ca²⁺-dependent proteolytic enzymes (caspases and calpains) as well as Ca²⁺- dependent gene expression changes involved in deleterious muscle injury and wasting processes. One would expect these effects to be exacerbated with an increased duration of MV. The present study also demonstrates that preventing RyRl leak with SI 07, a small molecule known to stabilize the RyRl-calstabinl interaction, can prevent muscle weakness induced by MV in mice. This concept is further proven by the lack of effect of SI 07 in calstabinl deficient mice as already reported in other forms of skeletal muscle and cardiac pathophysiology. As mentioned earlier, the RyR complex is a converging target in many pathophysiological situations, and all of them have certainly not yet been reported. This can be explained in part by the ubiquitous function of Ca²⁺ in cellular processes but also in the complexity and fragility of the RyR macromolecular complex. Therefore, the fact that RyRl is a potential mediator of muscle weakness in VIDD suggests that patients with comorbidities and/or confounding factors that may affect RyR function such as heart failure or aging, might have a greater vulnerability to VIDD.

In conclusion, this study demonstrates the pathophysiological role of RyRl in VIDD and strongly supports the hypothesis that preventing the RyRl -mediated SR Ca²⁺ leak induced by MV may provide a new therapeutic approach to prevent diaphragm muscle dysfunction in patients who require artificial respiratory support.

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3. T. Vassilakopoulos, B. J. Petrof, Ventilator-induced diaphragmatic dysfunction. Am J Respir Crit Care Med 169, 336 (Feb 1, 2004).

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6. A. C. Watson et al, Measurement of twitch transdiaphragmatic, esophageal, and endotracheal tube pressure with bilateral anterolateral magnetic phrenic nerve stimulation in patients in the intensive care unit. Crit Care Med 29, 1325 (Jul, 2001).

7. M. J. Tobin, F. Laghi, L. Brochard, Role of the respiratory muscles in acute respiratory failure of COPD: lessons from weaning failure. J Appl Physiol 107, 962 (Sep, 2009).

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9. R. A. Shanely et al, Mechanical ventilation- induced diaphragmatic atrophy is associated with oxidative injury and increased proteolytic activity. Am J Respir Crit Care

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10. M. A. Zergeroglu et al, Mechanical ventilation- induced oxidative stress in the diaphragm. J Appl Physiol 95, 1116 (Sep, 2003).

11. J. L. Betters et al, Trolox attenuates mechanical ventilation- induced diaphragmatic dysfunction and proteolysis. Am J Respir Crit Care Med 170, 1179 (Dec 1 ,

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12. D. J. Falk et al, Mechanical ventilation promotes redox status alterations in the diaphragm. J Appl Physiol 101, 1017 (Oct, 2006).

13. S. K. Powers et al, Mitochondria-targeted antioxidants protect against mechanical ventilation-induced diaphragm weakness. Crit Care Med 39, 1749 (Jul, 2011).

14. M. Picard et al, Mitochondrial dysfunction and lipid accumulation in the human diaphragm during mechanical ventilation. Am J Respir Crit Care Med 186, 1140 (Dec 1, 2012). 15. A. N. Belcastro, L. D. Shewchuk, D. A. Raj, Exercise-induced muscle injury: a calpain hypothesis. Mo I Cell Biochem 179, 135 (Feb, 1998).

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17. R. W. Jackman, S. C. Kandarian, The molecular basis of skeletal muscle atrophy. Am J Physiol Cell Physiol 287, C834 (Oct, 2004).

18. M. Chen, D. J. Won, S. Krajewski, R. A. Gottlieb, Calpain and mitochondria in ischemia/reperfusion injury. J Biol Chem 277, 29181 (Aug 9, 2002).

19. T. M. Scarabelli et al., Different signaling pathways induce apoptosis in endothelial cells and cardiac myocytes during ischemia/reperfusion injury. Circ Res 90, 745

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20. K. Maes, D. Testelmans, S. Powers, M. Decramer, G. Gayan-Ramirez, Leupeptin inhibits ventilator-induced diaphragm dysfunction in rats. Am J Respir Crit Care Med 1134 (Jun 1, 2007).

21. J. M. McClung et al., Caspase-3 regulation of diaphragm myonuclear domain during mechanical ventilation-induced atrophy. Am J Respir Crit Care Med 175, 150 (Jan 15, 2007).

22. J. M. McClung et al., Apocynin attenuates diaphragm oxidative stress and protease activation during prolonged mechanical ventilation. Crit Care Med 37, 1373 (Apr, 2009).

23. S. Mrozek et al., Rapid onset of specific diaphragm weakness in a healthy murine model of ventilator-induced diaphragmatic dysfunction. Anesthesiology 117, 560 (Sep, 2012).

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25. A. M. Bellinger, M. Mongillo, A. R. Marks, Stressed out: the skeletal muscle ryanodine receptor as a target of stress. J Clin Invest 118, 445 (Feb, 2008).

26. A. B. Brillantes et al., Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell 77, 513 (May 20, 1994).

27. S. Reiken et al., PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure. J Cell Biol 160, 919 (Mar 17, 2003). 28. C. W. Ward et al, Defects in ryanodine receptor calcium release in skeletal muscle from post-myocardial infarct rats. FASEB J \l, 1517 (Aug, 2003).

29. A. M. Bellinger et al, Remodeling of ryanodine receptor complex causes "leaky" channels: a molecular mechanism for decreased exercise capacity. Proc Natl Acad Sci USA 105, 2198 (Feb 12, 2008).

30. A. M. Bellinger et al., Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med 15, 325 (Mar, 2009).

31. D. C. Andersson et al., Ryanodine receptor oxidation causes intracellular calcium leak and muscle weakness in aging. Cell Metab 14, 196 (Aug 3, 2011).

32. S. Jaber et al., Alteration of the piglet diaphragm contractility in vivo and its recovery after acute hypercapnia. Anesthesiology 108, 651 (Apr, 2008).

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All publications, references, patents and patent applications cited herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

Claims

CLAIMS:

1. A method of treating ventilator-induced diaphragmatic dysfunction in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an agent capable of stabilizing RyRl-calstabinl interaction.

2. The method of claim 1 wherein the agent has the formula I:

3. The method of claim 1 wherein the agent is selected from the group consisting of compounds of the general Formula II:

wherein,

- T is O, CH2, NH, or S=(0₂)_n;

- n is 0, 1, or 2;

q is 0, 1, 2, 3, or 4;

each R is independently selected from the group consisting of H, halogen, -OH, -NH₂, -N0₂, -CN, -CF₃, -OCF₃, -N₃, -S0₃H, -S(=0)₂alkyl, -S(=0)alkyl, -OS(=0)₂CF₃, acyl, -O-acyl, alkyl, alkoxyl, alkylamino, alkylarylamino, alkylthio, cycloalkyl, alkylaryl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, -O-acyl, alkyl, alkoxyl, alkylamino, alkylarylamino, alkylthio, cycloalkyl, alkylaryl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be optionally substituted; Ri is selected from the group consisting of H, oxo, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted; R₂ is selected from the group consisting of H, -C(=0)R₅, -C(=S)R6, -S0₂R₇, -P(=0)RsR₉, -(CH₂)_m-Rio, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl; wherein each alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl may be optionally substituted and wherein m is 0, 1, 2, 3, or 4;

R₃ is selected from the group consisting of H, -C0₂Y, -C(=0)NHY, acyl, -O-acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted; and wherein Y is selected from the group consisting of H, alkyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl, and wherein each alkyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted;

R4 is selected from the group consisting of H, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted; R₅ is selected from the group consisting of -NR15R16, -(CH₂)zNRi₅Ri6, -NHNR15R16, -NHOH, -ORis, -C(=0)NHNRi₅Ri6, -CO2R15,

-CH₂X, acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted, and wherein z is 1, 2, 3, 4, 5, or 6;

5 is selected from the group consisting of -OR15, -NHNR15R16, -NHOH, -NR15R16, -CH₂X, acyl, alkenyl, alkyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;

R₇ is selected from the group consisting of -OR15, -NR15R16, -NHNR15R16, -NHOH, alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted; Rs and R9 independently are selected from the group consisting of OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;

Rio is selected from the group consisting of -NR15R16, OH, -SO2R11, -NHSO2R11, C(=0)(Ri₂), NHC=0(Ri₂), -OC=0(Ri₂), and -P(=0)Ri₃Ri₄;

R11 , Ri₂, Ri₃, and Ri₄ independently are selected from the group consisting of H, OH, NH₂, -NHNH₂, -NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted; X is selected from the group consisting of halogen, -CN, -C0₂Ri₅, -C(=0)NRi₅Ri₆, -NR15R16, -OR15, -S0₂R₇ and -P(=0)R₈R₉ and

Ri5 and Ri₆ independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, OH, NH₂, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted; and optionally R15 and Ri₆ together with the N to which they are bonded may form a heterocycle which may be substituted; the nitrogen in the benzothiazepine ring may optionally be a quaternary nitrogen;

and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes, and prodrugs thereof, or any combination thereof.

4. The method of claim 3 wherein the agent is selected from the group consisting of compounds of formula Il-k or II-k-1 : wherein:

- R, R' and R" are independently selected from the group consisting of H, halogen, -OH, -NH₂, -NO₂, -CN, -CF₃, -OCF₃, -N₃, -S0₃H, -S(=0)₂alkyl, -S(=0)alkyl, -OS(=0)₂CF₃, acyl, alkyl, alkoxyl, alkylamino, alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be substituted or unsubstituted;

- Ri8 is selected from the group consisting of H, -NR15R16,

-(C=0)ORi₅, -OR₁₅, alkyl, aryl, cycloalkyl, heterocyclyl, and at one labeling group; wherein each alkyl, aryl, cycloalkyl, and heterocyclyl may be substituted or unsubstituted;

and further wherein:

- q is 0, 1, 2, 3, or 4;

- p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

- n is 0, 1, or 2;

and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes and prodrugs thereof.

5. The method of claim 4 wherein the agent is selected from the group consisting of:

- compounds of formula Il-k wherein each R is independently selected from the group consisting of H, halogen, -OH, OMe, -NH₂, -N0₂, -CN, -CF₃, -OCF₃, -N₃, -S(=0)₂Ci-C₄alkyl, -S(=0)Ci-C₄alkyl, -S-Ci-C₄alkyl, -OS(=0)₂CF₃, Ph, -NHCH₂Ph, -C(=0)Me, -OC(=0)Me, morpholinyl and propenyl; and n is 0, 1 or 2; compounds of formula Il-k wherein R is H or is OMe at position 7 of the benzothiazepine ring; compounds of formula II-k-1 wherein R' and R" are independently selected from the group consisting of H, halogen, -OH, OMe, -NH₂, -N0₂, -CN, -CF₃, -OCF₃, -N₃, -S(=0)₂Ci-C₄alkyl, -S(=0)Ci-C₄alkyl, -S-Ci-C₄alkyl, -OS(=0)₂CF₃, Ph, -NHCH₂Ph, -C(=0)Me, -OC(=0)Me, morpholinyl and propenyl, and n is 0, 1 or 2; compounds of formula II-k-1 wherein R' is H or OMe, and R" is H; compounds of formula Il-k or II-k-1, wherein Ris is selected from the group consisting of -NR15R16,

-OR15, alkyl, aryl, and at one labeling group; and wherein each alkyl and aryl may be substituted or unsubstituted; compounds of formula Il-k or II-k-1 wherein, m is 1, and Ris is Ph, C(=0)OMe, C(=0)OH, aminoalkyl, NH₂, NHOH, or NHCbz; those compounds of formula Il-k or II-k-1 wherein m is 0, and Ris is C1-C4 alkyl, such as Me, Et, propyl, and butyl; compounds of formula Il-k or II-k-1 wherein m is 2, and Ris is pyrrolidine, piperidine, piperazine, or morpholine; compounds of Formula II-o:

wherein:

Re is -(Ci-C₆ alkyl)-phenyl, -(Ci-C₆ alkyl)-C(0)Rb, or substituted or unsubstituted -Ci-C₆ alkyl; and

- R_b is -OH or -0-(Ci-C₆ alkyl), and - wherein the phenyl or substituted alkyl is substituted with one or more of halogen, hydroxyl, -Ci-C₆ alkyl, -0-(Ci-C₆ alkyl), -NH₂, -NH(Ci-C₆ alkyl), -N(Ci-C₆ alkyl)₂, cyano, or dioxolane; and

S 107 having the formula III :

6. The method of claim 3 wherein the agent is selected from the group consisting of SI ,

S2, S3, S4, S5, S6, S7, S9, Sl l, S12, S13, S14, S19, S20, S22, S23, S24, S25, S26, S27, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S68, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, S100, S101, S102, S103, S104, S107, S108, S109, S110, Si l l, S112, S113, S114, S115, S116, S117, S118, S119, S120, S121, S122, S123, S136, S137, S138, S139, S140, S146, S147, S148, S149, S150, S151, S152, S153, S156, S157, S159, S160, S161, S166, S167, S182, S186, S189, S203, S217, S251, S252, S258, S277, S279, S282, S291, S293, S296, S301, S302, S306, S311, S312, S313, S318, S322, S324, S326, S331, S335, S337, S351, S352, S353, S354, S397, S398, S399, S423, S454, S463, S466, S470, S473, S477 and salts thereof.

7. The method of claim 1 wherein the agent is selected from the group of compounds of the general Formula IV:

wherein

- n is 0, 1, 2, 3, or 4;

- X is O, -NR₅ or -C(R₅)₂; each R is independently selected from the group consisting of Z, R₅, -OR₅, -SR₅, -N(R₅)₂, -NR₅C(=0)OR₅, -C(=0)N(R₅)₂, -C(=0)OR₅, -C(=0)R₅, -OC(=0)R₅, N0₂, CN, -CZ₃, OCZ₃, -N₃, and -P(=0)R₈R₉;

Ri and R₃ are each independently selected from the group consisting of oxo, R₅, -CH₂OR₅, -CH₂OC(=0)R₆, -C(=0)OR₅, -C(=0)NHR₅, -C(=0)R₅, and

-OC(=0)R₅;

R₂ is selected from the group consisting of R₅, -(C=0)R6, -(C=S)R6, and

(CH₂)_mRio, wherein m is 1, 2, 3, 4, 5, or 6; or

Rl and R₂ together with the carbon and nitrogen to which they are respectively attached, form an unsubstituted or substituted heterocycle; or

R₂ and R₃ together with the nitrogen and carbon to which they are respectively attached, form an unsubstituted or substituted heterocycle other than a piperazine; or

R₃ and R4 together with the carbon atoms to which they are respectively attached, form an unsubstituted or substituted cycloalkyl or heterocyclic ring; or

R4 is selected from the group consisting of R₅ and oxo;

each R₅ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, alkylaryl, and alkylheteroaryl;

- 5 is selected from the group consisting of R5, -(CH₂)t,NRi₃Ri₄, -NR₅OR₅, -OR₅,

-C(0)OR₅, -C(=0)NRi₃Ri₄, -(CH₂ )_CY, and -C(=0)R₅, wherein b is 0, 1, 2, 3, 4, 5, or 6 and c is 1, 2, 3, 4 or 5;

Rio is selected from the group consisting of R₅, -OR₅, -S0₂Rn, -C(=0)Ri₂, -NH(C=0)Ri3, -0(C=0)Ri2, and -P(=0)R₈R₉;

- Rs, R9, R11 and Ri₂ are independently selected from the group consisting of R₅,

OR₅, and -N(R₅)₂;

- Y is selected from the group consisting of Z, -C0₂R₅, -C(=0)NRi₃Ri₄, and -OR₅;

Z is a halogen selected from F, CI, Br and I;

Ri₃ and Ri₄ are independently selected from the group consisting of R₅, or Ri₃ and Ri₄ together with the N to which they are bonded may form an unsubstituted or substituted heterocycle; and

wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, alkylaryl, and alkylheteroaryl may be substituted or unsubstituted; wherein the nitrogen in the benzoxazepine ring may optionally be a quaternary nitrogen;

8. The method of claim 7 wherein the agent is selected from the group consisting of ARM136, ARM137, ARM138, ARM139, ARM140, ARM146, ARM147, ARM148, ARM149, ARM150, ARM151, ARM152, ARM153, ARM156, ARM157, ARM159, ARM160, ARM161, ARM166, ARM167, ARM182, ARM186, ARM187, ARM189, ARM 200, ARM203, ARM 205, ARM217, ARM251 , ARM252, ARM258, ARM277, ARM279, ARM282, ARM291, ARM293, ARM296, ARM301, ARM302, ARM306, ARM311, ARM312, ARM313, ARM318, ARM322, ARM324, ARM326, ARM331, ARM335, ARM337, ARM351, ARM352, ARM353, ARM354, ARM397, ARM398, ARM399, ARM423, ARM454, ARM463, ARM466, ARM470, ARM473 and ARM477.

9. The method of claim 1 wherein the subject suffers from respiratory failure and/or heart failure which can be aggravated by sepsis, metabolic disorder, neuromuscular diseases, or surgery along with post-surgical recovery.

10. The method of claim 1 wherein the subject suffers from a disease selected from the group consisting of Chronic Obstructive Pulmonary Disease (COPD), pneumonia, sepsis, Acute Respiratory Distress Syndrome (ARDS), Severe Acute Respiratory Syndrome (SARS) and cystic fibrosis (CF).

11. The method of claim 1 wherein the subject suffers from a trauma.

12. The method of claim 1 wherein ventilator-induced diaphragmatic dysfunction results from prolonged controlled mechanical ventilation (MV) greater than 12 hours.

13. The method of claim 1 wherein the agent is administered before MV, immediately after MV initiation, during MV, and/or immediately after MV.

14. A method for screening a plurality of test substances useful for treatment of ventilator- induced diaphragmatic dysfunction comprising the steps consisting of (a) testing each of the test substances for its ability stabilize the RyRl-calstabinl interaction and/or to reduce SR Ca2+ leak via RyRl channel and (b) and positively selecting the test substances capable of restoring said integrated stress response.