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Cachexia is a muscle wasting syndrome occurring in many advanced cancer patients. Cachexia significantly increases cancer morbidity and mortality. Cardiac atrophy and contractility deficits have been observed in patients and in animal models with cancer cachexia, which may contribute to cachexia pathophysiology. However, underlying contributors to decreased in vivo cardiac contractility are not well understood. In this study, we sought to distinguish heart-intrinsic changes from systemic factors contributing to cachexia-associated cardiac dysfunction. We hypothesized that isolated heart and cardiac myocyte functional deficits underlie in vivo contractile dysfunction. To test this hypothesis, isolated heart and cardiac myocyte function was measured in the colon-26 adenocarcinoma murine model of cachexia. Ex vivo perfused hearts from cachectic animals exhibited marked contraction and relaxation deficits during basal and pacing conditions. Isolated myocytes displayed significantly decreased peak contraction and relaxation rates, which was accompanied by decreased peak calcium and decay rates. This study uncovers significant organ and cellular-level functional deficits in cachectic hearts outside of the catabolic in vivo environment, which is explained in part by impaired calcium cycling. These data provide insight into physiological mechanisms of cardiomyopathy in cachexia, which is critical for the ultimate development of effective treatments for patients., (© 2021. The Author(s).)

The sarcomere is the functional unit of cardiac muscle, essential for normal heart function. To date, it has not been possible to study, in real time, thin filament-based activation dynamics in live cardiac muscle. We report here results from a cardiac troponin C (TnC) FRET-based biosensor integrated into the cardiac sarcomere via stoichiometric replacement of endogenous TnC. The TnC biosensor provides, for the first time, evidence of multiple thin filament activating ligands, including troponin I interfacing with TnC and cycling myosin, during a cardiac twitch. Results show that the TnC FRET biosensor transient significantly precedes that of peak twitch force. Using small molecules and genetic modifiers known to alter sarcomere activation, independently of the intracellular Ca 2+ transient, the data show that the TnC biosensor detects significant effects of the troponin I switch domain as a sarcomere-activating ligand. Interestingly, the TnC biosensor also detected the effects of load-dependent altered myosin cycling, as shown by a significant delay in TnC biosensor transient inactivation during the isometric twitch. In addition, the TnC biosensor detected the effects of myosin as an activating ligand during the twitch by using a small molecule that directly alters cross-bridge cycling, independently of the intracellular Ca 2+ transient. Collectively, these results aid in illuminating the basis of cardiac muscle contractile activation with implications for gene, protein, and small molecule-based strategies designed to target the sarcomere in regulating beat-to-beat heart performance in health and disease., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The heterotrimeric cardiac troponin complex is a key regulator of contraction and plays an essential role in conferring Ca 2+ sensitivity to the sarcomere. During ischemic injury, rapidly accumulating protons acidify the myoplasm, resulting in markedly reduced Ca 2+ sensitivity of the sarcomere. Unlike the adult heart, sarcomeric Ca 2+ sensitivity in fetal cardiac tissue is comparatively pH insensitive. Replacement of the adult cardiac troponin I (cTnI) isoform with the fetal troponin I (ssTnI) isoform renders adult cardiac contractile machinery relatively insensitive to acidification. Alignment and functional studies have determined histidine 132 of ssTnI to be the predominant source of this pH insensitivity. Substitution of histidine at the cognate position 164 in cTnI confers the same pH insensitivity to adult cardiac myocytes. An alanine at position 164 of cTnI is conserved in all mammals, with the exception of the platypus, which expresses a proline. Prolines are biophysically unique because of their innate conformational rigidity and helix-disrupting function. To provide deeper structure-function insight into the role of the TnC-TnI interface in determining contractility, we employed a live-cell approach alongside molecular dynamics simulations to ascertain the chemo-mechanical implications of the disrupted helix 4 of cTnI where position 164 exists. This important motif belongs to the critical switch region of cTnI. Substitution of a proline at position 164 of cTnI in adult rat cardiac myocytes causes increased contractility independent of alterations in the Ca 2+ transient. Free-energy perturbation calculations of cTnC-Ca 2+ binding indicate no difference in cTnC-Ca 2+ affinity. Rather, we propose the enhanced contractility is derived from new salt bridge interactions between cTnI helix 4 and cTnC helix A, which are critical in determining pH sensitivity and contractility. Molecular dynamics simulations demonstrate that cTnI A164P structurally phenocopies ssTnI under baseline but not acidotic conditions. These findings highlight the evolutionarily directed role of the TnI-cTnC interface in determining cardiac contractility., (Copyright © 2018. Published by Elsevier Inc.)

Molecular inotropy refers to cardiac contractility that can be modified to affect overall heart pump performance. Here we show evidence of a new molecular pathway for positive inotropy by a cardiac-restricted microRNA (miR). We report enhanced cardiac myocyte performance by acute titration of cardiac myosin-embedded miR-208a. The observed positive effect was independent of host gene myosin effects with evidence of negative regulation of cAMP-specific 3',5'-cyclic phosphodiesterase 4D (PDE4D) and the regulatory subunit of PKA (PRKAR1α) content culminating in PKA-site dependent phosphorylation of cardiac troponin I (cTnI) and phospholamban (PLN). Further, acute inhibition of miR-208a in adult myocytes in vitro increased PDE4D expression causing reduced isoproterenol-mediated phosphorylation of cTnI and PLN. Next, rAAV-mediated miR-208a gene delivery enhanced heart contractility and relaxation parameters in vivo. Finally, acute inducible increases in cardiac miR-208a in vivo reduced PDE4D and PRKAR1α, with evidence of increased content of several complementary miRs harboring the PDE4D recognition sequence. Physiologically, this resulted in significant cardiac cTnI and PLN phosphorylation and improved heart performance in vivo. As phosphorylation of cTnI and PLN is critical to myocyte function, titration of miR-208a represents a potential new mechanism to enhance myocardial performance via the PDE4D/PRKAR1α/PKA phosphoprotein signaling pathway.

The sarcomere is the functional unit of the heart. Alterations in sarcomere activation lead to disease states such as hypertrophic and restrictive cardiomyopathy (HCM/RCM). Mutations in many of the sarcomeric genes are causal for HCM/RCM. In most cases, these mutations result in increased Ca(2+) sensitivity of the sarcomere, giving rise to altered systolic and diastolic function. There is emerging evidence that small-molecule sarcomere neutralization is a potential therapeutic strategy for HCM/RCM. To pursue proof-of-concept, W7 was used here because of its well-known Ca(2+) desensitizer biochemical effects at the level of cardiac troponin C. Acute treatment of adult cardiac myocytes with W7 caused a dose-dependent (1-10 μM) decrease in contractility in a Ca(2+)-independent manner. Alkalosis was used as an in vitro experimental model of acquired heightened Ca(2+) sensitivity, resulting in increased live cell contractility and decreased baseline sarcomere length, which were rapidly corrected with W7. As an inherited cardiomyopathy model, R193H cardiac troponin I (cTnI) transgenic myocytes showed significant decreased baseline sarcomere length and slowed relaxation that were rapidly and dose-dependently corrected by W7. Langendorff whole heart pacing stress showed that R193H cTnI transgenic hearts had elevated end-diastolic pressures at all pacing frequencies compared with hearts from nontransgenic mice. Acute treatment with W7 rapidly restored end-diastolic pressures to normal values in R193H cTnI hearts, supporting a sarcomere intrinsic mechanism of dysfunction. The known off-target effects of W7 notwithstanding, these results provide further proof-of-concept that small-molecule-based sarcomere neutralization is a potential approach to remediate hyper-Ca(2+)-sensitive sarcomere function., (Copyright © 2016 the American Physiological Society.)

Cardiac gene delivery of parvalbumin (Parv), an EF-hand Ca(2+) buffer, has been studied as a therapeutic strategy for diastolic heart failure, in which slow Ca(2+) reuptake is an important contributor. A limitation of wild-type (WT) Parv is the significant trade-off between faster relaxation and blunted contraction amplitude, occurring because WT-Parv sequesters Ca(2+) too early in the cardiac cycle and prematurely truncates sarcomere shortening in the facilitation of rapid relaxation. We recently demonstrated that an E → Q substitution (ParvE101Q) at amino acid 12 of the EF-hand Ca(2+)/Mg(2+) binding loop disrupts bidentate Ca(2+) binding, reducing Ca(2+) affinity by 99-fold and increasing Mg(2+) affinity twofold. ParvE101Q caused faster relaxation and not only preserved contractility, but unexpectedly increased it above untreated myocytes. To gain mechanistic insight into the increased contractility, we focused here on amino acid 12 of the EF-hand motif. We introduced an E → D substitution (ParvE101D) at this site, which converts bidentate Ca(2+) coordination to monodentate coordination. ParvE101D decreased Ca(2+) affinity by 114-fold and increased Mg(2+) affinity 28-fold compared to WT-Parv. ParvE101D increased contraction amplitude compared to both untreated myocytes and myocytes with ParvE101Q, with limited improvement in relaxation. Additionally, ParvE101D increased spontaneous contractions after pacing stress. ParvE101D also increased Ca(2+) transient peak height and was diffusely localized around the Z-line of the sarcomere, suggesting a Ca(2+)-dependent mechanism of enhanced contractility. Sarcoplasmic reticulum Ca(2+) load was not changed with ParvE101D, but postpacing Ca(2+) waves were increased. Together, these data show that inverted Ca(2+)/Mg(2+) binding affinities of ParvE101D increase myocyte contractility through a Ca(2+)-dependent mechanism without altering sarcoplasmic reticulum Ca(2+) load and by increasing unstimulated contractions and Ca(2+) waves. ParvE101D provides mechanistic insight into how changes in the Ca(2+)/Mg(2+) binding affinities of parvalbumin's EF-hand motif alter function of cardiac myocytes. These data are informative in developing new Ca(2+) buffering strategies for the failing heart., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The hearts of mammalian hibernators maintain contractile function in the face of severe environmental stresses during winter heterothermy. To enable survival in torpor, hibernators regulate the expression of numerous genes involved in excitation-contraction coupling, metabolism, and stress response pathways. Understanding the basis of this transition may provide new insights into treatment of human cardiac disease. Few studies have investigated hibernator heart performance during both summer active and winter torpid states, and seasonal comparisons of whole heart function are generally lacking. We investigated the force-frequency relationship and the response to ex vivo ischemia-reperfusion in intact isolated hearts from 13-lined ground squirrels (Ictidomys tridecemlineatus) in the summer (active, July) and winter (torpid, January). In standard euthermic conditions, we found that winter hearts relaxed more rapidly than summer hearts at low to moderate pacing frequencies, even though systolic function was similar in both seasons. Proteome data support the hypothesis that enhanced Ca(2+) handling in winter torpid hearts underlies the increased relaxation rate. Additionally, winter hearts developed significantly less rigor contracture during ischemia than summer hearts, while recovery during reperfusion was similar in hearts between seasons. Winter torpid hearts have an increased glycogen content, which likely reduces development of rigor contracture during the ischemic event due to anaerobic ATP production. These cardioprotective mechanisms are important for the hibernation phenotype and highlight the resistance to hypoxic stress in the hibernator., (Copyright © 2015 the American Physiological Society.)

EF-hand proteins are ubiquitous in cell signaling. Parvalbumin (Parv), the archetypal EF-hand protein, is a high-affinity Ca(2+) buffer in many biological systems. Given the centrality of Ca(2+) signaling in health and disease, EF-hand motifs designed to have new biological activities may have widespread utility. Here, an EF-hand motif substitution that had been presumed to destroy EF-hand function, that of glutamine for glutamate at position 12 of the second cation binding loop domain of Parv (ParvE101Q), markedly inverted relative cation affinities: Mg(2+) affinity increased, whereas Ca(2+) affinity decreased, forming a new ultra-delayed Ca(2+) buffer with favorable properties for promoting cardiac relaxation. In therapeutic testing, expression of ParvE101Q fully reversed the severe myocyte intrinsic contractile defect inherent to expression of native Parv and corrected abnormal myocardial relaxation in diastolic dysfunction disease models in vitro and in vivo. Strategic design of new EF-hand motif domains to modulate intracellular Ca(2+) signaling could benefit many biological systems with abnormal Ca(2+) handling, including the diseased heart.

Restrictive cardiomyopathy (RCM) has been linked to mutations in the thin filament regulatory protein cardiac troponin I (cTnI). As the pathogenesis of RCM from genotype to clinical phenotype is not fully understood, transgenic (Tg) mice were generated with cardiac specific expression of an RCM-linked missense mutation (R193H) in cTnI. R193H Tg mouse hearts with 15% stoichiometric replacement had smaller hearts and significantly elevated end diastolic pressures (EDP) in vivo. Using a unique carbon microfiber-based assay, membrane intact R193H adult cardiac myocytes generated higher passive tensions across a range of physiologic sarcomere lengths resulting in significant Ca(2+) independent cellular diastolic tone that was manifest in vivo as elevated organ-level EDP. Sarcomere relaxation and Ca(2+) decay was uncoupled in isolated R193H Tg adult myocytes due to the increase in myofilament Ca(2+) sensitivity of tension, decreased passive compliance of the sarcomere, and adaptive in vivo changes in which phospholamban (PLN) content was decreased. Further evidence of Ca(2+) and mechanical uncoupling in R193H Tg myocytes was demonstrated by the biphasic response of relaxation to increased pacing frequency versus the negative staircase seen with Ca(2+) decay. In comparison, non-transgenic myocyte relaxation closely paralleled the accelerated Ca(2+) decay. Ca(2+) transient amplitude was also significantly blunted in R193H Tg myocytes despite normal mechanical shortening resulting in myocyte hypercontractility when compared to non-transgenics. These results identify for the first time that a single point mutation in cTnI, R193H, directly causes elevated EDP due to a myocyte intrinsic loss of compliance independent of Ca(2+) cycling or altered cardiac morphology. The compound influence of impaired relaxation and elevated EDP represents a clinically severe form of diastolic dysfunction similar to the hemodynamic state documented in RCM patients., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P<0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules.

Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration caused by lack of the cytoskeletal protein dystrophin. Dystrophin deficiency causes muscle membrane instability, skeletal muscle wasting, cardiomyopathy, and heart failure. Advances in palliative respiratory care have increased the incidence of heart disease in DMD patients, for which there is no cure or effective therapy. Here we have shown that chronic infusion of membrane-sealing poloxamer to severely affected dystrophic dogs reduced myocardial fibrosis, blocked increased serum cardiac troponin I (cTnI) and brain type natriuretic peptide (BNP), and fully prevented left-ventricular remodeling. Mechanistically, we observed a markedly greater primary defect of reduced cell compliance in dystrophic canine myocytes than in the mildly affected mdx mouse myocytes, and this was associated with a lack of utrophin upregulation in the dystrophic canine cardiac myocytes. Interestingly, after chronic poloxamer treatment, the poor compliance of isolated canine myocytes remained evident, but this could be restored to normal upon direct application of poloxamer. Collectively, these findings indicate that dystrophin and utrophin are critical to membrane stability-dependent cardiac myocyte mechanical compliance and that poloxamer confers a highly effective membrane-stabilizing chemical surrogate in dystrophin/utrophin deficiency. We propose that membrane sealant therapy is a potential treatment modality for DMD heart disease and possibly other disorders with membrane defect etiologies.

Current inotropic therapies used to increase cardiac contractility of the failing heart center on increasing the amount of calcium available for contraction, but their long-term use is associated with increased mortality due to fatal arrhythmias. Thus, there is a need to develop and explore novel inotropic therapies that can act via calcium-independent mechanisms. The purpose of this study was to determine whether fast alpha-myosin molecular motor gene transfer can confer calcium-independent positive inotropy in slow beta-myosin-dominant rabbit and human failing ventricular myocytes. To this end, we generated a recombinant adenovirus (AdMYH6) to deliver the full-length human alpha-myosin gene to adult rabbit and human cardiac myocytes in vitro. Fast alpha-myosin motor expression was determined by Western blotting and immunocytochemical analysis and confocal imaging. In experiments using electrically stimulated myocytes from ischemic failing hearts, AdMYH6 increased the contractile amplitude of failing human [23.9+/-7.8 nm (n=10) vs. AdMYH6 amplitude 78.4+/-16.5 nm (n=6)] and rabbit myocytes. The intracellular calcium transient amplitude was not altered. Control experiments included the use of a green fluorescent protein or a beta-myosin heavy chain adenovirus. Our data provide evidence for a novel form of calcium-independent positive inotropy in failing cardiac myocytes by fast alpha-myosin motor protein gene transfer.

Intracellular acidosis is a profound negative regulator of myocardial performance. We hypothesized that titrating myofilament calcium sensitivity by a single histidine substituted cardiac troponin I (A164H) would protect the whole animal physiological response to acidosis in vivo. To experimentally induce severe hypercapnic acidosis, mice were exposed to a 40% CO(2) challenge. By echocardiography, it was found that systolic function and ventricular geometry were maintained in cTnI A164H transgenic (Tg) mice. By contrast, non-Tg (Ntg) littermates experienced rapid and marked cardiac decompensation during this same challenge. For detailed hemodymanic assessment, Millar pressure-conductance catheterization was performed while animals were treated with a beta-blocker, esmolol, during a severe hypercapnic acidosis challenge. Survival and load-independent measures of contractility were significantly greater in Tg vs. Ntg mice. This assay showed that Ntg mice had 100% mortality within 5 min of acidosis. By contrast, systolic and diastolic function were protected in Tg mice during acidosis, and they had 100% survival. This study shows that, independent of any beta-adrenergic compensation, myofilament-based molecular manipulation of inotropy by histidine-modified troponin I maintains cardiac inotropic and lusitropic performance and markedly improves survival during severe acidosis in vivo.

Aims: Contractile dysfunction associated with myocardial ischaemia is a significant cause of morbidity and mortality in the elderly. Strategies to protect the aged heart from ischaemia-mediated pump failure are needed. We hypothesized that troponin I-mediated augmentation of myofilament calcium sensitivity would protect cardiac function in aged mice., Methods and Results: To address this, we investigated transgenic (Tg) mice expressing a histidine-substituted form of adult cardiac troponin I (cTnI A164H), which increases myofilament calcium sensitivity in a pH-dependent manner. Serial echocardiography revealed that Tg hearts showed significantly improved systolic function at 4 months, which was sustained for 2 years based on ejection fraction and velocity of circumferential fibre shortening. Age-related diastolic dysfunction was also attenuated in Tg mice as assessed by Doppler measurements of the mitral valve inflow and lateral annulus Doppler tissue imaging. During acute hypoxia, cardiac contractility significantly improved in aged Tg mice made evident by increased stroke volume, end systolic pressure, and +dP/dt compared with non-transgenic mice., Conclusion: This study shows that increasing myofilament function by means of a pH-responsive histidine button engineered into cTnI results in enhanced baseline heart function in Tg mice over their lifetime, and during acute hypoxia improves survival in aged mice by maintaining cardiac contractility.

Restrictive cardiomyopathy (RCM) is a debilitating disease characterized by impaired ventricular filling, reduced ventricular volumes, and severe diastolic dysfunction. Hypertrophic cardiomyopathy (HCM) is characterized by ventricular hypertrophy and heightened risk of premature sudden cardiac death. These cardiomyopathies can result from mutations in the same gene that encodes for cardiac troponin I (cTnI). Acute genetic engineering of adult rat cardiac myocytes was used to ascertain whether primary physiologic outcomes could distinguish between RCM and HCM alleles at the cellular level. Co-transduction of cardiac myocytes with wild-type (WT) cTnI and RCM/HCM linked mutants in cTnI's inhibitory region (IR) demonstrated that WT cTnI preferentially incorporated into the sarcomere over IR mutants. The cTnI IR mutants exhibited minor effects in single myocyte Ca(2+)-activated tension assays yet prolonged relaxation and Ca(2+) decay. In comparison RCM cTnI mutants in the helix-4/C-terminal region demonstrated a) hyper-sensitivity to Ca(2+) under loaded conditions, b) slowed myocyte mechanical relaxation and Ca(2+) transient decay, c) frequency-dependent Ca(2+)-independent diastolic tone, d) heightened myofilament incorporation and e) irreversible cellular contractile defects with acute diltiazem administration. For species comparison, a subset of cTnI mutants were tested in isolated adult rabbit cardiac myocytes. Here, RCM and HCM mutant cTnIs exerted similar effects of slowed myocyte relaxation and Ca(2+) transient decay but did not show variable phenotypes by cTnI region. This study highlights cellular contractile defects by cardiomyopathy mutant cTnIs that are allele and species dependent. The species dependent results in particular raise important issues toward elucidating a unifying mechanistic pathway underlying the inherited cardiomyopathies.

The cytosolic Ca(2+)/Mg(2+)-binding protein alpha-parvalbumin (alpha-Parv) has been shown to accelerate cardiac relaxation; however, beyond an optimal concentration range, alpha-Parv can also diminish contractility. Mathematical modeling suggests that increasing Parv's Mg(2+) affinity may lower the effective concentration of Parv ([Parv]) to speed relaxation and, thus, limit Parv-mediated depressed contraction. Naturally occurring alpha/beta-Parv isoforms show divergence in amino acid primary structure (57% homology) and cation-binding affinities, with beta-Parv having an estimated 16% greater Mg(2+) affinity and approximately 200% greater Ca(2+) affinity than alpha-Parv. We tested the hypothesis that, at the same or lower estimated [Parv], mechanical relaxation rate would be more significantly accelerated by beta-Parv than by alpha-Parv. Dahl salt-sensitive (DS) rats were used as an experimental model of diastolic dysfunction. Relaxation properties were significantly slowed in adult cardiac myocytes isolated from DS rats compared with controls: time from peak contraction to 50% relaxation was 57 +/- 2 vs. 49 +/- 2 (SE) ms (P < 0.05), validating this model system. DS cardiac myocytes were subsequently transduced with alpha- or beta-Parv adenoviral vectors. Upon Parv gene transfer, beta-Parv caused significantly faster relaxation than alpha-Parv (P < 0.05), even though estimated [beta-Parv] was approximately 10% of [alpha-Parv]. This comparative analysis showing distinct functional outcomes raises the prospect of utilizing naturally occurring Parv variants to address disease-associated slowed cardiac relaxation.

Elucidating the relative roles of cardiac troponin I (cTnI) and phospholamban (PLN) in beta-adrenergic-mediated hastening of cardiac relaxation has been challenging and controversial. To test the hypothesis that beta-adrenergic phosphorylation of cTnI has a prominent role in accelerating cardiac myocyte relaxation performance we used transgenic (Tg) mice bearing near complete replacement of native cTnI with a beta-adrenergic phospho-mimetic of cTnI whereby tandem serine codons 23/24 were converted to aspartic acids (cTnI S23/24D). Adult cardiac myocytes were isolated and contractility determined at physiological temperature under unloaded and loaded conditions using micro-carbon fibers. At baseline, cTnI S23/24D myocytes had significantly faster relaxation times relative to controls, and isoproterenol stimulation (Iso) had only a small effect to further speed relaxation in cTnI S23/24D myocytes (delta Iso: 7.2 ms) relative to the maximum Iso effect (31.2 ms) in control. The Ca(2+) transient decay rate was similarly accelerated by Iso in Tg and nontransgenic (Ntg) myocytes. Gene transfer of cTnI S23/24D to myocytes in primary culture showed comparable findings. Gene transfer of cTnI with both serines 23/24 converted to alanines (cTnI S23/24A), or gene transfer of slow skeletal TnI, both of which lack PKA phosphorylation sites, significantly blunted Iso-mediated enhanced relaxation compared with controls. Gene transfer of wild-type cTnI had no effect on relaxation. These findings support a key role of cTnI in myocyte relaxation and highlight a direct contribution of the myofilaments in modulating the dynamics of myocardial performance.

Inherited restrictive cardiomyopathy (RCM) is a debilitating disease characterized by a stiff heart with impaired ventricular relaxation. Mutations in cardiac troponin I (cTnI) were identified as causal for RCM. Acute genetic engineering of adult cardiac myocytes was used to identify primary structure/function effects of mutant cTnI. Studies focused on R193H cTnI owing to the poor prognosis of this allele. Compared with wild-type cTnI, R193H mutant cTnI more effectively incorporated into the sarcomere, where it exerted dose-dependent effects on basal and dynamic contractile function. Under loaded conditions, permeabilized myocyte Ca(2+) sensitivity of tension was increased, whereas the passive tension-extension relationship was not altered by R193H cTnI. Normal rod-shaped myocyte morphology acutely transitioned to a "short-squat" phenotype in concert with progressive stoichiometric incorporation of R193H in the absence of altered diastolic Ca(2+). The specific myosin inhibitor blebbistatin fully blocked this transition. Heightened Ca(2+) buffering by the R193H myofilaments, and not alterations in Ca(2+) handling by the sarcoplasmic reticulum, slowed the decay rate of the Ca(2+) transient. Incomplete mechanical relaxation conferred by R193H was exacerbated at increasing pacing frequencies independent of elevated diastolic Ca(2+). R193H cTnI-dependent mechanical tone caused acute remodeling to a quasicontracted state not elicited by other Ca(2+)-sensitizing proteins and is a direct correlate of the stiff heart characteristic of RCM in vivo. These results point toward targets downstream of Ca(2+) handling, notably thin filament regulation and actin-myosin interaction, in designing therapeutic strategies to redress the primary cell morphological and mechanical underpinnings of RCM.

Increased relative expression of the slow molecular motor of the heart (beta-myosin heavy chain [MyHC]) is well known to occur in many rodent models of cardiovascular disease and in human heart failure. The direct effect of increased relative beta-MyHC expression on intact cardiac myocyte contractility, however, is unclear. To determine the direct effects of increased relative beta-MyHC expression on cardiac contractility, we used acute genetic engineering with a recombinant adenoviral vector (AdMYH7) to genetically titrate beta-MyHC protein expression in isolated rodent ventricular cardiac myocytes that predominantly expressed alpha-MyHC (fast molecular motor). AdMYH7-directed beta-MyHC protein expression and sarcomeric incorporation was observed as soon as 1 day after gene transfer. Effects of beta-MyHC expression on myocyte contractility were determined in electrically paced single myocytes (0.2 Hz, 37 degrees C) by measuring sarcomere shortening and intracellular calcium cycling. Gene transfer-based replacement of alpha-MyHC with beta-MyHC attenuated contractility in a dose-dependent manner, whereas calcium transients were unaffected. For example, when beta-MyHC expression accounted for approximately 18% of the total sarcomeric myosin, the amplitude of sarcomere-length shortening (nanometers, nm) was depressed by 42% (151.0+/-10.7 [control] versus 87.0+/-5.4 nm [AdMYH7 transduced]); and genetic titration of beta-MyHC, leading to 38% beta-MyHC content, attenuated shortening by 57% (138.9+/-13.0 versus 59.7+/-7.1 nm). Maximal isometric cross-bridge cycling rate was also slower in AdMYH7-transduced myocytes. Results indicate that small increases of beta-MyHC expression (18%) have Ca2+ transient-independent physiologically relevant effects to decrease intact cardiac myocyte function. We conclude that beta-MyHC is a negative inotrope among the cardiac myofilament proteins.

We developed a mathematical model specific to rat ventricular myocytes that includes electrophysiological representation, ionic homeostasis, force production, and sarcomere movement. We used this model to interpret, analyze, and compare two genetic manipulations that have been shown to increase myocyte relaxation rates, parvalbumin (Parv) de novo expression, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) overexpression. The model was used to seek mechanistic insights into 1) the relative contribution of two mechanisms by which SERCA2a overexpression modifies Ca2+ sequestration, i.e., more pumps and an increase in the SERCA2a-to-phospholamban ratio, 2) the mechanisms behind postrest potentiation and how Parv and SERCA2a influence this response, and 3) why Parv myocytes retain their fast kinetics when endogenous SERCA2a is partially impaired by thapsigargin (a condition used to mimic diastolic dysfunction). The model was also utilized to predict whether Parv metal-binding characteristics might be modified to improve diastolic and systolic functions and whether Parv or SERCA2a might affect diastolic Ca2+ levels and myocyte energetics. One outcome of the model was to demonstrate a higher peak and total ATP consumption in SERCA2a myocytes and more even distribution of ATP throughout the cardiac cycle in Parv myocytes. This may have implications for failing hearts that are energetically compromised.

Hypertrophic cardiomyopathy mutations A63V and E180G in alpha-tropomyosin (alpha-Tm) have been shown to cause slow cardiac muscle relaxation. In this study, we used two complementary genetic strategies, gene transfer in isolated rat myocytes and transgenesis in mice, to ascertain whether parvalbumin (Parv), a myoplasmic calcium buffer, could correct the diastolic dysfunction caused by these mutations. Sarcomere shortening measurements in rat cardiac myocytes expressing the alpha-Tm A63V mutant revealed a slower time to 50% relengthening (T50R: 44.2+/-1.4 ms in A63V, 36.8+/-1.0 ms in controls; n=96 to 108; P<0.001) when compared with controls. Dual gene transfer of alpha-Tm A63V and Parv caused a marked decrease in T50R (29.8+/-1.0 ms). However, this increase in relaxation rate was accompanied with a decrease in shortening amplitude (114.6+/-4.4 nm in A63+Parv, 137.8+/-5.3 nm in controls). Using an asynchronous gene transfer strategy, Parv expression was reduced (from approximately 0.12 to approximately 0.016 mmol/L), slow relaxation redressed, and shortening amplitude maintained (T50R=33.9+/-1.6 ms, sarcomere shortening amplitude=132.2+/-7.0 nm in A63V+PVdelayed; n=56). Transgenic mice expressing the E180G alpha-Tm mutation and mice expressing Parv in the heart were crossed. In isolated adult myocytes, the alpha-Tm mutation alone (E180G+/PV-) had slower sarcomere relengthening kinetics than the controls (T90R: 199+/-7 ms in E180G+/PV-, 130+/-4 ms in E180G-/PV-; n=71 to 72), but when coexpressed with Parv, cellular relaxation was faster (T90R: 36+/-4 ms in E180G+/PV+). Collectively, these findings show that slow relaxation caused by alpha-Tm mutants can be corrected by modifying calcium handling with Parv.

Troponin is essential for the regulation of cardiac contraction. Troponin is a sarcomeric molecular switch, directly regulating the contractile event in concert with intracellular calcium signals. Troponin isoform switching, missense mutations, proteolytic cleavage, and posttranslational modifications are known to directly affect sarcomeric regulation. This review focuses on physiologically relevant covalent and noncovalent modifications in troponin as part of a thematic series on cardiac thin filament function in health and disease.

Thin filament proteins tropomyosin (Tm), troponin T (TnT), and troponin I (TnI) form an allosteric regulatory complex that is required for normal cardiac contraction. Multiple isoforms of TnT, Tm, and TnI are differentially expressed in both cardiac development and disease, but concurrent TnI, Tm, and TnT isoform switching has hindered assignment of cellular function to these transitions. We systematically incorporated into the adult sarcomere the embryonic/fetal isoforms of Tm, TnT, and TnI by using gene transfer. In separate experiments, greater than 90% of native TnI and 40-50% of native Tm or TnT were specifically replaced. The Ca(2+) sensitivity of tension development was markedly enhanced by TnI replacement but not by TnT or Tm isoform replacement. Titration of TnI replacement from >90% to <30% revealed a dominant functional effect of slow skeletal TnI to modulate regulation. Over this range of isoform replacement, TnI, but not Tm or TnT embryonic isoforms, influenced calcium regulation of contraction, and this identifies TnI as a potential target to modify contractile performance in normal and diseased myocardium.

On average, the human heart beats 100,000 times a day and it is in a person's best interest to have the heart move blood as efficiently as possible. For example, imagine a wet rag: squeezing the rag in your fist does not remove as much water as wringing the same rag between two hands. Thus, in hearts as in rags, torsional wringing, as opposed to squeezing, more thoroughly empties the heart of blood. Recent reports indicate that the activity and the distribution of myosin light chain kinase (MLCK) in the myocardium is key to this process. MLCK-dependent phosphorylation of the regulatory light chain (R-LC), a subunit of the myosin molecule, may lead to increased force and power of contraction. It is the asymmetric distribution of MLCK in the myocardium that allows for torsional wringing rather than squeezing. Specifically targeting MLCK expression in the heart might, in the future, lead to promising therapies that counteract cardiomyopathy.

Mutations in tropomyosin (Tm) have been linked to distinct inherited diseases of cardiac and skeletal muscle, hypertrophic cardiomyopathy (HCM), and nemaline myopathy (NM). How HCM and NM mutations in nearly identical Tm proteins produce the vastly divergent clinical phenotypes of heightened, prolonged cardiac muscle contraction in HCM and skeletal muscle weakness in NM is currently unknown. We report here a direct comparison of the effects of HCM (A63V) and NM (M9R) mutant Tm on membrane-intact myocyte contractile function as assessed by adenoviral gene transfer to fully differentiated cardiac muscle cells. Wild-type, and mutant HCM, and mutant NM proteins were expressed at similar levels in myocytes and incorporated into sarcomeres. Interestingly, HCM mutant Tm produced significantly longer contractions by slowing relaxation, whereas NM mutant Tm produced the opposite effect of accelerated muscle relaxation. We propose slowed relaxation caused by HCM mutant Tm can directly contribute to diastolic dysfunction seen in HCM even without secondary cardiac remodeling. Conversely, hastening of relaxation by NM mutant Tm may shift the force-frequency relationship in skeletal muscle and contribute to muscle weakness seen in NM. Together, these results implicate divergent, abnormal "turning off" of muscle contraction as a cellular basis for the differential pathogenesis of mutant Tm-associated HCM and NM.

Advancing maturation of stem cell‐derived cardiac muscle represents a major barrier to progress in cardiac regenerative medicine. Cardiac muscle maturation involves a myriad of gene, protein, and cell‐based transitions, spanning across all aspects of cardiac muscle form and function. We focused here on a key developmentally controlled transition in the cardiac sarcomere, the functional unit of the heart. Using a gene‐editing platform, human induced pluripotent stem cell (hiPSCs) were engineered with a drug‐inducible expression cassette driving the adult cardiac troponin I (cTnI) regulatory isoform, a transition shown to be a rate‐limiting step in advancing sarcomeric maturation of hiPSC cardiac muscle (hiPSC‐CM) toward the adult state. Findings show that induction of the adult cTnI isoform resulted in the physiological acquisition of adult‐like cardiac contractile function in hiPSC‐CMs in vitro. Specifically, cTnI induction accelerated relaxation kinetics at baseline conditions, a result independent of alterations in the kinetics of the intracellular Ca2+ transient. In comparison, isogenic unedited hiPSC‐CMs had no cTnI induction and no change in relaxation function. Temporal control of adult cTnI isoform induction did not alter other developmentally regulated sarcomere transitions, including myosin heavy chain isoform expression, nor did it affect expression of SERCA2a or phospholamban. Taken together, precision genetic targeting of sarcomere maturation via inducible TnI isoform switching enables physiologically relevant adult myocardium‐like contractile adaptations that are essential for beat‐to‐beat modulation of adult human heart performance. These findings have relevance to hiPSC‐CM structure‐function and drug‐discovery studies in vitro, as well as for potential future clinical applications of physiologically optimized hiPSC‐CM in cardiac regeneration/repair. [ABSTRACT FROM AUTHOR]