EF-hand proteins are ubiquitous in cell signaling. Parvalbumin (Parv), the archetypal EF-hand protein, is a high-affinity Ca2+ buffer in many biological systems. Given the centrality of Ca2+ 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: Mg2+ affinity increased, whereas Ca2+ affinity decreased, forming a new ultra-delayed Ca2+ 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 Ca2+ signaling could benefit many biological systems with abnormal Ca2+ handling, including the diseased heart.
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.
Recombinant adeno-associated virus (AAV) is a valuable and often used gene therapy vector. With increased demand for highly purified virus comes the need for a standardized purification procedure that is applicable across many serotypes and includes bioengineered viruses. Currently cesium chloride banding or affinity chromatography are the predominate forms of purification. These approaches expose the final purified virus to toxic contaminants or are highly capsid dependent and may require significant optimization to isolate purified AAV. These methods may also limit crude viral lysate processing volume resulting in a significant loss of viral titer. To circumvent these issues, we have developed an AAV purification protocol independent of toxic compounds, supernatant volume and capsid moiety. This purification method standardizes virus purification across native serotype and bioengineered mosaic capsids.
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.
Previously we have introduced a single cell system allowing long term culturing (1 week) of adult rat ventricular myocytes while maintaining their overall morphology, contractile behaviour and calcium-signalling. Here, we characterize the subcellular morphology of the myocytes, including the Golgi apparatus, endo-/sarcoplasmic reticulum (ER/SR), plasma membrane and mitochondria. Cells were isolated from adult rats following a standard enzymatic procedure. Organelles were labelled using targeted expression of fluorescent proteins, e.g. dsRed1 fused to the subunit VIII of human cytochrome C oxidase for mitochondria, YFP fused to a GPI-anchor for the plasma membrane, YFP fused to ts045G for the Golgi apparatus and dsRed2 fused to calreticulin for the ER/ SR. Complementing this we also applied fluorescent dyes; di-8-ANEPPS for the plasma membrane and MitoTracker Green for the mitochondria. 3-dimensional stacks of individual cells were acquired with a nipkow-disc based confocal microscope. Using both labelling approaches, the analysis of the plasma membrane illustrated a gradual loss of the t-tubules during culturing with cytosolic membrane fragments being present for extended time periods. Mitochondria, which are very prominent and densely packed in cardiac myocytes, underwent an apparent fusion of originally isolated mitochondria, possibly reflecting the loss of t-tubules. While the structure of the ER/SR remained unaltered, the Golgi apparatus underwent a significant redistribution during the culturing time from a wide cytosolic distribution to a perinuclear accumulation. We provide important evidence that cell morphology changes unavoidably occurring in adult cardiac myocytes during long term culture are highly reproducible and thus strongly support the application of such a single cell model in high-content screening applications.
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