Myosin Heavy Chain Isoform Switch Research Articles

Structural and Functional Changes in Skeletal Muscles in an A8V-Troponin C Hypertrophic Cardiomyopathy Knock-In Mouse Model

Missense mutations in the TNNC1 gene encoding cardiac and slow skeletal troponin C (c/ssTnC) are associated with phenotypic outcomes of hypertrophic cardiomyopathy (HCM). The impact of HCM c/ssTnC mutations in skeletal muscle structure and function is unknown; therefore, we examined the effect of an HCM-associated c/ssTnC A8V mutation on myosin heavy chain (MHC) isoform expression, contractile properties of different skeletal muscles, and cardiac autonomic function in a homozygous knock-in mouse (KI-TnC-A8V+/+). The distribution of MHC I/II isoforms in various muscles from 3-month old male WT and KI-TnC-A8V+/+ mice were analyzed by glycerol SDS-PAGE and the muscle/body weight ratios (M/BW) were recorded. Mechanics of contraction in isolated intact soleus muscle, running/endurance capacity and heart rate variability (HRV) were also measured. In comparison to the WT mice, MHC isoform switching was observed in diaphragm (increased MHCIIb, decreased MHCI) and soleus (decreased MHCIIa, increased MHCI) muscles of KI-TnC-A8V+/+ mice; while, the M/BW ratio was unchanged for all tested muscles. The force vs frequency relationship at 15Hz and the t1/2 of relaxation increased in the intact soleus muscle of KI-TnC A8V+/+ mice in comparison to the WT; whereas, no changes in maximal force, time-to-twitch peak, velocity and power output were observed. In endurance and HRV tests, KI-TnC-A8V+/+ mice had a lower running distance capacity and their global autonomic, cardiovagal, and baroreflex activities were significantly reduced compared to WT. In spite of the increase in type I fibers in soleus muscle of mice expressing c/ssTnC A8V, they display reduced endurance capacity. These findings suggest that the slow skeletal muscle is trying to compensate for the cardiac dysfunction caused by the c/ssTnC A8V+/+ mutation. Supported by NIH HL103840 (JRP).

Cardiac remodeling and physical training post myocardial infarction.

After myocardial infarction (MI), the heart undergoes extensive myocardial remodeling through the accumulation of fibrous tissue in both the infarcted and noninfarcted myocardium, which distorts tissue structure, increases tissue stiffness, and accounts for ventricular dysfunction. There is growing clinical consensus that exercise training may beneficially alter the course of post-MI myocardial remodeling and improve cardiac function. This review summarizes the present state of knowledge regarding the effect of post-MI exercise training on infarcted hearts. Due to the degree of difficulty to study a viable human heart at both protein and molecular levels, most of the detailed studies have been performed by using animal models. Although there are some negative reports indicating that post-MI exercise may further cause deterioration of the wounded hearts, a growing body of research from both human and animal experiments demonstrates that post-MI exercise may beneficially alter the course of wound healing and improve cardiac function. Furthermore, the improved function is likely due to exercise training-induced mitigation of renin-angiotensin-aldosterone system, improved balance between matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1, favorable myosin heavy chain isoform switch, diminished oxidative stress, enhanced antioxidant capacity, improved mitochondrial calcium handling, and boosted myocardial angiogenesis. Additionally, meta-analyses revealed that exercise-based cardiac rehabilitation has proven to be effective, and remains one of the least expensive therapies for both the prevention and treatment of cardiovascular disease, and prevents re-infarction.

Ranolazine prevents INaL enhancement and blunts myocardial remodelling in a model of pulmonary hypertension.

Pulmonary arterial hypertension (PAH) reflects abnormal pulmonary vascular resistance and causes right ventricular (RV) hypertrophy. Enhancement of the late sodium current (INaL) may result from hypertrophic remodelling. The study tests whether: (i) constitutive INaL enhancement may occur as part of PAH-induced myocardial remodelling; (ii) ranolazine (RAN), a clinically available INaL blocker, may prevent constitutive INaL enhancement and PAH-induced myocardial remodelling. PAH was induced in rats by a single monocrotaline (MCT) injection [60 mg/kg intraperitoneally (i.p.)]; studies were performed 3 weeks later. RAN (30 mg/kg bid i.p.) was administered 48 h after MCT and washed-out 15 h before studies. MCT increased RV systolic pressure and caused RV hypertrophy and loss of left ventricular (LV) mass. In the RV, collagen was increased; myocytes were enlarged with T-tubule disarray and displayed myosin heavy chain isoform switch. INaL was markedly enhanced; diastolic Ca(2+) was increased and Ca(2+) release was facilitated. K(+) currents were down-regulated and APD was prolonged. In the LV, INaL was enhanced to a lesser extent and cell Ca(2+) content was strongly depressed. Electrical remodelling was less prominent than in the RV. RAN completely prevented INaL enhancement and limited most aspects of PAH-induced remodelling, but failed to affect in vivo contractile performance. RAN blunted the MCT-induced increase in RV pressure and medial thickening in pulmonary arterioles. PAH induced remodelling with chamber-specific aspects. RAN prevented constitutive INaL enhancement and blunted myocardial remodelling. Partial mechanical unloading, resulting from an unexpected effect of RAN on pulmonary vasculature, might contribute to this effect.

A Transgenic Platform for Testing Drugs Intended for Reversal of Cardiac Remodeling Identifies a Novel 11βHSD1 Inhibitor Rescuing Hypertrophy Independently of Re-Vascularization

RationaleRescuing adverse myocardial remodeling is an unmet clinical goal and, correspondingly, pharmacological means for its intended reversal are urgently needed.ObjectivesTo harness a newly-developed experimental model recapitulating progressive heart failure development for the discovery of new drugs capable of reversing adverse remodeling.Methods and ResultsA VEGF-based conditional transgenic system was employed in which an induced perfusion deficit and a resultant compromised cardiac function lead to progressive remodeling and eventually heart failure. Ability of candidate drugs administered at sequential remodeling stages to reverse hypertrophy, enlarged LV size and improve cardiac function was monitored. Arguing for clinical relevance of the experimental system, clinically-used drugs operating on the Renin-Angiotensin-Aldosterone-System (RAAS), namely, the ACE inhibitor Enalapril and the direct renin inhibitor Aliskerin fully reversed remodeling. Remodeling reversal by these drugs was not accompanied by neovascularization and reached a point-of-no-return. Similarly, the PPARγ agonist Pioglitazone was proven capable of reversing all aspects of cardiac remodeling without affecting the vasculature. Extending the arsenal of remodeling-reversing drugs to pathways other than RAAS, a specific inhibitor of 11β-hydroxy-steroid dehydrogenase type 1 (11β HSD1), a key enzyme required for generating active glucocorticoids, fully rescued myocardial hypertrophy. This was associated with mitigating the hypertrophy-associated gene signature, including reversing the myosin heavy chain isoform switch but in a pattern distinguishable from that associated with neovascularization-induced reversal.ConclusionsA system was developed suitable for identifying novel remodeling-reversing drugs operating in different pathways and for gaining insights into their mechanisms of action, exemplified here by uncoupling their vascular affects.

Myosin Heavy Chain Isoform Switching in Skeletal Muscles in an A8V-Troponin C Hypertrophic Cardiomyopathy Knock-In Mouse Model

Missense mutations in the TNNC1 gene encoding cardiac and slow skeletal troponin C (c/ssTnC) are associated with phenotypic outcomes of hypertrophic cardiomyopathy (HCM). Since the impact of HCM c/ssTnC mutations during skeletal muscle development and function is unknown, we addressed whether changes in the myosin heavy chain (MHC) isoforms can occur outside of the heart. We hypothesized that the ratio of MHC I to MHC II in various skeletal muscles change due to an HCM-associated c/ssTnC A8V mutation in a knock-in homozygous mouse (KI-TnC-A8V+/+). The distribution of MHC I/II isoforms in diaphragm (D), soleus (S), tibialis anterior (TA), extensor digitorum longus (EDL) and quadriceps (Q) isolated from 3- and 9-month (mo) old male WT and KI-TnC-A8V+/+ mice were analyzed by glycerol SDS-PAGE. The muscle/body weight ratio (M/BW) and running capacity were also measured. At 3 mo, MHC isoform switching was observed in D (increased MHCIIb), S (decreased MHCIIa, increased MHCI) and EDL (increased MHCIIa/x, decreased MHCIIb) of KI-TnC-A8V+/+ compared to WT. TA M/BW is increased in 3- and 9-mo KI-TnC-A8V+/+ mice compared to WT at the same age. Pairwise comparison of M/BW from 3- vs 9-mo KI-TnC-A8V+/+ mice showed a significant decrease for TA and Q and an increase for EDL, and no change for any WT muscles. In running tests, KI-TnC-A8V+/+ mice had a lower capacity to run distances compared to WT (281 vs 413 meters). Thus, changes in MHC isoforms in the skeletal muscles of mice expressing c/ssTnC A8V are associated with reduced exercise endurance, suggesting that skeletal muscle remodeling may contribute to the development of HCM phenotype. Future studies will examine MHC isoforms at 9 mo and Ca2+-activated myofibrilar Mg2+-ATPase rates from skeletal muscles in KI-TnC-A8V+/+ mice.

The Emerging Role of miR-208a in the Heart

MicroRNAs (miRNAs) are a class of regulatory small RNAs that have fundamentally transformed our understanding of how gene networks are regulated representing one of the most exciting areas of the modern cardiology research. Among all known miRNAs, miR-208a is one of the most important heart-enriched miRNA playing a crucial role in the heart health and disease. miR-208a is a member of a miRNA family that also included miR-208b and is encoded by an intronic region of the Myh6 gene. Within the heart, miR-208a and miR-208b are involved in the regulation of the myosin heavy chain isoformswitch during development and in pathophysiological conditions. miR-208a is sufficient to induce arrhythmias, cardiac remodeling, and to regulate the expression of hypertrophy pathway components and the cardiac conduction system. Recently, the identification of miR-208a in the bloodstream has led to a great clinical interest to use this molecule as a potential noninvasive biomarker of myocardial injuries.

Metabolic Remodeling in the Hypertrophic Heart

When it comes to fuel for energy production, it is commonly accepted that the heart is an omnivore, capable of oxidizing a wide range of carbon substrates, and that this metabolic plasticity is necessary to maintain a high and variable workload in the midst of an ever-changing hormonal and nutritional state.1 It is also widely appreciated that shifts in substrate utilization in the heart occur in response to chronic metabolic and hemodynamic stresses.2–5 What is less clear, however, is whether such chronic metabolic shifts should be considered a cause or consequence in the pathogenesis of contractile dysfunction. In the case of diabetes mellitus, the heart exhibits an increased dependence on fatty acids for oxidative energy production, and this increase in lipid metabolism has been proposed to significantly contribute to the etiology of impaired cardiac function.6 Similarly, increased rates of fatty acid oxidation immediately after an ischemic event have been implicated in exacerbation of reperfusion injury.7 However, in both of these pathophysiological settings, compelling data to support a direct causal relationship between contractile abnormalities and metabolic dysregulation remain elusive. Article, see p 728 In response to pressure overload–induced cardiac hypertrophy, the heart reverts more toward a fetal-like metabolic profile, indicative of a decrease in fatty acid oxidation (concomitant with an increased reliance on carbohydrates for oxidative energy metabolism).3 It has been suggested that this substrate shift, which is associated with reactivation of other fetal-like hallmarks (eg, myosin heavy chain isoform switching), contributes to the progression to overt contractile failure.8 Dietary, pharmacological, and genetic strategies have been used in attempts to provide insight regarding the impact that substrate shifts have on pressure overload–induced remodeling; however, mixed results have been reported. …

Dimethylthiourea normalizes velocity-dependent, but not force-dependent, index of ventricular performance in diabetic rats: role of myosin heavy chain isozyme

Hydroxyl radicals and hydrogen peroxide are involved in the pathogenesis of systolic dysfunction in diabetic rats, but the precise mechanisms and the effect of antioxidant therapy in diabetic subjects have not been elucidated. We aimed to evaluate the effects of dimethylthiourea (DMTU), a potent hydroxyl radical scavenger, on both force-dependent and velocity-dependent indexes of cardiac contractility in streptozotocin (STZ)-induced early and chronic diabetic rats. Seventy-two hours and 8 wk after STZ (55 mg/kg) injection, diabetic rats were randomized to either DMTU (50 mg x kg(-1) x day(-1) ip) or vehicle treatment for 6 and 12 wk, respectively. All rats were then subjected to invasive hemodynamic studies. Maximal systolic elastance (E(max)) and maximum theoretical flow (Q(max)) were assessed by curve-fitting techniques in terms of the elastance-resistance model. Both normalized E(max) (E(maxn)) and afterload-adjusted Q(max) (Q(maxad)) were depressed in diabetic rats, concomitant with altered myosin heavy chain (MHC) isoform composition and its upstream regulators, such as myocyte enhancer factor-2 (MEF-2) and heart autonomic nervous system and neural crest derivatives (HAND). In chronic diabetic rats, DMTU markedly attenuated the impairment in Q(maxad) and normalized the expression of MEF-2 and eHAND and MHC isoform composition but exerted an insignificant benefit on E(maxn). Regarding preventive treatment, DMTU significantly ameliorated both E(maxn) and Q(maxad) in early diabetic rats. In conclusion, our study shows that DMTU has disparate effects on Q(maxad) and E(maxn) in chronic diabetic rats. The advantage of DMTU in chronic diabetic rats might involve normalization of MEF-2 and eHAND, as well as reversal of MHC isoform switch.

Artificial Selection for Whole Animal Low Intrinsic Aerobic Capacity Co-Segregates with Hypoxia-Induced Cardiac Pump Failure

Oxygen metabolism is a strong predictor of the general health and fitness of an organism. In this study, we hypothesized that a divergence in intrinsic aerobic fitness would co-segregate with susceptibility for cardiovascular dysfunction. To test this hypothesis, cardiac function was assessed in rats specifically selected over nineteen generations for their low (LCR) and high (HCR) intrinsic aerobic running capacity. As an integrative marker of native aerobic capacity, run time to exhaustion between LCR and HCR rats had markedly diverged by 436% at generation nineteen of artificial selection. In vivo assessment of baseline cardiac function by echocardiography and catheter-based conductance micromanometry showed no marked difference in cardiac performance. However, when challenged by exposure to acute hypoxia, cardiac pump failure occurred significantly earlier in LCR rats compared to HCR animals. Acute cardiac decompensation in LCR rats was exclusively due to the development of intractable irregular ventricular contractions. Analysis of isolated cardiac myocytes showed significantly slower sarcomeric relaxation and delayed kinetics of calcium cycling in LCR myocytes compared to HCR myocytes. This study also revealed that artificial selection for low native aerobic capacity is a novel pathologic stimulus that results in myosin heavy chain isoform switching in the heart as shown by increased levels of β-MHC in LCR rats. Together, these results provide evidence that alterations in sub-cellular calcium handling and MHC isoform composition are associated with susceptibility to compensatory cardiac remodeling and hypoxia induced pump failure in animals with low intrinsic aerobic capacity.

Functional overload in ground squirrel plantaris muscle fails to induce myosin isoform shifts

We performed 2 wk of mechanical overload by synergist ablation on plantaris muscles from a small rodent hibernator, Spermophilus lateralis. While this muscle displays prominent myosin heavy-chain (MyHC) isoform shifts during hibernation, sensitivity to mechanical loading as a stimulus for muscle mass and isoform plasticity has not been demonstrated. Squirrel muscles, whether during hibernation or not, potentially are less sensitive to mechanical unloading, but we hypothesized that increased loading would produce the typical mammalian response of greater plantaris mass and MyHC shifts. Mechanical overload produced a 50% increase in muscle mass but, surprisingly, no changes in MyHC isoform protein or mRNA expression, despite previously observed fast-to-slow MyHC isoform switching during hibernation. Citrate synthase enzyme activity, as well as mRNA expression of creatine kinase and the muscle growth factor myostatin, were all unchanged. The mRNA expression of critical muscle atrophy genes decreased by 50% during hypertrophy, including ubiquitin ligases MuRF1 and MAFbx, and the related transcription factor FOXO-1a. Insulin-like growth factor (IGF-1) and hypoxia-inducible factor (HIF-1alpha) mRNA expression was elevated by 400% and 150%. Fast-to-slow MyHC isoform shifts appear unnecessary to support the increased recruitment of the plantaris muscle, shifts which are seen in other rodent models. Our results are consistent with muscular activity during interbout arousals as a potential mechanism to preserve muscle mass, but illustrate the primary importance of other seasonal factors besides patterns of muscle activation which must act in concert to alter MyHC isoforms and muscle fiber type during hibernation.

Time course and mechanisms of left ventricular systolic and diastolic dysfunction in monocrotaline-induced pulmonary hypertension

Although pulmonary hypertension (PH) selectively overloads the right ventricle (RV), neuroendocrine activation and intrinsic myocardial dysfunction have been described in the left ventricle (LV). In order to establish the timing of LV dysfunction development in PH and to clarify underlying molecular changes, Wistar rats were studied 4 and 6 weeks after subcutaneous injection of monocrotaline (MCT) 60 mg/kg (MCT-4, n = 11; MCT-6, n = 11) or vehicle (Ctrl-4, n = 11; Ctrl-6, n = 11). Acute single beat stepwise increases of systolic pressure were performed from baseline to isovolumetric (LVPiso). This hemodynamic stress was used to detect early changes in LV performance. Neurohumoral activation was evaluated by measuring angiotensin-converting enzyme (ACE) and endothelin-1 (ET-1) LV mRNA levels. Cardiomyocyte apoptosis was evaluated by TUNEL assay. Extracellular matrix composition was evaluated by tenascin-C mRNA levels and interstitial collagen content. Myosin heavy chain (MHC) composition of the LV was studied by protein quantification. MCT treatment increased RV pressures and RV/LV weight ratio, without changing LV end-diastolic pressures or dimensions. Baseline LV dysfunction were present only in MCT-6 rats. Afterload elevations prolonged tau and upward-shifted end-diastolic pressure dimension relations in MCT-4 and even more in MCT-6. MHC-isoform switch, ACE upregulation and cardiomyocyte apoptosis were present in both MCT groups. Rats with severe PH develop LV dysfunction associated with ET-1 and tenascin-C overexpression. Diastolic dysfunction, however, could be elicited at earlier stages in response to hemodynamic stress, when only LV molecular changes, such as MHC isoform switch, ACE upregulation, and myocardial apoptosis were present.

Low Carbohydrate/High-Fat Diet Attenuates Cardiac Hypertrophy, Remodeling, and Altered Gene Expression in Hypertension

The effects of dietary fat intake on the development of left ventricular hypertrophy and accompanying structural and molecular remodeling in response to hypertension are not understood. The present study compared the effects of a high-fat versus a low-fat diet on development of left ventricular hypertrophy, remodeling, contractile dysfunction, and induction of molecular markers of hypertrophy (ie, expression of mRNA for atrial natriuretic factor and myosin heavy chain beta). Dahl salt-sensitive rats were fed either a low-fat (10% of total energy from fat) or a high-fat (60% of total energy from fat) diet on either low-salt or high-salt (6% NaCl) chow for 12 weeks. Hearts were analyzed for mRNA markers of ventricular remodeling and activities of the mitochondrial enzymes citrate synthase and medium chain acyl-coenzyme A dehydrogenase. Similar levels of hypertension were achieved with high-salt feeding in both diet groups (systolic pressure of approximately 190 mm Hg). In hypertensive rats fed low-fat chow, left ventricular mass, myocyte cross-sectional area, and end-diastolic volume were increased, and ejection fraction was decreased; however, these effects were not observed with the high-fat diet. Hypertensive animals on low-fat chow had increased atrial natriuretic factor mRNA, myosin heavy chain isoform switching (alpha to beta), and decreased activity of citrate synthase and medium chain acyl-coenzyme A dehydrogenase, which were all attenuated by high-fat feeding. In conclusion, increased dietary lipid intake can reduce cardiac growth, left ventricular remodeling, contractile dysfunction, and alterations in gene expression in response to hypertension.

CRNK Gene Transfer Improves Function and Reverses the Myosin Heavy Chain Isoform Switch During Post‐MI Remodeling

PYK2 is a Ca2+-dependent nonreceptor tyrosine kinase that is involved in left ventricular hypertrophy (LVH) and heart failure. We and others have previously investigated PYK2's function in vitro using cultured rat ventricular myocytes. However, the function of PYK2 in the in vivo adult heart remains unknown. Here we evaluated the effect of PYK2 inhibition following myocardial infarction (MI) using adenoviral (Adv) overexpression of the C-terminal domain of PYK2, known as CRNK. CRNK functions as a dominant-negative inhibitor, presumably by displacing PYK2 from focal adhesions and costameres. Male Sprague-Dawley rats (~300g) underwent permanent left coronary artery ligation. One wk post-MI, either Adv-GFP (n=35) or Adv-CRNK (n=28) was administered (1010 pfu, 0.1ml) via catheter-based, Optison®-mediated gene transfer. LV function and infarct size were evaluated by echocardiography 1 and 3 wk after gene transfer, and LV tissue was analyzed by real-time RT-PCR and Western blotting. CRNK expression was readily detected by Western blotting 1 wk following gene transfer. Adv-CRNK improved overall survival (P=0.03; Logrank Test) and LV fractional shortening (23±2% vs. 31±2% for Adv-GFP vs. Adv-CRNK infected animals, respectively). Whereas MI hearts exhibited increased β-, and decreased α-myosin heavy chain (MHC) mRNA espression characteristic of LVH, Adv-CRNK reversed the MHC isoenzyme switch (3.3±1.4 fold increase in αMHC; 0.4±0.1 fold decrease in βMHC; P<0.05 for both). In summary, CRNK gene transfer imparts a functional benefit and alters MHC gene expression suggesting an attenuation of LV remodeling post-MI. Supported by the Dr. Ralph and Marian Falk Medical Research Trust.

Functional Consequences of Sarcomeric Protein Abnormalities in Failing Myocardium

Sarcomeric protein abnormalities have been recognized for many years in heart failure due to dilated cardiomyopathy (DCM). In contrast, virtually nothing is known about myofilament abnormalities in heart failure occurring in association with diastolic dysfunction. With the exception of sarcomeric protein mutations that cause DCM, the most important mechanism of myofilament dysfunction in DCM is probably altered post-translational modification, in particular the phosphorylation state of troponins I and T and possibly myosin light chain. Other modifications, including oxidation and glycation, may also play a role. Myosin heavy chain isoform switching occurs in human heart failure, but its functional significance is uncertain. Myofilament abnormalities contribute significantly to myocardial dysfunction in DCM, although their relative importance compared with abnormal calcium handling is debated. One consistent functional abnormality in DCM is increased myofilament calcium sensitivity of tension generation, which contributes to slowed or incomplete relaxation. More recently, decreases in the optimal frequency of myofilament work and power generation have been recognized. These changes may contribute to depression of the force-frequency relation in DCM. Altered mechanoenergetics constitute one of the most important manifestations of myofilament dysfunction in heart failure. DCM and hemodynamic overload are associated with more economical and efficient energy utilization by the contractile machinery, which may be protective of the myocardium. This change is strongly associated with depressed myofibrillar ATPase activity. We speculate that the effectiveness of mechanical therapies such as resynchronization may at least in part be related to improved mechanical function without loss of this mechanoenergetic advantage.

Brief Exposure to Testosterone Is Sufficient to Induce Sex Differences in the Rabbit Masseter Muscle

In order to evaluate whether testosterone is responsible for inducing long-lasting sex differences in myosin heavy chain gene expression in the rabbit masseter muscle, we castrated young adult animals and administered supra-physiologic doses of testosterone for 3 or 6 weeks duration. Biopsies were taken from the masseter muscles of these animals at the time of castration and 3, 6 and 9 weeks later. Both immunoblot and immunohistochemical analyses of the myosin heavy chain (MyHC) isoform content of these muscle samples were performed. Exposure to testosterone for either duration resulted in a dramatic decrease in the content of the cardiac alpha MyHC isoform and a comparable increase in the content of the IIa MyHC isoform. The decrease in the cardiac alpha MyHC isoform content persisted for as long as 6 weeks after the end of treatment, but the increase in the content of the IIa MyHC isoform declined to normal during this time. Significant numbers of fibers were found containing both the cardiac alpha (and slow) and the IIa isoforms. Several fibers were encountered that contained both IIa and, presumably, the IIx isoform. Thus, a brief exposure to testosterone during postnatal maturation is able to produce a long lasting myosin heavy chain isoform switch that is similar in magnitude to that found during normal development.

Effects of myosin heavy chain isoform switching on Ca2+-activated tension development in single adult cardiac myocytes.

Cardiac myosin heavy chain (MHC) isoforms are known to play a key role in defining the dynamic contractile behavior of the heart during development. It remains unclear, however, whether cardiac MHC isoforms influence other important features of cardiac contractility, including the Ca2+ sensitivity of isometric tension development. To address this question, adult rats were treated chemically to induce the hypothyroid state and cause a transition in the ventricular cardiac MHC isoform expression pattern from predominantly the alpha-MHC isoform to exclusively the beta-MHC isoform. We found a significant desensitization in the Ca2+ sensitivity of tension development in beta-MHC-expressing ventricular myocytes (pCa50=5. 51+/-0.03, where pCa is -log[Ca2+], and pCa50 is pCa at which tension is one-half maximal) compared with that in predominantly alpha-MHC-expressing myocytes (pCa50=5.68+/-0.05). No differences between the 2 groups were observed in the steepness of the tension-pCa relationship or in the maximum isometric force generated. Instantaneous stiffness measurements were made that provide a relative measure of changes in the numbers of myosin crossbridges attached to actin during Ca2+ activation. Results showed that the relative stiffness-pCa relationship was shifted to the right in beta-MHC-expressing myocytes compared with the alpha-MHC-expressing cardiac myocytes (pCa50=5.47+/-0.05 versus 5.76+/-0.05, respectively). We conclude that MHC isoform switching in adult cardiac myocytes alters the Ca2+ sensitivity of stiffness and tension development. These results suggest that the activation properties of the thin filament are in part MHC isoform dependent in cardiac muscle, indicating an additional role for MHC isoforms in defining cardiac contractile function.

Atrial natriuretic factor and brain natriuretic peptide gene expression in the spontaneous hypertensive rat during postnatal development

The increase in natriuretic peptides (NP), atrial natriuretic factor (ANF), and brain natriuretic peptide (BNP) production and release by cardiocytes that occurs in hypertension has been considered to be a compensatory mechanism against ventricular overload. Studies on NP production in the spontaneously hypertensive rat (SHR), an experimental model of human hypertension, have produced controversial results and were carried out when hypertension was already established (> 17 weeks). At this time, age-related physiologic and molecular changes in cardiac muscle are difficult to separate from those related to hypertension, ie, increased ANF production and plasma levels. In addition, most of the studies used male rats because the rate of increase in arterial blood pressure—as well as the level to which it rises—is greater in males than in females. Studies of a similar nature using female SHR are not available. The aim of this work was to determine 1) whether ANF and BNP production and secretion increase with the development of hypertension in genetically hypertensive rats; 2) whether a sexual dimorphism in ANF and BNP production and secretion is present in the genetically hypertensive rat during the development of hypertension; and 3) whether the demand for ANF and BNP is the same from each chamber of the heart under these experimental conditions. Age-matched male and female SHR, Wistar-Kyoto (WKY), and Sprague Dawley (SD) rats at 2, 4, and 8 weeks of age were used. The normotensive SD were included to provide a wider basis for baseline findings, as WKY rats are not always a suitable control for SHR due to genetic variations. Natriuretic peptide plasma levels and tissue content were measured by radioimmunoassay. ANF, BNP, as well as α- and β-myosin heavy chain (MHC) mRNA were estimated by Northern blot analysis. Blood pressure (BP) of more than 150 mm Hg was found only in 8-week-old male SHR. Plasma immunoreactive (ir)ANF and irBNP increased significantly at puberty (8 weeks) in both male and female SHR. The earliest molecular change encountered during the development of hypertension was a significant increase in BNP mRNA in the right and left atria from both male and female 8-week-old SHR. In the ventricles from both male and female SHR, there was no increase in the ratio of left ventricular wet weight/body weight, no increase in ventricular ANF mRNA transcripts, and no myosin heavy chain isoform switch (a protein marker of hypertrophy). irBNP ventricular concentration, however, increased significantly in both male and female SHR, but only in female SHR was there a concomitant increase in BNP mRNA. These results suggest that 1) ANF and BNP production are not coordinated in all cardiac compartments during the development of hypertension; 2) upregulation of BNP in the atria from male and female SHR is the earliest event detected at 8 weeks; 3) the prehypertensive stage, in the genetically hypertensive female rats, is associated with an increase in ventricular irBNP concentration and BNP mRNA; 4) there is a dissociation between BP and plasma levels of NP; and 5) as well, there is a dissociation between NP gene expression and MHC isoform switch. The regulation of NP is not coordinated in either gender during the development of hypertension. The activation of the BNP gene in female SHR suggests that BNP might play an important role at the onset of hypertension.

Hormonal and neurogenic control of Na-K-ATPase and myosin isoforms in neonatal rat cardiac myocytes.

In the rat heart there is a postnatal switch in the expression of isoforms of both Na-K-ATPase and myosin heavy chain (MHC). Here we investigated factors controlling isoform changes in cultures of neonatal cardiomyocytes. In serum-free medium, the compositions of either Na-K-ATPase or MHC isoforms resembled the neonatal phenotype. Thyroid hormone induced the MHC isoform switch but increased expression of all Na-K-ATPase isoforms to various extents. Dexamethasone failed to induce the MHC switch and inhibited Na-K-ATPase alpha 1 isoform expression without inducing the other isoforms. With both hormones, the adult phenotype for both MHC and Na-K-ATPase was seen but with low Na-K-ATPase alpha 2. The paucity of alpha 2 protein was not predicted by studies of mRNA levels. In serum, there was a gradual decline of Na-K-ATPase alpha 3 and the appearance of alpha 2, but again at a relatively low level. Expression of Na-K-ATPase alpha 2 was significantly upregulated when cardiomyocytes were cocultured with sympathetic neurons from superior cervical ganglia, without changes in the MHC isoforms. Thus innervation is postulated to play a specific role in modulating Na-K-ATPase gene expression.

Gender‐related differences in the regulatory influence of thyroid hormone on the expression of myosin isoforms in young and old rats

The effects of 4 weeks of thyroid hormone (3.5.3'-triiodothyronine. T3) treatment on the expression of myosin heavy chain (MyHC) isoforms were examined in young (3-6 months; and old (20-24 months) female rats, and compared with those in age-matched male rats (Larsson et al. 1995). In control rats ageing was associated with a type IIA to I MyHC isoform switching in the slow-twitch soleus and a type IIB to IIX MyHC isoform switching in the fast-twitch extensor digitorum longus muscle (EDL). Gender- and muscle-specific differences were observed in the regulation of MyHC isoforms by T3. In the soleus, dramatic downregulation of the type I and upregulation of the type IIA MyHC isoform were observed in both females and males, but upregulation of the IIX MyHC isoform was observed only in male rats. In EDL, T3 treatment had no significant influence on the MyHC isoform composition in the males irrespective of the age of the animal. In the females, on the other hand, T3 treatment resulted in a significant MyHC transformation from IIA to IIB, probably via IIX myosin, in spite of the fact that type IIA mRNA has been reported to be downregulated in both females and males. It is concluded that the regulation of MyHC isoforms by thyroid hormone differs between females and males, presumably as a result of a gender-related difference in the translational or post-translational regulation of MyHC synthesis.

Left atrial mechanical and biochemical adaptation to pacing induced heart failure

To determine the left atrial mechanical and biochemical adaptations to congestive heart failure, 10 dogs with rapid atrial and ventricular pacing and seven control dogs were studied. Animals were instrumented with left atrial sonomicrometers and micromanometers and left atrial pressure-volume relationships were generated by phenylephrine boluses for maximum elastance (Emax) and end systolic elastance (Ees) calculations. Left atrial maximum volume, ejection fraction, and mean circumferential fibre shortening (Vcf) were compared at matched left atrial pressure. At necropsy, myosin heavy chain (MHC) isoforms from the left atrial body and appendage were separated with SDS-PAGE, stained with monoclonal antibodies to alpha and beta MHC, and quantified with laser densitometry. Left atrial ejection fraction and Vcf were significantly lower and maximum atrial volume and atrial systolic stroke volume were significantly greater in heart failure than in control. Emax was not significantly altered in heart failure, at 5.9(SD 2.9) v 4.5(1.6) mm Hg.ml-1 in controls. However, Vcf was lower (P < 0.05) and the A loop pressure-volume area (an index of eternal mechanical work performed by the left atrium) was greater (P < 0.05) in heart failure than in control dogs. The percent beta MHC in the left atrial body was greater in dogs with heart failure than in controls, at 42.6(9.8) v 17.3(9.0)%, P < 0.05. By contrast there was no significant beta MHC isoform switch in the left atrial appendage [14.4(7.6) v 17.9(9.7)%]. In this model of left atrial pressure and volume overload, there is significant upregulation of beta MHC in the left atrial body but not in the appendage and this isoform switch is associated with decreased velocity of left atrial contraction, increased atrial mechanical work, and unchanged force generation.