A peptide of the N terminus of GRK5 attenuates pressure-overload hypertrophy and heart failure.

Aberrant changes in gene expression underlie the pathogenesis and progression of pressure-overload heart failure, leading to maladaptive cardiac hypertrophy, ventricular remodeling, and contractile dysfunction. Signaling through the G protein Gq triggers maladaptation and heart failure, in part through the activation of G protein-coupled receptor kinase 5 (GRK5). Hypertrophic stimuli induce the accumulation of GRK5 in the nuclei of cardiomyocytes, where it regulates pathological gene expression through multiple transcription factors including NFAT. The nuclear targeting of GRK5 is mediated by an amino-terminal (NT) domain that binds to calmodulin (CaM). Here, we sought to prevent GRK5-mediated pathology in pressure-overload maladaptation and heart failure by expressing in cardiomyocytes a peptide encoding the GRK5 NT (GRK5nt) that encompasses the CaM binding domain. In cultured cardiomyocytes, GRK5nt expression abrogated Gq-coupled receptor-mediated hypertrophy, including attenuation of pathological gene expression and the transcriptional activity of NFAT and NF-κB. We confirmed that GRK5nt bound to and blocked Ca2+-CaM from associating with endogenous GRK5, thereby preventing GRK5 nuclear accumulation after pressure overload. We generated mice that expressed GRKnt in a cardiac-specific fashion (TgGRK5nt mice), which exhibited reduced cardiac hypertrophy, ventricular dysfunction, pulmonary congestion, and cardiac fibrosis after chronic transverse aortic constriction. Together, our data support a role for GRK5nt as an inhibitor of pathological GRK5 signaling that prevents heart failure.

GRK5 is a regulator of fibroblast activation and cardiac fibrosis.

Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor kinase 5 (GRK5) is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of nuclear factor of activated T cells (NFAT). Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts; however, its pathophysiological role in these crucial cardiac cells is unknown. We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits angiotensin II (AngII)-mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice led to decreased fibrosis and cardiac hypertrophy after chronic AngII infusion or after ischemic injury compared to nontransgenic littermate controls (NLCs). Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. These data demonstrate that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrate the potential beneficial effects of GRK5 inhibition in the context of cardiac disease.

G protein-coupled receptor kinases as therapeutic targets in the heart.

G protein-coupled receptors (GPCRs) are critical cellular sensors that mediate numerous physiological processes. In the heart, multiple GPCRs are expressed on various cell types, where they coordinate to regulate cardiac function by modulating critical processes such as contractility and blood flow. Under pathological settings, these receptors undergo aberrant changes in expression levels, localization and capacity to couple to downstream signalling pathways. Conventional therapies for heart failure work by targeting GPCRs, such as ß-adrenergic receptor and angiotensin II receptor antagonists. Although these treatments have improved patient survival, heart failure remains one of the leading causes of mortality worldwide. GPCR kinases (GRKs) are responsible for GPCR phosphorylation and, therefore, desensitization and downregulation of GPCRs. In this Review, we discuss the GPCR signalling pathways and the GRKs involved in the pathophysiology of heart disease. Given that increased expression and activity of GRK2 and GRK5 contribute to the loss of contractile reserve in the stressed and failing heart, inhibition of overactive GRKs has been proposed as a novel therapeutic approach to treat heart failure.

Alteration of myocardial GRK2 produces a global metabolic phenotype.

A vast body of literature has established GRK2 as a key player in the development and progression of heart failure. Inhibition of GRK2 improves cardiac function post injury in numerous animal models. In recent years, discovery of several non-canonical GRK2 targets has expanded our view of this kinase. Here, we describe the novel and exciting finding that cardiac GRK2 activity can regulate whole body metabolism. Transgenic mice with cardiac-specific expression of a peptide inhibitor of GRK2 (TgßARKct) display an enhanced obesogenic phenotype when fed a high fat diet (HFD). In contrast, mice with cardiac-specific overexpression of GRK2 (TgGRK2) show resistance to HFD induced obesity. White adipose tissue (WAT) mass was significantly enhanced in HFD fed TgßARKct mice. Furthermore, regulators of adipose differentiation were differentially regulated in WAT from mice with gain or loss of GRK2 function. Using complex metabolomics we found that cardiac GRK2 signaling altered myocardial BCAA and endocannabinoid metabolism and modulated circulating BCAA and endocannabinoid metabolite profiles on a HFD, and one of the BCAA metabolites identified here enhances adipocyte differentiation in vitro. Taken together, these results suggest that metabolic changes in the heart due to GRK2 signaling on a HFD control whole body metabolism.

Sarcomere-based genetic enhancement of systolic cardiac function in a murine model of dilated cardiomyopathy.

Diminished cardiac contractile function is a characteristic feature of dilated cardiomyopathy (DCM) and many other heart failure (HF) causing etiologies. We tested the hypothesis that targeting the sarcomere to increase cardiac contractility can effectively prevent the DCM phenotype in muscle-LIM protein knockout (MLP-/-) mice. The ablation of cardiac myosin binding protein C (MYBPC3-/-) protected the MLP-/- mice from developing the DCM phenotype. We examined the in vivo cardiac function and morphology of the resultant mouse model lacking both MLP and MYBPC3 (DKO) by echocardiography and pressure-volume catheterization and found a significant reduction in hypertrophy, as evidenced by normalized wall thickness and chamber dimensions, and improved systolic function, as evidenced by enhanced ejection fraction (~26% increase compared MLP-/- mice) and rate of pressure development (DKO 7851.0 ±â€¯504.8 vs. MLP-/- 4496.4 ±â€¯196.8 mmHg/s). To investigate the molecular basis for the improved DKO phenotype we performed mechanical experiments in skinned myocardium isolated from WT and the individual KO mice. Skinned myocardium isolated from DKO mice displayed increased Ca2+ sensitivity of force generation, and significantly accelerated rate of cross-bridge detachment (+63% compared to MLP-/-) and rate of XB recruitment (+58% compared to MLP-/-) at submaximal Ca2+ activations. The in vivo and in vitro functional enhancement of DKO mice demonstrates that enhancing the sarcomeric contractility can be cardioprotective in HF characterized by reduced cardiac output, such as in cases of DCM.

Dose-Dependent Effects of the Myosin Activator Omecamtiv Mecarbil on Cross-Bridge Behavior and Force Generation in Failing Human Myocardium.

BACKGROUND: Omecamtiv mecarbil (OM) enhances systolic function in vivo by directly binding the myosin cross-bridges (XBs) in the sarcomere. However, the mechanistic details governing OM-induced modulation of XB behavior in failing human myocardium are unclear. METHODS AND RESULTS: The effects of OM on steady state and dynamic XB behavior were measured in chemically skinned myocardial preparations isolated from human donor and heart failure (HF) left ventricle. HF myocardium exhibited impaired contractile function as evidenced by reduced maximal force, magnitude of XB recruitment (Pdf), and a slowed rate of XB detachment (krel) at submaximal Ca2+ activations. Ca2+ sensitivity of force generation (pCa50) was higher in HF myocardium when compared with donor myocardium, both prior to and after OM incubations. OM incubation (0.5 and 1.0 µmol/L) enhanced force generation at submaximal Ca2+ activations in a dose-dependent manner. Notably, OM induced a slowing in krel with 1.0 µmol/L OM but not with 0.5 µmol/L OM in HF myocardium. Additionally, OM exerted other differential effects on XB behavior in HF myocardium as evidenced by a greater enhancement in Pdf and slowing in the time course of cooperative XB recruitment (Trec), which collectively prolonged achievement of peak force development (Tpk), compared with donor myocardium. CONCLUSIONS: Our findings demonstrate that OM augments force generation but also prolongs the time course of XB transitions to force-bearing states in remodeled HF myocardium, which may extend the systolic ejection time in vivo. Optimal OM dosing is critical for eliciting enhanced systolic function without excessive prolongation of systolic ejection time, which may compromise diastolic filling.

Cardiac myosin binding protein-C Ser302 phosphorylation regulates cardiac ß-adrenergic reserve.

Phosphorylation of cardiac myosin binding protein-C (MyBP-C) modulates cardiac contractile function; however, the specific roles of individual serines (Ser) within the M-domain that are targets for ß-adrenergic signaling are not known. Recently, we demonstrated that significant accelerations in in vivo pressure development following ß-agonist infusion can occur in transgenic (TG) mouse hearts expressing phospho-ablated Ser282 (that is, TGS282A) but not in hearts expressing phospho-ablation of all three serines [that is, Ser273, Ser282, and Ser302 (TG3SA)], suggesting an important modulatory role for other Ser residues. In this regard, there is evidence that Ser302 phosphorylation may be a key contributor to the ß-agonist-induced positive inotropic responses in the myocardium, but its precise functional role has not been established. Thus, to determine the in vivo and in vitro functional roles of Ser302 phosphorylation, we generated TG mice expressing nonphosphorylatable Ser302 (that is, TGS302A). Left ventricular pressure-volume measurements revealed that TGS302A mice displayed no accelerations in the rate of systolic pressure rise and an inability to maintain systolic pressure following dobutamine infusion similar to TG3SA mice, implicating Ser302 phosphorylation as a critical regulator of enhanced systolic performance during ß-adrenergic stress. Dynamic strain-induced cross-bridge (XB) measurements in skinned myocardium isolated from TGS302A hearts showed that the molecular basis for impaired ß-adrenergic-mediated enhancements in systolic function is due to the absence of protein kinase A-mediated accelerations in the rate of cooperative XB recruitment. These results demonstrate that Ser302 phosphorylation regulates cardiac contractile reserve by enhancing contractile responses during ß-adrenergic stress.

Sarcomeric protein modification during adrenergic stress enhances cross-bridge kinetics and cardiac output.

Molecular adaptations to chronic neurohormonal stress, including sarcomeric protein cleavage and phosphorylation, provide a mechanism to increase ventricular contractility and enhance cardiac output, yet the link between sarcomeric protein modifications and changes in myocardial function remains unclear. To examine the effects of neurohormonal stress on posttranslational modifications of sarcomeric proteins, mice were administered combined α- and ß-adrenergic receptor agonists (isoproterenol and phenylephrine, IPE) for 14 days using implantable osmotic pumps. In addition to significant cardiac hypertrophy and increased maximal ventricular pressure, IPE treatment accelerated pressure development and relaxation (74% increase in dP/dtmax and 14% decrease in τ), resulting in a 52% increase in cardiac output compared with saline (SAL)-treated mice. Accelerated pressure development was maintained when accounting for changes in heart rate and preload, suggesting that myocardial adaptations contribute to enhanced ventricular contractility. Ventricular myocardium isolated from IPE-treated mice displayed a significant reduction in troponin I (TnI) and myosin-binding protein C (MyBP-C) expression and a concomitant increase in the phosphorylation levels of the remaining TnI and MyBP-C protein compared with myocardium isolated from saline-treated control mice. Skinned myocardium isolated from IPE-treated mice displayed a significant acceleration in the rate of cross-bridge (XB) detachment (46% increase) and an enhanced magnitude of XB recruitment (43% increase) at submaximal Ca2+ activation compared with SAL-treated mice but unaltered myofilament Ca2+ sensitivity of force generation. These findings demonstrate that sarcomeric protein modifications during neurohormonal stress are molecular adaptations that enhance in vivo ventricular contractility through accelerated XB kinetics to increase cardiac output.NEW & NOTEWORTHY Posttranslational modifications to sarcomeric regulatory proteins provide a mechanism to modulate cardiac function in response to stress. In this study, we demonstrate that neurohormonal stress produces modifications to myosin-binding protein C and troponin I, including a reduction in protein expression within the sarcomere and increased phosphorylation of the remaining protein, which serve to enhance cross-bridge kinetics and increase cardiac output. These findings highlight the importance of sarcomeric regulatory protein modifications in modulating ventricular function during cardiac stress.

Cardiac Myosin Binding Protein-C Phosphorylation Modulates Myofilament Length-Dependent Activation.

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is an important regulator of contractile function, however, its contributions to length-dependent changes in cross-bridge (XB) kinetics is unknown. Therefore, we performed mechanical experiments to quantify contractile function in detergent-skinned ventricular preparations isolated from wild-type (WT) hearts, and hearts expressing non-phosphorylatable cMyBP-C [Ser to Ala substitutions at residues Ser273, Ser282, and Ser302 (i.e., 3SA)], at sarcomere length (SL) 1.9 µm or 2.1µm, prior and following protein kinase A (PKA) treatment. Steady-state force generation measurements revealed a blunting in the length-dependent increase in myofilament Ca(2+)-sensitivity of force generation (pCa50) following an increase in SL in 3SA skinned myocardium compared to WT skinned myocardium. Dynamic XB behavior was assessed at submaximal Ca(2+)-activations by imposing an acute rapid stretch of 2% of initial muscle length, and measuring both the magnitudes and rates of resultant phases of force decay due to strain-induced XB detachment and delayed force rise due to recruitment of additional XBs with increased SL (i.e., stretch activation). The magnitude (P2) and rate of XB detachment (k rel) following stretch was significantly reduced in 3SA skinned myocardium compared to WT skinned myocardium at short and long SL, and prior to and following PKA treatment. Furthermore, the length-dependent acceleration of k rel due to decreased SL that was observed in WT skinned myocardium was abolished in 3SA skinned myocardium. PKA treatment accelerated the rate of XB recruitment (k df) following stretch at both SL's in WT but not in 3SA skinned myocardium. The amplitude of the enhancement in force generation above initial pre-stretch steady-state levels (P3) was not different between WT and 3SA skinned myocardium at any condition measured. However, the magnitude of the entire delayed force phase which can dip below initial pre-stretch steady-state levels (Pdf) was significantly lower in 3SA skinned myocardium under all conditions, in part due to a reduced magnitude of XB detachment (P2) in 3SA skinned myocardium compared to WT skinned myocardium. These findings demonstrate that cMyBP-C phospho-ablation regulates SL- and PKA-mediated effects on XB kinetics in the myocardium, which would be expected to contribute to the regulation of the Frank-Starling mechanism.

The contributions of cardiac myosin binding protein C and troponin I phosphorylation to ß-adrenergic enhancement of in vivo cardiac function.

KEY POINTS: ß-adrenergic stimulation increases cardiac myosin binding protein C (MyBP-C) and troponin I phosphorylation to accelerate pressure development and relaxation in vivo, although their relative contributions remain unknown. Using a novel mouse model lacking protein kinase A-phosphorylatable troponin I (TnI) and MyBP-C, we examined in vivo haemodynamic function before and after infusion of the ß-agonist dobutamine. Mice expressing phospho-ablated MyBP-C displayed cardiac hypertrophy and prevented full acceleration of pressure development and relaxation in response to dobutamine, whereas expression of phosphor-ablated TnI alone had little effect on the acceleration of contractile function in response to dobutamine. Our data demonstrate that MyBP-C phosphorylation is the principal mediator of the contractile response to increased ß-agonist stimulation in vivo. These results help us understand why MyBP-C dephosphorylation in the failing heart contributes to contractile dysfunction and decreased adrenergic reserve in response to acute stress. ß-adrenergic stimulation plays a critical role in accelerating ventricular contraction and speeding relaxation to match cardiac output to changing circulatory demands. Two key myofilaments proteins, troponin I (TnI) and myosin binding protein-C (MyBP-C), are phosphorylated following ß-adrenergic stimulation; however, their relative contributions to the enhancement of in vivo cardiac contractility are unknown. To examine the roles of TnI and MyBP-C phosphorylation in ß-adrenergic-mediated enhancement of cardiac function, transgenic (TG) mice expressing non-phosphorylatable TnI protein kinase A (PKA) residues (i.e. serine to alanine substitution at Ser23/24; TnI(PKA-)) were bred with mice expressing non-phosphorylatable MyBP-C PKA residues (i.e. serine to alanine substitution at Ser273, Ser282 and Ser302; MyBPC(PKA-)) to generate a novel mouse model expressing non-phosphorylatable PKA residues in TnI and MyBP-C (DBL(PKA-)). MyBP-C dephosphorylation produced cardiac hypertrophy and increased wall thickness in MyBPC(PKA-) and DBL(PKA-) mice, and in vivo echocardiography and pressure-volume catheterization studies revealed impaired systolic function and prolonged diastolic relaxation compared to wild-type and TnI(PKA-) mice. Infusion of the ß-agonist dobutamine resulted in accelerated rates of pressure development and relaxation in all mice; however, MyBPC(PKA-) and DBL(PKA-) mice displayed a blunted contractile response compared to wild-type and TnI(PKA-) mice. Furthermore, unanaesthesized MyBPC(PKA-) and DBL(PKA-) mice displayed depressed maximum systolic pressure in response to dobutamine as measured using implantable telemetry devices. Taken together, our data show that MyBP-C phosphorylation is a critical modulator of the in vivo acceleration of pressure development and relaxation as a result of enhanced ß-adrenergic stimulation, and reduced MyBP-C phosphorylation may underlie depressed adrenergic reserve in heart failure.

Molecular effects of the myosin activator omecamtiv mecarbil on contractile properties of skinned myocardium lacking cardiac myosin binding protein-C.

Decreased expression of cardiac myosin binding protein-C (cMyBP-C) in the myocardium is thought to be a contributing factor to hypertrophic cardiomyopathy in humans, and the initial molecular defect is likely abnormal cross-bridge (XB) function which leads to impaired force generation, decreased contractile performance, and hypertrophy in vivo. The myosin activator omecamtiv mecarbil (OM) is a pharmacological drug that specifically targets the myosin XB and recent evidence suggests that OM induces a significant decrease in the in vivo motility velocity and an increase in the XB duty cycle. Thus, the molecular effects of OM maybe beneficial in improving contractile function in skinned myocardium lacking cMyBP-C because absence of cMyBP-C in the sarcomere accelerates XB kinetics and enhances XB turnover rate, which presumably reduces contractile efficiency. Therefore, parameters of XB function were measured in skinned myocardium lacking cMyBP-C prior to and following OM incubation. We measured ktr, the rate of force redevelopment as an index of XB transition from both the weakly- to strongly-bound state and from the strongly- to weakly-bound states and performed stretch activation experiments to measure the rates of XB detachment (krel) and XB recruitment (kdf) in detergent-skinned ventricular preparations isolated from hearts of wild-type (WT) and cMyBP-C knockout (KO) mice. Samples from donor human hearts were also used to assess the effects of OM in cardiac muscle expressing a slow ß-myosin heavy chain (ß-MHC). Incubation of skinned myocardium with OM produced large enhancements in steady-state force generation which were most pronounced at low levels of [Ca(2+)] activations, suggesting that OM cooperatively recruits additional XB's into force generating states. Despite a large increase in steady-state force generation following OM incubation, parallel accelerations in XB kinetics as measured by ktr were not observed, and there was a significant OM-induced decrease in krel which was more pronounced in the KO skinned myocardium compared to WT skinned myocardium (58% in WT vs. 76% in KO at pCa 6.1), such that baseline differences in krel between KO and WT skinned myocardium were no longer apparent following OM-incubation. A significant decrease in the kdf was also observed following OM incubation in all groups, which may be related to the increase in the number of cooperatively recruited XB's at low Ca(2+)-activations which slows the overall rate of force generation. Our results indicate that OM may be a useful pharmacological approach to normalize hypercontractile XB kinetics in myocardium with decreased cMyBP-C expression due to its molecular effects on XB behavior.

Length-dependent changes in contractile dynamics are blunted due to cardiac myosin binding protein-C ablation.

Enhanced cardiac contractile function with increased sarcomere length (SL) is, in part, mediated by a decrease in the radial distance between myosin heads and actin. The radial disposition of myosin heads relative to actin is modulated by cardiac myosin binding protein-C (cMyBP-C), suggesting that cMyBP-C contributes to the length-dependent activation (LDA) in the myocardium. However, the precise roles of cMyBP-C in modulating cardiac LDA are unclear. To determine the impact of cMyBP-C on LDA, we measured isometric force, myofilament Ca(2+)-sensitivity (pCa50) and length-dependent changes in kinetic parameters of cross-bridge (XB) relaxation (k rel), and recruitment (k df) due to rapid stretch, as well as the rate of force redevelopment (k tr) in response to a large slack-restretch maneuver in skinned ventricular multicellular preparations isolated from the hearts of wild-type (WT) and cMyBP-C knockout (KO) mice, at SL's 1.9 µm or 2.1 µm. Our results show that maximal force was not significantly different between KO and WT preparations but length-dependent increase in pCa50 was attenuated in the KO preparations. pCa50 was not significantly different between WT and KO preparations at long SL (5.82 ± 0.02 in WT vs. 5.87 ± 0.02 in KO), whereas pCa50 was significantly different between WT and KO preparations at short SL (5.71 ± 0.02 in WT vs. 5.80 ± 0.01 in KO; p < 0.05). The k tr, measured at half-maximal Ca(2+)-activation, was significantly accelerated at short SL in WT preparations (8.74 ± 0.56 s(-1) at 1.9 µm vs. 5.71 ± 0.40 s(-1) at 2.1 µm, p < 0.05). Furthermore, k rel and k df were accelerated by 32% and 50%, respectively at short SL in WT preparations. In contrast, k tr was not altered by changes in SL in KO preparations (8.03 ± 0.54 s(-1) at 1.9 µm vs. 8.90 ± 0.37 s(-1) at 2.1 µm). Similarly, KO preparations did not exhibit length-dependent changes in k rel and k df. Collectively, our data implicate cMyBP-C as an important regulator of LDA via its impact on dynamic XB behavior due to changes in SL.

The contribution of cardiac myosin binding protein-c Ser282 phosphorylation to the rate of force generation and in vivo cardiac contractility.

Cardiac myosin binding protein-C phosphorylation plays an important role in modulating cardiac muscle function and accelerating contraction. It has been proposed that Ser282 phosphorylation may serve as a critical molecular switch that regulates the phosphorylation of neighbouring Ser273 and Ser302 residues, and thereby govern myofilament contractile acceleration in response to protein kinase A (PKA). Therefore, to determine the regulatory roles of Ser282 we generated a transgenic (TG) mouse model expressing cardiac myosin binding protein-C with a non-phosphorylatable Ser282 (i.e. serine to alanine substitution, TG(S282A)). Myofibrils isolated from TG(S282A) hearts displayed robust PKA-mediated phosphorylation of Ser273 and Ser302, and the increase in phosphorylation was identical to TG wild-type (TG(WT)) controls. No signs of pathological cardiac hypertrophy were detected in TG(S282A) hearts by either histological examination of cardiac sections or echocardiography. Baseline fractional shortening, ejection fraction, isovolumic relaxation time, rate of pressure development and rate of relaxation (τ) were unaltered in TG(S282A) mice. However, the increase in cardiac contractility as well as the acceleration of pressure development observed in response to ß-adrenergic stimulation was attenuated in TG(S282A) mice. In agreement with our in vivo data, in vitro force measurements revealed that PKA-mediated acceleration of cross-bridge kinetics in TG(S282A) myocardium was significantly attenuated compared to TG(WT) myocardium. Taken together, our data suggest that while Ser282 phosphorylation does not regulate the phosphorylation of neighbouring Ser residues and basal cardiac function, full acceleration of cross-bridge kinetics and left ventricular pressure development cannot be achieved in its absence.

Cardiac myosin binding protein-C: a novel sarcomeric target for gene therapy.

Through its ability to interact with both the thick and thin filament proteins within the sarcomere, cardiac myosin binding protein-C (cMyBP-C) regulates the contractile properties of the myocardium. The central regulatory role of cMyBP-C in heart function is emphasized by the fact that a large proportion of inherited hypertrophic cardiomyopathy cases in humans are caused by mutations in cMyBP-C. The primary dysfunction in cMyBP-C-related cardiomyopathies is likely to be abnormal myofilament contractile function; however, currently, there are no effective therapies for ameliorating these contractile defects. Thus, there is a compelling need to design novel therapies to restore normal contractile function in cMyBP-C-related cardiomyopathies. To this end, concepts gleaned from various structural, functional, and biochemical studies can now be utilized to engineer cMyBP-C proteins that, when incorporated into the sarcomere, can significantly improve contractile function. In this review, we discuss the rationale for cMyBP-C-based gene therapies that can be utilized to treat contractile dysfunction in inherited and acquired cardiomyopathies.

Intracellular acidosis and pH regulation in central respiratory chemoreceptors.

Dysfunctions of brainstem regions responsible for central CO2 chemoreception have been proposed as an underlying pathophysiology of Sudden Infant Death Syndrome (SIDS). We recorded respiratory motor output and intracellular pH (pHi) from chemosensitive neurons in an in vitro tadpole brainstem during normocapnia and hypercapnia. Flash photolysis of the H+ donor nitrobenzaldehyde was used to induce focal decreases in pHi alone. Hypercapnia and flash photolysis significantly decreased pHi from normocapnia. In addition, chemoreceptors did not regulate pHi during hypercapnia, but demonstrated significant pHi recovery when only pHi was reduced by flash photolysis. Respiration was stimulated by decreases in pHi (hypercapnia and flash photolysis) by decreases in burst cycle. These data represent our ability to load the brainstem with nitrobenzaldehyde without disrupting the respiration, to quantify changes in chemoreceptor pHi recovery, and to provide insights regarding mechanisms of human health conditions with racial/ethnic health disparities such as SIDS and Apnea of Prematurity (AOP).

Airway inflammation and central respiratory control: results from in vivo and in vitro neonatal rat.

In infants, respiratory infection elicits tachypnea. To begin to evaluate the role of brainstem cytokine expression in modulation of breathing pattern changes, we compared the pattern generated after endotracheal instillation of lipopolysaccharide (LPS) in in vivo rat pups to local pro-inflammatory cytokine injection in the nucleus tractus solitarius (nTS) in an in vitro en bloc brainstem spinal cord preparation. We hypothesized that both challenges would elicit similar changes in patterning of respiration. In anesthetized, spontaneously breathing rat pups, lipopolysaccharide (LPS) or saline was instilled in the airway of urethane-anesthetized rats (postnatal days 10-11). We recorded diaphragm EMG over the subsequent 2h and saw a 20-30% decrease in interburst interval (Te) at 20-80min post-injection in LPS-instilled animals with no significant change in Ti. In contrast, IL-1ß injections into the nTS of en bloc in vitro brainstem-spinal cord preparations from 0 to 5 day-old pups maintained Ti and caused an increase in Te as early as 20min later, decreasing frequency for 80-120min after injection. Our results suggest that the neonatal respiratory response to the cytokine IL-1ß mediated inflammatory response depends on the site of the inflammatory stimulus and that the direct effect of IL-1ß in the nTS is to slow rather than increase rate.