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Increased protein kinase C (PKC) activity is associated with heart failure, and can target multiple cardiac troponin I (cTnI) residues in myocytes, including S23/24, S43/45 and T144. In earlier studies, cTnI-S43D and/or -S45D augmented S23/24 and T144 phosphorylation, which suggested there is communication between clusters. This communication is now explored by evaluating the impact of phospho-mimetic cTnI S43/45D combined with S23/24D (cTnIS4D) or T144D (cTnISDTD). Gene transfer of epitope-tagged cTnIS4D and cTnISDTD into adult cardiac myocytes progressively replaced endogenous cTnI. Partial replacement with cTnISDTD or cTnIS4D accelerated the time to peak (TTP) shortening and time to 50% re-lengthening (TTR 50% ) on day 2, but peak shortening was only diminished by cTnIS4D. Extensive cTnIS4D replacement continued to accelerate TTP, and decrease shortening amplitude, while TTR 50% returned to baseline levels on day 4. In contrast, cTnISDTD modestly reduced shortening amplitude and continued to accelerate myocyte TTP and TTR 50% . These results indicate cTnIS43/45 communicates with S23/24 and T144, with S23/24 exacerbating and T144 attenuating the S43/45D-dependent functional deficit. In addition, more severe functional alterations in cTnIS4D myocytes were accompanied by higher levels of secondary phosphorylation compared to cTnISDTD. These results suggest that secondary phosphorylation helps to maintain steady-state contractile function during chronic cTnI phosphorylation at PKC sites., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A phospho-null Ala substitution at protein kinase C (PKC)-targeted cardiac troponin I (cTnI) S43/45 reduces myocyte and cardiac contractile function. The goal of the current study was to test whether cTnIS43/45N is an alternative, functionally conservative substitution in cardiac myocytes. Partial and more extensive endogenous cTnI replacement was similar at 2 and 4 days after gene transfer, respectively, for epitope-tagged cTnI and cTnIS43/45N. This replacement did not significantly change thin filament stoichiometry. In functional studies, there were no significant changes in the amplitude and/or rates of contractile shortening and re-lengthening after this partial (2 days) and extensive (4 days) replacement with cTnIS43/45N. The cTnIS43/45N substitution also was not associated with adaptive changes in the myocyte Ca(2+) transient or in phosphorylation of the protein kinase A and C-targeted cTnIS23/24 site. These results provide evidence that cTnIS43/45N is a functionally conservative substitution, and may be appropriate for use as a phospho-null in rodent models designed for studies on PKC modulation of cardiac performance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Traditional methods for DNA transfection are often inefficient and toxic for terminally differentiated cells, such as cardiac myocytes. Vector-based gene transfer is an efficient approach for introducing exogenous cDNA into these types of primary cell cultures. In this chapter, separate protocols for adult rat cardiac myocyte isolation and gene transfer with recombinant adenovirus are provided and are routinely utilized for studying the effects of sarcomeric proteins on myofilament function.

Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70-80% of endogenous cTnI replaced within 4days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca(2+) sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca(2+) transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: The mechanical environment is a key regulator of function in cardiomyocytes. We studied the role of substrate stiffness on the organization of sarcomeres and costameres in adult rat cardiomyocytes and further examined the resulting changes in cell shortening and calcium dynamics., Methods: Cardiomyocytes isolated from adult rats were plated on laminin-coated polydimethylsiloxane substrates of defined stiffness (255 kPa, 117 kPa, 27 kPa, and 7 kPa) for 48 h. Levels of α-actinin and β1 integrins were determined by immunofluoresence imaging and immunoblotting, both in the absence and presence of the phosphatase inhibitor calyculin A. Quantitative reverse transcriptase polymerase chain reaction was used to measure message levels of key structural proteins (α-actinin, α7 integrin, β1 integrin, vinculin). Sarcomere shortening and calcium dynamics were measured at 2, 24, and 48 h., Results: Overall cardiomyocyte morphology was similar on all substrates. However, well organized sarcomere structures were observed on only the stiffest (255 kPa) and the most compliant (7 kPa) substrates. Levels of α-actinin in cells were the same on all substrates, while message levels of structural proteins were up-regulated on substrates of intermediate stiffness. Inhibition of phosphatase activity blocked the degradation of contractile structures, but altered overall cardiomyocyte morphology. Shortening and calcium dynamics also were dependent on substrate stiffness; however, there was no clear causative relationship between the phenomena., Conclusions: Extracellular matrix stiffness can affect structural remodeling by adult cardiomyocytes, and the resulting contractile activity. These findings illuminate changes in cardiomyocyte function in cardiac fibrosis, and may suggest cardiac-specific phosphatases as a target for therapeutic intervention., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Significant up-regulation of the protein kinase Cβ(II) (PKCβ(II)) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCβ(II) modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCβ(II) protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCβ(II) was distributed in a peri-nuclear pattern and this expression resulted in diminished rates and amplitude of shortening and re-lengthening compared to controls and myocytes expressing dominant negative PKCβ(II) (PKCβDN). Similar decreases were observed in the Ca(2+) transient and the Ca(2+) decay rate slowed in response to caffeine in PKCβ(II)-expressing myocytes. Parallel phosphorylation studies indicated PKCβ(II) targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCβ inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCβ(II) expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCβ(II) increased Ca(2+)-activated, calmodulin-dependent kinase IIδ (CaMKIIδ) expression and enhanced both CaMKIIδ and protein kinase D (PKD) phosphorylation. Phosphorylation of both signaling targets also was resistant to acute inhibition by LY. These later results provide evidence PKCβ(II) modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Vitamin D(3) deficiency enhances cardiac contraction in experimental studies, yet paradoxically this deficiency is linked to congestive heart failure in humans. Activated vitamin D(3) (1alpha,25-dihydroxyvitamin D(3)) or calcitriol, decreases peak force and activates protein kinase C (PKC) in isolated perfused hearts. However, the direct influence of this hormone on adult cardiac myocyte contractile function is not well understood. Our aim is to investigate whether 1alpha,25-dihydroxyvitamin D(3) acutely modulates contractile function via PKC activation in adult rat cardiac myocytes. Sarcomere shortening and re-lengthening were measured in electrically stimulated myocytes isolated from adult rat hearts, and the vitamin D(3) response (10(-10) to 10(-7) M) was compared to shortening observed under basal conditions. Maximum changes in sarcomere shortening and relaxation were observed with 10(-9) M 1alpha,25-dihydroxyvitamin D(3). This dose decreased peak shortening, and accelerated contraction and relaxation rates within 5 min of administration, and changes in the Ca(2+) transient contributed to the peak shortening and relaxation effects. The PKC inhibitor, bis-indolylmaleimide (500 nM) largely blocked the acute influence of the most potent dose (10(-9) M) on contractile function. While peak shortening and shortening rate returned to baseline within 30 min, there was a sustained acceleration of relaxation that continued over 60 min. Phosphorylation of the Ca(2+) regulatory proteins, phospholamban, and cardiac troponin I correlated with the accelerated relaxation observed in response to acute application of 1alpha,25-dihydroxyvitamin D(3). Accelerated relaxation continued to be observed after chronic addition of 1alpha,25-dihydroxyvitamin D(3) (e.g. 2 days), yet this sustained increase in relaxation was not associated with increased phosphorylation of phospholamban or troponin I. These results provide evidence that 1alpha,25-dihydroxyvitamin D(3) directly modulates adult myocyte contractile function, and protein kinase C plays an important signaling role in the acute response. Phosphorylation of key Ca(2+) regulatory proteins by this kinase contributes to the enhanced relaxation observed in response to acute, but not chronic calcitriol.

Obscurin is a recently identified giant multidomain muscle protein (approximately 800 kDa) whose structural and regulatory functions remain to be defined. The goal of this study was to examine the effect of obscurin gene silencing induced by RNA interference on the dynamics of myofibrillogenesis and hypertrophic response to phenylephrine in cultured rat cardiomyocytes. We found that that the adenoviral transfection of short interfering RNA (siRNA) constructs targeting the first coding exon of obscurin sequence resulted in progressive depletion of cellular obscurin. Confocal microscopy demonstrated that downregulation of obscurin expression led to the impaired assembly of new myofibrillar clusters and considerable aberrations of the normal structure of the contractile apparatus. While the establishment of the initial periodic pattern of alpha-actinin localization remained mainly unaffected in siRNA-transfected cells, obscurin depletion did cause the defective lateral alignment of myofibrillar bundles, leading to their abnormal bifurcation, dispersal and multiple branching. Bending of immature myofibrils, apparently associated with the loss of their rigidity, a modified titin pattern, the absence of well-formed A-bands in newly formed contractile structures as documented by a diffuse localization of sarcomeric myosin labeling, and an occasional irregular periodicity of sarcomere spacing were typical of obscurin siRNA-treated cells. These results suggest that obscurin is indispensable for spatial positioning of contractile proteins and for the structural integration and stabilization of myofibrils, especially at the stage of myosin filament incorporation and A-band assembly. This demonstrates a vital role for obscurin in myofibrillogenesis and hypertrophic growth.

A component of multiorgan dysfunction in burned patients is heart failure. Burn trauma induces cytokine synthesis of interleukin (IL) 1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) which can negatively impact cardiac function. Infectious complications are common following severe burn injury. We hypothesized that burn injury and lipopolysaccharide (LPS) exposure independently influence peak cardiomyocyte contraction and cytokine secretion. Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 1, 6, 12, and 24 h after burn, cardiomyocytes were isolated and incubated with increasing LPS doses. Peak sarcomere shortening and contractile velocity parameters were recorded using a variable-rate video camera with sarcomere length detection software. Supernatants were assayed for IL-1beta, IL-6, and TNF-alpha by ELISA. Peak sarcomere shortening was decreased in the burn group at 1, 6, 12, and 24 h after burn. IL-1beta, IL-6, and TNF-alpha levels were increased in cardiomyocytes isolated 1 h after burn compared with sham controls, but returned to sham levels at 6, 12, and 24 h after burn. LPS exposure caused dose-dependent decreases in sarcomere shortening in sham and burn animals. LPS exposure did not produce increased cardiomyocyte cytokine expression. Burn injury diminished peak sarcomere shortening. Whereas exposure to LPS did not have an effect on cardiomyocyte cytokine expression, LPS significantly inhibited sarcomere shortening in a dose-dependent fashion. Combined burn and LPS exposure inhibited sarcomere shortening more than each alone. These results demonstrate that LPS exposure and burn injury independently decrease peak cardiac shortening. These decreases did not directly correlate with the levels of cytokines released in response to each stressor.

Obscurin is a newly identified giant muscle protein whose functions remain to be elucidated. In this study we used high-resolution confocal microscopy to examine the dynamics of obscurin localization in cultures of rat cardiac myocytes during the assembly and disassembly of myofibrils. Double immunolabeling of neonatal and adult rat cells for obscurin and sarcomeric alpha-actinin, the major protein of Z-lines, demonstrated that, during myofibrillogenesis, obscurin is intensely incorporated into M-band areas of A-bands and, to a lesser extent, in Z-lines of newly formed sarcomeres. Presarcomeric structural precursors of myofibrils were intensely immunopositive for alpha-actinin and, unlike mature myofibrils, weakly immunopositive or immunonegative for obscurin. This indicates that most of the obscurin assembles in developing myofibrils after abundant incorporation of alpha-actinin and that massive integration of obscurin occurs at more advanced stages of sarcomere assembly. Immunoreactivity for obscurin in the middle of A-bands and in Z-lines of sarcomeres bridged the gaps between individual bundles of newly formed myofibrils, suggesting that this protein appears to be directly involved in their primary lateral connection and registered alignment into larger clusters. Close sarcomeric localization of obscurin and titin suggests that they may interact during myofibril assembly. Interestingly, the laterally aligned striated pattern of obscurin formed at a stage when desmin, traditionally considered as a molecular linker responsible for the lateral binding and stabilization of myofibrils at the Z-bands, was still diffusely localized. During the disassembly of the contractile system in adult myocytes, disappearance of the cross-striated pattern of obscurin preceded the disorganization of registered alignment and intense breakdown of myofibrils. The cross-striated pattern of desmin typical of terminally differentiated myocytes disappeared before or simultaneously with obscurin. During redifferentiation, as in neonatal myocytes, sarcomeric incorporation of obscurin closely followed that of alpha-actinin and occurred earlier than the striated arrangement of desmin intermediate filaments. The presence of obscurin in the Z-lines and its later assembly into the A/M-bands indicate that it may serve to stabilize and align sarcomeric structure when myosin filaments are incorporated. Our data suggest that obscurin, interacting with other muscle proteins and possibly with the sarcoplasmic reticulum, may have a role as a flexible structural integrator of myofibrils during assembly and adaptive remodeling of the contractile apparatus., (Copyright The Histochemical Society, Inc.)

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.